Cerebral Angiodystonia

Zh Nevropatol Psikhiatr Im S S Korsakova. 1990;90(7):108-12.

Regional cerebral angiodystonia in the practice of a neuropathologist and therapist.

[Article in Russian]

Pokalev GM, Raspopina LA.

Altogether 108 patients with regional cerebral angiodystonia were examined using rheoencephalography, measurements of temporal and venous pressure and functional tests (nitroglycerin and bicycle ergometry). Three variants of abnormalities connected with regional cerebral angiodystonia were distinguished: dysfunction of the inflow, derangement of the venous outflow, and initial functional venous hypertonia. The patients were treated with nonmedicamentous therapy (electroanalgesia, magnetotherapy, iontotherapy).

Central Nervous System Disorders

Logo of nrr

Neural Regen Res. 2016 Dec; 11(12): 1888–1895. doi:  10.4103/1673-5374.195277 PMCID: PMC5270416

Extremely low frequency electromagnetic fields stimulation modulates autoimmunity and immune responses: a possible immuno-modulatory therapeutic effect in neurodegenerative diseases

Fabio Guerriero, M.D., Ph.D.1,2,* and Giovanni Ricevuti1,21Department of Internal Medicine and Medical Therapy, Section of Geriatrics, University of Pavia, Pavia, Italy 2Azienda di Servizi alla Persona, Istituto di Cura Santa Margherita of Pavia, Pavia, Italy *Correspondence to: Fabio Guerriero, ti.aivapidatisrevinu@10oreirreug.oibaf.

Author contributions: All authors contributed to developing the concepts, designing the structure, and writing/revising the manuscript, and approved the final version before submission and agree to be accountable. Author information ? Article notes ? Copyright and License information ? Accepted 2016 Nov 25. Copyright : © Neural Regeneration Research This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

Increasing evidence shows that extremely low frequency electromagnetic fields (ELF-EMFs) stimulation is able to exert a certain action on autoimmunity and immune cells. In the past, the efficacy of pulsed ELF-EMFs in alleviating the symptoms and the progression of multiple sclerosis has been supported through their action on neurotransmission and on the autoimmune mechanisms responsible for demyelination. Regarding the immune system, ELF-EMF exposure contributes to a general activation of macrophages, resulting in changes of autoimmunity and several immunological reactions, such as increased reactive oxygen species-formation, enhanced phagocytic activity and increased production of chemokines. Transcranial electromagnetic brain stimulation is a non-invasive novel technique used recently to treat different neurodegenerative disorders, in particular Alzheimer’s disease. Despite its proven value, the mechanisms through which EMF brain-stimulation exerts its beneficial action on neuronal function remains unclear. Recent studies have shown that its beneficial effects may be due to a neuroprotective effect on oxidative cell damage. On the basis of in vitro and clinical studies on brain activity, modulation by ELF-EMFs could possibly counteract the aberrant pro-inflammatory responses present in neurodegenerative disorders reducing their severity and their onset. The objective of this review is to provide a systematic overview of the published literature on EMFs and outline the most promising effects of ELF-EMFs in developing treatments of neurodegenerative disorders. In this regard, we review data supporting the role of ELF-EMF in generating immune-modulatory responses, neuromodulation, and potential neuroprotective benefits. Nonetheless, we reckon that the underlying mechanisms of interaction between EMF and the immune system are still to be completely understood and need further studies at a molecular level.Keywords: electromagnetic fields, Alzheimer’s disease, transcranial magnetic stimulation, autoimmunity, immunomodulation

Introduction

The etiology of neurodegenerative diseases is multifactorial. Genetic polymorphisms, increasing age and environmental cues are recognized to be primary risk factors. Although different neuronal cell populations are affected across diverse neurodegenerative disorders, hallmark protein modifications is a common feature that supports the differential disease diagnosis and provides a mechanistic basis to gauge disease progression (Bossy-Wetzel et al., 2004).

It is becoming increasingly clear that, particularly for chronic neurodegenerative disorders occurring late in life, a complex combination of risk factors can initiate disease development and modify proteins that have a physiological function into ones with pathological roles via a number of defined mechanisms (Moreno-Gonzalez and Soto, 2011).

Amyloid-beta plaques and tau protein tangles – hallmarks of the pathology – are most likely a non-specific result of the disease process, rather than a cause (Lee et al., 2007). A large body of evidence supports the direct contribution of inflammation in the development and progression of neurodegeneration (Tweedie et al., 2007). A common denominator in the occurrence of different pathogenic mechanisms is oxidative stress accompanied by redox dysregulation, which have a role in mitochondrial dysfunction, toxicity, missignalling by calcium, glial cell dysfunction and neuroinflammation itself. Each of these can influence one another at multiple different levels, and hence oxidative stress can both be secondary to them as well as have a primary part in their initiation (von Bernhardi and Eugenin, 2012).

In the last years, evidence are remarkably revealing that Alzheimer’s disease (AD) has an autoimmune component (D’Andrea, 2005). In older patients the presence of anti-neuronal autoantibodies in the serum frequently occurs; if blood-brain barrier (BBB) dysfunction comes up, these autoantibodies are able to reach their targets and determine deleterious effect (D’Andrea, 2003). In fact, a profound change in BBB permeability has been observed in AD. In these patients amyloid deposits have been observed in microvessels and this overload is associated with degenerating endothelium (decreased mitochondrial content, increased pinocytotic vesicles), damaged smooth muscle cells and pericytes, and basement membrane changes (focal necrosis, reduplication, increased collagen content, disintegrating) (Thomas et al., 1996; Wardlaw et al., 2003). All these components strengthen the possibility that the ‘major pathological role of amyloid in AD may be to inflict vascular damage’ and hence, impair BBB function (Franzblau et al., 2013; Attems and Jellinger, 2014).

Immunoglobulins (IGs) have been detected in serum, cerebrospinal fluid and amyloid plaques of patients with AD. IGs are associated with vessel-associated amyloid, which has been linked to a faulty BBB (Franzblau et al., 2013). As a consequence, the presence of neuronal autoantibodies associated with a BBB dysfunction seems to be a relevant part of AD neuropathology (Attems and Jellinger, 2014).

Additional data about relationship between autoimmune diseases (e.g., thyroid dysfunction, diabetes) and AD has been proven. In fact, patients with AD have a significant increase in the values of anti-thyroglobulin and anti-microsomial autoantibodies compared to healthy controls (Genovesi et al., 1996).

Moreover, typical features of autoimmunity have been associated with both AD and diabetes (e.g., high levels of advanced glycation end products and their receptor have been detected in tissues and in the circulation in both disease) (Mruthinti et al., 2006).

In summary, these data in the context of the underlying mechanisms of many autoimmune diseases indicated that AD has proven autoimmune mechanisms, which provide a link between vascular pathology (altered BBB function) and neuronal cell death. Furthermore, according to these data, BBB dysfunction precedes neuronal degeneration and dementia (Rhodin and Thomas, 2001).

Electromagnetic Brain Stimulation and Immunomodulation in Neurodegenerative Diseases

Over the past decades, neuroscientists and clinicians have been exploring the properties of the brain’s electromagnetic activity for both diagnostic and therapeutic purposes. In the 1990s, research on electromagnetic radiation was motivated by the need to better understand the potential harmful effects of environmental magnetic fields (Bennett, 1995; Bracken and Patterson, 1996); actually, it is becoming increasingly clear that interactions between magnetic fields and biological systems deserve to be studied in their own right because these interactions appear to be fundamental to life processes and could represent a therapeutic agent in several diseases.

In our opinion, one of the more striking observations related to the effects of EMFs on biological systems concerns the presence of a “window effect,” showing that biological effects occur only at particular combinations of frequency and field intensity (Panagopoulos and Margaritis, 2010). These effects have been reported especially for changes in calcium ion flux in cells and tissues. Related window effects are reports of signal-specific quantitative and qualitative response to EMFs in several different tissues (Azanza and del Moral, 1994).

ELF-EMFs interact readily with the central nervous system (CNS). While the high-frequency EMFs encountered in industry can expose workers to an increased risk of AD (Hakansson et al., 2003), amyotrophic lateral sclerosis and multiple sclerosis (MS) (Johansen, 2004), EMFs of weak and very weak intensity can exert interesting and proven therapeutic effects on the CNS (Sandyk, 1992; Sandyk and Iacono, 1994; Boggio et al., 2012). The level of radiation is typically in the range of 1 millitesla (mT) in most studies.

Transcranial magnetic brain stimulation (TMS) is a commonly-used neurostimulation and a neuromodulation technique, based on the principle of electromagnetic induction of an electrical field in the brain. This field can be of sufficient magnitude and density to depolarize neurons, and when TMS pulses are applied repetitively they can modulate cortical excitability, decreasing or increasing it, depending on the parameters of stimulation, even beyond the duration of the train of stimulation (Fregni and Pascual-Leone, 2007; Ridding and Rothwell, 2007).

The last decade has seen a rapid increase in the applications of TMS to study cognition, neurobehavioral relations and the pathophysiology of several neurologic and psychiatric disorders. Evidence has accumulated that demonstrates that TMS provides a valuable tool for modulating brain activity in a specific, distributed, cortico-subcortical network through control and manipulation of cognition, neuromotoricity and behavior (George et al., 2007; Guerriero et al., 2015).

Since the immune system plays a primary role in the control of many diseases and tumor growth, many laboratories have investigated the influence of ELF-EMF stimulation on blood mononuclear cells, various cellular components and cellular processes; other studies have examined electromagnetic effects on specific genes expressions and signal transduction pathways, but the experimental data obtained are currently controversial (Cossarizza et al., 1993; Onodera et al., 2003).

The mechanisms by which ELF-EMFs elicit cellular responses are somewhat still unknown, and it is still unclear which cellular components mediate these fields’ effects. However, there are several hypotheses to explain EMF interaction with the living matter.

It is assumed that some type of initial interaction occurs at the level of the cell membrane and that specific signal amplification processes carry the membrane-mediated effect into the cell (Frey, 1993). Molecular studies of the membrane signaling processes have shown, for example, that the involved cells can use mechanisms such as intracellular second-messenger (e.g., Ca2+, cyclic adenosine monophosphate [cAMP], cyclic guanosine monophosphate [cGMP]) cascades, positive feedback, and linear membrane channel-gating (Grundler et al., 1992). Some of the most important calcium-related processes such as synaptic neurotransmitter and synthesis and release and levels of cAMP (Matthews and Gersdorff, 1996), essential for the functioning of the neurons that are influenced by EMFs (Rosen, 1992). In addition, amplification via calcium flux could also provide the means by which the membrane-mediated effects of EMFs could be carried into the cell (Karabakhtsian et al., 1994).

As described below, EMFs proved to exert a certain immune function modulation. Modulation of neural activity by ELF-EMFs could possibly counteract the aberrant pro-inflammatory responses present in neurodegenerative and neuropsychiatric disorders reducing their severity and, possibly, their onset.

Thus, in the next sections we will address the influence of ELF-EMFs on autoimmunity and immune cells, supposing that ELF-EMF may act on the basis of mechanisms centered on immunomodulation. This could have particular relevance for the treatment of neurodegenerative disorders, such as AD.

Low-frequency Electromagnetic Fields Stimulation and Autoimmunity

Regarding a possible relationship between EMF and autoimmunity, the researches conducted by Sandyk and colleagues deserve great interest. In the 1990s, Sandyk amply demonstrated the efficacy of pulsed ELF-EMFs of a few mT in alleviating the symptoms of MS through their action on axonal and synaptic neurotransmission (Sandyk and Iacono, 1993; Sandyk and Dann, 1995). Weekly treatment administered for years with very weak ELF-EMFs can alter the clinical course of chronic progressive MS, arresting progression of the disease for as long as four years (Sandyk, 1995a, 1997). This observation prompts the hypothesis that, in addition to effects on axonal and synaptic neurotransmission, effects may also be exerted on the autoimmune mechanisms responsible for demyelination.

Other proposals that to use pulsed ELF-EMFs of a few mT aims to modify the autoimmune pathology of the disease by eliciting profound membrane changes (Bistolfi, 2002) (the so-called Marinozzi effect) (Marinozzi et al., 1982) in the MS plaque cells.

While the action of ELF fields of a few pT is characterized by an improvement in neurotransmission, the use of ELF fields of a few mT aims to exert an action of local immunomodulation on the cells of the MS plaque through the induction of the Marinozzi effect. It therefore follows that the targets of ELF fields in the mT range will be the plaque cells (T-lymphocytes, macrophagic monocytes, microglia cells and dendritic cells), those cells disseminated in the seemingly normal nervous tissue (macrophages and microglia cells) (Bistolfi, 2007).

More specifically, the target should be the plasma membrane of these cells, which is almost always carpeted with microvilli and protrusions of various types. Since the plasma membrane is central to the relationships among immune cells (Lassmann et al., 2007) and since the plasma membrane itself is the elective target of ELF-EMF, a possible induction of the Marinozzi effect could slow down the activity of autoimmune cells in the plaque. It may determine an effect of local (on the brain) or regional immunomodulation (on the entire CNS) (Baureus Koch et al., 2003).

In far 1998, Richards et al. (1998) expressed the hope that electromagnetic fields might find application in the therapy of MS, both to manage symptoms and to achieve long-term effects by eliciting beneficial changes in the immune system and in nerve regeneration.

Our personal hypothesis is that – as observed in MS – similar effects could be present and relevant during EMF brain stimulation in patients with other CNS neurodegenerative disorders and be responsible for their therapeutic effect.

Low-frequency Electromagnetic Fields Stimulation and Immunomodulation

ELF-EMF effects on macrophages, nitric oxide and heat shock proteins

Macrophages are responsible for eliminating infectious agents and other cellular debris (Tintut et al., 2002). The recruitment of monocytes/macrophages to inflammatory sites and neoplastic tissues and their activation therein is crucial to the success of an immune reaction, in part because further cell migration is intimately related to leukocyte function. Resting macrophages have low levels of phagocytic activity and become fully active through the binding of pathogens or by local cytokine release. Once activated, macrophages exhibit an increased level of phagocytic activity and an increased production of reactive oxygen species (ROS) enabling the killing of microbes within phagosomes. The first step is the phagocytosis of the infectious agent, which is then transferred to the phagosome where it is killed by ROS and reactive nitrogen oxide species. The main protagonist of this process is nitric oxide (NO), which in turn induces the formation of cGMP, which in turn triggers a cascade of intracellular signaling. In the other hand, ROS also act as a signaling molecule and targets a wide range of physiological pathways. Activation of these cellular pathways also causes the secretion of inflammatory cytokines including IL-1b and TNF-alpha (Laskin and Laskin, 2001). Therefore when stimulated with bacterial toxins, NO and ROS stimulate cells to synthesize heat shock proteins (HSPs) (Polla et al., 1996).

Several studies have shown the effect of ELF-EMFs on macrophages. Kawczyk-Krupka and colleagues aimed to determine the effect of ELF-EMFs on the physiological response of phagocytes to an infectious agent. Human monocytic leukemia cell lines were cultured and 50 Hz, 1 mT EMF was applied for 4–6 hours to cells induced with Staphylococcus aureus. The growth curve of exposed bacteria was lower than the control, while field application increased NO levels. The increase was more prominent for Staphylococcus aureus-induced cells and appeared earlier than the increase in cells without field application (Kawczyk-Krupka et al., 2002). Increased cGMP levels in response to field application were closely correlated with increased NO levels (Azanza and del Moral, 1994).

Another study on mouse macrophages after short-term (45 minutes) exposure to 50 Hz EMF at 1.0 mT showed a significant uptake of carboxylated latex beads in macrophages, suggesting EMFs stimulate the phagocytic activity of their macrophages (Frahm et al., 2006). Tetradecanoylphorbol acetate (TPA) was used as positive control to prove the activating capacity of cells, as TPA is known to activate the protein kinase C and induce cellular processes including pinocytosis and phagocytosis (Laskin et al., 1980). On the basis of these data, ELF-EMF seems to potentially play a role in decreasing the growth rate of bacteria and other pathogens eliminated by phagocytosis.

A significant increase of free radical production has been observed after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse macrophages (Aktan, 2004). To elucidate whether NADPH- or NADH-oxidase functions are influenced by EMF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. EMF-induced free radical production was not inhibited by DPI, whereas TPA-induced free radical production was diminished by approximately 70%. Since DPI lacks an inhibitory effect in EMF-exposed cells, 50 Hz EMF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, the oscillation in superoxide anion radical release in mouse macrophages suggests a cyclic pattern of NADH-oxidase activity (Rollwitz et al., 2004).

An important aspect of these phagocytic cells is that they produce high levels of free radicals in response to infection, and the effect of ELF-EMF on free radicals has been widely proposed as a probable direct mechanism for the action of ELF-EMF on the living systems (Simko and Mattsson, 2004).

NO, a free radical, is an intra-cellular and inter-cellular signaling molecule and it constitutes an important host defense effector for the phagocytic cells of the immune system. It is synthesized by NO synthase, which has two major types: “constitutive” and “inducible”. Inducible nitric oxide synthase (iNOS) is particularly expressed in macrophages and other phagocytic cells that are stimulated during an immune response to infection (Aktan, 2004). Although high concentration of NO can be beneficial as an antibacterial and antitumor agent, an excess of NO can be fatal and can lead to cell injury. For example the excessive activity of iNOS has detrimental effects on oligodendrocytes, cells responsible for the myelination of neuron in the CNS (Klostergaard et al., 1991). The roles of NO in the pathophysiology of disease are still being defined, but there is a growing body of evidence that the neutralization of iNOS activity may have a therapeutic value (Parmentier et al., 1999).

Some studies have focused on the potential toxicity of the ensuing high-output NO-synthesis serving as a mean to eliminate pathogens or tumor cells, but the expression of iNOS, contributes to local tissue destruction during chronic inflammation. NO increases the ability of monocytes to respond to chemotactic agents more effectively, and it is considered to be one of the principal effector molecules involved in macrophage-mediated cytotoxicity (Desai et al., 2003).

It has been observed that exposure to ELF-EMFs modifies both NOS and MCP-1 chemokine expression and that these modifications are related to each other and are furthermore mediated by increased NF-?B protein expression (Goodman et al., 1994). EMF represents a non-pharmacological inhibitor of NO and an inducer of MCP-1, the latter of which activates one of these molecules and leads to inhibition of the former and vice versa, establishing a mechanism that protects cells from excess stimulation and contributes to the regulation of cellular homeostasis (Biswas et al., 2001). Moreover in vitro study observed a slight decrease was observed in iNOS levels was observed in cells induced with Staphlococcus aureus after ELF-EMF stimulation (Azanza and del Moral, 1994).

HSPs are evolutionarily conserved proteins known to play a key role in cellular defense against the effect of stressors and their function in modulating apoptosis has been well assessed (Beere, 2004). Concerning the relationship between EMF stimulus and HSPs expressions, Goodman et al. (1994) first demonstrated that HSP expression was enhanced by exposure to electromagnetic fields. Tokalov and Gutzeit (2004) showed the effect of ELF-EMF on heat shock genes and demonstrated that even a low dose of ELF-EMF (10 mT) caused an increase in HSPs, especially hsp70, implying that the cell senses ELF-EMF as a physical stressor.

ELF-EMF stimulation and oxidative stress

Oxidative stress derives from two primary sources: 1) chronic ROS creation that is generated from the mitochondrial electron transport chain during normal cellular function; 2) high levels of acute ROS generation resulting from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, particularly associated with the activation of the CNS immune system (Barja, 1998). In both circumstances, oxidative stress comes up when an imbalance between ROS production and clearance of radical species occurs.

ROS have been implicated as second messengers that activate protein kinase cascades, although the means by which ROS regulate signal transduction remains unclear. ROS release and cytokine production, such as IL-1?, are common cell activation markers in immune relevant cells. ROS is involved in the activation of IL-1? signal transduction pathway (Li and Engelhardt, 2006). To neutralize the detrimental effects of ROS, cells have evolved a hierarchy of sophisticated antioxidant response mechanisms regulated by NF-E2-related factor 2 (Nrf2) transcription factor (Tasset et al., 2010).

Environmental factors including EMFs, stressors or diseases that augment the former or lower the latter can amplify and drive the process. Thus, in practical terms, oxidative stress is determined by excessive exposure to oxidant molecules when there is insufficient availability of antioxidant mechanisms, with the resulting free ROS oxidizing vulnerable cellular constituents, including proteins, nucleic acids and lipids, inducing microglial activation, inducing pro-inflammatory and suppressing anti-inflammatory cytokines and related signaling pathways and ultimately causing both synaptic and neuronal damage and dysfunction (Bonda et al., 2010). Whereas most environmental electromagnetic radiations cause oxidative stress in the brain (Sahin and Gumuslu, 2007), ELF-EMF seems to have an antioxidant and neuroprotective effect (Medina and Tunez, 2010).

As shown by Tunez et al. (2006), ELF-EMF induces the antioxidant pathway Nrf2, which is closely associated with its protective effect against neurotoxicity induced by 3-nitropropionic acid (3-NP) (Tunez et al., 2006). This effect may be due to the induction of Nrf2, increasing its concentration in the nucleus as a result, at least in part, on its translocation from the cytoplasm to the nucleus. These changes in antioxidant systems were associated with a reduction of cell and oxidative damage biomarkers. In fact given that Nrf2 regulates the expression of antioxidant protein systems, its decrease may plausibly be related to a reduction in antioxidant system levels. Thus, the depletion of Nrf2 showed that 3-NP induced a significant decrease in antioxidant enzyme activity in the striatum and an intense depletion of glutathione levels. This was accompanied by clear and intense oxidative damage characterized by lipid and protein oxidation, an increase in cell death and damage markers and neuronal loss. Thus, the reduction in Nrf2 in both cytoplasm and nucleus may have been due to significant cell loss induced by 3-NP (Tunez et al., 2006).

Animal studies have demonstrated that ELF-EMF exposure, in the form of TMS (60 Hz, 0.7 mT) applied to rats for 2 hours twice daily, can be neuroprotective (Tunez et al., 2006; Tasset et al., 2012). Administered prior to and after a toxic insult to the brain, for example in the systemic injection of 3-nitropropionic acid to induce an animal model of Huntington’s disease (Tunez and Santamaria, 2009), ELF-EMF can mitigate oxidative damage, elevate neurotrophic protein levels in brain and potentially augment neurogenesis (Arias-Carrion et al., 2004).

EMF 1.0 mT exposure of mouse macrophages showed a significant increase in extracellular IL-1b release after only 4 hours of exposure, which was continuously increased after 12–24 hours of exposure. This data suggests that EMF stimulation is able to increase cytokines in murine macrophages. Cossarizza and colleagues described the increased release of IL-2, IL-1, and IL-6 in peritoneal lymphocytes after long-term exposure to ELF-EMF (Cossarizza et al., 1989). On the other hand, investigation on cytokine production by Pessina et al. showed no effects after EMF on peritoneal blood cells (Pessina and Aldinucci, 1998).

Beyond these results, such studies reiterate the importance that the cellular effects of ELF-EMFs depend, in a large part, on their intensity and exposure time, as well as on the phenotype of the cellular target and interactions with intracellular structures. The level and timing of exposure can potentially be scheduled to optimize endogenous compensatory mechanisms following an adverse reaction.

ELF-EMF effects on pro-inflammatory chemokines

Chemokines are produced by a variety of cells including monocytes, T lymphocytes, neutrophils, fibroblasts, endothelial cells and epithelial cells (Murdoch and Finn, 2000). Chemokines play a relevant role in inflammatory events, such as trans-endothelial migration and accumulation of leucocytes at the site of damage. In addition, they modulate a number of biological responses, including enzyme secretion, cellular adhesion, cytotoxicity, T-cell activation and tissue regeneration (Zlotnik and Yoshie, 2000).

Since their discovery, chemokines have emerged as important regulators of leukocyte trafficking, and MCP-1, one of the best-studied chemokines, is known to exert multiple effects on target cells, such as increased cytosolic calcium levels, superoxide anion production, lysosomal enzyme release, production of anti-inflammatory cytokines and adhesion molecules in monocytes. MCP-1 is involved in the induction of polarized type Th2 responses and in the enhancement of IL-4 production. A possible feedback loop for Th2 activation would be the production of IL-4 and IL-13 by Th2, which stimulates MCP-1 production and leads to further recruitment of Th2 cells (Moser and Loetscher, 2001).

The fine control of inflammatory mediator levels is critical to neuronal homeostasis and health. For example, a deficiency in neuronal TGF-? signaling promotes neurodegeneration and AD, whereas augmented TGF-? can act as an anti-inflammatory cytokine and has potential neuroprotective action in AD and following other insults to the central nervous system (Ren et al., 1997).

Studies have shown the anti-inflammatory effects of ELF-EMF in vivo; for instance, Selvam used a coil system emitting a 5 Hz frequency to treat rats with rheumatoid arthritis for 90 minutes, producing significant anti-exhudative effects and resulting in the restoration of normal functional parameters (Vianale et al., 2008). This anti-inflammatory effect was reported to be partially mediated through the stabilizing action of ELF-EMF on cell membranes, reflected the restoration of intracellular Ca2+ levels in plasma lymphocytes (Selvam et al., 2007). Other investigators have suggested that ELF-EMF can interact with cells through mechanisms that involve extracellular calcium channels (Cho et al., 1999).

Moreover, incubating mononuclear cells with an iNOS inhibitor showed a significant reduction of iNOS and an increase of MCP-1 levels, and these effects are consistent with iNOS and MCP-1 level modifications observed in mononuclear cells exposed to ELF-EMF. Selective inhibition of the NF-?B signaling pathway by ELF-EMF may be involved in the decrease of chemokine production. If so, ELF-EMF exposure, interfering with many cellular processes, may be included in the plethora of stimuli that modulate NF-?B activation (including pro-inflammatory cytokines such as tumor necrosis factor-? and IL-1?, chemokines, phorbol 12-myristate 13-acetate, growth factors, lipopolysaccharide, ultraviolet irradiation, viral infection, as well as various chemical and physical stresses) (Vianale et al., 2008).

Lymphocyte activity and electrotaxis: a possible link to ELF-EMF stimulation

Recent studies have shown that cells can directionally respond to applied electric fields, in both in vitro and in vivo settings, a phenomenon called electrotaxis. However, the exact cellular mechanisms for sensing electrical signals are still not fully well understood, and it is thus far unknown how cells recognize and respond to electric fields, although some studies have suggested that electro-migration of some cell surface receptors and ion channels in cells could be involved (Cortese et al., 2014). Directed cell migration is essential to numerous physiological processes including immune responses, wound healing, cancer metastasis and neuron guidance (Kubes, 2002). Normal blood lymphocytes and monocytes respond to a steady electric field in Transwell assays. All lymphocyte subsets, including naive and memory CD4+, CD8+ T cells and B cells migrated toward the cathode. Electrotaxisis highly directional and the uniform migration of circulating lymphocytes suggests that other leukocyte subsets (e.g., tissue memory cells) may undergo electrotaxis as well.

Lymphocytes respond to electric fields with activation of Erk-kinases and Akt, which are involved in chemo-attractant receptor signaling and in electrotactic signaling in other cells (Sotsios et al., 1999; Zhao et al., 2006). Activation of these pathways suggests that electrotaxis and chemotaxis engage common intracellular cell motility programs in responding lymphocytes. In fact, electric field exposure induces Erk1/2 and Akt activation in lymphocytes, consistent with the activation of the MAPK and PI3K signaling pathways implicated in coordinated cell motility. Furthermore, it has been proven that an applied electric field induced the electrotactic migration of endogenous lymphocytes to mouse skin (Lin et al., 2008). These data thus define electrotaxis andpotentially present an additional mechanism for the control of lymphocyte and monocyte migration.

ELF-EMFs can either inhibit or stimulate lymphocyte activity as a function not only of the exposure (Petrini et al., 1990), but also of the biological conditions to the cells are exposed, with mitogen-activated cells being more responsive than resting cells (Conti et al., 1986). To explain this ambivalence of the effects of ELF magnetic fields on the immune system, Marino and colleagues have presented the hypothesis that the biological effects of ELF magnetic fields are governed by non-linear laws, and that deterministic responses may therefore occur that are both real and inconsistent, thereby yielding two conflicting types of results (Marino et al., 2000). A particular role in the interaction of ELF-EMFs with lymphocytes seems to be played by the mobilization of intracellular Ca2+from the calciosomes and of extracellular Ca2+ through the membrane channels (Conti et al., 1985). The action of ELF-EMFs on lymphoid cells, however, can also be exerted on the functions of the plasma membrane: the duration of the ligand-receptor bond (Chiabrera et al., 1984), the clustering of membrane proteins (Bersani et al., 1997), the activity of enzymatic macro-molecules (Lindstrom et al., 2001), and the active ion pumps (Ca2+ ATPase and Na+ K+ATPase).

Conclusions

Several studies have shown that ELF-EMF exposure is able to activate primary monocytes and macrophages from different species and also in cell lines. This activation potential is comparable to the activation by certain chemicals resulting in physiologically relevant cellular responses.

In the past, several findings have demonstrated the efficacy of pulsed ELF-EMFs of a few mT in alleviating the symptoms of MS through their action on synaptic neurotransmission and autoimmunity (by determining cell membrane changes in plaques).

Moreover, ELF-EMF exposure contributes to a general activation of macrophages, resulting in changes of numerous immunological reactions, such as increased ROS formation, in an enhanced phagocytic activity, and in an increased IL-1? release. Therefore, we can deduce that EMFs activate physiological functions of immune cells. However, the underlying mechanisms of interaction between EMF and immune system are still to be completely understood and need further studies at the molecular level.

Animal studies have demonstrated that ELF-EMF exposure, in the form of transcranial magnetic stimulation (60 Hz, 0.7 mT) applied to rats for 2 hours twice daily, has been seen to be neuroprotective (Sahin and Gumuslu, 2007; Medina and Tunez, 2010).

The effects of low flux density magnetic fields are exerted on altered functional states, in the sense of hyper- or hypo-function, rather than on normal functional states. The neurophysiological interpretation is that neurotransmission is favored at various sites: partially synapses, the cerebellum, and interhemisphere transcallosal connections, an idea which is strongly supported by the rapid regression seen in certain symptoms in patients with MS (Sandyk, 1995b). Based on all these evidences such effect could be attributed to the correction of perturbations of synaptic conductivity and immunomodulation (Bistolfi, 2007), resulting in clinical therapeutic effect as observed in neurodegenerative disorders such as AD (Mruthinti et al., 2006; Attems and Jellinger, 2014).

However, so far there is still no general agreement on the exact biological effect elicited by EMFs on the physical mechanisms that may be behind their interaction with biological systems. Of course the biological effects of EMFs are dependent on frequency, amplitude, timing and length of exposure, but are also related to intrinsic susceptibility and responsiveness of different cell types (Tenuzzo et al., 2006). Level and timing of exposure can be potentially scheduled to optimize endogenous compensatory mechanisms following an adverse challenge.

In the light of results reviewed here, we conclude that there is growing evidence of the potential role of EMFs in biological modulation of autoimmunity, immune functions and oxidative stress. As a consequence, the hypothesis that ELF-EMFs explicit their therapeutic effect through modulation of immune relevant cells is of clear interest, in particular in neurodegenerative diseases.

It is notable to underline that the effects of ELF-EMFs are not unique as they depend on their intensity, exposure time and cellular targets; further efforts towards more scheduled and well defined level and timing of exposure should be warranted.

Hence, it is necessary to proceed with substantial research on this issue, paying particular attention to the choice of the appropriate biological model and controlled experimental conditions.

Footnotes

Conflicts of interest: The authors report no conflicts of interest in this work. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References

  • Aktan F. iNOS-mediated nitric oxide production and its regulation. Life Sci. 2004;75:639–653. [PubMed]
  • Arias-Carrion O, Verdugo-Diaz L, Feria-Velasco A, Millan-Aldaco D, Gutierrez AA, Hernandez-Cruz A, Drucker-Colin R. Neurogenesis in the subventricular zone following transcranial magnetic field stimulation and nigrostriatal lesions. J Neurosci Res. 2004;78:16–28. [PubMed]
  • Attems J, Jellinger KA. The overlap between vascular disease and Alzheimer’s disease-lessons from pathology. BMC Med. 2014;12:206.[PMC free article] [PubMed]
  • Azanza MJ, del Moral A. Cell membrane biochemistry and neurobiological approach to biomagnetism. Prog Neurobiol. 1994;44:517–601. [PubMed]
  • Barja G. Mitochondrial free radical production and aging in mammals and birds. Ann N Y Acad Sci. 1998;854:224–238. [PubMed]
  • Baureus Koch CL, Sommarin M, Persson BR, Salford LG, Eberhardt JL. Interaction between weak low frequency magnetic fields and cell membranes. Bioelectromagnetics. 2003;24:395–402. [PubMed]
  • Beere HM. “The stress of dying”: the role of heat shock proteins in the regulation of apoptosis. J Cell Sci. 2004;117:2641–2651. [PubMed]
  • Bennett WR. Electromagnetic fields and power lines. Sci Am. 1995;2:68–77.
  • Bersani F, Marinelli F, Ognibene A, Matteucci A, Cecchi S, Santi S, Squarzoni S, Maraldi NM. Intramembrane protein distribution in cell cultures is affected by 50 Hz pulsed magnetic fields. Bioelectromagnetics. 1997;18:463–469. [PubMed]
  • Bistolfi F. Are microvilli and cilia sensors of electromagnetic fields? Physica Medica. 2002;XVIII:85–94.
  • Bistolfi F. Extremely low-frequency pulsed magnetic fields and multiple sclerosis: effects on neurotransmission alone or also on immunomodulation? Building a working hypothesis. Neuroradiol J. 2007;20:676–693. [PubMed]
  • Biswas SK, Sodhi A, Paul S. Regulation of nitric oxide production by murine peritoneal macrophages treated in vitro with chemokine monocyte chemoattractant protein 1. Nitric Oxide. 2001;5:566–579. [PubMed]
  • Boggio PS, Ferrucci R, Mameli F, Martins D, Martins O, Vergari M, Tadini L, Scarpini E, Fregni F, Priori A. Prolonged visual memory enhancement after direct current stimulation in Alzheimer’s disease. Brain Stimul. 2012;5:223–230. [PubMed]
  • Bonda DJ, Wang X, Perry G, Nunomura A, Tabaton M, Zhu X, Smith MA. Oxidative stress in Alzheimer disease: a possibility for prevention. Neuropharmacology. 2010;59:290–294. [PubMed]
  • Bossy-Wetzel E, Schwarzenbacher R, Lipton SA. Molecular pathways to neurodegeneration. Nat Med. 2004;10(Suppl):S2–9. [PubMed]
  • Bracken TD, Patterson RM. Variability and consistency of electric and magnetic field occupational exposure measurements. J Expo Anal Environ Epidemiol. 1996;6:355–374. [PubMed]
  • Chiabrera A, Grattarola M, Viviani R. Interaction between electromagnetic fields and cells: microelectrophoretic effect on ligands and surface receptors. Bioelectromagnetics. 1984;5:173–191. [PubMed]
  • Cho MR, Thatte HS, Silvia MT, Golan DE. Transmembrane calcium influx induced by ac electric fields. FASEB J. 1999;13:677–683. [PubMed]
  • Conti P, Gigante GE, Cifone MG, Alesse E, Fieschi C. Effect of electromagnetic field on two calcium dependent biological systems. J Bioelectr. 1985;4:227–236.
  • Conti P, Gigante GE, Cifone MG, Alesse E, Fieschi C, Bologna M, Angeletti PU. Mitogen dose-dependent effect of weak pulsed electromagnetic field on lymphocyte blastogenesis. FEBS Lett. 1986;199:130–134. [PubMed]
  • Cortese B, Palama IE, D’Amone S, Gigli G. Influence of electrotaxis on cell behaviour. Integr Biol. 2014;6:817–830. [PubMed]
  • Cossarizza A, Monti D, Bersani F, Paganelli R, Montagnani G, Cadossi R, Cantini M, Franceschi C. Extremely low frequency pulsed electromagnetic fields increase interleukin-2 (IL-2) utilization and IL-2 receptor expression in mitogen-stimulated human lymphocytes from old subjects. FEBS Lett. 1989;248:141–144. [PubMed]
  • Cossarizza A, Angioni S, Petraglia F, Genazzani AR, Monti D, Capri M, Bersani F, Cadossi R, Franceschi C. Exposure to low frequency pulsed electromagnetic fields increases interleukin-1 and interleukin-6 production by human peripheral blood mononuclear cells. Exp Cell Res. 1993;204:385–387. [PubMed]
  • D’Andrea MR. Evidence linking neuronal cell death to autoimmunity in Alzheimer’s disease. Brain Res. 2003;982:19–30. [PubMed]
  • D’Andrea MR. Add Alzheimer’s disease to the list of autoimmune diseases. Med Hypotheses. 2005;64:458–463. [PubMed]
  • Desai A, Miller MJ, Huang X, Warren JS. Nitric oxide modulates MCP-1 expression in endothelial cells: implications for the pathogenesis of pulmonary granulomatous vasculitis. Inflammation. 2003;27:213–223.[PubMed]
  • Frahm J, Lantow M, Lupke M, Weiss DG, Simko M. Alteration in cellular functions in mouse macrophages after exposure to 50 Hz magnetic fields. J Cell Biochem. 2006;99:168–177. [PubMed]
  • Franzblau M, Gonzales-Portillo C, Gonzales-Portillo GS, Diamandis T, Borlongan MC, Tajiri N, Borlongan CV. Vascular damage: a persisting pathology common to Alzheimer’s disease and traumatic brain injury. Med Hypotheses. 2013;81:842–845. [PMC free article] [PubMed]
  • Fregni F, Pascual-Leone A. Technology insight: noninvasive brain stimulation in neurology-perspectives on the therapeutic potential of rTMS and tDCS. Nat Clini Prac Neurol. 2007;3:383–393. [PubMed]
  • Frey AH. Electromagnetic field interactions with biological systems. FASEB J. 1993;7:272–281. [PubMed]
  • Genovesi G, Paolini P, Marcellini L, Vernillo E, Salvati G, Polidori G, Ricciardi D, de Nuccio I, Re M. Relationship between autoimmune thyroid disease Rand Alzheimer’s disease. Panminerva Med. 1996;38:61–63.[PubMed]
  • George MS, Nahas Z, Borckardt JJ, Anderson B, Foust MJ, Burns C, Kose S, Short EB. Brain stimulation for the treatment of psychiatric disorders. Curr Opin Psychiat. 2007;20:250–254. discussion 247-259. [PubMed]
  • Goodman R, Blank M, Lin H, Dai R, Khorkava O, Soo L, Weisbrot D, Henderson A. Increased levels of hsp70 transcripts induced when cells are exposed to low frequency electro-magnetic fields. Bioelectrochem Bioenerg. 1994;33:115–120.
  • Grundler W, Kaiser F, Keilmann F, Walleczek J. Mechanisms of electromagnetic interaction with cellular systems. Naturwissenschaften. 1992;79:551–559. [PubMed]
  • Guerriero F, Botarelli E, Mele G, Polo L, Zoncu D, Renati P, Sgarlata C, Rollone M, Ricevuti G, Maurizi N, Francis M, Rondanelli M, Perna S, Guido D, Mannu P. An innovative intervention for the treatment of cognitive impairment-Emisymmetric bilateral stimulation improves cognitive functions in Alzheimer’s disease and mild cognitive impairment: an open-label study. Neuropsychiatr Dis Treat. 2015;11:2391–2404.[PMC free article] [PubMed]
  • Hakansson N, Gustavsson P, Johansen C, Floderus B. Neurodegenerative diseases in welders and other workers exposed to high levels of magnetic fields. Epidemiology. 2003;14:420–426. discussion 427-428. [PubMed]
  • Johansen C. Electromagnetic fields and health effects–epidemiologic studies of cancer, diseases of the central nervous system and arrhythmia-related heart disease. Scand J Work Environ Health. 2004;30(Suppl 1):1–30. [PubMed]
  • Karabakhtsian R, Broude N, Shalts N, Kochlatyi S, Goodman R, Henderson AS. Calcium is necessary in the cell response to EM fields. FEBS Lett. 1994;349:1–6. [PubMed]
  • Kawczyk-Krupka A, Sieron A, Shani J, Czuba ZP, Krol W. Biological effects of extremely low-frequency magnetic fields on stumlated macrophages J774-2 in cell culture. Electromagn Biol Med. 2002;21:141–153.
  • Klostergaard J, Leroux ME, Hung MC. Cellular models of macrophage tumoricidal effector mechanisms in vitro. Characterization of cytolytic responses to tumor necrosis factor and nitric oxide pathways in vitro. J Immunol. 1991;147:2802–2808. [PubMed]
  • Kubes P. The complexities of leukocyte recruitment. Semin Immunol. 2002;14:65–72. [PubMed]
  • Laskin DL, Laskin JD. Role of macrophages and inflammatory mediators in chemically induced toxicity. Toxicology. 2001;160:111–118. [PubMed]
  • Laskin DL, Laskin JD, Weinstein IB, Carchman RA. Modulation of phagocytosis by tumor promoters and epidermal growth factor in normal and transformed macrophages. Cancer Res. 1980;40:1028–1035.[PubMed]
  • Lassmann H, Bruck W, Lucchinetti CF. The immunopathology of multiple sclerosis: an overview. Brain Pathol. 2007;17:210–218. [PubMed]
  • Lee HG, Zhu X, Castellani RJ, Nunomura A, Perry G, Smith MA. Amyloid-beta in Alzheimer disease: the null versus the alternate hypotheses. J Pharmacol Exp Ther. 2007;321:823–829. [PubMed]
  • Li Q, Engelhardt JF. Interleukin-1beta induction of NFkappaB is partially regulated by H2O2-mediated activation of NFkappaB-inducing kinase. J Biol Chem. 2006;281:1495–1505. [PubMed]
  • Lin F, Baldessari F, Gyenge CC, Sato T, Chambers RD, Santiago JG, Butcher EC. Lymphocyte electrotaxis in vitro and in vivo. J Immunol. 2008;181:2465–2471. [PMC free article] [PubMed]
  • Lindstrom E, Still M, Mattsson MO, Mild KH, Luben RA. ELF magnetic fields initiate protein tyrosine phosphorylation of the T cell receptor complex. Bioelectrochemistry (Amsterdam, Netherlands) 2001;53:73–78.[PubMed]
  • Marino AA, Wolcott RM, Chervenak R, Jourd’Heuil F, Nilsen E, Frilot C., 2nd Nonlinear response of the immune system to power-frequency magnetic fields. Am J Physiol Regul Integr Comp Physiol. 2000;279:R761–768. [PubMed]
  • Marinozzi G, Benedetto A, Brandimarte B, Ripani M, Carpano S, Camporiondo MP. Effetti dei campi magnetici pulsanti su colture cellulari. Giorn Ital Oncol. 1982;2:87–100.
  • Matthews G, Gersdorff H. Calcium dependence of neurotransmitter release. Semin Neurosci. 1996;8:329–334.
  • Medina FJ, Tunez I. Huntington’s disease: the value of transcranial meganetic stimulation. Curr Med Chem. 2010;17:2482–2491. [PubMed]
  • Moreno-Gonzalez I, Soto C. Misfolded protein aggregates: mechanisms, structures and potential for disease transmission. Semin Cell Dev Biol. 2011;22:482–487. [PMC free article] [PubMed]
  • Moser B, Loetscher P. Lymphocyte traffic control by chemokines. Nat Immunol. 2001;2:123–128. [PubMed]
  • Mruthinti S, Schade RF, Harrell DU, Gulati NK, Swamy-Mruthinti S, Lee GP, Buccafusco JJ. Autoimmunity in Alzheimer’s disease as evidenced by plasma immunoreactivity against RAGE and Abeta42: complication of diabetes. Curr Alzheimer Res. 2006;3:229–235. [PubMed]
  • Murdoch C, Finn A. Chemokine receptors and their role in inflammation and infectious diseases. Blood. 2000;95:3032–3043. [PubMed]
  • Onodera H, Jin Z, Chida S, Suzuki Y, Tago H, Itoyama Y. Effects of 10-T static magnetic field on human peripheral blood immune cells. Radiat Res. 2003;159:775–779. [PubMed]
  • Panagopoulos DJ, Margaritis LH. The identification of an intensity ‘window’ on the bioeffects of mobile telephony radiation. Int J Radiat Biol. 2010;86:358–366. [PubMed]
  • Parmentier S, Bohme GA, Lerouet D, Damour D, Stutzmann JM, Margaill I, Plotkine M. Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury. Br J Pharmacol. 1999;127:546–552.[PMC free article] [PubMed]
  • Pessina GP, Aldinucci C. Pulsed electromagnetic fields enhance the induction of cytokines by peripheral blood mononuclear cells challenged with phytohemagglutinin. Bioelectromagnetics. 1998;19:445–451.[PubMed]
  • Petrini M, Polidori R, Ambrogi F. Effects of different low-frequency electro-magnetic fields on lymphocyte activation: at which cellular level? J Bioelectr. 1990;9:159–166.
  • Polla BS, Kantengwa S, Francois D, Salvioli S, Franceschi C, Marsac C, Cossarizza A. Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. Proc Natl Acad Sci U S A. 1996;93:6458–6463. [PMC free article] [PubMed]
  • Ren RF, Hawver DB, Kim RS, Flanders KC. Transforming growth factor-beta protects human hNT cells from degeneration induced by beta-amyloid peptide: involvement of the TGF-beta type II receptor. Brain Res Mol Brain Res. 1997;48:315–322. [PubMed]
  • Rhodin JA, Thomas T. A vascular connection to Alzheimer’s disease. Microcirculation. 2001;8:207–220. [PubMed]
  • Richards TL, Lappin MS, Lawrie FW, Stegbauer KC. Bioelectromagnetic applications for multiple sclerosis. Phys Med Rehabil Clin N Am. 1998;9:659–674. [PubMed]
  • Ridding MC, Rothwell JC. Is there a future for therapeutic use of transcranial magnetic stimulation? Nat Rev Neurosci. 2007;8:559–567.[PubMed]
  • Rollwitz J, Lupke M, Simko M. Fifty-hertz magnetic fields induce free radical formation in mouse bone marrow-derived promonocytes and macrophages. Biochim Biophys Acta. 2004;1674:231–238. [PubMed]
  • Rosen AD. Magnetic field influence on acetylcholine release at the neuromuscular junction. Am J Physiol. 1992;262:C1418–1422. [PubMed]
  • Sahin E, Gumuslu S. Immobilization stress in rat tissues: alterations in protein oxidation, lipid peroxidation and antioxidant defense system. Comp Biochem Physiol C Toxicol Pharmacol. 2007;144:342–347.[PubMed]
  • Sandyk R. Successful treatment of multiple sclerosis with magnetic fields. Int J Neurosci. 1992;66:237–250. [PubMed]
  • Sandyk R. Long term beneficial effects of weak electromagnetic fields in multiple sclerosis. Int J Neurosci. 1995a;83:45–57. [PubMed]
  • Sandyk R. Chronic relapsing multiple sclerosis: a case of rapid recovery by application of weak electromagnetic fields. Int J Neurosci. 1995b;82:223–242. [PubMed]
  • Sandyk R. Treatment with electromagnetic fields reverses the long-term clinical course of a patient with chronic progressive multiple sclerosis. Int J Neurosci. 1997;90:177–185. [PubMed]
  • Sandyk R, Iacono RP. Resolution of longstanding symptoms of multiple sclerosis by application of picoTesla range magnetic fields. Int J Neurosci. 1993;70:255–269. [PubMed]
  • Sandyk R, Iacono RP. Multiple sclerosis: improvement of visuoperceptive functions by picoTesla range magnetic fields. Int J Neurosci. 1994;74:177–189. [PubMed]
  • Sandyk R, Dann LC. Resolution of Lhermitte’s sign in multiple sclerosis by treatment with weak electromagnetic fields. Int J Neurosci. 1995;81:215–224. [PubMed]
  • Selvam R, Ganesan K, Narayana Raju KV, Gangadharan AC, Manohar BM, Puvanakrishnan R. Low frequency and low intensity pulsed electromagnetic field exerts its antiinflammatory effect through restoration of plasma membrane calcium ATPase activity. Life Sci. 2007;80:2403–2410. [PubMed]
  • Simko M, Mattsson MO. Extremely low frequency electromagnetic fields as effectors of cellular responses in vitro: possible immune cell activation. J Cell Biochem. 2004;93:83–92. [PubMed]
  • Sotsios Y, Whittaker GC, Westwick J, Ward SG. The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes. J Immunol. 1999;163:5954–5963. [PubMed]
  • Tasset I, Medina FJ, Jimena I, Aguera E, Gascon F, Feijoo M, Sanchez-Lopez F, Luque E, Pena J, Drucker-Colin R, Tunez I. Neuroprotective effects of extremely low-frequency electromagnetic fields on a Huntington’s disease rat model: effects on neurotrophic factors and neuronal density. Neuroscience. 2012;209:54–63. [PubMed]
  • Tasset I, Perez-De La Cruz V, Elinos-Calderon D, Carrillo-Mora P, Gonzalez-Herrera IG, Luna-Lopez A, Konigsberg M, Pedraza-Chaverri J, Maldonado PD, Ali SF, Tunez I, Santamaria A. Protective effect of tert-butylhydroquinone on the quinolinic-acid-induced toxicity in rat striatal slices: role of the Nrf2-antioxidant response element pathway. Neurosignals. 2010;18:24–31. [PubMed]
  • Tenuzzo B, Chionna A, Panzarini E, Lanubile R, Tarantino P, Di Jeso B, Dwikat M, Dini L. Biological effects of 6 mT static magnetic fields: a comparative study in different cell types. Bioelectromagnetics. 2006;27:560–577. [PubMed]
  • Thomas T, Thomas G, McLendon C, Sutton T, Mullan M. beta-Amyloid-mediated vasoactivity and vascular endothelial damage. Nature. 1996;380:168–171. [PubMed]
  • Tintut Y, Patel J, Territo M, Saini T, Parhami F, Demer LL. Monocyte/macrophage regulation of vascular calcification in vitro. Circulation. 2002;105:650–655. [PubMed]
  • Tokalov SV, Gutzeit HO. Weak electromagnetic fields (50 Hz) elicit a stress response in human cells. Environ Res. 2004;94:145–151. [PubMed]
  • Tunez I, Santamaria A. Model of Huntington’s disease induced with 3-nitropropionic acid. Rev Neurol. 2009;48:430–434. [PubMed]
  • Tunez I, Drucker-Colin R, Jimena I, Medina FJ, Munoz Mdel C, Pena J, Montilla P. Transcranial magnetic stimulation attenuates cell loss and oxidative damage in the striatum induced in the 3-nitropropionic model of Huntington’s disease. J Neurochem. 2006;97:619–630. [PubMed]
  • Tweedie D, Sambamurti K, Greig NH. TNF-alpha inhibition as a treatment strategy for neurodegenerative disorders: new drug candidates and targets. Curr Alzheimer Res. 2007;4:378–385. [PubMed]
  • Vianale G, Reale M, Amerio P, Stefanachi M, Di Luzio S, Muraro R. Extremely low frequency electromagnetic field enhances human keratinocyte cell growth and decreases proinflammatory chemokine production. Br J Dermatol. 2008;158:1189–1196. [PubMed]
  • von Bernhardi R, Eugenin J. Alzheimer’s disease: redox dysregulation as a common denominator for diverse pathogenic mechanisms. Antioxid Redox Signal. 2012;16:974–1031. [PubMed]
  • Wardlaw JM, Sandercock PA, Dennis MS, Starr J. Is breakdown of the blood-brain barrier responsible for lacunar stroke, leukoaraiosis, and dementia? Stroke. 2003;34:806–812. [PubMed]
  • Zhao M, Song B, Pu J, Wada T, Reid B, Tai G, Wang F, Guo A, Walczysko P, Gu Y, Sasaki T, Suzuki A, Forrester JV, Bourne HR, Devreotes PN, McCaig CD, Penninger JM. Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN. Nature. 2006;442:457–460. [PubMed]
  • Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity. 2000;12:121–127. [PubMed]

Articles from Neural Regeneration Research are provided here courtesy of Medknow Publications Behav Brain Funct. 2015; 11: 26.  Published online 2015 Sep 7. doi: 10.1186/s12993-015-0070-z

Mechanisms and therapeutic applications of electromagnetic therapy in Parkinson’s disease.

Maria Vadalà, Annamaria Vallelunga, Lucia Palmieri, Beniamino Palmieri, Julio Cesar Morales-Medina, and Tommaso Iannitticorresponding author
Department of General Surgery and Surgical Specialties, University of Modena and Reggio Emilia Medical School, Surgical Clinic, Modena, Italy
Department of Medicine and Surgery, Centre for Neurodegenerative Diseases (CEMAND), University of Salerno, Salerno, Italy
Department of Nephrology, University of Modena and Reggio Emilia Medical School, Surgical Clinic, Modena, Italy
Centro de Investigación en Reproducción Animal, CINVESTAV-Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico
Department of Neuroscience, Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK
Maria Vadalà, Email: moc.liamg@aladav.yram.
Contributor Information.
corresponding authorCorresponding author. Author information ? Article notes ? Copyright and License information ?
Received 2015 Jan 5; Accepted 2015 Jul 22.
Copyright © Vadalà et al. 2015
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Abstract
Electromagnetic therapy is a non-invasive and safe approach for the management of several pathological conditions including neurodegenerative diseases. Parkinson’s disease is a neurodegenerative pathology caused by abnormal degeneration of dopaminergic neurons in the ventral tegmental area and substantia nigra pars compacta in the midbrain resulting in damage to the basal ganglia. Electromagnetic therapy has been extensively used in the clinical setting in the form of transcranial magnetic stimulation, repetitive transcranial magnetic stimulation, high-frequency transcranial magnetic stimulation and pulsed electromagnetic field therapy which can also be used in the domestic setting. In this review, we discuss the mechanisms and therapeutic applications of electromagnetic therapy to alleviate motor and non-motor deficits that characterize Parkinson’s disease.Keywords: Parkinson’s disease, Electromagnetic therapy, Transcranial magnetic stimulation, Repetitive transcranial magnetic stimulation, High-frequency transcranial magnetic stimulation, Pulsed electromagnetic field therapy

Background
Parkinson’s disease

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases worldwide, second only to Alzheimer’s disease (AD) [1]. PD is accompanied by the impairment of the cortico-subcortical excitation and inhibition systems, hence belonging to the involuntary movement diseases [2]. PD is caused by progressive loss of structure and function of dopaminergic neurons in the ventral tegmental area and substantia nigra pars compacta in the midbrain with subsequent damage to the basal ganglia (BG) [3]. Cumulative evidence supports the hypothesis that PD is the result of complex interactions among genetic abnormalities, environmental toxins and mitochondrial dysfunction [4–6]. The mechanisms of neuronal degeneration characterizing PD have been studied extensively and include a complex interplay among multiple pathogenic processes including oxidative stress, protein aggregation, excitotoxicity and impaired axonal transport [7]. The increasing number of genes and proteins critical in PD is unraveling a complex network of molecular pathways involved in its etiology, suggesting that common mechanisms underlie familial and sporadic PD, the two forms of this pathology. While the sporadic form is the most common (90–95% of PD cases), only 5–10% of PD cases are familial [8, 9]. At least ten distinct loci are responsible for rare Mendelian forms of PD and mutations in five genes have been linked to familial PD [10]. PD is characterized by motor and non-motor symptoms. The main motor symptoms include bradykinesia, tremor at rest (tremor affecting the body part that is relaxed or supported against gravity and not involved in purposeful activities [11]), rigidity and postural instability [12–17]. However, motor symptoms are now considered as the “tip of the iceberg” of PD clinical manifestations. PD non-motor symptoms include cognitive decline, decrease in sleep efficiency, increased wake after sleep onset, sleep fragmentation, and vivid dreams as well as neuropsychiatric symptoms such as depression and psychosis, [18–23]. Pain syndrome and autonomic dysfunctions have also been observed in PD patients [24–26].

Neuroimaging and genes: towards a personalized medicine for Parkinson’s disease

Several research groups have begun to perform genome-wide association studies (GWAS) on data or index measures derived from brain images, with the final goal of finding new genetic variants that might account for abnormal variations in brain structure and function that increase the risk of a given disease. Numerous genes have been identified using GWAS and have been associated with PD. They include alpha-synuclein, vacuolar protein sorting-associated protein 35, human leukocyte antigen family, leucine-rich repeat kinase 2 and acid ?-glucosidase [27–29]. Neuroimaging associates individual differences in the human genome to structural and functional variations into the brain. Van der Vegt and colleagues reported structural and functional brain mapping studies that have been performed in individuals carrying a mutation in specific PD genes including PARK1, PARK2, PARK6, PARK7, PARK8, and discussed how this “neurogenetics-neuroimaging approach” provides unique means to study key PD pathophysiological aspects [30]. In addition, neuroimaging of presymptomatic (non-manifesting) mutation carriers has emerged as a valuable tool to identify mechanisms of adaptive motor reorganization at the preclinical stage that may prevent or delay PD clinical manifestation [30]. Neuroimaging may be useful to study the effectiveness of electromagnetic therapy in PD patients.

Available therapies for Parkinson’s disease

PD treatment includes the use of pharmacological agents such as the dopaminergic agent l-3,4-dihy-droxy-phenylalanine (Levodopa or l-dopa) and stereotactic brain surgery which are associated with numerous side effects [31]. For example, the on-and-off phenomenon includes profound diurnal fluctuations in the psychomotor state of PD patients treated with l-dopa [32]. Furthermore, l-dopa loses effectiveness over time and can induce motor fluctuations such as the “wearing off” effect and dyskinesia [33]. While l-dopa metabolites are neurotoxic [33], the search for alternate, non-dopaminergic therapies to overcome the l-dopa-induced side effects has positioned adenosine A2A receptor (A2AR) antagonists as a promising therapeutic option for PD treatment [34]. Despite the favorable features of A2AR antagonists, their pharmacological properties, such as poor oral bioavailability and the lack of blood–brain barrier permeability, constitute a major problem to their clinical application [35]. Furthermore, regular physiotherapy and instrumental rehabilitation that have been employed to manage PD symptoms, such as tremor, slowness and difficulty in walking, are only moderately helpful [36]. Electromagnetic therapy has also been extensively used for PD treatment and may represent a promising therapeutic option for this condition since it promotes a lasting improvement in motor and non-motor symptoms [37–41].

Electromagnetic therapy background

Electromagnetic therapy includes the use of six groups of electromagnetic fields as previously described [42, 43] and summarized below:

Static/permanent magnetic fields can be created by various permanent magnets as well as by passing direct current through a coil.
Transcranial magnetic stimulation (TMS) utilizes frequencies in the range 1–200 Hz.
Low-frequency electromagnetic fields mostly utilize 60 Hz (in the US and Canada) and 50 Hz (in Europe and Asia) frequencies in distribution lines.
Pulsed radiofrequency fields utilize frequencies in the range 12–42 MHz.
Millimeter waves refer to very high-frequency in the range 30–100 GHz.
Pulsed electromagnetic fields (PEMFs) utilize frequencies in the range 5–300 Hz with very specific shapes and amplitudes.
Electromagnetic therapy is defined as the use of time-varying electromagnetic fields of low-frequency values (3 Hz–3 kHz) that can induce a sufficiently strong current to stimulate living tissue [44]. Electromagnetic fields can penetrate all tissues including the epidermis, dermis, and subcutaneous tissue, as well as tendons, muscles and bones [45]. The amount of electromagnetic energy used and its effect on the target organ depends on the size, strength and duration of treatment [44]. Electromagnetic fields can be divided into two categories: static and time-varying. Electromagnetic therapy falls into two categories: (1) hospital use which includes TMS, repetitive transcranial magnetic stimulation (rTMS) and high-frequency TMS and (2) home use including PEMF therapy.

Aim and searching criteria

We searched Pubmed/Medline using the keywords “Parkinson’s Disease” combined with “electromagnetic therapy”, “TMS”, “rTMS”, “high-frequency TMS” or “PEMF” and we included articles published between 1971 and 2015. This article aims to review the state of the art of electromagnetic therapy for treatment of PD.

Transcranial magnetic stimulation
TMS is a safe and non-invasive method of electrical stimulation of neurons in the human cerebral cortex, modifying neuronal activity locally and at distant sites when delivered in series of pulses [46]. TMS is also a useful tool to investigate various aspects of human neurophysiology, particularly corticospinal function, in health and disease [47]. An electromagnetic field generator sends a current with a peak amplitude of about 8,000 A that lasts about 1 ms, through an induction coil placed on the scalp [48]. TMS is based on the principle of electromagnetic induction, as discovered by Faraday in 1838. The current flowing briefly in the iron coil placed over a patient’s head generates an electromagnetic field that penetrates the scalp and skull reaching the brain where it induces a secondary ionic current. The site of stimulation of the brain is the point along its length at which sufficient current passes through its membrane to cause depolarization [49]. TMS can be used to determine several parameters associated to different aspects of cortical excitability: (1) the resting motor threshold or active motor threshold which reflects membrane properties; (2) the silent period, which is a quiescent phase in the electromyogram (EMG), is partially of cortical origin and is related to the function of gamma-aminobutyric acid receptors; (3) the short intracortical inhibition and facilitation which occur when a subthreshold stimulus precedes a suprathreshold stimulus by less than 5 ms or 8–30 ms, respectively. The peak of electromagnetic field strength is related to the magnitude of the current and the number of turns of wire in the coil [50]. The electrical current is rapidly turned on and off in the coil through the discharge of electronic components called the capacitors.

Transcranial magnetic stimulation in Parkinson’s disease

TMS clinical applications were first reported by Barker and colleagues who stimulated the brain, spinal cord and peripheral nerves using TMS with low or no pain [51]. Following this work, several TMS protocols that evidenced the correlation of TMS with peripheral EMG and monitored the modulation of TMS-induced motor evoked potentials (MEPs), were described [52–54]. For example, Cantello and coworkers studied the EMG potentials evoked in the bilateral first dorsal interosseus muscle by electromagnetic stimulation of the corticomotoneuronal descending system in 10 idiopathic PD patients without tremor but with rigidity with asymmetric body involvement and 10 healthy controls [55]. The threshold to cortical stimulation measured on the rigid side of PD patients was lower than on the contralateral side or than normal values. PD patients’ MEPs on the rigid side were larger compared to controls when the cortical stimulus was at rest or during slight tonic contraction of the target muscle [55]. Several clinical trials have pointed out the therapeutic efficacy of TMS in PD patients [3, 31, 56, 57]. For example, biomagnetic measurements performed using magnetoencephalography (MEG) in 30 patients affected by idiopathic PD exposed to TMS evidenced that 60% of patients did not exhibit tremor, muscular ache or dyskinesias for at least 1 year after TMS therapy [58]. The patients’ responses to TMS included a feeling of relaxation, partial or complete disappearance of muscular ache and l-dopa-induced dyskinesias as well as rapid reversal of visuospatial impairment [58]. Additional MEG measurements in PD patients also showed abnormal brain functions including slowing of background activity (increased theta and decreased beta waves) and increased alpha band connectivity [59]. These changes may reflect abnormalities in specific networks and neurotransmitter systems, and could be useful for differential diagnosis and treatment monitoring.

Repetitive transcranial magnetic stimulation
rTMS is a non-invasive technique of brain stimulation based on electromagnetic induction [60]. rTMS has the potential to alter cortical excitability depending on the duration and mode of stimulation [61]. The electromagnetic pulse easily passes through the skull, and causes small electrical currents that stimulate nerve cells in the targeted brain region [62]. Since this type of pulse generally does not reach further than two inches into the brain, it is possible to selectively target specific brain areas [62]. Generally, the patient feels a slight knocking or tapping on the head as the pulses are administered. rTMS frequencies of around 1 Hz induce an inhibitory effect on cortical excitability [63] and stimulus rates of more than 5 Hz generate a short-term increase in cortical excitability [64]. rTMS induces a MEP of the muscles of the lower extremities by stimulating the motor and supplementary motor area (SMA) of the cerebral cortex [31].

Repetitive transcranial magnetic stimulation in Parkinson’s disease

Several studies have reported the efficacy of rTMS on PD motor symptoms [65–69]. These effects are primarily directed at surface cortical regions, since the dopaminergic deficiency in PD is localized to the subcortical BG. The BG comprises a group of interconnected deep brain nuclei, i.e. the caudate and putamen, globus pallidus, substantia nigra and the subthalamic nucleus (STN) that, through their connections with the thalamus and the cortex, primarily influence the involuntary components of movement and muscle tone [70]. Several studies have documented the long-term effects of rTMS applied to PD patients for several days, rather than single sessions [71–73]. For instance, Shimamoto and coworkers applied rTMS on a broad area including the left and right motor, premotor and SMAs in nine PD patients for a period of 2 months, and observed improvements in the Unified Parkinson’s Disease Rating Scale (UPDRS), a rating scale used to follow PD progression [74]. A further trial in PD patients reported a shortened interruption of voluntary muscle contraction, defined cortical silent period, suggesting a disturbed inhibitory mechanism in the motor cortex [57]. PD patients show altered activation patterns in the SMA and overall less cortico-cortical excitability [75–81] that play a key role in motor selection in sequentially structured tasks, including handwriting. In a randomized controlled trial with a crossover design in PD patients, rTMS applied over the SMA influenced several key aspects of handwriting, e.g. vertical size and axial pressure, at least in the short term [82]. Ten PD patients treated with rTMS, evidenced short-term changes in functional fine motor task performance. rTMS over the SMA compensated for cortico-striatal imbalance and enhanced cortico-cortical connections. This treatment improved PD patients deficits such as reduction in speed during the writing task and decrease in letter size (micrographia).

Two mechanisms have been proposed to explain how cortically directed rTMS may improve PD symptoms: (1) rTMS induces brain network changes and positively affects the BG function; (2) rTMS directed to cortical sites compensates for PD-associated abnormal changes in cortical function [60]. Indeed, in support of the former mechanism, rTMS might modulate cortical areas, such as the prefrontal cortex and primary motor cortex, which are substantially connected to both the striatum and STN via glutamatergic projection, and thus indirectly modulate the release of dopamine in the BG [83]. Several TMS/functional imaging studies have demonstrated the effects of rTMS on BG and an increase in dopamine in the BG after rTMS applied to the frontal lobe [84].

rTMS can also transiently disrupt the function of a cortical target creating a temporary “virtual brain lesion” [85–87]. Mottaghy and coworkers have studied the ability of rTMS to produce temporary functional lesions in the BG, an area involved in working memory, and correlated these behavioral effects with changes in regional cerebral blood flow in the involved neuronal network [88]. Functional imaging and TMS studies in PD subjects have shown altered cortical physiology in areas associated to the BG such as the SMA, dorsolateral prefrontal cortex and primary motor cortex [57, 89], characterized by excessive corticospinal output at rest, concomitant to, or resulting from a reduced intracortical inhibition [60]. These altered changes in cortical function in PD patients might avoid the suppression of competing motor areas and therefore decrease the motor system performance, resulting in symptoms such as tonic contractions and rigidity [89].

rTMS has not only been applied to a motor area of the brain but has also been used to target PD non-motor deficits. For example, in a study involving six PD patients with mild cognitive impairment, a cognitive dysfunction defined by deficits in memory, rTMS was delivered over the frontal region at 1.2 times the motor threshold (minimum stimulation intensity) of the right abductor pollicis brevis muscle [3]. Over a period of 3 months, rTMS was performed for a total of 1200 stimulations. Improvement in neuropsychological tests (the trail-making test part B and the Wisconsin card-sorting test) was observed in all patients. In addition, an improvement in subjective symptoms and objective findings were also observed by the subjects, their families, and the therapists. The changes observed in PD subjects included “faster reactions”, “better body movement and smoother standing-up and movement”, “more active”, “more cheerful”, and “more expressive”. An increase in the amount of conversation, an increase in the neural mechanisms of mutual understanding within daily living and an improvement in responses to visitors were also noted, if compared to baseline. Additionally, changes such as better hand usage while eating and better sleep were also observed.

Cognitive dysfunction is often seen in PD patients with major depression and its neural basis could be the functional failure of the frontostriatal circuit [3, 90]. Ten days of rTMS in the frontal cortex can effectively alleviate PD-associated depression as shown by an open trial reporting a significant decrease in the Hamilton Depression Rating Scale (HDRS) scores [91]. A further double blind, sham stimulation-controlled, randomized study, involving 42 idiopathic PD patients affected by major or minor depression undergoing rTMS for 10 days, evidenced a mean decrease in HDRS and Beck depression inventory after therapy [92].

In opposition to the above mentioned positive reports concerning the efficacy of rTMS in PD patients, a lack of effectiveness of rTMS on objective or subjective symptoms has also been described. For example, in a study involving 85 idiopathic PD patients, no significant differences in clinical features were observed between patients receiving rTMS and sham stimulation [65]. Moreover, total and motor score of UPDRS were improved by rTMS and sham stimulation in the same manner. Despite this improvement, PD patients treated with rTMS revealed signs of depression, reporting no subjective benefits. In another randomized crossover study, 10 patients affected by idiopathic PD received rTMS to the SMA which resulted in subclinical worsening of complex and preparatory movement [93]. The rTMS protocol was not tolerated by 2 out of 10 patients. Furthermore, this study showed that, following rTMS, subtle regional disruption can persist for over 30 min, raising safety concerns. A further randomized crossover study involving 11 patients with idiopathic PD, treated with rTMS over the motor cortex, did not show any therapeutic effect on concurrent fine movement in PD [94].

In summary, conflicting findings regarding the efficacy of rTMS in PD have been reported and they can be explained by differences in stimulation parameters, including intensity, frequency, total number of pulses, stimulation site and total number of sessions. Therefore, further studies comparing different parameters are required.High-frequency transcranial magnetic stimulation
High-frequency TMS consists of continuous high-frequency stimulation of specific brain regions, including the motor cortex, cerebellum and BG, through implanted large four-contact electrodes connected to a pulse generator and positioned into the center of the target region [70]. Such stimulation induces an electrical field that spreads and depolarizes neighboring membranes of cell bodies, afferent and efferent axons, depending on neuronal element orientation and position in the field and on stimulation parameters [95]. Optimal clinical results are obtained by using pulses of 60–200 ms duration and 1–5 V amplitude, delivered in the STN at 120–180 Hz [96]. For example, high-frequency TMS produces a transient blockade of spontaneous STN activity, defined HFS-induced silence. During HFS-induced silence, the persistent Na+ current is totally blocked and the Ca2+-mediated responses are strongly reduced, suggesting that T- and L-type Ca2+ currents are transiently depressed by high-frequency TMS [97].Indeed, recent evidence suggests that the stimulation of the motor cortex, the cerebellum and the BG not only produces inhibitory and excitatory effects on local neurons, but also influences afferent and efferent pathways. Therefore, the mechanism of action of high-frequency TMS depends on changes in neural activity generated in the stimulated, afferent and efferent nuclei of the BG and motor cortex [98].High-frequency transcranial magnetic stimulation in Parkinson’s disease

In the first PD patients treated with high-frequency TMS in 1993, motor symptoms, tremor, rigidity and akinesia improved significantly allowing to decrease the administration of l-dopa by a mean of 55% [99]. Since then, several thousands of patients worldwide have been fitted with high-frequency TMS implants achieving marked improvements in their symptoms, making this method the reference procedure for advanced PD [100]. The time course of improvement following high-frequency TMS treatment differs for different cardinal symptoms of PD [101]. For instance, rigidity and resting tremor decrease immediately, within a few seconds after high-frequency TMS [102]. Different clinical effects are observed in PD patients depending on the site of stimulation [103]. For example, stimulation of the ventral intermediate nucleus of the thalamus can dramatically relieve PD-associated tremor [104]. Similarly, stimulation of the STN or globus pallidus interna (GPi) can substantially reduce rigidity, tremor, and gait difficulties in patients affected by idiopathic PD [105]. Stimulation of the GPi also reduces all of the major PD motor manifestations, including the reduction of l-dopa-induced dyskinesias and involuntary movements produced by individual doses of dopaminergic medications that can limit treatment efficacy [106]. Thalamic stimulation in the region of the ventral intermediate nucleus reduces limb tremor but it has little effect on other manifestations of the disease [107]. In order to explain the beneficial effects of high-frequency TMS, two fundamental mechanisms have been proposed by Garcia and coworkers: silencing and excitation of STN neurons [95]. They reported that high-frequency TMS using stimulus parameters that yield therapeutic effects has a dual effect, i.e. it suppresses spontaneous activity and drives STN neuronal activity. High-frequency TMS switches off a pathological disrupted activity in the STN (i.e. silencing of STN neurons mechanism) and imposes a new type of discharge in the upper gamma-band frequency (60–80 Hz range) that is endowed with beneficial effects (i.e. excitation of STN neurons mechanism) [95]. This improvement generated by high-frequency TMS is due to parallel non-exclusive actions, i.e. silencing of ongoing activity and generation of an activity pattern in the gamma range [108]. There is an important advantage in silencing spontaneous activity and generating a pattern: the signal to noise ratio and the functional significance of the new signal are enhanced [109].

Techniques and preparations employed to study the mechanisms of high-frequency TMS include electrophysiological techniques, measurement of neurotransmitter release in vivo, post-mortem immunohistochemistry of a metabolic marker such as cytochrome oxidase and imaging studies in vivo [95]. Such results consistently show a post-stimulus period of reduced neuronal firing followed by the slow recovery of spontaneous activity. High-frequency TMS, at frequencies >50 Hz, applied to the STN of PD patients undergoing functional stereotactic procedures [110–112], to the STN of rats in vivo [113, 114] and rat STN slices in vitro [97, 115, 116], produces a period of neuronal silence of hundreds of milliseconds to tens of seconds. During brief high-frequency TMS in PD patients off medication and in the murine model of parkinsonism obtained by acute injections of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine for 5 consecutive days, a reduced STN activity, as response to stimulation, is observed at 5–14 Hz and this response is frequency-dependent [114]. High-frequency TMS has two main advantages: (a) it reduces the time a patient spends in the “off” state because the individual dose of these profound diurnal fluctuations leaves a person slow, shaky, stiff, and unable to rise from a chair; (b) it allows the reduction of medications and their consequent side effects [117].

Pulsed electromagnetic field therapy
PEMF therapy is a non-static energy delivery system, characterized by electromagnetic fields inducing microcurrents in the target body tissues [118]. These microcurrents elicit specific biological responses depending on field parameters such as intensity, frequency and waveform [119]. The benefits of PEMF therapy have been observed in several clinical studies for treatment of several medical conditions including knee osteoarthritis [120], shoulder impingement syndrome [121], lower back pain [122, 123], multiple sclerosis [124, 125], cancer [121, 123, 125, 126], PD [127], AD [128] and reflex sympathetic dystrophy syndrome [129]. A large number of PEMF therapy devices contains user-friendly software packages with pre-recorded programs with the ability to modify programs depending on the patient’s needs [43, 130–132]. Examples of PEMF devices are the Curatron® (Amjo Corp, West Chester, PA, USA), Seqex® system (S.I.S.T.E.M.I. Srl, Trento, Italy), MRS 2000®, iMRS®, QRS® (all produced by Swiss Bionic Solutions Schweiz GmbH, Dulliken, Switzerland) and TESLA Stym (Iskra Medical, Ljubljana, Slovenia).

Pulsed electromagnetic field therapy in Parkinson’s disease

In October 2008 the Food and Drug Administration approved the use of PEMF therapy for treatment of major depressive disorder in PD patients who failed to achieve satisfactory improvement from very high dosages of antidepressant medications [133, 134]. Several studies reported PEMF therapy improved cognitive functions and motor symptoms. For example, an investigation involving three elderly PD patients with cognitive impairment assessed the effect of PEMF therapy on macrosomatognosia, a disorder of the body image in which the patient perceives a part or parts of his body as disproportionately large [135]. After receiving PEMF therapy, PD patients’ drawings showed reversal of macrosomatognosia (assessed by Draw-a-Person test) with reduction of the right parietal lobe dysfunction. Furthermore, PEMF therapy applied to a 49-year-old male PD patient with stage 3 disease, as assessed by Hoehn and Yahr scale, resulted in a marked improvement in motor and non-motor symptoms such as mood swings, sleeplessness, pain and sexual and cognitive dysfunctions, suggesting that PEMF therapy should be tested in large cohorts of PD patients as monotherapy and should also be considered as a treatment modality for de novo diagnosed PD patients [136]. PEMF therapy was also effective in improving visuospatial deficits in four PD patients, as assessed by the clock-drawing test [137]. Moreover, PEMF therapy improved PD-associated freezing (a symptom manifesting as a sudden attack of immobility usually experienced during walking) in 3 PD patients through the facilitation of serotonin neurotransmission at both junctional and non-junctional neuronal target sites [127].

Discussion
Although many studies on electromagnetic therapy included only a small number of participants, several investigations suggest that this therapy is effective in treating PD patients’ motor and non-motor symptoms. In the development of electromagnetic therapies, it is important to clarify the pathophysiological mechanisms underlying the symptoms to treat in order to determine the appropriate brain region to target. Thus, in the future, electromagnetic therapy must tend towards a more personalized approach, tailored to the specific PD patient’s symptoms. All the types of electromagnetic therapy described in this review can be used in combination with pharmacological and non-pharmacological therapies but this approach is understudied in PD patients. Therefore, specific protocols should be designed and tested in combination with other therapies in future controlled trials in patients affected by PD.

Transcranial magnetic stimulation

TMS increases the release of dopamine in the striatum and frontal cortex, which in turn improves PD symptoms including motor performance [138]. Furthermore, TMS applied in the prefrontal cortex induces the release of endogenous dopamine in the ipsilateral caudate nucleus as observed by positron emission tomography in healthy human subjects [89]. TMS application results in partial or complete disappearance of muscular pain and l-dopa-induced dyskinesia as well as regression of visuospatial impairment. This clinical improvement is followed by MEG improvement and normalization recorded after TMS, suggesting that TMS has an immediate and beneficial effect on corticostriatal interactions that play an important role in the pathophysiology of PD [58]. Cerasa and coworkers observed that repetitive TMS applied over the inferior frontal cortex reduced the amount of dyskinesia induced by a supramaximal single dose of levodopa in PD patients, suggesting that this area may play a key role in controlling the development of dyskinesia [139]. The mechanism underlying TMS effectiveness in PD remains an unanswered question due to the complexity of behavioral and neuroendocrine effects exerted by the TMS when applied to biological systems and their potential impact on neurotransmitter functions [140]. The effect of TMS differs depending on the stage of the disease, the age of disease onset, the amount of cerebral atrophy and genetic factors [37]. TMS has a low cost and is simple to operate and portable, opening the possibility for patients to perform at home stimulation which could be of high relevance in the elderly and in patients who are severely disabled. As far as side effects are concerned, the muscles of the scalp, jaw or face may contract or tingle during the procedure and mild headache or brief lightheadedness may occur [141, 142]. A recent large-scale study on the safety of TMS found that most side effects, such as headaches or scalp discomfort, were mild or moderate, and no seizures occurred [143]. Although evidence shows that TMS exerts complex cellular, systemic and neuroendocrine effects on biological systems impacting neurotransmitter functions [58], future controlled studies in larger cohorts of patients and with a long term follow-up are needed to further clarify the mechanisms underlying TMS efficacy in PD patients.

Repetitive transcranial magnetic stimulation

rTMS can be defined as a safe and non-invasive technique of brain stimulation which allows to specifically treat PD with low-frequency electromagnetic pulses [60]. As opposed to high-frequency TMS, which can induce convulsions in healthy subjects, rTMS does not affect the electroencephalogram pattern [71, 144]. Slow waves have been induced by rTMS over the right prefrontal area, a brain area involved in executive dysfunction that is observed in early stages of PD and is characterized by deficits in internal control of attention, set shifting, planning, inhibitory control, dual task performance, decision-making and social cognition tasks [3, 145]. rTMS applied to PD patients, enhances not only executive function, but also motor function, subjective symptoms and objective findings [3]. rTMS also increases cognitive function and other symptoms associated to the prefrontal area in PD patients [146]. In PD patients, therapeutic efficacy and long-term benefits of rTMS are obtained following multiple regular sessions rather than single sessions, but side effects associated to this therapy still warrant investigation in large controlled trials.

High-frequency magnetic stimulation

The observations that STN activity is disorganized in PD patients and that a lesion or chemical inactivation of STN neurons ameliorate motor symptoms led to the hypothesis that high-frequency TMS silences STN neurons and, by eliminating a pathological pattern, alleviates PD symptoms [147–151]. Garcia and colleagues proposed another hypothesis suggesting that high-frequency TMS suppresses not only the pathological STN activity but also imposes a new activity on STN neurons [95]. They proposed that high-frequency TMS excites the stimulated structure and evokes a regular pattern time-locked to the stimulation, overriding the pathological STN activity. As a consequence, high-frequency TMS removes the STN spontaneous activity and introduces a new and regular pattern that improves the dopamine-deficient network [95]. Elahi and coworkers found that high-frequency TMS modulates the excitability of the targeted brain regions and produces clinically significant motor improvement in PD patients [66]. This improvement is due to parallel non-exclusive actions, i.e. silencing of ongoing activity and generation of an activity pattern in the high gamma range [152]. Several clinical studies reported positive clinical results following high-frequency TMS in l-dopa-responsive forms of PD, including patients with selective brain dopaminergic lesions [153]. It remains unclear whether the mechanisms of action of high-frequency TMS and l-dopa are similar or they could be even synergic. However, high-frequency TMS improves the l-dopa-sensitive cardinal motor symptoms of PD patients with benefits similar to those given by l-dopa, though with reduced motor complications [154, 155]. The interactions with the dopaminergic system seem to be a key factor explaining the efficacy of both treatments [156]. High-frequency TMS changes dopamine lesion-induced functional alterations in the BG of PD animal models and gives an insight into the mechanisms underlying its antiparkinsonian effects [114, 157, 158]. The intrinsic capacity of the BG to generate oscillations and change rapidly from a physiological to a pathogenic pattern is crucial; the next step will be to identify how high-frequency TMS is propagated inside the BG. Disadvantages of this therapy are the high cost and limited availability of the devices to specialized medical centers, limited knowledge of potential long-term side effects and the necessity to employ highly trained personnel.

Pulsed electromagnetic fields

PEMF therapy improves PD symptoms including tremor, slowness of movement and difficulty in walking [159]. It is non-invasive, safe and improves PD patients’ quality of life [124, 160]. PEMF therapy, employed for PD treatment, supports the body’s own healing process for 4–6 h after therapy session [161–163]. It can be used at home and applied to the entire body or locally to target a specific body area and, if compared with dopaminergic systemic therapy, e.g. l-dopa, it can offer an alternative treatment avoiding systemic side effects such as hepatotoxicity and nephrotoxicity.

Conclusions
Electromagnetic therapy opens a new avenue for PD treatment. Each electromagnetic therapy technique described in this review can be applied according to a single protocol or as a combination of different protocols specifically tailored to the PD patient’s needs. Beyond the necessity to choose coil or electrode size and placement, there is a variety of parameters that have to be taken into account when designing electromagnetic therapy approaches and they include stimulation intensity, duration, frequency, pattern, electrode polarity and size. Furthermore, electromagnetic therapy can also be combined with pharmacological or non-pharmacological treatments, e.g. physical therapy and cognitive tasks, to produce additive or potentiated clinical effects. In conclusion, electromagnetic therapy represents a non-invasive, safe and promising approach that can be used alone or combined with conventional therapies for the challenging treatment of PD motor and non-motor symptoms.

Authors’ contributions
MV, AV, LP, BP, JCMM, and TI contributed equally to this review. All authors read and approved the final manuscript.

Acknowledgements

JCMM thanks CONACyT, México for membership. The authors thank Iskra Medical (Stegne 23, 1000 Ljubljana, Slovenia) for supporting the open access publication of this article.

Compliance with ethical guidelines

Competing interests The authors declare that they have no competing interests.

Contributor Information
Maria Vadalà, Email: moc.liamg@aladav.yram.

Annamaria Vallelunga, Email: moc.liamg@airamannaagnulellav.

Lucia Palmieri, Email: moc.liamg@ireimlap.aicul.

Beniamino Palmieri, Email: ti.erominu@ireimlap.

Julio Cesar Morales-Medina, Email: xm.vatsevnic@mselaromj.

Tommaso Iannitti, Email: moc.liamg@ittinnai.osammot.

References
1. Granado N, Ares-Santos S, Moratalla R. Methamphetamine and Parkinson’s disease. Parkinsons Dis. 2013;1:1–10.
2. Popa LCA, Constantinescu A, Popescu CD. Differences of cortical excitability between Parkinson’s disease patients and healthy subjects. A comparative TMS study. Romanian J Neurol. 2012;11:1.
3. Furukawa T, Izumi S, Toyokura M, Masakado Y. Effects of low-frequency repetitive transcranial magnetic stimulation in Parkinson’s disease. Tokai J Exp Clin Med. 2009;34(3):63–71. [PubMed]
4. Desplats P, Patel P, Kosberg K, Mante M, Patrick C, Rockenstein E, et al. Combined exposure to Maneb and Paraquat alters transcriptional regulation of neurogenesis-related genes in mice models of Parkinson’s disease. Mol Neurodegener. 2012;7:49. doi: 10.1186/1750-1326-7-49. [PMC free article] [PubMed] [Cross Ref]
5. Subramaniam SR, Chesselet MF. Mitochondrial dysfunction and oxidative stress in Parkinson’s disease. Prog Neurobiol. 2013;106–107:17–32. doi: 10.1016/j.pneurobio.2013.04.004. [PMC free article] [PubMed] [Cross Ref]
6. Vallelunga A, Ragusa M, Di Mauro S, Iannitti T, Pilleri M, Biundo R, et al. Identification of circulating microRNAs for the differential diagnosis of Parkinson’s disease and Multiple System Atrophy. Front Cell Neurosci. 2014;8:156. doi: 10.3389/fncel.2014.00156. [PMC free article] [PubMed] [Cross Ref]
7. Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol. 2009;7(1):65–74. doi: 10.2174/157015909787602823. [PMC free article] [PubMed] [Cross Ref]
8. Dauer W, Przedborski S. Parkinson’s disease: mechanisms and models. Neuron. 2003;39(6):889–909. doi: 10.1016/S0896-6273(03)00568-3. [PubMed] [Cross Ref]
9. Valente EM, Salvi S, Ialongo T, Marongiu R, Elia AE, Caputo V, et al. PINK1 mutations are associated with sporadic early-onset parkinsonism. Ann Neurol. 2004;56:336–341. doi: 10.1002/ana.20256. [PubMed] [Cross Ref]
10. Polymeropoulos MHLC, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science. 1997;276(5321):2045–2047. doi: 10.1126/science.276.5321.2045. [PubMed] [Cross Ref]
11. Chou KL. Diagnosis and management of the patient with tremor. Med Health R I. 2004;87(5):135–138. [PubMed]
12. Dauer W, Przedborski S. Parkinson’s disease: mechanisms and models. Neuron. 2003;39(6):889–909. doi: 10.1016/S0896-6273(03)00568-3. [PubMed] [Cross Ref]
13. McGeer PL, McGeer EG. Inflammation and neurodegeneration in Parkinson’s disease. Parkinsonism Relat Disord. 2004;10(1):S3–S7. doi: 10.1016/j.parkreldis.2004.01.005. [PubMed] [Cross Ref]
14. Mendez I, Viñuela A, Astradsson A, Mukhida K, Hallett P, Robertson H, et al. Dopamine neurons implanted into people with Parkinson’s disease survive without pathology for 14 years. Nat Med. 2008;14(5):507–509. doi: 10.1038/nm1752. [PMC free article] [PubMed] [Cross Ref]
15. Richardson PJ, Kase H, Jenner PG. Adenosine A2A receptor antagonists as new agents for the treatment of Parkinson’s disease. Trends Pharmacol Sci. 1997;18(9):338–344. doi: 10.1016/S0165-6147(97)01096-1. [PubMed] [Cross Ref]
16. Schapira AH, Bezard E, Brotchie J, Calon F, Collingridge GL, Ferger B, et al. Novel pharmacological targets for the treatment of Parkinson’s disease. Nat Rev Drug Discov. 2006;5(10):845–854. doi: 10.1038/nrd2087. [PubMed] [Cross Ref]
17. Bezard E, Gerlach I, Moratalla R, Gross CE, Jork R. 5-HT1A receptor agonist-mediated protection from MPTP toxicity in mouse and macaque models of Parkinson’s disease. Neurobiol Dis. 2006;23(1):77–86. doi: 10.1016/j.nbd.2006.02.003. [PubMed] [Cross Ref]
18. Poryazova RG, Zachariev ZI. REM sleep behavior disorder in patients with Parkinson’s disease. Folia Med (Plovdiv) 2005;47(1):5–10. [PubMed]
19. Eisensehr I, v Lindeiner H, Jäger M, Noachtar S. REM sleep behavior disorder in sleep-disordered patients with versus without Parkinson’s disease: is there a need for polysomnography? J Neurol Sci. 2001;186(1–2):7–11. doi: 10.1016/S0022-510X(01)00480-4. [PubMed] [Cross Ref]
20. Kales A, Ansel RD, Markham CH, Scharf MB, Tan TL. Sleep in patients with Parkinson’s disease and normal subjects prior to and following levodopa administration. Clin Pharmacol Ther. 1971;12(2):397–406. [PubMed]
21. Factor SA, McAlarney T, Sanchez-Ramos JR, Weiner WJ. Sleep disorders and sleep effect in Parkinson’s disease. Mov Disord Off J Mov Disord Soc. 1990;5(4):280–285. doi: 10.1002/mds.870050404. [PubMed] [Cross Ref]
22. Lees AJ, Blackburn NA, Campbell VL. The nighttime problems of Parkinson’s disease. Clin Neuropharmacol. 1988;11(6):512–519. doi: 10.1097/00002826-198812000-00004. [PubMed] [Cross Ref]
23. Comella CL, Nardine TM, Diederich NJ, Stebbins GT. Sleep-related violence, injury, and REM sleep behavior disorder in Parkinson’s disease. Neurology. 1998;51(2):526–529. doi: 10.1212/WNL.51.2.526. [PubMed] [Cross Ref]
24. Chaudhuri KR, Healy DG, Schapira AH, FmedSci Non-motor symptoms of Parkinson’s disease: diagnosis and management. Lancet Neurol. 2006;5(3):235–245. doi: 10.1016/S1474-4422(06)70373-8. [PubMed] [Cross Ref]
25. Lieberman A. Depression in Parkinson’s disease—a review. Acta Neurol Scand. 2006;113(1):1–8. doi: 10.1111/j.1600-0404.2006.00536.x. [PubMed] [Cross Ref]
26. Poewe W. Non-motor symptoms in Parkinson’s disease. Eur J Neurol. 2008;15(1):14–20. doi: 10.1111/j.1468-1331.2008.02056.x. [PubMed] [Cross Ref]
27. Trinh J, Farrer M. Advances in the genetics of Parkinson disease. Nat Rev Neurol. 2013;9(8):445–454. doi: 10.1038/nrneurol.2013.132. [PubMed] [Cross Ref]
28. Lubbe S, Morris HR. Recent advances in Parkinson’s disease genetics. J Neurol. 2014;261(2):259–266. doi: 10.1007/s00415-013-7003-2. [PubMed] [Cross Ref]
29. Taymans JM, Baekelandt V. Phosphatases of alpha-synuclein, LRRK2, and tau: important players in the phosphorylation-dependent pathology of Parkinsonism. Front Genet. 2014;5:382. doi: 10.3389/fgene.2014.00382. [PMC free article] [PubMed] [Cross Ref]
30. van der Vegt JP, van Nuenen BF, Bloem BR, Klein C, Siebner HR. Imaging the impact of genes on Parkinson’s disease. Neuroscience. 2009;164(1):191–204. doi: 10.1016/j.neuroscience.2009.01.055. [PubMed] [Cross Ref]
31. Kimura H, Kurimura M, Kurokawa K, Nagaoka U, Arawaka S, Wada M, et al. A comprehensive study of repetitive transcranial magnetic stimulation in Parkinson’s disease. ISRN Neurol. 2011;2011:845453. doi: 10.5402/2011/845453. [PMC free article] [PubMed] [Cross Ref]
32. Lees AJ. The on-off phenomenon. J Neurol Neurosurg Psychiatry. 1989;52(1):29–37. doi: 10.1136/jnnp.52.Suppl.29. [PMC free article] [PubMed] [Cross Ref]
33. Hattoria N, Wanga M, Taka H, Fujimura T, Yoritaka A, Kubo S, et al. Toxic effects of dopamine metabolism in Parkinson’s disease. Parkinsonism Relat Disord. 2009;15(1):S35–S38. doi: 10.1016/S1353-8020(09)70010-0. [PubMed] [Cross Ref]
34. Belcastro V, Tozzi A, Tantucci M, Costa C, Di Filippo M, Autuori A, et al. A2A adenosine receptor antagonists protect the striatum against rotenone-induced neurotoxicity. Exp Neurol. 2009;217(1):231–234. doi: 10.1016/j.expneurol.2009.01.010. [PubMed] [Cross Ref]
35. Benabid AL, Chabardes S, Mitrofanis J, Pollak P. Deep brain stimulation of the subthalamic nucleus for the treatment of Parkinson’s disease. Lancet Neurol. 2009;8(1):67–81. doi: 10.1016/S1474-4422(08)70291-6. [PubMed] [Cross Ref]
36. Wang Z, Che PL, Du J, Ha B, Yarema KJ. Static magnetic field exposure reproduces cellular effects of the Parkinson’s disease drug candidate ZM241385. PLoS One. 2010;5(11):e13883. doi: 10.1371/journal.pone.0013883. [PMC free article] [PubMed] [Cross Ref]
37. Anderkova L, Rektorova I. Cognitive effects of repetitive transcranial magnetic stimulation in patients with neurodegenerative diseases—clinician’s perspective. J Neurol Sci. 2014;339(1–2):15–25. doi: 10.1016/j.jns.2014.01.037. [PubMed] [Cross Ref]
38. Caspar S. Invasive and non-invasive stimulation in Parkinson’s disease. Germany: Department of Clinical Neurophysiol; 2011.
39. Sandyk R. Weak magnetic fields as a novel therapeutic modality in Parkinson’s disease. Int J Neurosci. 1992;66(1–2):1–15. [PubMed]
40. Sandyk R. Treatment with weak electromagnetic fields restores dream recall in a parkinsonian patient. Int J Neurosci. 1997;90(1–2):75–86. doi: 10.3109/00207459709000627. [PubMed] [Cross Ref]
41. Vonloh M, Chen R, Kluger B. Safety of transcranial magnetic stimulation in Parkinson’s disease: a review of the literature. Parkinsonism Relat Disord. 2013;19(6):573–585. doi: 10.1016/j.parkreldis.2013.01.007. [PMC free article] [PubMed] [Cross Ref]
42. Wade B. A review of pulsed electromagnetic field (PEMF) mechanisms at a cellular level: a rationale for clinical use. Am J Health Res. 2013;1(3):51–55. doi: 10.11648/j.ajhr.20130103.13. [Cross Ref]
43. Markov MS. Expanding use of pulsed electromagnetic field therapies. Electromagn Biol Med. 2007;26(3):257–274. doi: 10.1080/15368370701580806. [PubMed] [Cross Ref]
44. Weintraub MI. Magnetotherapy: historical background with a stimulating future. Phys Rehabil Med. 2004;16(2):95–108.
45. De Loecker W, Cheng N, Delport PH. Emerging electromagnetic medicine. New York: Springer; 1990. Effects of pulsed electromagnetic fields on membrane transport; pp. 45–57.
46. Wassermann EM, Lisanby SH. Therapeutic application of repetitive transcranial magnetic stimulation: a review. Clin Neurophysiol Off J Int Fed Clin Neurophysiol. 2001;112(8):1367–1377. doi: 10.1016/S1388-2457(01)00585-5. [PubMed] [Cross Ref]
47. Wassermann EM, Grafman J, Berry C, Hollnagel C, Wild K, Clark K, et al. Use and safety of a new repetitive transcranial magnetic stimulator. Electroencephalogr Clin Neurophysiol. 1996;101(5):412–417. doi: 10.1016/0924-980X(96)96004-X. [PubMed] [Cross Ref]
48. Edwards MJ, Talelli P, Rothwell JC. Clinical applications of transcranial magnetic stimulation in patients with movement disorders. Lancet Neurol. 2008;7(9):827–840. doi: 10.1016/S1474-4422(08)70190-X. [PubMed] [Cross Ref]
49. Kobayashi M, Pascual-Leone A. Transcranial magnetic stimulation in neurology. Lancet Neurol. 2003;2:145–156. doi: 10.1016/S1474-4422(03)00321-1. [PubMed] [Cross Ref]
50. Rudiak D, Marg E. Finding the depth of magnetic brain stimulation: a re-evaluation. Electroencephalogr Clin Neurophysiol. 1994;93(5):358–371. doi: 10.1016/0168-5597(94)90124-4. [PubMed] [Cross Ref]
51. Barker AT, Jalinous R, Freeston IL. Non-invasive magnetic stimulation of human motor cortex. Lancet. 1985;1(8437):1106–1107. doi: 10.1016/S0140-6736(85)92413-4. [PubMed] [Cross Ref]
52. Fuhr P, Agostino R, Hallett M. Spinal motor neuron excitability during the silent period after cortical stimulation. Electroencephalogr Clin Neurophysiol. 1991;81(4):257–262. doi: 10.1016/0168-5597(91)90011-L. [PubMed] [Cross Ref]
53. Inghilleri M, Berardelli A, Cruccu G, Manfredi M. Silent period evoked by transcranial stimulation of the human cortex and cervicomedullary junction. J Physiol. 1993;466:521–534. [PMC free article] [PubMed]
54. Farzan F, Barr MS, Hoppenbrouwers SS, Fitzgerald PB, Chen R, Pascual-Leone A, et al. The EEG correlates of the TMS-induced EMG silent period in humans. Neuroimage. 2013;83:120–134. doi: 10.1016/j.neuroimage.2013.06.059. [PMC free article] [PubMed] [Cross Ref]
55. Cantello R, Gianelli M, Bettucci D, Civardi C, De Angelis MS, Mutani R. Parkinson’s disease rigidity: magnetic motor evoked potentials in a small hand muscle. Neurology. 1991;41(9):1449–1456. doi: 10.1212/WNL.41.9.1449. [PubMed] [Cross Ref]
56. Khedr EM, Farweez HM, Islam H. Therapeutic effect of repetitive transcranial magnetic stimulation on motor function in Parkinson’s disease patients. Eur J Neurol. 2003;10(5):567–572. doi: 10.1046/j.1468-1331.2003.00649.x. [PubMed] [Cross Ref]
57. Lefaucheur JP. Motor cortex dysfunction revealed by cortical excitability studies in Parkinson’s disease: influence of antiparkinsonian treatment and cortical stimulation. Clin Neurophysiol. 2005;116(2):244–253. doi: 10.1016/j.clinph.2004.11.017. [PubMed] [Cross Ref]
58. Anninos P, Adamopoulos A, Kotini A, Tsagas N, Tamiolakis D, Prassopoulos P. MEG evaluation of Parkinson’s diseased patients after external magnetic stimulation. Acta Neurol Belg. 2007;107(1):5–10. [PubMed]
59. Stam CJ. Use of magnetoencephalography (MEG) to study functional brain networks in neurodegenerative disorders. J Neurol Sci. 2010;289(1–2):128–134. doi: 10.1016/j.jns.2009.08.028. [PubMed] [Cross Ref]
60. Fregni F, Simon DK, Wu A, Pascual-Leone A. Non-invasive brain stimulation for Parkinson’s disease: a systematic review and meta-analysis of the literature. J Neurol Neurosurg Psychiatry. 2005;76(12):1614–1623. doi: 10.1136/jnnp.2005.069849. [PMC free article] [PubMed] [Cross Ref]
61. Hallett M. Transcranial magnetic stimulation: a primer. Neuron. 2007;55(2):187–199. doi: 10.1016/j.neuron.2007.06.026. [PubMed] [Cross Ref]
62. Greenberg BD, Malone DA, Friehs GM, Rezai AR, Kubu CS, Malloy PF, et al. Three-year outcomes in deep brain stimulation for highly resistant obsessive-compulsive disorder. Neuropsychopharmacol Off Publ Am Coll Neuropsychopharmacol. 2006;31(11):2384–2393. doi: 10.1038/sj.npp.1301165. [PubMed] [Cross Ref]
63. Chen R, Classen J, Gerloff C, Celnik P, Wassermann EM, Hallett M, et al. Depression of motor cortex excitability by low-frequency transcranial magnetic stimulation. Neurology. 1997;48(5):1398–1403. doi: 10.1212/WNL.48.5.1398. [PubMed] [Cross Ref]
64. Pascual-Leone A, Valls-Solé J, Wassermann EM, Hallett M. Responses to rapid-rate transcranial magnetic stimulation of the human motor cortex. Brain J Neurol. 1994;117(Pt 4):847–858. doi: 10.1093/brain/117.4.847. [PubMed] [Cross Ref]
65. Okabe S, Ugawa Y, Kanazawa I. 0.2-Hz repetitive transcranial magnetic stimulation has no add-on effects as compared to a realistic sham stimulation in Parkinson’s disease. Mov Disord. 2003;18(4):382–388. doi: 10.1002/mds.10370. [PubMed] [Cross Ref]
66. Elahi B, Chen R. Effect of transcranial magnetic stimulation on Parkinson motor function—systematic review of controlled clinical trials. Mov Disord. 2009;24(3):357–363. doi: 10.1002/mds.22364. [PubMed] [Cross Ref]
67. Wang M, Ping GU, Xiao-wei MA, Yan-min LI. Effects of low frequency repetitive transcranial magnetic stimulation on motor function and affective disorder in patients with Parkinson’s disease. Chin J Geriatr. 2009;28:729–732.
68. Niu X, G Y. Observation of repetitively transcranial magnetic stimulation in the treatment of depression induced by Parkinson’s disease. Chin J Pract Nerv Dis. 2012;15:11–13.
69. Shirota Y, Ohtsu H, Hamada M, Enomoto H, Ugawa Y. Supplementary motor area stimulation for Parkinson disease: a randomized controlled study. Neurology. 2013;80(15):1400–1405. doi: 10.1212/WNL.0b013e31828c2f66. [PubMed] [Cross Ref]
70. Pizzolato G, Mandat T. Deep brain stimulation for movement disorders. Mini Rev Art Front Integr Neurosci. 2012;6(2):1–5.
71. Boutros NN, Berman RM, Hoffman R, Miano AP, Campbell D, Ilmoniemi R. Electroencephalogram and repetitive transcranial magnetic stimulation. Depress Anxiety. 2000;12(3):166–169. doi: 10.1002/1520-6394(2000)12:3<166::AID-DA8>3.0.CO;2-M. [PubMed] [Cross Ref]
72. Fregni F, Boggio PS, Valle AC, Rocha RR, Duarte J, Ferreira MJ, et al. A sham-controlled trial of a 5-day course of repetitive transcranial magnetic stimulation of the unaffected hemisphere in stroke patients. Stroke. 2006;37(8):2115–2122. doi: 10.1161/01.STR.0000231390.58967.6b. [PubMed] [Cross Ref]
73. Fox MD, Liu H, Pascual-Leone A. Identification of reproducible individualized targets for treatment of depression with TMS based on intrinsic connectivity. Neuroimage. 2013;66:151–160. doi: 10.1016/j.neuroimage.2012.10.082. [PMC free article] [PubMed] [Cross Ref]
74. Shimamoto H, Takasaki K, Shigemori M, Imaizumi T, Ayabe M, Shoji H. Therapeutic effect and mechanism of repetitive transcranial magnetic stimulation in Parkinson’s disease. J Neurol. 2001;248(3):III48–III52. doi: 10.1007/PL00007826. [PubMed] [Cross Ref]
75. Eckert T, Peschel T, Heinze HJ, Rotte M. Increased pre-SMA activation in early PD patients during simple self-initiated hand movements. J Neurol. 2006;253(2):199–207. doi: 10.1007/s00415-005-0956-z. [PubMed] [Cross Ref]
76. Buhmann C, Glauche V, Stürenburg HJ, Oechsner M, Weiller C, Büchel C. Pharmacologically modulated fMRI–cortical responsiveness to levodopa in drug-naive hemiparkinsonian patients. Brain. 2003;126(Pt 2):451–461. doi: 10.1093/brain/awg033. [PubMed] [Cross Ref]
77. Ceballos-Baumann AO, Boecker H, Bartenstein P, von Falkenhayn I, Riescher H, Conrad B, et al. A positron emission tomographic study of subthalamic nucleus stimulation in Parkinson disease: enhanced movement-related activity of motor-association cortex and decreased motor cortex resting activity. Arch Neurol. 1999;56(8):997–1003. doi: 10.1001/archneur.56.8.997. [PubMed] [Cross Ref]
78. Jahanshahi M, Jenkins IN, Brown RG, Marsden CD, Passingham RE, Brooks DJ. Self-initiated versus externally triggered movements. I. An investigation using measurement of regional cerebral blood flow with PET and movement-related potentials in normal and Parkinson’s disease subjects. Brain. J Neurol. 1995;118(Pt 4):913–933. [PubMed]
79. Jenkins IH, Fernandez W, Playford ED, Lees AJ, Frackowiak RS, Passingham RE, et al. Impaired activation of the supplementary motor area in Parkinson’s disease is reversed when akinesia is treated with apomorphine. Ann Neurol. 1992;32(6):749–757. doi: 10.1002/ana.410320608. [PubMed] [Cross Ref]
80. Playford ED, Jenkins IH, Passingham RE, Nutt J, Frackowiak RSJ, Brooks DJ. Impaired mesial frontal and putamen activation in Parkinson’s disease: a positron emission tomography study. Ann Neurol. 1992;32(2):151–161. doi: 10.1002/ana.410320206. [PubMed] [Cross Ref]
81. Rascol O, Sabatini U, Chollet F, Fabre N, Senard JM, Montastruc JL, et al. Normal activation of the supplementary motor area in patients with Parkinson’s disease undergoing long-term treatment with levodopa. J Neurol Neurosurg Psychiatry. 1994;57(5):567–571. doi: 10.1136/jnnp.57.5.567. [PMC free article] [PubMed] [Cross Ref]
82. Randhawa BK, Farley BG, Boyd LA. Repetitive transcranial magnetic stimulation improves handwriting in Parkinson’s disease. Parkinsons Dis. 2013;2013:751925. [PMC free article] [PubMed]
83. Morari M, Marti M, Sbrenna S, Fuxe K, Bianchi C, Beani L. Reciprocal dopamine-glutamate modulation of release in the basal ganglia. Neurochem Int. 1998;33(5):383–397. doi: 10.1016/S0197-0186(98)00052-7. [PubMed] [Cross Ref]
84. Keck ME, Welt T, Müller MB, Erhardt A, Ohl F, Toschi N, et al. Repetitive transcranial magnetic stimulation increases the release of dopamine in the mesolimbic and mesostriatal system. Neuropharmacology. 2002;43(1):101–109. doi: 10.1016/S0028-3908(02)00069-2. [PubMed] [Cross Ref]
85. Grafman J, Pascual-Leone A, Alway D, Nichelli P, Gomez-Tortosa E, Hallett M. Induction of a recall deficit by rapid-rate transcranial magnetic stimulation. Neuroreport. 1994;5(9):1157–1160. doi: 10.1097/00001756-199405000-00034. [PubMed] [Cross Ref]
86. Jahanshahi M, Profice P, Brown RG, Ridding MC, Dirnberger G, Rothwell JC. The effects of transcranial magnetic stimulation over the dorsolateral prefrontal cortex on suppression of habitual counting during random number generation. Brain. 1998;121(Pt 8):1533–1544. doi: 10.1093/brain/121.8.1533. [PubMed] [Cross Ref]
87. Pascual-Leone A, Bartres-Faz D, Keenan JP. Transcranial magnetic stimulation: studying the brain-behaviour relationship by induction of ‘virtual lesions’ Philos Trans R Soc Lond B Biol Sci. 1999;354(1387):1229–1238. doi: 10.1098/rstb.1999.0476. [PMC free article] [PubMed] [Cross Ref]
88. Mottaghy FM, Krause BJ, Kemna LJ, Töpper R, Tellmann L, Beu M, et al. Modulation of the neuronal circuitry subserving working memory in healthy human subjects by repetitive transcranial magnetic stimulation. Neurosci Lett. 2000;280(3):167–170. doi: 10.1016/S0304-3940(00)00798-9. [PubMed] [Cross Ref]
89. Strafella AP, Paus T, Barrett J, Dagher A. Repetitive transcranial magnetic stimulation of the human prefrontal cortex induces dopamine release in the caudate nucleus. J Neurosci. 2001;21(15):RC157. [PubMed]
90. Gessler M, Bruns GA. A physical map around the WAGR complex on the short arm of chromosome 11. Genomics. 1989;5(1):43–55. doi: 10.1016/0888-7543(89)90084-0. [PubMed] [Cross Ref]
91. Dragasevic N, Potrebic A, Damjanovi? A, Stefanova E, Kosti? VS. Therapeutic efficacy of bilateral prefrontal slow repetitive transcranial magnetic stimulation in depressed patients with Parkinson’s disease: an open study. Mov Disord Off J Mov Disord Soc. 2002;17(3):528–532. doi: 10.1002/mds.10109. [PubMed] [Cross Ref]
92. Fregni F, Santos CM, Myczkowski ML, Rigolino R, Gallucci-Neto J, Barbosa ER, et al. Repetitive transcranial magnetic stimulation is as effective as fluoxetine in the treatment of depression in patients with Parkinson’s disease. J Neurol Neurosurg Psychiatry. 2004;75(8):1171–1174. doi: 10.1136/jnnp.2003.027060. [PMC free article] [PubMed] [Cross Ref]
93. Boylan LS, Pullman SL, Lisanby SH, Spicknall KE, Sackeim HA. Repetitive transcranial magnetic stimulation to SMA worsens complex movements in Parkinson’s disease. Clin Neurophysiol. 2001;112(2):259–264. doi: 10.1016/S1388-2457(00)00519-8. [PubMed] [Cross Ref]
94. Ghabra MB, Hallett M, Wassermann EM. Simultaneous repetitive transcranial magnetic stimulation does not speed fine movement in PD. Neurology. 1999;52(4):768–770. doi: 10.1212/WNL.52.4.768. [PubMed] [Cross Ref]
95. Garcia L, D’Alessandro G, Bioulac B, Hammond C. High-frequency stimulation in Parkinson’s disease: more or less? Trends Neurosci. 2005;28(4):209–216. doi: 10.1016/j.tins.2005.02.005. [PubMed] [Cross Ref]
96. Moro E, Esselink RJA, Xie J, Hommel M, Benabid AL, Pollak P. The impact on Parkinson’s disease of electrical parameter settings in STN stimulation. Neurology. 2002;59(5):706–713. doi: 10.1212/WNL.59.5.706. [PubMed] [Cross Ref]
97. Beurrier C, Bioulac B, Audin J, Hammond C. High-frequency stimulation produces a transient blockade of voltage-gated currents in subthalamic neurons. J Neurophysiol. 2001;85(4):1351–1356. [PubMed]
98. McIntyre CC, Savasta M, Walter BL, Vitek JL. How does deep brain stimulation work? Present understanding and future questions. J Clin Neurophysiol. 2004;21(1):40–50. doi: 10.1097/00004691-200401000-00006. [PubMed] [Cross Ref]
99. Krack P, Batir A, Van Blercom N, Chabardes S, Fraix V, Ardouin C, et al. Five-year follow-up of bilateral stimulation of the subthalamic nucleus in advanced Parkinson’s disease. N Engl J Med. 2003;349(20):1925–1934. doi: 10.1056/NEJMoa035275. [PubMed] [Cross Ref]
100. Maltete D, Jodoin N, Karachi C, Houeto JL, Navarro S, Cornu P, et al. Subthalamic stimulation and neuronal activity in the substantia nigra in Parkinson’s disease. J Neurophysiol. 2007;97(6):4017–4022. doi: 10.1152/jn.01104.2006. [PubMed] [Cross Ref]
101. Kita H, Tachibana Y, Nambu A, Chiken S. Balance of monosynaptic excitatory and disynaptic inhibitory responses of the globus pallidus induced after stimulation of the subthalamic nucleus in the monkey. J Neurosci Off J Soc Neurosci. 2005;25(38):8611–8619. doi: 10.1523/JNEUROSCI.1719-05.2005. [PubMed] [Cross Ref]
102. Zhao XD, Cao YQ, Liu HH, Li FQ, You BM, Zhou XP. Long term high frequency stimulation of STN increases dopamine in the corpus striatum of hemiparkinsonian rhesus monkey. Brain Res. 2009;1286:230–238. doi: 10.1016/j.brainres.2009.06.069. [PubMed] [Cross Ref]
103. Putzke JD, Wharen RE, Wszolek ZK, Turk MF, Strongosky AJ, Uitti RJ. Thalamic deep brain stimulation for tremor-predominant Parkinson’s disease. Parkinsonism Relat Disord. 2003;10(2):81–88. doi: 10.1016/j.parkreldis.2003.09.002. [PubMed] [Cross Ref]
104. Dipti P, Yogesh B, Kain AK, Pauline T, Anju B, Sairam M, et al. Lead induced oxidative stress: beneficial effects of Kombucha tea. Biomed Environ Sci. 2003;16(3):276–282. [PubMed]
105. Anderson VC, Burchiel KJ, Hogarth P, Favre J, Hammerstad JP. Pallidal vs subthalamic nucleus deep brain stimulation in Parkinson disease. Arch Neurol. 2005;62(4):554–560. doi: 10.1001/archneur.62.4.554. [PubMed] [Cross Ref]
106. Peppe A, Pierantozzi M, Altibrandi MG, Giacomini P, Stefani A, Bassi A, et al. Bilateral GPi DBS is useful to reduce abnormal involuntary movements in advanced Parkinson’s disease patients, but its action is related to modality and site of stimulation. Eur J Neurol Off J Eur Fed Neurol Soc. 2001;8(6):579–586. [PubMed]
107. Benabid AL, Pollak P, Gao D, Hofmann D, Limousin P, Gay E, et al. Chronic electrical stimulation of the ventralis intermedius nucleus of the thalamus as a treatment of movement disorders. J Neurosurg. 1996;84(2):203–214. doi: 10.3171/jns.1996.84.2.0203. [PubMed] [Cross Ref]
108. Brown P, Mazzone P, Oliviero A, Altibrandi MG, Pilato F, Tonali PA, et al. Effects of stimulation of the subthalamic area on oscillatory pallidal activity in Parkinson’s disease. Exp Neurol. 2004;188(2):480–490. doi: 10.1016/j.expneurol.2004.05.009. [PubMed] [Cross Ref]
109. Hassani OK, Fèger J. Effects of intrasubthalamic injection of dopamine receptor agonists on subthalamic neurons in normal and 6-hydroxydopamine-lesioned rats: an electrophysiological and c-Fos study. Neuroscience. 1999;92(2):533–543. doi: 10.1016/S0306-4522(98)00765-9. [PubMed] [Cross Ref]
110. Filali M, Hutchison WD, Palter VN, Lozano AM, Dostrovsky JO. Stimulation-induced inhibition of neuronal firing in human subthalamic nucleus. Exp Brain Res. 2004;156(3):274–281. doi: 10.1007/s00221-003-1784-y. [PubMed] [Cross Ref]
111. Lozano AM, Dostrovsky J, Chen R, Ashby P. Deep brain stimulation for Parkinson’s disease: disrupting the disruption. Lancet Neurol. 2002;1(4):225–231. doi: 10.1016/S1474-4422(02)00101-1. [PubMed] [Cross Ref]
112. Welter ML, Houeto JL, Bonnet AM, Bejjani PB, Mesnage V, Dormont D, et al. Effects of high-frequency stimulation on subthalamic neuronal activity in parkinsonian patients. Arch Neurol. 2004;61(1):89–96. doi: 10.1001/archneur.61.1.89. [PubMed] [Cross Ref]
113. Burbaud P, Gross C, Bioulac B. Effect of subthalamic high frequency stimulation on substantia nigra pars reticulata and globus pallidus neurons in normal rats. J Physiol Paris. 1994;88(6):359–361. doi: 10.1016/0928-4257(94)90029-9. [PubMed] [Cross Ref]
114. Tai CH, Boraud T, Bezard E, Bioulac B, Gross C, Benazzouz A. Electrophysiological and metabolic evidence that high-frequency stimulation of the subthalamic nucleus bridles neuronal activity in the subthalamic nucleus and the substantia nigra reticulata. FASEB J Off Publ Fed Am Soc Exp Biol. 2003;17(13):1820–1830. [PubMed]
115. Garcia L, Audin J, D’Alessandro G, Bioulac B, Hammond C. Dual effect of high-frequency stimulation on subthalamic neuron activity. J Neurosci Off J Soc Neurosci. 2003;23(25):8743–8751. [PubMed]
116. Lee KH, Chang SY, Roberts DW, Kim U. Neurotransmitter release from high-frequency stimulation of the subthalamic nucleus. J Neurosurg. 2004;101(3):511–517. doi: 10.3171/jns.2004.101.3.0511. [PubMed] [Cross Ref]
117. Jaggi JL, Umemura A, Hurtig HI, Siderowf AD, Colcher A, Stern MB, et al. Bilateral stimulation of the subthalamic nucleus in Parkinson’s disease: surgical efficacy and prediction of outcome. Stereotact Funct Neurosurg. 2004;82(2–3):104–114. doi: 10.1159/000078145. [PubMed] [Cross Ref]
118. Holden KR (2012) Biological effects of pulsed electromagnetic field (PEMF) therapy. Med News
119. Siskin BF, Walker J. Therapeutic aspects of electromagnetic fields for soft-tissue healing. In: Blank M, editor. Electromagnetic fields: biological interactions and mechanisms. Washington, DC: American Chemical Society; 1995. pp. 277–285.
120. Iannitti T, Fistetto G, Esposito A, Rottigni V, Palmieri B. Pulsed electromagnetic field therapy for management of osteoarthritis-related pain, stiffness and physical function: clinical experience in the elderly. Clin Interv Aging. 2013;8:1289–1293. doi: 10.2147/CIA.S35926. [PMC free article] [PubMed] [Cross Ref]
121. Aktas I, Akgun K, Cakmak B. Therapeutic effect of pulsed electromagnetic field in conservative treatment of subacromial impingement syndrome. Clin Rheumatol. 2007;26(8):1234–1239. doi: 10.1007/s10067-006-0464-2. [PubMed] [Cross Ref]
122. Thomas AW, Graham K, Prato FS, McKay J, Forster PM, Moulin DE, et al. A randomized, double-blind, placebo-controlled clinical trial using a low-frequency magnetic field in the treatment of musculoskeletal chronic pain. Pain Res Manage J Can Pain Soc (journal de la societe canadienne pour le traitement de la douleur) 2007;12(4):249–258. [PMC free article] [PubMed]
123. Lee PB, Kim YC, Lim YJ, Lee CJ, Choi SS, Park SH, et al. Efficacy of pulsed electromagnetic therapy for chronic lower back pain: a randomized, double-blind, placebo-controlled study. J Int Med Res. 2006;34(2):160–167. doi: 10.1177/147323000603400205. [PubMed] [Cross Ref]
124. Lappin MS, Lawrie FW, Richards TL, Kramer ED. Effects of a pulsed electromagnetic therapy on multiple sclerosis fatigue and quality of life: a double-blind, placebo controlled trial. Altern Ther Health Med. 2003;9(4):38–48. [PubMed]
125. Richards TL, Lappin MS, Acosta-Urquidi J, Kraft GH, Heide AC, Lawrie FW, et al. Double-blind study of pulsing magnetic field effects on multiple sclerosis. J Altern Complement Med. 1997;3(1):21–29. doi: 10.1089/acm.1997.3.21. [PubMed] [Cross Ref]
126. Barbault A, Costa FP, Bottger B, Munden RF, Bomholt F, Kuster N, et al. Amplitude-modulated electromagnetic fields for the treatment of cancer: discovery of tumor-specific frequencies and assessment of a novel therapeutic approach. J Exp Clin Cancer Res. 2009;28:51. doi: 10.1186/1756-9966-28-51. [PMC free article] [PubMed] [Cross Ref]
127. Sandyk R. Freezing of gait in Parkinson’s disease is improved by treatment with weak electromagnetic fields. Int J Neurosci. 1996;85(1–2):111–124. doi: 10.3109/00207459608986356. [PubMed] [Cross Ref]
128. Arendash GW, Sanchez-Ramos J, Mori T, Mamcarz M, Lin X, Runfeldt M, et al. Electromagnetic field treatment protects against and reverses cognitive impairment in Alzheimer’s disease mice. J Alzheimers Dis. 2010;19(1):191–210. [PubMed]
129. Ericsson AD, Hazlewood CF, Markov M, Crawford F. Biological effects of EMF’s. Greece: KOS; 2004. Specific Biochemical changes in circulating lymphocytes following acute ablation of symptoms in Reflex Sympathetic Dystrophy (RSD): a pilot study; pp. 683–688.
130. Yost MG, Liburdy RP. Time-varying and static magnetic fields act in combination to alter calcium signal transduction in the lymphocyte. FEBS Lett. 1992;296(2):117–122. doi: 10.1016/0014-5793(92)80361-J. [PubMed] [Cross Ref]
131. Edmonds DT. Larmor precession as a mechanism for the detection of static and alternating magnetic fields. Bioelectrochem Bioenerg. 1993;30:3–12. doi: 10.1016/0302-4598(93)80057-2. [Cross Ref]
132. Liboff AR, Cherng S, Jenrow KA, Bull A. Calmodulin-dependent cyclic nucleotide phosphodiesterase activity is altered by 20 microT magnetostatic fields. Bioelectromagnetics. 2003;24(1):32–38. doi: 10.1002/bem.10063. [PubMed] [Cross Ref]
133. Demitrack MA, Thase ME. Clinical significance of transcranial magnetic stimulation (TMS) in the treatment of pharmacoresistant depression: synthesis of recent data. Psychopharmacol Bull. 2009;42(2):5–38. [PubMed]
134. Liboff AR (2004) Signal shapes in electromagnetic therapies: a primer. In: Rosch PJ, Markov MS (eds) Bioelectromagnetic medicine. Marcel Dekker, NY, pp 17–37
135. Sandyk R. Reversal of a body image disorder (macrosomatognosia) in Parkinson’s disease by treatment with AC pulsed electromagnetic fields. Int J Neurosci. 1998;93(1–2):43–54. doi: 10.3109/00207459808986411. [PubMed] [Cross Ref]
136. Sandyk R. A drug naive parkinsonian patient successfully treated with weak electromagnetic fields. Int J Neurosci. 1994;79(1–2):99–110. [PubMed]
137. Sandyk R. Reversal of visuospatial deficit on the Clock Drawing Test in Parkinson’s disease by treatment with weak electromagnetic fields. Int J Neurosci. 1995;82(3–4):255–268. doi: 10.3109/00207459508999805. [PubMed] [Cross Ref]
138. Ben-Shachar D, Belmaker RH, Grisaru N, Klein E. TMS induces alterations in brain monoamines. J Neural Trans. 1997;104:191–197. doi: 10.1007/BF01273180. [PubMed] [Cross Ref]
139. Cerasa A, Koch G, Donzuso G, Mangone G, Morelli M, Brusa L, et al. A network centred on the inferior frontal cortex is critically involved in levodopa-induced dyskinesias. Brain. 2015;138(2):414–427. doi: 10.1093/brain/awu329. [PubMed] [Cross Ref]
140. Keck ME, Welt T, Post A, Müller MB, Toschi N, Wigger A, et al. Neuroendocrine and behavioral effects of repetitive transcranial magnetic stimulation in a psychopathological animal model are suggestive of antidepressant-like effects. Neuropsychopharmacol Off Publ Am Coll Neuropsychopharmacol. 2001;24(4):337–349. doi: 10.1016/S0893-133X(00)00191-3. [PubMed] [Cross Ref]
141. Fitzgerald PB, Brown TL, Marston NA, Daskalakis ZJ, De Castella A, Kulkarni J. Transcranial magnetic stimulation in the treatment of depression: a double-blind, placebo-controlled trial. Arch Gen Psychiatry. 2003;60(10):1002–1008. [PubMed]
142. Loo CK, Mitchell PB, Croker VM, Malhi GS, Wen W, Gandevia SC, et al. Double-blind controlled investigation of bilateral prefrontal transcranial magnetic stimulation for the treatment of resistant major depression. Psychol Med. 2003;33(1):33–40. doi: 10.1017/S0033291702006839. [PubMed] [Cross Ref]
143. Janicak PG, O’Reardon RJ, et al. Transcranial magnetic stimulation in the treatment of major depressive disorder: a comprehensive summary of safety experience from acute exposure, extended exposure, and during reintroduction treatment. J Clin Psychiatry. 2008;69(2):222–232. doi: 10.4088/JCP.v69n0208. [PubMed] [Cross Ref]
144. Wassermann EM. Risk and safety of repetitive transcranial magnetic stimulation: report and suggested guidelines from the International Workshop on the Safety of Repetitive Transcranial Magnetic Stimulation, June 5–7, 1996. Electroencephalogr Clin Neurophysiol. 1998;108(1):1–16. doi: 10.1016/S0168-5597(97)00096-8. [PubMed] [Cross Ref]
145. Dirnberger G, Jahanshahi M. Executive dysfunction in Parkinson’s disease: a review. J Neuropsychol. 2013;7(2):193–224. doi: 10.1111/jnp.12028. [PubMed] [Cross Ref]
146. Narayanan NS, Rodnitzky RL, Uc EY. Prefrontal dopamine signaling and cognitive symptoms of Parkinson’s disease. Rev Neurosci. 2013;24(3):267–278. doi: 10.1515/revneuro-2013-0004. [PMC free article] [PubMed] [Cross Ref]
147. Aziz TZ, Peggs D, Sambrook MA, Crossman AR. Lesion of the subthalamic nucleus for the alleviation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism in the primate. Mov Disord Off J Mov Disord Soc. 1991;6(4):288–292. doi: 10.1002/mds.870060404. [PubMed] [Cross Ref]
148. Benazzouz A, Gross C, Féger J, Boraud T, Bioulac B. Reversal of rigidity and improvement in motor performance by subthalamic high-frequency stimulation in MPTP-treated monkeys. Eur J Neurosci. 1993;5(4):382–389. doi: 10.1111/j.1460-9568.1993.tb00505.x. [PubMed] [Cross Ref]
149. Bergman H, Wichmann T, DeLong MR. Reversal of experimental parkinsonism by lesions of the subthalamic nucleus. Science. 1990;249(4975):1436–1438. doi: 10.1126/science.2402638. [PubMed] [Cross Ref]
150. Lang AE. Surgery for Parkinson disease: a critical evaluation of the state of the art. Arch Neurol. 2000;57(8):1118–1125. doi: 10.1001/archneur.57.8.1118. [PubMed] [Cross Ref]
151. Levy R, Lang AE, Dostrovsky JO, Pahapill P, Romas J, Saint-Cyr J, et al. Lidocaine and muscimol microinjections in subthalamic nucleus reverse Parkinsonian symptoms. Brain J Neurol. 2001;124(Pt 10):2105–2118. doi: 10.1093/brain/124.10.2105. [PubMed] [Cross Ref]
152. Hashimoto T, Elder CM, Okun MS, Patrick SK, Vitek JL. Stimulation of the subthalamic nucleus changes the firing pattern of pallidal neurons. J Neurosci Off J Soc Neurosci. 2003;23(5):1916–1923. [PubMed]
153. Lacombe E, Carcenac C, Boulet S, Feuerstein C, Bertrand A, Poupard A, et al. High-frequency stimulation of the subthalamic nucleus prolongs the increase in striatal dopamine induced by acute l-3,4-dihydroxyphenylalanine in dopaminergic denervated rats. Eur J Neurosci. 2007;26(6):1670–1680. doi: 10.1111/j.1460-9568.2007.05747.x. [PMC free article] [PubMed] [Cross Ref]
154. Benabid AL, Krack PP, Benazzouz A, Limousin P, Koudsie A, Pollak P. Deep brain stimulation of the subthalamic nucleus for Parkinson’s disease: methodologic aspects and clinical criteria. Neurology. 2000;12(6):S40–S44. [PubMed]
155. Welter ML, Houeto J, Tezenas du Montcel S, Mesnage V, Bonnet AM, Pillon B, et al. Clinical predictive factors of subthalamic stimulation in Parkinson’s disease. Brain J Neurol. 2002;125(Pt 3):575–583. doi: 10.1093/brain/awf050. [PubMed] [Cross Ref]
156. Stoffers D, Bosboom JL, Wolters E, Stam CJ, Berendse HW. Dopaminergic modulation of cortico-cortical functional connectivity in Parkinson’s disease: an MEG study. Exp Neurol. 2008;213(1):191–195. doi: 10.1016/j.expneurol.2008.05.021. [PubMed] [Cross Ref]
157. Degos B, Deniau JM, Thierry AM, Glowinski J, Pezard L, Maurice N. Neuroleptic-induced catalepsy: electrophysiological mechanisms of functional recovery induced by high-frequency stimulation of the subthalamic nucleus. J Neurosci Off J Soc Neurosci. 2005;25(33):7687–7696. doi: 10.1523/JNEUROSCI.1056-05.2005. [PubMed] [Cross Ref]
158. Salin P, Manrique C, Forni C, Kerkerian-Le Goff L. High-frequency stimulation of the subthalamic nucleus selectively reverses dopamine denervation-induced cellular defects in the output structures of the basal ganglia in the rat. J Neurosci. 2002;22(12):5137–5148. [PubMed]
159. Poulet E, Haesebaert F, Saoud M, Suaud-Chagny MF, Brunelin J. Treatment of schizophrenic patients and rTMS. Psychiatr Danub. 2010;22(1):S143–S146. [PubMed]
160. Markov MS (2007) History of Pulsed Electro Magnetic Field Therapy. PEMF Systems Inc
161. Sklar B (2014) Announcing the iMRS from swiss bionic solutions. Relax Restore Massage
162. Sklar B (2009) MRS 2000 + the revolutionary “sawtooth” wave impulse. Relax and Restore Massage Services
163. Andras V (1999) Proof of ion transport due to application of QRS System Salut-II. Quantron Medizin GmbH zHd Dr Fischer Nußloch Neuropsychiatr Dis Treat. 2015 Sep 18;11:2391-404. doi: 10.2147/NDT.S90966. eCollection 2015.

An innovative intervention for the treatment of cognitive impairment-Emisymmetric bilateral stimulation improves cognitive functions in Alzheimer’s disease and mild cognitive impairment: an open-label study.

Guerriero F1, Botarelli E2, Mele G2, Polo L2, Zoncu D2, Renati P3, Sgarlata C4, Rollone M5, Ricevuti G6, Maurizi N4, Francis M4, Rondanelli M7, Perna S7, Guido D8, Mannu P2. . Author information
1Department of Internal Medicine and Medical Therapy, Section of Geriatrics, University of Pavia, Pavia, Italy ; Agency for Elderly People Services, Santa Margherita Hospital, Pavia, Italy ; Ambra Elektron, Italian Association of Biophysics for the Study of Electromagnetic Fields in Medicine, Turin, Italy.
2Ambra Elektron, Italian Association of Biophysics for the Study of Electromagnetic Fields in Medicine, Turin, Italy.
3Ambra Elektron, Italian Association of Biophysics for the Study of Electromagnetic Fields in Medicine, Turin, Italy ; Alberto Sorti Research Institute, Medicine and Metamolecular Biology, Turin, Italy.
4Department of Internal Medicine and Medical Therapy, Section of Geriatrics, University of Pavia, Pavia, Italy.
5Agency for Elderly People Services, Santa Margherita Hospital, Pavia, Italy.
6Department of Internal Medicine and Medical Therapy, Section of Geriatrics, University of Pavia, Pavia, Italy ; Agency for Elderly People Services, Santa Margherita Hospital, Pavia, Italy.
7Department of Public Health, Experimental and Forensic Medicine, Section of Human Nutrition, Endocrinology and Nutrition Unit, University of Pavia, Pavia, Italy.
8Agency for Elderly People Services, Santa Margherita Hospital, Pavia, Italy ; Department of Public Health, Experimental and Forensic Medicine, Biostatistics and Clinical Epidemiology Unit, University of Pavia, Pavia, Italy. Abstract
BACKGROUND AND AIMS:
In the last decade, the development of different methods of brain stimulation by electromagnetic fields (EMF) provides a promising therapeutic tool for subjects with impaired cognitive functions. Emisymmetric bilateral stimulation (EBS) is a novel and innovative EMF brain stimulation, whose working principle is to introduce very weak noise-like stimuli through EMF to trigger self-arrangements in the cortex of treated subjects, thereby improving cognitive faculties. The aim of this pilot study was to investigate in patients with cognitive impairment the effectiveness of EBS treatment with respect to global cognitive function, episodic memory, and executive functions. METHODS:
Fourteen patients with cognitive decline (six with mild cognitive impairment and eight with Alzheimer’s disease) underwent three EBS applications per week to both the cerebral cortex and auricular-specific sites for a total of 5 weeks. At baseline, after 2 weeks and 5 weeks, a neuropsychological assessment was performed through mini-mental state examination, free and cued selective reminding tests, and trail making test. As secondary outcomes, changes in behavior, functionality, and quality of life were also evaluated. RESULTS:
After 5 weeks of standardized EBS therapy, significant improvements were observed in all neurocognitive assessments. Mini-mental state examination score significantly increased from baseline to end treatment (+3.19, P=0.002). Assessment of episodic memory showed an improvement both in immediate and delayed recalls (immediate recall =+7.57, P=0.003; delayed recall =+4.78, P<0.001). Executive functions significantly improved from baseline to end stimulation (trail making test A -53.35 seconds; P=0.001). Of note, behavioral disorders assessed through neuropsychiatric inventory significantly decreased (-28.78, P<0.001). The analysis concerning the Alzheimer’s disease and mild cognitive impairment group confirmed a significant improvement of cognitive functions and behavior after EBS treatment. CONCLUSION:
This pilot study has shown EBS to be a promising, effective, and safe tool to treat cognitive impairment, in addition to the drugs presently available. Further investigations and controlled clinical trials are warranted. KEYWORDS:
Alzheimer’s disease; Emisymmetric bilateral stimulation; cognitive decline; pulsed electromagnetic fields J Alzheimer’s Dis.  2012;32(2):243-66. doi: 10.3233/JAD-2012-120943.

Transcranial electromagnetic treatment against Alzheimer’s disease: why it has the potential to trump Alzheimer’s disease drug development.

Arendash GW.

Source

Department of Cell Biology, University of South Florida, Tampa, FL, USA. arendash@cas.usf.edu

Abstract

The universal failure of pharmacologic interventions against Alzheimer’s disease (AD) appears largely due to their inability to get into neurons and the fact that most have a single mechanism-of-action. A non-invasive, neuromodulatory approach against AD has consequently emerged: transcranial electromagnetic treatment (TEMT). In AD transgenic mice, long-term TEMT prevents and reverses both cognitive impairment and brain amyloid-B (AB) deposition, while TEMT even improves cognitive performance in normal mice. Three disease-modifying and inter-related mechanisms of TEMT action have been identified in the brain: 1) anti-AB aggregation, both intraneuronally and extracellularly; 2) mitochondrial enhancement; and 3) increased neuronal activity. Long-term TEMT appears safe in that it does not impact brain temperature or oxidative stress levels, nor does it induce any abnormal histologic/anatomic changes in the brain or peripheral tissues. Future TEMT development in both AD mice and normal mice should involve head-only treatment to discover the most efficacious set of parameters for achieving faster and even greater cognitive benefit. Given the already extensive animal work completed, translational development of TEMT could occur relatively quickly to “proof of concept” AD clinical trials. TEMT’s mechanisms of action provide extraordinary therapeutic potential against other neurologic disorders/injuries, such as Parkinson’s disease, traumatic brain injury, and stroke.

PLoS One. 2012; 7(4): e35751. Published online 2012 April 25. doi:  10.1371/journal.pone.0035751 PMCID: PMC3338462

Electromagnetic Treatment to Old Alzheimer’s Mice Reverses B-Amyloid Deposition, Modifies Cerebral Blood Flow, and Provides Selected Cognitive Benefit

Gary W. Arendash,1,2,* Takashi Mori,3 Maggie Dorsey,4 Rich Gonzalez,5 Naoki Tajiri,6 and Cesar Borlongan61

Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, United States of America, 2 The Florida Alzheimer’s Disease Research Center, Tampa, Florida, United States of America, 3 Departments of Biomedical Sciences and Pathology, Saitama Medical Center and Saitama Medical University, Kawagoe, Saitama, Japan, 4 The University of South Florid Health Byrd Alzheimer’s Institute, Tampa, Florida, United States of America, 5 SAI of Florida, Redington Beach, Florida, United States of America, 6 Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, Florida, United States of America Efthimios M. C. Skoulakis, Editor Received December 27, 2011; Accepted March 22, 2012.

Copyright.   This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Link to original article:

Abstract

Few studies have investigated physiologic and cognitive effects of “long-term” electromagnetic field (EMF) exposure in humans or animals. Our recent studies have provided initial insight into the long-term impact of adulthood EMF exposure (GSM, pulsed/modulated, 918 MHz, 0.25–1.05 W/kg) by showing 6+ months of daily EMF treatment protects against or reverses cognitive impairment in Alzheimer’s transgenic (Tg) mice, while even having cognitive benefit to normal mice. Mechanistically, EMF-induced cognitive benefits involve suppression of brain B-amyloid (AB) aggregation/deposition in Tg mice and brain mitochondrial enhancement in both Tg and normal mice. The present study extends this work by showing that daily EMF treatment given to very old (21–27 month) Tg mice over a 2-month period reverses their very advanced brain A? aggregation/deposition. These very old Tg mice and their normal littermates together showed an increase in general memory function in the Y-maze task, although not in more complex tasks. Measurement of both body and brain temperature at intervals during the 2-month EMF treatment, as well as in a separate group of Tg mice during a 12-day treatment period, revealed no appreciable increases in brain temperature (and no/slight increases in body temperature) during EMF “ON” periods. Thus, the neuropathologic/cognitive benefits of EMF treatment occur without brain hyperthermia. Finally, regional cerebral blood flow in cerebral cortex was determined to be reduced in both Tg and normal mice after 2 months of EMF treatment, most probably through cerebrovascular constriction induced by freed/disaggregated A? (Tg mice) and slight body hyperthermia during “ON” periods. These results demonstrate that long-term EMF treatment can provide general cognitive benefit to very old Alzheimer’s Tg mice and normal mice, as well as reversal of advanced A? neuropathology in Tg mice without brain heating. Results further underscore the potential for EMF treatment against AD.

Introduction

Despite the best efforts of pharmaceutical industry and academia, no new drugs against Alzheimer’s Disease (AD) have been developed since 2003 [1]. Moreover, currently available drugs (acetylcholinesterase inhibitors and/or N-metyle D-aspartate (NMDA) antagonists) only treat/mask AD symptoms for about one year, if at all – none of them directly slow or lessen AD pathogenesis itself. In view of the universal failure of every major drug trial to alter the course of AD, it is time to think outside the “pharmaceutical box” by considering non-pharmaceutical approaches that are safe, disease modifying, and can be expeditiously explored to treat AD. We propose high frequency electromagnetic field (EMF) treatment could be that approach, based on several epidemiologic studies [2], [3] and our recently completed EMF studies in Alzheimer’s transgenic (Tg) mice [4], [5].

In humans, high frequency EMF exposure/treatment studies have essentially involved “cell phone level” EMF parameters (pulsed, modulated and primarily GSM), in large part because of initial concerns that high frequency EMF exposure may induce health problems such as brain cancer [6], [7]. However, the recent 13-nation INTERPHONE study [8], as well as analytic findings from NIEHS [9] and numerous epidemiologic studies [10][12], all collectively conclude that there is no consistent evidence that long-term exposure of adults or children/adolescents to cell phone level EMFs causes brain tumors, or very likely any other health problems for that matter. In concert with these studies alleviating safety issues related to high frequency EMF exposure, dozens of studies have investigated potential cognitive and physiologic (i.e., EEG, cerebral blood flow, and auditory processing) effects of cell phone level EMF exposure. With rare exception [13], [14], these studies only involved brief (3–120 minute), single EMF exposure at GMS, CW, or UMTS cell phone parameters given to normal subjects. Not surprisingly, recent reviews/meta-analyses find these “acute” exposure studies to result in no significant beneficial or impairing effects on cognitive performance [15], [16]. Nonetheless, several PET studies have reported that acute, single-exposure EMF treatment can affect regional cerebral blood flow [17], [18] and increase brain glucose utilization [19], thus suggesting that even acute high frequency EMF treatment can affect brain neuronal activity.

Although results from acute, single EMF exposure studies are insightful, they are most probably not indicative of the physiologic and cognitive effects of long-term/daily EMF exposure (i.e. the EMF exposure typical of cell phone users or the repeated EMF treatments almost certainly required for any clinical EMF applications). In this context, no controlled human studies have investigated the “long-term” effects of high frequency EMF treatment in normal or AD subjects over weeks, months, or years. Nonetheless, two epidemiologic studies have provided initial human evidence that years of high frequency EMF exposure are associated with cognitive benefit. One of these studies found that heavy cell phone use over several years resulted in better performance of normal subjects on a word interference test [2], while the other study reported that long-term cell phone users (>10 years) had a 30–40% decreased risk of hospitalization due to AD and vascular dementia [3].

The lack of controlled human studies investigating cognitive effects of “long-term” EMF exposure/treatment has at least been partially negated by our highly controlled EMF treatment studies in AD Tg mice and littermate non-transgenic (NT) mice [4], [5]. In the first long-term, high frequency EMF treatment study evaluating cognition in adult humans or animals [4], we reported that treatment (at cell phone levels of 918 MHz/0.25–1.05 W/kg; pulsed and modulated) over 7–9 months prevented or reversed cognitive impairment in AD Tg mice bearing the APPsw mutation. Even normal mice showed EMF-induced cognitive enhancement in that initial study. For AD mice, the primary mechanism of cognitive benefit appears to be a suppression of brain A? aggregation into neuritic plaques, presumably resulting in greater A? efflux from the brain [4]. Moreover, the cognitive benefits of long-term EMF treatment to both AD mice and normal mice occurs without any evidence of tissue abnormalities in either the brain or peripheral tissues, without any evidence of increased oxidative stress in the brain, and without any increase in DNA damage to circulating blood cells. Thus, long-term EMF treatment in mice appears safe in having no deleterious side effects across multiple sensitive markers of brain/body function.

In a second study that involved AD Tg mice bearing the APPsw+PS1 double mutation, we reported that daily EMF treatment for one month enhances the impaired brain mitochondrial function of these AD mice, as well as the brain mitochondrial function of normal mice [5]. These EMF-induced mitochondrial enhancements occurred through “non-thermal” mechanisms because brain temperatures were either stable or decreased during and after daily high frequency EMF treatments. Since this EMF-induced mitochondrial enhancement in AD mice was linked to dramatic 5–10 fold elevations in soluble A? within the same mitochondria, EMF treatment disaggregated toxic A? oligomers therein, apparently resulting in very high monomeric A? levels (which are innocuous to mitochondrial function). Our mitochondrial function results in Dragicevic et al. [5] collectively suggest that brain mitochondrial enhancement may be a primary mechanism through which long-term EMF treatment provides cognitive benefit to both AD mice and NT mice.

In a third study, we have most recently reported that two months of daily EMF treatment enhances neuronal activity in the entorhinal cortex of aged Alzheimer’s Tg mice and littermate NT mice [20]. This EMF-induced enhancement of neuronal activity was temporally linked to cognitive benefit in the same animals. Based on these results, we have suggested that EMF treatment could be a viable approach to counter the neuronal hypo-activity that occurs very early in AD pathogenesis [20].

It is noteworthy that our prior EMF studies [4], [5], [20] identified the first biologic mechanisms that could explain the EMF-induced cognitive benefits, which we also reported in normal and Alzheimer’s Tg mice (i.e., anti-A? aggregation, mitochondrial enhancement, and enhanced neuronal activity). The fact that our long-term EMF treatment involves pulsed, modulated GSM signal is important because a recent, comprehensive review concluded that EMF-induction of biologic effects occurs primarily with GSM-type modulation and a pulsed signal – not continuous wave or UMTS fields [21].

Our initial behavioral study in AD Tg mice involved long-term EMF treatment to young adult APPsw mice (from 2–7.5 months of age), as well as to older APPsw adults (from 5–13.5 months of age) [4]. Inasmuch as A? pathology was not yet well established when treatment began for these mice, the beneficial effects reported were most relevant to human EMF treatment in pre-symptomatic/prodromal AD or in mild cognitive impairment (MCI), the prelude to AD. The present study extends our earlier findings by evaluating the impact of long-term EMF treatment given to very old 21–26 month-old APPsw and APPsw+PS1 mice, both of which bear much heavier brain A? burdens/A? levels than the APPsw mice in our initial work. In these aged mice with advanced A? pathology, we evaluated an array of behavioral, neuropathologic, and physiologic measures to get a clearer understanding of how long-term EMF treatment might impact the aged and heavily A?-burdened brain. We report a profound ability of long-term EMF treatment to reverse brain A? deposition, induce changes in regional cerebral blood flow, and provide selected cognitive benefits – all without induction of brain hyperthermia.

Results

Behavioral assessment during long-term EMF treatment

In Study I, behavioral testing of aged Tg and NT mice between 1 and 2 months into daily EMF treatment indicated no deleterious effects of EMF treatment on sensorimotor function (Table 1). For both Tg and NT mice, general activity/exploratory behavior was unaffected by EMF treatment, as indexed by open field activity and Y-maze choices made. As well, balance and agility abilities were not impacted in either Tg or NT mice by EMF treatment, as indexed by balance beam and string agility performance. In both of these tasks, however, an overall effect of genotype was presence, with Tg mice having poorer balance/agility compared to NT mice irrespective of EMF treatment (p<0.002). Finally, visual acuity testing in the visual cliff task at the end of behavioral testing (2 months into EMF treatment) indicated no deleterious effects of EMF treatment on vision in either Tg or NT mice.

Table 1

Table 1

Sensorimotor measures in NT and Tg mice given long-term EMF treatment.

For cognitive-based tasks/measures, EMF effects were task specific with benefits observed in the Y-maze task, but no effects in either the circular platform or radial arm water maze (RAWM) tasks. In the Y-maze alternation task of general mnemonic function, both Tg and NT mice being given EMF treatment showed near-significance increases in percent alternation compared to their respective controls (Fig. 1A, left). Because there was no difference in performance of Tg and NT mice, these genotypic groups were combined to determine if an overall EMF treatment effect was present. Indeed, a significant increase in spontaneous alternation percentage was evident irrespective of genotype (Fig. 1A, right), indicating a beneficial effect of EMF treatment on general mnemonic function. In the circular platform task of spatial/reference memory, Tg mice were impaired vs. NT controls during the final (2nd block) of testing, irrespective of whether they were receiving EMF treatment or not (Fig. 1B). Furthermore, EMF treatment did not improve the poor performance (e.g, high escape latencies) of both Tg and NT mice in this task.

Figure 1

Figure 1

Cognitive performance of non-transgenic (NT) and APPsw transgenic (Tg) mice in the Y-maze task of spontaneous alternation (Fig. 1A) and the circular platform task of spatial/reference memory (Fig. 1B).

For the RAWM task of working memory, all animals were tested prior to the start of EMF treatment to establish baseline performance levels and to determine if a transgenic effect was present. Throughout pre-treatment RAWM testing, both Tg and NT mice showed the high escape latencies typically seen during the naïve first trial (T1), as exemplified by the last block of pre-treatment testing (Fig. 2A). By contrast, Tg mice showed a severe working memory impairment compared to NT mice at individual test blocks and overall, as exemplified by their substantially higher escape latencies during working memory Trial 5 (T5) for the last block of pre-treatment testing (Fig. 2A). Following completion of pre-treatment testing, Tg mice were divided into two sub-groups balanced in RAWM performance (as were NT mice), with one sub-group receiving EMF treatment and the other group not. Ensuing RAWM testing at both 1 month and 1.5 months into EMF treatment continued to show substantially impaired working memory (T5) performance in Tg mice vs. NT controls, irrespective of whether they were receiving EMF treatment or not (Figs. 2B, C). The similar T5 working memory impairment of Tg+EMF mice and Tg controls (evident during individual blocks and overall) is exemplified by the last block of testing, as shown in Figs. 2B and C.

Figure 2

Figure 2

Working memory in the radial arm water maze (RAWM) task pre-treatment, 1 month, and 1.5 months into EMF treatment for the naïve first trial (T1) and working memory trial (T5) of APPsw transgenic (Tg) and non-transgenic (NT) mice.

Thus, EMF-induced cognitive benefits to very old AD and NT mice were selective in enhancing general mnemonic function (Y-maze alternation), but not impacting spatial reference learning/memory (circular platform) or working memory (radial arm water maze).

Body/brain temperature recording during long-term EMF treatment

Study I

Body and brain temperature measurements were attained from aged animals in Study I before start of EMF treatment (control) and at 1, 3, and 6 weeks into treatment (final temperature measurements were unfortunately not taken at the 2-month termination point of this study). Throughout the 6-week study period, body and brain temperature recordings indicated very stable temperature in control NT and control APPsw (Tg) mice not being given EMF treatment (Fig. 3). By contrast, body temperature for both EMF-treated NT and Tg mice was modestly elevated by 0.5–0.9°C during ON periods compared to OFF periods, from 1 week into EMF treatment onward through treatment. For Tg mice, this increase in body temperature during ON periods was evident even on the first day of EMF treatment. During EMF OFF periods for both NT and Tg mice, body temperature always came back down to their pre-treatment levels. Since body temperature before start of EMF treatment was identical for Tg mice (but not NT mice) to be given EMF or sham treatment, temperature comparisons between these two groups throughout the EMF treatment period also revealed that the elevated body temperatures of Tg mice during ON periods always came back down to sham control levels during OFF periods.

Figure 3

Figure 3

Body and brain temperature measurements for non-transgenic (NT) and APPsw transgenic (Tg) mice recorded prior to the start of EMF treatment (control), and at 1 Day, 1 week, 3 weeks, and 6 weeks into EMF treatment.

As indicated in Fig. 3, brain temperature in control NT and Tg mice was usually 0.3–0.4°C lower than body temperature, which is typically the case for rodents [22]. As with body temperatures, brain temperature measurements in control NT and Tg mice (not given EMF treatment) were very stable throughout the study. In EMF-treated NT mice, elevations of 0.3–0.4°C in brain temperature during ON periods were evident and significant by 3 weeks into treatment (Fig. 3). In EMF-treated Tg mice, however, only trends for a slight increase in brain temperature were present during ON periods. Thus, even with peripheral increases in temperature during ON periods, brain temperature remained stable or was only elevated minimally through 6 weeks of EMF exposure. Following any brain temperature elevations during ON periods, brain temperature always returned to pre-treatment levels during OFF periods.

Study II

For the aged APPsw+PS1 (Tg) mice in Study II, body and brain temperature measurements were taken before the start of EMF treatment, as well as at 5 and 12 days into treatment (Fig. 4). Though still modest, the difference between body and brain temperature measurements for control APPsw+PS1 mice throughout this study was larger (0.7–0.9°C) than for the body/brain temperature differences of APPsw mice throughout Study I. Despite receiving the same daily EMF exposure as APPsw mice in Study I, APPsw+PS1 mice in this study showed no significant increase in body or brain temperature during ON periods at 5 and 12 days into EMF treatment. For all time points evaluated, there were no differences between EMF-treated and control Tg mice in either body or brain temperature.

Figure 4

Figure 4

Body and brain temperature measurements for APPsw+PS1 transgenic (Tg) mice recorded prior to the start of EMF treatment (control), as well as at 5 days and 12 days into EMF treatment.

Cerebral blood flow measurements during long-term and sub-chronic EMF treatment

Laser Doppler measurements of regional cerebral blood flow (rCBF) in cerebral cortex were performed at 2 months into EMF treatment for Study I and at 12 days into EMF treatment for Study II. In Study I, control NT and Tg mice (not being given EMF treatment) had very consistent rCBF readings between their sham ON and OFF periods (Fig. 5A). Although NT+EMF mice exhibited no change in rCBF between ON and OFF periods, Tg mice showed a significant 13% decrease in rCBF during the ON period vs. OFF period (Fig. 5A). The decreased rCBF present in Tg mice during the ON period was even greater (?25%) in relation to rCBF in control Tg mice during their sham ON period. Visual inspect of the data in Fig. 5A revealed rCBF measurements during both OFF and ON periods to be lower in EMF-treated mice compared to control (sham-treated) mice irrespective of genotype. This, in addition to no genotypic differences in rCBF being present for EMF-treated or control mice, warranted combination of individual animal data from both genotypes to determine the main effect of EMF during OFF and ON periods (Fig. 5B). A significant decrease in rCBF was present not only during ON periods for EMF vs. control mice, but also present during OFF periods as well. Thus, EMF effects on rCBF were present not only during ON periods, but also during OFF periods, at 2 months into EMF treatment.

Figure 5

Figure 5

Regional cerebral blood flow (rCBF) in cerebral cortex of NT and Tg mice in Studies I and II obtained by Laser Doppler measurements at the end of their 2 month and 12-day EMF treatment periods, respectively.

rCBF measurements in Study II only involved Tg mice and at a shorter 12-days into the same daily EMF exposure. As shown in Fig. 5C, control Tg mice had stable and similar rCBF measurements during OFF and sham ON periods. By contrast, a nearly significant (p=0.10) reduction in rCBF (?19%) was present in EMF-treated Tg mice during their ON period vs. OFF period at 12 days into EMF exposure. Supportive that a true EMF-induced decrease in rCBF had indeed occurred, 4 out of five Tg+EMF mice had decreases of 7–46% in rCBF during the ON period compared to the OFF period. The significantly higher rCBF present in EMF-treated mice vs. control Tg mice during the OFF period was due to several EMF-treated mice with high rCBF readings during both OFF and ON periods.

AB immunohistochemistry

After two months of EMF treatment, the very old (23–28 months old) APPsw and NT mice in Study I were euthanized and their brains processed for quantitative analysis of A? deposition. As expected, NT mice exhibited no human A? immunostaining in their brains irrespective of treatment. Very old Tg controls (Tg), however, had extremely high levels of A? deposition in both their hippocampus and entorhinal cortex, bearing A? burdens of 11–12% in these two brain areas (Fig. 6B). In sharp contrast, Tg mice that had received two months of EMF treatment exhibited substantial decreases in A? burden within both hippocampus (?30%) and entorhinal cortex (?24%) compared to Tg controls (Fig. 6B). Thus, EMF treatment reversed pre-existing A? deposition/plaque formation. Fig. 6A shows representative photomicrographs of typical A? immunostained-plaques from 23–28 months old Tg and Tg+EMF mice, underscoring the substantial reduction in A? deposition present in brains of very old Tg mice given a two-month period of daily EMF treatment. Analysis of plasma samples taken at euthanasia revealed no effects of EMF treatment on plasma A?1–40 (4620±442 pg/ml for Tg vs. 4885±920 pg/ml for Tg+EMF; p=0.78) or A?1–42 (1452±120 pg/ml for Tg vs. 1175±251 pg/ml; p=0.30).

Figure 6

Figure 6

Brain A deposition in APPsw transgenic (Tg) mice at 2 months after EMF treatment (Study I).

Discussion

We have previously reported that long-term (>6 months) EMF exposure at cell phone level frequencies and SAR levels can protect against or reverse cognitive impairment in Alzheimer’s Tg mice, while even having cognitive benefit to normal mice [4]. Moreover, we previously provided the first mechanistic insight into long-term EMF treatment by reporting the ability of such treatment to suppress brain A aggregation/deposition in AD mice, while enhancing brain mitochondrial function and neuronal activity in both Tg and normal mice [4], [5], [20]. The present study extends the above works by administering long-term (2 months) of daily EMF treatment to very old Alzheimer’s Tg mice and showing that such treatment can reverse their very advanced brain  aggregation/deposition while providing selected cognitive benefit to both Tg and normal mice. Moreover, these neuropathologic and cognitive benefits occurred without appreciable increases in brain temperature, indicating involvement of non-thermal brain mechanisms (i.e., A? anti-aggregation, mitochondrial enhancement, neuronal activity). Finally, the present study is the first to determine the effects of long-term EMF exposure on rCBF, and in the same animals evaluated for cognitive, neuropathologic, and body/brain temperature endpoints. Our finding of an EMF-induced decrease in cortical blood flow raises several interesting mechanisms of action that merit consideration.

Cognitive and AB deposition effects of EMF treatment

Two months of cell phone level EMF treatment (e.g., GSM, 918 MHz, 0.25–1.05 W/kg, pulsed and modulated) improved the cognitive performance of very old (23–27 month old) Tg and NT mice combined in the Y-maze task of spontaneous alternation. This task evaluates general mnemonic function and is not associated with brain A? levels/deposition [23]. Thus, generalized mechanisms irrespective of genotype, such as the brain mitochondrial enhancement present by one month into EMF treatment [5], are most likely involved. The present Y-maze results are consistent with our initial study [4] wherein we found Y-maze spontaneous alternation to be significantly increased in NT mice given long-term EMF treatment. By contrast, long-term EMF treatment was not able to reverse the cognitive impairment in two tasks wherein performance is linked to brain A levels/deposition – the circular platform task of spatial/reference memory and RAWM task of working memory [23]. The RAWM task, in particular, is very sensitive to brain A deposition, with poorer working memory performance highly correlated with extent of A deposition in both hippocampus and cortex.

Although the very old Tg mice of this study had extraordinarily high brain A burdens (11–12%) that were substantially reduced (24–30%) by EMF treatment, this large quantitative reduction in A? deposition was apparently not sufficient for cognitive benefit to become manifest in tasks linked to brain A levels/deposition. A longer EMF treatment period or more effective EMF parameters is probably needed to attain widespread behavioral benefit in these very old Tg mice. In our initial study [4], 6–7 months of daily EMF treatment was required to manifest widespread cognitive benefit in younger Tg mice bearing only around 2% brain A? burdens. Parenthetically, animals in the present study were given double the daily EMF exposure (two 2-hour periods) compared to our initial study (two 1-hour periods). For both studies, a more effective removal of A from the brain through greater EMF-induced ? disaggregation and ensuing greater removal of resultant soluble A from the brain into the blood would appear to be key to realization of earlier cognitive benefits.

It is important to underscore that an absolute reduction in brain “soluble” A? is not required to get EMF-induced cognitive benefits, as we have repeatedly demonstrated for various AD therapeutics including EMF treatment [4], [24], [25]. This is because the disaggregating action of EMF treatment on brain A? (from insoluble to soluble forms) appears to shift most soluble A? from the cognitive-impairing “oligomeric” form to smaller (innocuous) dimeric/monomeric forms. That is the probable reason why we observed brain mitochondrial enhancement in aged Tg mice given long-term (1 month) EMF treatment despite those treated mice having 5–10× higher soluble A? in their brain mitochondria (i.e., most of this soluble A? was in innocuous monomeric/dimeric forms) [5]. Such enhanced levels of monomeric/soluble A? are also consistent with the lack of EMF-induced reductions in plasma A? levels observed in the present study, as well as in our earlier EMF study [4].

Prior to our recent study showing cognitive efficacy of “cell phone-level” EMF exposure administered daily for >6 months to Tg and normal mice [4], animal studies investigating cognitive effects of cell phone level EMF exposure involved “normal” mice/rats receiving daily “head-only” [26][28] or “full body” [29] EMF exposure for a relatively short 4–14 days. No cognitive benefits were reported in those studies, nor did longer 2- or 6-month periods of daily head-only EMF exposure impact cognitive performance in normal rats [28]. However, a 5-week period of cell phone level EMF exposure to immature (3 weeks old) rats did improve their rate of learning in the Morris water maze task [30]. It is important to note that future rodent studies emphasize “head-only” EMF exposure over many months and utilize a comprehensive array of cognitive measures/tasks (not simply a single measure/task).

In humans, all cell phone level EMF studies investigating cognitive function have been unilateral and involved either single EMF exposure [15], [16] or daily EMF exposure for 6–27 days [13], [14], with no cognitive effects being reported in either case. However, one study did report that heavy cell phone users evaluated over a 2-year period performed better in a word interference test [2]. Clearly, there is a critical need for long-term, well-controlled EMF studies in humans to evaluate cognitive effects in both normal and cognitive-impaired individuals.

Body/brain temperature and cerebral blood flow effects of EMF treatment

Before our own recent work [4], [5] and the present study, only one prior animal study investigated the effects of EMF exposure on body/brain temperature and/or cerebral blood flow [31]. That study, involving a single head-only GSM exposure for 18 minutes to anesthetized rats, was at very high frequency (2000 MHz) and very high SAR levels (10–263 W/kg). This acute EMF exposure increased brain temperature in a dose-dependent fashion (by 1–12°C), and increased cortical cerebral blood flow (by 30–70%). In humans, no studies investigating EMF effects on brain temperature have apparently been done in living individuals, and EMF effects on cerebral blood flow have only involved a single, unilateral EMF exposure, with inconsistent results [16]. Thus, for both animals and humans, there had previously been no investigations into long-term EMF effects on brain temperature or cerebral blood flow.

Regarding temperature, our recent studies [4], [5] have investigated both acute and long-term body/brain temperature effects of EMF treatment (i.e., GSM, pulse/modulated at 918 MHz and 0.25–1.05 W/kg), with the following findings: 1) a single day of EMF treatment has no effect on body or brain temperature of either AD Tg or normal mice during ON periods; 2) At 8–9 months into daily EMF treatment, body temperature of both Tg and NT mice is elevated by approximately 1°C during ON periods; and 3) At 1 month into daily EMF treatment, body temperature of aged Tg and NT mice is elevated by around 1°C during ON periods while brain temperatures are either stable (NT mice) or decreased (Tg mice) during ON periods. For both long-term EMF studies in 2) and 3), body temperature always returned back down to normal levels during OFF periods.

The present work extends our aforementioned initial findings by performing two separate temperature-monitoring studies in order to evaluated sub-chronic (12 days) and long-term (6 weeks) effects of daily EMF treatment on both body and brain temperature measurements in very old AD mice and normal mice. During multiple temperature measurements taken over a 6-week period in very old mice that had been behaviorally tested, small (but significant) increases of around 0.5°C in body temperature were evident in both Tg and normal mice. This small increase of <1°C in body temperature during ON periods of long-term EMF treatment is very consistent with that seen in our prior studies [4], [5]. Despite these small, but significant increases in body temperature during ON periods, brain temperature for Tg and normal mice remained stable or was only elevated 0.3–0.4°C through 6 weeks of exposure – far below what would be needed to incur brain/physiologic damage [32]. Thus, the EMF-induced cognitive benefits in mice that we have reported both in our prior report [4] and presently are apparently due to non-thermal brain mechanisms – several of which we have already identified (see last section).

In the sub-chronic (12-day) EMF treatment study, very old APPsw+PS1 (Tg) mice exhibited no change in body or brain temperature during ON periods at both 5 days and 12 days into EMF treatment. This is somewhat in contrast to the long-term study, wherein a significant increase in body temperature during ON periods was already present at 1 week into EMF treatment, although no change in brain temperature occurred (same as in sub-chronic study). The only difference between the two studies, other than temperature recording points, was that double Tg (APPsw+PS1) mice were used in the sub-chronic study, which would have even greater brain A? burdens than the APPsw mice used in the long-term study.

At 2 months into daily EMF treatment in the long-term study, Tg mice (but not normal mice) exhibited a significant 13% decrease in rCBF during ON vs. OFF periods. This EMF-induced reduction in rCBF was even greater (?25%) compared to control Tg mice during sham ON periods. The difference between Tg and NT mice is brain production and aggregation/deposition of A? in Tg mice. Earlier studies have provided evidence that EMF treatment increases neuronal activity [16], [19], [21], [33], [34]. As mentioned previously, our very recent findings show that long-term EMF treatment does indeed increase neuronal activity in Tg and NT mice, irrespective of genotype [20]. Since intraneuronal A? is synaptically released in greater amounts during increased neuronal activity [35], there is presumably greater efflux of this soluble/monomeric A? out of the brain and into the blood during EMF exposure. Inasmuch as vascular A? is a well-known constrictor of smooth muscle in resistance vessels (e.g., arterioles), we believe that this enhanced presence of cerebrovascular A? due to EMF exposure induces cerebral vasoconstriction and the resulting decreases in rCBF that were observed in Tg mice.

Also in the long-term (2 months) study, rCBF was reduced even during OFF periods in both Tg and normal mice being given EMF treatment. Indeed, when both genotypes were combined to investigate main effects of EMF treatment, rCBF was significantly decreased during both ON (?23%) and OFF (?16%) periods. Clearly, some non-specific EMF mechanism is reducing rCBF during OFF periods in both Tg and NT mice. For example, this may be a continuing auto-regulatory response to limit brain heating due to the slight body hyperthermia present during ON periods. Along this line, body hyperthermia (such as that induced by exercise) has been shown to decrease cerebral blood flow in humans by 18% [36], [37]. The reductions in rCBF presently observed during both ON and OFF periods of long-term EMF treatment in Tg and NT mice are consistent with several human PET studies reporting that rCBF is reduced during single exposure EMF treatment [18], [38].

Similar to rCBF results from the long-term EMF study, evaluation of rCBF at 12 days into EMF treatment for APPsw+PS1 (Tg) mice in the sub-chronic study revealed a near significant 19% decrease in rCBF during ON periods. Indeed, 4 of 5 Tg-treated mice exhibited rCBF decreases of 7–46%. Since there was no increase in body temperature during ON periods, there was no need for themoregulatory mechanisms to limit CBF to the brain. However, it is likely that during ON periods, elevated vascular A? caused a modest vasoconstriction in the brain and the ensuing decrease in CBF that was observed.

Mechanisms of long-term EMF action and evidence for EMF safety

Results from the present study, in concert with those from our prior three studies [4], [5], [20], are beginning to provide critical mechanistic insight into the ability of long-term, high frequency EMF exposure to benefit cognitive function in normal and AD mice. Fig. 7 summarizes our current understanding of those mechanisms, which are relevant to human long-term EMF exposure as well. Although this summary diagram is the result of long-term studies involving GMS-modulated and pulsed EMF treatment at specific parameters (918 MHz, 0.25–1.05 W/kg), different combinations of frequency/SAR levels will likely provide more robust mechanistic actions within this circuit and expand it, resulting in greater or more rapid cognitive benefit.

Figure 7

Figure 7

Summary diagram depicting both confirmed and proposed mechanisms of long-term EMF action in normal mice and Alzheimer’s transgenic (Tg) mice.

As depicted in Fig. 7 for AD mice, high frequency EMF treatment would appear to exert two complementary actions that ultimately result in enhanced A? removal/efflux from the brain: 1) prevention and reversal of brain A? aggregation/deposition [4], and 2) increased neuronal/EEG activity [16], [20], [19][21], [33], [34]. EMF treatment’s suppression of extracellular and intracellular A? aggregation, combined with enhanced synaptic release of intra-neuronal A? during increased neuronal activity [35], result in soluble monomergic forms of free A? in the brain parenchyma – A? forms that can be readily transported across the blood-brain barrier [39] and into the blood for eventual degradation. As previously mentioned, soluble/monomeric A? is a powerful vasoconstrictor [40], [41], which is probably key to the substantial decrease in rCBF present during EMF ON periods in Tg mice. Since A? is not a factor for EMF effects in normal mice, normal mice incur a less robust, generalized decrease in CBF through some as yet unidentified mechanism (e.g., compensatory to EMF-induced increases in body temperature). Similarly, long-term EMF treatment to Tg mice induces large enhancements in brain mitochondrial function due to disaggregation of mitochondrial-impairing oligomeric A? in neurons, with a lesser enhancement present in normal mice due to an as yet unidentified mechanism [5].

All of the aforementioned EMF mechanisms occur in mice with only a slight (or no) increase in brain temperature [5] and no increase in brain oxidative stress/damage [4]. Indeed, examination of both peripheral and brain tissues from animals given daily EMF treatment for over 8 months has revealed no tissue abnormalities [4], including no increase in DNA damage to blood cells from these same animals [Cao et al., unpublished observations]. The lack of deleterious brain and peripheral effects in such long-term EMF studies, in combination with recent epidemiologic human studies also reporting no consistent evidence for EMF-induced health problems [10][12], underscores the mounting evidence that high frequency EMF treatment over long periods of time, could be a safe and novel disease-modifying therapeutic against AD.

Materials and Methods

Ethics statement

All animal procedures were performed in AAALAC-certified facilities under protocol #R3258, approved by the University of South Florida Institutional Animal Care and Use Committee.

Animals

For both studies of this work, a total of 41 aged mice derived from the Florida Alzheimer’s Disease Research Center’s colony were included. Each mouse had a mixed background of 56.25% C57, 12.5% B6, 18.75% SJL, and 12.5% Swiss-Webster. All mice were derived from a cross between heterozygous mice carrying the mutant APPK670N, M671L gene (APPsw) with heterozygous PS1 (Tg line 6.2) mice, which provided offspring consisting of APPsw+PS1, APPsw, PS1, and NT genotypes. After weaning and genotyping of these F10 and F11 generation offspring, APPsw and NT mice were selected for a long-term behavioral study (Study I), while APPsw+PS1 mice were selected for a follow-up, shorter duration temperature/cerebral blood flow-monitoring study (Study II) – aged APPsw were not available for the ensuing Study II. All mice were housed individually after genotyping, maintained on a 12-hour dark and 12-hour light cycle with ad libitum access to rodent chow and water.

Study I: Two-month EMF Treatment Study

At 21–26 months of age, APPsw Tg mice (n=17) and NT littermates (n=10) were first evaluated in RAWM task of working memory (see Behavioral testing protocols) to establish baseline cognitive performance for both genotypes prior to EMF treatment. Based on pretreatment performance in the RAWM task, Tg and NT groups were each divided into two performance-balanced sub-groups as follows: Tg controls (n=8), Tg+EMF (n=9), NT controls (n=5), and NT+EMF (n=5). Tg and NT mice to be exposed to EMFs had their cages placed within a large Faraday cage, which contained an EMF generator antenna that provided two 2-hour periods of EMF treatment per day (see EMF treatment protocol). At 22–27 months of age (one month into EMF treatment), all mice were started on a one-month series of behavioral tasks. EMF treatment was continued during the one-month behavioral testing period, with all testing performed during “OFF” periods in between the two daily EMF treatments. Body and brain temperature measurements were performed just prior to initiation of EMF treatment and at 1, 3, and 6 weeks into EMF treatment (see Body/brain temperature determinations). Doppler recordings of rCBF were taken at 2 months in EMF treatment (see rCBF determinations). On the day following rCBF measurements, animals were euthanized at 23–28 months of age, during which a blood sample was taken and brains were perfused with isotonic phosphate-buffered saline (PBS). The caudal brain was then paraffin-embedded and processed for A? immunohistochemical staining, while the remaining forebrain was sagitally bisected and dissected into hippocampus and cortical areas that were quick-frozen for neurochemical analyses. Plasma was analyzed for both A?1–40 and A?1–42.

Study II: 12-day EMF Treatment Study

At 22 months of age, 11 APPsw+PS1 Tg mice were divided into two groups of 5–6 mice each. One group was placed into the faraday cage for two daily EMF exposures exactly as for mice in the 2-month EMF Treatment Study (see EMF treatment protocol). The other group served as EMF controls, housed in a completely separate room with an identical environment without EMF treatment. Body and brain temperature recordings were taken from all mice just prior to onset of the first EMF treatment, as well as on the 5th day and 12th day into EMF treatment. Concurrent with temperature recording on Day 12, cerebral blood flow measurements were also taken.

EMF treatment protocol

Tg and NT mice given EMF treatment were individually housed in cages within a large Faraday cage, which also housed the antenna of an EMF generator providing two 2-hour periods of electromagnetic waves per day (early morning and late afternoon). Each EMF exposure was at 918 MHz frequency, involved modulation with Gaussian minimal-shift keying (GMSK) signal, and was pulsed/non-continuous with carrier bursts repeated every 4.6 ms, giving a pulse repetition rate of 217 Hz. The electrical field strength varied between 17 and 35 V/m. This resulted in calculated SAR levels that varied between 0.25 and 1.05 W/kg. Calculated SAR values have been shown to correspond closely with measured SAR values [42]. SAR was calculated from the below equation, with ? (0.88 sec/m) and ? (1030 kg/m3) values attained from Nightingale et al. [43]:

equation image

?=mean electrical conductivity of mouse brain tissue.

?=mass density of mouse brain.

E=electrical field strength.

For the 2-month and 12-day periods of EMF treatment given to mice in Study’s I and II, respectively, cages of individually-housed mice were maintained within the Faraday cage (1.2×1.2×1.2 m3) and arranged in a circular pattern. Each cage was approximately 26 cm from a centrally located EMF-emitting antenna. The antenna was connected to a Hewlett–Packard ESG D4000A digital signal generator (Houston, TX, USA) set to automatically provide two 2-hour exposures per day. With a 12-hour light ON/OFF cycle, the 2-hour daily exposures occurred in early morning and late afternoon of the lights on period. Sham-treated control Tg and NT mice were located in a completely separate room, with identical room temperature as in the EMF exposure room and with animals individually housed in cages that were arranged in the same circular pattern.

Behavioral Testing Protocols

Prior to EMF treatment, all mice in Study I were behaviorally tested for 10 days in RAWM task of working memory to determine baseline cognitive performance in this task. Daily EMF treatment was then started, with behavioral testing initiated at one month into EMF treatment and occurring between early morning and late afternoon EMF treatments. One-day tasks of sensorimotor function were initially carried out (open field activity, balance beam, string agility), followed by a one-day Y-maze task (locomotor activity, spontaneous alternation), then RAWM Test I (4 days), circular platform performance (4 days), RAWM Test II (4 days), then finally the visual cliff test of visual acuity (1 day). Although the methodologies for all of these tasks have been previous described and are well established [44][46], a brief description of each task is provided below:

Open field activity

Open field activity was used to measure exploratory behavior and general activity. Mice were individually placed into an open black box 81×81 cm with 28.5-cm high walls. This area was divided by white lines into 16 squares measuring 20×20 cm. Lines crossed by each mouse over a 5-minute period were counted.

Balance beam

Balance beam was used to measure balance and general motor function. The mice were placed on a 1.1-cm wide beam, suspended above a padded surface by two identical columns. Attached at each end of the beam was an escape platform. Mice were placed on the beam in a perpendicular orientation and were monitored for a maximum of 60 secs. The time spent by each mouse on the beam before falling or reaching one of the platforms was recorded for each of three successive trials. If a mouse reached one of the escape platforms, a time of 60 secs was assigned for that trial. The average of all three trials was utilized.

String agility

String agility was used to assess forepaw grip capacity and agility. Mice were placed in the center of a taut cotton string suspended above a padded surface between the same two columns as in the balance beam task. Mice were allowed to grip the string with only their forepaws and then released for a maximum of 60 secs. A rating system, ranging between 0 and 5, was employed to assess string agility for a single 60-sec trial.

Y-maze spontaneous alternation

Y-maze spontaneous alternation was used to measure general activity and basic mnemonic function. Mice were allowed 5 minute to explore a black Y-maze with three arms. The number and sequence of arm choices were recorded. General activity was measured as the total number of arm entries, while basic mnemonic function was measured as a percentage of spontaneous alternation (the ratio of arm choices different from the previous two choices divided by the total number of entries).

Circular platform

Circular platform was used to measure spatial/reference learning and memory. Mice were placed on a 69-cm circular platform with 16 equally spaced holes on the periphery of the platform. Underneath only one of the 16 holes was a box filled with bedding to allow the mouse to escape from aversive stimuli (e.g. two 150-W flood lamps hung 76 cm above the platform and one high-speed fan 15 cm above the platform). Each mouse was administered one 5-minute trial per day to explore the area. For the single trial administered on each of four test days, mice were placed in the center of the platform facing away from their escape hole (which differed for each mouse). Escape latency was measured (maximum of 300 secs) each day. Data was statistically analyzed in two 2-day blocks.

RAWA

RAWA task of spatial working memory involved use of an aluminum insert, placed into a 100 cm circular pool to create 6 radially distributed swim arms emanating from a central circular swim area. An assortment of 2-D and 3-D visual cues surrounded the pool. The latency and number of errors prior to locating which one of the 6 swim arms contained a submerged escape platform (9 cm diameter) was determined for 5 trials/day over 10 days of pre-treatment testing. There was a 30-minute time delay between the 4th trial and the 5th trial (T5; memory retention trial). The platform location was changed daily to a different arm, with different start arms for each of the 5 trials semi-randomly selected from the remaining 5 swim arms. During each trial (60-sec maximum), the mouse was returned to that trial’s start arm upon swimming into an incorrect arm and the number of seconds required to locate the submerged platform was recorded. If the mouse did not find the platform within a 60-sec trial, it was guided to the platform for the 30-sec stay. The latency and number of errors during Trial 1 (T1) are chance performance since the animal does not know where the submerged platform is for the first trial of any given day. Latency and errors during the last trial (Trial 5; T5) of any given day are considered indices of working memory and are temporally similar to the standard registration/recall testing of specific items used clinically in evaluating AD patients. Data for T1 and T5 were statistically analyzed in two-day blocks, as well as overall, for the 10-day of pretreatment RAWM testing, the 4-day of RAWM Test I, and the 4-day of RAWM Test II. Because the final block of testing is most representative of true working memory potential in this task, results from the last 2-day block of testing are presented for all three RAWM test periods.

Visual Cliff

Visual Cliff was utilized on the last day of behavioral testing to evaluate vision/depth perception. A wooden box has two horizontal surfaces, both of which have the same bold pattern, but one surface of which is 10–12 inches below the other. A sheet of clear Plexiglass is placed across the entire horizontal surface, providing the visual appearance of a cliff. An animal with poor vision/depth perception cannot detect the “cliff” and will move without hesitation across the cliff, resulting in a score of “1?. An animal with good vision will pause/hesitate at the cliff before crossing it and is scored a “2?.

Body/brain temperature determinations

For body/brain temperature determinations of mice in both Studies I and II, body temperature was taken via rectal probe and brain temperature via temporalis muscle probe. Prior studies have demonstrated that temporalis muscle temperature very accurately reflects brain temperature in rodents [47], [48]. Temperature determinations during EMF treatment (ON periods) were taken near the end of the morning EMF treatment, while temperature determinations during OFF periods were in early afternoon (mid-way between the two daily EMF treatments). Each measurement only took a couple of minutes for each mouse.

rCBF determinations

In cerebral cortex, rCBF measurements during the ON period were taken near the end of either the morning EMF treatment session (Study I) or the afternoon treatment session (Study II). rCBF measurements during the OFF period were taken in early afternoon, mid-way between both EMF treatment sessions. For each measurement, anesthetized (equithesin 300 mg/kg i.p.) animals underwent rCBF measurement using laser Doppler flowmetry (PF-5010, Periflux system, Järfälla, Sweden) with relative flow values expressed as perfusion units [49], [50]. All rCBF measurements were conducted with the animal fixed in a Kopf stereotaxic apparatus, with the probe placed at the level of the dura directly above a small skull opening. Using a micromanipulator, two probes (probe 411, 0.45 mm in diameter) were positioned to cortical coordinates of 1.3 mm posterior to the bregma and 2.8 mm to each side of midline on the intact skull, being careful to avoid pial vessels after reflection of the skin overlying the calvarium. Because mouse skull and subarachnoid space are very thin, transcranial measurements of rCBF are consistent with craniectomy measurements [51]. The rCBF of both hemispheres were continuously measured for 15 minutes and averaged for each determination. All rCBF data was continuously stored in a computer and analyzed using the Perimed data acquisition and analysis system.

A  immunohistochemistry and image analysis

[ratio]

At the level of the posterior hippocampus (bregma 2.92 mm to 3.64 mm), five 5 µm sections (150 µm apart) were taken from each mouse brain using a sliding microtome (REM-710, Yamato Kohki Industrial, Asaka, Saitama, Japan). Immunohistochemical staining was performed following the manufacturer’s protocol using aVectastainABC Elite kit (Vector Laboratories, Burlingame, CA) coupled with the diaminobenzidine reaction, except that the biothinylated secondary antibody step was omitted. Used as the primary antibody was a biothinylated human A? monoclonal antibody (clone 4G8; 1200, Covance Research Products, Emeryville, CA). Brain sections were treated with 70% formic acid prior to the pre-blocking step. 0.1 M PBS (pH 7.4) or normal mouse serum (isotype control) was used instead of primary antibody or ABC reagent as a negative control. Quantitative image analysis was done based on previously validated method [52]. Images were acquired using an Olympus BX60 microscope with an attached digital camera system (DP-70, Olympus, Tokyo, Japan), and the digital image was routed into a Windows PC for quantitative analysis using SimplePCI software (Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan). Images of five 5-µm sections (150 µm apart) through both anatomic regions of interest (hippocampus and entorhinal cortex) were captured from each animal, and a threshold optical density was obtained that discriminated staining from background. Each region of interest was manually edited to eliminate artifacts, with A? burden data reported as percentage of immune-labeled area captured (positive pixels) relative to the full area captured (total pixels). Each analysis was done by a single examiner blinded to sample identities.

Plasma A levels

A 1–40 and 1–42 levels were determined from plasma samples by using ELISA kits (KHB3482 for 40, KHB3442 for 42, Invitrogen, CA). Standard and samples were mixed with detection antibody and loaded on the antibody pre-coated plate as the designated wells. HRP-conjugated antibody was added after wash, and substrates were added for colorimetric reaction, which was then stopped with sulfuric acid. Optical density was obtained and concentrations were calculated according a standard curve.

Statistical Analysis

Data analysis of physiologic and neurohistologic measurements, as well as all one-day behavioral measures, were performed using ANOVA followed by Fisher’s LSD post hoc test. For the multiple-day behavioral tasks (RAWM and circular platform), initial ANOVA analysis of 2-day blocks and overall were followed by analysis of post hoc pair-by-pair differences between groups via the Fisher LSD test. For temperature and blood flow measurements within the same animal, paired t-tests were employed. All data are presented as mean ± SEM, with significant group differences being designated by p<0.05 or higher level of significance.

Acknowledgments

We gratefully acknowledge the graphic skills of Loren Glover for figure preparations.

Footnotes

Competing Interests: Co-author Dr. Cesar Borlongan is a PLoS ONE Editorial Board member. Co-author Richard Gonzalez is founder and CEO of a small electronics company, SAI of Florida, Redington Beach, Florida 33708. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Funding: This work was supported by funds from the NIA-designated Florida Alzheimer’s Disease Research Center (AG025711) to G.A., the USF/Byrd Alzheimer’s Institute to G.A., and a USF Interdisciplinary Research Development Grant to G.A. and C.V.B. N.T. is a recipient of the 2011 Alzheimer’s Drug Discovery Foundation Young Investigator Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1. Gravitz L. A tangled web of targets. Nature. 2011;475:S9–S11. [PubMed] 2. Arns M, Luijtelaar G, Sumich A, Hamilton R, Gordon E. Electroencephalographic, personality, and executive function measures associated with frequent mobile phone use. Int J Neurosci. 2007;117:1341–1360. [PubMed] 3. Schüz J, Waldemar G, Olsen J, Johansen C. Risks for central nervous system diseases among mobile phone subscribers: a Danish retrospective cohort study. PLoS One. 2009;4:e4389. [PMC free article] [PubMed] 4. Arendash GW, Sanchez-Ramos J, Mori T, Mamcarz M, Lin X, et al. Electromagnetic field treatment protects against and reverses cognitive impairment in Alzheimer’s mice. J Alzheimers Dis. 2010;19:191–210. [PubMed] 5. Dragicevic N, Bradshaw PC, Mamcarz M, Lin X, Wang L, et al. Long-term electromagnetic field treatment enhances brain mitochondrial function of both Alzheimer’s transgenic mice and normal mice: a mechanism for electromagnetic field-induced cognitive benefit? Neuroscience. 2011;185:135–149. [PubMed] 6. Hardell L, Carlberg M, Soderqvist F, Hansson Mild K. Meta-analysis of long-term mobile phone use and the association with brain tumours. Int J Oncol. 2008;32:1097–1103. [PubMed] 7. Khurana VG, Teo C, Kundi M, Hardell L, Carlberg M. Cell phones and brain tumors: a review including the long-term epidemiologic data. Surg Neurol. 2009;72:205–215. [PubMed] 8. INTERPHONE Study Group. Brain tumour risk in relation to mobile telephone use: results of the INTERPHONE international case-control study. Int J Epidemiol. 2010;39:675–694. [PubMed] 9. Swerdlow AJ, Feychting M, Green A, Kheifets L, Savitz DA. Mobile phones, brain tumours and the Interphone Study: Where are we now? 2011. National Institute of Environmental Health Sciences (NIEHS), Available: http://dx.doi.org/10.1289/ehp.1103693. 10. Valberg PA, van Deventer TE, Repacholi MH. Workgroup report: base stations and wireless networks-radiofrequency (RF) exposures and health consequences. Environ Health Perspect. 2007;115:416–424. [PMC free article] [PubMed] 11. Krewski D, Glickman BW, Habash RW, Habbick B, Lotz WG, et al. Recent advances in research on radiofrequency fields and health: 2001–2003. J Toxicol Environ Health B Crit Rev. 2007;10:287–318. [PubMed] 12. Aydin D, Feychting M, Schüz J, Tynes T, Andersen TV, et al. Mobile phone use and brain tumors in children and adolescents: a multicenter case-control study. J Natl Cancer Inst. 2011;103:1264–1276. [PubMed] 13. Besset A, Espa F, Dauvilliers Y, Billiard M, de Seze R. No effect on cognitive function from daily mobile phone use. Bioelectromagnetics. 2005;26:102–108. [PubMed] 14. Fritzer G, Goder R, Friege L, Wachter J, Hansen V, et al. Effects of short- and long-term pulsed radiofrequency electromagnetic fields on night sleep and cognitive functions in healthy subjects. Bioelectromagnetics. 2007;28:316–325. [PubMed] 15. Barth A, Ponocny I, Gnambs T, Winker R. No effects of short-term exposure to mobile phone electromagnetic fields on human cognitive performance: A meta-analysis. Bioelectromagnetics. 2011 doi: 10.1002/bem.20697. [PubMed] 16. Kwon MS, Hämäläinen H. Effects of mobile phone electromagnetic fields: critical evaluation of behavioral and neurophysiological studies. Bioelectromagnetics. 2011;32:253–272. [PubMed] 17. Huber R, Treyer V, Schuderer J, Berthold T, Buck A, et al. Exposure to pulse-modulated radio frequency electromagnetic fields affects regional cerebral blood flow. Eur J Neurosci. 2005;21:1000–1006. [PubMed] 18. Aalto S, Haarala C, Brück A, Sipilä H, Hämäläinen H, et al. Mobile phone affects cerebral blood flow in humans. J Cereb Blood Flow Metab. 2006;26:885–890. [PubMed] 19. Volkow ND, Tomasi D, Wang GJ, Vaska P, Fowler JS, et al. Effects of cell phone radiofrequency signal exposure on brain glucose metabolism. JAMA. 2011;305:808–813. [PMC free article] [PubMed] 20. Mori T, Arendash GW. Long-term electromagnetic field treatment increases brain neuronal activity: linkage to cognitive benefit and therapeutic implications for Alzheimer’s disease. J Alzheimer’s Dis and Parkinsonism. 2011;1:2. Available: http://dx.doi.org/10.4172/2161-0460.1000102. 21. Juutilainen J, Hoyto A, Kumlin T, Naarala J. Review of possible modulation-dependent biological effects of radiofrequency fields. Bioelectromagnetics. 2011;32:511–534. [PubMed] 22. DeBow S, Colbourne F. Brain temperature measurement and regulation in awake and freely moving rodents. Methods. 2003;30:167–171. [PubMed] 23. Leighty RE, Nilsson LN, Potter H, Costa DA, Low MA, et al. Use of multimetric statistical analysis to characterize and discriminate between the performance of four Alzheimer’s transgenic mouse lines differing in A? deposition. Behav Brain Res. 2004;153:107–121. [PubMed] 24. Olcese JM, Cao C, Mori T, Mamcarz MB, Maxwell A, et al. Protection against cognitive deficits and markers of neurodegeneration by long-term oral administration of melatonin in a transgenic model of Alzheimer disease. J Pineal Res. 2009;47:82–96. [PubMed] 25. Echeverria V, Zeitlin R, Burgess S, Patel S, Barman A, et al. Cotinine reduces amyloid-? aggregation and improves memory in Alzheimer’s disease mice. J Alzheimers Dis. 2011;24:817–835. [PubMed] 26. Dubreuil D, Jay T, Edeline JM. Does head-only exposure to GSM-900 electromagnetic fields affect the performance of rats in spatial learning tasks? Behav Brain Res. 2002;129:203–210. [PubMed] 27. Dubreuil D, Jay T, Edeline JM. Head-only exposure to GSM 900-MHz electromagnetic fields does not alter rat’s memory in spatial and non-spatial tasks. Behav Brain Res. 2003;145:51–61. [PubMed] 28. Ammari M, Jacquet A, Lecomte A, Sakly M, Abdelmelek H, et al. Effect of head-only sub-chronic and chronic exposure to 900-MHz GSM electromagnetic fields on spatial memory in rats. Brain Inj. 2008;22:1021–1029. [PubMed] 29. Sienkiewicz ZJ, Blackwell RP, Haylock RG, Saunders RD, Cobb BL. Low-level exposure to pulsed 900 MHz microwave radiation does not cause deficits in the performance of a spatial learning task in mice. Bioelectromagnetics. 2000;21:151–158. [PubMed] 30. Kumlin T, Iivonen H, Miettinen P, Juvonen A, van Groen T, et al. Mobil phone radiation and the developing brain: behavioral and morphological effects in juvenile rats. Radiat Res. 2007;168:471–479. [PubMed] 31. Masuda H, Hirata A, Kawai H, Wake K, Watanabe S, et al. Local exposure of the rat cortex to radiofrequency electromagnetic fields increases local cerebral blood flow along with temperature. J Appl Physiol. 2011;110:142–148. [PubMed] 32. Van Leeuwen GM, Lagendijk JJ, Van Leersum BJ, Zwamborn AP, Hornsleth SN, et al. Calculation of change in brain temperatures due to exposure to a mobile phone. Phys Med Biol. 1999;44:2367–2379. [PubMed] 33. Tattersall JE, Scott IR, Wood SJ, Nettell JJ, Bevir MK, et al. Effects of low intensity radiofrequency electromagnetic fields on electrical activity in rat hippocampal slices. Brain Res. 2001;904:43–53. [PubMed] 34. Cook CM, Saucier DM, Thomas AW, Prato FS. Changes in human EEG alpha activity following exposure to two different pulsed magnetic field sequences. Bioelectromagnetics. 2009;30:9–20. [PubMed] 35. Cirrito JR, Yamada KA, Finn MB, Sloviter RS, Bales KR, et al. Synaptic activity regulates interstitial fluid amyloid-? levels in vivo. Neuron. 2005;48:913–922. [PubMed] 36. Nybo L, Møller K, Volianitis S, Nielsen B, Secher NH. Effects of hyperthermia on cerebral blood flow and metabolism during prolonged exercise in humans. J Appl Physiol. 2002;93:58–64. [PubMed] 37. Nelson MD, Haykowsky MJ, Stickland MK, Altamirano-Diaz LA, Willie CK, et al. Reductions in cerebral blood flow during passive heat stress in humans: partitioning the mechanisms. J Physiol. 2011;589:4053–4064. [PMC free article] [PubMed] 38. Haarala C, Aalto S, Hautzel H, Julkunen L, Rinne JO, et al. Effects of a 902 MHz mobile phone on cerebral blood flow in humans: a PET study. Neuroreport. 2003;14:2019–2023. [PubMed] 39. Ito S, Ohtsuki S, Kamiie J, Nezu Y, Terasaki T. Cerebral clearance of human amyloid-? peptide (1–40) across the blood-brain barrier is reduced by self-aggregation and formation of low-density lipoprotein receptor-related protein-1 ligand complexes. J Neurochem. 2007;103:2482–2490. [PubMed] 40. Arendash GW, Su GC, Crawford FC, Bjugstad KB, Mullan M. Intravascular ?-amyloid infusion increases blood pressure: implications for a vasoactive role of ?-amyloid in the pathogenesis of Alzheimer’s disease. Neurosci Lett. 1999;268:17–20. [PubMed] 41. Paris D, Humphrey J, Quadros A, Patel N, Crescentini R, et al. Vasoactive effects of A? in isolated human cerebrovessels and in a transgenic mouse model of Alzheimer’s disease: role of inflammation. Neurol Res. 2003;25:642–651. [PubMed] 42. Kubacki R, Sobiech J, Sedek E. Model for investigation of microwave energy absorbed by young and mature living animals. 2007. 2nd International Conference on Electromagnetic Fields, Health, and Environment. Wroclaw, Poland. 43. Nightingale NR, Goodridge VD, Sheppard RJ, Christie JL. The dielectric properties of the cerebellum, cerebrum and brain stem of mouse brain at radiowave and microwave frequencies. Phys Med Biol. 1983;28:897–903. [PubMed] 44. Arendash GW, Schleif W, Rezai-Zadeh K, Jackson EK, Zacharia LC, et al. Caffeine protects Alzheimer’s mice against cognitive impairment and reduces brain A? production. Neuroscience. 2006;142:941–952. [PubMed] 45. Arendash GW, Jensen MT, Salem N, Jr, Hussein N, Cracchiolo J, et al. A diet high in omega-3 fatty acids does not improve or protect cognitive performance in Alzheimer’s transgenic mice. Neuroscience. 2007;149:286–302. [PubMed] 46. Arendash GW, Mori T, Cao C, Mamcarz M, Runfeldt M, et al. Caffeine reverses cognitive impairment and decreases brain amyloid-? levels in aged Alzheimer’s disease mice. J Alzheimers Dis. 2009;17:661–680. [PubMed] 47. Shimizu H, Chang LH, Litt L, Zarow G, Weinstein PR. Effect of brain, body, and magnet bore temperatures on energy metabolism during global cerebral ischemia and reperfusion monitored by magnetic resonance spectroscopy in rats. Magn Reson Med. 1997;37:833–839. [PubMed] 48. Brambrink AM, Kopacz L, Astheimer A, Noga H, Heimann A, et al. Control of brain temperature during experimental global ischemia in rats. J Neurosci Methods. 1999;92:111–122. [PubMed] 49. Borlongan CV, Lind JG, Dillon-Carter O, Yu G, Hadman M, et al. Bone marrow grafts restore cerebral blood flow and blood brain barrier in stroke rats. Brain Res. 2004;1010:108–116. [PubMed] 50. Borlongan CV, Lind JG, Dillon-Carter O, Yu G, Hadman M, et al. Intracerebral xenografts of mouse bone marrow cells in adult rats facilitate restoration of cerebral blood flow and blood-brain barrier. Brain Res. 2004;1009:26–33. [PubMed] 51. Hara H, Huang PL, Panahian N, Fishman MC, Moskowitz MA. Reduced brain edema and infarction volume in mice lacking the neuronal isoform of nitric oxide synthase after transient MCA occlusion. J Cereb Blood Flow Metab. 1996;16:605–611. [PubMed] 52. Mori T, Rezai-Zadeh K, Koyama N, Arendash GW, Yamaguchi H, et al. Tannic acid is a natural ?-secretase inhibitor that prevents cognitive impairment and mitigates Alzheimer-like pathology in transgenic mice. J Biol Chem. 2012;287:6912–6927. [PMC free article] [PubMed]


Altern Ther Health Med.  2011 Nov-Dec;17(6):22-8. Long-term Effects of Bio-electromagnetic-energyregulation Therapy on Fatigue in Patients With Multiple Sclerosis. Ziemssen T, Piatkowski J, Haase R. Abstract Background Electromagnetic-field therapy has beneficial short-term effects in multiple sclerosis (MS) patients with major fatigue, but long-term data are lacking. Primary Study Objectives To evaluate the long-term effects of a specific electromagnetic therapy device (Bio-Electromagnetic- Energy-Regulation [BEMER]) on MS-related fatigue, we designed a crossover control of a previously performed randomized controlled trial and a long-term open-label follow-up trial. Design and Setting: Crossover and open-label follow-up trials at a single neurological outpatient center. Participants Patients with relapsing-remitting MS who had major fatigue (N = 37 patients). Intervention After a previous randomized controlled trial (exposure to low-frequency pulsed magnetic fields for 8 min twice daily or to placebo treatment for 12 wk), a crossover from control to treatment for another 12 weeks, followed by an openlabel follow-up trial to 3 years, were done. Primary Outcome Measures The outcome criteria were the Modified Fatigue Impact Scale (MFIS), Fatigue Severity Scale (FSS), German long version of the Center for Epidemiologic Studies Depression Scale (CES-D), Multiple Sclerosis Functional Scale (MSFC), and Expanded Disability Status Scale (EDSS). Results Patients previously on placebo during the randomized controlled trial experienced significant reductions in fatigue after crossing over to treatment. The MFIS and FSS scores were significantly lower in the open-label group than in the control subjects after follow-up. Participation in the open-label treatment was the strongest predictor of low fatigue outcome after followup. Electromagnetic-field therapy was well tolerated. Conclusions In this long-term study, a beneficial effect of long-term BEMER therapy on MS fatigue was demonstrated. Electromagnetic-field therapy may be a useful therapeutic modality in MS patients with severe fatigue.  J Recept Signal Transduct Res. 2010 Aug;30(4):214-26. Electromagnetic fields: mechanism, cell signaling, other bioprocesses, toxicity, radicals, antioxidants and beneficial effects. Kovacic P, Somanathan R. Department of Chemistry, San Diego State University, San Diego, California, USA. pkovacic@sundown.sdsu.edu Abstract Electromagnetic fields (EMFs) played a role in the initiation of living systems, as well as subsequent evolution. The more recent literature on electrochemistry is documented, as well as magnetism. The large numbers of reports on interaction with living systems and the consequences are presented. An important aspect is involvement with cell signaling and resultant effects in which numerous signaling pathways participate. Much research has been devoted to the influence of man-made EMFs, e.g., from cell phones and electrical lines, on human health. The degree of seriousness is unresolved at present. The relationship of EMFs to reactive oxygen species (ROS) and oxidative stress (OS) is discussed. There is evidence that indicates a relationship involving EMFs, ROS, and OS with toxic effects. Various articles deal with the beneficial aspects of antioxidants (AOs) in countering the harmful influence from ROS-OS associated with EMFs. EMFs are useful in medicine, as indicated by healing bone fractures. Beneficial effects are recorded from electrical treatment of patients with Parkinson’s disease, depression, and cancer. J Altern Complement Med.  2009 May;15(5):507-11. Effect of BEMER magnetic field therapy on the level of fatigue in patients with multiple sclerosis: a randomized, double-blind controlled trial. Piatkowski J, Kern S, Ziemssen T. Source Neurological Outpatient Center Reichenbachstrasse, Dresden, Germany. Abstract OBJECTIVES: Electromagnetic field therapy has been reported to be beneficial in patients with multiple sclerosis (MS) with significant fatigue. This study was designed to evaluate the long-term effects of Bio-Electro-Magnetic-Energy-Regulation (BEMER) on MS-related fatigue. DESIGN: This was a monocenter, patient- and rater-blinded, placebo-controlled trial. PATIENTS: There were 37 relapsing-remitting patients with MS with significant fatigue in the study. INTERVENTION: The intervention consisted of BEMER magnetic field treatment for 8 minutes twice daily in comparison to placebo for 12 weeks. OUTCOME MEASURES: The primary outcome criterion was change in the Modified Fatigue Impact Scale (MFIS) between baseline and 12 weeks. The secondary outcome criteria were changes of the Fatigue Severity Scale (FSS), a general depression scale-long version (ADS-L), Multiple Sclerosis Functional Scale (MSFC), and the Expanded Disability Status Scale (EDSS). RESULTS: There was evidence of a significant difference of MFIS value (primary outcome criterion) after 12 weeks in favor of the verum group (26.84 versus 36.67; p = 0.024). In addition, FSS values were significantly lower in the verum group after 12 weeks (3.5 versus 4.7; p = 0.016). After 6 weeks’ follow-up, verum and placebo groups did not differ in experienced fatigue (MFIS, FSS). Regarding the subscales of the MFIS, there was a significant decrease in physical (p = 0.018) and cognitive (p = 0.041), but not in psychologic subscales only in the verum group regarding the timepoints baseline and 12 weeks. BEMER therapy was well tolerated. DISCUSSION: In this pilot study, we were able to demonstrate a beneficial effect of BEMER intervention on MS fatigue. As this was only a pilot study, trials with more patients and longer duration are mandatory to describe long-term effects. Biolectromagn Biol Med. 2007;26(4):305-9. The autistic syndrome and endogenous ion cyclotron resonance: state of the art. Crescentini F. Department of Bioelectromagnetic Research, I.R.P. L’Aquila, Pescara, Italy. The autistic syndrome is a multigenic disease whose expression is different according to the level of involvement of different structures in the central nervous system. The pathogenesis is unknown. No completely effective medical therapy has yet been demonstrated. Accepting the request of the families of eight autistic children in Lomazzo, Milan and Naples, we used ion cyclotron resonance (Seqex(R) therapy) therapeutic support after many other therapies had been already carried out on these patients. After regimens consisting of 20-30 treatments with ICR, improvements were noted in all cases. Int J Neurosci. 2006 Jul;116(7):775-826. Serotonergic mechanisms in amyotrophic lateral sclerosis. Sandyk R. The Carrick Institute for Clinical Ergonomics Rehabilitation, and Applied Neurosciences, School of Engineering Technologies State University of New York at Farmingdale, Farmingdale, New York 11735, USA. rsandyk@optonline.net Serotonin (5-HT) has been intimately linked with global regulation of motor behavior, local control of motoneuron excitability, functional recovery of spinal motoneurons as well as neuronal maturation and aging. Selective degeneration of motoneurons is the pathological hallmark of amyotrophic lateral sclerosis (ALS). Motoneurons that are preferentially affected in ALS are also densely innervated by 5-HT neurons (e.g., trigeminal, facial, ambiguus, and hypoglossal brainstem nuclei as well as ventral horn and motor cortex). Conversely, motoneuron groups that appear more resistant to the process of neurodegeneration in ALS (e.g., oculomotor, trochlear, and abducens nuclei) as well as the cerebellum receive only sparse 5-HT input. The glutamate excitotoxicity theory maintains that in ALS degeneration of motoneurons is caused by excessive glutamate neurotransmission, which is neurotoxic. Because of its facilitatory effects on glutaminergic motoneuron excitation, 5-HT may be pivotal to the pathogenesis and therapy of ALS. 5-HT levels as well as the concentrations 5-hydroxyindole acetic acid (5-HIAA), the major metabolite of 5-HT, are reduced in postmortem spinal cord tissue of ALS patients indicating decreased 5-HT release. Furthermore, cerebrospinal fluid levels of tryptophan, a precursor of 5-HT, are decreased in patients with ALS and plasma concentrations of tryptophan are also decreased with the lowest levels found in the most severely affected patients. In ALS progressive degeneration of 5-HT neurons would result in a compensatory increase in glutamate excitation of motoneurons. Additionally, because 5-HT, acting through presynaptic 5-HT1B receptors, inhibits glutamatergic synaptic transmission, lowered 5-HT activity would lead to increased synaptic glutamate release. Furthermore, 5-HT is a precursor of melatonin, which inhibits glutamate release and glutamate-induced neurotoxicity. Thus, progressive degeneration of 5-HT neurons affecting motoneuron activity constitutes the prime mover of the disease and its progression and treatment of ALS needs to be focused primarily on boosting 5-HT functions (e.g., pharmacologically via its precursors, reuptake inhibitors, selective 5-HT1A receptor agonists/5-HT2 receptor antagonists, and electrically through transcranial administration of AC pulsed picotesla electromagnetic fields) to prevent excessive glutamate activity in the motoneurons. In fact, 5HT1A and 5HT2 receptor agonists have been shown to prevent glutamate-induced neurotoxicity in primary cortical cell cultures and the 5-HT precursor 5-hydroxytryptophan (5-HTP) improved locomotor function and survival of transgenic SOD1 G93A mice, an animal model of ALS. Neuron. 2005 Jan 20;45(2):181-3.

Toward establishing a therapeutic window for rTMS by theta burst stimulation.

Paulus W.

Department of Clinical Neurophysiology, University of Goettingen, D-37075 Goettingen, Germany.

In this issue of Neuron, Huang et al. show that a version of the classic theta burst stimulation protocol used to induce LTP/LTD in brain slices can be adapted to a transcranial magnetic stimulation (TMS) protocol to rapidly produce long lasting (up to an hour), reversible effects on motor cortex physiology and behavior. These results may have important implications for the development of clinical applications of rTMS in the treatment of depression, epilepsy, Parkinson’s, and other diseases.

Wiad Lek. 2003;56(9-10):434-41.

Application of variable magnetic fields in medicine-15 years experience.

[Article in Polish]

Sieron A, Cieslar G.

Katedra i Klinika Chorob Wewnetrznych, Angiologii i Medycyny Fizykalnej SAM, ul. Batorego 15, 41-902 Bytom. sieron@mediclub.pl

The results of 15-year own experimental and clinical research on application of variable magnetic fields in medicine were presented. In experimental studies analgesic effect (related to endogenous opioid system and nitrogen oxide activity) and regenerative effect of variable magnetic fields with therapeutical parameters was observed. The influence of this fields on enzymatic and hormonal activity, free oxygen radicals, carbohydrates, protein and lipid metabolism, dielectric and rheological properties of blood as well as behavioural reactions and activity of central dopamine receptor in experimental animals was proved. In clinical studies high therapeutic efficacy of magnetotherapy and magnetostimulation in the treatment of osteoarthrosis, abnormal ossification, osteoporosis, nasosinusitis, multiple sclerosis, Parkinson’s disease, spastic paresis, diabetic polyneuropathy and retinopathy, vegetative neurosis, peptic ulcers, colon irritable and trophic ulcers was confirmed.

Adv Anat Embryol Cell Biol. 2003;173:III-IX, 1-77.

Electric field-induced effects on neuronal cell biology accompanying dielectrophoretic trapping.

Heida T.

University of Twente, Faculty of Electrical Engineering, Mathematics and Computer Science, Laboratory of Measurement and Instrumentation, Laboratory of Biomedical Engineering, P.O. Box 217, 7500 AE Enschede, The Netherlands. t.heida@el.utwente.nl

Abstract

Trapping neuronal cells may aid in the creation of the cultured neuron probe. The aim of the development of this probe is the creation of the interface between neuronal cells or tissue in a (human) body and electrodes that can be used to stimulate nerves in the body by an external electrical signal in a very selective way. In this way, functions that were (partially) lost due to nervous system injury or disease may be restored. First, a direct contact between cultured neurons and electrodes is created. This is realized using a microelectrode array (MEA) which can be fabricated using standard photolithographic and etching methods. Section 1 gives an overview of the human nervous system, methods for functional recovery focused on the cultured neuron probe, and the prerequisites for culturing neurons on a microelectrode array. An important aspect in the selective stimulation of neuronal cells is the positioning of cells or a small group of cells on top of each of the electrode sites of the MEA. One of the most efficient methods for trapping neuronal cells is to make use of di-electrophoresis (DEP). Dielectrophoretic forces are created when (polarizable) cells are located in nonuniform electric fields. Depending on the electrical properties of the cells and the suspending medium, the DEP force directs the cells towards the regions of high field strength (positive dielectrophoresis; PDEP) or towards regions of minimal field intensities (negative dielectrophoresis; NDEP). Since neurons require a physiological medium with a sufficient concentration of Na+, the medium conductivity is rather high (~ 1.6 S/m). The result is that negative dielectrophoretic forces are created over the entire frequency range. With the use of a planar quadrupole electrode sturcture negative forces are directed so that in the center of this structure cell can be collected. The process of trapping cortical rat neurons is described in Sect. 2 theoretically and experimentally. Medium and cell properties are frequency-dependent due to relaxation processes, which have a direct influence on the strength of the dielectrophorectic force. On the other hand, the nonideal material properties of the gold electrodes and glass substrate largely determine the electric field strength created inside the medium. Especially, the electrode-medium interface results in a significant loss of the imput signal at lower frequencies (< 1 MHz), and thus a reduction of the electric field strength inside the medium. Furthermore, due to the high medium conductivity, the electric field causes Joule heating. Local temperature rises result in local gradients in fluid density, which induces fluid flow. The electrode-medium interface and induced fluid flow are theoretically investigated with the use of modeling techniques such as finite elements modeling. Experimental and theoretical results agreed with each other on the occurrence of the effects described in this section. For the creation of the cultured neuron probe, preservation of cell viability during the trapping process is a prerequisite. Cell viability of dielectrophoretically trapped neurons has to be investigated. The membrane potential induced by the external field plays a crucial role in preservation of cell viability. The membrane can effectively be represented by a capaticance in parallel woth a low conductance; with increasing frequency and /or decreasing field strength the induced membrane potential decreases. At high induced membrane potentials ths representation for the membrane is no longer valid. At this point membrane breakdown occurs and the normally insulating membrane becomes conductive and permeable. The creation of electropores has been proposed in literature to be the cause of this high permeability state. Pores may grow or many small pores may be created which eventually may lead to membrane rupture, and thus cell death. Membrane breakdown may be reversible, but a chemical imbalance created during the high permeability state may still exist after the resealing of the membrane. This may cause cell death after several hours or even days after field application. Section 3 gives a detailed description of membrane breakdown. Since many investigations on electroporation of lipid bilayers and cell membranes are based on uniform electric fields, a finite element model is used to investigate induced membrane potentials in the nonuniform field created by the quadropole electrode structure. Modeling results are presented in cmbination with the results of breakdown experiments using four frequencies in the range from 100 kHz to 1MHz. Radomly positioned neuronals cells were exposed to stepwise increasing electric field strengths. The field strength at which membrane rupture occurred gives an indication of the maximum induced membrane potential. Due to the nonuniformity of the electric field, cell collapse was expected to be position-dependent. However, at 100 kHz cells collapsed at a break down level of about 0.4 V, in contradistinction to findings at higher frequencies where more variation in breakdown levels were found. Model simulations were able to explain the experimental results. For examining whether the neuronal cells trapped by dielectrophoresis were still viable after the trapping process, the frequency range was divided into two ranges. First, a high frequency (14 MHz) and a rather low signal amplitude (3 Vpp) were used to trap cells. At this high frequency the field-induced membrane potential is small according to the theoretical model, and therefore no real damage is expected. The experimental analysis included the investigation of the growth of the neurons, number and length of the processes (dendrites and axons), and the number of outgrowing (~ viable) versus nonoutgrowing (~ nonviable) neural cells. The experimental results agreed with the expectation. The effect of the use of driving signals with lower frequencies and/or higher amplitudes on cell viability was investigated using a staining method as described in the second part of Sect. 4. Survival chances are not directly linked to the estimated maximum induced membrane potential. The frequency of the dield plays an important role, decreasing frequency lowering the chance of survival. A lower frequency limit of 100 kHz is preferable at field strengths less than 80 k V/m, while with increasing field strength this limit shifts towards higher frequencies. The theoretical and experimental results presented in this review form the inception of the development of new electrode structures for trapping neuronal cells on top of each of the electrodes of the MEA. New ways to investigate cell properties and the phenomenon of electroporation using electrokinetic methods were developed that can be exploited in future research linking cell biology to technology.

Curr Opin Neurol. 2000 Aug;13(4):397-405.

Recent advances in amotrophic lateral sclerosis.

Al-Chalabi A, Leigh PN.

Department of Neurology, Guy’s King’s and St Thomas’ School of Medicine and Institute of Psychiatry, De Crespigny Park, London, UK.

The mechanisms by which mutations of the SOD1 gene cause selective motor neuron death remain uncertain, although interest continues to focus on the role of peroxynitrite, altered peroxidase activity of mutant SOD1, changes in intracellular copper homeostasis, protein aggregation, and changes in the function of glutamate transporters leading to excitotoxicity. Neurofilaments and peripherin appear to play some part in motor neuron degeneration, and amyotrophic lateral sclerosis is occasionally associated with mutations of the neurofilament heavy chain gene. Linkage to several chromosomal loci has been established for other forms of familial amyotrophic lateral sclerosis, but no new genes have been identified. In the clinical field, interest has been shown in the population incidence and prevalence of amyotrophic lateral sclerosis and the clinical variants that cause diagnostic confusion. Transcranial magnetic stimulation has been used to detect upper motor neuron damage and to explore cortical excitability in amyotrophic lateral sclerosis, and magnetic resonance imaging including proton magnetic resonance spectroscopy and diffusion weighted imaging also provide useful information on the upper motor neuron lesion. Aspects of care including assisted ventilation, nutrition, and patient autonomy are addressed, and underlying these themes is the requirement to measure quality of life with a new disease-specific instrument. Progress has been made in developing practice parameters. Riluzole remains the only drug to slow disease progression, although interventions such as non-invasive ventilation and gastrostomy also extend survival.

Int J Neurosci. 1994 Jun;76(3-4):185-225.

Alzheimer’s disease: improvement of visual memory and visuoconstructive performance by treatment with picotesla magnetic fields.

Sandyk R.

NeuroCommunication Research Laboratories, Danbury, CT 06811.

Impairments in visual memory and visuoconstructive functions commonly occur in patients with Alzheimer’s disease (AD). Recently, I reported that external application of electromagnetic fields (EMF) of extremely low intensity (in the picotesla range) and of low frequency (in the range of 5Hz-8Hz) improved visual memory and visuoperceptive functions in patients with Parkinson’s disease. Since a subgroup of Parkinsonian patients, specifically those with dementia, have coexisting pathological and clinical features of AD, I investigated in two AD patients the effects of these extremely weak EMF on visual memory and visuoconstructive performance. The Rey-Osterrieth Complex Figure Test as well as sequential drawings from memory of a house, a bicycle, and a man were employed to evaluate the effects of EMF on visual memory and visuoconstructive functions, respectively. In both patients treatment with EMF resulted in a dramatic improvement in visual memory and enhancement of visuoconstructive performance which was associated clinically with improvement in other cognitive functions such as short term memory, calculations, spatial orientation, judgement and reasoning as well as level of energy, social interactions, and mood. The report demonstrates, for the first time, that specific cognitive symptoms of AD are improved by treatment with EMF of a specific intensity and frequency. The rapid improvement in cognitive functions in response to EMF suggests that some of the mental deficits of AD are reversible being caused by a functional (i.e., synaptic transmission) rather than a structural (i.e., neuritic plaques) disruption of neuronal communication in the central nervous system.

Acupunct Electrother Res. 1992;17(2):107-48.

Common factors contributing to intractable pain and medical problems with insufficient drug intake in areas to be treated, and their pathogenesis and treatment: Part I. Combined use of medication with acupuncture, (+) Qi gong energy-stored material, soft laser or electrical stimulation.

Omura Y, Losco BM, Omura AK, Takeshige C, Hisamitsu T, Shimotsuura Y, Yamamoto S, Ishikawa H, Muteki T, Nakajima H, et al.

Heart Disease Research Foundation, New York.

Most frequently encountered causes of intractable pain and intractable medical problems, including headache, post-herpetic neuralgia, tinnitus with hearing difficulty, brachial essential hypertension, cephalic hypertension and hypotension, arrhythmia, stroke, osteo-arthritis, Minamata disease, Alzheimer’s disease and neuromuscular problems, such as Amyotrophic Lateral Sclerosis, and cancer are often found to be due to co-existence of 1) viral or bacterial infection, 2) localized microcirculatory disturbances, 3) localized deposits of heavy metals, such as lead or mercury, in affected areas of the body, 4) with or without additional harmful environmental electro-magnetic or electric fields from household electrical devices in close vicinity, which create microcirculatory disturbances and reduced acetylcholine. The main reason why medications known to be effective prove ineffective with intractable medical problems, the authors found, is that even effective medications often cannot reach these affected areas in sufficient therapeutic doses, even though the medications can reach the normal parts of the body and result in side effects when doses are excessive. These conditions are often difficult to treat or may be considered incurable in both Western and Oriental medicine. As solutions to these problems, the authors found some of the following methods can improve circulation and selectively enhance drug uptake: 1) Acupuncture, 2) Low pulse repetition rate electrical stimulation (1-2 pulses/second), 3) (+) Qi Gong energy, 4) Soft lasers using Ga-As diode laser or He-Ne gas laser, 5) Certain electro-magnetic fields or rapidly changing or moving electric or magnetic fields, 6) Heat or moxibustion, 7) Individually selected Calcium Channel Blockers, 8) Individually selected Oriental herb medicines known to reduce or eliminate circulatory disturbances. Each method has advantages and limitations and therefore the individually optimal method has to be selected. Applications of (+) Qi Gong energy stored paper or cloth every 4 hours, along with effective medications, were often found to be effective, as Qigongnized materials can often be used repeatedly, as long as they are not exposed to rapidly changing electric, magnetic or electro-magnetic fields. Application of (+) Qi Gong energy-stored paper or cloth, soft laser or changing electric field for 30-60 seconds on the area above the medulla oblongata, vertebral arteries or endocrine representation area at the tail of pancreas reduced or eliminated microcirculatory disturbances and enhanced drug uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

Int J Neurosci. 1991 Aug;59(4):259-62.

Age-related disruption of circadian rhythms: possible relationship to memory impairment and implications for therapy with magnetic fields.

Sandyk R, Anninos PA, Tsagas N.

Department of Psychiatry, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY 10461.

Disorganization of circadian rhythms, a hallmark of aging, may be related causally to the progressive deterioration of memory functions in senescence and possibly Alzheimer’s disease (AD). In experimental animals, disruption of circadian rhythms produces retrograde amnesia by interfering with the circadian organization of memory processes. The circadian system is known to be synchronized to external 24 h periodicities of ambient light by a neural pathway extending from the retina to the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. There is also evidence that the earth’s magnetic field is a time cue (“Zeitgeber”) of circadian organization and that shielding of the ambient magnetic field leads to disorganization of the circadian rhythms in humans. Since aging is associated with a delay of the circadian rhythm phase, and since light, which phase advances circadian rhythms, mimics the effects of magnetic fields on melatonin secretion, we postulate that application of magnetic fields might improve memory functions in the elderly as a result of resynchronization of the circadian rhythms. Moreover, since the circadian rhythm organization is more severely disrupted in patients with AD, it is possible that magnetic treatment might prove useful also in improving memory functions in these patients. If successful, application of magnetic fields might open new avenues in the management of memory disturbances in the elderly and possibly in AD.

Zh Nevropatol Psikhiatr Im S S Korsakova. 1990;90(7):108-12.

Regional cerebral angiodystonia in the practice of a neuropathologist and therapist.

[Article in Russian]

Pokalev GM, Raspopina LA.

Altogether 108 patients with regional cerebral angiodystonia were examined using rheoencephalography, measurements of temporal and venous pressure and functional tests (nitroglycerin and bicycle ergometry). Three variants of abnormalities connected with regional cerebral angiodystonia were distinguished: dysfunction of the inflow, derangement of the venous outflow, and initial functional venous hypertonia. The patients were treated with nonmedicamentous therapy (electroanalgesia, magnetotherapy, iontotherapy).

Rev Neurol. 2004 Feb 16-29;38(4):374-80.

Transcranial magnetic stimulation. Applications in cognitive neuroscience.

[Article in Spanish]

Calvo-Merino B, Haggard P.

Institute of Movement Neuroscience, University College, Londres, UK. b.calvo@ion.ucl.ac.uk

OBJECTIVE: In this review we trace some of the mayor developments in the use of transcranial magnetic stimulation (TMS) as a technique for the investigation of cognitive neuroscience. Technical aspects of the magnetic stimulation are also reviewed.

DEVELOPMENT: Among the many methods now available for studying activity of the human brain, magnetic stimulation is the only technique that allows us to interfere actively with human brain function. At the same time it provides a high degree of spatial and temporal resolution. Standard TMS applications (central motor conduction time, threshold and amplitude of motor evoked potentials) allow the evaluation of the motor conduction in the central nervous system and more complex TMS applications (paired pulse stimulation, silent period) permit study the mechanisms of diseases causing changes in the excitability of cortical areas. These techniques also allow investigation into motor disorder, epilepsy, cognitive function and psychiatric disorders.

CONCLUSIONS: Transcranial magnetic stimulation applications have an important place among the investigative tools to study cognitive functions and neurological and psychiatric disorders. Even so, despite the many published research and clinical studies, a systematic study about the possible diagnostic value and role in neurocognitive rehabilitation of TMS testing need to be realized to offer new possibilities of future applications.

Neuroreport. 2005 Nov 7;16(16):1849-1852.

Repetitive transcranial magnetic stimulation over the right dorsolateral prefrontal cortex affects strategic decision-making.

Wout MV, Kahn RS, Sanfey AG, Aleman A.

aDepartment of Psychonomics, Helmholtz Research Institute, University of Utrecht bDepartment of Psychiatry, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht cBCN NeuroImaging Center, Groningen, The Netherlands dDepartment of Psychology, University of Arizona, Tucson, Arizona, USA.

Although decision-making is typically seen as a rational process, emotions play a role in tasks that include unfairness. Recently, activation in the right dorsolateral prefrontal cortex during offers experienced as unfair in the Ultimatum Game was suggested to subserve goal maintenance in this task. This is restricted to correlational evidence, however, and it remains unclear whether the dorsolateral prefrontal cortex is crucial for strategic decision-making. The present study used repetitive transcranial magnetic stimulation in order to investigate the causal role of the dorsolateral prefrontal cortex in strategic decision-making in the Ultimatum Game. The results showed that repetitive transcranial magnetic stimulation over the right dorsolateral prefrontal cortex resulted in an altered decision-making strategy compared with sham stimulation. We conclude that the dorsolateral prefrontal cortex is causally implicated in strategic decision-making in healthy human study participants.

Trends Cogn Sci. 2005 Nov;9(11):503-5. Epub 2005 Sep 21.

Recharging cognition with DC brain polarization.

Wassermann EM, Grafman J.

Brain Stimulation Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

Electrical direct current (DC) has been applied to the human head throughout history for various reasons and with claims of behavioral effects and clinical benefits. This technique has recently been rediscovered and its effects validated with modern quantitative techniques and experimental designs. Despite the very weak current used, DC polarization applied to specific brain areas can alter verbal fluency, motor learning and perceptual thresholds, and can be used in conjunction with transcranial magnetic stimulation. Compact and safe, this old technique seems poised to allow major advances cognitive science and therapy.

J ECT. 2005 Jun;21(2):88-95.

Transcranial magnetic stimulation in persons younger than the age of 18.

Quintana H.

Department of Psychiatry, Division of Child and Adolescent Psychiatry, Louisiana State University Health Science Center, School of Medicine, New Orleans, Louisiana 70112-2822, USA. Hquint@lsuhsc.edu

OBJECTIVES: To review the use of transcranial magnetic stimulation (single-pulse TMS, paired TMS, and repetitive TMS [rTMS]) in persons younger than the age of 18 years. I discuss the technical differences, as well as the diagnostic, therapeutic, and psychiatric uses of TMS/rTMS in this age group.

METHODS: I evaluated English-language studies from 1993 to August 2004 on nonconvulsive single-pulse, paired, and rTMS that supported a possible role for the use of TMS in persons younger than 18. Articles reviewed were retrieved from the MEDLINE database and Clinical Scientific index.

RESULTS: The 48 studies reviewed involved a total of 1034 children ages 2 weeks to 18 years; 35 of the studies used single-pulse TMS (980 children), 3 studies used paired TMS (20 children), and 7 studies used rTMS (34 children). Three studies used both single and rTMS. However, the number of subjects involved was not reported.

CONCLUSIONS: Single-pulse TMS, paired TMS, and rTMS in persons younger than 18 has been used to examine the maturation/activity of the neurons of various central nervous system tracts, plasticity of neurons in epilepsy, other aspects of epilepsy, multiple sclerosis, myoclonus, transcallosal inhibition, and motor cortex functioning with no reported seizure risk. rTMS has been applied to psychiatric disorders such as ADHD, ADHD with Tourette’s, and depression. Adult studies support an antidepressant effect from repetitive TMS, but there is only one study that has been reported on 7 patients that used rTMS to the left dorsal prefrontal cortex on children/adolescents with depression (5 of the 7 subjects treated responded). Although there are limited studies using rTMS (in 34 children), these studies did not report significant adverse effects or seizures. Repetitive TMS safety, ethical, and neurotoxicity concerns also are discussed.

Biol Psychiatry. 2005 Jun 15;57(12):1597-600.

Transcranial magnetic stimulation-evoked cortical inhibition: a consistent marker of attention-deficit/hyperactivity disorder scores in tourette syndrome.

Gilbert DL, Sallee FR, Zhang J, Lipps TD, Wassermann EM.

Division of Neurology, Cincinnati Children’s Hospital Medical Center and University of Cincinnati, OH 45229-3039, USA. d.gilbert@cchmc.org

BACKGROUND: Prior case-control studies using Transcranial Magnetic Stimulation (TMS) to probe the neural inhibitory circuitry of Attention Deficit Hyperactivity Disorder (ADHD), Tourette Syndrome (TS), and Obsessive Compulsive Disorder (OCD), have yielded conflicting results. Using regression analysis in TS patients with tics, ADHD, and/or OCD symptoms, all ranging from none to severe, we previously found that TMS-evoked short interval intracortical inhibition (SICI) correlated inversely with ADHD scores. We sought to validate this observation.

METHODS: We used regression to estimate the consistency of the association between ADHD symptom scores and TMS-evoked SICI at two separate visits in 28 children and adults with TS.

RESULTS: ADHD scores correlated significantly and consistently with SICI, particularly in patients not taking dopamine receptor blockers (r=.60 and r=.58). Hyperactivity, not inattention, scores accounted for ADHD-related variance in SICI.

CONCLUSIONS: SICI reliably reflects the severity of hyperactivity in children and adults with TS.

Child Adolesc Psychiatr Clin N Am. 2005 Jan;14(1):1-19, v.

Emerging brain-based interventions for children and adolescents: overview and clinical perspective.

Hirshberg LM, Chiu S, Frazier JA.

The NeuroDevelopment Center, 260 West Exchange Street, Suite 302, Providence, RI 02903, USA. lhirshberg@neruodevelopmentcenter.com

Electroencephalogram biofeedback (EBF), repetitive transcranial magnetic stimulation (rTMS), and vagal nerve stimulation (VNS) are emerging interventions that attempt to directly impact brain function through neurostimulation and neurofeedback mechanisms. This article provides a brief overview of each of these techniques, summarizes the relevant research findings, and examines the implications of this research for practice standards based on the guidelines for recommending evidence based treatments as developed by the American Academy of Child and Adolescent Psychiatry for attention deficit hyperactivity disorder (ADHD). EBF meets the “Clinical Guidelines” standard for ADHD, seizure disorders, anxiety, depression, and traumatic brain injury. VNS meets this same standard for treatment of refractory epilepsy and meets the lower “Options” standard for several other disorders. rTMS meets the standard for “Clinical Guidelines” for bipolar disorder, unipolar disorder, and schizophrenia. Several conditions are discussed regarding the use of evidence based thinking related to these emerging interventions and future directions.

Curr Med Res Opin. 2003;19(2):125-30.

Repetitive transcranial magnetic stimulation (rTMS): new tool, new therapy and new hope for ADHD.

Acosta MT, Leon-Sarmiento FE.

Department of Neurology, Children’s National Medical Center, Washington, DC, USA.

Attention-deficit hyperactivity disorder (ADHD) is the most common developmental disorder that is associated with environmental and genetic factors. Neurobiological evidence suggests that fronto-striatum-cerebellum circuit abnormalities, mainly in the right hemisphere, are responsible for most of the disturbed sensorimotor integration; dopamine seems to be the main neurochemical alteration underlying these morphological abnormalities. Different conventional treatments have been employed on ADHD; however, repetitive transcranial magnetic stimulation (rTMS), a new and useful option for the clinical/research investigation of several neuropsychiatric disorders involving dopamine circuits, has yet to be considered as a therapeutic tool and possible drug-free option for ADHD. Here the authors explore the available evidence that makes this tool a rational therapeutic possibility for patients with ADHD, calling attention to safety issues, while highlighting the potentials of such an approach and the new hope it may bring for patients, parents, researchers and clinicians. The authors advocate carefully conducted clinical trials to investigate efficacy, safety, cost-effectiveness and clinical utility of rTMS for ADHD patients – in comparison to both placebo and standard treatments.

Clin Neurophysiol. 2003 Nov;114(11):2036-42.

Disturbed transcallosally mediated motor inhibition in children with attention deficit hyperactivity disorder (ADHD).

Buchmann J, Wolters A, Haessler F, Bohne S, Nordbeck R, Kunesch E.

Department of Child and Adolescence Neuropsychiatry, Centre of Nerve Disease, University of Rostock, Gehlsdorfer Strasse 20, 18147 Rostock, Germany.

OBJECTIVE: The aim of this study was to investigate mechanisms of motor-cortical excitability and inhibition which may contribute to motor hyperactivity in children with attention deficit hyperactivity disorder (ADHD).

METHODS: Using transcranial magnetic stimulation (TMS), involvement of the motor cortex and the corpus callosum was analysed in 13 children with ADHD and 13 sex- and age-matched controls. Contralateral silent period (cSP) and transcallosally mediated ipsilateral silent period (iSP) were investigated.

RESULTS: Resting motor threshold (RMT), amplitudes of motor evoked potentials (MEP) and cSP were similar in both groups whereas iSP-latencies were significantly longer (p<0.05) and their duration shorter (p<0.01) in the ADHD group. For the ADHD group iSP duration tended to increase and iSP latency to decrease with age (n.s.). Conners-Scores did neither correlate with iSP-latencies and -duration nor with children’s age.

CONCLUSIONS: The shortened duration of iSP in ADHD children could be explained by an imbalance of inhibitory and excitatory drive on the neuronal network between cortex layer III-the projection site of transcallosal motor-cortical fibers-and layer V, the origin of the pyramidal tract. The longer iSP-latencies might be the result of defective myelination of fast conducting transcallosal fibers in ADHD. iSP may be a useful supplementary diagnostic tool to discriminate between ADHD and normal children.

J Child Neurol. 2001 Dec;16(12):891-4.

Subjective reactions of children to single-pulse transcranial magnetic stimulation.

Garvey MA, Kaczynski KJ, Becker DA, Bartko JJ.

Pediatric Movement Disorders Unit, Pediatrics and Developmental Neuropsychiatry Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-1255, USA. garveym@intra.nimh.nih.gov

Single-pulse transcranial magnetic stimulation is a useful tool to investigate cortical function in childhood neuropsychiatric disorders. Magnetic stimulation is associated with a shock-like sensation that is considered painless in adults. Little is known about how children perceive the procedure. We used a self-report questionnaire to assess children’s subjective experience with transcranial magnetic stimulation. Normal children and children with attention-deficit hyperactivity disorder (ADHD) underwent transcranial magnetic stimulation in a study of cortical function in ADHD. Subjects were asked to rate transcranial magnetic stimulation on a 1 to 10 scale (most disagreeable = 1, most enjoyable = 10) and to rank it among common childhood events. Thirty-eight subjects completed transcranial magnetic stimulation; 34 said that they would repeat it. The overall rating for transcranial magnetic stimulation was 6.13, and transcranial magnetic stimulation was ranked fourth highest among the common childhood events. These results suggest that although a few children find transcranial magnetic stimulation uncomfortable, most consider transcranial magnetic stimulation painless. Further studies are necessary to confirm these findings.

Int J Neurosci. 1994 Jun;76(3-4):185-225.

Alzheimer’s disease: improvement of visual memory and visuoconstructive performance by treatment with picotesla range magnetic fields.

Sandyk R.

NeuroCommunication Research Laboratories, Danbury, CT 06811.

Impairments in visual memory and visuoconstructive functions commonly occur in patients with Alzheimer’s disease (AD). Recently, I reported that external application of electromagnetic fields (EMF) of extremely low intensity (in the picotesla range) and of low frequency (in the range of 5Hz-8Hz) improved visual memory and visuoperceptive functions in patients with Parkinson’s disease. Since a subgroup of Parkinsonian patients, specifically those with dementia, have coexisting pathological and clinical features of AD, I investigated in two AD patients the effects of these extremely weak EMF on visual memory and visuoconstructive performance. The Rey-Osterrieth Complex Figure Test as well as sequential drawings from memory of a house, a bicycle, and a man were employed to evaluate the effects of EMF on visual memory and visuoconstructive functions, respectively. In both patients treatment with EMF resulted in a dramatic improvement in visual memory and enhancement of visuoconstructive performance which was associated clinically with improvement in other cognitive functions such as short term memory, calculations, spatial orientation, judgement and reasoning as well as level of energy, social interactions, and mood. The report demonstrates, for the first time, that specific cognitive symptoms of AD are improved by treatment with EMF of a specific intensity and frequency. The rapid improvement in cognitive functions in response to EMF suggests that some of the mental deficits of AD are reversible being caused by a functional (i.e., synaptic transmission) rather than a structural (i.e., neuritic plaques) disruption of neuronal communication in the central nervous system.

Int J Neurosci. 1991 Aug;59(4):259-62.

Age-related disruption of circadian rhythms: possible relationship to memory impairment and implications for therapy with magnetic fields.

Sandyk R, Anninos PA, Tsagas N.

Department of Psychiatry, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY 10461.

Disorganization of circadian rhythms, a hallmark of aging, may be related causally to the progressive deterioration of memory functions in senescence and possibly Alzheimer’s disease (AD). In experimental animals, disruption of circadian rhythms produces retrograde amnesia by interfering with the circadian organization of memory processes. The circadian system is known to be synchronized to external 24 h periodicities of ambient light by a neural pathway extending from the retina to the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. There is also evidence that the earth’s magnetic field is a time cue (“Zeitgeber”) of circadian organization and that shielding of the ambient magnetic field leads to disorganization of the circadian rhythms in humans. Since aging is associated with a delay of the circadian rhythm phase, and since light, which phase advances circadian rhythms, mimics the effects of magnetic fields on melatonin secretion, we postulate that application of magnetic fields might improve memory functions in the elderly as a result of resynchronization of the circadian rhythms. Moreover, since the circadian rhythm organization is more severely disrupted in patients with AD, it is possible that magnetic treatment might prove useful also in improving memory functions in these patients. If successful, application of magnetic fields might open new avenues in the management of memory disturbances in the elderly and possibly in AD.

Clin EEG Neurosci. 2004 Jan;35(1):4-13.

Current status of the utilization of antileptic treatments in mood, anxiety and aggression: drugs and devices.

Barry JJ, Lembke A, Bullock KD.

Department of Psychiatry, Stanford University Medical Center, 401 Quarry Road MC 5723, Stanford, CA 94305, USA. jbarry@leland.stanford.edu

Interventions that have been utilized to control seizures in people with epilepsy have been employed by the psychiatric community to treat a variety of disorders. The purpose of this review will be to give an overview of the most prominent uses of antiepileptic drugs (AEDs) and devices like the Vagus Nerve Stimulator (VNS) and Transcranial Magnetic Stimulation (TMS) in the treatment of psychiatric disease states. By far, the most prevalent use of these interventions is in the treatment of mood disorders. AEDs have become a mainstay in the effective treatment of Bipolar Affective Disorder (BAD). The U.S. Food and Drug Administration has approved the use of valproic acid for acute mania, and lamotrigine for BAD maintenance therapy. AEDs are also effectively employed in the treatment of anxiety and aggressive disorders. Finally, VNS and TMS are emerging as possibly useful tools in the treatment of more refractory depressive illness.

Am J Psychiatry. 2004 Jan;161(1):93-8.

Low-field magnetic stimulation in bipolar depression using an MRI-based stimulator.

Rohan M, Parow A, Stoll AL, Demopulos C, Friedman S, Dager S, Hennen J, Cohen BM, Renshaw PF.

Brain Imaging Center, McLean Hospital, Belmont, MA 02478, USA. mrohan@mclean.harvard.edu

OBJECTIVE: Anecdotal reports have suggested mood improvement in patients with bipolar disorder immediately after they underwent an echo-planar magnetic resonance spectroscopic imaging (EP-MRSI) procedure that can be performed within clinical MR system limits. This study evaluated possible mood improvement associated with this procedure.

METHOD: The mood states of subjects in an ongoing EP-MRSI study of bipolar disorder were assessed by using the Brief Affect Scale, a structured mood rating scale, immediately before and after an EP-MRSI session. Sham EP-MRSI was administered to a comparison group of subjects with bipolar disorder, and actual EP-MRSI was administered to a comparison group of healthy subjects. The characteristics of the electric fields generated by the EP-MRSI scan were analyzed.

RESULTS: Mood improvement was reported by 23 of 30 bipolar disorder subjects who received the actual EP-MRSI examination, by three of 10 bipolar disorder subjects who received sham EP-MRSI, and by four of 14 healthy comparison subjects who received actual EP-MRSI. Significant differences in mood improvement were found between the bipolar disorder subjects who received actual EP-MRSI and those who received sham EP-MRSI, and, among subjects who received actual EP-MRSI, between the healthy subjects and the bipolar disorder subjects and to a lesser extent between the unmedicated bipolar disorder subjects and the bipolar disorder subjects who were taking medication. The electric fields generated by the EP-MRSI scan were smaller (0.7 V/m) than fields used in repetitive transcranial magnetic stimulation (rTMS) treatment of depression (1-500 V/m) and also extended uniformly throughout the head, unlike the highly nonuniform fields used in rTMS. The EP-MRSI waveform, a 1-kHz train of monophasic trapezoidal gradient pulses, differed from that used in rTMS.

CONCLUSIONS: These preliminary data suggest that the EP-MRSI scan induces electric fields that are associated with reported mood improvement in subjects with bipolar disorder. The findings are similar to those for rTMS depression treatments, although the waveform used in EP-MRSI differs from that used in rTMS. Further investigation of the mechanism of EP-MRSI is warranted.

Psychiatry Res. 2004 Sep 30;128(2):199-202.

Repetitive transcranial magnetic stimulation as an add-on therapy in the treatment of mania: a case series of eight patients.

Saba G, Rocamora JF, Kalalou K, Benadhira R, Plaze M, Lipski H, Januel D.

Unite de recherche clinique, secteur III de Ville Evrard, 5, Rue du Dr Delafontaine, Saint-Denis, 93200 France. urcve@free.fr

The aim of this study is to assess the efficacy of repetitive transcranial magnetic stimulation (rTMS) as an add-on therapy in the treatment of manic bipolar patients. Eight patients were enrolled in an open trial. They received fast rTMS (five trains of 15 s, 80% of the motor threshold, 10 Hz) over the right dorsolateral prefrontal cortex (DLPFC). They were evaluated using the Mania Assessment Scale (MAS) and the Clinical Global Impression (CGI) at baseline and at day 14. All patients were taking medication during the treatment trial. There was a significant improvement of manic symptoms at the end of the trial. No side effects were reported. The results show a significant improvement of mania when patients are treated with fast rTMS over the right DLPFC. However, these results have to be interpreted with caution since they derive from an open case series and all the subjects were taking psychotropic medication during rTMS treatment. Double-blind controlled studies with a sham comparison condition should be conducted to investigate the efficiency of this treatment in manic bipolar disorders.

J Affect Disord. 2004 Mar;78(3):253-7.

Treatment of bipolar mania with right prefrontal rapid transcranial magnetic stimulation.

Michael N, Erfurth A.

Mood Disorders Unit, Department of Psychiatry, University of Muenster, Albert-Schweitzer-Str. 11, 48129 Muenster, Germany.

BACKGROUND: Transcranial magnetic stimulation (TMS) has been suggested for the treatment of a variety of CNS disorders including depression and mania.

METHODS: Nine bipolar (I) in-patients diagnosed with mania were treated with right prefrontal rapid TMS in an open and prospective study. Eight of nine patients received TMS as add-on treatment to an insufficient or only partially effective drug therapy.

RESULTS: During the 4 weeks of TMS treatment a sustained reduction of manic symptoms as measured by the Bech-Rafaelsen mania scale (BRMAS) was observed in all patients.

LIMITATIONS: Due to the open and add-on design of the study, a clear causal relationship between TMS treatment and reduction of manic symptoms cannot be established.

CONCLUSIONS: Our data suggest that right prefrontal rapid TMS is safe and efficacious in the add-on treatment of bipolar mania showing laterality opposed to the proposed effect of rapid TMS in depression.

Bipolar Disord. 2003 Feb;5(1):40-7.

Left prefrontal transcranial magnetic stimulation (TMS) treatment of depression in bipolar affective disorder: a pilot study of acute safety and efficacy.

Nahas Z, Kozel FA, Li X, Anderson B, George MS.

Brain Stimulation Laboratory, Department of Psychiatry, Medical University of South Carolina, Charleston 29425, USA.

OBJECTIVES: Repetitive transcranial magnetic stimulation (rTMS) has been shown to improve depressive symptoms. We designed and carried out the following left prefrontal rTMS study to determine the safety, feasibility, and potential efficacy of using TMS to treat the depressive symptoms of bipolar affective disorder (BPAD).

METHODS: We recruited and enrolled 23 depressed BPAD patients (12 BPI depressed state, nine BPII depressed state, two BPI mixed state). Patients were randomly assigned to receive either daily left prefrontal rTMS (5 Hz, 110% motor threshold, 8 sec on, 22 sec off, over 20 min) or placebo each weekday morning for 2 weeks. Motor threshold and subjective rating scales were obtained daily, and blinded Hamilton Rating Scale for Depression (HRSD) and Young Mania Rating Scales (YMRS) were obtained weekly.

RESULTS: Stimulation was well tolerated with no significant adverse events and with no induction of mania. We failed to find a statistically significant difference between the two groups in the number of antidepressant responders (>50% decline in HRSD or HRSD <10 – 4 active and 4 sham) or the mean HRSD change from baseline over the 2 weeks (t = -0.22, p = 0.83). Active rTMS, compared with sham rTMS, produced a trend but not statistically significant greater improvement in daily subjective mood ratings post-treatment (t = 1.58, p = 0.13). The motor threshold did not significantly change after 2 weeks of active treatment (t = 1.11, p = 0.28).

CONCLUSIONS: Daily left prefrontal rTMS appears safe in depressed BPAD subjects, and the risk of inducing mania in BPAD subjects on medications is small. We failed to find statistically significant TMS clinical antidepressant effects greater than sham. Further studies are needed to fully investigate the potential role, if any, of TMS in BPAD depression.

CNS Drugs. 2002;16(1):47-63.

The Bech-Rafaelsen Mania Scale in clinical trials of therapies for bipolar disorder: a 20-year review of its use as an outcome measure.

Bech P.

Psychiatric Research Unit, WHO Collaborating Centre for Mental Health, Frederiksborg General Hospital, Hillerod, Denmark. pebe@fa.dk

Over the last two decades the Bech-Rafaelsen Mania Scale (MAS) has been used extensively in trials that have assessed the efficacy of treatments for bipolar disorder. The extent of its use makes it possible to evaluate the psychometric properties of the scale according to the principles of internal validity, reliability, and external validity. Studies of the internal validity of the MAS have demonstrated that the simple sum of the 11 items of the scale is a sufficient statistic for the assessment of the severity of manic states. Both factor analysis and latent structure analysis (the Rasch analysis) have been used to demonstrate this. The total score of the MAS has been standardised such that scores below 15 indicate hypomania, scores around 20 indicate moderate mania, and scores around 28 indicate severe mania. The inter-observer reliability has been found to be high in a number of studies conducted in various countries. The MAS has shown an acceptable external validity, in terms of both sensitivity and responsiveness. Thus, the MAS was found to be superior to the Clinical Global Impression scale with regard to responsiveness, and sensitivity has been found to be adequate, with the MAS able to demonstrate large drug-placebo differences. Based on pretreatment scores, trials of antimanic therapies can be classified into: (i) ultrashort (1 week) therapy of severe mania; (ii) short-term therapy (3 to 8 weeks) of moderate mania; (iii) short-term therapy of hypomanic or mixed bipolar states; and (iv) long-term (12 months) therapy of bipolar states. The responsiveness of MAS is such that the scale has been able to demonstrated that typical antipsychotics are effective as an ultrashort therapy of severe mania; that lithium and anticonvulsants are effective in the short-term therapy of moderate mania; and that atypical antipsychotics, electroconvulsive therapy (ECT) and transcranial magnetic stimulation seem to have promising effects in the short-term therapy of moderate mania. In contrast, the scale has been used to demonstrate that calcium antagonists (e.g. verapamil) are ineffective in the treatment of mania. MAS has also been used to add to the literature on the evidence-based effect of lithium as a short-term therapy for hypomania or mixed bipolar states and as a long-term therapy of bipolar states.

Altern Ther Health Med. 2006 Sep-Oct;12(5):42-9

Regenerative effects of pulsed magnetic field on injured peripheral nerves.

  • Mert T,
  • Gunay I,
  • Gocmen C,
  • Kaya M,
  • Polat S.

Department of Biophysics, University of Cukurova School of Medicine, Adana, Turkey.

Previous studies confirm that pulsed magnetic field (PMF) accelerates functional recovery after a nerve crush lesion. The contention that PMF enhances the regeneration is still controversial, however. The influence of a new PMF application protocol (trained PMF) on nerve regeneration was studied in a model of crush injury of the sciatic nerve of rats. To determine if exposure to PMF influences regeneration, we used electrophysiological recordings and ultrastructural examinations. After the measurements of conduction velocity, the sucrose-gap method was used to record compound action potentials (CAPs) from sciatic nerves. PMF treatment during the 38 days following the crush injury enhanced the regeneration. Although the axonal ultrastructures were generally normal, slight to moderate myelin sheath degeneration was noted at the lesion site. PMF application for 38 days accelerated nerve conduction velocity, increased CAP amplitude and decreased the time to peak of the CAP. Furthermore, corrective effects of PMF on. the abnormal characteristics of sensory nerve fibers were determined. Consequently, long-periodic trained-PMF may promote both morphological and electrophysiological properties of the injured nerves. In addition, corrective effects of PMF on sensory fibers may be considered an important finding for neuropathic pain therapy.

Bioelectromagnetics. 2005 Jan;26(1):20-7.

Pulsed electromagnetic fields induce peripheral nerve regeneration and endplate enzymatic changes.

De Pedro JA, Perez-Caballer AJ, Dominguez J, Collia F, Blanco J, Salvado M.

Department of Orthopaedics, University Hospital of Salamanca, Salamanca, Spain. jpedrom@usal.es

An experimental study was carried out in rats with the purpose of demonstrating the capacity of pulsed electromagnetic fields (PEMFs) to stimulate regeneration of the peripheral nervous system (PNS). Wistar and Brown Norway (BN) rats were used. Direct sciatic nerve anastomoses were performed after section or allograft interposition. Treatment groups then received 4 weeks of PEMFs. Control groups received no stimulation. The evaluation of the results was carried out by quantitative morphometric analysis, demonstrating a statistically significant increase in regeneration indices (P < 0.05) in the stimulated groups (9000 +/- 5000 and 4000 +/- 6000) compared to the non-stimulated groups (2000 +/- 4000 and 700 +/- 200). An increase of NAD specific isocitrate dehydrogenase (IDH) activity was found along with an increase in the activity of acetyl cholinesterase at the motor plate. The present study might lead to the search for new alternatives in the stimulation of axonal regenerative processes in the PNS and other possible clinical applications. 2004 Wiley-Liss, Inc.

Spine. 2003 Dec 15;28(24):2660-6.

Exposure to pulsed magnetic field enhances motor recovery in cats after spinal cord injury.

Crowe MJ, Sun ZP, Battocletti JH, Macias MY, Pintar FA, Maiman DJ.

Neuroscience Research Laboratories, The Clement J. Zablocki VA Medical Center, Milwaukee, WI 53295, USA. mcrowe@mcw.edu

STUDY DESIGN: Animal model study of eight healthy commercial cats was conducted.

OBJECTIVE: To determine whether pulsed electromagnetic field (PMF) stimulation results in improvement of function after contusive spinal cord injury in cats. SUMMARY OF

BACKGROUND DATA: PMF stimulation has been shown to enhance nerve growth, regeneration, and functional recovery of peripheral nerves. Little research has been performed examining the effects of PMF stimulation on the central nervous system and no studies of PMF effects on in vivo spinal cord injury (SCI) models have been reported.

MATERIALS AND METHODS: PMF stimulation was noninvasively applied for up to 12 weeks to the midthoracic spine of cats with acute contusive spinal cord injury. The injury was produced using a weight-drop apparatus. Motor functions were evaluated with the modified Tarlov assessment scale. Morphologic analyses of the injury sites and somatosensory-evoked potential measurements were conducted to compare results between PMF-stimulated and control groups.

RESULTS: There was a significant difference in locomotor recovery between the PMF-stimulated and control groups. Although not statistically significant, PMF-stimulated spinal cords demonstrated greater sparing of peripheral white matter and smaller lesion volumes compared to controls. Somatosensory-evoked potential measurements indicated that the PMF-stimulated group had better recovery of preinjury waveforms than the control group; however, this observation also was not statistically significant because of the small sample size.

CONCLUSIONS: This preliminary study indicates that pulsed magnetic fields may have beneficial effects on motor function recovery and lesion volume size after acute spinal cord injury.

J Neurosci Res. 1999 Jan 15;55(2):230-7.

Electromagnetic fields influence NGF activity and levels following sciatic nerve transection.

Longo FM, Yang T, Hamilton S, Hyde JF, Walker J, Jennes L, Stach R, Sisken BF.

Department of Neurology, UCSF/VAMC, San Francisco, California, USA. LFM@itsa.UCSF.edu

Pulsed electromagnetic fields (PEMF) have been shown to increase the rate of nerve regeneration. Transient post-transection loss of target-derived nerve growth factor (NGF) is one mechanism proposed to signal induction of early nerve regenerative events. We tested the hypothesis that PEMF alter levels of NGF activity and protein in injured nerve and/or dorsal root ganglia (DRG) during the first stages of regeneration (6-72 hr). Rats with a transection injury to the midthigh portion of the sciatic nerve on one side were exposed to PEMF or sham control PEMF for 4 hr/day for different time periods. NGF-like activity was determined in DRG, in 5-mm nerve segments proximal and distal to the transection site and in a corresponding 5-mm segment of the contralateral nonoperated nerve. NGF-like activity of coded tissue samples was measured in a blinded fashion using the chick DRG sensory neuron bioassay. Overall, PEMF caused a significant decrease in NGF-like activity in nerve tissue (P < 0.02, repeated measures analysis of variance, ANOVA) with decreases evident in proximal, distal, and contralateral nonoperated nerve. Unexpectedly, transection was also found to cause a significant (P=0.001) 2-fold increase in DRG NGF-like activity between 6 and 24 hr postinjury in contralateral but not ipsilateral DRG. PEMF also reduced NGF-like activity in DRG, although this decrease did not reach statistical significance. Assessment of the same nerve and DRG samples using ELISA and NGF-specific antibodies confirmed an overall significant (P < 0.001) decrease in NGF levels in PEMF-treated nerve tissue, while no decrease was detected in DRG or in nerve samples harvested from PEMF-treated uninjured rats. These findings demonstrate that PEMF can affect growth factor activity and levels, and raise the possibility that PEMF might promote nerve regeneration by amplifying the early postinjury decline in NGF activity.

Neurosci Behav Physiol. 1998 Sep-Oct;28(5):594-7.

Magnetic and electrical stimulation in the rehabilitative treatment of patients with organic lesions of the nervous system.

Tyshkevich TG, Nikitina VV.

A. L. Polenov Russian Science Research Neurosurgical Institute, St. Petersburg.

Studies were performed on 89 patients with organic lesions of the nervous system in which the leading clinical symptoms consisted of paralysis and pareses. Patients received complex treatment, including pulsed magnetic fields and an electrical stimulation regime producing multilevel stimulation. A control group of 49 patients with similar conditions was included, and these patients received only sinusoidal currents. Combined treatment with magnetic and electrical stimulation was more effective, as indicated by radiographic and electromyographic investigations.

Arch Otolaryngol Head Neck Surg. 1998 Apr;124(4):383-9.

Effect of pulsed electromagnetic stimulation on facial nerve regeneration.

Byers JM, Clark KF, Thompson GC.

Department of Otorhinolaryngology, University of Oklahoma Health Sciences Center, Oklahoma City, USA.

OBJECTIVE: To determine if exposure to electromagnetic fields influences regeneration of the transected facial nerve in the rat.

DESIGN AND METHODS: The left facial nerve was transected in the tympanic section of the fallopian canal in 24 rats randomly assigned to 2 groups. The cut ends of the facial nerve were reapproximated without sutures within the fallopian canal to maximize the potential for regeneration. Rats in the experimental group (n= 12) were then exposed to pulsed electromagnetic stimulation (0.4 millitesla at 120 Hz) for 4 hours per day, 5 days per week, for 8 weeks. Rats in the control group (n=12) were handled in an identical manner without pulsed electromagnetic stimulation. Four other rats were given sham operations in which all surgical procedures were carried out except for the actual nerve transection. Two of these rats were placed in each group. Nerve regeneration was evaluated using electroneurography (compound action potentials), force of whisker and eyelid movements, and voluntary facial movements before and at 2-week intervals after transection. Histological evaluation was performed at 10 weeks after transection. Each dependent variable was analyzed using a 2-way analysis of variance with 1 between variable (groups) and 1 within repeated measures variable (days after transection).

RESULTS: Statistical analysis indicated that N1 (the negative deflection of depolarization phase of the muscle and/or nerve fibers) area, N1 amplitude, and N1 duration, as well as absolute amplitude of the compound action potentials, were all significantly greater 2 weeks after transection in the experimental than in the control group of rats. The force of eye and whisker movements after electrical stimulation was statistically greater in the experimental group of rats 4 weeks after transection. Voluntary eye movements in the experimental group were significantly better at 5 and 10 weeks, while whisker movements were better at 3 and 10 weeks. There was no statistical difference between the 2 groups for any histological variable.

CONCLUSION: Results of this study indicate that pulsed electromagnetic stimulation enhances early regeneration of the transected facial nerve in rats.

J Cell Biochem. 1993 Apr;51(4):387-93.

Beneficial effects of electromagnetic fields.

Bassett CA.

Bioelectric Research Center, Columbia University, Riverdale, New York 10463.

Selective control of cell function by applying specifically configured, weak, time-varying magnetic fields has added a new, exciting dimension to biology and medicine. Field parameters for therapeutic, pulsed electromagnetic field (PEMFs) were designed to induce voltages similar to those produced, normally, during dynamic mechanical deformation of connective tissues. As a result, a wide variety of challenging musculoskeletal disorders have been treated successfully over the past two decades. More than a quarter million patients with chronically ununited fractures have benefitted, worldwide, from this surgically non-invasive method, without risk, discomfort, or the high costs of operative repair. Many of the athermal bioresponses, at the cellular and subcellular levels, have been identified and found appropriate to correct or modify the pathologic processes for which PEMFs have been used. Not only is efficacy supported by these basic studies but by a number of double-blind trials. As understanding of mechanisms expands, specific requirements for field energetics are being defined and the range of treatable ills broadened. These include nerve regeneration, wound healing, graft behavior, diabetes, and myocardial and cerebral ischemia (heart attack and stroke), among other conditions. Preliminary data even suggest possible benefits in controlling malignancy.

Bioelectromagnetics. 1993;14(4):353-9.

Pretreatment of rats with pulsed electromagnetic field enhances regeneration of the sciatic nerve.

Kanje M, Rusovan A, Sisken B, Lundborg G.

Department of Animal Physiology, University of Lund, Sweden.

Regeneration of the sciatic nerve was studied in rats pretreated in a pulsed electromagnetic field (PEMF). The rats were exposed between a pair of Helmholtz coils at a pulse repetition rate of 2 pps at a field density of 60 or 300 microT. The PEMF treatment was then discontinued. After an interval of recovery, regeneration of the sciatic nerve was initiated by a crush lesion. Regeneration of sensory fibers was measured by the “pinch test” after an additional 3-6 days. A variety of PEMF pretreatments including 4 h/day for 1-4 days or exposure for 15 min/day during 2 days resulted in an increased regeneration distance, measured 3 days after the crush lesion. This effect could be demonstrated even after a 14-day recovery period. In contrast, pretreatment for 4 h/day for 2 days at 60 microT did not affect the regeneration distance. The results showed that PEMF pretreatment conditioned the rat sciatic nerve in a manner similar to that which occurs after a crush lesion, which indicates that PEMF affects the neuronal cell body. However, the mechanism of this effect remains obscure.

Brain Res. 1989 Apr 24;485(2):309-16.

Stimulation of rat sciatic nerve regeneration with pulsed electromagnetic fields.

Sisken BF, Kanje M, Lundborg G, Herbst E, Kurtz W.

Center for Biomedical Engineering, University of Kentucky, Lexington 40506.

The effects of pulsed electromagnetic fields (PEMF) on rat sciatic nerve regeneration after a crush lesion were determined. The rats were placed between a pair of Helmholtz coils and exposed to PEMF of frequency 2 Hz and magnetic flux density of 0.3 mT. A 4 h/day treatment for 3-6 days increased the rate of nerve regeneration by 22%. This stimulatory effect was independent of the orientation of the coils. Exposure times of 1 h/day-10 h/day were equally effective in stimulating nerve regeneration. Rats exposed to PEMF for 4 h/day for 7 days before crush, followed by 3 days after crush without PEMF, also showed significantly increased regeneration. This pre-exposure ‘conditioning’ effect suggests that PEMF influences regeneration indirectly.

J Hand Surg [Br]. 1984 Jun;9(2):105-12.

An experimental study of the effects of pulsed electromagnetic field (Diapulse) on nerve repair.

Raji AM.

This study investigates the effects of a pulsed electromagnetic field (PEMF) (Diapulse) on experimentally divided and sutured common peroneal nerves in rats. Evidence is presented to show that PEMF accelerates recovery of use of the injured limb and enhances regeneration of damaged nerves.

Clin Orthop Relat Res. 1983 Dec;(181):283-90.

Effect of weak, pulsing electromagnetic fields on neural regeneration in the rat.

Ito H, Bassett CA.

The short- and long-term effects of pulsed electromagnetic fields (PEMFs) on the rate and quality of peripheral nerve regeneration were studied. High bilateral transections of rat sciatic nerves were surgically approximated (a 1-mm gap was left) and shielded with a Silastic sleeve. Animals were exposed to PEMFs for two to 14 weeks after operation. Three groups of 20 rats each (control rats and rats undergoing 12- and 24-hour/day PEMF exposure) were killed at two weeks. Histologically, regenerating axons had penetrated the distal stump nearly twice as far in the PEMF-exposed animals as in the control animals. Return of motor function was judged two to 14 weeks after operation by the load cell-measured, plantar-flexion force produced by neural stimulation proximal to the transection site. Motor function returned earlier in experimental rats and to significantly higher load levels than in control rats. Nerves from animals functioning 12-14 weeks after operation had less interaxonal collagen, more fiber-containing axis cylinders, and larger fiber diameters in the PEMF-exposed group than in the control rats. Histologic and functional data indicate that PEMFs improve the rate and quality of peripheral nerve regeneration in the severed rat sciatic nerve by a factor of approximately two.

Paraplegia. 1976 May;14(1):12-20.

Experimental regeneration in peripheral nerves and the spinal cord in laboratory animals exposed to a pulsed electromagnetic field.

Wilson DH, Jagadeesh P.

Peripheral nerve section and suture was performed in 132 rats. Postoperatively half the animals were exposed to a pulsed electromagnetic field each day and half were kept as controls. Nerve conduction studies, histology and nerve fibre counts all indicated an increased rate of regeneration in the treated animals. A similar controlled study of spinal cord regeneration following hemicordotomy in cats has been started, and preliminary results indicate that when the animals are sacrificed three months after the hemicordotomy, the pulsed electromagnetic therapy has induced nerve fibre regeneration across the region of the scar.

Altern Ther Health Med. 2006 Sep-Oct;12(5):42-9

Regenerative effects of pulsed magnetic field on injured peripheral nerves.

Mert T, Gunay I, Gocmen C, Kaya M, Polat S.

Department of Biophysics, University of Cukurova School of Medicine, Adana, Turkey.

Previous studies confirm that pulsed magnetic field (PMF) accelerates functional recovery after a nerve crush lesion. The contention that PMF enhances the regeneration is still controversial, however. The influence of a new PMF application protocol (trained PMF) on nerve regeneration was studied in a model of crush injury of the sciatic nerve of rats. To determine if exposure to PMF influences regeneration, we used electrophysiological recordings and ultrastructural examinations. After the measurements of conduction velocity, the sucrose-gap method was used to record compound action potentials (CAPs) from sciatic nerves. PMF treatment during the 38 days following the crush injury enhanced the regeneration. Although the axonal ultrastructures were generally normal, slight to moderate myelin sheath degeneration was noted at the lesion site. PMF application for 38 days accelerated nerve conduction velocity, increased CAP amplitude and decreased the time to peak of the CAP. Furthermore, corrective effects of PMF on. the abnormal characteristics of sensory nerve fibers were determined. Consequently, long-periodic trained-PMF may promote both morphological and electrophysiological properties of the injured nerves. In addition, corrective effects of PMF on sensory fibers may be considered an important finding for neuropathic pain therapy.

Neurorehabil Neural Repair. 2004 Mar;18(1):42-6.

Pulsed magnetic field therapy in refractory neuropathic pain secondary to peripheral neuropathy: electrodiagnostic parameters–pilot study.

Weintraub MI, Cole SP.

New York Medical College, Briarcliff Manor, New York 10510, USA.

CONTEXT: Neuropathic pain (NP) from peripheral neuropathy (PN) arises from ectopic firing of unmyelinated C-fibers with accumulation of sodium and calcium channels. Because pulsed electromagnetic fields (PEMF) safely induce extremely low frequency (ELF) quasirectangular currents that can depolarize, repolarize, and hyperpolarize neurons, it was hypothesized that directing this energy into the sole of one foot could potentially modulate neuropathic pain.

OBJECTIVE: To determine if 9 consecutive 1-h treatments in physician’s office (excluding weekends) of a pulsed signal therapy can reduce NP scores in refractory feet with PN.

DESIGN/SETTING/PATIENTS: 24 consecutive patients with refractory and symptomatic PN from diabetes, chronic inflammatory demyelinating polyneuropathy (CIDP), pernicious anemia, mercury poisoning, paraneoplastic syndrome, tarsal tunnel, and idiopathic sensory neuropathy were enrolled in this nonplacebo pilot study. The most symptomatic foot received therapy. Primary endpoints were comparison of VAS scores at the end of 9 days and the end of 30 days follow-up compared to baseline pain scores. Additionally, Patients’ Global Impression of Change (PGIC) questionnaire was tabulated describing response to treatment. Subgroup analysis of nerve conduction scores, quantified sensory testing (QST), and serial examination changes were also tabulated. Subgroup classification of pain (Serlin) was utilized to determine if there were disproportionate responses.

INTERVENTION: Noninvasive pulsed signal therapy generates a unidirectional quasirectangular waveform with strength about 20 gauss and a frequency about 30 Hz into the soles of the feet for 9 consecutive 1-h treatments (excluding weekends). The most symptomatic foot of each patient was treated.

RESULTS: All 24 feet completed 9 days of treatment. 15/24 completed follow-up (62%) with mean pain scores decreasing 21% from baseline to end of treatment (P=0.19) but with 49% reduction of pain scores from baseline to end of follow-up (P<0.01). Of this group, self-reported PGIC was improved 67% (n=10) and no change was 33% (n=5). An intent-to-treat analysis based on all 24 feet demonstrated a 19% reduction in pain scores from baseline to end of treatment (P=0.10) and a 37% decrease from baseline to end of follow-up (P<0.01). Subgroup analysis revealed 5 patients with mild pain with nonsignificant reduction at end of follow-up. Of the 19 feet with moderate to severe pain, there was a 28% reduction from baseline to end of treatment (P<0.05) and a 39% decrease from baseline to end of follow-up (P<0.01). Benefit was better in those patients with axonal changes and advanced CPT baseline scores. The clinical examination did not change. There were no adverse events or safety issues.

CONCLUSIONS: These pilot data demonstrate that directing PEMF to refractory feet can provide unexpected short term analgesic effects in more than 50% of individuals. The role of placebo is not known and was not tested. The precise mechanism is unclear yet suggests that severe and advanced cases are more magnetically sensitive. Future studies are needed with randomized placebo-controlled design and longer treatment periods.

Arch Phys Med Rehabil. 2003 May;84(5):736-46.

Static magnetic field therapy for symptomatic diabetic neuropathy: a randomized double-blind, placebo-controlled trial.

Weintraub MI, Wolfe GI, Barohn RA, Cole SP, Parry GJ, Hayat G, Cohen JA, Page JC, Bromberg MB, Schwartz SL; Magnetic Research Group.

Department of Neurology, New York Medical College, Valhalla, NY, USA. miwneuro@pol.net

OBJECTIVE: To determine if constant wearing of multipolar, static magnetic (450G) shoe insoles can reduce neuropathic pain and quality of life (QOL) scores in symptomatic diabetic peripheral neuropathy (DPN).

DESIGN: Randomized, placebo-control, parallel study.

SETTING: Forty-eight centers in 27 states.

PARTICIPANTS: Three hundred seventy-five subjects with DPN stage II or III were randomly assigned to wear constantly magnetized insoles for 4 months; the placebo group wore similar, unmagnetized device.

INTERVENTION: Nerve conduction and/or quantified sensory testing were performed serially.

MAIN OUTCOME MEASURES: Daily visual analog scale scores for numbness or tingling and burning and QOL issues were tabulated over 4 months. Secondary measures included nerve conduction changes, role of placebo, and safety issues. Analysis of variance (ANOVA), analysis of covariance (ANCOVA), and chi-square analysis were performed.

RESULTS: There were statistically significant reductions during the third and fourth months in burning (mean change for magnet treatment, -12%; for sham, -3%; P<.05, ANCOVA), numbness and tingling (magnet, -10%; sham, +1%; P<.05, ANCOVA), and exercise-induced foot pain (magnet, -12%; sham, -4%; P<.05, ANCOVA). For a subset of patients with baseline severe pain, statistically significant reductions occurred from baseline through the fourth month in numbness and tingling (magnet, -32%; sham, -14%; P<.01, ANOVA) and foot pain (magnet, -41%; sham, -21%; P<.01, ANOVA).

CONCLUSIONS: Static magnetic fields can penetrate up to 20mm and appear to target the ectopic firing nociceptors in the epidermis and dermis. Analgesic benefits were achieved over time.

Neurosci Behav Physiol. 2003 Oct;33(8):745-52.

The use of pulsed electromagnetic fields with complex modulation in the treatment of patients with diabetic polyneuropathy.

Musaev AV, Guseinova SG, Imamverdieva SS.

Science Research Institute of Medical Rehabilitation, Baku, Azerbaidzhan.

Clinical and electroneuromyographic studies were performed in 121 patients with diabetic polyneuropathy (DPN) before and after courses of treatment with pulsed electromagnetic fields with complex modulation (PEMF-CM) at different frequencies (100 and 10 Hz). Testing of patients using the TSS and NIS LL scales demonstrated a correlation between the severity and frequency of the main subjective and objective effects of disease and the stage of DPN. The severity of changes in the segmental-peripheral neuromotor apparatus–decreases in muscle bioelectrical activity, the impulse conduction rate along efferent fibers of peripheral nerves, and the amplitude of the maximum M response–depended on the stage of DPN and the duration of diabetes mellitus. The earliest and most significant electroneuromyographic signs of DPN were found to be decreases in the amplitude of the H reflex and the Hmax/Mmax ratio in the muscles of the lower leg. Application of PEMF-CM facilitated regression of the main clinical symptoms of DPN, improved the conductive function of peripheral nerves, improved the state of la afferents, and improved the reflex excitability of functionally diverse motoneurons in the spinal cord. PEMF-CM at 10 Hz was found to have therapeutic efficacy, especially in the initial stages of DPN and in patients with diabetes mellitus for up to 10 years.

Vopr Kurortol Fizioter Lech Fiz Kult. 1993 Sep-Oct;(5):38-41.

The use of combined methods of magnetoelectrotherapy in treating polyneuropathies.

[Article in Russian]

A comparative evaluation by such parameters as alleviation of pain syndrome, improvement of peripheral resistance and vegetotrophic processes, a decline in pareses and sensory disorders has been performed in 3 groups of patients: group 1 underwent benzohexonium electrophoresis, group 2 benzohexonium electrophoresis in the magnetic field produced by the unit “Polyus-I” followed by low-frequency electrotherapy with bipolar impulse current, group 3 benzohexonium electrophoresis in the magnetic field from the unit “ADMT-Magnipuls” followed by low-frequency electrotherapy with bipolar impulse current. The best clinical and physiological results were reported in group 3 patients.

Wiad Lek. 2003;56(9-10):434-41.

Application of variable magnetic fields in medicine–15 years experience.

[Article in Polish]

Sieron A, Cieslar G.

Katedra i Klinika Chorob Wewnetrznych, Angiologii i Medycyny Fizykalnej SAM, ul. Batorego 15, 41-902 Bytom. sieron@mediclub.pl

The results of 15-year own experimental and clinical research on application of variable magnetic fields in medicine were presented. In experimental studies analgesic effect (related to endogenous opioid system and nitrogen oxide activity) and regenerative effect of variable magnetic fields with therapeutical parameters was observed. The influence of this fields on enzymatic and hormonal activity, free oxygen radicals, carbohydrates, protein and lipid metabolism, dielectric and rheological properties of blood as well as behavioural reactions and activity of central dopamine receptor in experimental animals was proved. In clinical studies high therapeutic efficacy of magnetotherapy and magnetostimulation in the treatment of osteoarthrosis, abnormal ossification, osteoporosis, nasosinusitis, multiple sclerosis, Parkinson’s disease, spastic paresis, diabetic polyneuropathy and retinopathy, vegetative neurosis, peptic ulcers, colon irritable and trophic ulcers was confirmed.

Klin Med (Mosk). 1996;74(5):39-41.

Magentotherapy in the comprehensive treatment of vascular complications of diabetes mellitus.

[Article in Russian]

Kirillov IB, Suchkova ZV, Lastushkin AV, Sigaev AA, Nekhaeva TI.

320 diabetes mellitus (DM) patients were exposed to impulsed magnetic field, 100 control DM patients received conservative therapy alone. 270 patients had microangiopathy, macroangiopathy was diagnosed in 50 patients. Good and satisfactory results of magnetotherapy in combination with conservative methods were achieved in 74% of patients versus 28% in control group. Metabolism stabilization resulted in some patients in reduced blood sugar. Use of magnetic field produced faster and longer response than conservative therapy.

Vestn Oftalmol. 1990 Sep-Oct;106(5):54-7.

Effectiveness of magnetotherapy in optic nerve atrophy.  A preliminary study.

[Article in Russian]

Zobina LV, Orlovskaia LS, Sokov SL, Sabaeva GF, Konde LA, Iakovlev AA.

Magnetotherapy effects on visual functions (vision acuity and field), on retinal bioelectric activity, on conductive vision system, and on intraocular circulation were studied in 88 patients (160 eyes) with optic nerve atrophy. A Soviet Polyus-1 low-frequency magnetotherapy apparatus was employed with magnetic induction of about 10 mT, exposure 7-10 min, 10-15 sessions per course. Vision acuity of patients with its low (below 0.04 diopters) values improved in 50 percent of cases. The number of patients with vision acuity of 0.2 diopters has increased from 46 before treatment to 75. Magnetotherapy improved ocular hemodynamics in patients with optic nerve atrophy, it reduced the time of stimulation conduction along the vision routes and stimulated the retinal ganglia cells. The maximal effect was achieved after 10 magnetotherapy sessions. A repeated course carried out in 6-8 months promoted a stabilization of the process.

Int J Neurosci. 1998 Apr;93(3-4):239-50.

Treatment with AC pulsed electromagnetic fields normalizes the latency of the visual evoked response in a multiple sclerosis patient with optic atrophy.

Sandyk R.

Department of Neuroscience at the Institute for Biomedical Engineering and Rehabilitation Services of Touro College, Dix Hills, NY 11746, USA.

Visual evoked response (VER) studies have been utilized as supportive information for the diagnosis of multiple sclerosis (MS) and may be useful in objectively monitoring the effects of various therapeutic modalities. Delayed latency of the VER, which reflects slowed impulse transmission in the optic pathways, is the most characteristic abnormality associated with the disease. Brief transcranial applications of AC pulsed electromagnetic fields (EMFs) in the picotesla flux density are efficacious in the symptomatic treatment of MS and may also reestablish impulse transmission in the optic pathways. A 36 year old man developed an attack of right sided optic neuritis at the age of 30. On presentation he had blurring of vision with reduced acuity on the right and fundoscopic examination revealed pallor of the optic disc. A checkerboard pattern reversal VER showed a delayed latency to right eye stimulation (P100 = 132 ms; normal range: 95-115 ms). After he received two successive applications of AC pulsed EMFs of 7.5 picotesla flux density each of 20 minutes duration administered transcranially, there was a dramatic improvement in vision and the VER latency reverted to normal (P100= 107 ms). The rapid improvement in vision coupled with the normalization of the VER latency despite the presence of optic atrophy, which reflects chronic demyelination of the optic nerve, cannot be explained on the basis of partial or full reformation of myelin. It is proposed that in MS synaptic neurotransmitter deficiency is associated with the visual impairment and delayed VER latency following optic neuritis and that the recovery of the VER latency by treatment with pulsed EMFs is related to enhancement of synaptic neurotransmitter functions in the retina and central optic pathways. Recovery of the VER latency in MS patients may have important implications with respect to the treatment of visual impairment and prevention of visual loss. Specifically, repeated pulsed applications of EMFs may maintain impulse transmission in the optic nerve and thus potentially sustain its viability.

Altern Ther Health Med. 2003 Jul-Aug;9(4):38-48.

Effects of a pulsed electromagnetic therapy on multiple sclerosis fatigue and quality of life: a double-blind, placebo-controlled trial.

Lappin MS, Lawrie FW, Richards TL, Kramer ED.

Energy Medicine Developments, (North America), Inc., Burke, Va., USA.

CONTEXT: There is a growing literature on the biological and clinical effects of pulsed electromagnetic fields. Some studies suggest that electromagnetic therapies may be useful in the treatment of chronic illnesses. This study is a follow-up to a placebo controlled pilot study in which multiple sclerosis (MS) patients exposed to weak, extremely low frequency pulsed electromagnetic fields showed significant improvements on a composite symptom measure.

OBJECTIVE: To evaluate the effects of a pulsed electromagnetic therapy on MS related fatigue, spasticity, bladder control, and overall quality of life.

DESIGN: A multi-site, double-blind, placebo controlled, crossover trial. Each subject received 4 weeks of the active and placebo treatments separated by a 2-week washout period. SETTING: The University of Washington Medical Center in Seattle Wash, the Neurology Center of Fairfax in Fairfax, Va, and the headquarters of the Multiple Sclerosis Association of America in Cherry Hill, NJ.

SUBJECTS: 117 patients with clinically definite MS.

INTERVENTION: Daily exposure to a small, portable pulsing electromagnetic field generator.

MAIN OUTCOME: The MS Quality of Life Inventory (MSQLI) was used to assess changes in fatigue, bladder control, spasticity, and a quality of life composite.

RESULTS: Paired t-tests were used to assess treatment differences in the 117 subjects (81% of the initial sample) who completed both treatment sessions. Improvements in fatigue and overall quality of life were significantly greater on the active device. There were no treatment effects for bladder control and a disability composite, and mixed results for spasticity.

CONCLUSIONS: Evidence from this randomized, double-bind, placebo controlled trial is consistent with results from smaller studies suggesting that exposure to pulsing, weak electromagnetic fields can alleviate symptoms of MS. The clinical effects were small, however, and need to be replicated. Additional research is also needed to examine the possibility that ambulatory patients and patients taking interferons for their MS may be most responsive to this kind of treatment.

Phys Med Rehabil Clin N Am. 1998 Aug;9(3):659-74.

Bioelectromagnetic applications for multiple sclerosis.

Richards TL, Lappin MS, Lawrie FW, Stegbauer KC.

Department of Radiology, University of Washington, Seattle, USA.

There are EM effects on biology that are potentially both harmful and beneficial. We have reviewed applications of EM fields that are relevant to MS. It is possible that EM fields could be developed into a reproducible therapy for both symptom management and long-term care for MS. The long-term care for MS would have to include beneficial changes in the immune system and in nerve regeneration.

Mult Scler. 2005 Jun;11(3):302-5.

Effect of pulsed magnetic field therapy on the level of fatigue in patients with multiple sclerosis–a randomized controlled trial.

Mostert S, Kesselring J.

Department of Neurology, Rehabilitation Centre, CH 7317 Valens, Switzerland.

Twenty-five multiple sclerosis patients, taking part in a rehabilitation program, were randomly assigned to treatment with pulsed magnetic field therapy (PMFT) or to sham therapy in order to study the additional effect of PMFT as part of a multimodal neurological rehabilitation program on fatigue. Patients demographic and disease specific characteristics were recorded. Level of fatigue was measured by fatigue severity scale (FSS) at entrance and discharge and with a visual analog scale (VAS) immediate before and after a single treatment session. The ‘Magnetic Cell Regeneration’ system by Santerra was used for PMFT. A single treatment lasted 16 minutes twice daily over 3-4 weeks and consisted of relaxed lying on a PMF mattress. Sham intervention was conducted in an identical manner with the PMF-device off. Patients and statistics were blinded. Level of fatigue measured by FSS was high at entrance in both treatment group (TG) and control group (CG) (5.6 versus 5.5). Over time of rehabilitation fatigue was reduced by 18% in TG and 7% in CG which was statistically not significant. There was a statistically significant immediate effect of the single treatment session which 18% reduction of fatigue measured by VAS in TG versus 11% in CG. Because of a high ‘placebo effect’ of simple bed rest, a only small and short lasting additional effect of PMFT and high costs of a PMF-device, we cannot recommend PMFT as an additional feature of a multimodal neurological rehabilitation program in order to reduce fatigue level of MS-patients.

Int J Neurosci. 1997 Nov;92(1-2):95-102.

Treatment with electromagnetic fields improves dual-task performance (talking while walking) in multiple sclerosis.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

Multiple sclerosis (MS) is associated with an increased risk of falling resulting from visual disturbances, difficulties with gait and balance, apraxia of gait and peripheral neuropathy. These factors often interact synergistically to compromise the patient’s gait stability. It has long been recognized that walking involves a cognitive component and that simultaneous cognitive and motor operations (dual-task) such as talking while walking may interfere with normal ambulation. Talking while walking reflects an example of a dual-task which is frequently impaired in MS patients. Impaired dual-task performance during walking may compromise the patient’s gait and explain why in some circumstances, MS patients unexpectedly lose their balance and fall. Frontal lobe dysfunction, which commonly occurs in MS patients, may disrupt dual-task performance and increase the risk of falling in these patients. This report concerns a 36 old man with remitting-progressive MS with an EDSS score of 5.5 who experienced marked increase in spasticity in the legs and trunk and worsening of his gait and balance, occasionally resulting in falling, when talking while walking. His gait and balance improved dramatically after he received two successive transcranial treatments, each of 45 minutes, with AC pulsed electromagnetic fields (EMFs) of 7.5 picotesla flux density. Simultaneously, there was improvement in dual-task performance to the extent that talking while walking did not adversely affect his ambulation. In addition, neuropsychological testing revealed an almost 5-fold increase in word output on the Thurstone’s Word-Fluency Test, which is sensitive to frontal lobe dysfunction. It is suggested that facilitation of dual-task performance during ambulation contributes to the overall improvement of gait and balance observed in MS patients receiving transcranial treatment with AC pulsed EMFs.

Int J Neurosci. 1997 Aug;90(3-4):177-85.

Treatment with electromagnetic fields reverses the long-term clinical course of a patient with chronic progressive multiple sclerosis.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

It is estimated that 10-20% of patients with multiple sclerosis (MS) have a chronic progressive (CP) course characterized by an insidious onset of neurological deficits followed by steady progression of disability in the absence of symptomatic remission. To date no therapeutic modality has proven effective in reversing the clinical course of CP MS although there are indications that prolonged treatment with picotesla electromagnetic fields (EMFs) alters the clinical course of patients with CP MS. A 40 year-old woman presented in December of 1992 with CP MS with symptoms of spastic paraplegia, loss of trunk control, marked weakness of the upper limbs with loss of fine and gross motor hand functions, severe fatigue, cognitive deficits, mental depression, and autonomic dysfunction with neurogenic bladder and bowel incontinence. Her symptoms began at the age of 18 with weakness of the right leg and fatigue with long distance walking and over the ensuing years she experienced steady deterioration of functions. In 1985 she became wheelchair dependent and it was anticipated that within 1-2 years she would become functionally quadriplegic. In December of 1992 she began experimental treatment with EMFs. While receiving regularly weekly transcortical treatments with AC pulsed EMFs in the picotesla range intensity she experienced during the first year improvement in mental functions, return of strength in the upper extremities, and recovery of trunk control. During the second year she experienced the return of more hip functions and recovery of motor functions began in her legs. For the first time in years she can now initiate dorsiflexion of her ankles and actively extend her knees voluntarily. Over the past year she started to show signs of redevelopment of reciprocal gait. Presently, with enough function restored in her legs, she is learning to walk with a walker and is able to stand unassisted and maintain her balance for a few minutes. She also regained about 80% of functions in the upper limbs and hands. Most remarkably, there was no further progression of the disease during the 4 years course of magnetic therapy. This patient’s clinical recovery cannot be explained on the basis of a spontaneous remission. It is suggested that pulsed applications of picotesla EMFs affect the neurobiological and immunological mechanisms underlying the pathogenesis of CP MS.

Int J Neurosci. 1997 Aug;90(3-4):145-57.

Resolution of sleep paralysis by weak electromagnetic fields in a patient with multiple sclerosis.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

Sleep paralysis refers to episodes of inability to move during the onset of sleep or more commonly upon awakening. Patients often describe the sensation of struggling to move and may experience simultaneous frightening vivid hallucinations and dreams. Sleep paralysis and other manifestations of dissociated states of wakefulness and sleep, which reflect deficient monoaminergic regulation of neural modulators of REM sleep, have been reported in patients with multiple sclerosis (MS). A 40 year old woman with remitting-progressive multiple sclerosis (MS) experienced episodes of sleep paralysis since the age of 16, four years prior to the onset of her neurological symptoms. Episodes of sleep paralysis, which manifested at a frequency of about once a week, occurred only upon awakening in the morning and were considered by the patient as a most terrifying experience. Periods of mental stress, sleep deprivation, physical fatigue and exacerbation of MS symptoms appeared to enhance the occurrence of sleep paralysis. In July of 1992 the patient began experimental treatment with AC pulsed applications of picotesla intensity electromagnetic fields (EMFs) of 5Hz frequency which were applied extracerebrally 1-2 times per week. During the course of treatment with EMFs the patient made a dramatic recovery of symptoms with improvement in vision, mobility, balance, bladder control, fatigue and short term memory. In addition, her baseline pattern reversal visual evoked potential studies, which showed abnormally prolonged latencies in both eyes, normalized 3 weeks after the initiation of magnetic therapy and remained normal more than 2.5 years later. Since the introduction of magnetic therapy episodes of sleep paralysis gradually diminished and abated completely over the past 3 years. This report suggests that MS may be associated with deficient REM sleep inhibitory neural mechanisms leading to sleep paralysis secondary to the intrusion of REM sleep atonia and dream imagery into the waking state. Pineal melatonin and monoaminergic neurons have been implicated in the induction and maintenance of REM sleep and the pathogenesis of sleep paralysis and it is suggested that resolution of sleep paralysis in this patient by AC pulsed applications of EMFs was related to enhancement of melatonin circadian rhythms and cerebral serotoninergic neurotransmission.

Int J Neurosci. 1997 Jun;90(1-2):59-74.

Immediate recovery of cognitive functions and resolution of fatigue by treatment with weak electromagnetic fields in a patient with multiple sclerosis.

Sandyk R.

Department of Neuroscience, Institute for Biomedical Engineering, Dix Hills, NY, USA.

Cognitive deficits are common among patients with multiple sclerosis (MS). The pathogenetic mechanisms underlying the cognitive impairment in MS are unknown and there is presently no effective therapeutic modality which has shown efficacy in improving cognitive deficits in MS. A 53 year old college professor with a long history of secondary progressive MS experienced, over the preceding year, noticeable deterioration in cognitive functions with difficulties in short and long term memory, word finding in spontaneous speech, attention and concentration span. Unable to pursue his academic activities, he was considering early retirement. Mental examination disclosed features of subcortical and cortical dementia involving frontal lobe, left hemispheric and right hemispheric dysfunction. Almost immediately following the extracerebral application of AC pulsed electromagnetic fields (EMFs) of 7.5 picotesla intensity and a 4-Hz sinusoidal wave, the patient experienced a heightend sense of well being, which he defined as enhancement of cognitive functions with a feeling “like a cloud lifted off my head.” He reported heightend clarity of thinking and during the application of EMFs he felt that words were formed faster and he experienced no difficulty finding the appropriate words. His speech was stronger and well modulated and he felt “energized” with resolution of his fatigue. There was improvement in manual dexterity and handwriting and testing of constructional praxis demonstrated improvement in visuospatial, visuoperceptive and visuomotor functions. It is suggested that some of the cognitive deficits associated with MS, which are caused by synaptic disruption of neurotransmitter functions, may be reversed through pulsed applications of picotesla range EMFs.

Int J Neurosci. 1996 Oct;87(1-2):5-15.

Suicidal behavior is attenuated in patients with multiple sclerosis by treatment with electromagnetic fields.

Sandyk R.

NeuroCommunication Research Laboratories, Danbury, CT 06811, USA.

A marked decrease in the levels of serotonin (5-HT) and its metabolite (5-HIAA) has been demonstrated in postmortem studies of suicide victims with various psychiatric disorders. Depression is the most common mental manifestation of multiple sclerosis (MS) which accounts for the high incidence of suicide in this disease. CSF 5-HIAA concentrations are reduced in MS patients and nocturnal plasma melatonin levels were found to be lower in suicidal than in nonsuicidal patients. These findings suggest that the increased risk of suicide in MS patients may be related to decreased 5-HT functions and blunted circadian melatonin secretion. Previous studies have demonstrated that extracerebral applications of pulsed electromagnetic fields (EMFs) in the picotesla range rapidly improved motor, sensory, affective and cognitive deficits in MS. Augmentation of cerebral 5-HT synthesis and resynchronization of circadian melatonin secretion has been suggested as a key mechanism by which these EMFs improved symptoms of the disease. Therefore, the prediction was made that this treatment modality would result in attenuation of suicidal behavior in MS patients. The present report concerns three women with remitting-progressive MS who exhibited suicidal behavior during the course of their illness. All patients had frequent suicidal thoughts over several years and experienced resolution of suicidal behavior within several weeks after introduction of EMFs treatment with no recurrence of symptoms during a follow-up of months to 3.5 years. These findings demonstrate that in MS pulsed applications of picotesla level EMFs improve mental depression and may reduce the risk of suicide by a mechanism involving the augmentation of 5-HT neurotransmission and resynchronization of circadian melatonin secretion.

Int J Neurosci. 1996 Jul;86(1-2):79-85.

Effect of weak electromagnetic fields on body image perception in patients with multiple sclerosis.

Sandyk R.

NeuroCommunication Research Laboratories, Danbury, CT 06811, USA.

Cerebellar ataxia is one of the most disabling symptoms of multiple sclerosis (MS) and also one of the least responsive to pharmacotherapy. However, cerebellar symptoms often improve dramatically in MS patients by brief, extracerebral applications of picotesla flux electromagnetic fields (EMFs). This report concerns two MS patients with chronic disabling ataxia who experienced rapid improvement in gait and balance after receiving a series of treatments with EMFs. To assess whether improvement in cerebellar gait is accompanied by changes in body image perception, a parietal lobe function, both patients were administered the Human Figure Drawing Test before and after a series of brief treatments with EMFs. Prior to application of EMFs these patients’ free drawings of a person showed a figure with a wide-based stance characteristic of cerebellar ataxia. After receiving a series of EMFs treatments both patients demonstrated a change in body image perception with the drawings of the human figure showing a normal stance. These findings demonstrate that in MS improvement in cerebellar symptoms by pulsed applications of picotesla EMFs is associated with changes in the body image.

Int J Neurosci. 1996 Jul;86(1-2):67-77.

Treatment with weak electromagnetic fields attenuates carbohydrate cravings in a patients with multiple sclerosis.

Sandyk R.

NeuroCommunication Research Laboratories, Danbury, CT 06811, USA.

Pharmacological studies have implicated serotonergic (5-HT) neurons in the regulation of food intake and food preference. It has been shown that the urge to consume carbohydrate rich foods is regulated by 5-HT activity and that carbohydrate craving is triggered by 5-HT deficiency in the medical hypothalamus. Ingestion of carbohydrate foods stimulates insulin secretion which accelerates the uptake of tryptophan, the precursor of 5-HT and melatonin, into the brain and pineal gland, respectively. Thus, carbohydrate craving might be considered a form of “self medication” aimed at correcting an underlying dysfunction of cerebral 5-HT and pineal melatonin functions. A 51 year old woman with remitting-progressive MS experienced carbohydrate craving during childhood and adolescence and again in temporal association with the onset of her first neurological symptoms at the age of 45. Carbohydrate craving, which resembled the pattern observed in patients with seasonal affective disorder (SAD), was attenuated by a series of extracranial AC pulsed applications of picotesla (10(-12) Tesla) flux intensity electromagnetic fields (EMFs). It is suggested that AC pulsed EMFs applications activated retinal mechanisms which, through functional interactions with the medial hypothalamus, initiated an increased release of 5-HT and resynchronization of melatonin secretion ultimately leading to a decrease in carbohydrate craving. The occurrence of carbohydrate craving in early life may have increased the patient’s vulnerability to viral infection given the importance of 5-HT and melatonin in immunomodulation and the regulation of the integrity of the blood brain barrier. The recurrence of this craving in temporal relation to the onset of neurological symptoms suggests that 5-HT deficiency and impaired pineal melatonin functions are linked to the timing of onset of the clinical symptoms of the disease. The report supports the role of experimental factors in the pathophysiology of MS.

Int J Neurosci. 1995 Nov;83(1-2):81-92.

Resolution of dysarthria in multiple sclerosis by treatment with weak electromagnetic fields.

Sandyk R.

NeuroCommunication Research Laboratories, Danbury, CT 06811, USA.

It has been reported that 50% or more of patients diagnosed with multiple sclerosis (MS) exhibit speech impairment (dysarthria) which in some cases can be exceedingly disabling. Currently there is no effective medical treatment for the dysarthria of MS which occurs as a result of lesions to the cerebellum and its outflow tracts. It was reported recently that extracranial application of brief AC pulsed electromagnetic fields (EMFs) in the picotesla (pT) range intensity produced in patients with MS sustained improvement in motor functions including cerebellar symptomatology. This communication concerns two MS patients with a chronic progressive course who exhibited severe dysarthria which improved already during the initial treatment with pulsed EMFs and which resolved completely 3-4 weeks later. Since application of EMFs has been shown to alter: (a) the resting membrane potential and synaptic neurotransmitter release through an effect involving changes in transmembrane calcium flux; and (b) the secretion of pineal melatonin which in turn influences the synthesis and release of serotonin (5-HT) and gamma-amino butyric acid (GABA) in the cerebellum, it is suggested that the immediate improvement of the dysarthria occurred as a result of changes in cerebellar neurotransmitter functions particularly 5-HT and GABA rather than from remyelination.

Int J Neurosci. 1995 Jun;82(3-4):223-42.

Chronic relapsing multiple sclerosis: a case of rapid recovery by application of weak electromagnetic fields.

Sandyk R.

NeuroCommunication Research Laboratories, Danbury, CT 06811, USA.

A 54 year-old woman was diagnosed with multiple sclerosis (MS) in 1985 at the age of 45 after she developed diplopia, slurred speech, and weakness in the right leg. A Magnetic Resonance Imaging (MRI) scan obtained in 1985 showed several areas of plaque formation distributed in the periventricular white matter and centrum semiovale bilaterally. Coincident with slow deterioration in her condition since 1990 a second MRI scan was obtained in 1991 which showed a considerable increase in the number and size of plaques throughout both cerebral hemispheres, subcortical white matter, periventricularly and brainstem. In 1994, the patient received treatment with Interferon beta- 1b (Betaseron) for 6 months with no improvement in symptoms. However, following two successive extracranial applications of pulsed electromagnetic fields (EMFs) in the picotesla (pT) range each of 20 minutes duration the patient experienced an immediate improvement in symptoms most dramatically in gait, balance, speech, level of energy, swallowing, mood, and vision. On a maintenance program of 3 treatments per month the patient’s only symptom is mild right foot and leg weakness. The report points to the unique efficacy of externally applied pT range EMFs in the symptomatic treatment of MS, indicates a lack of an association between the extent of demyelinating plaques on MRI scan and rate and extent of recovery in response to EMFs, and supports the notion that dysfunction of synaptic conductivity due to neurotransmitter deficiency particularly of serotonin (5-HT) contributes more significantly to the development of MS symptoms than the process of demyelination which clinically seems to represent an epiphenomenon of the disease.

Int J Neurosci. 1994 Dec;79(3-4):199-212.

Weak electromagnetic fields attenuate tremor in multiple sclerosis.

Sandyk R, Dann LC.

NeuroCommunication Research Laboratories, Danbury, CT 06811, USA.

It has been estimated that about 75% of patients diagnosed with multiple sclerosis (MS) have tremor which can be exceedingly disabling. The most common tremor observed in patients with MS is a cerebellar intention tremor (‘kinetic tremor’) although postural tremor (‘static tremor’) is also common and often extremely incapacitating. Currently there is no effective medical treatment for the tremor of MS which, in some severe cases, may be abolished by stereotactic thalamotomy. It was reported recently that extracranial application of brief AC pulsed electromagnetic fields (EMFs) in the picotesla (pT) range produced improvement in motor and cognitive functions in patients with MS. The present communication concerns three MS patients with a chronic progressive course of the disease (mean age: 39.3 +/- 8.3 years; mean duration of illness: 11.3 +/- 3.2 years) in whom brief external applications of pulsed EMFs of 7.5 pT intensity reduced intention and postural tremors resulting in significant functional improvement. The report suggests that these extremely low intensity EMFs are beneficial also in the treatment of tremors in MS and that this treatment may serve as an alternative method to stereotactic thalamotomy in the management of tremor in MS. The mechanisms by which EMFs attenuate the tremors of MS are complex and are thought to involve augmentation of GABA and serotonin (5-HT) neurotransmission in the cerebellum and its outflow tracts.

Therapeutic effects of alternating current pulsed electromagnetic fields in multiple sclerosis.

Sandyk R. Dep. of Neuroscience, Institute for Biomedical Engineering and Rehab Services of Touro College, Dix Hills, New York.

Multiple sclerosis is the third most common cause of severe disability in patients between the ages of 15 and 50 years. The cause of the disease and its pathogenesis remain unknown. The last 20 years have seen only meager advances in the development of effective treatments for the disease. No specific treatment modality can cure the disease or alter its long-term course and eventual outcome. Moreover, there are no agents or treatments that will restore premorbid neuronal function. A host of biological phenomena associated with the disease involving interactions among genetic, environmental, immunologic, and hormonal factors, cannot be explained on the basis of demyelination alone and therefore require refocusing attention on alternative explanations, one of which implicates the pineal gland as pivotal. The pineal gland functions as a magnetoreceptor organ. This biological property of the gland provided the impetus for the development of a novel and highly effective therapeutic modality, which involves transcranial applications of alternating current (AC) pulsed electromagnetic fields flux density. This review summarizes recent clinical work on the effects of transcranially applied pulsed electromagnetic fields for the symptomatic treatment of the disease.

J In Biologic Effects of Light 1998 Symposium

Pulsing magnetic field effects on brain electrical activity in multiple sclerosis.

Richards TL, Acosta-Urquidi,

Multiple sclerosis (MS) is a disease of the central nervous system. Clinical symptoms include central fatigue, impaired bladder control, muscle weakness, sensory deficits, impaired cognition, and others. The cause of MS is unknown, but from histologic, immunologic, and radiologic studies, we know that there are demyelinated brain lesions (visible on magnetic resonance images) that contain immune cells such as macrophages and T-cells (visible on microscopic analysis of brain sections). Recently, a histologic study has also shown that widespread axonal damage occurs in MS along with demyelination. What is the possible connection between MS and bio-electromagnetic fields? We recently published a review entitled “Bio-electromagnetic applications for multiple sclerosis,” which examined several scientific studies that demonstrated the effects of electromagnetic fields on nerve regeneration, brain electrical activity (electro-encephalography), neurochemistry, and immune system components. All of these effects are important for disease pathology and clinical symptoms in multiple sclerosis (MS). EEG was measured in this study in order to test our hypothesis that the pulsing magnetic device affects the brain electrical activity, and that this may be a mechanism for the effect we have observed on patient-reported symptoms. The EEG data reported previously were measured only during resting and language conditions. The purpose of the current study was to measure the effect of the electromagnetic device on EEG activity during and after photic stimulation with flashing lights. After photic stimulation, there was a statistically significant increase in alpha EEG magnitude that was greater in the active group compared to the placebo group in electrode positions P3, T5, and O1 (analysis of variance p<.001, F=14, DF = 1,16). In the comparison between active versus placebo, changes measured from three electrode positions were statistically significantly even after multiple comparison correction.

Treatment with weak electromagnetic fiels improves fatigue associated with multiple sclerosis.

Sandyk R. NeuroCommunication Research Laboratories, Danbury, CT, USA

It is estimated that 75-90% of patients with multiple sclerosis (MS) experience fatigue at some point during the course of the disease and that in about half of these patients, subjective fatigue is a primary complaint. In the majority of patients fatigue is present throughout the course of the day being most prominent in the mid to late afternoon. Sleepiness is not prominent, but patients report that rest may attenuate fatigability. The pathophysiology of the fatigue of MS remains unknown. Delayed impulse conduction in demyelinated zones may render transmission in the brainstem reticular formation less effective. In addition, the observation that rest may restore energy and that administration of pemoline and amantadine, which increase the synthesis and release of monoamines, often improve the fatigue of MS suggest that depletion of neurotransmitter stores in damaged neurons may contribute significantly to the development of fatigue in these patients. The present report concerns three MS patients who experienced over several years continuous and debilitating fatigue throughout the course of the day. Fatigue was exacerbated by increased physical activity and was not improved by rest. After receiving a course of treatments with picotesla flux electromagnetic fields (EMFs), which were applied extracranially, all patients experienced improvement in fatigue. Remarkably, patients noted that several months after initiation of treatment with EMFs they were able to recover, after a short period of rest, from fatigue which followed increased physical activity. These observations suggest that replenishment of monoamine stores in neurons damaged by demyelination in the brainstem reticular formation by periodic applications of picotesla flux intensity EMFs may lead to more effective impulse conduction and thus to improvement in fatigue including rapid recovery of fatigue after rest.

Int J Neurosci. 1998 Jul;95(1-2):107-13.

Yawning and stretching–a behavioral syndrome associated with transcranial application of electromagnetic fields in multiple sclerosis.

Sandyk R.

Department of Neuroscience at the Institute for Biomedical Engineering and Rehabilitation Services of Touro College, Dix Hills, NY 11746, USA.

Intracerebral administration of adrenocorticotropic hormone (ACTH) elicits in experimental animals a yawning stretching behavior which is believed to reflect an arousal response mediated through the septohippocampal cholinergic neurons. A surge in plasma ACTH levels at night and just prior to awakening from sleep is also associated in humans with yawning and stretching behavior. Recurrent episodes of uncontrollable yawning and body stretching, identical to those observed upon awakening from physiological sleep, occur in a subset of patients with multiple sclerosis (MS) during transcranial therapeutic application of AC pulsed electromagnetic fields of picotesla flux density. This behavioral response has been observed exclusively in young female patients who are fully ambulatory with a relapsing remitting course of the disease who also demonstrate a distinctly favorable therapeutic response to magnetic stimulation. ACTH is employed for the treatment of MS due to its immunomodulatory effects and a surge in its release in response to AC pulsed magnetic stimulation could explain some of the mechanism by which these fields improve symptoms of the disease.

Int J Neurosci. 1997 Jan;89(1-2):39-51.

Progressive cognitive improvement in multiple sclerosis from treatment with electromagnetic fields.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

It has long been recognized that cognitive impairment occurs in patients with multiple sclerosis (MS) particularly among patients with a chronic progressive course. MS is considered a type of “subcortical dementia” in which cognitive and behavioral abnormalities resemble those observed in patients with a frontal lobe syndrome. The Bicycle Drawing Test is employed for the neuropsychological assessment of cognitive impairment specifically that of mechanical reasoning and visuographic functioning. It also provides clues concerning the patient’s organizational skills which are subserved by the frontal lobes. Extracerebral pulsed applications of picotesla flux intensity electromagnetic fields (EMFs) have been shown to improve cognitive functions in patients with MS. I present three patients with long standing symptoms of MS who, on the initial baseline, pretreatment Bicycle Drawing Test, exhibited cognitive impairment manifested by omissions of essential details and deficient organizational skills. All patients demonstrated progressive improvement in their performance during treatment with EMFs lasting from 6-18 months. The improvement in cognitive functions, which occurred during the initial phases of the treatment, was striking for the changes in organizational skills reflecting frontal lobe functions. These findings demonstrate that progressive recovery of cognitive functions in MS patients are observed over time through continued administration of picotesla flux intensity EMFs. It is believed that the beneficial cognitive effects of these EMFs are related to increased synaptic neurotransmission and that the progressive cognitive improvement noted in these patients is associated with slow recovery of synaptic functions in monoaminergic neurons of the frontal lobe or its projections from subcortical areas.

Wiad Lek. 2003;56(9-10):434-41.

Application of variable magnetic fields in medicine–15 years experience.

[Article in Polish]

Sieron A, Cieslar G.

Katedra i Klinika Chorob Wewnetrznych, Angiologii i Medycyny Fizykalnej SAM, ul. Batorego 15, 41-902 Bytom. sieron@mediclub.pl

The results of 15-year own experimental and clinical research on application of variable magnetic fields in medicine were presented. In experimental studies analgesic effect (related to endogenous opioid system and nitrogen oxide activity) and regenerative effect of variable magnetic fields with therapeutical parameters was observed. The influence of this fields on enzymatic and hormonal activity, free oxygen radicals, carbohydrates, protein and lipid metabolism, dielectric and rheological properties of blood as well as behavioural reactions and activity of central dopamine receptor in experimental animals was proved. In clinical studies high therapeutic efficacy of magnetotherapy and magnetostimulation in the treatment of osteoarthrosis, abnormal ossification, osteoporosis, nasosinusitis, multiple sclerosis, Parkinson’s disease, spastic paresis, diabetic polyneuropathy and retinopathy, vegetative neurosis, peptic ulcers, colon irritable and trophic ulcers was confirmed.

Ann Neurol. 2005 Oct 20; [Epub ahead of print]

Altered plasticity of the human motor cortex in Parkinson’s disease.

Ueki Y, Mima T, Ali Kotb M, Sawada H, Saiki H, Ikeda A, Begum T, Reza F, Nagamine T, Fukuyama H.

Human Brain Research Center, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan.

Interventional paired associative stimulation (IPAS) to the contralateral peripheral nerve and cerebral cortex can enhance the primary motor cortex (M1) excitability with two synchronously arriving inputs. This study investigated whether dopamine contributed to the associative long-term potentiation-like effect in the M1 in Parkinson’s disease (PD) patients. Eighteen right-handed PD patients and 11 right-handed age-matched healthy volunteers were studied. All patients were studied after 12 hours off medication with levodopa replacement (PD-off). Ten patients were also evaluated after medication (PD-on). The IPAS comprised a single electric stimulus to the right median nerve at the wrist and subsequent transcranial magnetic stimulation of the left M1 with an interstimulus interval of 25 milliseconds (240 paired stimuli every 5 seconds for 20 minutes). The motor-evoked potential amplitude in the right abductor pollicis brevis muscle was increased by IPAS in healthy volunteers, but not in PD patients. IPAS did not affect the motor-evoked potential amplitude in the left abductor pollicis brevis. The ratio of the motor-evoked potential amplitude before and after IPAS in PD-off patients increased after dopamine replacement. Thus, dopamine might modulate cortical plasticity in the human M1, which could be related to higher order motor control, including motor learning. Ann Neurol 2006.

Int J Neurosci. 1999 Aug;99(1-4):139-49.

AC pulsed electromagnetic fields-induced sexual arousal and penile erections in Parkinson’s disease.

Sandyk R.

Department of Neuroscience at the Institute for Biomedical Engineering and Rehabilitation Services, Touro College, Bay Shore, NY 11706, USA.

Sexual dysfunction is common in patients with Parkinson’s disease (PD) since brain dopaminergic mechanisms are involved in the regulation of sexual behavior. Activation of dopamine D2 receptor sites, with resultant release of oxytocin from the paraventricular nucleus (PVN) of the hypothalamus, induces sexual arousal and erectile responses in experimental animals and humans. In Parkinsonian patients subcutaneous administration of apomorphine, a dopamine D2 receptor agonist, induces sexual arousal and penile erections. It has been suggested that the therapeutic efficacy of transcranial administration of AC pulsed electromagnetic fields (EMFs) in the picotesla flux density in PD involves the activation of dopamine D2 receptor sites which are the principal site of action of dopaminergic pharmacotherapy in PD. Here, 1 report 2 elderly male PD patients who experienced sexual dysfunction which was recalcitrant to treatment with anti Parkinsonian agents including selegiline, levodopa and tolcapone. However, brief transcranial administrations of AC pulsed EMFs in the picotesla flux density induced in these patients sexual arousal and spontaneous nocturnal erections. These findings support the notion that central activation of dopamine D2 receptor sites is associated with the therapeutic efficacy of AC pulsed EMFs in PD. In addition, since the right hemisphere is dominant for sexual activity, partly because of a dopaminergic bias of this hemisphere, these findings suggest that right hemispheric activation in response to administration of AC pulsed EMFs was associated in these patient with improved sexual functions

Int J Neurosci. 1999 Apr;97(3-4):225-33.

Treatment with AC pulsed electromagnetic fields improves olfactory function in Parkinson’s disease.

Sandyk R.

Department of Neuroscience at the Institute for Biomedical Engineering and Rehabilitation Services of Touro College, Dix Hills, NY 11746, USA.

Olfactory dysfunction is a common symptom of Parkinson’s disease (PD). It may manifest in the early stages of the disease and infrequently may even antedate the onset of motor symptoms. The cause of olfactory dysfunction in PD remains unknown. Pathological changes characteristic of PD (i.e., Lewy bodies) have been demonstrated in the olfactory bulb which contains a large population of dopaminergic neurons involved in olfactory information processing. Since dopaminergic drugs do not affect olfactory threshold in PD patients, it has been suggested that olfactory dysfunction in these patients is not dependent on dopamine deficiency. I present two fully medicated Parkinsonian patients with long standing history of olfactory dysfunction in whom recovery of smell occurred during therapeutic transcranial application of AC pulsed electromagnetic fields (EMFs) in the picotesla flux density. In both patients improvement of smell during administration of EMFs occurred in conjunction with recurrent episodes of yawning. The temporal association between recovery of smell and yawning behavior is remarkable since yawning is mediated by activation of a subpopulation of striatal and limbic postsynaptic dopamine D2 receptors induced by increased synaptic dopamine release. A high density of dopamine D2 receptors is present in the olfactory bulb and tract. Degeneration of olfactory dopaminergic neurons may lead to upregulation (i.e., supersensitivity) of postsynaptic dopamine D2 receptors. Presumably, small amounts of dopamine released into the synapses of the olfactory bulb during magnetic stimulation may cause activation of these supersensitive receptors resulting in enhanced sense of smell. Interestingly, in both patients enhancement of smell perception occurred only during administration of EMFs of 7 Hz frequency implying that the release of dopamine and activation of dopamine D2 receptors in the olfactory bulb was partly frequency dependent. In fact, weak magnetic fields have been found to cause interaction with biological systems only within narrow frequency ranges (i.e., frequency windows) and the existence of such frequency ranges has been explained on the basis of the cyclotron resonance model.

Int J Neurosci. 1998 Sep;95(3-4):255-69.

Reversal of the bicycle drawing direction in Parkinson’s disease by AC pulsed electromagnetic fields.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

The Draw-a-Bicycle Test is employed in neuropsychological testing of cognitive skills since the bicycle design is widely known and also because of its complex structure. The Draw-a-Bicycle Test has been administered routinely to patients with Parkinson’s disease (PD) and other neurodegenerative disorders to evaluate the effect of transcranial applications of AC pulsed electromagnetic fields (EMFs) in the picotesla flux density on visuoconstructional skills. A seminal observation is reported in 5 medicated PD patients who demonstrated reversal of spontaneous drawing direction of the bicycle after they received a series of transcranial treatments with AC pulsed EMFs. In 3 patients reversal of the bicycle drawing direction was observed shortly after the administration of pulsed EMFs while in 2 patients these changes were observed within a time lag ranging from several weeks to months. All patients also demonstrated a dramatic clinical response to the administration of EMFs. These findings are intriguing because changes in drawing direction do not occur spontaneously in normal individuals as a result of relateralization of cognitive functions. This report suggests that administration of AC pulsed EMFs may induce in some PD patients changes in hemispheric dominance during processing of a visuoconstructional task and that these changes may be predictive of a particularly favourable response to AC pulsed EMFs therapy.

Int J Neurosci. 1998 May;94(1-2):41-54.

Transcranial AC pulsed applications of weak electromagnetic fields reduces freezing and falling in progressive supranuclear palsy: a case report.

Sandyk R.

Department of Neuroscience, Institute for Biomedical Engineering and Rehabilitation Services, Touro College, Dix Hills, NY 11746, USA.

Freezing is a common and disabling symptom in patients with Parkinsonism. It affects most commonly the gait in the form of start hesitation and sudden immobility often resulting in falling. A higher incidence of freezing occurs in patients with progressive supranuclear palsy (PSP) which is characterized clinically by a constellation of symptoms including supranuclear ophthalmoplegia, postural instability, axial rigidity, dysarthria, Parkinsonism, and pseudobulbar palsy. Pharmacologic therapy of PSP is currently disappointing and the disease progresses relentlessly to a fatal outcome within the first decade after onset. This report concerns a 67 year old woman with a diagnosis of PSP in whom freezing and frequent falling were the most disabling symptoms of the disease at the time of presentation. Both symptoms, which were rated 4 on the Unified Parkinson Rating Scale (UPRS) which grades Parkinsonian symptoms and signs from 0 to 4, with 0 being normal and 4 being severe symptoms, were resistant to treatment with dopaminergic drugs such as levodopa, amantadine, selegiline and pergolide mesylate as well as with the potent and highly selective noradrenergic reuptake inhibitor nortriptyline. Weekly transcranial applications of AC pulsed electromagnetic fields (EMFs) of picotesla flux density was associated with approximately 50% reduction in the frequency of freezing and about 80-90% reduction in frequency of falling after a 6 months follow-up period. At this point freezing was rated 2 while falling received a score of 1 on the UPRS. In addition, this treatment was associated with an improvement in Parkinsonian and pseudobulbar symptoms with the difference between the pre-and post EMF treatment across 13 measures being highly significant (p < .005; Sign test). These results suggest that transcranial administration AC pulsed EMFs in the picotesla flux density is efficacious in the treatment of PSP.

J Neurosci. 1998 Feb;93(1-2):43-54.

Reversal of a body image disorder (macrosomatognosia) in Parkinson’s disease by treatment with AC pulsed electromagnetic fields.

Sandyk R.

Department of Neuroscience, Institute for Biomedical Engineering and Rehabilitation Services of Touro College, Dix Hills, NY 11746, USA.

Macrosomatognosia refers to a disorder of the body image in which the patient perceives a part or parts of his body as disproportionately large. Macrosomatognosia has been associated with lesions in the parietal lobe, particularly the right parietal lobe, which integrates perceptual-sensorimotor functions concerned with the body image. It has been observed most commonly in patients with paroxysmal cerebral disorders such as epilepsy and migraine. The Draw-a-Person-Test has been employed in neuropsychological testing to identify disorders of the body image. Three fully medicated elderly Parkinsonian patients who exhibited, on the Draw-a-Person Test, macrosomatognosia involving the upper limbs are presented. In these patients spontaneous drawing of the figure of a man demonstrated disproportionately large arms. Furthermore, it was observed that the arm affected by tremor or, in the case of bilateral tremor, the arm showing the most severe tremor showed the greatest abnormality. This association implies that dopaminergic mechanisms influence neuronal systems in the nondominant right parietal lobe which construct the body image. After receiving a course of treatments with AC pulsed electromagnetic fields (EMFs) in the picotesla flux density applied transcranially, these patients’ drawings showed reversal of the macrosomatognosia. These findings demonstrate that transcranial applications of AC pulsed EMFs affect the neuronal systems involved in the construction of the human body image and additionally reverse disorders of the body image in Parkinsonism which are related to right parietal lobe dysfunction.

Int J Neurosci. 1997 Nov;92(1-2):63-72.

Speech impairment in Parkinson’s disease is improved by transcranial application of electromagnetic fields.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

A 52 year old fully medicated physician with juvenile onset Parkinsonism experienced 4 years ago severe “on-off” fluctuations in motor disability and debilitating speech impairment with severe stuttering which occurred predominantly during “on-off” periods. His speech impairment improved 20%-30% when sertraline (75 mg/day), a serotonin reuptake inhibitor, was added to his dopaminergic medications which included levodopa, amantadine, selegiline and pergolide mesylate. A more dramatic and consistent improvement in his speech occurred over the past 4 years during which time the patient received, on a fairly regular basis, weekly transcranial treatments with AC pulsed electromagnetic fields (EMFs) of picotesla flux density. Recurrence of speech impairment was observed on several occasions when regular treatments with EMFs were temporarily discontinued. These findings demonstrate that AC pulsed applications of picotesla flux density EMFs may offer a nonpharmacologic approach to the management of speech disturbances in Parkinsonism. Furthermore, this case implicates cerebral serotonergic deficiency in the pathogenesis of Parkinsonian speech impairment which affects more than 50% of patients. It is believed that pulsed applications of EMFs improved this patient’s speech impairment through the facilitation of serotonergic transmission which may have occurred in part through a synergistic interaction with sertraline.

Int J Neurosci. 1997 Oct;91(3-4):189-97.

Treatment with AC pulsed electromagnetic fields improves the response to levodopa in Parkinson’s disease.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

A 52 year old fully medicated Parkinsonian patient with severe disability (stage 4 on the Hoehn & Yahr disability scale) became asymptomatic 10 weeks after he received twice weekly transcranial treatments with AC pulsed electromagnetic fields (EMFs) of picotesla flux density. Prior to treatment with EMFs, his medication (Sinemet CR) was about 50% effective and he experienced end-of-dose deterioration and diurnal-related decline in the drug’s efficacy. For instance, while his morning medication was 90% effective, his afternoon medication was only 50% effective and his evening dose was only 30% effective. Ten weeks after introduction of treatment with EMFs, there was 40% improvement in his response to standard Sinemet medication with minimal change in its efficacy during the course of the day or evening. These findings demonstrate that intermittent, AC pulsed applications of picotesla flux density EMFs improve Parkinsonian symptoms in part by enhancing the patient’s response to levodopa. This effect may be related to an increase in the capacity of striatal DA neurons to synthesize, store and release DA derived from exogenously supplied levodopa as well as to increased serotonin (5-HT) transmission which has been shown to enhance the response of PD patients to levodopa. Since decline in the response to levodopa is a phenomenon associated with progression of the disease, this case suggests that intermittent applications of AC pulsed EMFs of picotesla flux density reverse the course of chronic progressive PD.

Int J Neurosci. 1997 Sep;91(1-2):57-68.

Reversal of cognitive impairment in an elderly parkinsonian patient by transcranial application of picotesla electromagnetic fields.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

A 74 year old retired building inspector with a 15 year history of Parkinson’s disease (PD) presented with severe resting tremor in the right hand, generalized bradykinesia, difficulties with the initiation of gait with freezing, mental depression and generalized cognitive impairment despite being fully medicated. Testing of constructional abilities employing various drawing tasks demonstrated drawing impairment compatible with severe left hemispheric dysfunction. After receiving two successive transcranial applications, each of 20 minutes duration, with AC pulsed electromagnetic fields (EMFs) of 7.5 picotesla flux density and frequencies of 5Hz and 7Hz respectively, his tremor remitted and there was dramatic improvement in his drawing performance. Additional striking improvements in his drawing performance occurred over the following two days after he continued to receive daily treatments with EMFs. The patient’s drawings were subjected to a Reliability Test in which 10 raters reported 100% correct assessment of pre- and post drawings with all possible comparisons (mean 2 = 5.0; p < .05). This case demonstrates in PD rapid reversal of drawing impairment related to left hemispheric dysfunction by brief transcranial applications of AC pulsed picotesla flux density EMFs and suggests that cognitive deficits associated with Parkinsonism, which usually are progressive and unaffected by dopamine replacement therapy, may be partly reversed by administration of these EMFs. Treatment with picotesla EMFs reflects a “cutting edge” approach to the management of cognitive impairment in Parkinsonism.

Int J Neurosci. 1997 Jun;90(1-2):75-86.

Treatment with weak electromagnetic fields restores dream recall in a parkinsonian patient.

Sandyk R.

Department of Neuroscience, Institute for Biomedical Engineering and Rehabilitation Services, Touro College, Dix Hills, NY 11746, USA.

Absent or markedly reduced REM sleep with cessation of dream recall has been documented in numerous neurological disorders associated with subcortical dementia including Parkinson’s disease, progressive supranuclear palsy and Huntington’s chorea. This report concerns a 69 year old Parkinsonian patient who experienced complete cessation of dreaming since the onset of motor disability 13 years ago. Long term treatment with levodopa and dopamine (DA) receptor agonists (bromocriptine and pergolide mesylate) did not affect dream recall. However, dreaming was restored after the patient received three treatment sessions with AC pulsed picotesla range electromagnetic fields (EMFs) applied extracranially over three successive days. Six months later, during which time the patient received 3 additional treatment sessions with EMFs, he reported dreaming vividly with intense colored visual imagery almost every night with some of the dreams having sexual content. In addition, he began to experience hypnagogic imagery prior to the onset of sleep. Cessation of dream recall has been associated with right hemispheric dysfunction and its restoration by treatment with EMFs points to right hemispheric activation, which is supported by improvement in this patient’s visual memory known to be subserved by the right temporal lobe. Moreover, since DA neurons activate REM sleep mechanisms and facilitate dream recall, it appears that application of EMFs enhanced DA activity in the mesolimbic system which has been implicated in dream recall. Also, since administration of pineal melatonin has been reported to induce vivid dreams with intense colored visual imagery in normal subjects and narcoleptic patients, it is suggested that enhanced nocturnal melatonin secretion was associated with restoration of dream recall in this patient. These findings demonstrate that unlike chronic levodopa therapy, intermittent pulsed applications of AC picotesla EMFs may induce in Parkinsonism reactivation of reticular-limbic-pineal systems involved in the generation of dreaming.

Int J Neurosci. 1996 Nov;87(3-4):209-17.

Brief communication: electromagnetic fields improve visuospatial performance and reverse agraphia in a parkinsonian patient.

Sandyk R.

Department of Neuroscience, Touro College, Dix Hills, NY 11746, USA.

A 73 year old right-handed man, diagnosed with Parkinson’s disease (PD) in 1982, presented with chief complaints of disabling resting and postural tremors in the right hand, generalized bradykinesia and rigidity, difficulties with the initiation of gait, freezing of gait, and mild dementia despite being fully medicated. On neuropsychological testing the Bicycle Drawing Test showed cognitive impairment compatible with bitemporal and frontal lobe dysfunction and on attempts to sign his name he exhibited agraphia. After receiving two successive treatments, each of 20 minutes duration, with AC pulsed electromagnetic fields (EMFs) of 7.5 picotesla intensity and 5 Hz frequency sinusoidal wave, his drawing to command showed improvement in visuospatial performance and his signature became legible. One week later, after receiving two additional successive treatments with these EMFs each of 20 minutes duration with a 7 Hz frequency sinusoidal wave, he drew a much larger, detailed and visuospatially organized bicycle and his signature had normalized. Simultaneously, there was marked improvement in Parkinsonian motor symptoms with almost complete resolution of the tremors, start hesitation and freezing of gait. This case demonstrates the dramatic beneficial effects of AC pulsed picotesla EMFs on neurocognitive processes subserved by the temporal and frontal lobes in Parkinsonism and suggest that the dementia of Parkinsonism may be partly reversible.

Int J Neurosci. 1996 Mar;85(1-2):111-24.

Freezing of gait in Parkinson’s disease is improved by treatment with weak electromagnetic fields.

Sandyk R.

NeuroCommunication Research Laboratories, Danbury, CT 06811, USA.

Freezing, a symptom characterized by difficulty in the initiation and smooth pursuit of repetitive movements, is a unique and well known clinical feature of Parkinson’s disease (PD). It usually occurs in patients with long duration and advanced stage of the disease and is a major cause of disability often resulting in falling. In PD patients freezing manifests most commonly as a sudden attack of immobility usually experienced during walking, attempts to turn while walking, or while approaching a destination. Less commonly it is expressed as arrest of speech or handwriting. The pathophysiology of Parkinsonian freezing, which is considered a distinct clinical feature independent of akinesia, is poorly understood and is believed to involve abnormalities in dopamine and norepinephrine neurotransmission in critical motor control areas including the frontal lobe, basal ganglia, locus coeruleus and spinal cord. In general, freezing is resistant to pharmacological therapy although in some patients reduction or increase in levodopa dose may improve this symptom. Three medicated PD patients exhibiting disabling episodes of freezing of gait are presented in whom brief, extracerebral applications of pulsed electromagnetic fields (EMFs) in the picotesla range improved freezing. Two patients had freezing both during “on” and “off” periods while the third patient experienced random episodes of freezing throughout the course of the day. The effect of each EMFs treatment lasted several days after which time freezing gradually reappeared, initially in association with “off” periods. These findings suggest that the neurochemical mechanisms underlying the development of freezing are sensitive to the effects of EMFs, which are believed to improve freezing primarily through the facilitation of serotonin (5-HT) neurotransmission at both junctional (synaptic) and nonjunctional neuronal target sites.

Int J Neurosci. 1998 Apr;93(3-4):239-50.

Treatment with AC pulsed electromagnetic fields normalizes the latency of the visual evoked response in a multiple sclerosis patient with optic atrophy.

Sandyk R.

Department of Neuroscience at the Institute for Biomedical Engineering and Rehabilitation Services of Touro College, Dix Hills, NY 11746, USA.

Visual evoked response (VER) studies have been utilized as supportive information for the diagnosis of multiple sclerosis (MS) and may be useful in objectively monitoring the effects of various therapeutic modalities. Delayed latency of the VER, which reflects slowed impulse transmission in the optic pathways, is the most characteristic abnormality associated with the disease. Brief transcranial applications of AC pulsed electromagnetic fields (EMFs) in the picotesla flux density are efficacious in the symptomatic treatment of MS and may also reestablish impulse transmission in the optic pathways. A 36 year old man developed an attack of right sided optic neuritis at the age of 30. On presentation he had blurring of vision with reduced acuity on the right and fundoscopic examination revealed pallor of the optic disc. A checkerboard pattern reversal VER showed a delayed latency to right eye stimulation (P100 = 132 ms; normal range: 95-115 ms). After he received two successive applications of AC pulsed EMFs of 7.5 picotesla flux density each of 20 minutes duration administered transcranially, there was a dramatic improvement in vision and the VER latency reverted to normal (P100= 107 ms). The rapid improvement in vision coupled with the normalization of the VER latency despite the presence of optic atrophy, which reflects chronic demyelination of the optic nerve, cannot be explained on the basis of partial or full reformation of myelin. It is proposed that in MS synaptic neurotransmitter deficiency is associated with the visual impairment and delayed VER latency following optic neuritis and that the recovery of the VER latency by treatment with pulsed EMFs is related to enhancement of synaptic neurotransmitter functions in the retina and central optic pathways. Recovery of the VER latency in MS patients may have important implications with respect to the treatment of visual impairment and prevention of visual loss. Specifically, repeated pulsed applications of EMFs may maintain impulse transmission in the optic nerve and thus potentially sustain its viability.

Int J Neurosci, 66(3-4):209-35 1992 Oct

Magnetic fields in the therapy of parkinsonism.

Sandyk R NeuroCommunication Research Laboratories, Danbury, CT 06811.

In a recent Editorial published in this Journal, I presented a new and revolutionary method for the treatment of Parkinson’s disease (PD). I reported that extracranial treatment with picoTesla magnetic fields (MF) is a highly effective, safe, and revolutionary modality in the symptomatic management of PD. My conclusion was based on experience gained following the successful treatment of over 20 Parkinsonian patients, two of whom had levodopa-induced dyskinesias. None of the patients developed side effects during a several month period of follow-up. In the present communication, I present two reports. The first concerns four Parkinsonian patients in whom picoTesla MF produced a remarkable and sustained improvement in disability. Three of the patients had idiopathic PD and the fourth patient developed a Parkinsonian syndrome following an anoxic episode. In all patients, treatment with MF was applied as an adjunct to antiParkinsonian medication. The improvement noted in these patients attests to the efficacy of picoTesla MF as an additional, noninvasive modality in the therapy of the disease. The second report concerns two demented Parkinsonian patients in whom treatment with picoTesla MF rapidly reversed visuospatial impairment as demonstrated by the Clock Drawing Test. These findings demonstrate, for the first time, the efficacy of these MF in the amelioration of cognitive deficits in Parkinson’s disease. Since Alzheimer’s pathology frequently coexists with the dementia of Parkinsonism, these observations underscore the potential efficacy of picoTesla MF in the treatment of dementias of various etiologies.

Centimeter Waves

Biofizika. 2009 Mar-Apr;54(2):256-9.

The role of protein kinase SAPK/JNK in cell responses to low-intensity nonionizing radiation.

[Article in Russian]

Cherenkov DA, Novoselova EG, Khrenov MO, Glushkova OV, Lunin SM, Novoselova TV, Fesenko EE.

Abstract

The effect of low-intensity lases light (0.2 mW/cm2, 632.8 nm, exposure time 1 min) or centimeter waves (8.15-18 GHz, 1 W/cm2, exposure time 1 h) on PhosphoSAPK/JNK production in mice lymphocytes was investigated. Normal isolated spleen lymphocytes or cells incubated previously with geldanamycin, an inhibitor of heat shock protein 90 (HSP90), were used in the experiments. A significant stimulation of PhosphoSAPK/JNK production in lymphocytes after treatment with laser light or microwaves has been shown in both cell models. It was proposed that the activation of SAPK/JNK signal pathway plays one of the central roles in cellular stress response to low-power nonionizing radiation.

Biofizika. 2008 Jan-Feb;53(1):93-9.

The role of heat shock proteins HSP90 in the response of immune cells to centimeter microwaves.

[Article in Russian]

Glushkova OV, Novoselova EG, Khrenov MO, Novoselova TV, Cherenkov DA, Lunin SM, Fesenko EE.

Abstract

The effects of low-level electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1 h) on the production of heat shock proteins, several cytokines, and nitric oxide in isolated mouse macrophages and lymphocytes were examined both under normal conditions and after the treatment of the cells with geldanamycin (GA), a depressor of activity of the heat shock protein 90 (Hsp90). The irradiation of cells without GA induced the production of Hsp70, nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-10 (IL-10), and the tumor necrosis factor -alpha (TNF-alpha). No changes in the production of Hsp90 in irradiated cells were observed, but intracellular locations of Hsp25 and Hsp70 altered. The preliminary treatment of cells with GA did not remove the effects of microwaves: in these conditions, the synthesis of all cytokines tested, nitric oxide, as well as total and membrane amount of Hsp70, and the amount of Hsp25 in the cytoplasm and cytoskeleton increased. Moreover, the exposure of cells incubated with GA resulted in the reduction of Hsp90-alpha production.

Biofizika. 2007 Sep-Oct;52(5):938-46.

Effects of centimeter waves on the immune system of mice in endotoxic shock.

[Article in Russian]

Glushkova OV, Novoselova EG, Cherenkov DA, Novoselova TV, Lunin SM, Khrenov MO, Parfeniuk SB, Fesenko EE.

Abstract

The effects of centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 10 days; MW) on the production of the tumor necrosis factor alpha, interleukin-lalpha, interleukin-1beta, interleukin-2, and the expression of interleukin-6, interleukin-10, interferon-gamma, nitric oxide and HSP27, HSP72 and HSP90alpha in mice irradiated before or after LPS injection were studied. An acute endotoxic model was produced by a single LPS injection. The effects of microwaves on nitric oxide, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were dependent on the functional status of exposed animals. Thus, an exposure of healthy mice to microwaves for 10 days was followed by a decrease in nitric oxide and interferon-gamma production, and an increase in the production of the tumor necrosis factor-alpha and interleukin-6. On the contrary, an exposure to MW before intoxication resulted in an increase in the synthesis of nitric oxide and interferon-gamma as well as a decrease in the concentration of the tumor necrosis factor-alpha and interleukin-6 in blood of mice in endotoxic shock. When microwave exposure was used after LPS injection, it did not provide any protective effect, and preliminary irradiation enhanced the resistance of the organism to endotoxic shock.

Biofizika. 2007 Sep-Oct;52(5):888-92.

The role of transcription factors in the response of mouse lymphocytes to low-level electromagnetic and laser radiations.

[Article in Russian]

Khrenov MO, Cherenkov DA, Glushkova OV, Novoselova TV, Lunin SM, Parfeniuk SB, Lysenko EA, Novoselova EG, Fesenko EE.

Abstract

The effects of low-intensity laser radiation (LILR, 632.8 nm, 0.2 mW/cm2) and low-intensity electromagnetic waves (LIEW, 8.15 – 18 GHz, 1 MW/cm2) on the production of transcription factors in lymphocytes from NMRI male mice were examined. The total level of NF-KB and its phosphorylated metabolite Phospho-NF-kappaB, as well as the regulatory protein IkappaB-alpha were determined in spleen lymphocytes subjected to laser or microwave radiations. The proteins were determined by immunoblotting. Laser light induced a lowering in the level of NF-kappaB and IkappaB-alpha. By contrast, irradiation with electromagnetic waves resulted in a significant increase in the amount of NF-kappaB and IkappaB-alpha. The phosphorylated form of NF-kappaB did not noticeably change under either of the two kinds of radiation. The results showed that electromagnetic waves activate the production of both NF-kappaB and the regulatory protein IkappaB-alpha and these data confirm the stress character of the response of spleen lymphocytes to low-level microwaves of the centimeter range.

Biofizika. 2004 May-Jun;49(3):545-50.

A comparison of the effects of millimeter and centimeter waves on tumor necrosis factor production in mouse cells.

[Article in Russian]

Sinotova OA, Novoselova EG, Glushkova OV, Fesenko EE.

Abstract

The effects of millimeter (40 GHz) and centimeter (8.15-18.00 GHz) low-intensity waves on the production of tumor necrosis factor (TNE) in macrophages and lymphocytes from exposed mice as well as in exposed isolated cells were compared. It was found that the dynamics of TNF secretory activity of cells varies depending on the frequency and duration of exposure. The application of millimeter waves induced a nonmonotonous course of the dose-effect curve for TNF changes in macrophages and splenocytes. Alternately, a stimulation and a decrease in TNF production were observed following the application of millimeter waves. On the contrary, centimeter waves provoked an activation in cytokine production. It is proposed that, in contrast to millimeter waves, the single application of centimeter waves to animals (within 2 to 96 h) or isolated cells (within 0.5 to 2.5 h) induced a much more substantial stimulation of immunity.

Biofizika. 2003 May-Jun;48(3):511-20.

Effect of low intensity of electromagnetic radiation in the centimeter and millimeter range on proliferative and cytotoxic activity of murine spleen lymphocytes.

[Article in Russian]

Oga? VB, Novoselova EG, Fesenko EE.

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

Abstract

It was found that single total-body exposure to electromagnetic centimeter waves (8.15-18 GHz, 1 microW/cm2, 5 h) stimulated the proliferation of mouse T and B splenic lymphocytes. The same effects were observed upon in vivo treatment of rats for 5 h with millimeter waves (42.2 GHz, amplitude modulation 10 Hz, 1 microW/cm2). The whole-body irradiation with centimeter or millimeter waves did not cause any significant changes in natural activity of killer cells. The cellular responses induced by the irradiation of isolated animal cells in vitro did not coincide with those revealed after the total-body irradiation of animals. Thus, the in vitro irradiation of natural killer cells to millimeter waves for 1 h increased their cytotoxic activity whereas, after treatment to centimeter waves for the same time, the activity of killer cells did not change. On the contrary, irradiation of T and B lymphocytes with millimeter waves (42.2 GHz, amplitude modulation 10 Hz, 1 microW/cm2, 1 h) suppressed the blasttransformation of cells. The results show a higher immunostimulative potential of centimeter waves as compared to millimeter waves.

Biofizika. 2003 Mar-Apr;48(2):281-8.

Immunocorrective effect of low intensity radiation of ultrahigh frequency on carcinogenesis in mice.

[Article in Russian]

Glushkova OV, Novoselova EG, Sinotova OA, Fesenko EE.

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

Abstract

The effect of low-intensity centimeter electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1.5 h daily, 20 days) on the production of tumor necrosis factor, intreleukin-2, and interleukin-3 and the expression of the heat shock protein 72 in healthy and tumor-bearing mice was measured. A significant increase in tumor necrosis factor production and a slight reduction of interleukin-2 concentration were observed after exposure to microwaves; we consider these effects as adaptive response. The interleukin-3 production in healthy mice was not affected by microwaves. Low-intensity centimeter waves induced antitumoral resistance in tumor-bearing mice. Thus, exposure of tumor-bearing mice led to a significant rise in the tumor necrosis factor production and the normalization of both interleukin-2 and interleukin-3 concentration. We assume that the significant immunomodulating effect of low-density centimeter microwaves can be used for immunocorrection and suppression of tumor growth.

Biofizika. 2002 Mar-Apr;47(2):376-81.

Immunomodulating effect of electromagnetic waves on production of tumor necrosis factor in mice with various rates of neoplasm growth.

[Article in Russian]

Glushkova OV, Novoselova EG, Sinotova OA, Vrublevskaia VV, Fesenko EE.

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

Abstract

The effects of low-density centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 14 days; MW) on tumor necrosis factor production in macrophages of mice with different growth rate of a cancer solid model produced after hypodermic injection of Ehrlich carcinoma ascites cells into hind legs were studied. After irradiation, an increase in the concentration of tumor necrosis factor in immunocompetent cells of healthy and, specially, of tumor-bearing animals was observed; and the effect of stimulation was higher upon exposure of mice carrying rapidly growing tumors. We suggest that the significant immunomodulating effect of low-density microwaves can be utilized for tumor growth suppression.

Biofizika. 2002 Jan-Feb;47(1):78-82.

Effect of electromagnetic waves in the centimeter range on the production of tumor necrosis factor and interleukin-3 in immunized mice.

[Article in Russian]

Sinotova OA, Novoselova EG, Oga? VB, Glushkova OV, Fesenko EE.

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

Abstract

The effect of prolonged treatment with weak microwaves on the production of tumor necrosis factor and interleukin-3 in peritoneal macrophages and T cells of male NMRI mice twice immunized by affinity-purified carboanhydrase was studied. Against the back ground of a high titer of antibody production, a significant increase in the production of tumor necrosis factor in peritoneal macrophages and splenic T lymphocytes of immunized mice was revealed, and a much stronger effect was observed for irradiated immunized animals. A tendency to increased secretion of interleukin-3 for unirradiated and irradiated immunized animals was found; in the latter group of animals, the effect being more pronounced. The stimulation of production of the cytokins, especially tumor necrosis factor, by combination of antigenic stimulation and microwaves can be used in adjuvant therapy of various immune diseases.

Biofizika. 2001 Jan-Feb;46(1):131-5.

Effect of centimeter microwaves and the combined magnetic field on the tumor necrosis factor production in cells of mice with experimental tumors.

[Article in Russian]

Novoselova EG, Oga? VB, Sorokina OV, Novikov VV, Fesenko EE.

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

Abstract

The effect of fractionated exposure to low-intensity microwaves (8.15-18 GHz, 1 microW/cm2, 1.5 h daily for 7 days) and combined weak magnetic field (constant 65 1 microT; alternating–100 nT, 3-10 Hz) on the production of tumor necrosis factor in macrophages of mice with experimental solid tumors produced by transplantation of Ehrlich ascites carcinoma was studied. It was found that exposure of mice to both microwaves and magnetic field enhanced the adaptive response of the organism to the onset of tumor growth: the production of tumor necrosis factor in peritoneal macrophages of tumor-bearing mice was higher than in unexposed mice.

Biofizika. 1999 Jul-Aug;44(4):737-41.

Stimulation of murine natural killer cells by weak electromagnetic waves in the centimeter range.

[Article in Russian]

Fesenko EE, Novoselova EG, Semiletova NV, Agafonova TA, Sadovnikov VB.

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

Abstract

Irradiation with electromagnetic waves (8.15-18 GHz, 1 Hz within, 1 microW/cm2) in vivo increases the cytotoxic activity of natural killer cells of rat spleen. In mice exposed for 24-72 h, the activity of natural killer cells increased by 130-150%, the increased level of activity persisting within 24 h after the cessation of treatment. Microwave irradiation of animals in vivo for 3.5 and 5 h, and a short exposure of splenic cells in vitro did not affect the activity of natural killer cells.

Biofizika. 1998 Nov-Dec;43(6):1132-3.

Stimulation of production of tumor necrosis factor by murine macrophages when exposed in vio and in vitro to weak electromagnetic waves in the centimeter range.

[Article in Russian]

Novoselova ET, Fesenko EE.

Abstract

Whole-body microwave sinusoidal irradiation of male NMRI mice, exposure of macrophages in vitro, and preliminary irradiation of culture medium with 8.15-18 GHz (1 Hz within) at a power density of 1 microW/cm2 caused a significant enhancement of tumor necrosis factor production in peritoneal macrophages. The role of microwaves as a factor interfering with the process of cell immunity is discussed.

Lik Sprava. 1992 Oct;(10):69-71.

The brain function of animals exposed to the action of centimeter electromagnetic waves.

[Article in Russian]

Smolia AL, Bezdol’naia IS.

Abstract

It was established that centimeter electromagnetic waves (EMW) are a biologically active factor. Dynamic of changes of behavioural reactions under the effect of EMW evidences instability of the functional state of the brain EMW densities of 1000, 1500 mW/cm2 produce a response characterized by inhibition of motor activity.

Vopr Kurortol Fizioter Lech Fiz Kult. 1992 Mar-Apr;(2):3-7.

The immunological and hormonal effects of combined exposure to a bitemporal ultrahigh-frequency electrical field and to decimeter waves at different sites.

[Article in Russian]

Sidorov VF, Pershin SB, Frenkel’ ID, Bobkova AS, Korovkina EG.

Abstract

Bitemporal UHF electric field is shown to enhance glucocorticoid adrenal function unlike inhibition of the thyroid function suppressing a primary immune response (PIR) in the productive phase. The combined exposure to bitemporal UHF electric field and decimeter waves of the adrenals doubles glucocorticoid synthesis abolishing the inhibitory action of the UHF therapy on thyroid function resultant in much more suppressed PIR. Both modalities inhibit thymic production. Decimeter waves alone are less effective. The exposure of the thyroid to decimeter waves initiated PIR by 2.5-fold activation of medullar lymphocytes and by a 80% increase in the thymic function. No response was achieved in combined action on the thyroid of the electric field and decimeter waves.

Tsitologiia. 1988 Nov;30(11):1345-9.

Effect of microwaves on the expression by thymocytes of various surface membrane markers.

[Article in Russian]

Evstropov VM, Melikhova ON.

Abstract

A study was made of the effects of microwave irradiation of different intensity within decimeter and centimeter ranges in vitro on the guinea-pig thymocyte-induced receptor expression to their own and rabbit erythrocytes. Besides, effects of decimeter waves on mice thymocyte-induced expression of Thy-1 antigen were studied. Microwaves were found to modulate the thymocyte-induced expression of the membrane surface markers under study.

Cataract Extraction – Lens Implantation

Vestn Oftalmol. 2002 May-Jun;118(3):15-7.

Laser magnetotherapy after cataract extraction with implantation of intraocular lens.

[Article in Russian]

Maksimov VIu, Zakharova NV, Maksimova IS, Golushkov GA, Evseev SIu.

Effects of low-intensive laser and alternating magnetic field on the course of the postoperative period were studied in patients with exudative reaction after extracapsular cataract extraction with implantation of intraocular lens (IOL). The results are analyzed for 148 eyes with early exudative reaction after IOL implantation (136 patients aged 42-75 years). The patients were observed for up to 6 months. The treatment efficiency was evaluated by the clinical picture of inflammatory reaction, visual acuity, and results of biochemical analysis of the lacrimal fluid (the ratio of lipid peroxidation products to antioxidants in cell membrane). The course of the postoperative period was more benign and recovery sooner in patients of the main group in comparison with the control.

Carpal Tunnel Syndrome

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J Adv Res. 2017 Jan; 8(1): 45–53. Published online 2016 Nov 21. doi:  10.1016/j.jare.2016.11.001 PMCID: PMC5144749

Pulsed magnetic field versus ultrasound in the treatment of postnatal carpal tunnel syndrome: A randomized controlled trial in the women of an Egyptian population

Dalia M. Kamel,a,b,? Nashwa S. Hamed,c Neveen A. Abdel Raoof,d and Sayed A. Tantawyb,eaDepartment of Physical Therapy for Obstetrics and Gynecology, Faculty of Physical Therapy, Cairo University, P.O. Box 12612, Giza, Egypt bDepartment of Physical Therapy, Faculty of Medical and Health Sciences. Ahlia University, P.O. Box 10878, Manama, Bahrain cDepartment of Physical Therapy for Neuromuscular Disorders and Its Surgery, Faculty of Physical Therapy, Cairo University, P.O. Box 12612, Giza, Egypt dDepartment of Basic Science for Physical Therapy, Faculty of Physical Therapy, Cairo University, P.O. Box 12612, Giza, Egypt eCenter of Radiation, Oncology and Nuclear Medicine, Cairo University, Giza, Egypt Dalia M. Kamel: moc.oohay@lemakailad_rd: ge.ude.uc@lemakailad_rd: hb.ude.ailha@attiwehsd ?Corresponding author. Fax: +20237617692; +973 17290083.Department of Physical Therapy for Obstetrics and GynecologyFaculty of Physical TherapyCairo UniversityP.O. Box 12612GizaEgypt ; Email: moc.oohay@lemakailad_rd, ; Email: ge.ude.uc@lemakailad_rd, ; Email: hb.ude.ailha@attiwehsd Author information ? Article notes ? Copyright and License information ? Received 2016 May 25; Revised 2016 Nov 4; Accepted 2016 Nov 14. Copyright © 2016 Production and hosting by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Graphical abstract

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Abbreviations: CTS, carpal tunnel syndrome; PEMF, pulsed electromagnetic magnetic field; US, ultrasound; MMDL, median motor distal latency; MSDL, median sensory distal latency; VAS, visual analogue scale; EMG, electromyography; MSDL, median segmental sensory distal latency; NCSs, nerve conduction studies; CTSQ, carpal tunnel syndrome questionnaire; MSCV, median sensory conduction velocity; MMCV, median motor conduction velocity; NCV, nerve conduction velocity Keywords: Carpal tunnel syndrome, Electromagnetic field, Pulsed ultrasound, Pregnancy, Postnatal, Pain, Nerve conduction velocity

Abstract

The aim of this study was to compare the effects of pulsed electromagnetic field versus pulsed ultrasound in treating patients with postnatal carpal tunnel syndrome. The study was a randomized, double-blinded trial. Forty postnatal female patients with idiopathic carpal tunnel syndrome were divided randomly into two equal groups. One group received pulsed electromagnetic field, with nerve and tendon gliding exercises for the wrist, three times per week for four weeks. The other group received pulsed ultrasound and the same wrist exercises. Pain level, sensory and motor distal latencies and conduction velocities of the median nerve, functional status scale and hand grip strength were assessed pre- and post-treatment. There was a significant decrease (P < 0.05) in pain level, sensory and motor distal latencies of the median nerve, and significant increase (P < 0.05) in sensory and motor conduction velocities of the median nerve and hand grip strength in both groups, with a significant difference between the two groups in favour of pulsed electromagnetic field treatment. However, the functional status scale showed intergroup no significant difference (P > 0.05). In conclusion, while the symptoms were alleviated in both groups, pulsed electromagnetic field was more effective than pulsed ultrasound in treating postnatal carpal tunnel syndrome.

Introduction

Carpal tunnel syndrome (CTS) is the most common entrapment neuropathy, which results from median nerve compression [1], [2]. Prevalence of CTS in the general population is 3.8% when diagnosed clinically and 2.7% when diagnosed neurophysiologically [3]. Women are more susceptible to CTS, with a 70% incidence rate, especially middle-aged women [4]. CTS is a common complaint during pregnancy, as the existing data show the prevalence rate of CTS during pregnancy to be as high as 62% [5], [6]. CTS usually develops in the second half of pregnancy because of fluid retention, due to decreased venous circulation, which causes swelling of tissues [7]. Another factor that increases CTS rates during pregnancy is hormonal alterations, including increased oestrogen, aldosterone, and cortisol levels. In addition, increased levels of prolactin are strongly correlated with CTS symptoms worsening during the night, which coincides with the prolactin circadian rhythm [8]. Further, release of relaxin can lead to relaxation of the transverse carpal ligament, leading to its flattening, and subsequent compression of the median nerve [9]. Although most pregnant women experience symptom relief following delivery, a significant percentage continue to have some level of complaint up to three years after giving birth [10]. The most typical symptoms of CTS are numbness and tingling in the distribution of the median nerve, burning sensation, pain, as well as loss of grip strength and dexterity [11].

There are several therapeutic options for patients with CTS depending on various factors, including the stage of the disease, the severity of the symptoms, and patients’ preferences. Non-surgical intervention is recommended as the first-line treatment, in cases of mild to moderate CTS. Surgery is reserved for patients with severe CTS, and those who have experienced failure of conservative treatment. The same treatment strategy is used for postnatal patients with CTS [12].

Non-surgical treatment modalities used for the management of CTS are numerous and include medical and physical therapy. Primary physical therapy interventions are splinting, nerve and tendon gliding exercises, acupuncture, low-level laser, and ultrasound with or without phonophoresis. Electromagnetic therapy is less widely used than these other therapies as currently there is limited research into the effects of electromagnetic therapy on CTS [13].

To our knowledge, no study has yet compared magnetic field therapy (which has limited research supporting its use), and ultrasound (which is among the most common treatments for CTS), in postnatal women, a population with a high incidence of CTS. Thus, our aim was to investigate which modality gives better results in treating CTS.

Subjects and methods

Subjects

The initial sample was pregnant women clinically diagnosed with CTS in their third trimester; they were recruited and screened for eligibility in this study (Fig. 1). After the approval of the Research Ethical Committee P.T.REC/012/001211, of the Faculty of Physical Therapy, Cairo University, and clinical trial registration in Clinicaltrial.gov with identifier number NCT02745652, subjects were selected from the obstetric, orthopaedic and neurological outpatient clinics in Al Kasr Al Ani Hospitals and the Faculty of Physical Therapy, Cairo University. Patients were advised to wear a hand splint until giving birth and come back three months after delivery for baseline measures and initiation of treatment.

Fig. 1

Fig. 1 Flowchart of the patients.

An informed consent form was signed by each subject prior to starting the study. Participants were randomly assigned into two groups using a random number table, and the selection process was performed by a third party not involved in the research. The study was double-blinded and the participants were randomized into the following two equal groups: group A (n = 20), who received pulsed electromagnetic field (PEMF), and group B (n = 20), who received pulsed ultrasound (US). Both groups received nerve and tendon gliding exercises for 5 min. Treatment in both groups was conducted for four weeks, three times per week with a total of 12 treatment sessions. The study started in May 2014 and ended in March 2015.

The following inclusion and exclusion criteria were designed to select a relatively homogeneous group of patients.

Inclusion criteria were unilateral affection, mild to moderate CTS with positive electro-diagnostic findings of prolonged median motor distal latency (MMDL) above 4 ms, and prolonged median sensory distal latency (MSDL) above 3.5 ms [14]. Positive both or either Phalen’s and Tinel’s tests, both tests have high percentages of sensitivity (73% and 67% respectively), and specificity (40% and 30% respectively), for CTS diagnosis [15]. Lastly, subjects reported pain intensity of more than five on the visual analogue scale (VAS).

Exclusion criteria for the study were electro-neurographic and clinical signs of axonal degeneration of the median nerve [14], and orthopaedic or neurological disorders of the neck or the upper limb such as cervical radiculopathy, pronator teres syndrome or double crush syndrome. Patients with pre-existing CTS before their most recent pregnancy, current pregnancy, diabetic neuropathy and thoracic outlet syndrome were excluded. Further exclusion criteria were wasting of thenar muscles, ulnar neuropathy, rheumatoid arthritis, previous fractured carpal bone, and previous surgery in the forearm, especially transverse ligament release.

Assessment was done before and after four weeks of intervention for both groups using the following.

  • 1. Visual Analogue Scale (VAS). It is considered a valid way of assessing pain, and allows graphic representation and numerical analysis of the collected data.
  • 2. Computerized Electromyography (EMG). Tonnies neuroscreen plus (version 1.59 Art, No: 780918 Erich Jaeger, Inc. Hoechberg, Germany) with Food and Drug Administration (FDA) registration No. 9615102, was used for assessment of the nerve conduction studies (NCSs). MMDL was recorded through wrist stimulation, and proximal latency through elbow stimulation. Both patient’s and room temperature were monitored so as not to affect the recording procedures, and the patient’s skin was cleaned with alcohol 70% to decrease its resistance. An active electrode (one-centimetre disc recording, either platinum or disposable) was placed over the belly of the abductor pollicis brevis, half the distance between the metacarpophalangeal joint of the thumb and midpoint of the distal wrist crease, while a reference electrode was placed on the distal phalanx of the thumb. For the wrist, a stimulation electrode (cathode distal) was placed 2 cm proximal to the distal wrist crease between the flexor carpi radialis and the palmaris longus tendons. For the elbow, the stimulating electrode was applied at the elbow crease, just medial to the biceps tendon. A ground electrode was placed between the stimulating and recording electrodes using a Velcro strap. Then median motor conduction velocity (MMCV) was calculated. MSDL measuring points were the active electrode, which is a ring electrode placed on the mid-portion of the proximal phalanx of the index finger (or middle finger), and the reference electrode, which is a ring electrode placed on the mid-portion of the middle phalanx of the index finger, with 2.5 cm distance between the two poles (anode is proximal to cathode). Wrist stimulation was performed at a distance of 14 cm from the ring electrodes (anti-dromic). Percutaneous stimuli were delivered until a supra-maximal response was obtained. Median sensory conduction velocity (MSCV) was calculated on the basis of the latency and the distance between the stimulating and recording electrode. For motor studies, pulse duration was 0.2 ms, filter settings were 10–10,000 Hz, sweep speed was 2–5 m/s per division, and sensitivity was 1000–5000 ?v per division. For sensory studies, pulse duration was 0.05 ms, filter settings were 20–2000 Hz, sweep speed was 1–2 m/s per division, and sensitivity was 5–10 ?v per division [16].
  • 3. Hand grip dynamometer. A hydraulic hand dynamometer (“SH5001” SAEHAN Corporation, Masan, South Korea) was used to detect hand grip strength and for measuring the maximum isometric strength of the hand and forearm muscles in kilograms (kg). It is a simple and commonly used test of general strength level [17]. The average of three trials of the affected hand was recorded.
  • 4. Functional status scale. This is a part of the Carpal Tunnel Syndrome Questionnaire (CTSQ) [18]. It asks about eight functional activities such as writing, buttoning of clothes, gripping of a telephone handle. Each functional activity is scaled from one to five, where one means none or never and five means very severe.
  • 5. Phalen test. The result of the test is positive if numbness or paresthesia develops in the median nerve distribution after flexion of the wrist for 60 s.
  • 6. Tinel test. The test is positive if numbness develops in the median nerve distribution after tapping on the volar aspect of the wrist over the course of the median nerve.

Treatment sessions occurred three times per week for four weeks, as follows.

  • 1. All patients in both groups performed nerve and tendon gliding and median nerve gliding exercises [19]. Tendon gliding exercises were done in five steps (straight, hook, fist, table top and straight fist). Median nerve gliding exercises were performed in six steps (fist, straight, wrist extension, wrist and fingers extension, supination, and gentle stretch of thumb). During these exercises, the neck and the shoulder were in a neutral position, and the elbow was in supination and 90 degrees of flexion. At each step, the patient maintained each position for five seconds, for 10 repetitions at each session. These exercises were performed in each session, three times/week for four weeks.
  • 2. PEMF Group treatment protocol used Pulsed Magnetic Field (automatic PTM Quattro PRO, code # F9020079, ASA S.r.l Company, Arcugnano [VI], Italy). This is an ASA magnetic device for magneto-therapy, which has an appliance, motorized bed, and applicable large solenoids, which can be moved to four different positions according to the treatment area, and an additional small solenoid of 30 cm diameter for hand treatment. Patients in this group received pulsed electromagnetic field therapy at frequency 50 Hz and intensity 80 gauss for 30 min. The patient was in sitting position, while the forearm rested on the bed inside the solenoid in a supination position. Safety was evaluated in the PEMF group by recording adverse effects, both those that lead to cessation of treatment (dropouts), and those that did not.
  • 3. US Group treatment protocol used Therapeutic Ultrasound (Phyaction 190 I, Uniphy P.O. Box 558.5600 AN Eindhoven, Netherlands). Pulsed mode US was applied over the volar surface of the forearm (the carpal tunnel area) for 15 min per session with a frequency of 1 MHz and intensity of 1.0 W/cm2 [20].

Outcome measures

Outcomes recorded before and after the four-week treatment course were pain intensity, median motor distal latency (MMDL) and median sensor distal latency (MSDL), Median sensory conduction velocity (MSCV), median motor conduction velocity (MMCV), the Tinel’s test, Phalen’s test, hand grip strength and the functional status scale.

Statistical analysis

All the collected data were tabulated and imported into SPSS version 18 to calculate both descriptive and inferential statistics. Descriptive analysis was performed in terms of mean, standard deviation and percentages. While inferential statistics were in the form of a Paired t-test to determine the difference within each group, an unpaired t-test was done to determine the difference in pre- and post-treatment between both groups. In addition, nonparametric statistics in the form of the Mann–Whitney test was performed to compare intergroup differences for the Tinel’s sign, Phalen’s test, VAS and functional status scale while intragroup differences were done by Kolmogorov Smirnov test. Furthermore, the work demographic data were tested by Chi-square test. Statistical significance was established at the conventional (P < 0.05) with confidence interval (CI) of 95%.

Results

This study included 55 pregnant women with unilateral idiopathic CTS. Of the 55 patients, five did not fulfil the inclusion criteria and were excluded from the study. The exclusions were due to pre-pregnancy diabetes mellitus (two cases), severe CTS with delayed MMDL equalling 9.5 ms (one case), and another two cases diagnosed with thoracic outlet syndrome. In addition, another five patients experienced greatly alleviated CTS symptoms after giving birth and chose to withdraw from the study. These patients all experienced significant postnatal weight loss with a mean difference of 5.5 kg (P = 0.0001). Lastly, one patient did not return at the three-month follow-up. During the study, there were four additional cases lost to follow-up, two cases from each treatment group. Thus, the final sample consisted of 40 patients, 20 in each group. The demographic data for both groups were tested pre-intervention to confirm homogeneity and no significant difference was found (P > 0.05) (Table 1).

Table 1

Table 1 Demographic data of subjects in both groups.

The comparisons of intragroup mean values of all variables in both groups, before and after end of the treatment showed a significant intragroup improvement in both groups (Table 2). Furthermore, Table 3 summarizes the intragroup differences for the Tinel’s test, Phalen’s test, VAS, and the functional status scale.

Table 2

Table 2 Comparison of mean pre and post treatment in both groups.

Table 3

Table 3 Tinel’s and Phalen’s tests, VAS, and functional status scale in both groups.

Clinical outcomes

Pain (VAS), showed significant improvement at the end of treatment in both groups, PEMF and US groups (P = 0.0001 and 0.021), respectively. PEMF leads to a 4.93 point reduction in VAS, while the US group had a 1.3 point reduction with a significant difference in the rate of improvement (P = 0.0001) in favour of PEMF (Table 3). Pre-treatment, the Tinel’s test was positive in 15 (75%) of the PEMF group and 17 (85%) of the US group and these numbers decreased significantly after treatment to 5 (25%) and 6 (30%) subjects, respectively. There was non-significant difference (P = 0.727) between the groups at the end of treatment (Table 3). The same was true for the Phalen’s test, as positive results were observed in 13 (65%) and 14 (70%) in both PEMF and US groups, respectively, and were reduced significantly to 4 (20%) and 6 (30%), respectively. There was a non-significant difference (P = 0.471) between the groups at the end of treatment (Table 3).

Hand grip strength showed significant improvement in both groups at the end of the intervention periods (Table 2), and PEMF showed a significantly higher level of improvement (P = 0.017, CI 0.32–2.68) in comparison with the US group’s hand grip strength. The functional status scores showed significant improvement intragroup (P = 0.0001) in both groups but there was non-significant difference (P = 0.414) between groups (Table 3).

Electrophysiological outcomes

Both MSDL and MMDL were significantly decreased, and MSCV and MMCV were significantly improved, in both groups at the end of the treatment (P < 0.05) (Table 2). PEMF showed significant intergroup differences in both MSDL (P = 0.001, CI ?2.23?(?1.42)) and MSCV (P = 0.0001, CI 15.3–20.03), with mean differences of 1.83 and 17.63 respectively, in comparison with the US group. In addition, both MMDL (P = 0.007, CI ?1.10?(?0.25)) and MMCV (P = 0.0001, CI 3.8–7.9) showed significant differences in favour of the PEMF group with mean differences of 0.67 and 5.86, respectively.

Discussion

CTS is a painful, debilitating condition; it has many therapeutic options, but no single treatment modality has been definitively established as superior to any other [21]. The results from conservative treatments vary, and there is no widespread agreement on the best method of treatment. Likewise, the results of surgery, with either an open or endoscopic transverse carpal ligament release, are inconsistent [22].

Forty postnatal women who developed CTS during their third trimester were involved in this study and were divided randomly into one of two treatment protocols: PEMF or therapeutic US. The data showed greater alleviation of disease symptoms with PEMF in comparison with therapeutic US in all outcome measures except for the functional status scale, which showed no significant difference between the two groups.

In the current study, five cases from the initial antenatal sample had their CTS symptoms diminish in the first two weeks after delivery. They all had significant postnatal weight loss (P = 0.0001), so their CTS regression was likely strongly related to their weight loss [23]. However, the rest of the women participants still had CTS postnatally, which is consistent with the fact that a significant percentage of women still have CTS symptoms up to three or more years after delivery, and continue to wear splints [10].

Additionally, CTS is associated with hand-intensive activities such as housework and typing, which may contribute to the higher incidence in women [24]. This is consistent with the current study, in which the participants were either housewives or administrative workers, in addition to being caregivers of their new-born child.

The Phalen’s and Tinel’s tests are clinical tests for CTS; both have high sensitivity and specificity [15]. In the current study, even though not all the enrolled patients had positive results in both these clinical tests, they were still given treatment in both groups. This was because, while not all pregnant women exhibit CTS symptoms, most, if not all, exhibit impaired median nerve function [25]. In fact, these clinical signs were found to be positive in a higher percentage of pregnant women to confirm CTS diagnosis, compared to neurophysiological indicators [26].

Both groups performed nerve and tendon gliding exercises as they are commonly employed for treating symptoms of CTS and are believed to improve axonal transport and nerve conduction [27]. The benefits of these exercises are prevention of adhesion formation even if the wrist is immobilized [28], reduction of pressure in the carpal tunnel, and maximization of the relative excursion of the median nerve and the flexor tendons [29]. These benefits were consistent with what was observed in the current study.

The superior intergroup improvements that were recorded in the PEMF group are attributable to the effects of PEMF on pain perception in the form of neuron firing, calcium ion movement, endorphin levels, acupuncture action, and nerve regeneration [30], [31]. A gating response with simultaneous stimulation of the A? fibres produces an inhibitory anti-nociceptive effect on C fibres, which is compatible with the Melzack–Wall hypothesis [31].

The PEMF group showed increased median nerve distal latency and nerve conduction velocity (NCV) that can be attributed to the stimulation of endothelial release of fibroblast growth factor beta–2 (FGF–2) [32], which stimulates neurotrophic factors and improves the micro-environment of the tissues, leading to regeneration of the nerve [33]. In the available literature, there is limited research on PEMF treatment for CTS [13]; nevertheless, a few studies support the current findings. In such studies, pilot data of static [34] and dynamic PEMF [35], [36] directed to the carpal tunnel region revealed significantly reduced neuropathic pain. Another research trial applied combined static and dynamic magnetic fields for 4 h per day over two months. There was significant pain reduction, but only mild improvement in objective neuronal functions in the magnetic treatment group versus placebo [37]. This mode of treatment was not appropriate in the current study because of the need to avoid long-term exposure of the newborn to PEMF at home. Despite there being no prior recorded side effects with treatment by magnetic therapy [38], patients were instructed not to bring their babies during sessions. They were also instructed to report side effects at any time, such as dizziness, headache, metallic taste in the mouth, or seizures. Fortunately, no patient in the PEMF group reported any of these side effects.

In contrast to the previously mentioned studies that found significant improvement with PEMF treatment, two small randomized trials [39], [40] concluded that there were no differences between the PEMF treatment and placebo groups. Both groups experienced insignificant improvement in symptoms. These results may be due to the treatment short duration (two weeks of PEMF application) in these studies.

Despite the intergroup superior effect of PEMF, the US group also exhibited significant intragroup improvements. These improvements are attributable to the ultrasonic thermal effects, leading to an increase in blood flow, local metabolism and tissue regeneration, and reduced inflammation, oedema and pain, thereby facilitating the recovery of nerve compression [41]. There is an inverse relationship between fibre size and sensitivity to US; hence, C fibres are more sensitive than A fibres. This selective absorption by smaller fibres may lead to a decrease in pain transmission [42]. Furthermore, the current study used deep, pulsed US (1 MHz and intensity of 1.0 W/cm2) over the carpal tunnel for 15 min, since superficial, continuous US was found to be no more effective than placebo US, and did not improve median nerve conduction [43], [44].

In addition, deep pulsed US has been shown to decrease pain and paresthesia symptoms, reduce sensory loss, and improve median NCV and strength when compared with placebo US [43], [45]. This form of US treatment can also provide a positive effect on sensation and patient-reported symptoms [43]. In the current study, this was captured by the functional status scale, which showed no significant difference between the two groups.

Conclusions

It can be concluded that PEMF has a significant and superior effect on CTS in postnatal women, as compared to therapeutic US. This superior effect was found in the reduction in pain, improvement in the electrophysiological studies, and hand grip strength. There are no reported side effects, discomforts, or known health risks from PEMF therapy, and it is generally accepted that brief exposure to this modality is safe [38], [46]. PEMF has lower treatment costs than surgery [47], but its cost effectiveness in comparison with other therapeutic options needs further investigation. There is a need to develop a treatment guideline for CTS, which includes a combination of different modalities and techniques.

Limitations

The current study had some limitations that should be addressed in future research, such as the small sample size. The literature lacks information about the standard PEMF dose for CTS, so a comparison of different PEMF doses is also needed. In addition, the current study did not investigate the long-term effect of the interventions.

Conflict of interest

The authors have declared no conflict of interest.

Footnotes

Peer review under responsibility of Cairo University.

figure fx1

References

1. Ashraf A., Daghaghzadeh A., Naseri M., Nasiri A., Fakheri M. A study of interpolation method in diagnosis of carpal tunnel syndrome. Ann Ind Acad Neurol. 2013;16(4):623–626. [PMC free article] [PubMed] 2. Hashempur H., Homayouni K., Ashraf A., Salehi A., Taghizadeh M., Heydari M. Effect of Linum usitatissimum L. (linseed) oil on mild and moderate carpal tunnel syndrome: a randomized, double blind, placebo-controlled clinical trial. DARU. 2014;22:43. [PubMed] 3. Atroshi I., Gummesson C., Johnsson R., Ornstein E., Ranstam J., Rosen I. Prevalence of carpel tunnel syndrome in a general population. JAMA. 1999;282(2):153–158.[PubMed] 4. Charles J., Fahridin S., Britt H. Carpal tunnel syndrome. Aust Fam Physician. 2009;38(9):665. [PubMed] 5. Bahrami H., Rayegani M., Fereidouni M., Baghbani M. Prevalence and severity of carpal tunnel syndrome (CTS) during pregnancy. Electromyogr Clin Neurophysiol. 2005;45(2):123–125. [PubMed] 6. Pazzaglia C., Caliandro P., Aprile I., Mondelli M., Foschini M., Tonali A. Multicenter study on carpal tunnel syndrome and pregnancy incidence and natural course. Acta Neurochir Suppl. 2005;92:35–39. [PubMed] 7. Voitk J., Mueller C., Farlinger E., Johnston U. Carpal tunnel syndrome in pregnancy. Can Med Assoc J. 1983;128:277–281. [PubMed] 8. Snell J., Coysh L., Snell J. Carpal tunnel syndrome presenting in the peurperium. Practitioner. 1980;224(1340):191–193. [PubMed] 9. Nicholas G., Noone B., Graham P. Carpal tunnel syndrome in pregnancy. Hand. 1971;3:80–83. [PubMed] 10. Monelli M., Rossi S., Monti E., Aprile I., Caliandro P., Pazzaglia C. Long term follow-up of carpal tunnel syndrome during pregnancy: a cohort study and review of the literature. Electromyogr Clin Neuorophysiol. 2007;47(6):259–271. [PubMed] 11. Ablove H., Ablove S. Prevalence of carpal tunnel syndrome in pregnant women. WMJ. 2009;108(4):194–196. [PubMed] 12. Hashempur M., Naseri M., Ashraf A. Carpal tunnel syndrome in lactation: a challenging issue. Women’s Health Bull. 2015;2(4):e31414. 13. Carlson H., Colbert A., Frydl J., Arnall E., Elliot M., Carlson N. Current options for nonsurgical management of carpal tunnel syndrome. Int J Clin Rheumtol. 2010;5(1):129–142. [PubMed] 14. Kouyoumdjian A. Carpal tunnel syndrome: age, nerve conduction severity and duration of symptomatology. Arq Neuro-Psiquiatr. 1999;57(2B):382–386. [PubMed] 15. Naranjo A., Ojeda S., Mendoza D., Francisco F., Quevedo J.C., Erausquin C. What is the diagnostic value of ultrasonography compared to physical evaluation in patients with idiopathic carpal tunnel syndrome? Clin Exp Rhemutatol. 2007;25:853–859.[PubMed] 16. Graham B. The value added by electro-diagnostic testing in the diagnosis of carpal tunnel syndrome. J Bone Joint Surg Am. 2008;90(2587):2587–2593. [PubMed] 17. Richards L., Palmiter P. Grip strength measurement: a critical review of tools, methods, and clinical utility. Crit Rev Phys Rehab Med. 1996;8:87–109. 18. Levine W., Simmons P., Koris J., Daltroy H., Hohl G., Fossel H. A self-administered questionnaire for the assessment severity of symptoms and functional status in carpal tunnel syndrome. J Bone Joint Surg Am. 1993;75A:1585–1592. [PubMed] 19. Totten A., Hunter M. Therapeutic techniques to enhance nerve gliding in thoracic outlet syndrome and carpal tunnel syndrome. Hand Clin. 1991;7:505–520. [PubMed] 20. Baysal O., Altay Z., Ozcan C., Ertem K., Yologlu S., Kayhan A. Comparison of three conservative treatment protocols in carpal tunnel syndrome. Int J Clin Pract. 2006;60(7):820–828. [PubMed] 21. Verdugo j., Salinas A., Castillo L., Cea G. Surgical versus nonsurgical treatment for carpal tunnel syndrome. Cochrance Database Syst Rev. 2008;(4) Art. No.: CD001552.[PubMed] 22. Breuer B., Sperber K., Wallenstein S. Clinically significant placebo analgesic response in a pilot trial of botulinum B in patients with hand pain and carpal tunnel syndrome. Pain Med. 2006;7(1):16–24. [PubMed] 23. Finsen V., Zeitlmann H. Carpal tunnel syndrome during pregnancy. Scand J Plast Reconstr Surg Hand Surg. 2006;40(1):41–45. [PubMed] 24. Tang X., Zhuang L., Lu Z. Carpal tunnel syndrome: a retrospective analysis of 262 cases and a one to one matched case control study of 61 women pairs in relationship between manual housework and carpal tunnel syndrome. Chin Med J (Engl) 1999;112:44–48. [PubMed] 25. Tupkovic E., Nisic M., Kendic S. Median nerve: neurophysiological parameters in the third trimester of pregnancy. Bosn J Basic Med Sci. 2007;7(1):84–89. [PubMed] 26. Padua L., Aprile I., Caliandro P., Mondelli M., Pasqualetti P., Tonali P.A. Carpal tunnel syndrome in pregnancy: multiperspective follow-up of untreated cases. Neurology. 2002;59(10):1643–1646. [PubMed] 27. Butler D., Gifford L. The concept of adverse mechanical tension in the nervous system, part 2; examination and treatment. Physiotherapy. 1989;75:629–636. 28. Szabo M., Bay K., Sharkey A. Median nerve displacement through the carpal canal. J Hand Surg [Am] 1994;19:901–906. [PubMed] 29. Rempel D., Manojlovic R., Levinsohn G., Bloom T., Gordon L. The effect of wearing a flexible wrist splint on carpal tunnel pressure during repetitive hand activity. J Hand Surg [Am] 1994;19:106–110. [PubMed] 30. Lee B., Kim C., Lim J., Lee J., Choi S., Park H. Efficacy of pulsed electromagnetic therapy for chronic lower back pain: a randomized, double-blind, placebo-controlled study. J Int Med Res. 2006;34(2):160–167. [PubMed] 31. Thamsborg G., Florescu A., Oturai P., Fallentin E., Tritsaris K., Dissing S. Treatment of knee osteoarthritis with pulsed electromagnetic fields: a randomized, double blind, placebo-controlled study. Osteoarthritis Cartilage. 2005;13:575–581. [PubMed] 32. Tepper M., Callaghan J., Chang I., Galiano D., Bhatt A., Baharestani S. Electromagnetic fields increase in vitro and in vivo angiogenesis through endothelial release of FGF-2. FASEB J. 2004;18:1231–1233. [PubMed] 33. Midura J., Ibiwoye O., Powell A., Sakai Y., Dochring T., Grabiner D. PEMF treatments enhance the healing of fibular osteotomies. J Orthop Res. 2005;23(5):1035–1046. [PubMed] 34. Weintraub I. Neuromagnetic treatment of pain in refractory carpal tunnel syndrome: an electrophysiological placebo analysis. J Back Musculoskeletal Rehabil. 2002;15:77–81. [PubMed] 35. Weintraub I., Cole S.P. Pulsed magnetic field therapy in refractory carpal tunnel syndrome: electro diagnostic parameters––pilot study. J Back Musculoskeletal Rehabil. 2005;18:79–83. 36. Weintraub I., Cole P. Time-varying, biaxial magnetic stimulation in refractory carpal tunnel syndrome: a novel treatment. Pilot Study: Semin Integr Med. 2005;3:123–128. 37. Weintraub I., Cole P. A randomized controlled trial of the effects of a combination of static and dynamic magnetic fields on carpal tunnel syndrome. Pain Med. 2008;9(5):493–504. [PubMed] 38. Colbert P., Markov S., Banerji M., Pilla A. Magnetic mattress pad use in patients with fibromyalgia: a randomized double-blind pilot study. J Back Musculoskelet Rehab. 1999;13:19–31. 39. Carter R., Aspy B., Mold J. The effectiveness of magnet therapy for treatment of wrist pain attributed to carpal tunnel syndrome. J Fam Pract. 2002;51(1):38–40.[PubMed] 40. Colbert P., Markov S., Carlson N., Gregory L., Carlson H., Elmer J. Static magnetic field therapy for carpal tunnel syndrome: a feasibility study. Arch Phys Med Rehabil. 2010;91(7):1098–1104. [PubMed] 41. Gerritsen A., de Krom C., Struijs A., Scholten J., de Vet C., Bouter M. Conservative treatment options for carpal tunnel syndrome: a systematic review of randomized controlled trials. J Neurol. 2002;249:272–280. [PubMed] 42. Young R., Henneman E. Reversible block of nerve conduction by ultrasound. Arch Neurol. 1961;4:83–89. [PubMed] 43. Ebenbichler R., Resch L., Nicolakis P., Wiesinger F., Uhl F., Ghanem H. Ultrasound treatment for treating the carpal tunnel syndrome: randomised “sham” controlled trial. BMJ. 1998 Mar 7;316(7133):731–735. [PubMed] 44. Oztas O., Turan B., Bora I., Karakaya K. Ultrasound therapy effect in carpal tunnel syndrome. Arch Phys Med Rehabil. 1998 Dec;79(12):1540–1544. [PubMed] 45. O’Connor D., Marshall S.C., Massy-Westropp N. Non-surgical treatment (other than steroid injection) for carpal tunnel syndrome. Cochrane Database Syst Rev. 2003;1:CD003219. [PubMed] 46. Trock D. Electromagnetic fields and magnets: investigational treatment for musculoskeletal disorders. Rheum Dis Clin North Am. 2000;26(1):51–62. [PubMed] 47. Rubik B. Bioelectromagnetics and the future of medicine. Admin Radiol J. 1997;16(8):38–46. [PubMed] . Adv Med Sci. Oct 29:1-5. [Epub ahead of print]

Comparison of the long – term effectiveness of physiotherapy programs with low – level laser therapy and pulsed magnetic field in patients with carpal tunnel syndrome.

Dakowicz A, Kuryliszyn-Moskal A, Koszty?a-Hojna B, Moskal D, Latosiewicz R.

Source

Department of Rehabilitation, Medical University of Bialystok, Bialystok, Poland.

Abstract

Purpose: The aim of the study was to compare the long term effects of low – level laser therapy (LLLT) and pulsed magnetic field (PMF) in the rehabilitation of patients with carpal tunnel syndrome (CTS).Methods: The study included 38 patients with idiopathic CTS, confirmed by electroneurographic (ENG) examination. All patients were randomly assigned to 2 groups: group L (18 patients) treated with LLLT a

nd group M (20 patients) with PMF therapy. Clinical assessment, including day and night pain, the presence of paresthesia, functional tests (Phalen, Tinel, armband tests) and pain severity according to the Visual Analogue Scale (VAS) was conducted before treatment, after the first series of 10 sessions, after a two-week break, after the second series of 10 sessions and six months after the last series.

Results: After LLLT a significant reduction of day and night pain was observed at each stage of treatment and 6 months after the last series (p<0.05). However, in group M, a significant reduction of both day and night pain was demonstrated only after the second series (p<0.05). A reduction of the incidence of Phalen’s symptoms were noticed in both groups, however, only in group L the improvement was significant (p<0.05). In groups L and M a significant reduction of pain intensity was observed at every stage of treatment (p<0.05).

Conclusions: Although after LLL as well as PMF therapy clinical improvement was observed, the most significant differences were registered after the second series and persisted for up to 6 months in both groups.

Arch Phys Med Rehabil. 2010 Jul;91(7):981-1004.

Carpal tunnel syndrome. Part I: effectiveness of nonsurgical treatments–a systematic review.

Huisstede BM, Hoogvliet P, Randsdorp MS, Glerum S, van Middelkoop M, Koes BW.

Department of General Practice, Erasmus Medical Center, Rotterdam, The Netherlands. b.huisstede@erasmusmc.nl

Abstract

OBJECTIVE: To review literature systematically concerning effectiveness of nonsurgical interventions for treating carpal tunnel syndrome (CTS).

DATA SOURCES: The Cochrane Library, PubMed, EMBASE, CINAHL, and PEDro were searched for relevant systematic reviews and randomized controlled trials (RCTs).

STUDY SELECTION: Two reviewers independently applied the inclusion criteria to select potential studies.

DATA EXTRACTION: Two reviewers independently extracted the data and assessed the methodologic quality.

DATA SYNTHESIS: A best-evidence synthesis was performed to summarize the results of the included studies. Two reviews and 20 RCTs were included. Strong and moderate evidence was found for the effectiveness of oral steroids, steroid injections, ultrasound, electromagnetic field therapy, nocturnal splinting, and the use of ergonomic keyboards compared with a standard keyboard, and traditional cupping versus heat pads in the short term. Also, moderate evidence was found for ultrasound in the midterm. With the exception of oral and steroid injections, no long-term results were reported for any of these treatments. No evidence was found for the effectiveness of oral steroids in long term. Moreover, although higher doses of steroid injections seem to be more effective in the midterm, the benefits of steroids injections were not maintained in the long term. For all other nonsurgical interventions studied, only limited or no evidence was found.

CONCLUSIONS: The reviewed evidence supports that a number of nonsurgical interventions benefit CTS in the short term, but there is sparse evidence on the midterm and long-term effectiveness of these interventions. Therefore, future studies should concentrate not only on short-term but also on midterm and long-term results.

Pain Med. 2008 Jul-Aug;9(5):493-504.

A randomized controlled trial of the effects of a combination of static and dynamic magnetic fields on carpal tunnel syndrome.

Weintraub MI, Cole SP.

Department of Neurology, New York Medical College, Valhalla, New York, USA. miwneuro@pol.net

Abstract

OBJECTIVE: To determine if a physics-based combination of simultaneous static and time-varying dynamic magnetic field stimulation to the wrist 4 hours/day for 2 months can reduce subjective neuropathic pain and influence objective electrophysiologic parameters of patients with carpal tunnel syndrome (CTS).

METHODS: Randomized, double-blinded, placebo-controlled trial of 36 symptomatic hands. Primary endpoints were visual analog scale (VAS) and neuropathic pain scale (NPS) scores at baseline and 2 months and a Patient’s Global Impression of Change (PGIC) questionnaire at the end of 2 months. Secondary endpoints were neurologic examination, median nerve distal latencies (compound muscle action potential [CMAP]/sensory nerve action potential [SNAP]), dynamometry, pinch gauge readings, and current perception threshold (CPT) scores. An “active” device was provided gratis at the end of the study, with 15 subjects voluntarily remaining within the open protocol an additional 2-10 months and using the preselected primary and secondary parameters.

RESULTS: (two months). Of the 31 hands, 25 (13 magnet, 12 sham) had moderate to severe pain (VAS > 4). The VAS and PGIC revealed a nonsignificant pain reduction. NPS analyses (anova) demonstrated a statistically significant reduction of “deep” pain (35% downward arrow vs 12% upward arrow, P = 0.018), NPS Total Composite (decreases of 42% vs 24%, P = 0.042), NPS Total Descriptor Score (NPS 8; 43% vs 24%), and NPS 4 (42% vs 11%). Motor strength, CMAP/SNAP, and CPT scores were not significantly changed. Of the 15 hands with up to 10 months of active PEMF (pulsed electromagnetic fields) exposure, there was objective improvement in nerve conduction (CMAP = 53%, SNAP = 40%, >1 SD), and subjective improvement on examination (40%), pain scores (50%), and PGIC (70%). No detectable changes in motor strength and CPT.

CONCLUSIONS: PEMF exposure in refractory CTS provides statistically significant short- and longterm pain reduction and mild improvement in objective neuronal functions. Neuromodulation appears to influence nociceptive-C and large A-fiber functions, probably through ion/ligand binding.

Cardiovascular Disease

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Sci Rep. 2016; 6: 30783. Published online 2016 Jul 29. doi:  10.1038/srep30783 PMCID: PMC4965791 PMID: 27470078

Noninvasive low-frequency electromagnetic stimulation of the left stellate ganglion reduces myocardial infarction-induced ventricular arrhythmia

Songyun Wang,1,* Xiaoya Zhou,1,* Bing Huang,1 Zhuo Wang,1 Liping Zhou,1 Menglong Wang,1 Lilei Yu,a,1 andHong Jiangb,11Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute of Wuhan University, Wuhan, 430060, Hubei, China aEmail: moc.361@ieliluyuhwbEmail: moc.361@gnohgnaijuhw*These authors contributed equally to this work. Author information ? Article notes ? Copyright and License information ? Disclaimer Received 2016 Jan 29; Accepted 2016 Jul 11. Copyright © 2016, Macmillan Publishers Limited This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Abstract

Noninvasive magnetic stimulation has been widely used in autonomic disorders in the past few decades, but few studies has been done in cardiac diseases. Recently, studies showed that low-frequency electromagnetic field (LF-EMF) might suppress atrial fibrillation by mediating the cardiac autonomic nervous system. In the present study, the effect of LF-EMF stimulation of left stellate ganglion (LSG) on LSG neural activity and ventricular arrhythmia has been studied in an acute myocardium infarction canine model. It is shown that LF-EMF stimulation leads to a reduction both in the neural activity of LSG and in the incidence of ventricular arrhythmia. The obtained results suggested that inhibition of the LSG neural activity might be the causal of the reduction of ventricular arrhythmia since previous studies have shown that LSG hyperactivity may facilitate the incidence of ventricular arrhythmia. LF-EMF stimulation might be a novel noninvasive substitute for the existing implant device-based electrical stimulation or sympathectomy in the treatment of cardiac disorders.

Previous studies have demonstrated that the activation and remodeling of left stellate ganglion (LSG) induced by myocardial infarction1,2 might be the immediate triggering mechanisms of ventricular arrhythmia (VA) and sudden cardiac death3,4, and suppressing LSG neural activity might be a feasible antiarrhythmic therapy5. In the past decades, LSG denervation and blocking have been shown to be benefit for reducing VA6. However, undesirable side effects, such as cervical injury and Horner’s syndrome, have limited the clinic use of LSG denervation or blocking. Therefore, exploring a novel noninvasive approach is necessary.

Transcranial magnetic stimulation (TMS), a neurostimulation and neuromodulation technique based on the principle of electromagnetic induction of an electric field in the brain, has been proposed for treatment of a variety of neurological disorders. Previous studies has shown that TMS might mediate the cardiac rhythm by modulating the autonomic nervous system7. Scherlag et al.8 showed that exposure the vagal trunks or the chest to the low-frequency magnetic field (LF-EMF) might suppress atrial fibrillation, whereas exposure to the high-frequency field might induce atrial fibrillation by autonomic modulating. Recently, Yu et al.9 further demonstrated that LF-EMF stimulation of the vagal trunks or chest might suppress atrial fibrillation by inhibiting the neural activity of atrial ganglionated plexus. In this study, we hypothesized that exposure LSG to the LF-EMF might inhibit the LSG neural activity, thereby reducing VAs after acute myocardial infarction6.

Results

LSG was exposed to intermittent LF-EMF stimulation before left anterior descending artery occlusion in LF-EMF group (Fig. 1A–C). Both the blood pressure and heart rate were kept at a stable level during the LF-EMF stimulation. No visible damage was shown in LSG or cardiac tissue after 90?min LF-EMF treatment. All dogs developed ECG ST-segment and/or T-wave changes acutely after ligating the left anterior descending artery.

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Open in a separate windowFigure 1

Schematic representation of the position of the LF-EMF (A), stimulus pattern (B) and the experimental design flow chart (C). LSG, left stellate ganglion; LF-EMF, low-frequency electromagnetic field; LAD, left anterior descending artery; MAP, monophasic action potential; HRV, heart rate variability; VA, ventricular arrhythmia.

Effect of LF-EMF stimulation on myocardial infarction-induced VAs

Figure 2A shows the representative examples of VAs in the Control group and LF-EMF group. As compared to the Control group, both the number of ventricular premature beat (VPB) and the number of non-sustained ventricular tachycardia (VT) were significantly decreased (Fig. 2B,C). Furthermore, the incidence of sustained VT/VF was significantly suppressed (75.0% vs 12.5%, P?<?0.05, Fig. 2D) in the LF-EMF group.

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Figure 2

Representative examples (A) and the incidence (BE) of AMI-induced VAs in the Control group (n?=?8) and EMF group (n?=?8). *P?<?0.05 and **P?<?0.05 as compared to the Control group. AMI, acute myocardial infarction; VPB, ventricular premature beats; VT, ventricular tachycardia; VF, ventricular fibrillation; other abbreviations as in Fig. 1.

Effect of LF-EMF stimulation on MAP

Figure 3A–F demonstrates the effect of LF-EMF on action potential duration at 90% repolarization (APD90Fig. 3A–C), pacing cycle length of action potential duration alternans (PCL, Fig. 3D–F) and the maximal slope of the restitute curve (SmaxFig. 3G–I). As compared to group baseline, no significant change was shown in APD90, PCL or Smax obtained from different sites of left ventricle in the Control group, whereas a significant change was shown in APD90, PCL and Smax of those sites both at 30?min and 90?min after LF-EMF stimulation in the LF-EMF group (Fig. 3A–F).

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Figure 3

Effect of LF-EMF stimulation on APD90 (A,B), PCL (C,D) and Smax (E,F) in the Control group (n?=?8) and EMF group (n?=?8). *P?<?0.05 and **P?<?0.05 as compared to the group baseline; #P?<?0.05 and ##P?<?0.05 as compared to the Control group. LVA, left ventricular apex; LVM, the median of left ventricle; LVB, left ventricular base; MAP, monophasic action potential; APD, action potential duration; APD90, monophasic action potential duration determined at 90% of repolarization; PCL, pacing cycle length of APD alternans; BH, baseline; Smax, the maximal slope of the restitution curve, other abbreviations are identical to Fig. 1.

Effect of LF-EMF stimulation on heart rate variability

Figure 4 demonstrates that both low frequency component (LF) and the ratio between LF the high component (LF/HF) were significantly decreased by LF-EMF stimulation both at 30?min and 90?min later but not by sham LF-EMF stimulation as compared to group baseline. In comparison with group baseline, acute myocardial infarction resulted in a significant change in LF (2.54?±?0.23?ms2 vs 1.72?±?0.12?ms2, P?<?0.01, Fig. 4A), high frequency component (HF, 1.01?±?0.08?ms2 vs 1.43?±?0.18?ms2, P?<?0.01, Fig. 4B) and LF/HF (2.51?±?0.34 vs 1.20?±?0.20, P?<?0.01, Fig. 4C) in the Control group, whereas those were kept at a normal level in the LF-EMF group (LF, 1.52?±?0.1?1?ms2 vs 1.68?±?0.10?ms2; HF, 1.43?±?0.12?ms2 vs 1.48?±?0.13?ms2; LF/HF, 1.06?±?0.10 vs 1.14?±?0.19, all P?>?0.05, Fig. 4A–C).

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Figure 4

Effect of LF-EMF stimulation on LF (A), HF (B) and LF/HF (C) in the Control group (n?=?8) and EMF group (n?=?8). *P?<?0.05 and **P?<?0.01 vs group baseline; #P?<?0.05 and ##P?<?0.05 as compared to the Control group. LF, low frequency; HF, high frequency; LF/HF, the ratio between LF and HF; BH, baseline. Other abbreviations are identical to those in Fig. 1.

Effect of LF-EMF stimulation on serum norepinephrine and LSG function

In comparison with group baseline, serum norepinephrine was decreased from 180.3?±?6.8?pg/ml to 162.5?±?5.8?pg/ml at 30?min later and to 160.3?±?5.2?pg/ml at 90?min later in the LF-EMF group, whereas kept a stable level in the Control group (Fig. 5A). Furthermore, the systolic blood pressure increase in response to LSG stimulation was kept a baseline level in the Control group (Fig. 5B), whereas significantly attenuated by LF-EMF in the LF-EMF group at a voltage of 20–30?V as compared to group baseline (Fig. 5C). Take 25?V for example, the maximal systolic blood pressure increase induced by LSG stimulation was decreased from 88.3?±?15.4% to 43.1?±?6.2% (P?<?0.01) at 90?min later, whereas kept at about 90% in the Control group (Fig. 5B,C).

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Figure 5

Effect of LF-EMF stimulation on serum NE (A) and LSG function (B,C) in the Control group (n?=?8) and EMF group (n?=?8). NS, P?>?0.05, *P?<?0.05 and **P?<?0.01 as compared to the Control group at the same time point. NE, norepinephrine. Other abbreviations are alike to those in Fig. 1.

Effect of LF-EMF stimulation on the neural activity of LSG

Figure 6A shows the representative examples of LSG neural activity at baseline, 30?min after LF-EMF stimulation, 90?min after LF-EMF stimulation and 15?min after acute myocardial infarction. Figure 6B,Cdemonstrates that no significant difference was shown both in the frequency and the amplitude of LSG neural activity between the Control group and the LF-EMF group. As compared to group baseline, LF-EMF stimulation resulted in a significant decrease in LSG neural activity at 30?min and 90?min later, whereas no significant change was caused by sham LF-EMF stimulation (Fig. 6B,C). Furthermore, as compared to baseline, the neural activity was significantly increased after acute myocardial infarction in the Control group (Frequency: 62.5?±?5.2impulse/min vs 112.2?±?8.1impulse/min, P?<?0.01; Amplitude: 0.18?±?0.03?mV vs 0.33?±?0.05?mV, P?<?0.01) but kept at a comparable level in the LF-EMF group (Frequency: 60.8?±?4.8impulse/min vs 65.6?±?4.8impulse/min, P?>?0.05; Amplitude: 0.19?±?0.02?mV vs 0.18?±?0.02?mV, P?>?0.05).

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Open in a separate windowFigure 6

Representative examples (A) and quantitative analysis (B,C) of LSG neural activity in the Control group (n?=?8) and EMF group (n?=?8). **P?<?0.01 as compared to group baseline; #P?<?0.05 and ##P?<?0.05 as compared to the Control group. All abbreviations are identical to Figs 1 and ?and22.

Discussion

In the present study, we applied LF-EMF at the body surface of LSG. Both the ventricular electrophysiological parameters (APD90, PCL, Smax) and autonomic neural activity (serum norepinephrine, LSG function and LSG neural activity) were significantly affected by LF-EMF stimulation. Furthermore, the acute myocardial infarction-induced increased neural activity of LSG was significantly attenuated and the VAs was significantly reduced by LF-EMF. These findings suggested that exposure the LSG to LF-EMF might significantly reduce the neural activity of LSG, therefore reducing the incidence of VAs.

Previous studies have shown that activation of LSG facilitates, whereas inhibition of LSG protects against VAs4,10. In the past two decades, TMS has been widely used in clinical neurology11,12. Amounts of studies have shown that high-frequency stimulation increases cortical excitability, whereas low-frequency stimulation decreases neuronal excitability11,12. Recently, studies also demonstrated that TMS might affect the cardiac rhythm by modulating the autonomic nervous system7. Scherlag et al.8 showed that high-frequency magnetic stimulation of the vagal nerves might induce atrial tachycardia and atrial fibrillation, which was eliminated after propranolol and atropine injection. Low-frequency stimulation of the vagal nerves, however, reduced the heart rate and decreased the voltage required to induce atrioventricular conduction block8. Furthermore, recent study demonstrated that exposure the heart to the LF-EMF might significantly suppress atrial fibrillation and the mechanism might be by modulating the neural activity of atrial ganglionated plexus9. In the present study, we found that exposure the LSG to the LF-EMF significantly reduced the serum norepinephrine, neural activity of LSG and VAs. All these indicate that noninvasive LF-EMF might reduce VAs by facilitating the autonomic rebalance, but what underlie the beneficial effects of LF-EMF on LSG was poorly defined.

In the present study, we suggested some possible mechanisms underlying the suppressing of LSG neural activity. Firstly, TMS, as an effective treatment for patients with neural disorders, has been implicated long-lasting therapeutic effects after the cessation of TMS treatment13. Most researchers have contributed these effects to be long-term depression (LTD) and long-term potentiation (LTP) cause the duration of the effects seemed to implicate changes in synaptic plasticity13. LTD is caused by low-frequency stimulation or the stimulation of a postsynaptic neuron, whereas LTP is caused by high-frequency stimulation or the stimulation of a presynaptic neuron13. Ca++ signal, which is known to regulate membrane excitability and modulate second messengers related to multiple receptors and signal transduction pathways, has been shown to be the major determinant whether LTD or LTD arises14,15. Recently, Scherlag et al.8 also suggested that LTP or LTD was existed cause exposure the chest to the low-frequency electromagnetic field for 35?mins might result in the suppression of atrial fibrillation for 3 to 4?hours after the application of LF-EMF. In the present study, we also found that pretreatment with LF-EMF might significantly attenuated the acute myocardial infarction-induced activation of LSG neural activity and VAs, suggesting that LTP or LTD might be a potential explain for the salutary effects of LF-EMF stimulation. Secondly, previous studies have shown that TMS might also affect the expression levels of various receptors and other neuromediators, such as ?-adrenoreceptors, dopamine11,16,17. In the present study, serum norepinephrine was significantly decreased after exposure to the LF-EMF, indicating that modulating the neurotransmitters might be one of the underlying mechanisms underlying the salutary effects of LF-EMF stimulation. Thirdly, previous studies also showed that TMS might also modulate dentritic sprouting (axon growth) and the density of synaptic contacts, and the authors suggested that these results are associated with the Brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB) signaling system18,19. BDNF, as the most abundant neurotrophin in the brain, was reported to be a major contributor to the N-methyl-D-aspartate receptor-dependent LTP and LTD processes20. Wang et al.21 demonstrated that low-frequency TMS might reduce BDNF levels. High-frequency stimulation, however, might increase serum BDNF levels and the affinity of BDNF for TrkB receptors. Furthermore, previous studies also showed that trancranial stimulation might result in the changes in neural-related proteins, such as c-fos and tyrosine hydroxylase, which are closely related with the neural remodeling processes6,13,20. Autonomic neural remodeling, however, plays a key role in the initiation and maintenance of VAs4,10. All these implicate that modulating autonomic neural remodeling might be another mechanism of the antiarrhythmic effect of LF-EMF stimulation. Fourthly, the above mainly shows the underlying mechanisms of LF-EMF stimulation, but how can the LSG perceive the LF-EMF remains unknown. During the past few decades, many mechanisms, which might provide the basis for how the animals detect magnetic fields, have been proposed22. However, the magnetoreceptors have not been identified with certainty in any animal, and the mode of transduction for the magnetic sense remains unknown23. Recently, Xie et al. hypothesized that the putative magnetoreceptor, the iron-sulphur cluster protein, might combine with the magnetoreception-related photoreceptor cryptochromes to form the basis of magnetoreception in animals and this was corroborated in pigeon retina24. Furthermore, Zhang et al. further showed that the cells which had been transfected iron-sulphur cluster protein might response to the remote magnetic stimulation25. All these indicate that the iron-sulphur cluster protein might be the potential magnetoreceptor for the animals to detect the magnetic fields.

Though the present study showed wonderful results, but there are some limitations in this study. First, anesthesia with pentobarbital might affect the autonomic nervous system. However, this could be counteracted cause anesthesia was maintained continuously during the whole surgery and conducted in a same fashion in both groups. Second, the coil used in this study is too large to achieve LSG-targeted stimulation without affecting the surrounding tissues. It would be a great step forward if the coils could be technically improved. Third, we only observed the effect of LF-EMF in acute canine model. Fourth, we mainly focused on the autonomic nervous system imbalance, one of the major contributors of post-infarction VAs, cause we intervened the LSG with LF-EMF in this study. It’s a great limitation that some other major factors, like area at risk, infarct size, degree of collateral flow and the possibility of any preconditioning pathway were not involved in this study. However, previous studies have shown that LSG activation might facilitate the incidence of VAs, whereas pre-emptive or post-ischemic/infarction LSG inhibition by blockage or denervation might decrease the incidence of VAs and improve the infarct size, collateral flow, contractile force both in animals26,27,28,29 and patients30,31. Furthermore, studies have shown that LSG stimulation might increase the likelihood of early or delayed afterdepolarization development and the initiation of reentry, thereby resulting in the incidence of VAs32,33,34. In this study, LF-EMF stimulation of the LSG might significantly inhibit the neural activity of LSG, thereby reducing the incidence of VAs. Therefore, it’s reasonable to refer that improving the above factors might also be the potential mechanisms underlying the beneficial effects of LF-EMF stimulation, but further studies with optimized parameters and all-round considerations are required in the future.

In conclusion, the present study showed that LF-EMF stimulation might significantly reduce the neural function and neural activity of LSG. Exposure the LSG to the LF-EMF might be a feasible method for preventing the acute myocardial infarction-induced VAs. However, larger studies with optimized parameters should be done in the chronic models to verify the beneficial effect of LF-EMF stimulation.

Methods

Animal preparation

Sixteen canines weighing between 20 and 25?kg were included in this study. The experiments were approved by the Animal Ethics Committee of Wuhan University under approval number 2015–0445 and followed the guidelines outlined by the Care and Use of Laboratory Animals of the National Institutes of Health. All surgeries were performed under anesthesia with sodium pentobarbital at an initial dose of 30?mg/kg and a maintenance dose of 60?mg/h. The depth of anesthesia was evaluated by monitoring corneal reflexes, jaw tone, and alterations in cardiovascular indices. The body surface electrocardiogram was recorded throughout the experiment with a computer-based Lab System (Lead 2000B, Jingjiang Inc., Wuhan, China). The core body temperature of the dogs was kept at 36.5?±?1.5?°C. Left thoracotomy was conducted at the fourth intercostal space. At the end of the experiment, canines were a lethal dose of pentobarbital (100?mg/kg, iv).

LF-EMF

Repeated LF-EMF was supplied by the magnetic stimulation machine (YRD CCY-I, YiRuiDe Inc., Wuhan, China) with the curve 8 coil located at the body surface of the LSG (Fig. 1A). The LSG was stimulated by intermittent (8?s ON, 10?s OFF) LF-EMF stimulation with the frequency set at 1?HZ and intensity at approximately 90% of motor threshold (Fig. 1B). Motor threshold was defined as the lowest electromagnetic intensity that induced muscle contractions in the proximal forepaw and shoulder.

Monophasic action potential recording

Monophasic action potentials from the left ventricle were recorded with a custom-made Ag–AgCl catheter. A dynamic steady state pacing protocol (S1S1) was performed to determine action potential duration alternans35. The pulse train was delivered at an initial cycle length slightly shorter than the sinus cycle length and the drive train of stimuli was maintained for 30?s to ensure a steady state, and then a 2-min interruption was taken to minimize the pacing memory effects. After that, another pulse train with the PCL decreased by 10?ms was delivered until action potential duration alternans appeared. Action potential duration alternans was defined as ?APD90?10?ms for ?5 consecutive beats36. The monophasic action potential recordings were analyzed by the LEAD 2000B work station system (Lead 2000B, Jingjiang Inc. China). The APD90 was defined as the 90% repolarization duration and the diastolic interval was the time interval from the previous APD90 point to the activation time of the following beat. As described in previous studies, the dynamic action potential duration restitution curves were constructed from (Diastolic interval, APD90) pairs using Origin 8.0 (OriginLab, Co., Northampton, MA, USA)35,37. Slope of the shortest diastolic interval was defined as Smax.

Measurements of heart rate variability

Spectral power for heart rate variability was analyzed on 5-minute electrocardiogram recording segments and an autoregressive algorithm was used to analyze digitized signals from the electrocardiographic recordings. The following power spectral variables were determined: HF, LF and LF/HF38.

Neural recording from the LSG

To record the neural activity of the LSG, one tungsten-coated microelectrode was inserted into the fascia of the LSG and one ground lead was connected to the chest wall. The signal of the LSG was recorded with a PowerLab data acquisition system (8/35, AD Instruments, Australia) and amplified by an amplifier (DP-304, Warner Instruments, Hamden, CT, USA). The band-pass filters were set at 300?Hz to 1?kHz and the amplification ranges from 30 to 50 times39. The neural activity, deflections with a signal-to-noise ratio greater than 3:1, was manually determined as described in our previous studies39,40,41.

LSG function

LSG function was measured as the LSG stimulation-induced maximal change in systolic blood pressure as described in our previous study38. High frequency stimulation (20?Hz, 0.1?ms pulse duration) was applied to the LSG using a stimulator (Grass-S88; Astro-Med, West Warwick, RI, USA). The voltage ranged from 20?V to 30?V and increased by 5?V. To eliminate the residual effect of the LSG stimulation, each stimulation should be less than 30?s and the next stimulation should be not be taken until the blood pressure returned to a normal level.

Blood sampling

Venous blood samples were collected. Serum was separated by centrifuging at 3000?rpm for 15?min at 4?°C, and stored at ?80?°C until assayed. The serum norepinephrine level was measured with a canine-specific high sensitivity ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing City, China)38.

Measurement of the acute myocardial infarction-induced VAs

The left anterior descending coronary artery was ligated at approximately 2.5?centimeters away from its origin to induce acute myocardial infarction. The incidence and duration of the VAs induced by acute myocardial infarction during the first hour was analyzed. The VAs recorded on the ECG were defined as following42: VPBs, identifiable premature QRS complexes; VT, three or more consecutive VPBs; non-sustained VT, VT terminating spontaneously within 30?s; sustained VT, VT sustained for more than 30?s; and VF, a tachycardia with random ECG morphology and an associated loss of arterial blood pressure that degenerates into ventricular asystole.

Experimental protocol

Sixteen dogs were randomly divided into LF-EMF group (n?=?8, with LF-EMF) and Control group (n?=?8, with sham LF-EMF). LF-EMF (1?HZ; stimulation time 8?s; interstimulus interval, 5?s) was delivered to the surface area of LSG for 90?minutes. As shown in Fig. 1C, monophasic action potential, heart rate variability, serum norepinephrine, LSG function and LSG neural activity were measured at baseline, 30?min and 90?min after LF-EMF treatment. Measurements of heart rate variability and LSG neural activity were repeated at 15?min after acute myocardial infarction. Furthermore, the incidence of VAs was recorded during the first hour after acute myocardial infarction.

Statistical analysis

Continuous variables are presented as the mean?±?SEM and were analyzed by t test, one-way ANOVA, or two-way repeated-measures ANOVA with a Bonferroni posthoc test. To compare the incidence of VF between groups, Fisher’s exact test was used. All data was analyzed by GraphPad Prism version 5.0 software (GraphPad Software, Inc., San Diego, CA), and two-tailed P???0.05 was considered significant.

Additional Information

How to cite this article: Wang, S. et al. Noninvasive low-frequency electromagnetic stimulation of the left stellate ganglion reduces myocardial infarction-induced ventricular arrhythmia. Sci. Rep. 6, 30783; doi: 10.1038/srep30783 (2016).

Acknowledgments

This work was supported by the grants from National Natural Science Foundation of China No. 81270339, No. 81300182, No. 81530011, No. 81570463, grant from the Natural Science Foundation of Hubei Province No. 2013CFB302, and grants from the Fundamental Research Funds for the Central Universities No. 2042014kf0110 and No. 2042015kf0187.

Footnotes

Author Contributions S.W. and X.Z. wrote the main manuscript text and prepared figures; B.H., Z.W., L.Z. and M.W. performed experiments and anlalyzed data; L.Y. and H.J. designed the project and revised the paper. All authors reviewed and approved the final version.

References

  • Ajijola O. A. & Shivkumar K. Neural remodeling and myocardial infarction: the stellate ganglion as a double agent. Journal of the American College of Cardiology. 59, 962–964 (2012). [PMC free article] [PubMed]
  • Han S. et al. . Electroanatomic remodeling of the left stellate ganglion after myocardial infarction. Journal of the American College of Cardiology. 59, 954–961 (2012). [PMC free article] [PubMed]
  • Saffitz J. E. Sympathetic neural activity and the pathogenesis of sudden cardiac death. Heart rhythm.5, 140–141 (2008). [PubMed]
  • Zhou S. et al. . Spontaneous stellate ganglion nerve activity and ventricular arrhythmia in a canine model of sudden death. Heart rhythm. 5, 131–139 (2008). [PubMed]
  • Hayase J., Patel J., Narayan S. M. & Krummen D. E. Percutaneous stellate ganglion block suppressing VT and VF in a patient refractory to VT ablation. Journal of cardiovascular electrophysiology. 24, 926–928 (2013). [PMC free article] [PubMed]
  • Funamizu H., Ogiue-Ikeda M., Mukai H., Kawato S. & Ueno S. Acute repetitive transcranial magnetic stimulation reactivates dopaminergic system in lesion rats. Neuroscience letters. 383, 77–81 (2005). [PubMed]
  • Cabrerizo M. et al. . Induced effects of transcranial magnetic stimulation on the autonomic nervous system and the cardiac rhythm. ScientificWorldJournal. 2014, 349718 (2014). [PMC free article][PubMed]
  • Scherlag B. J. et al. . Magnetism and cardiac arrhythmias. Cardiol Rev. 12, 85–96 (2004). [PubMed]
  • Yu L. et al. . The use of low-level electromagnetic fields to suppress atrial fibrillation. Heart rhythm: the official journal of the Heart Rhythm Society. 12, 809–817 (2015). [PubMed]
  • Chen P. S. et al. . Sympathetic nerve sprouting, electrical remodeling and the mechanisms of sudden cardiac death. Cardiovasc Res. 50, 409–416 (2001). [PubMed]
  • Kobayashi M. & Pascual-Leone A. Transcranial magnetic stimulation in neurology. Lancet Neurol.2, 145–156 (2003). [PubMed]
  • Wassermann E. M. & Lisanby S. H. Therapeutic application of repetitive transcranial magnetic stimulation: a review. Clin Neurophysiol. 112, 1367–1377 (2001). [PubMed]
  • Chervyakov A. V., Chernyavsky A. Y., Sinitsyn D. O. & Piradov M. A. Possible Mechanisms Underlying the Therapeutic Effects of Transcranial Magnetic Stimulation. Front Hum Neurosci. 9, 303 (2015). [PMC free article] [PubMed]
  • Gaetani R. et al. . Differentiation of human adult cardiac stem cells exposed to extremely low-frequency electromagnetic fields. Cardiovasc Res. 82, 411–420 (2009). [PubMed]
  • Waxman S. G. & Zamponi G. W. Regulating excitability of peripheral afferents: emerging ion channel targets. Nat Neurosci. 17, 153–163 (2014). [PubMed]
  • Frye R. E., Rotenberg A., Ousley M. & Pascual-Leone A. Transcranial magnetic stimulation in child neurology: current and future directions. J Child Neurol. 23, 79–96 (2008). [PMC free article][PubMed]
  • Hemond C. C. & Fregni F. Transcranial magnetic stimulation in neurology: what we have learned from randomized controlled studies. Neuromodulation. 10, 333–344 (2007). [PubMed]
  • Amassian V. E., Stewart M., Quirk G. J. & Rosenthal J. L. Physiological basis of motor effects of a transient stimulus to cerebral cortex. Neurosurgery. 20, 74–93 (1987). [PubMed]
  • Di Lazzaro V., Ziemann U. & Lemon R. N. State of the art: Physiology of transcranial motor cortex stimulation. Brain Stimul 1, 345–362 (2008). [PubMed]
  • Ziemann U. et al. . TMS and drugs revisited 2014. Clin Neurophysiol (2014). [PubMed]
  • Wang H. Y. et al. . Repetitive transcranial magnetic stimulation enhances BDNF-TrkB signaling in both brain and lymphocyte. The Journal of neuroscience: the official journal of the Society for Neuroscience. 31, 11044–11054 (2011). [PMC free article] [PubMed]
  • Wiltschko R. W., W. Provides a comprehensive review of magbetoreception research and its history up to 1995. Spinger (1995).
  • Johnsen S. & Lohmann K. J. The physics and neurobiology of magnetoreception. Nat Rev Neurosci.6, 703–712 (2005). [PubMed]
  • Qin S. et al. . A magnetic protein biocompass. Nat Mater (2015).
  • Long X., Ye J., Zhao D. & Zhang S. J. Magnetogenetics: remote non-invasive magnetic activation of neuronal activity with a magnetoreceptor. Sci. Bull. 1–13 (2015). [PMC free article] [PubMed]
  • Kingma J. G., Simard D., Voisine P. & Rouleau J. R. Influence of cardiac decentralization on cardioprotection. PLoS One. 8, e79190 (2013). [PMC free article] [PubMed]
  • Jones C. E., Devous M. D. Sr., Thomas J. X. Jr. & DuPont E. The effect of chronic cardiac denervation on infarct size following acute coronary occlusion. Am Heart J. 95, 738–746 (1978).[PubMed]
  • Thomas J. X. Jr., Randall W. C. & Jones C. E. Protective effect of chronic versus acute cardiac denervation on contractile force during coronary occlusion. Am Heart J. 102, 157–161 (1981).[PubMed]
  • Yokoyama M. et al. . An experimental study on the role of coronary collateral development in preservation and improvement of contractile force in the ischemic myocardium. Jpn Circ J. 42, 1249–1256 (1978). [PubMed]
  • Biagini A. et al. . Treatment of perinfarction recurrent ventricular fibrillation by percutaneous pharmacological block of left stellate ganglion. Clin Cardiol. 8, 111–113 (1985). [PubMed]
  • Hartikainen J., Mustonen J., Kuikka J., Vanninen E. & Kettunen R. Cardiac sympathetic denervation in patients with coronary artery disease without previous myocardial infarction. Am J Cardiol. 80, 273–277 (1997). [PubMed]
  • Huffaker R., Lamp S. T., Weiss J. N. & Kogan B. Intracellular calcium cycling, early afterdepolarizations, and reentry in simulated long QT syndrome. Heart Rhythm. 1, 441–448, doi: 10.1016/j.hrthm.2004.06.005 (2004). [PubMed] [Cross Ref]
  • Priori S. G., Mantica M. & Schwartz P. J. Delayed afterdepolarizations elicited in vivo by left stellate ganglion stimulation. Circulation. 78, 178–185 (1988). [PubMed]
  • Shimizu W. & Antzelevitch C. Cellular basis for the ECG features of the LQT1 form of the long-QT syndrome: effects of beta-adrenergic agonists and antagonists and sodium channel blockers on transmural dispersion of repolarization and torsade de pointes. Circulation. 98, 2314–2322 (1998).[PubMed]
  • He B. et al. . Effects of ganglionated plexi ablation on ventricular electrophysiological properties in normal hearts and after acute myocardial ischemia. Int J Cardiol. 168, 86–93 (2013). [PubMed]
  • Banville I., Chattipakorn N. & Gray R. A. Restitution dynamics during pacing and arrhythmias in isolated pig hearts. Journal of cardiovascular electrophysiology. 15, 455–463 (2004). [PubMed]
  • He B. et al. . Effects of low-intensity atrial ganglionated plexi stimulation on ventricular electrophysiology and arrhythmogenesis. Autonomic neuroscience: basic & clinical. 174, 54–60 (2013). [PubMed]
  • Huang B. et al. . Left renal nerves stimulation facilitates ischemia-induced ventricular arrhythmia by increasing nerve activity of left stellate ganglion. Journal of cardiovascular electrophysiology. 25(2014). [PubMed]
  • Yu L. et al. . Low-level transcutaneous electrical stimulation of the auricular branch of the vagus nerve: a noninvasive approach to treat the initial phase of atrial fibrillation. Heart rhythm. 10, 428–435 (2013). [PubMed]
  • Wang S. et al. . Spinal cord stimulation suppresses atrial fibrillation by inhibiting autonomic remodeling. Heart rhythm. 13, 274–281 (2016). [PubMed]
  • Wang S. et al. . Spinal cord stimulation protects against ventricular arrhythmias by suppressing left stellate ganglion neural activity in an acute myocardial infarction canine model. Heart rhythm. 12, 1628–1635 (2015). [PubMed]
  • Walker M. J. et al. . The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia infarction, and reperfusion. Cardiovascular research 22, 447–455 (1988). [PubMed]

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Biosci Rep. 2016 Dec; 36(6): e00420. Published online 2016 Dec 5. Prepublished online 2016 Oct 25. doi:  10.1042/BSR20160082 PMCID: PMC5137536

Novel protective effects of pulsed electromagnetic field ischemia/reperfusion injury rats

Fenfen Ma,*,1 Wenwen Li,‡,1 Xinghui Li, Ba Hieu Tran,§ Rinkiko Suguro,§ Ruijuan Guan, Cuilan Hou, Huijuan Wang,? Aijie Zhang, Yichun Zhu, and YiZhun Zhu?¶,2*Department of Pharmacy, Shanghai Pudong Hospital, Fudan University, Shanghai 201399, China Shanghai Institute of Immunology & Department of Immunobiology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China Shanghai Key Laboratory of Bioactive Small Molecules and Research Center on Aging and Medicine, Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China §Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China ?Longhua Hospital, Shanghai University of Tradition Chinese Medicine, Shanghai 201203,China Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore 119228, Singapore 1These authors contributed equally to the article. 2To whom correspondence should be addressed (email nc.ude.naduf@zyuhz). Author information ? Article notes ? Copyright and License information ? Received 2016 Mar 17; Revised 2016 Oct 11; Accepted 2016 Oct 17. Copyright © 2016 The Author(s) This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY).

Abstract

Extracorporeal pulsed electromagnetic field (PEMF) has shown the ability to regenerate tissue by promoting cell proliferation. In the present study, we investigated for the first time whether PEMF treatment could improve the myocardial ischaemia/reperfusion (I/R) injury and uncovered its underlying mechanisms.

In our study, we demonstrated for the first time that extracorporeal PEMF has a novel effect on myocardial I/R injury. The number and function of circulating endothelial progenitor cells (EPCs) were increased in PEMF treating rats. The in vivo results showed that per-treatment of PEMF could significantly improve the cardiac function in I/R injury group. In addition, PEMF treatment also reduced the apoptosis of myocardial cells by up-regulating the expression of anti-apoptosis protein B-cell lymphoma 2 (Bcl-2) and down-regulating the expression of pro-apoptosis protein (Bax). In vitro, the results showed that PEMF treatment could significantly reduce the apoptosis and reactive oxygen species (ROS) levels in primary neonatal rat cardiac ventricular myocytes (NRCMs) induced by hypoxia/reoxygenation (H/R). In particular, PEMF increased the phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS), which might be closely related to attenuated cell apoptosis by increasing the releasing of nitric oxide (NO). Therefore, our data indicated that PEMF could be a potential candidate for I/R injury.Keywords: apoptosis, Bax, B-cell lymphoma 2 (Bcl-2), ischaemia/reperfusion (I/R) injury, pulsed electromagnetic field (PEMF)

INTRODUCTION

Hypertension, arrhythmia, myocardial infarction (MI) and myocardial ischaemia/reperfusion (I/R) injury are all the most common cardiac diseases, which are the major causes of mortality in the world [1]. Among them, myocardial I/R injury is the most important cause of cardiac damage. Its pathological process is closely related to postoperative complications [2,3] caused by coronary artery vascular formation, coronary revascularization and heart transplantation. After myocardium suffered severe ischaemia, restoration of the blood flow is a prerequisite for myocardial salvage [2]. However, reperfusion may induce oxidative stress [4], inflammatory cell infiltration and calcium dysregulation [5]. All these players contribute to the heart damage such as contraction and arrhythmias [6], generally named myocardial I/R injury. Recently, more and more evolving therapies have been put into use for I/R injury.

Pulsed electromagnetic field (PEMF) is the most widely tested and investigated technique in the various forms of electromagnetic stimulations for wound healing [7], alleviating traumatic pain and neuronal regeneration [8,9]. The rats were randomly divided into PEMF-treated (5 mT, 25 Hz, 1 h daily) and control groups. They hypothesized the possible mechanism that PEMF would increase the myofibroblast population, contributing to wound closure during diabetic wound healing. It is a non-invasive and non-pharmacological intervention therapy. Recent studies indicated that PEMF also stimulated angiogenesis in patients with diabetes [10], and could improve arrhythmia, hypertension and MI [1]. The MI rats were exposed to active PEMF for 4 cycles per day (8 min/cycle, 30±3 Hz, 6 mT) after MI induction. In vitro, PEMF induced the degree of human umbilical venous endothelial cells tubulization and increased soluble pro-angiogenic factor secretion [VEGF and nitric oxide (NO)] [7]. However, the role of PEMF in ischaemia and reperfusion diseases remains largely unknown. Our study aimed to investigate the effects of PEMF preconditioning on myocardial I/R injury and to investigate the involved mechanisms.

In our study, we verified the cardioprotective effects of PEMF in myocardial I/R rats and the anti-apoptotic effects of PEMF in neonatal rat cardiac ventricular myocytes (NRCMs) subjected to hypoxia/reoxygenation (H/R). We hypothesized that PEMF treatment could alleviate myocardial I/R injury through elevating the protein expression of B-cell lymphoma 2 (Bcl-2), phosphorylation of protein kinase B (Akt). Meanwhile, it could decrease Bax. We emphatically made an effort to investigate the MI/R model and tried to uncover the underlying mechanisms.

MATERIALS AND METHODS

Animals

Male, 12-week-old Sprague Dawley (SD) rats (250–300 g) were purchased from Shanghai SLAC Laboratory Animal. Animals were housed in an environmentally controlled breeding room and given free access to food and water supplies. All animals were handled according to the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH). Experimental procedures were managed according to the Institutional Aminal Care and Use Committee (IACUC), School of Pharmacy, Fudan University.

The measurement of blood pressure in SHR rats

At the end of 1 week treatment with PEMF, the rats were anesthetized with chloral hydrate (350 mg/kg, i.p.), the right common carotid artery (CCA) was cannulated with polyethylene tubing for recording of the left ventricle pressures (MFlab 200, AMP 20130830, Image analysis system of physiology and pathology of Fudan University, Shanghai, China).

Myocardial I/R injury rat model and measurement of infarct size

All the rats were divided into three groups: (1) Sham: The silk was put under the left anterior descending (LAD) without ligation; (2) I/R: Hearts were subjected to ischaemia for 45 min and then reperfusion for 4 h; (3) I/R + PEMF: PEMF device was provided by Biomobie Regenerative Medicine Technology. The I/R rats were pre-exposed to active PEMF for 2 cycles per day (8 min per cycle), whereas other two groups were housed with inactive PEMF generator. I/R was performed by temporary ligation of the LAD coronary artery for 45 min through an incision in the fourth intercostal space under anaesthesia [11]. Then, the ligature was removed after 45 min of ischaemia, and the myocardium was reperfused for 4 h. Ischaemia and reperfusion were confirmed and monitored by electrocardiogram (ECG) observation. The suture was then tightened again, and rats were intravenously injected with 2% Evans Blue (Sigma–Aldrich). After explantation of the hearts, the left ventricles were isolated, divided into 1 mm slices, and subsequently incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma–Aldrich) in 0.9% saline at 37°C for 25 min, to distinguish infarcted tissue from viable myocardium. These slices were flushed with saline and then fixed in 10% paraformaldehyde in PBS (pH 7.4) for 2 h. Next, the slices were placed on a glass slice and photographed by digital camera, the ImageJ software (NIH) was used in a blind fashion for analysis. Infarct size was expressed as a ratio of the infarct area and the area at risk [12].

Pulsed electromagnetic field treatment

PEMF were generated by a commercially available healing device (length × width × height: 7 cm × 5cm × 3cm) purchased from Biomoble Regenerative Medicine Technology. The adapter input voltage parameter is approximately 100–240 V and output parameter is 5 V. Fields were asymmetric and consisted of 4.5 ms pulses at 30±3 Hz, with an adjustable magnetic field strength range (X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis 0.06±0.02 mT). The I/R rats were housed in custom designed cages and exposed to active PEMF for 2 cycles per time (8 min for 1 cycle), whereas the I/R rats were housed in identical cages with inactive PEMF generator. For in vitro study, culture dishes were directly exposed to PEMF for 1–2 cycles as indicated (8 min for 1 cycle, 30 Hz, X-axis 0.22 mT, Y-axis 0.20 mT, Z-axis 0.06 mT) [1]. The background magnetic field in the room area of exposure animals/samples and controls is 0 mT.

Detection of myocardium apoptosis

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay was applied to analyse cardiomyocyte apoptosis. Heart samples were first fixed in 10% formalin and then paraffin embedded at day 14. Then, the hearts were cut into 5 ?m sections. TUNEL staining was carried out as described previously [12]. When apoptosis occurred, cells would look green.

Determination of myocardial enzymes in plasma

Blood samples were collected after haemodynamic measurement and centrifuged at 3000 g for 15 min to get the plasma. Creatine kinase (CK), lactate dehydrogenase (LDH), creatine kinase isoenzyme-MB (CKMB) and ?-hydroxybutyrate dehydrogenase (HBDH) were quantified by automatic biochemical analyzer (Cobas 6000, Roche). All procedures were performed according to the manufacturer’s protocols.

Myocardium cells morphology via TEM

At the end of the experiment, sections from myocardial samples of left ventricular were immediately fixed overnight in glutaraldehyde solution at 4°C and then incubated while protected from light in 1% osmium tetroxide for 2 h. After washing with distilled water for three times (5 min each), specimens were incubated in 2% uranyl acetate for 2 h at room temperature and then dehydrated in graded ethanol concentrations. Finally, sections were embedded in molds with fresh resin. The changes in morphology and ultrastructure of the myocardial tissues were observed and photographed under a TEM [13].

Scal-1+/flk-1+ cells counting of endothelial progenitor cells

We applied antibodies to the stem cell antigen-1 (Sca-1) and fetal liver kinase-1 (flk-1) to sign endothelial progenitor cells (EPCs) as described before, and used the isotype specific conjugated anti-IgG as a negative control. The amount of Scal-1+/flk-1+ cells would be counted by flow cytometry technique [14].

Measurement of nitric oxide concentration and Western blotting

Plasma concentrations of NO were measured with Griess assay kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. The expressions of Bax, Bcl-2, p-Akt, Akt, p-endothelial nitric oxide synthase (eNOS), eNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed using Western blot as described recently [15]. Proteins were measured with Pierce BCA Protein Assay Kit (Thermo). Hippocampal protein lysates (50 mg/well) were separated using (SDS/PAGE) under reducing conditions. Following electrophoresis, the separated proteins were transferred to a PVDF membrane (Millipore). Subsequently, non-specific proteins were blocked using blocking buffer (5% skim milk or 5% BSA in T-TBS containing 0.05% Tween 20), followed by overnight incubation with primary rabbit anti-rat antibodies specific for target proteins as mentioned before (Cell Signaling Technology) at 4°C. Blots were rinsed three times (5 min each) with T-TBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000, Proteintech) for 2 h at room temperature. The blots were visualized by using enhanced chemiluminescence (ECL) method (Thermo). GAPDH was applied to be the internal control protein. Intensity of the tested protein bands was quantified by densitometry.

Cell culture

Primary neonatal rat cardiac ventricular myocytes (NRCMs) were collected as previously described [15]. Briefly, the ventricles of new born SD rats (1–3 days old) were minced and digested with 0.125% trypsin. Isolated cardiomyocytes were cultured in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12, Life Technologies) supplemented with 10% (v/v) FBS (Life Technologies), 100 units/ml penicillin and 100 mg/ml streptomycin. The following experiments used spontaneously beating cardiomyocytes 48–72 hours after plating. (37°C with 5% CO2).

Cell treatment (hypoxia/reoxygenation)

NRCMs were prepared according to the methods recently described [15]. To establish the H/R model, the cells were cultured in DMEM/F-12 without glucose and serum. The cells were exposed to hypoxia (99% N2+5% CO2) for 8 h, followed by reoxygenation for 16 h. The cells were pretreated with PEMF for 30 min before the H/R procedure. The control group was cultured in DMEM/F-12 with low glucose (1000 mg/l) and 2% serum under normoxic air conditions for the corresponding times.

Cell viability assays

The viability of NRCMs cultured in 96-well plates was measured by using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies) according to the manufacturer’s instructions. The absorbance of CCK-8 was obtained with a microplate reader at 450 nm.

Measurement of intracellular reactive oxygen species levels

Reactive oxygen species (ROS) levels in NRVMs were determined by dihydroethidium (DHE, Sigma–Aldrich) fluorescence using confocal microscopy (Zeiss, LSM 710). After different treatments, cells were washed with D-PBS and incubated with DHE (10 ?mol/l) at 37°C for 30 min in the dark. Then, residual DHE was removed by PBS-washing. Fluorescent signals were observed (excitation, 488 nm; emission, 610 nm) under a laser confocal microscope (Zeiss).

Data analysis

All the data were presented as means ± S.E.M. Differences were compared by one-way ANOVA analysis by using SPSS software version 19.0 (SPSS) and P value <0.05 was taken as statistically significant.

RESULTS

PEMF could lower blood pressure under treatment of certain PEMF intensity in SHR rat model (double-blind)

To determine whether PEMF has any effects on blood pressure of SHR rats, we treated SHR rats with different PEMF intensity 1–4 cycles per day for 7 days and measured the blood pressure changes via CCA. We observed that PEMF treatment could significantly lower the blood pressure in the Bioboosti WIN235 and WI215-stimulating groups than that in non-treated ones (Figures 1A and ?and1B).1B). But Bioboosti WIN221 and WC65 treating groups did not have any effects on the blood pressure in SHR rats, compared with the non-treated ones (Figures 1C and ?and1D).1D). Fields were asymmetric and consisted of 4.5 ms pulses at 30±3 Hz, with an adjustable magnetic field strength range (X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis 0.06±0.02 mT). The I/R rats were housed in custom designed cages and exposed to active PEMF for 2 cycles per time (8 min for 1 cycle), whereas the I/R rats were housed in identical cages with inactive PEMF generator.

Figure 1

Figure 1The effect of PEMF on SHR rats in vivo. PEMF could lower the blood pressure in SHR rats. At day 7 treatment with different intensity PEMF, blood pressure was recorded via CCA [1(A), 1(B), 1(C) and 1(D)]. Data were represented as the mean ±

According to this result, we chose Bioboosti WIN235 as our needed PEMF to carry out the following experiments.

PEMF treatment could observably improve the abundance of EPCs

Amplifying EPCs abundance and function is an active focus of research on EPCs-mediated neovascularization after I/R. Thus, the number of circulating EPCs was identified by Sca-1/flk-1 dual positive cells as described. We determined that PEMF treatment could remarkably increase the number of Scal-1+/flk-1+ cells in peripheral blood at postoperative days 7 and 14 (Figure 2).

Figure 2

Figure 2The effect of PEMF on the number of Scal-1+/flk-1+ cells after treating EPSc for 7 and 14 days. PEMF treatment notably increased the number of Scal-1+/flk-1+ cells after treating EPSc for 7 and 14 days. Data were represented as the mean

Preliminary assessment of PEMF showed great protective effect against myocardial infarction/reperfusion injury (MI/RI) rat model

To examine the effect of PEMF on myocardial I/R, male SD rats were divided into three groups: Sham, I/R and I/R+ PEMF (2 cycles per day, 8 min per cycle) per day until 28 days. We observed that PEMF stimulation could significantly decrease four plasma myocardial enzymes (LDH, CK, CKMB and HBDH) in I/R rats (Figure 3A). Additionally, we found that pre-stimulating PEMF could improve the cardiac morphology via TEM, compared with I/R+ PEMF group. TEM revealed the rupture of muscular fibres, together with mitochondrial swelling, and intracellular oedema in Group I/R. The shape of nucleus was irregular, with evidence of mitochondrial overflow after cell death. Compared with Group I/R+ PEMF, less muscular fibres were ruptured, with mild swelling of mitochondria, mild intercellular oedema and less cell death. In Group Sham, the ruptured muscular fibres, mitochondrial or intracellular oedema and dead cells were not observed (Figure 3B). To further confirm protective effect of PEMF, we measured the MI size by applying TTC and Evans Blue staining in all three groups. The MI area in I/R+ PEMF group could be reduced, compared with the model rats in I/R group (Figure 3C).

Figure 3

Figure 3Protective effect of PEMF on I/R rats in vivo. Plasma myocardial enzymes (LDH, CK, HBDH and CKMB) content was quantified by automatic biochemical analyzer (A) (n=18 in each group). Changes on cardiac cell morphology via TEM (B) (n=6 in

In vivo, PEMF dramatically reduced cell apoptosis induced by I/R injury

As H/R of cardiomyocytes contributed to cell death, we also detected the effect on myocardial apoptosis by using TUNEL kit, as shown in Figure 4(A). We uncovered that PEMF pretreating could dramatically decrease apoptosis of myocardial cells in I/R + PEMF group, compared with I/R group. In addition, we also found that PEMF treatment could significantly increase the expression of anti-apoptosis protein Bcl-2, p-eNOS and p-Akt and down-regulated the expression of pro-apoptosis protein Bax in the heart tissue, as shown in Figure 4(B).

Figure 4

Figure 4Apoptotic cardiomyocyte was identified by TUNEL analysis, apoptotic cardiomyocyte appears green whereas TUNEL-negative appears blue (A), photomicrographs were taken at ×200 magnification. Apoptosis-related protein Bcl-2, Bax, p-Akt level of different

The effect of PEMF on cell viability in neonatal rat cardiac ventricular myocytes

To further investigate whether PEMF has the same effect in vitro, we simulated the I/R injury model in vitro. We applied NRCMs and hypoxia incubator to mimic myocardial I/R injury via H/R as described in the section ‘Materials and Methods’. We found that PEMF treatment (2 cycles) could remarkably improve cell viability, compared with the H/R group (Figure 5). For in vitro study, culture dishes were directly exposed to PEMF for 1–2 cycles as indicated (8 min for 1 cycle, 30±3 Hz, X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis 0.06±0.02 mT).

Figure 5

Figure 5NRCMs viability measured by CCK-8 assay at the end of the treatment for 72 h. PEMF treatment enhanced the cell viability of hypoxia NRCMs. Data were represented as the mean ± S.E.M.

Specific-density PEMF could decrease intracellular ROS levels of primary cardiomyocytes subjected to hypoxia/reperfusion

As shown in Figure 6(A), NRCMs that were subjected to H/R increased significantly the ROS level, whereas the ROS level had been decreased in PEMF group (2 cycles), in contrast with the H/R group. Representative images of the ROS level were displayed in Figure 6(B). At the same time, we identified the effect on NRCMs apoptosis after suffering H/R by using TUNEL kit. As shown in Figure 6(C), cell apoptosis in the H/R group was aggravated, whereas PEMF treatment could reduce the cell death. Representative images of TUNEL staining were shown in Figure 6(D).

Figure 6

Figure 6PEMF protected Neonatal rat cardiac ventricular myocytes (NRCMs) from hypoxia/reoxygenation (H/R)-induced apoptosis via decreasing ROS levelat the end of the treatment for 72 h in vitro.

Effect of PEMF on NO releasing via Akt/eNOS pathway

Cultured NRCMs were treated with PEMF stimulation for 1 to 2 cycles and the supernatant and cell lysate were collected. When cells suffered H/R, intracellular levels of p-Akt, p-eNOS and Bcl-2 were decreased, whereas PEMF treatment could increase the phosphorylation of Akt, p-eNOS and Bcl-2 (Figures 7A–7C). The expression of Bax was increased when cells subjected to H/R whereas PEMF treatment reversed such increase (Figure 7C). Western blot analysis was shown in Figure 7(D) for p-Akt/Akt, Figure 7(E) for p-eNOS/eNOS, Figure 7(F) for Bcl-2 and Figure 7(G) for Bax.

Figure 7

Figure 7The related protein expression about the effect of PEMF on apoptosis induced by hypoxia/reoxygenationat the end of the treatment for 72 h in vitro. PEMF increased the phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), and the expressionGo to:

DISCUSSION

Our present study provides the first evidence that PEMF has novel functions as follows: (1) We treated SHR rats with different PEMF intensity (8 min for 1 cycle, 30±3 Hz, X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis 0.06±0.02 mT) 1–4 cycles per day for 7 days. PEMF can lower blood pressure under treatment of certain PEMF intensity in SHR rat model (double-blind). (2) PEMF has a profound effect on improving cardiac function in I/R rat model. (3) PEMF plays a vital role in inhibiting cardiac apoptosis via Bcl-2 up-regulation and Bax down-regulation. (4) In vitro, PEMF treatment also has a good effect on reducing ROS levels by Akt/eNOS pathway to release NO and improving cell apoptosis in NRCMs subjected to hypoxia.

Many previous studies showed that extracorporeal PEMF-treated(5 mT, 25 Hz, 1 h daily) could enhance osteanagenesis, skin rapture healing and neuronal regeneration, suggesting its regenerative potency [8,16,17]. And some researchers had found that PEMF therapy (8 min/cycle, 30±3 Hz, 6 mT) could improve the myocardial infarct by activating VEGF–Enos [18] system and promoting EPCs mobilized to the ischaemic myocardium [1,19]. Consistent with the previous work, our present study demonstrated that PEMF therapy could significantly alleviate cardiac dysfunction in I/R rat model.

Recent evidence suggest that circulating EPCs can be mobilized endogenously in response to tissue ischaemia or exogenously by cytokine stimulation and the recruitment of EPCs contributes to the adult blood vessels formation [19,20,21]. We hypothesized that PEMF could recruit more EPCs to the vessels. To confirm our hypothesis, we applied antibodies to the Sca-1 and flk-1 to sign EPC. The results indicated that PEMF could remarkably increase the number of EPCs in the PEMF group, compared with the I/R group.

Previous evidence indicated that when heart suffered I/R, cardiac apoptosis would be dramatically aggravated [2224]. Myocardial apoptosis plays a significant role in the pathogenesis of myocardial I/R injury. We assumed that PEMF might play its role in improving cardiac function through inhibiting cell apoptosis. The Bcl-2 family is a group of important apoptosis-regulating proteins that is expressed on the mitochondrial outer membrane, endoplasmic reticulum membrane and nuclear membrane. Overexpression of Bcl-2 proteins blocks the pro-apoptosis signal transduction pathway, thereby preventing apoptosis caused by the caspase cascade [25]. The role Bax plays in autophagy is a debatable. Recently, new genetic and biochemical evidence suggest that Bcl-2/Bcl-xL may affect apoptosis through its inhibition of Bax [26]. Overexpression of Bax protein promotes the apoptosis signal pathway. In the present study, we applied TUNEL staining to find that PEMF has a perfect effect on cardiac cell apoptosis by regulating apoptosis-related proteins Bcl-2 and Bax [25,26,27,28].

To verify our findings in the rat model, we mimicked I/R condition in vitro by hypoxia exposure in NRCMs. Results showed that not only in vivo, hypoxia could induce cell apoptosis in vitro. And we also found that PEMF treatment could significantly alleviate cell apoptosis induced by hypoxia. At the basal level, ROS play an important role in mediating multiple cellular signalling cascades including cell growth and stress adaptation. Conversely, excess ROS can damage tissues by oxidizing important cellular components such as proteins, lipids and DNA, as well as activating proteolytic enzymes such as matrix metalloproteinases [29]. Previous studies showed that when cells were subjected to hypoxia, the intracellular ROS level would be sharply increased, and the overproduction of ROS would result in cell damage [19,30,31]. In the present study, PEMF treatment could prominently down-regulate ROS levels. We also investigated how PEMF reduced the intracellular ROS level.

NO appears to mediate distinct pathways in response to oxidative stress via AKt–eNOS pathway [32,33]. NO is identified as gaseous transmitters. In vascular tissue, NO is synthesized from L-arginine by nitric oxide synthase (NOS) and it is considered to be the endothelium-derived relaxing factor. Evidence show that the NO generation in endothelium cells was damaged in hypertensive patients [34]. NO could also prevent platelet activation and promote vascular smooth muscle cells proliferation [35]. NO generation from eNOS is considered to be endothelium-derived relaxing and ROS-related factor [36,37]. Some researchers found that bradykinin limited MI induced by I/R injury via Akt/eNOS signalling pathway in mouse heart [38]. And bradykinin inhibited oxidative stress-induced cardiomyocytes senescence by acting through BK B2 receptor induced NO release [39]. Such evidence indicated that Akt phosphorylation could activate eNOS, which lead to NO releasing, and resulted in ROS reducing. In the present study, we found that PEMF decreased ROS via Akt/eNOS pathway.

In conclusion, this is the first study suggesting that PEMF treatment could improve cardiac dysfunction through inhibiting cell apoptosis. Furthermore, in vitro, we first clarified PEMF still plays a profound effect on improving cell death and removing excess ROS via regulating apoptosis-related proteins and Akt/eNOS pathway. All these findings highlight that PEMF would be applied as a potentially powerful therapy for I/R injury cure.

Acknowledgments

We thank all of the members of the Laboratory of Pharmacology of Chen Y., Ding Y.J. for their technical assistance.

Abbreviations

Aktprotein kinase B
BaxBcl-2 associated X protein
Bcl-2B-cell lymphoma 2
CCAcommon carotid artery
CCK-8Cell Counting Kit-8
CKcreatine kinase
CKMBcreatine kinase isoenzyme-MB
DAPI4,6?-diamidino-2?-phenylindole
DHEdihydroethidium
DMEM/F12Dulbecco’s modified Eagle’s medium/F-12
dUTPdeoxyuridine triphosphate
eNOSendothelial nitric oxide synthase
EPCsendothelial progenitor cells
flk-1fetal liver kinase-1
GAPDHglyceraldehyde-3-phosphate dehydrogenase
HBDH?-hydroxybutyrate dehydrogenase
H/Rhypoxia/reoxygenation
HRPhorseradish peroxidase
I/Rischaemia/reperfusion
LADleft anterior descending
LDHlactate dehydrogenase
MImyocardial infarction
MI/Rmyocardial infarction/reperfusion
MI/RImyocardial infarction/reperfusion injury
NRCMsneonatal rat cardiac ventricular myocytes
PEMFpulsed electromagnetic field
ROSreactive oxygen species
Sca-1stem cell antigen-1
SDSprague Dawley
SHRspontaneously hypertensive rats
TTC2,3,5-triphenyltetrazolium chloride
TUNELterminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling
VEGFvascular endothelial growth factor

AUTHOR CONTRIBUTION

Fenfen Ma designed and performed experiments on MI/RI rat model, histological stain and Western blot. Wenwen Li assisted the in vivo experiments, validated the effect in vitro experiments, analysed data and wrote the manuscript. Xinghui Li interpreted data and formatted manuscript. Rinkiko Suguro, Ruijuan Guan, Cuilan Hou, Huijuan Wang and Aijie Zhang interpreted data and edited manuscript. Yichun Zhu and YiZhun Zhu proposed the idea and supervised the project.

FUNDING

This work was supported by the key laboratory program of the Education Commission of Shanghai Municipality [grant number ZDSYS14005].

References

1. Hao C.N., Huang J.J., Shi Y.Q., Cheng X.W., Li H.Y., Zhou L., Guo X.G., Li R.L., Lu W., Zhu Y.Z., Duan J.L. Pulsed electromagnetic field improves cardiac function in response to myocardial infarction. Am. J. Transl. Res. 2014;6:281–290. [PMC free article] [PubMed] 2. Eltzschig H.K., Eckle T. Ischemia and reperfusion–from mechanism to translation. Nat. Med. 2011;17:1391–1401. doi: 10.1038/nm.2507. [PMC free article] [PubMed] [Cross Ref] 3. Thygesen K., Alpert J.S., Jaffe A.S., Simoons M.L., Chaitman B.R., White H.D. Third universal definition of myocardial infarction. Nat. Rev. Cardiol. 2012;9:620–633. doi: 10.1038/nrcardio.2012.122.[PubMed] [Cross Ref] 4. Nah D.Y., Rhee M.Y. The inflammatory response and cardiac repair after myocardial infarction. Korean Circ. J. 2009;39:393–398. doi: 10.4070/kcj.2009.39.10.393. [PMC free article] [PubMed] [Cross Ref] 5. Yellon D.M., Hausenloy D.J. Myocardial reperfusion injury. N. Engl. J. Med. 2007;357:1121–1135. doi: 10.1056/NEJMra071667. [PubMed] [Cross Ref] 6. Herron T.J., Milstein M.L., Anumonwo J., Priori S.G., Jalife J. Purkinje cell calcium dysregulation is the cellular mechanism that underlies catecholaminergic polymorphic ventricular tachycardia. Heart Rhythm. 2010;7:1122–1128. doi: 10.1016/j.hrthm.2010.06.010. [PMC free article] [PubMed] [Cross Ref] 7. Kim S.S., Shin H.J., Eom D.W., Huh J.R., Woo Y., Kim H., Ryu S.H., Suh P.G., Kim M.J., Kim J.Y., et al. Enhanced expression of neuronal nitric oxide synthase and phospholipase C-gamma1 in regenerating murine neuronal cells by pulsed electromagnetic field. Exp. Mol. Med. 2002;34:53–59. doi: 10.1038/emm.2002.8. [PubMed] [Cross Ref] 8. Tepper O.M., Callaghan M.J., Chang E.I., Galiano R.D., Bhatt K.A., Baharestani S., Gan J., Simon B., Hopper R.A., Levine J.P., Gurtner G.C. Electromagnetic fields increase in vitro and in vivo angiogenesis through endothelial release of FGF-2. FASEB J. 2004;18:1231–1233. [PubMed] 9. Weintraub M.I., Herrmann D.N., Smith A.G., Backonja M.M., Cole S.P. Pulsed electromagnetic fields to reduce diabetic neuropathic pain and stimulate neuronal repair: a randomized controlled trial. Arch. Phys. Med. Rehabil. 2009;90:1102–1109. doi: 10.1016/j.apmr.2009.01.019. [PubMed] [Cross Ref] 10. Graak V., Chaudhary S., Bal B.S., Sandhu J.S. Evaluation of the efficacy of pulsed electromagnetic field in the management of patients with diabetic polyneuropathy. Int. J. Diab. Dev. Ctries. 2009;29:56–61. doi: 10.4103/0973-3930.53121. [PMC free article] [PubMed] [Cross Ref] 11. Kin H., Zhao Z.Q., Sun H.Y., Wang N.P., Corvera J.S., Halkos M.E., Kerendi F., Guyton R.A., Vinten-Johansen J. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc. Res. 2004;62:74–85. doi: 10.1016/j.cardiores.2004.01.006.[PubMed] [Cross Ref] 12. Yao L.L., Huang X.W., Wang Y.G., Cao Y.X., Zhang C.C., Zhu Y.C. Hydrogen sulfide protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by preventing GSK-3beta-dependent opening of mPTP. Am. J. Physiol. Heart. Circ. Physiol. 2010;298:H1310–H1319. doi: 10.1152/ajpheart.00339.2009. [PubMed] [Cross Ref] 13. Zhikun G., Liping M., Kang G., Yaofeng W. Structural relationship between microlymphatic and microvascullar blood vessels in the rabbit ventricular myocardium. Lymphology. 2013;46:193–201.[PubMed] 14. Tsai S.H., Huang P.H., Chang W.C., Tsai H.Y., Lin C.P., Leu H.B., Wu T.C., Chen J.W., Lin S.J. Zoledronate inhibits ischemia-induced neovascularization by impairing the mobilization and function of endothelial progenitor cells. PLoS ONE. 2012;7:e41065. doi: 10.1371/journal.pone.0041065.[PMC free article] [PubMed] [Cross Ref] 15. Jin S., Pu S.X., Hou C.L., Ma F.F., Li N., Li X.H., Tan B., Tao B.B., Wang M.J., Zhu Y.C. Cardiac H2S generation is reduced in ageing diabetic mice. Oxid. Med. Cell. Longev. 2015;2015:758358.[PMC free article] [PubMed] 16. Cheing G.L., Li X., Huang L., Kwan R.L., Cheung K.K. Pulsed electromagnetic fields (PEMF) promote early wound healing and myofibroblast proliferation in diabetic rats. Bioelectromagnetics. 2014;35:161–169. doi: 10.1002/bem.21832. [PubMed] [Cross Ref] 17. Weintraub M.I., Herrmann D.N., Smith A.G., Backonja M.M., Cole S.P. Pulsed electromagnetic fields to reduce diabetic neuropathic pain and stimulate neuronal repair: a randomized controlled trial. Arch. Phys. Med. Rehabil. 2009;90:1102–1109. doi: 10.1016/j.apmr.2009.01.019. [PubMed] [Cross Ref] 18. Li J., Zhang Y., Li C., Xie J., Liu Y., Zhu W., Zhang X., Jiang S., Liu L., Ding Z. HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism. Cardiovasc. Res. 2013;99:674–684. doi: 10.1093/cvr/cvt139. [PubMed] [Cross Ref] 19. Goto T., Fujioka M., Ishida M., Kuribayashi M., Ueshima K., Kubo T. Noninvasive up-regulation of angiopoietin-2 and fibroblast growth factor-2 in bone marrow by pulsed electromagnetic field therapy. J. Orthop. Sci. 2010;15:661–665. doi: 10.1007/s00776-010-1510-0. [PubMed] [Cross Ref] 20. Asahara T., Masuda H., Takahashi T., Kalka C., Pastore C., Silver M., Kearne M., Magner M., Isner J.M. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ. Res. 1999;85:221–228. doi: 10.1161/01.RES.85.3.221. [PubMed] [Cross Ref] 21. Takahashi T., Kalka C., Masuda H., Chen D., Silver M., Kearney M., Magner M., Isner J.M., Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat. Med. 1999;5:434–438. doi: 10.1038/8462. [PubMed] [Cross Ref] 22. Freude B., Masters T.N., Robicsek F., Fokin A., Kostin S., Zimmermann R., Ullmann C., Lorenz-Meyer S., Schaper J. Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J. Mol. Cell Cardiol. 2000;32:197–208. doi: 10.1006/jmcc.1999.1066. [PubMed] [Cross Ref] 23. Martindale J.J., Fernandez R., Thuerauf D., Whittaker R., Gude N., Sussman M.A., Glembotski C.C. Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6. Circ. Res. 2006;98:1186–1193. doi: 10.1161/01.RES.0000220643.65941.8d. [PubMed] [Cross Ref] 24. Yu L., Lu M., Wang P., Chen X. Trichostatin A ameliorates myocardial ischemia/reperfusion injury through inhibition of endoplasmic reticulum stress-induced apoptosis. Arch. Med. Res. 2012;43:190–196. doi: 10.1016/j.arcmed.2012.04.007. [PubMed] [Cross Ref] 25. Maiuri M.C., Criollo A., Tasdemir E., Vicencio J.M., Tajeddine N., Hickman J.A., Geneste O., Kroemer G. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L) Autophagy. 2007;3:374–376. doi: 10.4161/auto.4237.[PubMed] [Cross Ref] 26. Lindqvist L.M., Heinlein M., Huang D.C., Vaux D.L. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak. Proc. Natl. Acad. Sci. U.S.A. 2014;111:8512–8517. doi: 10.1073/pnas.1406425111. [PMC free article] [PubMed] [Cross Ref] 27. Chandna S., Suman S., Chandna M., Pandey A., Singh V., Kumar A., Dwarakanath B.S., Seth R.K. Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of gamma-radiation. Int. J. Rad. Biol. 2013;89:1017–1027. doi: 10.3109/09553002.2013.825059. [PubMed][Cross Ref] 28. Xu M., Zhou B., Wang G., Wang G., Weng X., Cai J., Li P., Chen H., Jiang X., Zhang Y. miR-15a and miR-16 modulate drug resistance by targeting bcl-2 in human colon cancer cells. Zhonghua Zhong Liu Za Zhi. 2014;36:897–902. [PubMed] 29. Zuo L., Best T.M., Roberts W.J., Diaz P.T., Wagner P.D. Characterization of reactive oxygen species in diaphragm. Acta Physiol. (Oxf.) 2015;213:700–710. doi: 10.1111/apha.12410. [PubMed] [Cross Ref] 30. Kalogeris T., Bao Y., Korthuis R.J. Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning. Redox Biol. 2014;2:702–714. doi: 10.1016/j.redox.2014.05.006.[PMC free article] [PubMed] [Cross Ref] 31. Levraut J., Iwase H., Shao Z.H., Vanden H.T., Schumacker P.T. Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation. Am. J. Physiol. Heart Circ. Physiol. 2003;284:H549–H558. doi: 10.1152/ajpheart.00708.2002. [PubMed] [Cross Ref] 32. Dong R., Chen W., Feng W., Xia C., Hu D., Zhang Y., Yang Y., Wang D.W., Xu X., Tu L. Exogenous bradykinin inhibits tissue factor induction and deep vein thrombosis via activating the eNOS/phosphoinositide 3-kinase/Akt signaling pathway. Cell. Physiol. Biochem. 2015;37:1592–1606. doi: 10.1159/000438526. [PubMed] [Cross Ref] 33. Jin R.C., Loscalzo J. Vascular nitric oxide: formation and function. J. Blood Med. 2010;2010:147–162.[PMC free article] [PubMed] 34. Taddei S., Virdis A., Mattei P., Ghiadoni L., Sudano I., Salvetti A. Defective L-arginine-nitric oxide pathway in offspring of essential hypertensive patients. Circulation. 1996;94:1298–1303. doi: 10.1161/01.CIR.94.6.1298. [PubMed] [Cross Ref] 35. Tang E.H., Vanhoutte P.M. Endothelial dysfunction: a strategic target in the treatment of hypertension? Pflugers Arch. 2010;459:995–1004. doi: 10.1007/s00424-010-0786-4. [PubMed] [Cross Ref] 36. Beltowski J., Jamroz-Wisniewska A. Hydrogen sulfide and endothelium-dependent vasorelaxation. Molecules. 2014;19:21183–21199. doi: 10.3390/molecules191221183. [PubMed] [Cross Ref] 37. Wu D., Hu Q., Liu X., Pan L., Xiong Q., Zhu Y.Z. Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes. Nitric Oxide. 2015;46:204–212. doi: 10.1016/j.niox.2014.11.006. [PubMed] [Cross Ref] 38. Li Y.D., Ye B.Q., Zheng S.X., Wang J.T., Wang J.G., Chen M., Liu J.G., Pei X.H., Wang L.J., Lin Z.X., et al. NF-kappaB transcription factor p50 critically regulates tissue factor in deep vein thrombosis. J. Biol. Chem. 2009;284:4473–4483. doi: 10.1074/jbc.M806010200. [PMC free article] [PubMed] [Cross Ref] 39. Dong R., Xu X., Li G., Feng W., Zhao G., Zhao J., Wang D.W., Tu L. Bradykinin inhibits oxidative stress-induced cardiomyocytes senescence via regulating redox state. PLoS ONE. 2013;8:e77034. doi: 10.1371/journal.pone.0077034. [PMC free article] [PubMed] [Cross Ref] Am J Transl Res. 2014; 6(3): 281–290. Published online May 15, 2014

Pulsed electromagnetic field improves cardiac function in response to myocardial infarctionChang-Ning Hao,1,3,*Jing-Juan Huang,1,2,*Yi-Qin Shi,1,3Xian-Wu Cheng,3Hao-Yun Li,1Lin Zhou,1Xin-Gui Guo,2Rui-Lin Li,1,2Wei Lu,5,*Yi-Zhun Zhu,4,* and Jun-Li Duan1,2,*1Department of Gerontology, Xin Hua Hospital, Shanghai Jiaotong University, Kongjiang Road 1665, Shanghai 200092, China 2Department of Cardiology, Hua Dong Hospital, Fudan University, West Yan’an Road 221, Shanghai 200040, China 3Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan 4Department of Pharmacology, School of Pharmacy, Fudan University, Zhang-Heng Road 826, Shanghai 201203, China 5National Lab for Infrared Physics, Shanghai Institute of Technical Physics, Chinese Academy of Sciences, Yu-Tian Road 500, Shanghai 200083, China Address correspondence to: Jun-Li Duan, Department of Cardiology, Hua Dong Hospital, Fudan University, West Yan’an Road 221, Shanghai 200040, China. E-mail: moc.361@hxilnujnaud; Dr. Wei Lu, National Lab for Infrared Physics, Shanghai Institute of Technical Physics, Chinese Academy of Sciences, Yu-Tian Road 500, Shanghai 200083, China. E-mail: nc.ca.ptis.liam@iewul; Dr. Yi-Zhun Zhu, Department of Pharmacology, School of Pharmacy, Fudan University, Zhang-Heng Road 826, Shanghai 201203, China. E-mail: moc.liamg@uhznuhziy*Equal contributors. Author information  Article notes Copyright and License information Received February 3, 2014; Accepted April 18, 2014. AJTR Copyright © 2014

Introduction

Coronary artery disease is a leading cause of morbidity and mortality in modern society. Massive loss of cardiac muscle after several ischemic episodes lead to compromised cardiac function, remodeling and low quality life of patients. A growing body of evidence in experimental models of cardiac injury suggests that early re-establishment of blood perfusion to the injured myocardium would restrict infarct expansion, prevent cardiac remodeling and maintain cardiac function [13]. Although several strategies for therapeutic angiogenesis including the delivery of growth factors, gene therapy and stem cell implantation have been investigated, unsolvable theoretical limitations are still remaining [48]. For instance, the limited survival of implanted stem cell, uncontrolled angiogenesis and others [911]. Therefore, a safe, effective and non-invasive treatment for myocardial ischemia may be an ideal approach.

The therapeutic efficacy of various forms of electromagnetic stimulations, including capacitative coupling, direct current, combined magnetic fields, and pulsed electromagnetic field (PEMF), have been intensely investigated [12]. Among them, extracorporeal PEMF is the most widely tested techniques in the topic of osteanagenesis [13], skin rapture healing [14] and neuronal regeneration [15,16]. Recently, several study also indicated that PEMF exhibited the capability to stimulate angiogenesis and endothelial proliferation [1719], however the detailed mechanism remains modest understood.

In the present study, we investigated whether extracorporeal PEMF therapy was able to rescue ischemic myocardium through inhibiting cardiac apoptosis as well as promoting postnatal neovascularization in a rat model of myocardial infarction (MI).

Material and methods

Animals

Male Sprague-Dawley (SD) rats weighing 250-300 g were provided by Sino-British SIPPR/BK Laboratory Animal (Shanghai, China). Animals were housed with controlled temperature (22-25°C) and lighting (08:00-20:00 light, 20:00-08:00 dark), and free access to tap water and standard rat chow. All the animals in this work received humane care in compliance with institutional guidelines for health and care of experimental animals of Shanghai Jiao Tong University.

MI model

All rats (n=36) were subjected to permanent left anterior descending artery ligation to establish MI model. Briefly, left thoracotomy and pericardiectomy were performed, and the hearts were gently exteriorized. Left anterior descending artery was ligated 4 mm below the left atrium with a 5-0 silk suture. The chest wall was then closed and the animals were returned to home cages. MI rats were then randomly divided into PEMF treated and untreated groups.

PEMF treatment

PEMF were generated by a commercially available healing device purchased from Biomobie Regenerative Medicine Technology (Shanghai, China). Fields were asymmetric and consisted of 4.5 ms pulses at 30 ± 3 Hz, with a magnetic flux density increasing from 0 to 5 mT in 400 ?s. The MI rats were housed in custom-designed cages and exposed to active PEMF for 4 cycles per day (8 minutes for 1 cycle), while the control rats were housed in identical cages with inactive PEMF generator. For in vitro study, culture dishes were directly exposed to PEMF for 1-4 cycles as indicated (8 minutes for 1 cycle, 30 ± 3 Hz, 5 mT).

Echocardiography

Trans-thoracic echocardiographic analysis was performed using an animal specific instrument (VisualSonics, Vevo770; VisualSonicsInc, Toronto, Canada), at postoperative day 7, 14 and 28. Rats were anesthetized with 10% chloral hydrate solution. After shaving the chest, pre-warmed ultrasound transmission gel was applied to the chest and two dimensional-directed M-mode and Doppler echocardiographic studies were carried out. The ejection fraction (EF) and fractional shortening (FS) were used to assess left ventricular systolic function. All measurements were averaged for consecutive cardiac cycles and triplicated.

Capillary density

Capillary density in peri-infarcted zone (PIZ) was determined by anti-CD31 staining (R&D Systems, San Diego, CA, USA). Briefly, 14 days after MI, rats were euthanized and hearts were perfused with a 0.9% NaCl solution followed by 4% solution of paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4), and then dissected and fixed in this solution for 24 h. Next, samples were washed, dehydrated in a graded ethanol series and embedded in paraffin. 5 ?m-sections were cut transversely at 200 ?m intervals from into 5 slices from the ligation site to the apex. Endothelial capillaries were identified by goat anti-rat antibody of CD31 (5 ?g/ml, Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA), and followed by a secondary antibody (Invitrogen, Carlsbad, CA, USA). Capillary density was determined by counting of 10 randomly selected fields and is expressed as numbers of capillary/field (×400 magnification) [20,21].

Enzyme-linked immunosorbent assay (ELISA)

The concentration of vascular endothelial growth factor (VEGF) and nitric oxide (NO) contained in conditional media of cultured HUVECs was measured using ELISA kit purchased from R&D Systems (San Diego, CA, USA). The concentrations of VEGF contained in PIZ was determined by ELISA kits purchased from Raybiotech (Norcross, GA, USA) [22].

Western blotting

PIZ tissue and HUVECs were homogenized with ice-cold homogenizing buffer (20 ?l/gram tissue, 50 mmol/l Tris-HCl, 150 mmol/l NaCl, 1 mmol/l EDTA, and 0.5 mmol/l Triton X-100, pH 7.4) and protease inhibitor cocktail (5 mM, Roche, Berlin, Germany). Proteins were measured with Pierce BCA Protein Assay Kit (Thermo, Asheville, North Carolina, USA). Hippocampal protein lysates (50 mg/well) were separated using SDS-PAGE under reducing conditions. Following electrophoresis, the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, Massachusetts, USA). Subsequently, nonspecific proteins were blocked using blocking buffer (5% nonfat dried milk in T-TBS containing 0.05% Tween 20), followed by incubation with primary rabbit anti-rat antibodies specific for phospho-Akt (p-Akt), total Akt, hypoxic-inducible factor (HIF)-1? (Santa Cruz, California, USA), phospho-endothelial nitric oxide synthase (p-eNOS), total eNOS and ?-actin (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. Blots were washed four times with 0.1% Tween 20 in PBS and incubated with HRP-conjugated secondary antibody (1/5000; Biochain, Newark, California, USA) for 1 h at room temperature. The bands were visualized using enhanced chemiluminescence method (Bioimaging System; Syngene, Cambridge, UK). Intensity of the tested protein bands was quantified by densitometry.

Detection of apoptosis

Heart samples were fixed in 10% formalin and then paraffin embedded at day 14. Then, the hearts were cut into 5-?m sections. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was carried out using a commercially available kit according to the manufacturer’s instructions (Promega, Madison, Wisconsin, USA). Nuclei were stained by DAPI (Roche) [23]. Three mid-ventricular sections of each heart (from the apex to the base) were analyzed. Ten fields in the PIZ were randomly selected from each section for the calculation of the percentage of apoptotic nuclei (apoptotic nuclei/total nuclei) and the obtained ratios were averaged for statistical analysis.

Isolation of circulating endothelial progenitor cells (EPCs)

Circulating EPCs were obtained by cardiac puncture after animals were anesthetized. Peripheral blood-derived mononuclear cells (PB-MNCs) were then purified by Histopaque-1083 (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation at 400 g for 30 min. The mononuclear layer was then collected and re-suspended in endothelial growth medium-2 (EGM-2, Clonetics, San Diego, CA, USA). Antibodies to the stem cell antigen-1 (Sca-1) and Flk-1 were used to mark EPC as described before), and the isotype specific conjugated anti-IgG was used as a negative control. Sca-1+ and Flk-1+ cells were gated in the mononuclear cell fraction.

EPC migration assay

Migratory activity of PB-EPCs from PEMF-treated and untreated rats was evaluated by a 24-well modified Boyden chamber assay (Transwell, Corning, NY, USA) [24]. After cultured with EGM-2 for 4 days, PB-EPCs were trypsinized and 5×105 cells in 100 ?l of EBM-2 with 0.1% BSA in placed in the upper compartments. 50 ng/mL recombinant vascular endothelial growth factor (VEGF, Clonetics) in 600 ?L of chemotaxis buffer (serum-free EBM-2, 0.1% BSA) was added to the lower compartment. The chamber was incubated at 37°C for 6 hrs. The cells were then fixed and stained with hematoxylin and eosin (H&E). Non-migrated cells on the filter’s upper surface were removed using a cotton swab. The numbers of migrated cells were counted in 4 random high-power fields (HPF, ×400 magnification) and averaged for each sample.

Tube formation assay

Matrigel-Matrix (BD Biosciences, Franklin Lakes, New Jersey, USA) was inserted in the well of a 48-well cell culture plate and a number of 5×104 EPCs or HUVECs were seeded [25].

After incubation in EGM-2, images of tube morphology was taken and tube number was counted at random under four low power fields (magnifications ×40) per sample. Capillary tube branch points were counted in six randomly selected fields per well, and used as an index for tube formation.

Cell culture

Human umbilical vein endothelial cells (HUVECs, passage 3) were purchased from Clonetics (San Diego, CA, USA) and EGM-2 in a humidified atmosphere of 5% CO2 and 95% air. HUVECs were reseeded into plates coated with Matrix gel and stimulated for 1-4 cycles of PEMF stimulation (5.5 mT, 8 minutes per cycle). Supernatant and cell lysates were collected at 24 hrs after reseeding. Additionally, HUVECs-formed vasculature was quantified by calculating its length under microscopic photography 24 hrs after reseeding [26].

Statistical analysis

Data are expressed as means ± standard deviation (SD). Student’s t-test was used for statistical analyses. SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA) was used. A value of p<0.05 was considered significant.Go to:

Results

PEMF promotes cardiac function after MI

To determine whether PEMF could increase myocardial function in MI rats, echocardiographic studies were carried out at postoperative day 7, 14 and 28. We observed that PEMF had no effects on body weight and heart rates when compared with control group (Table 1). Meanwhile, higher EF and FS values were detected in PEMF-treated rats than control (Figure 1), indicating that PEMF preserves left ventricular contractility after MI damage.

Figure 1

Figure 1 Echocardiography after PEMF therapy. All rats were subjected to MI and randomly separated to control and PEMF group. The data of (A) ejection fractions and (B) fractional shorting in both groups collected in day 7, 14 and 28. Values are mean ±

Table 1

Table 1 Effect of PEMF on cardiac functions of MI rats

PEMF enhances angiogenesis in PIZ

To examine whether the changes in the cardiac function are associated with changes in capillary EC formation, we measured capillary densities of PEMF and control rats in PIZ through anti-CD31 immunofluorescence staining at postoperative day 14. Representative photomicrographs are shown in Figure 2A. Quantitative analyses by counting the CD31+ capillary ECs revealed that PEMF treatment significantly increases capillary densities in PIZ than control rats (Figure 2B). PEMF treatment also increased the protein levels of VEGF and HIF-1? in damaged hearts (Figure 2C and ?and2D),2D), as well as enhancing the phosphorylation of Akt signal pathway in ischemic myocardium at postoperative day 14 (Figure 2E).

Figure 2

Figure 2 Pro-angiogenic effect of PEMF in ischemic myocardium. A: Immunofluorescence staining of CD31-positive cells in the infarct border zone at postoperative day 14 in PEMF-treated and control rats. B: Quantitative analyses of capillary density between 2 groups

Protective effect of PEMF to MI-induced cardiac apoptosis

We evaluated the effect of PEMF on the survival of myocardium in response to hypoxia in vivo at postoperative day 7. The number of TUNEL positive nucleus in PIZ significantly increased in PEMF-treated rats compared with the non-treated ones (Figure 3), indicating that PEMF treatment decreases the susceptibility of cardiomyocytes to hypoxic damage.

Figure 3

Figure 3 Anti-apoptotic benefit of PEMF in damaged myocardium. A: TUNEL staining for cardiac cell apoptosis (green) and DAPI (blue) for nuclear staining in the border zone 14 days after AMI (×400 magnification). B: Quantitative analysis of the TUNEL-positive

PEMF augments EPC-mediated neovascularization

EPC-mediated neovascularization after myocardial infarction supported their therapeutic potential [27]. Thus, the strategy to amplify EPC abundance and function is an active focus of research. The number of circulating EPCs was identified by stem cell antigen-1 (Sca-1)/fetal liver kinase-1 (flk-1) dual positive cells as described. We found that PEMF treatment increased the number of Sca-1+/flk-1+ cells in peripheral blood at postoperative day 7 and 14 (Figure 4A). Additionally, EPCs isolated from PEMF-treated rats exhibited enhanced tube formative capacity and migratory ability when compared with control ones in vitro (Figure 4B and ?and4C),4C), which suggesting that PEMF increases the abundance and regenerative capacity of EPCs.

Figure 4

Figure 4 PEMF enhanced circulating endothelial progenitor cells (EPCs) function in MI Rats. 7 and 14 days after AMI induction, peripheral blood was collected from rats in both groups. A: Quantitative analysis of Sca-1/flk-1 dual positive PB-EPCs isolated from

Pro-angiogenic beneficial of PEMF in vitro

Cultured HUVECs were treated with PEMF stimulation for 1 to 4 cycles and the supernatant and cell lysate were collected. PEMF promoted VEGF and NO releasing from cultured HUVECs in a dose-dependent manner (Figure 5A and ?and5B).5B). Additionally, the phosphorylation of eNOS in HUVECs was also enhanced in response to PEMF following a dose dependent manner (Figure 5C). Finally, the HUVEC-formed tubes were lengthened by PEMF in a dose dependent manner (Figure 6).

Figure 5

Figure 5 Enhancement of the expression of VEGF and nitric oxide in PEMF-treated HUVECs. PEMF stimulated vascular endothelial growth factors secretion concentration dependently. Bar graph of the concentrations of (A) VEGF and (B) nitric oxide released from HUVECs

Figure 6

Figure 6 Effects of PEMF on tube formation of cultured HUVECs. Representative images of tube formation in HUVECs by stimulated PEMF for 1-4 cycles and quantitative analysis of tube length formed by PEMF-treated HUVECs. Values are mean ± SEM; n=4. *meansGo to:

Discussion

Major findings of our study are: (1) PEMF prevents cardiomyocytes against hypoxia-induced apoptosis and preserves cardiac systolic function in a rat MI model; (2) PEMF induces angiogenesis and vasculogenesis through activating VEGF-eNOS system and promoting EPCs mobilized to the ischemic myocardium.

We demonstrated that PEMF treatment preserved the cardiac systolic function after MI and prevented cardiac apoptosis. Previous report demonstrated that PEMF treatment activated voltage-gated calcium channels (VGCC) [28], which is crucial for maintaining cardiac contractility and cell survival [29,30]. Increased intracellular Ca2+produced by PEMF-mediated VGCC activation may lead to increase of NO through the action of eNOS, which is dominant modulator to prevent cardiomyocytes from apoptosis and enhance revascularization in PIZ after MI [31]. Consistent with the previous work, we demonstrated that the HIF-1?/Akt axis was activated in PIZ in PEMF rats. In addition, PEMF induced eNOS phosphorylation in vitro, which is a key molecular served in the survival pathway in both myocardium and endothelial cell lineage [32].

Another possible mechanism in cardiac protecting effect of PEMF is to stimulate neovascularization. Increasing evidence suggests that neovascularization limits infarct expansion and extension, improves cardiac remodeling [1,2]. Recent data demonstrated that PEMF stimulation induced angiogenesis and amplified endothelial cells function [17,20]. Some researchers believe that PEMF induces cellular proliferation, as evidenced by cAMP activation and uptake of tritiated thymidine [33]. In present study, we demonstrated that the capillary density in PIZ was increased after PEMF treatment. Moreover, PEMF therapy triggered the Akt/HIF-1?/VEGF cascade was activated in ischemic myocardium. In in vitro study, we confirmed PEMF-treated HUVECs released more VEGF and NO, which are the key factors response to endothelial proliferation and survival, suggesting that PEMF activates both autocrine and paracrine function of mature endothelial cells. Furthermore, Tepper and colleagues also reported that PEMF stimulated fibroblastic growth factor-2 (FGF-2) releasing and augment angiogenesis [14].

Recent evidence indicates that adult blood vessels may result from not only expansion of existing endothelial cells (angiogenesis), but also the recruitment of endothelial progenitor cells or EPCs (vasculogenesis) [24]. We hypothesized that besides mature endothelial cells, PEMF might also act as a stimulator of progenitor (EPC). To confirm the hypothesis, we examined the effect of PEMF on ex vivo angiogenesis. Our data demonstrated the number of Sca-1/flk-1 dual positive EPCs in peripheral blood increased in response to PEMF. Using the well-established Matrigel assay, we demonstrated that PEMF was able to dramatically enhance the tube formative capacity of either EPCs or mature endothelial cells in vitro. PEMF also accelerated the migratory ability of EPCs. Moreover, Goto et al reported that PEMF stimulation up-regulated the expression of angiopoietin-2 and FGF-2 in bone marrow, suggesting PEMF could promote the regenerative capacity of myeloid-derived cells (such as EPCs) in damaged tissue when recruited. From all these findings, we conclude that PEMF sufficiently re-establishes blood supply to the ischemic and hypoxic cardiomyocytes via enhancing both angiogenesis and vasculogenesis.

In conclusion, our findings indicate that extracorporeal PEMF treatment increases cardiac systolic function through inhibiting cardiac apoptosis and stimulating neovascularization in PIZ. These findings suggest that PEMF deserves further consideration of investigation in its regulation on the signaling pathway and new clinical strategies for ischemic vascular diseases.Go to:

Acknowledgements

This work was supported by the Shanghai Science and Technology Committee (11 nm 0503600), the China National Natural Science Foundation (11374213) and Foundation of National Lab for Infrared Physics (200901).Go to:

Disclosure of conflict of interest

The authors have nothing to disclose.Go to:

References

1. Hynes B, Kumar AH, O’Sullivan J, Buneker CK, Leblond AL, Weiss S, Schmeckpeper J, Martin K, Caplice NM. Potent endothelial progenitor cell-conditioned media-related anti-apoptotic, cardiotrophic, and pro-angiogenic effects post-myocardial infarction are mediated by insulin-like growth factor-1. Eur Heart J.2013;34:782–789. [PubMed] 2. Zhang S, Zhao L, Shen L, Xu D, Huang B, Wang Q, Lin J, Zou Y, Ge J. Comparison of Various Niches for Endothelial Progenitor Cell Therapy on Ischemic Myocardial Repair Coexistence of Host Collateralization and Akt-Mediated Angiogenesis Produces a Superior Microenvironment. Arterioscler Thromb Vasc Biol.2012;32:910–923. [PubMed] 3. Hao CN, Shi YQ, Huang JJ, Li HY, Huang ZH, Cheng XW, Lu W, Duan JL. The power combination of blood-pressure parameters to predict the incidence of plaque formation in carotid arteries in elderly. Int J Clin Exp Med. 2013;6:461–469. [PMC free article] [PubMed] 4. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature. 2005;438:967–974. [PubMed] 5. Hiasa K, Ishibashi M, Ohtani K, Inoue S, Zhao Q, Kitamoto S, Sata M, Ichiki T, Takeshita A, Egashira K. Gene Transfer of Stromal Cell-Derived Factor-1? Enhances Ischemic Vasculogenesis and Angiogenesis via Vascular Endothelial Growth Factor/Endothelial Nitric Oxide Synthase-Related Pathway Next-Generation Chemokine Therapy for Therapeutic Neovascularization. Circulation. 2004;109:2454–2461. [PubMed] 6. Duan JL, Hao CN, Lu W, Han L, Pan ZH, Gu Y, Liu PJ, Tao R, Shi YQ, Du YY. A new method for assessing variability of 24 h blood pressure and its first application in 1526 elderly men. Clin Exp Pharmacol Physiol.2009;36:1093–1098. [PubMed] 7. Nishiyama K, Takaji K, Kataoka K, Kurihara Y, Yoshimura M, Kato A, Ogawa H, Kurihara H. Id1 gene transfer confers angiogenic property on fully differentiated endothelial cells and contributes to therapeutic angiogenesis. Circulation. 2005;112:2840–2850. [PubMed] 8. Hao CN, Huang ZH, Shi YQ, Lu W, Duan JL. A new index to predict the incidence of cerebral infarction.CNS Neurosci Ther. 2011;17:783–784. [PubMed] 9. Zheng J, Xu DF, Li K, Wang HT, Shen PC, Lin M, Cao XH, Wang R. Neonatal exposure to fluoxetine and fluvoxamine alteres spine density in mouse hippocampal CA1 pyramidal neurons. Int J Clin Exp Pathol.2011;4:162–168. [PMC free article] [PubMed] 10. Olivetti G, Capasso JM, Meggs LG, Sonnenblick EH, Anversa P. Cellular basis of chronic ventricular remodeling after myocardial infarction in rats. Circ Res. 1991;68:856–869. [PubMed] 11. Liu AJ, Zang P, Guo JM, Wang W, Dong WZ, Guo W, Xiong ZG, Wang WZ, Su DF. Involvement of Acetylcholine-?7nAChR in the Protective Effects of Arterial Baroreflex against Ischemic Stroke. CNS Neurosci Ther. 2012;18:918–926. [PubMed] 12. Chalidis B, Sachinis N, Assiotis A, Maccauro G. Stimulation of bone formation and fracture healing with pulsed electromagnetic fields: biologic responses and clinical implications. Int J Immunopathol Pharmacol.2011;24:17–20. [PubMed] 13. Cheing GL, Li X, Huang L, Kwan RL, Cheung KK. Pulsed electromagnetic fields (PEMF) promote early wound healing and myofibroblast proliferation in diabetic rats. Bioelectromagnetics. 2014;35:161–169. [PubMed] 14. Kim SS, Shin HJ, Eom DW, Huh JR, Woo Y, Kim H, Ryu SH, Suh PG, Kim MJ, Kim JY, Koo TW, Cho YH, Chung SM. Enhanced expression of neuronal nitric oxide synthase and phospholipase C-gamma1 in regenerating murine neuronal cells by pulsed electromagnetic field. Exp Mol Med. 2002;34:53–59. [PubMed] 15. Weintraub MI, Herrmann DN, Smith AG, Backonja MM, Cole SP. Pulsed electromagnetic fields to reduce diabetic neuropathic pain and stimulate neuronal repair: a randomized controlled trial. Arch Phys Med Rehabil.2009;90:1102–1109. [PubMed] 16. Tepper OM, Callaghan MJ, Chang EI, Galiano RD, Bhatt KA, Baharestani S, Gan J, Simon B, Hopper RA, Levine JP, Gurtner GC. Electromagnetic fields increase in vitro and in vivo angiogenesis through endothelial release of FGF-2. FASEB J. 2004;18:1231–1233. [PubMed] 17. Yuan Y, Wei L, Li F, Guo W, Li W, Luan R, Lv A, Wang H. Pulsed magnetic field induces angiogenesis and improves cardiac function of surgically induced infarcted myocardium in Sprague-Dawley rats. Cardiology.2010;117:57–63. [PubMed] 18. Pan Y, Dong Y, Hou W, Ji Z, Zhi K, Yin Z, Wen H, Chen Y. Effects of PEMF on microcirculation and angiogenesis in a model of acute hindlimb ischemia in diabetic rats. Bioelectromagnetics. 2013;34:180–188.[PubMed] 19. Delle Monache S, Alessandro R, Iorio R, Gualtieri G, Colonna R. Extremely low frequency electromagnetic fields (ELF-EMFs) induce in vitro angiogenesis process in human endothelial cells. Bioelectromagnetics.2008;29:640–648. [PubMed] 20. Duan J, Murohara T, Ikeda H, Sasaki K, Shintani S, Akita T, Shimada T, Imaizumi T. Hyperhomocysteinemia impairs angiogenesis in response to hindlimb ischemia. Arterioscler Thromb Vasc Biol. 2000;20:2579–2585.[PubMed] 21. Duan J, Murohara T, Ikeda H, Katoh A, Shintani S, Sasaki K, Kawata H, Yamamoto N, Imaizumi T. Hypercholesterolemia inhibits angiogenesis in response to hindlimb ischemia: nitric oxide-dependent mechanism.Circulation. 2000;102:III370–376. [PubMed] 22. Sun Y, Gui H, Li Q, Luo ZM, Zheng MJ, Duan JL, Liu X. MicroRNA-124 protects neurons against apoptosis in cerebral ischemic stroke. CNS Neurosci Ther. 2013;19:813–819. [PubMed] 23. Li L, Zhao L, Yi-Ming W, Yu YS, Xia CY, Duan JL, Su DF. Sirt1 hyperexpression in SHR heart related to left ventricular hypertrophy. Can J Physiol Pharmacol. 2009;87:56–62. [PubMed] 24. Cheng XW, Kuzuya M, Kim W, Song H, Hu L, Inoue A, Nakamura K, Di Q, Sasaki T, Tsuzuki M, Shi GP, Okumura K, Murohara T. Exercise training stimulates ischemia-induced neovascularization via phosphatidylinositol 3-kinase/Akt-dependent hypoxia-induced factor-1 alpha reactivation in mice of advanced age. Circulation.2010;122:707–716. [PMC free article] [PubMed] 25. Assmus B, Honold J, Schachinger V, Britten MB, Fischer-Rasokat U, Lehmann R, Teupe C, Pistorius K, Martin H, Abolmaali ND, Tonn T, Dimmeler S, Zeiher AM. Transcoronary transplantation of progenitor cells after myocardial infarction. N Engl J Med. 2006;355:1222–1232. [PubMed] 26. Huang ZH, Guo W, Zhang LL, Song SW, Hao CN, Duan JL. Donepezil protects endothelial cells against hydrogen peroxide-induced cell injury. CNS Neurosci Ther. 2012;18:185–187. [PubMed] 27. Pall ML. Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects. J Cell Mol Med. 2013;17:958–965. [PMC free article] [PubMed] 28. Fanelli C, Coppola S, Barone R, Colussi C, Gualandi G, Volpe P, Ghibelli L. Magnetic fields increase cell survival by inhibiting apoptosis via modulation of Ca2+ influx. FASEB J. 1999;13:95–102. [PubMed] 29. Bers DM, Perez-Reyes E. Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release. Cardiovasc Res. 1999;42:339–360. [PubMed] 30. Li J, Zhang Y, Li C, Xie J, Liu Y, Zhu W, Zhang X, Jiang S, Liu L, Ding Z. HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism. Cardiovasc Res.2013;99:674–684. [PubMed] 31. Hopper RA, VerHalen JP, Tepper O, Mehrara BJ, Detch R, Chang EI, Baharestani S, Simon BJ, Gurtner GC. Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF-A independent mechanism. Bioelectromagnetics. 2009;30:189–197. [PubMed] 32. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest. 1999;103:1231–1236. [PMC free article] [PubMed] 33. Goto T, Fujioka M, Ishida M, Kuribayashi M, Ueshima K, Kubo T. Noninvasive up-regulation of angiopoietin-2 and fibroblast growth factor-2 in bone marrow by pulsed electromagnetic field therapy. J Orthop Sci. 2010;15:661–665. [PubMed]

Bioelectromagnetics. 2010 May;31(4):296-301.

Effects of weak static magnetic fields on endothelial cells.

Martino CF, Perea H, Hopfner U, Ferguson VL, Wintermantel E.

Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado, USA. martino@colorado.edu

Abstract

Pulsed electromagnetic fields (PEMFs) have been used extensively in bone fracture repairs and wound healing. It is accepted that the induced electric field is the dose metric. The mechanisms of interaction between weak magnetic fields and biological systems present more ambiguity than that of PEMFs since weak electric currents induced by PEMFs are believed to mediate the healing process, which are absent in magnetic fields. The present study examines the response of human umbilical vein endothelial cells to weak static magnetic fields. We investigated proliferation, viability, and the expression of functional parameters such as eNOS, NO, and also gene expression of VEGF under the influence of different doses of weak magnetic fields. Applications of weak magnetic fields in tissue engineering are also discussed. Static magnetic fields may open new venues of research in the field of vascular therapies by promoting endothelial cell growth and by enhancing the healing response of the endothelium.

Eur J Appl Physiol. 2007 Nov;101(4):495-502. Epub 2007 Aug 3.

Short-term effects of pulsed electromagnetic fields after physical exercise are dependent on autonomic tone before exposure.

Grote V, Lackner H, Kelz C, Trapp M, Aichinger F, Puff H, Moser M.

Institute of Noninvasive Diagnosis, JOANNEUM RESEARCH, Weiz, Austria.

Abstract

The therapeutic application of pulsed electromagnetic fields (PEMFs) can accelerate healing after bone fractures and also alleviate pain according to several studies. However, no objective criteria have been available to ensure appropriate magnetic field strength or type of electromagnetic field. Moreover, few studies so far have investigated the physical principles responsible for the impact of electromagnetic fields on the human body. Existing studies have shown that PEMFs influence cell activity, the autonomic nervous system and the blood flow. The aim of this study is to examine the instantaneous and short-term effects of a PEMF therapy and to measure the impact of different electromagnetic field strengths on a range of physiological parameters, especially the autonomic nervous systems, determined by heart rate variability (HRV) as well as their influence on subjects’ general feeling of well-being. The study comprised experimental, double-blind laboratory tests during which 32 healthy male adults (age: 38.4+/-6.5 years) underwent four physical stress tests at standardised times followed by exposure to pulsed magnetic fields of varying intensity [HPM, High Performance magnetic field; Leotec; pulsed signal; mean intensity increase: zero (placebo), 0.005, 0.03 and 0.09 T/s]. Exposure to electromagnetic fields after standardised physical effort significantly affected the very low frequency power spectral components of HRV (VLF; an indicator for sympathetically controlled blood flow rhythms). Compared to placebo treatment, exposure to 0.005 T/s resulted in accelerated recovery after physical strain. Subjects with lower baseline VLF power recovered more quickly than subjects with higher VLF when exposed to higher magnetic field strengths. The application of electromagnetic fields had no effect on subjects’ general feeling of well-being. Once the magnetic field exposure was stopped, the described effects quickly subsided. PEMF exposure has a short-term dosage-dependent impact on healthy subjects. Exposure to PEMF for 20 min resulted in more rapid recovery of heart rate variability, especially in the very low frequency range after physical strain. The study also showed the moderating influence of the subjects’ constitutional VLF power on their response to PEMF treatment. These findings have since been replicated in a clinical study and should be taken into consideration when PEMF treatment is chosen.

Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Sep-Oct;(5):9-11.

Rehabilitative medical technology for the correction of microcirculatory disorders in patients with arterial hypertension.

[Article in Russian]

Kul’chitskaia DB.

Abstract

The study with the use of laser Doppler flowmetry has revealed pathological changes in the microcirculatory system of patients with arterial hypertension. Their treatment with a low-frequency magnetic field showed that its effect on microcirculation depends on the regime and site of application of magnetotherapy as well as its combination with other physical factors. Frontal application of the magnetic field had the most pronounced beneficial effect on dynamic characteristics of microcirculation. Pulsed regime of magnetotherapy was more efficacious than conventional one. Amplipulse magnetotherapy produced better results than monotherapy.

Bioelectromagnetics. 2007 Jan;28(1):64-8.

A pilot investigation of the effect of extremely low frequency pulsed electromagnetic fields on humans’ heart rate variability.

Baldi E, Baldi C, Lithgow BJ.

Diagnostic and Neurosignal Processing Research Group, Electrical & Computer System Engineering, Monash University, Victoria, Australia. Emilio.Baldi@eng.monash.edu.au

Abstract

The question whether pulsed electromagnetic field (PEMF) can affect the heart rhythm is still controversial. This study investigates the effects on the cardiocirculatory system of ELF-PEMFs. It is a follow-up to an investigation made of the possible therapeutic effect ELF-PEMFs, using a commercially available magneto therapeutic unit, had on soft tissue injury repair in humans. Modulation of heart rate (HR) or heart rate variability (HRV) can be detected from changes in periodicity of the R-R interval and/or from changes in the numbers of heart-beat/min (bpm), however, R-R interval analysis gives only a quantitative insight into HRV. A qualitative understanding of HRV can be obtained considering the power spectral density (PSD) of the R-R intervals Fourier transform. In this study PSD is the investigative tool used, more specifically the low frequency (LF) PSD and high frequency (HF) PSD ratio (LF/HF) which is an indicator of sympatho-vagal balance. To obtain the PSD value, variations of the R-R time intervals were evaluated from a continuously recorded ECG. The results show a HR variation in all the subjects when they are exposed to the same ELF-PEMF. This variation can be detected by observing the change in the sympatho-vagal equilibrium, which is an indicator of modulation of heart activity. Variation of the LF/HF PSD ratio mainly occurs at transition times from exposure to nonexposure, or vice versa. Also of interest are the results obtained during the exposure of one subject to a range of different ELF-PEMFs. This pilot study suggests that a full investigation into the effect of ELF-PEMFs on the cardiovascular system is justified.

Georgian Med News. 2006 Jun;(135):109-13.

Influence of treatment with variable magnetic field of low frequency in low mountain environment on cardiohemodynamic index of patients with arterial hypertension.

[Article in Russian]

Tarkhan-Mouravi ID, Purtseladze NA.

Abstract

Pathological changes in function and action of cardiovascular system is the significant link in formation and progression of arterial hypertension. 68 patients were investigated. From these patients in 32 first stage of mentioned pathology, while in 36 – the II degree was found. It is established that treatment of arterial hypertension by variable magnetic field of low frequency in low mountain environment causes decrease of systolic, diastolic and heart dynamic blood pressure, normalizes heart index and pulse rate; decreases peripheral vascular specific resistance, increases amount of upset index accelerated of blood flow on the region “lung-ear”, improves electrocardiological data. Mentioned pathological displacements were more expressed at the first stage of arterial hypertension.

Bioelectromagnetics. 2005 Apr;26(3):161-72.

Decreased plasma levels of nitric oxide metabolites, angiotensin II, and aldosterone in spontaneously hypertensive rats exposed to 5 mT static magnetic field.

Okano H, Masuda H, Ohkubo C.

Department of Environmental Health, National Institute of Public Health, Tokyo 108-8638, Japan. okano@niph.go.jp

Previously, we found that whole body exposure to static magnetic fields (SMF) at 10 mT (B(max)) and 25 mT (B(max)) for 2-9 weeks suppressed and delayed blood pressure (BP) elevation in young, stroke resistant, spontaneously hypertensive rats (SHR). In this study, we investigated the interrelated antipressor effects of lower field strengths and nitric oxide (NO) metabolites (NO(x) = NO(2)(-) + NO(3)(-)) in SHR. Seven-week-old male rats were exposed to two different ranges of SMF intensity, 0.3-1.0 mT or 1.5-5.0 mT, for 12 weeks. Three experimental groups of 20 animals each were examined: (1) no exposure with intraperitoneal (ip) saline injection (sham-exposed control); (2) 1 mT SMF exposure with ip saline injection (1 mT); (3) 5 mT SMF exposure with ip saline injection (5 mT). Arterial BP, heart rate (HR), skin blood flow (SBF), plasma NO metabolites (NO(x)), and plasma catecholamine levels were monitored. SMF at 5 mT, but not 1 mT, significantly suppressed and retarded the early stage development of hypertension for several weeks, compared with the age matched, unexposed (sham exposed) control. Exposure to 5 mT resulted in reduced plasma NO(x) concentrations together with lower levels of angiotensin II and aldosterone in SHR. These results suggest that SMF may suppress and delay BP elevation via the NO pathways and hormonal regulatory systems.

Auton Neurosci. 2003 Apr 30;105(1):53-61.

Can extremely low frequency alternating magnetic fields modulate heart rate or its variability in humans?

Kurokawa Y, Nitta H, Imai H, Kabuto M.

Environmental Health Science Region, National Institute for Environmental Studies, 16-2 Onogawa, Ibaraki Tsukuba 305-0053, Japan.kurokawa@nies.go.jp

Abstract

This study is a reexamination of the possibility that exposure to extremely low frequency alternating magnetic field (ELF-MF) may influence heart rate (HR) or its variability (HRV) in humans. In a wooden room (cube with 2.7-m sides) surrounded with wire, three series of experiments were performed on 50 healthy volunteers, who were exposed to MFs at frequencies ranging from 50 to 1000 Hz and with flux densities ranging from 20 to 100 microT for periods ranging from 2 min to 12 h. In each experiment, six indices of HR/HRV were calculated from the RR intervals (RRIs): average RRI, standard deviation of RRIs, power spectral components in three frequency ranges (pVLF, pLF and pHF), and the ratio of pLF to pHF. Statistical analyses of results revealed no significant effect of ELF-MFs in any of the experiments, and suggested that the ELF-MF to which humans are exposed in their daily lives has no acute influence on the activity of the cardiovascular autonomic nervous system (ANS) that modulates the heart rate.

Klin Med (Mosk). 2003;81(1):24-7.

Clinico-functional efficacy of medicinal and photon stabilization in patients with angina pectoris.

[Article in Russian]

Vasil’ev AP, Senatorov IuN, Strel’tsova NN, Gorbunova TIu.

Modification of erythrocytic membrane and the trend in clinicofunctional indices were studied in 90 patients with angina of effort (FC I-IV) in the course of treatment with a combination of membranoprotective drugs (group 1), magneto-laser radiation (group 2) and imitation of laser radiation (group 3). In patients of groups 1 and 2 the treatment resulted in stabilization of cell membrane accompanied with a hypotensive effect and increased exercise tolerance due to more effective cardiac performance.

Saudi Med J. 2002 May;23(5):517-20.

The effect of magneto-treated blood autotransfusion on central hemodynamic values and cerebral circulation in patients with essential hypertension.

Alizade IG, Karayeva NT.

Department of Cardiology, Hospital of Ministry of Internal Affairs, Baku, Azerbaijan.

OBJECTIVE: The work was carried out to study the effect of magneto-treated blood autotransfusion on the values of central and cerebral hemodynamics in patients with essential hypertension.

METHODS: Sixty-six patients with stage II essential hypertension aged 31-60 years who underwent magneto-treated blood autotransfusion were evaluated and treated, at the Cardiology Department, Hospital of Ministry of Internal Affairs of the Azerbaijan Republic, over a period of 8 years. The diagnosis was based on clinical examination and generally accepted criteria of essential hypertension stages proposed in 1978 by the World Health Organization.

RESULTS: Sixty-six patients with stage II essential hypertension with stable drop in blood pressure, simultaneously showed a positive clinical effect. Central hemodynamic changes in the process of magneto-treated blood autotransfusion were different and depended on the initial state of circulation. High clinical effect showed the patients with hyperkinetic type of hemodynamics. Their blood pressure were significantly lower than the patients with hypokinetic type of circulation.

CONCLUSION: Rheoencephalographic study demonstrated that magneto-treated blood autotransfusion possessed insignificant effect on cerebral hemodynamics, mainly expressed by the reduction of arterial blood flow tension in the patients with hypokinetic type of hemodynamics.

Ter Arkh. 2001;73(10):70-3.

Changes in blood rheological properties in patients with hypertension.

[Article in Russian]

Shabanov VA, Terekhina EV, Kostrov VA.

AIM: To study hemorheology in patients with essential hypertension (EH), to improve EH treatment in terms of blood rheology.

MATERIAL AND METHODS: Blood rheology, microcirculation, lipid plasm spectrum, central hemodynamics were studied in 90 patients with mild and 83 patients with moderate or severe EH as well as 30 healthy controls before and after treatment (hypotensive drugs, essential phospholipids, intravenous laser blood radiation, plasmapheresis).

RESULTS: Hemorrheological disorders (subnormal deformability of the red cells and elastoviscosity of their membranes, disk-spherical transformation and hyperaggregation of blood cells, high dynamic viscosity) correlated with the disease severity, arterial pressure and total peripheral vascular resistance. Long-term (1-1.5 years) hypotensive therapy, especially with combination of beta-blockers with diuretics, has a negative effect on blood rheology. Optimisation of EH treatment in terms of blood rheology consists in using essential phospholipids in stable hypertension, intravenous laser radiation in complicated hypertension, plasmapheresis in drug-resistant hypertension. Such an approach not only significantly improves hemorheology but also provides good clinical and hypotensive effects in 75-80% patients.

CONCLUSION: Blood viscodynamics should be taken into consideration in individual treatment of hypertensive patients.

Med Tr Prom Ekol. 2001;(6):20-3.

Influence of low-frequency magnetotherapy and HF-puncture on the heart rhythm in hypertensive workers exposed to vibration.

[Article in Russian]

Drobyshev VA, Loseva MI, Sukharevskaia TM, Michurin AI.

Abstract

The authors present results concerning use of low-frequency magnetic fields and HF-therapy for correction of vegetative homeostasis in workers with variable length of service, exposed to vibration, having early forms of arterial hypertension. The most positive changes of vegetative status and central hemodynamics are seen in workers with low length of service.

Vopr Kurortol Fizioter Lech Fiz Kult. 2001 Mar-Apr;(2):11-5.

Therapeutic complexes of physical factors in mild arterial hypertension.

[Article in Russian]

Kniazeva TA, Nikiforova TI.

Three therapeutic complexes were compared clinically in patients with mild arterial hypertension. Complex 1 consisted of dry air–radon baths, bicycle exercise and exposure of the renal projection area to decimetric electromagnetic field. Its efficacy was 90%, mechanism of the hypotensive action is reduction of enhanced activity of the sympathico-adrenal and renin-angiotensin-aldosterone systems, improvement of water-mineral metabolism and lipid peroxidation. Complex 2 consisted of dry effervescent baths, anaprilin electrophoresis with sinusoidal modulated currents and exposure of the renal projection area to low-frequency alternating magnetic field. Its efficacy was 80%. It affects renin-angiotensin-aldosterone system, water-mineral metabolism and lipid peroxidation. Complex 3 consisted of electric sleep, laser therapy and general sodium chloride baths. Its efficacy was 63%. The effect was due to inhibition of high sympathico-adrenal system.

Klin Med (Mosk). 2000;78(3):23-5.

Characteristics of microcirculation and vascular responsiveness in elderly patients with hypertension and ischemic heart disease.

[Article in Russian]

Abramovich SG.

Microcirculation and vascular responsiveness were studied in 52 patients with arterial hypertension and ischemic heart disease versus 48 healthy elderly persons. The patients were found to have defects of the end blood flow in all links of microcirculation, longer and more severe vasoconstriction of conjunctival and skin vessels in response to norepinephrine and cold stimulation tests.

Vopr Kurortol Fizioter Lech Fiz Kult. 2000 May-Jun;(3):9-11.

The use of low-frequency magnetotherapy and EHF puncture in the combined treatment of arterial hypertension in vibration-induced disease.

[Article in Russian]

Drobyshev VA, Filippova GN, Loseva MI, Shpagina LA, Shelepova NV, Zhelezniak MS.

Combination of EHF therapy + magnetotherapy + drugs results in faster and persistent hypotensive and analgetic effect compared to standard drug therapy, potentiates action of vascular drugs on cerebral and peripheral circulation, reduces dose of hypotensive drugs in patients with arterial hypertension and vibration disease.

Crit Rev Biomed Eng. 2000;28(1-2):339-47.

The use of millimeter wavelength electromagnetic waves in cardiology.

Lebedeva AYu.

2nd Department of urgent cardiology at State Clinical Hospital, Russian State Medical University, Moscow.

Abstract

This paper concerns the problems of the use of millimeter wavelength electromagnetic waves for the treatment of cardiovascular disease. The prospects for this use are considered.

Vopr Kurortol Fizioter Lech Fiz Kult. 1999 Sep-Oct;(5):7-9.

The characteristics of the geroprotective action of magnetotherapy in elderly patients with combined cardiovascular pathology.

[Article in Russian]

Abramovich SG, Fedotchenko AA, Koriakina AV, Pogodin KV, Smirnov SN.

Central hemodynamics, diastolic and pumping functions of the heart, myocardial reactivity, microcirculation and biological age of cardiovascular system were studied in 66 elderly patients suffering from hypertension and ischemic heart disease. The patients received systemic magnetotherapy which produced a geroprotective effect as shown by improved microcirculation, myocardial reactivity, central hemodynamics reducing biological age of cardiovascular system and inhibiting its ageing.

Neuropsychobiology. 1998 Nov;38(4):251-6.

No effects of pulsed high-frequency electromagnetic fields on heart rate variability during human sleep.

Mann K, Roschke J, Connemann B, Beta H.

Department of Psychiatry, University of Mainz, Germany.

The influence of pulsed high-frequency electromagnetic fields emitted by digital mobile radio telephones on heart rate during sleep in healthy humans was investigated. Beside mean RR interval and total variability of RR intervals based on calculation of the standard deviation, heart rate variability was assessed in the frequency domain by spectral power analysis providing information about the balance between the two branches of the autonomic nervous system. For most parameters, significant differences between different sleep stages were found. In particular, slow-wave sleep was characterized by a low ratio of low- and high-frequency components, indicating a predominance of the parasympathetic over the sympathetic tone. In contrast, during REM sleep the autonomic balance was shifted in favor of the sympathetic activity. For all heart rate parameters, no significant effects were detected under exposure to the field compared to placebo condition. Thus, under the given experimental conditions, autonomic control of heart rate was not affected by weak-pulsed high-frequency electromagnetic fields.

Vopr Kurortol Fizioter Lech Fiz Kult. 1998 Jul-Aug;(4):31-6.

The combined action of infrared radiation and permanent and alternating magnetic fields in experimental atherosclerosis.

[Article in Russian]

Zubkova SM, Varakina NI, Mikhailik LV, Bobkova AS, Maksimov EB.

Paravertebral exposure to infrared radiation (0.87 micron, 5 mW) and permanent magnetic field in combination with one- and two-semiperiodic alternative magnetic fields (50 Hz, 15-30 mT) was studied in respect to the action on adaptive reactions in animals with experimental atherosclerosis. Complex consisting of infrared radiation, permanent magnetic field and one-semiperiodic pulse alternative magnetic field was most effective in restoration of vasomotor-metabolic and immune disturbances accompanying development of atherosclerosis.

Bioelectromagnetics. 1998;19(2):98-106.

Nocturnal exposure to intermittent 60 Hz magnetic fields alters human cardiac rhythm.

Sastre A, Cook MR, Graham C.

Midwest Research Institute, Kansas City, Missouri 64110, USA. Asastre@mriresearch.org

Abstract

Heart rate variability (HRV) results from the action of neuronal and cardiovascular reflexes, including those involved in the control of temperature, blood pressure and respiration. Quantitative spectral analyses of alterations in HRV using the digital Fourier transform technique provide useful in vivo indicators of beat-to-beat variations in sympathetic and parasympathetic nerve activity. Recently, decreases in HRV have been shown to have clinical value in the prediction of cardiovascular morbidity and mortality. While previous studies have shown that exposure to power-frequency electric and magnetic fields alters mean heart rate, the studies reported here are the first to examine effects of exposure on HRV. This report describes three double-blind studies involving a total of 77 human volunteers. In the first two studies, nocturnal exposure to an intermittent, circularly polarized magnetic field at 200 mG significantly reduced HRV in the spectral band associated with temperature and blood pressure control mechanisms (P = 0.035 and P = 0.02), and increased variability in the spectral band associated with respiration (P = 0.06 and P = 0.008). In the third study the field was presented continuously rather than intermittently, and no significant effects on HRV were found. The changes seen as a function of intermittent magnetic field exposure are similar, but not identical, to those reported as predictive of cardiovascular morbidity and mortality. Furthermore, the changes resemble those reported during stage II sleep. Further research will be required to determine whether exposure to magnetic fields alters stage II sleep and to define further the anatomical structures where field-related interactions between magnetic fields and human physiology should be sought.

Vopr Kurortol Fizioter Lech Fiz Kult. 1998 Jan-Feb;(1):16-8.

A comparative evaluation of the effect of an extremely high-frequency electromagnetic field on cerebral hemodynamics in hypertension patients exposed in different reflexogenic areas.

[Article in Russian]

Sokolov BA, Bezruchenko SV, Kunitsyna LA.

A single session and multiple sinocarotid and temporal exposures to EHF electromagnetic field in patients with stage I and II hypertension had different effects on cerebral circulation Variants of the above treatment are proposed.

Vopr Kurortol Fizioter Lech Fiz Kult. 1997 Jan-Feb;(1):8-11.

Prognostic criteria of the efficacy of magnetic and magnetic-laser therapy in patients with the initial stages of hypertension.

[Article in Russian]

Zadionchenko VS, Sviridov AA, Adasheva TV, Demicheva OIu, Bagatyrova KM, Beketova IL.

Study of the efficacy of a course of exposures to travelling pulsed magnetic field and magnetic laser sessions in 97 patients with stages I-II essential hypertension showed a high efficacy of travelling pulsed magnetic field in patients with hyperkinetic hemodynamics and initially just slightly shifted blood rheology and platelet hemostasis. Magnetic laser therapy is more effective in patients with eukinetic and hypokinetic hemodynamics and initially sharply expressed disorders of blood rheology and platelet hemostasis.

Biofizika. 1996 Jul-Aug;41(4):944-8.

Effect of a “running” pulse magnetic field on certain humoral indicators and physical ability to work in patients with neurocirculatory hypo- and hypertension.

[Article in Russian]

Orlov LL, Pochechueva GA, Makoeva LD.

The influence of “running” impulse magnetic field in patients with neurocirculatory hypo- and hypertension was studied. It has been determined that magnetotherapy in all patients increased physical load tolerability and at the same time produced different effects on hemodynamics (lowering blood pressure in hypertension and increasing it in hypotension). In patients with neurocirculatory hypotension the slightly expressed positive clinical effect was obtained, that makes “running” impulse magnetic field therapy useless in this pathology. At the same time in patients with neurocirculatory hypertension “running” impulse magnetic field therapy resulted in significant improvement of physical tolerability, improvement of patients general condition, blood pressure decrease, lowering of pressor power generation concentration, correcting effect on aldosterone blood content. These data witness for the usefulness of this method in treatment of patients with neurocirculatory hypertension.

Ter Arkh. 1996;68(5):63-7.

The therapeutic correction of disorders in thrombocyte-vascular hemostasis and of changes in the rheological properties of the blood in patients with arterial hypertension.

[Article in Russian]

Zadionchenko VS, Bagatyrova KM, Adasheva TV, Timofeeva NIu, Zaporozhets TP.

158 patients with essential hypertension received beta-adrenoblockers and were exposed to travelling impulse magnetic field, magnetolaser radiation. The study of platelet-vessel hemostasis and blood rheology revealed a relation of good clinical response and increased exercise tolerance with initial platelet dysfunction and rheological disorders which underwent positive changes in the course of treatment

Vopr Kurortol Fizioter Lech Fiz Kult. 1996 Mar-Apr;(2):5-8.

The effect of exposure to magnetics and lasers on the clinical status and the electrophysiological indices of the heart in patients with cardiac arrhythmias.

[Article in Russian]

Budnar’ LN, Antiuf’ev VF, Oranskii IE, Bekhter TV.

Magnetolaser radiation has a considerable influence on electrophysiological condition of the sinus node and sinoatrial zone. There are cases when patients with sick sinus syndrome get rid of arrhythmia. The treatment is safe and promising for further studies.

Vestn Khir Im I I Grek. 1996;155(5):37-9.

The potentials of laser and electromagnetic-laser therapy in the treatment of patients with arteriosclerosis obliterans of the vessels of the lower extremities.

[Article in Russian]

Galimzianov FV.

A comparative analysis of the laser and electromagnetic laser therapy was performed in the complex treatment of patients with obliterating atherosclerosis of the lower extremity vessels. Laser treatment exerts a therapeutic effect related with its influence upon microcirculation. The effectiveness of complex treatment becomes higher when using a combination of laser therapy with the impulse electromagnetic therapy of complex modulation at the expense of improvement of the regional blood circulation in all links of the vasculature.

Vopr Kurortol Fizioter Lech Fiz Kult. 1996 Mar-Apr;(2):8-10.

The effect of a low-frequency alternating magnetic field on the autonomic system in children with primary arterial hypertension.

[Article in Russian]

Konova OM, Khan MA.

The paper provides cardiointervalographic data assessing autonomic nervous system (ANS) function in children with primary arterial hypertension exposed to low-frequency alternating magnetic field. Favourable effects of such magnetotherapy manifest in attenuation of sympathetic and vagotonic symptoms.

Lik Sprava. 1996 Jan-Feb;(1-2):58-62.

The clinico-biochemical, functional, immunological and cellular characteristics of the body reactions in patients with the initial stages of hypertension to the effect of a magnetic field.

[Article in Ukrainian]

Myloslavs’kyi DK, Koval’ SM, Sheremet MS.

The article presents a comprehensive evaluation of major clinical, laboratory and functional indices in the time course of magnetotherapy as well as during administration of such treatments. The most promising alternative appears to be that involving the use of immunologic and cellular parameters as markers of efficacy of therapeutic action of magnetic fields in early stages of hypertensive disease. Causes for effectiveness and ineffectiveness of the above treatment option are analyzed, approaches to eliminating those are outlined, the main indications and contraindications are determined, merits and demerits of magnetotherapy are drawn attention to.

Vopr Kurortol Fizioter Lech Fiz Kult. 1994 May-Jun;(3):10-2.

The effect of the joint use of plasmapheresis and magnetic treatment of the blood on the indices of blood rheology and hemodynamics in hypertension patients.

[Article in Russian]

Alizade IG, Karaeva NT.

The results are presented obtained on combined application of plasmapheresis and magnetic blood treatment as regards hemorheology and hemodynamics in 41 patients with essential hypertension stage II. The course introduction of the above combined treatment led to positive shifts in arterial pressure irrespective of the patients’ hemodynamic type, in blood density, elasticity and dynamic properties.

Vopr Kurortol Fizioter Lech Fiz Kult. 1994 Jan-Feb;(1):8-9.

The efficacy of low-intensity exposures in hypertension.

[Article in Russian]

Kniazeva TA, Otto MP, Markarov GS, Donova OM, Markarova IS.

One hundred hypertensive subjects with labile and stable disease were exposed to low-intensity low-frequency electrostatic field generated by the unit “Infita-A”. In labile hypertension, the field produces a hypotensive effect, improves myocardial contractility, increases myocardial and coronary reserves due to reduced peripheral resistance and stimulation of myocardial propulsion. Therapeutic response to the treatment is attributed to normalization of deep brain structure functioning.

Vopr Kurortol Fizioter Lech Fiz Kult. 1994 Mar-Apr;(2):18-20.

The effect of a low-frequency magnetic field on erythrocyte membrane function and on the prostanoid level in the blood plasma of children with parasystolic arrhythmia.

[Article in Russian]

Vasil’eva EM, Danilova NV, Smirnov IE, Kupriianova OO, Gordeeva GF.

As shown by clinical and biochemical evidence on 23 parasystolic children, the treatment with low-frequency magnetic field improves humoral and cellular processes participating in cardiac rhythm regulation. There is activation of Ca, Mg-ATPase in the red cells, a reduction of plasma thromboxane levels. Red cell phospholipid composition insignificantly change. Further courses of magnetotherapy may lower the risk of recurrent arrhythmia.

Vopr Kurortol Fizioter Lech Fiz Kult. 1993 Sep-Oct;(5):4-9.

Changes in intracellular regeneration and the indices of endocrine function and cardiac microcirculation in exposure to decimeter waves.

[Article in Russian]

Korolev IuN, Geniatulina MS, Popov VI.

Abstract

An electron-microscopic study of rabbit heart with experimental myocardial infarction revealed that extracardiac exposure to decimetric waves (DW) activated intracellular regeneration in the myocardium. This was associated with enhanced circulation and endocrine activity in the heart. Most pronounced regeneration was registered in adrenal exposure, the effect of the parietal exposure being somewhat less.

Vopr Kurortol Fizioter Lech Fiz Kult. 1993 Sep-Oct;(5):22-5.

The use of magnetics and laser therapy in treating obliterating vascular disease of the extremities.

[Article in Russian]

Kirillov IuB, Shval’b PG, Lastushkin AV, Sigaev AA, Kachinskii AE, Shashkova SN.

The paper presents the results of treatment received by 60 patients suffering from lower limb vascular obliteration stage IIA-III. The treatment involved combined use of magnetic field and laser irradiation. Peripheral circulation and central hemodynamics were evaluated rheographically and using ultrasound Doppler sphygmomanometry. Combined application of the above two modalities produced a greater effect on central hemodynamics compared to them introduced alone.

Ter Arkh. 1993;65(1):44-9.

The comparative efficacy of nondrug and drug methods of treating hypertension.

[Article in Russian]

Ivanov SG.

Effectiveness of some physical therapeutic factors (constant magnetic field, impulse currents) and new hypotensive drugs (tobanum, prinorm, ormidol, minipress, arifon, arilix) was compared in the treatment of essential hypertension stage II. It is suggested that nonpharmaceutical therapy can regulate functions, correct hemodynamic and microcirculatory disorders, produce therapeutic effect without side effects typical for drugs.

Lik Sprava. 1992 May;(5):40-3.

The effect of combined treatment with the use of magnetotherapy on the systemic hemodynamics of patients with ischemic heart disease and spinal osteochondrosis.

[Article in Russian]

Dudchenko MA, Vesel’skii ISh, Shtompel’ VIu.

The authors examined 66 patients with ischemic heart disease and concomitant cervico-thoracic osteochondrosis and 22 patients without osteochondrosis. Differences were revealed in values of the systemic hemodynamics with prevalence of the hypokinetic type in patients with combined pathology. Inclusion of magnetotherapy in the treatment complex of patients with ischemic heart disease and osteochondrosis favours clinical improvement, normalization of indices of central and regional blood circulation.

Lik Sprava. 1992 Oct;(10):32-5.

A comparative evaluation of the efficacy of quantum methods for treating hypertension patients.

[Article in Ukrainian]

Nykul TD, Karpenko VV, Voitovych NS, Karmazyna OM.

A study is presented of the effect of laser and microwave resonance therapy on the hemodynamics and hemorheology in 56 patients with hypertensive disease. The hypotensive effect of intravascular laser therapy is related to the positive changes, reduction of blood viscosity and general peripheral vascular resistance. The effect of low molecular electromagnetic radiation on acupuncture points favoured clear reduction of peripheral vessel resistance. Combination of laser and microwave resonance therapy produces a positive effect due to potentiation of these methods and, thus, influencing the systems of hemodynamics, hemostasis and hemorheology.

Vopr Kurortol Fizioter Lech Fiz Kult. 1992 Sep-Dec;(5-6):13-8.

The effect of decimeter waves on the metabolism of the myocardium and its hormonal regulation in rabbits with experimental ischemia.

[Article in Russian]

Frenkel’ ID, Zubkova SM, Liubimova NN, Popov VI.

Abstract

Biochemical and morphometric methods were employed to study the effect of decimetric waves (460 MHz, 10 and 120 mW/cm2) in cardiac and thyroid exposure on oxygen metabolism, myocardial microcirculation and contractility, thyroid and adrenal hormonal activity, kallikrein-kinin system activity in rabbits with experimental myocardial ischemia. Hypoxia discontinued in all the treatment regimens, but the exposure of the heart (field density 10 mW/sm2) had the additional effect on lipid peroxidation which reduced in the serum and normalized in the myocardium, on myocardial contractility, kallikrein-kinin system and on the adrenal and thyroid hormones.

Vopr Kurortol Fizioter Lech Fiz Kult. 1992 May-Jun;(3):14-7.

Magnetotherapy in obliterating vascular diseases of the lower extremities.

[Article in Russian]

Kirillov IuB, Shval’b PG, Lastushkin AV, Baranov VM, Sigaev AA, Zueva GV, Karpov EI.

The investigators have developed a polymagnetic system “Avrora-MK-01” employing running impulse magnetic field to treat diseases of the leg vessels by the action on peripheral capillary bed. At a pregangrene stage a positive effect on peripheral capillaries was achieved in 75-82% of the patients treated.

Kardiologiia. 1991 Feb;31(2):67-70.

Optimization of the treatment of patients with hypertensive disease from the rheological viewpoint.

[Article in Russian]

Shabanov VA, Kitaeva ND, Levin GIa, Karsakov VV, Kostrov VA.

The efficacy of various modes of correcting rheological disorders (membrane-protective agents, laser irradiation, plasmapheresis was compared in hypertensive patients. In 30% of the patients, the conventional antihypertensive therapy was demonstrated to deteriorate hemorheological parameters, which was due to its atherogenic impact on the blood lipid spectrum. Essential phospholipids, laser irradiation, and plasmapheresis, which are supplemented to the multimodality therapy promote a significant improvement of hemorheological parameters, which makes it possible to recommend them for management of hypertensive patients with a stable (essential phospholipids), complicated (laser irradiation), and refractory (plasmapheresis) course.

Khirurgiia (Mosk). 1990 Nov;(11):41-3.

Outpatient electromagnetic therapy combined with hyperbaric oxygenation in arterial occlusive diseases.

[Article in Russian]

Reut NI, Kononova TI.

The authors first applied hyperbaric oxygenation (HBO) in the outpatient clinic in 1968. Barotherapy was conducted in 107 outpatients whose ages ranged from 27 to 80 years; they had various stages of the disease of 5- to 20-year history. In 70 patients treated for obliterating diseases of the vessels by HBO in a complex with magnetotherapy by means of magnetophors, the remission lasted 1-2 years; patients treated by HBO alone had a 3-8 month remission. A prolonged positive effect was produced in 64 patients. The suggested effective and safe method is an additional one to the existing means of treating this serious and progressive disease, which can be applied successfully in outpatient clinics.

Ter Arkh. 1990;62(9):71-4.

The magnetotherapy of hypertension patients.

[Article in Russian]

Ivanov SG, Smirnov VV, Solov’eva FV, Liashevskaia SP, Selezneva LIu.

A study was made of the influence of the constant MKM2-1 magnets on patients suffering from essential hypertension. Continuous action of the magnetic field, created by such magnets, on the patients with stage II essential hypertension was noted to result in a decrease of arterial pressure without the occurrence of any side effects and in a simultaneous reduction of the scope of drug administration. Apart from that fact, magnetotherapy was discovered to produce a beneficial effect on the central hemodynamics and microcirculation. The use of the MKM2-1 magnets may be regarded as a feasible method of the treatment of essential hypertension patients at any medical institution.

Patol Fiziol Eksp Ter. 1989 May-Jun;(3):59-61.

Changes of central hemodynamics in rats with spontaneous hypertension under the effect of a low-frequency magnetic field.

[Article in Russian]

Buiavykh AG, Stukanov AF.

It was established that a course of exposures of the renal region of rats with spontaneous hypertension to the effect of low-frequency magnetic field influenced the central hemodynamic parameters significantly, which was displayed by reduction of total peripheral vascular resistance and normalization of the cardiac output.

Cancer

Electromagn Biol Med. 2018;37(3):155-168. doi: 10.1080/15368378.2018.1499031. Epub 2018 Jul 18.

Resonant interaction between electromagnetic fields and proteins: A possible starting point for the treatment of cancer.

Calabrò E1,2, Magazù S1,3,4,5.

Author information

1 a Department of Mathematical and Informatics Sciences , Physical Sciences and Earth Sciences of Messina University , Messina , Italy. 2 e CISFA – Interuniversity Consortium of Applied Physical Sciences (Consorzio Interuniversitario di Scienze Fisiche Applicate) , Messina , Italy. 3 b Le Studium, Loire Valley Institute for Advanced Studies, Orléans & Tours , Orléans , France. 4 c Centre de Biophysique Moleculaire (CBM), rue Charles Sadron, Laboratoire Interfaces, Confinement, Matériaux et Nanostructures (ICMN) – UMR 7374 CNRS , Université d’Orléans , Orleans , France. 5 d Istituto Nazionale di Alta Matematica “F. Severi” – INDAM – Gruppo Nazionale per la Fisica Matematica – GNFM , Rome , Italy.

Abstract

Samples of human hemoglobin, bovine serum albumin, lysozyme and myoglobin were used as prototype of proteins to investigate their response to exposure to high frequency electromagnetic fields (HF-EMFs), in order to study possible application to the treatment of cancer. To this aim, Fourier-transform infrared spectroscopy was used in the infrared region. The most evident result which appeared after 3 h exposure to HF-EMFs was a significant increase in intensity of the Amide I band and of CH2 bending vibrations, showing that the proteins aligned toward the direction of the field. In addition, proteins’ unfolding and aggregation occurred after exposure to HF-EMFs. These findings can be explained assuming a resonance interaction between the natural frequencies of proteins and HF-EMFs, which can induce iperpolarization of cells. Given that cancerous tissues were found to have natural frequencies different from natural frequencies of normal tissues, we can hypothesize to irradiate cancerous tissues using EMFs at natural frequencies of cancer cells, causing resonant interaction with cellular membrane channels, inducing increasing of ions’ flux across cellular channels and damaging the cellular functions of cancer cells.

KEYWORDS:

Electromagnetic fields; FTIR spectroscopy; cancer treatment; membrane channel; proteins; resonanceIntegr Biol (Camb). 2017 Dec 11;9(12):979-987. doi: 10.1039/c7ib00116a.

High-frequency irreversible electroporation targets resilient tumor-initiating cells in ovarian cancer.

Rolong A1, Schmelz EM, Davalos RV.

Author information

1 Virginia Tech – Wake Forest University School of Biomedical Engineering and Sciences, Blacksburg, VA 24061, USA. davalos@vt.edu.

Abstract

We explored the use of irreversible electroporation (IRE) and high-frequency irreversible electroporation (H-FIRE) to induce cell death of tumor-initiating cells using a mouse ovarian surface epithelial (MOSE) cancer model. Tumor-initiating cells (TICs) can be successfully destroyed using pulsed electric field parameters common to irreversible electroporation protocols. Additionally, high-frequency pulses seem to induce cell death of TICs at significantly lower electric fields suggesting H-FIRE can be used to selectively target TICs and malignant late-stage cells while sparing the non-malignant cells in the surrounding tissue. We evaluate the relationship between threshold for cell death from H-FIRE pulses and the capacitance of cells as well as other properties that may play a role on the differences in the response to conventional IRE versus H-FIRE treatment protocols. Sci Rep. 2016 Jan 29;6:19451. doi: 10.1038/srep19451.

Constructal approach to cell membranes transport: Amending the ‘Norton-Simon’ hypothesis for cancer treatment.

Lucia U1, Ponzetto A2, Deisboeck TS3,4.

Author information

  • 1Dipartimento Energia, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy.
  • 2Department of Medical Sciences, University of Torino, Corso A.M. Dogliotti 14, 10126 Torino, Italy.
  • 3Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
  • 4ThinkMotu LLC, Wellesley, MA 02481, USA.

Abstract

To investigate biosystems, we propose a new thermodynamic concept that analyses ion, mass and energy flows across the cell membrane. This paradigm-shifting approach has a wide applicability to medically relevant topics including advancing cancer treatment. To support this claim, we revisit ‘Norton-Simon’ and evolving it from an already important anti-cancer hypothesis to a thermodynamic theorem in medicine. We confirm that an increase in proliferation and a reduction in apoptosis trigger a maximum of ATP consumption by the tumor cell. Moreover, we find that positive, membrane-crossing ions lead to a decrease in the energy used by the tumor, supporting the notion of their growth inhibitory effect while negative ions apparently increase the cancer’s consumption of energy hence reflecting a growth promoting impact. Our results not only represent a thermodynamic proof of the original Norton-Simon hypothesis but, more concretely, they also advance the clinically intriguing and experimentally testable, diagnostic hypothesis that observing an increase in negative ions inside a cell in vitro, and inside a diseased tissue in vivo, may indicate growth or recurrence of a tumor. We conclude with providing theoretical evidence that applying electromagnetic field therapy early on in the treatment cycle may maximize its anti-cancer efficacy. J Orthop Surg Res. 2015; 10: 104. Published online 2015 Jul 7. doi:  10.1186/s13018-015-0247-z PMCID: PMC4496869

Nanosecond pulsed electric field inhibits proliferation and induces apoptosis in human osteosarcoma

Xudong Miao,# Shengyong Yin,# Zhou Shao, Yi Zhang, and Xinhua Chen

corresponding author

The Department of Orthopedics, the Second Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang Province 310003 China The Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital, Zhejiang University, Collaborative Innovation Center for Diagnosis Treatment of Infectious Diseases, 79 Qinchun Road, Hangzhou, Zhejiang Province 310003 China The Department of Gynecology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province 310000 China Xinhua Chen, Phone: +86-571-87236570, Email: nc.ude.ujz@nehc_auhnix.

corresponding author

Corresponding author. #Contributed equally. Author information ? Article notes ? Copyright and License information ? Received 2015 Jun 11; Accepted 2015 Jun 29. Copyright © Miao et al. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Objective

Recent studies suggest that nanosecond pulsed electric field (nsPEF) is a novel minimal invasive and non-thermal ablation method that can induce apoptosis in different solid tumors. But the efficacy of nsPEF on bone-related tumors or bone metastasis is kept unknown. The current study investigates antitumor effect of nsPEF on osteosarcoma MG-63 cells in vitro.

Method

MG-63 cells were treated with nsPEF with different electric field strengths (0, 10, 20, 30, 40, and 50 kV/cm) and different pulse numbers (0, 6, 12, 18, 24, and 30 pulses). The inhibitory effect of nsPEF on the growth of MG-63 cells was measured by Cell Counting Kit-8 (CCK-8) assay at different time points (0, 3, 12, 24, and 48 h post nsPEF treatment). The apoptosis was analyzed by Hoechst stain, in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), and flow cytometric analysis. The expression of osteoprotegerin (OPG), receptor activator of NF-kB ligand (RANKL), and tumor necrosis factor a (TNF-a) was examined by reverse-transcription polymerase chain reaction (RT-PCR) and western blot.

Results

The CCK-8 assay showed that nsPEF induced a distinct electric field strength- and pulse number-dependent reduction of cell proliferation. For treatment parameter optimizing, the condition 40 kV/cm and 30 pulses at 24 h post nsPEF achieved the most significant apoptotic induction rate. Hoechst, TUNEL, and flow cytometric analysis showed that the cell apoptosis was induced and cells were arrested in the G0/G1 phase. PCR and western blot analysis demonstrated that nsPEF up-regulated OPG expression had no effect on RANKL, increased OPG/RANKL ratio.

Conclusion

NsPEF inhibits osteosarcoma growth, induces apoptosis, and affects bone metabolism by up-regulating OPG, indicating nsPEF-induced apoptosis in osteosarcoma MG-63 cells. NsPEF has potential to treat osteosarcoma or bone metastasis. When nsPEF is applied on metastatic bone tumors, it might be beneficial by inducing osteoblastic differentiation without cancer proliferation. In the future, nsPEF might be one of the treatments of metastatic bone tumor.Keywords: Osteosarcoma, MG-63 cells, Nanosecond pulsed electric field, Apoptosis

Introduction

Osteosarcoma is a malignant bone tumor with high occurrence in children and young adolescents. Retrospective review showed that in the past 30 years, osteosarcoma had a poor prognosis and there was no significant improvement of disease-free survival and the stagnated situation has not improved even with the aggressive use of neoadjuvant chemotherapy and radiation therapy [1]. Patients did not benefit from overtreatment, and as a result, a high rate of lung metastasis, recurrence, and pathological fracture frequently occur, keeping osteosarcoma still one of the lowest survival rates in pediatric cancers [2]. Thus, new therapeutic strategy needs to be developed.

Nanosecond pulsed electric field (nsPEF) is an innovative electric ablation method based on high-voltage power technology, which came into medical application in the last decade [3]. NsPEF accumulates the electric field energy slowly and releases it into the tumor in ultra-short nanosecond pulses, altering electrical conductivity and permeability of the cell membrane, causing both cell apoptosis and immune reaction [4].Quite different from any other traditional local ablation method, nsPEF accumulate less Joule heating and showed no hyperthermic effects [5], indicating unique advantage over other thermal therapies such as radiofrequency, cryoablation, microwave, and interstitial laser; nsPEF can be used alone and so avoid the side effect caused by chemotherapy or percutaneous ethanol injection [6].

We have used nsPEF to ablate tumor and showed the equal outcome as the radical resection with proper indication [7]. Clinical trials and pre-clinical studies from different groups proved that nsPEF has direct antitumor effects by inhibiting proliferation and causing apoptosis in human basal cell carcinoma [8, 9], cutaneous papilloma, squamous cell carcinoma [10], melanoma [11, 12], hepatocellular tumor [13], pancreatic tumor [14], colon tumor [15, 16], breast cancer [17, 18], salivary adenoid cystic carcinoma [19], oral squamous cell carcinoma [20], et al. Local ablation with nsPEF indicates the noticeable advantage of not only eliminating original tumors but also inducing an immune reaction, e.g., enhance macrophage [21] and T cell infiltration [22] and induce an immune-protective effect against recurrences of the same cancer [23]. The characteristic of electric field on bone metabolism [24] is extremely helpful for osteosarcoma patients with pathological fracture which leads to poor prognosis [25, 26].

Considering osteosarcoma is especially prevalent in children and young adults during quick osteoblastic differentiation [1, 2], unstable RB gene and p53 gene are commonly involved in this malignant transformation process [27]; we hypothesize that nsPEF affects osteosarcoma growth by targeting the Wnt/?-catenin signaling pathway, a key signaling cascade involved in osteosarcoma pathogenesis. Here, we investigate nsPEF-induced changes on human osteosarcoma MG-63 cells to determine (1) the dose-effect relationship and time-effect relationship of nsPEF on osteosarcoma cell growth and apoptosis induction and (2) the nsPEF effect on the osteosarcoma cell; osteoblast specific gene and protein expression (receptor activator of NF-?B ligand (RANKL) and osteoprotegerin (OPG)) were measured along with the production of the pro-inflammatory cytokine tumor necrosis factor a (TNF-a).

Materials and methods

Cell lines and cell culture

MG-63 human osteosarcoma cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS, SAFC Biosciences, Lenexa, KS, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin (Sigma, Aldrich, St. Louis, MO, USA). Cells were kept in a humidified atmosphere of 5 % CO2 at 37 °C.

The nsPEF treatment and dose-effect exam

The nsPEF treatment system was made by Leibniz Institute for Plasma Science and Technology, Germany, and an nsPEF generator with duration of 100 ns was applied. Varied electric fields were released in a cell treatment system from 10 to 60 kV/cm. Waveforms were monitored with a digital phosphor oscilloscope (DPO4054, Tektronix, USA) equipped with a high voltage probe (P6015A, Tektronix, USA). MG-63 human osteosarcoma cells were harvested with trypsin and resuspended in fresh DMEM with 10 % FBS to a concentration of 5.0 × 106 cells/mL. Five hundred microliters of cell suspension were placed into a sterile electroporation cuvette (Bio-Rad, US, 0.1-cm gap). Cells were exposed to 100 pulses at 0, 10, 20, 30, 40, 50, and 60 kV/cm electric field strengths, respectively. Under the 50 kV/cm electric field strength, the different pulse numbers were applied (0, 6, 12, 18, 24, and 30 pulses). The experiments were repeated for three times. After incubation for 24 h, cells were calculated by Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan).

Measurement of apoptosis with TUNEL assay, Hoechst stain, and flow cytometry

At different hours after nsPEF treatment (40 kV/cm, 30 pulses), the treated cells were incubated for 0, 3, 12, 24, and 48 h to determine single-cell apoptosis using the assay of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) with In Situ Cell Death Detection Kit (Millipore, USA) and Hoechst stain kit (Beyotime, Shanghai, China) according to the manufacturer’s instruction, as previously described [14]. Under different electric field strengths and with different pulses, the treated cells were incubated for 24 h to detect cell apoptosis by Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). The cell cycle was also analyzed as previously described [14].

Reverse-transcription polymerase chain reaction

Reverse-transcription polymerase chain reaction (RT-PCR) was performed for assessing the expression of OPG, RANKL, and TNF-a. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a house keeping gene, was used as the internal control to calculate the comparative expression. Total RNA was extracted using TRIzol reagent (Sangon, Shanghai, China). The first strand cDNA synthesis from 1 mg of RNA was performed using SuperScript II Reverse Transcriptase (Invitrogen) and Oligo dT primer (Promega, Madison, WI, USA) according to the manufacturer’s instructions. PCR was performed using the oligunucleotides listed as the following. The specific primers were made by Sangon, Shanghai, China, which were listed as the following: RANK: F: CAGGAGACCTAGCTACAGA, R: CAAGGTCAAGAGCATGGA, 95 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min; OPG (264 bp): F: AGTGGGAGCAGAAGACAT, R: TGGA CCTGGTTACCTATC, 95 °C, 1 min; 57 °C, 1 min; 72 °C, 1 min; TNF-a: F: GTGGCAGTCTCAAACTGA, R: TATGGAAAGGGGCACTGA, 94 °C, 40 s; 55 °C, 40 s; 72 °C, 40 s; GAPDH: F: CAG CGACACCCACTCCTC, R: TGAGGTCCA CCACCCTGT, 94 °C, 1 min; 57 °C, 1 min; 72 °C, 1 min.

Western blotting analysis

MG-63 cells (5 × 105) were plated and treated with different doses of nsPEF. Cells were then lysed with a lysis buffer and then quantified. The equal amounts of protein were loaded, and electrophoresis was applied on a 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis mini-gel. Proteins were transferred to a PVDF membrane and blocked with casein PBS and 0.05 % Tween-20 for 1 h at room temperature. Membranes were incubated with mouse monoclonal OPG, anti-OPG (1:500), RANKL (1:200), TNF-a (1:300), GAPDH (1:1000) antibodies which were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibody was purchased from Zhongshan (Zhongshan Golden Bridge, Beijing, China.). The protein expression was visualized with enhanced chemiluminescence reagent (ECL kit, Amersham, UK).

Statistical analysis

Statistical significance was determined using Student’s t test, using SPSS 13.0. P < 0.05 was considered to indicate a statistically significant result.

Results

NsPEF parameter optimizing by CCK-8 and flow cytometry

CCK-8 assay was used to calculate the IC50 values, and flow cytometry was used to detect apoptosis. There were significant growth inhibition and apoptosis induction in a dose-dependent manner following nsPEF treatment for 24 h. MG-63 cell growth was inhibited in an electric field strength- and pulse number-dependent manner. There was significant (P > 0.001) growth inhibition when electric field strength was 40–50 kV/cm (Fig. 1a) and when pulse number was 30 (Fig. 1d) vs control. Cells were treated by nsPEF and then incubated for 24 h. Apoptotic and dead cells were analyzed by flow cytometry using dual staining with propidium iodide (PI) and Annexin V-FITC. NsPEF induced viable apoptotic cells stained with Annexin. The apoptotic cell rate is significantly increased when electric field strength was 40–50 kV/cm (Fig. 1b, c) and when pulse number was 30 (Fig. 1e, f).

Fig. 1

Fig. 1 NsPEF treatment parameter optimizing by CCK-8 and flow cytometry. After 24 h post nsPEF, CCK-8 assay was used to calculate the IC50 values under different electric field strengths (a) and different pulse numbers (d). The flow cytometry was used to detect

Apoptosis induction at different times post nsPEF treatment

To determine the effects of nsPEF on the induction of apoptosis in MG-63 cells, the Annexin V assay was performed. After 40 kV/cm and 30 pulses of nsPEF treatment, the control and treated cells were stained with Hoechst 33528 (Fig. 2a upper lane) and TUNEL (Fig. 2a lower lane). The statistical analysis of the positive apoptotic cells were counted and shown in Fig. 2b at different hours (0, 3, 12, 24, and 48 h). Apoptotic cells induced by nsPEF treatment were recognized by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), detecting DNA fragmentation by labeling the terminal end of nucleic acids. The number or percentages of apoptotic cells detected following nsPEF treatment was shown in Fig. 2b. The quantitative analysis showed the percentages of apoptotic cells detected following nsPEF treatment which were 2.6 % (0 h), 8.8 % (3 h), 21 % (12 h), 42 % (24 h), and 15 % (48 h) without nsPEF treatment. The apoptotic induction 12 and 24 h post nsPEF treatment showed significance (P = 0.01243, 0.00081, respectively, vs control). The cell cycle was analyzed by flow cytometry (Fig. 2c) and statistically analyzed in Fig. 2d, which indicates that nsPEF arrest cells in the G0/G1 phase (Fig. 2d).

Fig. 2

Fig. 2 Apoptosis induction at different times post nsPEF treatment. After 40 kV/cm and 30 pulses of nsPEF treatment, the control and treated cells were stained with Hoechst 33528 (aupper lane) and TUNEL (alower lane). The statistical analysis of the positive

The effect of nsPEF on OPG/RANKL, TNF-? gene, and protein expression

With 30 pulses, 24 h post treatment, PCR and western blot were used to determine the different electric field strengths on cell OPG/RANKL, TNF-? gene (Fig. 3a), and the corresponding protein expression (Fig. 3b). NsPEF significantly increased OPG transcription and protein expression at 20–50 kV/cm (Fig. 3a, c). RANKL was almost undetectable both in the control and nsPEF-treated MG-63 cells (Fig. 3a, c). NsPEF slightly down-regulated TNF-a (Fig. 3a, c). The OPG is important in the regulation of bone formation. PCR results showed that the nsPEF-treated cells demonstrated a significantly up-regulation of OPG transcription. Western blot analysis confirmed that nsPEF stimulated osteoprotegerin protein production in the MG-63 cells.

Fig. 3

Fig. 3 The nsPEF effect on gene and protein expression. With 30 pulses, 24 h post treatment, PCR and western blot were used to determine the different electric field strengths on cell OPG/RANKL, TNF-a gene (a), and protein expression (b). NsPEF significantly

Discussion

The primary bone malignancy osteosarcoma is still a challenge for orthopedics. For patients who are not suitable for radical resection, the minimal invasive ablation techniques can be used as an alternative to surgery. NsPEF has been proved to be a novel non-thermal ablation method which can activate a protection immune response [2123]. According to the Clinical Practice Guidelines in Oncology of the National Comprehensive Cancer Network (NCCN), local ablation can be used for curative or palliative intent, either alone or in combination with immunotherapy or chemotherapy [11]. The effect of systemic chemotherapy may be enhanced by the physiological changes produced by ablation [11]. Furthermore, ablation can sometimes be used as a complement to surgery [13].

A number of studies have demonstrated that local ablation is effective in osteosarcoma [2830]. To our best knowledge, the application of nsPEF in osteosarcoma has never been reported. The bone-related tumor study is extremely important because many solid tumors tend to have metastasis in bones. The present study applies a new ablation methodology in osteosarcoma and identifies its molecular target. Our data suggest that nsPEF had direct effects on osteosarcoma cells, including the inhibition of tumor cell proliferation and induction of apoptosis. These results are consistent with previous reports. NsPEF inhibits cell proliferation and induces apoptosis in tumor cells [11, 16].

The development of osteoclasts is controlled by cytokine synthesized by osteoblasts like receptor activator of NF-?B ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor ? (TNF-a) [31].The extension of the current study is the investigation of nsPEF’s effect on bone resorption when nsPEF is in its ablation dosage. OPG is a member of the tumor necrosis factor receptor family. It has multiple biological functions such as regulation of bone turnover. OPG can block the interaction between RANKL and the RANK receptor [31]. NsPEF increased OPG expression in MG-63 in in vitro assays. Our data indicate that nsPEF up-regulated the OPG expression. Bone remodeling can be assessed by the relative ratio of OPG to RANKL [32]. NsPEF had no effect on RANKL expression. Defined as a potent bone-resorbing factor, TNF-a is responsible for stimulating bone resorption. TNF-? exerts its osteoclastogenic effect by activating NF-?B with RANKL [33]. Our results show that in osteosarcoma MG-63, in addition to apoptosis induction, nsPEF can regulate bone metabolism through adjusting OPG/RANKL ratio.

TNF-a expression still needs further investigation due to the weak expression. But, it is the key cytokine that we assume which would change the local inflammatory microenvironment in the ablation zone.

The limit of the current study

In this in vitro study, the MG-63 osteosarcoma cell line is used as a model system. Therefore, results obtained from cultured cells only gave hints for the nsPEF treatment of osteosarcoma. The current results need to be tested in an in vivo osteosarcoma model, e.g., MG-63 cell xenografts.

Conclusion

NsPEF can be considered as a potential therapeutic intervention to suppress bone remodeling and osteoclast activity involved in osteosarcoma. Further in vivo studies are required to optimize the dosing regimen of nsPEF to fully study its antitumor potential in the bone microenvironment.

Acknowledgments

All authors acknowledge Dr.Karl H. Shoenbach, Dr. Stephen Beebe, and Mr. Frank Reidy from Old Dominion University for their kind support.

Financial support

This research is supported by National Natural Science Foundation of China (Nos. 81372425 and 81371658), National S & T Major Project (No. 2012ZX10002017), Zhejiang Natural Science Foundation (LY13H180003), and Xinjiang Cooperation Project (2014KL002).

Footnotes

Xudong Miao and Shengyong Yin contributed equally to this work.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

XM and SY carried out the molecular genetic studies and drafted the manuscript. ZS carried out the immunoassays. YZ participated in the design of the study and performed the statistical analysis. XC conceived of the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.

References

1. Kansara M, Teng MW, Smyth MJ, Thomas DM. Translational biology of osteosarcoma. Nat Rev Cancer.2014;14(11):722–35. doi: 10.1038/nrc3838. [PubMed] [Cross Ref] 2. Stokke J, Sung L, Gupta A, Lindberg A, Rosenberg AR. Systematic review and meta-analysis of objective and subjective quality of life among pediatric, adolescent, and young adult bone tumor survivors. Pediatr Blood Cancer. 2015 Mar 27. doi: 10.1002/pbc.25514. [Epub ahead of print] [PMC free article][PubMed] 3. Deng J, Schoenbach KH, Buescher ES, Hair PS, Fox PM, Beebe SJ. The effects of intense submicrosecond electrical pulses on cells. Biophys J. 2003;84(4):2709–14. doi: 10.1016/S0006-3495(03)75076-0. [PMC free article] [PubMed] [Cross Ref] 4. Chen X, Chen X, Schoenbach KH, Zheng S, Swanson RJ. Comparative study of long- and short-pulsed electric fields for treating melanoma in an in vivo mouse model. In Vivo. 2011;25(1):23–7. [PubMed] 5. Pliquett U, Nuccitelli R. Measurement and simulation of Joule heating during treatment of B-16 melanoma tumors in mice with nanosecond pulsed electric fields. Bioelectrochemistry. 2014;100:62–8. doi: 10.1016/j.bioelechem.2014.03.001. [PubMed] [Cross Ref] 6. Nuccitelli R, Tran K, Sheikh S, Athos B, Kreis M, Nuccitelli P. Optimized nanosecond pulsed electric field therapy can cause murine malignant melanomas to self-destruct with a single treatment. Int J Cancer.2010;127(7):1727–36. doi: 10.1002/ijc.25364. [PMC free article] [PubMed] [Cross Ref] 7. Yin S, Chen X, Hu C, Zhang X, Hu Z, Yu J, et al. Nanosecond pulsed electric field (nsPEF) treatment for hepatocellular carcinoma: a novel locoregional ablation decreasing lung metastasis. Cancer Lett.2014;346(2):285–91. doi: 10.1016/j.canlet.2014.01.009. [PubMed] [Cross Ref] 8. Nuccitelli R, Wood R, Kreis M, Athos B, Huynh J, Lui K, et al. First-in-human trial of nanoelectroablation therapy for basal cell carcinoma: proof of method. Exp Dermatol. 2014;23(2):135–7. doi: 10.1111/exd.12303. [PMC free article] [PubMed] [Cross Ref] 9. Garon EB, Sawcer D, Vernier PT, Tang T, Sun Y, Marcu L, et al. In vitro and in vivo evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for human malignancies. Int J Cancer. 2007;121(3):675–82. doi: 10.1002/ijc.22723. [PubMed] [Cross Ref] 10. Yin D, Yang WG, Weissberg J, Goff CB, Chen W, Kuwayama Y, et al. Cutaneous papilloma and squamous cell carcinoma therapy utilizing nanosecond pulsed electric fields (nsPEF) PLoS One.2012;7(8):e43891. doi: 10.1371/journal.pone.0043891. [PMC free article] [PubMed] [Cross Ref] 11. Chen X, Kolb JF, Swanson RJ, Schoenbach KH, Beebe SJ. Apoptosis initiation and angiogenesis inhibition: melanoma targets for nanosecond pulsed electric fields. Pigment Cell Melanoma Res.2010;23(4):554–63. doi: 10.1111/j.1755-148X.2010.00704.x. [PubMed] [Cross Ref] 12. Guo F, Yao C, Li C, Mi Y, Peng Q, Tang J. In vivo evidences of nanosecond pulsed electric fields for melanoma malignancy treatment on tumor-bearing BALB/c nude mice. Technol Cancer Res Treat.2014;13(4):337–44. [PubMed] 13. Chen X, Zhuang J, Kolb JF, Schoenbach KH, Beebe SJ. Long term survival of mice with hepatocellular carcinoma after pulse power ablation with nanosecond pulsed electric fields. Technol Cancer Res Treat.2012;11(1):83–93. [PubMed] 14. Ren Z, Chen X, Cui G, Yin S, Chen L, Jiang J, et al. Nanosecond pulsed electric field inhibits cancer growth followed by alteration in expressions of NF-?B and Wnt/?-catenin signaling molecules. PLoS One.2013;8(9):e74322. doi: 10.1371/journal.pone.0074322. [PMC free article] [PubMed] [Cross Ref] 15. Hall EH, Schoenbach KH, Beebe SJ. Nanosecond pulsed electric fields (nsPEF) induce direct electric field effects and biological effects on human colon carcinoma cells. DNA Cell Biol. 2005;24(5):283–91. doi: 10.1089/dna.2005.24.283. [PubMed] [Cross Ref] 16. Hall EH, Schoenbach KH, Beebe SJ. Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells. Apoptosis. 2007;12(9):1721–31. doi: 10.1007/s10495-007-0083-7. [PubMed] [Cross Ref] 17. Wu S, Wang Y, Guo J, Chen Q, Zhang J, Fang J. Nanosecond pulsed electric fields as a novel drug free therapy for breast cancer: an in vivo study. Cancer Lett. 2014;343(2):268–74. doi: 10.1016/j.canlet.2013.09.032. [PubMed] [Cross Ref] 18. Wu S, Guo J, Wei W, Zhang J, Fang J, Beebe SJ. Enhanced breast cancer therapy with nsPEFs and low concentrations of gemcitabine. Cancer Cell Int. 2014;14(1):98. doi: 10.1186/s12935-014-0098-4.[PMC free article] [PubMed] [Cross Ref] 19. Qi W, Guo J, Wu S, Su B, Zhang L, Pan J, et al. Synergistic effect of nanosecond pulsed electric field combined with low-dose of pingyangmycin on salivary adenoid cystic carcinoma. Oncol Rep.2014;31(5):2220–8. [PubMed] 20. Wang J, Guo J, Wu S, Feng H, Sun S, Pan J, et al. Synergistic effects of nanosecond pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro. PLoS One. 2012;7(8):e43213. doi: 10.1371/journal.pone.0043213. [PMC free article] [PubMed] [Cross Ref] 21. Chen X, Yin S, Hu C, Chen X, Jiang K, Ye S, et al. Comparative study of nanosecond electric fields in vitro and in vivo on hepatocellular carcinoma indicate macrophage infiltration contribute to tumor ablation in vivo. PLoS One. 2014;9(1):e86421. doi: 10.1371/journal.pone.0086421. [PMC free article] [PubMed][Cross Ref] 22. Nuccitelli R, Tran K, Lui K, Huynh J, Athos B, Kreis M, et al. Non-thermal nanoelectroablation of UV-induced murine melanomas stimulates an immune response. Pigment Cell Melanoma Res. 2012;25(5):618–29. doi: 10.1111/j.1755-148X.2012.01027.x. [PMC free article] [PubMed] [Cross Ref] 23. Chen R, Sain NM, Harlow KT, Chen YJ, Shires PK, Heller R, et al. A protective effect after clearance of orthotopic rat hepatocellular carcinoma by nanosecond pulsed electric fields. Eur J Cancer.2014;50(15):2705–13. doi: 10.1016/j.ejca.2014.07.006. [PubMed] [Cross Ref] 24. Greenebaum B. Induced electric field and current density patterns in bone fractures. Bioelectromagnetics.2012;33(7):585–93. doi: 10.1002/bem.21722. [PubMed] [Cross Ref] 25. Salunke AA, Chen Y, Tan JH, Chen X, Khin LW, Puhaindran ME. Does a pathological fracture affect the prognosis in patients with osteosarcoma of the extremities?: a systematic review and meta-analysis. Bone Joint J. 2014;96-B(10):1396–403. doi: 10.1302/0301-620X.96B10.34370. [PubMed] [Cross Ref] 26. Sun L, Li Y, Zhang J, Li H, Li B, Ye Z. Prognostic value of pathologic fracture in patients with high grade localized osteosarcoma: a systemic review and meta-analysis of cohort studies. J Orthop Res.2015;33(1):131–9. doi: 10.1002/jor.22734. [PubMed] [Cross Ref] 27. Rubio R, Gutierrez-Aranda I, Sáez-Castillo AI, Labarga A, Rosu-Myles M, Gonzalez-Garcia S, et al. The differentiation stage of p53-Rb-deficient bone marrow mesenchymal stem cells imposes the phenotype of in vivo sarcoma development. Oncogene. 2013;32(41):4970–80. doi: 10.1038/onc.2012.507. [PubMed][Cross Ref] 28. Lerman DM, Randall RL. Local control of metastatic sarcoma. Curr Opin Pediatr. 2015;27(1):3–8. doi: 10.1097/MOP.0000000000000170. [PubMed] [Cross Ref] 29. Yu Z, Geng J, Zhang M, Zhou Y, Fan Q, Chen J. Treatment of osteosarcoma with microwave thermal ablation to induce immunogenic cell death. Oncotarget. 2014;5(15):6526–39. [PMC free article] [PubMed] 30. Saumet L, Deschamps F, Marec-Berard P, Gaspar N, Corradini N, Petit P, et al. Radiofrequency ablation of metastases from osteosarcoma in patients under 25 years: the SCFE experience. Pediatr Hematol Oncol.2015;32(1):41–9. doi: 10.3109/08880018.2014.926469. [PubMed] [Cross Ref] 31. Aoyama E, Kubota S, Khattab HM, Nishida T, Takigawa M. CCN2 enhances RANKL-induced osteoclast differentiation via direct binding to RANK and OPG. Bone. 2015;73:242–8. doi: 10.1016/j.bone.2014.12.058. [PubMed] [Cross Ref] 32. Tudpor K, van der Eerden BC, Jongwattanapisan P, Roelofs JJ, van Leeuwen JP, Bindels RJ, et al. Thrombin receptor deficiency leads to a high bone mass phenotype by decreasing the RANKL/OPG ratio.Bone. 2015;72:14–22. doi: 10.1016/j.bone.2014.11.004. [PubMed] [Cross Ref] 33. Walsh MC, Choi Y. Biology of the RANKL-RANK-OPG system in immunity, bone, and beyond. Front Immunol. 2014;5:511. doi: 10.3389/fimmu.2014.00511. [PMC free article] [PubMed] [Cross Ref]

Low Intensity and Frequency Pulsed Electromagnetic Fields Selectively Impair Breast Cancer Cell Viability

Sara Crocetti,1,2 Christian Beyer,3 Grit Schade,4 Marcel Egli,5 Jürg Fröhlich,3 and Alfredo Franco-Obregón2,6,* Ilya Ulasov, Editor 1Department of Environmental Science, University of Siena, Siena, Italy 2Institute of Biomechanics, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland 3Electromagnetic Fields and Microwave Electronics Laboratory, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland 4Amphasys AG, Technopark Luzern, Root D4, Switzerland 5The Center of Competence in Aerospace Biomedical Science and Technology, Lucerne University of Applied Sciences and Arts, Hergiswil, Switzerland 6Department of Surgery, National University Hospital, Singapore, Singapore University of Chicago, United States of America * E-mail: hc.zhte.tseh@ocnarfCompeting Interests: One of the authors, Grit Shade, is an employee of Amphasys, the company that provided the authors with the prototype of the Impedance Flow Cytometer utilized to conduct some of the experiments in the manuscript. GS provided technical support only. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Conceived and designed the experiments: AFO JF SC. Performed the experiments: SC. Analyzed the data: AFO SC. Contributed reagents/materials/analysis tools: ME JF GS. Wrote the paper: AFO SC CB. Realized PEMFs device and provided technical support: JF CB. Provided IFC instrument, technical support and help with analysis and interpretation of the IFC results: GS. Received November 27, 2012; Accepted July 22, 2013. Copyright notice This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Introduction

There is a growing interest in the use of electromagnetic fields as an anticancer treatment [1][5]. The search for new therapeutic strategies is particularly active in the field of oncology where standard antineoplastic treatments, based on chemotherapeutic drugs and/or radiotherapy, possess potentially detrimental secondary effects and on their own often fall short of providing a complete and resilient recovery. Fueling this recent interest is the fact that extremely low-frequency and low-intensity pulsed electromagnetic fields (PEMFs) have been shown to be innocuous, possibly even beneficial [4], [6][7], to normal cell types. On the other hand, certain malignant cell classes have been shown to be particularly vulnerable to their effects [5], [8][10]. A potential value of extremely low frequency PEMFs hence lies in their use as an adjuvant treatment to more traditional chemo- and radiotherapies with the aim of reducing their dosage, mitigating any harmful secondary side effects and enhancing patient prognosis. Despite recent successes, however, the types of signals applied and cancer classes tested varied widely, producing a wide range of killing efficiencies and succeeding in forestalling concurrence in this area of research [1], [3][5]. A clear determination of the types of cancer most susceptible to PEMFs and their subsequent optimization for targeted killing will be needed before they can be used to selectively remove cancer cells from a heterogeneous population of malignant and healthy cells.

Here we show that the ability of ultra-low intensity and frequency PEMFs to selectively kill breast cancer cells depends exquisitely on field parameters. MCF-7 breast cancer cells are selectively vulnerable to PEMFs within a discrete window of PEMF signal parameters and times of exposure with resolutions of mTeslas and tens of minutes, respectively. Using five independent means of monitoring cancer cell death we obtained identical findings; selective killing of MCF7 cells was best achieved with PEMFs of 3 mT peak-to-peak magnitude, at a pulse frequency of 20 Hz and duration of exposure of only 60 minutes per day. By stark contrast, this same pulsing paradigm (cytotoxic to MCF-7s) was innocuous to normal MCF-10 breast cells. PEMF-based therapeutic strategies might thus provide a manner to control certain classes of cancer while minimally implicating healthy tissues.

Materials and Methods

Cell lines

Human adenocarcinoma MCF7 cells and human not tumorigenic MCF10 cells were provided by ATCC (Manassas, VA, USA). MCF7 cells were grown in D-MEM (Life Technologies Corporation, Gibco, Paisley, United Kingdom) supplemented with fetal calf serum (10%) (Life Technologies Corporation,Gibco, Paisley, United Kingdom), L-glutamine (1%) (Life Technologies Corporation, Gibco, Paisley, United Kingdom) and penicillin-streptomycin (1%) (Sigma-Aldrich, St. Louis, MO, USA). MCF10 cells were cultured in D-MEM/F12 (Life Technologies Corporation, Gibco, Paisley, United Kingdom) supplemented with fetal calf serum (5%) (Life Technologies Corporation, Gibco, Paisley, United Kingdom), EGF (0.02%) (Peprotech, NJ, USA), hydrocortisone (0.05%) (Sigma-Aldrich, St. Louis, MO, USA), insulin (0.1%) (Sigma-Aldrich, St. Louis, MO, USA) and penicillin-streptomycin (1%) (Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained at 37°C in a standard tissue culture incubator (Vitaris AG, Baar, Switzerland) in an atmosphere of 95% humidity and 5% CO2.

PEMFs exposure system

The PEMF exposure setup, described in Text S1 and illustrated in Figure S1 A-C, was housed inside a standard cell culture incubator (Vitaris AG, Baar, Switzerland) providing a humidified environment at 37°C, but lacking CO2 regulation. The cells were exposed to an asymmetric pulsed magnetic field while continuously monitoring the field strength and temperature. The non-exposed (control) cells were placed within the same incubator for identical periods, but shielded from the magnetic fields by a mu metal enclosure surrounding the coils. Thus, all cells were exposed to the same climate and temperature.

PEMFs treatment

MCF7 and MCF10 cells were seeded in T25 flasks (SPL Life Sciences, Korea) at concentrations of 6.5×105cells/ml and 6.7×105 cells/ml, respectively. After 24 hours of being plated the cells were washed with PBS (Life Technologies Corporation, Gibco, Paisley, United Kingdom), given fresh medium and exposed to PEMFs for the first of three daily trials; media was not changed from this point onward. An asymmetric pulsed magnetic field of 6 ms interval at a repetition rate of 20 and 50 Hz were applied at flux densities of 2.0, 3.0 and 5.0 mT (peak-to-peak) for 1 hour/day for three days. Whereas exposure to PEMFs at a repetition rate of 20 Hz caused a significant increase in cancer cells death that was dependent on PEMF amplitude, PEMFs applied at a repetition rate of 50 Hz did not produce any noticeable effects over cell viability and were not dealt with further in this manuscript (Figure S2 A-B). To test for effects of different exposure durations, cells were exposed to PEMFs of 3 mT magnitude and at a repetition rate of 20 Hz for 30, 60 or 90 minutes per days for one, two or three days. Cells were collected and analyzed on the first, second or third day for analysis, depending on the assay being conducted.

Trypan blue assay

[ratio]

After a given PEMF exposure protocol, cells were detached, spun down at 1200 rcf for 5 min, resuspended in 1 ml of PBS and incubated in trypan blue at 11 (Sigma-Aldrich, St. Louis, MO, USA). A homogeneous suspension of cells was then deposited into a Burker chamber (BRAND GMBH + CO KG, Wertheim Germany), viewed microscopically and counted. The percentage of dead cells was obtained by calculating the ratio of trypan blue positive cells in treated and untreated samples. In some cases cells were allowed to recover for up to 48 hours after their last PEMF exposure. These cells were then detached, stained with trypan blue (Sigma-Aldrich, St. Louis, MO, USA) and the number of dead cells calculated relative to control.

Apoptosis determination by DNA strand break detection

Apoptosis was measured by means of an Apo-direct kit (BD biosciences, Allschwil, Switzerland) that labels DNA strand breaks using FITC-dUTP. After each treatment 5×105cells were collected and then fixed and stained accordingly to the manufacturer’s instructions. The assay was run on a FACS Calibur (BD Biosciences, Allschwil, Switzerland) flow cytometer using the positive and negative controls provided in the kit as well as an additional positive (death) control given by exposing MCF7 or MCF10 cells to 1 mM H2O2 overnight. H2O2 applied in this manner resulted in 87% ± 2% (+/– SD, n=4) and 82% ± 3% (+/– SD, n=4) lethality in MCF7 and MCF10 cells, respectively. The FITC fluorescence (520 nm) was detected in the FL1 channel and quantifies the amount of DNA strand breaks. For each measurement, 20,000 cells were acquired and analyzed by Flow Jo software (vers. 7.6.5) (Tree Star Inc. ON, USA).

Analysis of cellular electrical properties by means of Impedance microflow cytometer

Impedance flow cytometry (IFC) was conducted on a prototype provided by Amphasys AG (Root Längenbold (LU), Switzerland). Concisely, the apparatus consists of a microfluidic chip, outfitted with a pair of microelectrodes that measure changes of electrical impedance as cells pass through dual interrogation points in response to an alternating current at four user-defined frequencies in the mid frequency (MF) and high frequency (HF) bands [11][15]. The obtained data (amplitude, phase and cell velocity) were automatically converted into a standard FCS3 format and analyzed with Flow Jo (vers. 7.6.5) (Tree Star Inc. ON, USA).

After treatment cells were collected, resuspended in PBS at a concentration of 4–5×106 cells/ml and pumped through the chip at a maximum velocity of 1 cm per second, 500–1000 cells per second. For each measurement, 20,000 cells were analyzed at a frequency of 0.5 MHz to monitor apoptosis [11][13], [15] or 9 MHz to determine metabolic status [11][14], [16][17]. Each sample was run in parallel with polystyrene beads (8 µm) (Sigma-Aldrich, St. Louis, MO, USA) to obtain a standard signal response over the entire frequency spectrum, establishing a set point.

Apoptosis determination by Annexin V staining

An Annexin V/Propidium iodide (BD biosciences, Allschwil, Switzerland) assay was used to monitor the progression of apoptosis at distinct stages. Monitoring the dual staining pattern of Annexin V (FITC- conjugated) and propidium iodide (PI) allowed for the identification of early (Annexin V + and PI -) and late apoptosis as well as cells having undergone necrosis (dead cells, Annexin V and PI +). After each treatment, 3×105cells were collected and stained as specified by the manufacturer’s instructions. Staining was assayed on a FACS Calibur (BD Biosciences, Allschwil, Switzerland), recording 20,000 cells for each measurement. Fluorescence was detected in the FL1 and FL2 channels for FITC (Annexin V) and PI, respectively. Data were acquired and analyzed by Flow Jo software (vers. 7.6.5) (Tree Star Inc. ON, USA).

Statistical analyses

All histogram data were presented as mean ± SD (standard deviation) of at least 3 independent experimental runs (range=3 to 5) consisting of between 1 to 3 replicates for each biological parameter analyzed. The exact number of measurements in each presented data point is reported in the figure legend and is indicated in brackets (n). Statistics were performed using the Wilcoxon Rank-Sum Test (two-tailed) comparing each treated sample to relative control (sham-exposed sample) for all the cell lines used. A p-value <0.05 was considered statistically significant (*) and a p-value < 0.005 was considered highly significant (**).

Results

PEMFs increase breast cancer cell death as detected by Trypan Blue inclusion

Our objective was to devise a set of treatment protocols that could potentially translate into the clinical arena to slow cancer growth, while proving harmless to healthy tissues. We focused on a breast cancer cell model given our previous success using PEMFs to slow their growth [8]. To ascertain the sensitivity of normal and cancer cells to PEMFs we exposed MCF7 breast cancer cells and their normal breast epithelial counterparts, MCF10s, to PEMFs of magnitudes between 2 mT and 5 mT and at a repetition rate of 20 Hz for 1h per day for three days. Following the last exposure (day 3) all samples were harvested and stained with trypan blue to quantify cell death and compared to otherwise identically treated control (non-exposed) cultures. A highly significant reduction in the percentage of surviving MCF7 cells was observed in response to exposure to 3 mT PEMFs. By contrast, exposure of identical MCF-7 cultures to PEMFs of either 2 mT or 5 mT amplitudes resulted in less significant levels of cell death (Fig 1A). On the other hand, exposure to 3 mT PEMFs, which proved the most cytotoxic to MCF-7 cancer cells, was innocuous to “wild type” MCF10 cells (as were 2 and 5 mT PEMFs) and moreover, appeared to have even accentuated their survival (mitigating resting levels of apoptosis) relative to unexposed cells (also see Figure S5). We next sought to determine the best exposure interval to 3 mT PEMFs to kill breast cancer cells. Figure 1B depicts cell death as a function of duration of exposure to 3 mT PEMFs (20 Hz). Cells were exposed to 3 mT PEMFs for either 30, 60 or 90 minutes per day for 3 days before assaying for cell death. MCF7 cells were most susceptible to PEMF exposures of 60 minutes duration, whereas exposure times 50% shorter (30 minutes) or 50% longer (90 minutes) than this resulted in significantly less amounts of cell killing (Fig 1B). Once again, MCF10 cell viability was not compromised by PEMF exposure of any duration. Indeed, PEMFs appeared to make MCF10 cells more resistant to undergoing apoptosis, particularly in response to the 60-minute exposure regimen that proved most cytotoxic to MCF7 cells (Figure S5). The data thus reveals a discrete set of PEMF parameters (magnitude, frequency and duration of exposure) that are most cytotoxic to breast cancer cells, whereas the identical set of PEMFs parameters were apparently harmless to non-malignant cell types (also seeFigures S3 and S4).

Figure 1

Figure 1Trypan blue detection of dead cells after exposure to PEMFs for 3 consecutive days.

To ascertain whether the PEMFs-induced cytotoxicity reported here is a cumulative response or requires a threshold level of cellular insult to become evident, we treated cells with 3 mT PEMFs for either 60 or 90 minutes per day for 1, 2, or 3 days and next quantified the total number of dead and living cells. Whereas in the unexposed cultures total cell number steadily increased throughout the three days of trial, exposure to 60 or 90 minutes of PEMFs per day either totally abrogated or slowed the increase in cell number, respectively (Fig 2). On the other hand, the absolute number of dead (trypan blue positive) cells did not scale down in proportion to the decrease in total cell number as might be expected if cell proliferation was simply being slowed, but instead, increased. Notably, on the third day, in response to 60 minutes of daily exposure to PEMFs (3 mT), the total number of cells in the culture decreased, whereas the total number of dead cells increased, by –40% (+/–6% (SD); n=12) ((total cells in control sample – total cell in treated sample)/total cells in control sample)) and +20% (+/–13% (SD); n=12) ((dead cells in control sample – dead cell in treated sample)/dead cells in control sample)), respectively, indicating heightened cytotoxicity in response to PEMFs. Figure 3 shows that the increase in cell loss with time is greatest in cultures treated for 60 minutes per day, rather than 90 minutes per day.

Figure 2

Figure 2Time course in the development of cell death in response to PEMF exposure.

Figure 3Box plots depicting the increase in cell death after 1, 2 or 3 days of consecutive PEMF treatment

Table 1

Table 1Dead cells/total cells in MCF7 cells after 3 mT PEMFs treatment for 60 min/day for 3 days.

Table 2

Table 2Dead cells/total cells in MCF7 cells after 3 mT PEMFs treatment for 90 min/day for 3 days.

Assessment of PEMF-induced apoptosis by detecting DNA strand breaks

Our Flow Cytometric (FCM) determination of apoptosis was assayed with identical PEMF parameters (days of consecutive exposure, durations of exposure, field amplitudes and frequency) as those utilized for trypan blue assessment of killing efficiency with identical results. Figure 4A shows an overlay of MCF7 cells exposed to PEMFs of three distinct intensities (2, 3 or 5 mT) for 60 minutes per day. A shift to the right (greater FL1-H values) of a cell population reflects greater DNA damage. As previously demonstrated, MCF7 cancer cells are particularly vulnerable to 3 mT PEMFs. Figure 4B shows the extent of 3 mT PEMF-induced DNA strand breaks following 30, 60 or 90 minutes exposures per day. Once again, 60 minutes of 3 mT PEMFs for three consecutive days gave the greatest DNA damage in MCF7 cancer cells. And, once again, stronger fields (5 mT) or longer exposures (90 minutes per day) were less cytotoxic to MCF7 cells (Fig 4A-D). Further paralleling our trypan blue results, MCF10 normal breast epithelial cells were not harmed by any of the PEMF paradigms tested, particularly those observed to be especially cytotoxic to MCF7 cells. Indeed, a slight protective effect (a leftward shift to lower FL1-H values) was again discerned in MCF10 cells in response to the PEMF parameters that were most cytotoxic to MCF7 breast cancer cells (Fig 4E; see also Figure S5). To investigate if the previously described increase in DNA fragmentation observed in MCF7 cells after 3 days of PEMF treatment was cumulative with time, we stained cells after 1, 2 or 3 consecutive days of exposure to either 60 or 90 minute of 3 mT PEMFs. Although PEMF-induced DNA damage increased with time, it only really obtained significance from control levels after the third day and was particularly pronounced in response to 60-minute daily exposures (figure 5 A-D). Our FCM analysis thus corroborates and strengthens our trypan blue results, indicating that treatment with 3 mT PEMFs for 60 minutes per day were most effective at killing MCF7 breast cancer cells while leaving healthy cell classes (MCF10) unharmed.

Figure 4

Figure 4FCM determination of PEMF-induced DNA damage in MCF7 (cancer) and MCF10 (non-tumorigenic).

Figure 5

Figure 5Time course of apoptosis induction by PEMFs in MCF7 cells determined by FCM.

Determination of PEMF-induced apoptosis by Impedance Flow Cytometry

Impedance Flow Cytometry (IFC) assesses real-time cell viability by monitoring cellular electrical properties in behaving cells [11][13], [15]. In the dot plot generated from monitoring the entire cell population’s electrical characteristics at a scan frequency of 0.5 MHz dead cells reside in the far lower left quadrant (low impedance phase and magnitude values). PEMFs produced a shift in MCF7 cells to the lower left quadrant, particularly in response to 3 mT PEMFs, which gave the greatest separation between living (right) and dying (left) cells (Fig 6A). Figure 6B shows the results of MCF7 cells exposed to 3 mT PEMFs for either 30, 60 or 90 minutes per day for three days. In agreement with our previous trypan blue and FCM assessment of apoptosis, cells exposed to 60 minutes of 3 mT PEMFs per day exhibited the greatest percentage of dead cells as detected by IFC (Fig 6 C-D). In stark contrast, yet in further confirmation of our previous results, MCF10 cells were slightly benefitted by these same PEMF parameters (Fig 6E, see also Figure S5).

Figure 6

Figure 6Post-PEMF apoptosis determination by impedance flow cytometry (IFC) at 0.5 MHz.

Assessment of cell metabolic status after PEMF treatment with IFC

At higher scan frequencies the IFC discerns metabolic status [11][14], [16]. At a scan frequency of 9 MHz the IFC detects two populations of cells, the right-most population (higher phase values) reflects cells experiencing the initial stages of metabolic stress [11][14], [16][17]. After three days of exposing MCF7 cells to PEMFs the magnitude of right-most population augmented, the greatest right-shift coinciding exactly with those parameters (3 mT, 20 Hz, for 60 min/day for 3 days) producing the greatest cell death in response to PEMFs (Fig 7 A-D). And, once again, MCF10 normal breast cells were apparently benefitted by PEMFs as determined by IFC analysis at 9 MHz (Fig 7 D, see also Figure S5). Due to the relatively broad scope of the phenotype (metabolic stress) the effect is the largest we have measured in response to PEMFs (see next, see also Figure S5).

Figure 7

Figure 7MCF7 and MCF10 cell metabolic status analyzed by IFC at 9 MHz.

To independently validate that IFC effectively detects apoptosis and metabolic status in our cell system we treated MCF7 cancer and MCF10 normal cells with 1 mM H2O2 to evoke cell death to an extent of 87% ± 2% (+/– SD, n=4) and 82% ± 3% (+/– SD, n=4), respectively. When analyzed by IFC at a scan frequency of 0.5 MHz cells treated with H2O2 were displaced to the far lower left quadrant (Fig 8A; cf Fig 6A-D). Also, confirming that a cell population undergoing the initial stages of metabolic stress is indeed shifted to the right (in IFC scans at 9 MHz) we obtained an analogous right-shift in MCF7 cells after overnight exposure to 1 mM H2O2 (Fig 8B; cf Fig 7A-D). Hence, IFC does appear to be a viable method to monitor cancer cell viability.

Figure 8

Figure 8Independent corroboration that IFC detects impaired cells at 0.5 MHz and 9 MHz.

Assessment of PEMF-induced apoptosis by Annexin V staining

To further corroborate our trypan blue, FCM and IFC data demonstrating the induction of apoptosis in MCF7 cancer cells in response to PEMF exposure, we performed Annexin V/PI assays, discriminating cells in early apoptosis (Annexin V+/PI-) from dead and damaged cells (propidium iodide +). MCF7 (cancer) and MCF10 (normal) cells were directly exposed to the PEMFs paradigms we previously found to be most cytotoxic to MCF7 cells, 3 mT for 60 minutes per day. Figure 9A shows that PEMF treatment resulted in a 13% increase in Annexin V+ MCF7 cells relative to control, quantitatively agreeing with our other PEMF-induced cytotoxic assessments assayed with trypan blue (treated – control: 11% dead cells), FCM (treated – control: 14% dead cells), IFC at scan frequency of 0.5 MHz (treated – control: 16% dead cells) and IFC at scan frequency of 9 MHz (treated – control: 25%). As previously demonstrated with all the other apoptosis assays we performed, MCF10 cells were not adversely affected by these same PEMF parameters (Fig 9B) (also see Figure S5).

Figure 9

Figure 9Assessment of PEMF-induced apoptosis by Annexin V assay.

Discussion

Motivated by studies demonstrating the safety of very low frequency and intensity PEMFs [4], [6] and extending from our previous work [8], demonstrating that MCF7 cancer cells are selectively vulnerable to 20 Hz pulsed electromagnetic fields, we investigated the effects of PEMFs on human breast epithelial cells of malignant (MCF7) and non-malignant (MCF10) phenotypes. Cytotoxic sensitivity to certain PEMFs parameters was entirely restricted to the malignant phenotype and exhibited a clear dependency on the duration, frequency and intensity of the PEMFs employed. Specifically, breast cancer cells of the MCF7 lineage were most vulnerable to PEMFs of 3 mT magnitude, at a repetition rate of 20 Hz and for an exposure interval of 60 minutes per day (Fig 1 A-C). These same PEMF parameters, although cytotoxic to MCF7 cells, were slightly protective to non-malignant breast epithelial cells of an identical host lineage, MCF10 (see Figure S5). For these experiments we limited our analysis to within three days of exposure to remain within the realm of a clinically feasible therapeutic strategy. Three days was also chosen as an appropriate end point to our analysis as it avoided the overgrowth of control cells. In a tissue culture paradigm such as ours, staying below cell confluence would minimize the potential contributions of cell density/contact-induced changes in biochemical status or nutrient deprivation to our measured differences. The possibility hence remains, that increasing the number of days of exposure to PEMFs may enhance the specificity and efficiency of cancer cell killing. The design of longer time course experiments will be the focus of our future studies. Nonetheless, our results, although relatively modest are sufficiently provocative (in terms of their reproducibility and selectivity) to merit future studies aimed at further evolving this approach and yet, are consistent with previous studies demonstrating that sensitivity to electromagnetic fields depends on the signal parameters used as well as the type of cells exposed to the fields [5], [7], [9], [18][19].

For this study we focused our attention on PEMF parameters that: 1) could practically translate into the clinical arena with reference to duration of exposure and 2) were innocuous to healthy cell classes collaterally exposed to PEMFs during clinical treatment. Our results are notable given that: 1) our most effective exposure time to induce cancer cell (MCF7) death was only one hour per exposure rather than 3–72 hours as previously reported [5],[20][21] and; 2) the field paradigms we designed were apparently innocuous to normal cells (MCF10). As of yet, we have not achieved complete “selective” killing with PEMFs. Although this objective might be achieved with further fine-tuning of the PEMF parameters (exposure magnitude, duration, signal shape, number of days of treatment) we cannot then exclude the possibility that other tissues type might then be implicated in the death pool. Quite notable, however, were the diametrically opposed responses of MCF7 (cancer) and MCF10 (normal) cells to PEMFs, widening the cytotoxic gap between exposed cancer and exposed normal cells. Potentially, PEMFs might prove useful as a non-invasive adjuvant treatment to be combined with other common anti-cancer therapies.

The selective killing of cancer cells with PEMFs was corroborated by four independent methodologies using five different analytical paradigms, covering the full gambit of stages leading to ultimate cell death. Firstly, our trypan blue results gave the number of cells in a late stage of cell dying known as “postapoptotic necrosis” or “secondary necrosis” (Fig 1 A-B, 2 A-D and 3 A-B) [18], [22][23]. Secondly, our FCM analysis detected DNA breaks prior to cell death [17], [24] and occurring downstream of calcium-stimulated caspase activation (Fig 4 A-E and 5 A-D) [25]. Thirdly, we investigated the progression of apoptosis using Impedance Flow Cytometry (IFC) that detects changes in the electrical properties of cells reflecting physiological status [11][17], [24], [26][27] at two frequencies: 1) 0.5 MHz, to ascertain the number of cells having undergone apoptosis (Fig 6 A-E) [11][13], [15]and 2) 9 MHz, to monitor changes that coincide with the onset of cellular stress (Fig 7 A-E) [11][14], [16][17]. Several recent publications have supported the value of IFC to gauge cell viability [11][17], [27]. Finally, we employed an Annexin V/PI assay to distinguish early apoptotic cells from damaged or already dead cells (Fig 9 A-B) [28][29]. In all five assays of cell viability identical PEMF parameters produced the greatest degree of cell damage to MCF-7 breast cancer cells, 3 mT intensity for 60 minutes a day, demonstrating a clear and discrete window of vulnerability of MCF7 cells to PEMFs of given characteristics. Stronger fields, longer exposures, or higher frequencies to these empirically determined values (3 mT, 20 Hz, 60 minutes exposures per day) were less cytotoxic to MCF7 cells, clearly demonstrating the importance of field optimization for the eventual killing of malignant cell classes with PEMFs.

A clear window of vulnerability of cancer cells to PEMFs exists; more is not necessarily better. That weaker fields, or less exposure to them, are less lethal, upon first impression, might seem somewhat intuitive. However, the fact that stronger, or longer, exposure to fields is less efficient at killing, implies some specifically of biological action, rather than a straightforward dose-dependent accumulation of generalized damage over a susceptible cell status. The validity of the described window effect is implicitly substantiated within the context of our data presented herein, the fact that five independent assays (four distinct methodologies) of measuring cell viability gave the identical result and produced similar magnitudes of cell death (also see Figure S5). The cytotoxic-dependency on exposure duration was so robust that it was also apparent when examining the time course in the development of cytotoxicity during three days of consecutive PEMF exposure. That is, 60-minute daily exposures to PEMFs gave greater ratios of cell death (figure 3) and greater amounts of DNA fragmentation (figure 5) than 90 minutes of daily exposure. Moreover, the PEMF parameters that were most cytotoxic to MCF7 breast cancer cells proved most beneficial to MCF10 normal breast cells. Similar window effects have been reported in the field of electromagnetics and have been openly discussed in the literature, yet there are no accepted models to explain their existence [19], [30][31]. Within the Protection Guidelines Report of the International Commission on Non-Ionizing Radiation [30] it is stated, “Interpretation of several observed biological effects of AM (amplitude modulated) electromagnetic fields is further complicated by the apparent existence of “windows” of response in both the power density and frequency domains. There are no accepted models that adequately explain this phenomenon, which challenges the traditional concept of a monotonic relationship between the field intensity and the severity of the resulting biological effects.”

At this juncture, however, the relative contributions of an actual slowing of cell proliferation and the induction of cell death to the overall effect of PEMFs is unclear (cf figure 2), as is the rate and extent of absorption of dead cells by the culture after their demise. Therefore, although cell cycle withdrawal possibly resulting from PEMFs may contribute to observations reported here, the most directly measurable effect is that of induced apoptosis. Nonetheless, the capacity of PEMFs to slow the proliferation of a cancer cell class also would be positive clinical outcome and of relevance in advancing PEMF-based anti-cancer therapies.

The molecular mechanisms whereby cancerous (MCF7) cells are compromised yet, healthy (MCF10) cells are not fully understood and yet, of utmost importance for the ultimate development of PEMF-based strategies to combat cancer and will be the focus of our future investigations. We speculate that the window effect observed in this study results from changes in intracellular calcium handling in response to PEMF exposure. Calcium signaling is renowned for its multimodal effects relying on intracellular calcium increments that: 1) result from both calcium influx across the cell surface membrane and release from intracellular membrane-delimited compartments; 2) are simultaneously coded in space, time and holding level; 3) exhibit negative- and positive-feedback regulatory mechanisms and; 4) are coordinated by dynamic changes in membrane organization [32][33]. As a commonly reported consequence of PEMF exposure is elevations of intracellular calcium level [34] one possibility is that PEMFs mediate their effects via influencing intracellular calcium signaling pathways. In the context of this report 3 mT PEMFs at a frequency of 20 Hz for 60 minutes per day would create the “correct” combination of calcium signals that would most effectively result in cell death. Indeed, it has been previously shown that chelating or augmenting intracellular calcium accordingly spares or compromises MCF7 survival, respectively [35][37]. The shift to the right observed at 9 MHz in IFC (Fig 4 A-D) likely reflects changes in membrane complexity and cytoplasmic reorganization (change in whole-cell capacitance) [11][14], [16][17] that coincide with the establishment of cytomorphological features that reflect the modulation of biochemical pathways that, in turn, regulate the delicate balance between cell proliferation and apoptosis including, modifications in mitochondrial metabolism downstream of changes in intracellular calcium levels [16][17], [33], [38]. Future studies of ours will focus on the effects of PEMFs over cytosolic calcium increments.

Non-malignant MCF10 cells were unaffected, or even fortified, by the PEMF paradigms producing the greatest damage in MCF7 cells, revealing another level of specificity of action and supporting the possibility that it may be ultimately feasible to selectively remove cancer cells from an organism without implicating normal tissues in the death pool using PEMF-based technologies (Figs 1 A-B, ?,4E,4E, ?,6E,6E, ?,7E,7E, ?,9B9B and ). The immunity of MCF10 cells to PEMFs might suggest that their endogenous calcium homeostatic mechanisms are capable of buffering, or even exploiting, small increments in intracellular calcium concentrations, whereas MCF7 cells are not able to withstand even modest perturbations in cytosolic calcium levels, a supposition that is supported by recently published studies[36][37]. In further support for such a calcium-dependent mechanism of preferential killing of malignant cells it has been shown that Panaxydol, a derivative of Panax ginseng that induces sustained elevations in cytosolic calcium, preferentially induces apoptosis in cancer cells (including MCF7s) but not normal cells [39]. Such a selective calcium-dependent mechanism of cancer cell killings may eventually help in the refining of PEMF-based technologies to better execute the preferential killing of breast cancer cells in clinical settings.

Supporting Information

Figure S1

PEMF exposure system.

(PNG)Click here for additional data file.(291K, png)

Figure S2

Trypan blue staining of MCF7 cancer cells exposed to pulsed electromagnetic fields (PEMFs) at a frequency of 50 Hz.

(TIF)Click here for additional data file.(403K, tif)

Figure S3

Trypan blue staining of normal (human breast MCF10 and murine muscle C2C12) and cancer (human breast MCF7) cells exposed to PEMFs.

(TIF)Click here for additional data file.(531K, tif)

Figure S4

Growth rate of MCF7 cancer cells after PEMF-treatment or in control cultures after 3 days.

(TIF)Click here for additional data file.(1.4M, tif)

Figure S5

Consistent diametrically opposed responses of non-tumorigenic MCF10 and cancer MCF7 cells to PEMF treatment observed across 5 different assays of cell viability.

(TIF)Click here for additional data file.(228K, tif)

Figure S6

Reversibility of the cytotoxic effects of PEMFs.

(TIF)Click here for additional data file.(224K, tif)

Figure S7

FCM determination of DNA strand breaks in MCF7 cancer cells after PEMF exposure.

(TIF)Click here for additional data file.(489K, tif)

Figure S8

Observed range of sample responses in MCF7 cancer cells after exposure to the PEMF parameters producing the greatest cytotoxicity (3mT, 20 Hz, 60 minutes per day for three days).

(TIF)Click here for additional data file.(1.0M, tif)

Text S1

Description of PEMF Exposure System.

(DOC)Click here for additional data file.(29K, doc)

Text S2

Supplementary figure legends.

(DOC)Click here for additional data file.(42K, doc)

Acknowledgments

We would like to acknowledge Dr Malgorzata Kisielow and Ms Anette Schütz of the Flow Cytometry Laboratory of the ETH and University of Zürich for expert technical assistance during the FCM acquisition and analysis. Finally, we would like to thank the Statistical Consulting group of the ETH for their assistance in elaborating our statistical analysis.

Funding Statement

This study was partially supported by the Swiss Federal Office of Public Health (http://www.bag.admin.ch/) under the mandate number 11.003272, “Effects of pulsed electromagnetic fields on the proliferation of different mechano-sensitive cell types”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.

References

1. Barbault A, Costa FP, Bottger B, Munden RF, Bomholt F, et al. (2009) Amplitude-modulated electromagnetic fields for the treatment of cancer: Discovery of tumor-specific frequencies and assessment of a novel therapeutic approach. J Exper Clin Cancer Res 28: 51–61 [PMC free article] [PubMed] 2. Blackman CF (2012) Treating cancer with amplitude-modulated electromagnetic fields: a potential paradigm shift, again? Br J Cancer 106: 241–2 [PMC free article] [PubMed] 3. Cameron IL, Sun LZ, Short N, Hardman WE, Williams CD (2005) Therapeutic Electromagnetic Field (TEMF) and gamma irradiation on human breast cancer xenograft growth, angiogenesis and metastasis. Cancer Cell Int 5: 23. [PMC free article] [PubMed] 4. Elson EI (2009) The little explored efficacy of magnetic fields in cancer treatment and postulation of the mechanism of action. Electromagn Biol Med 28: 275–82 [PubMed] 5. Zimmerman JW, Pennison MJ, Brezovich I, Yi N, Yang CT, et al. (2012) Cancer cell proliferation is inhibited by specific modulation frequencies. Br J Cancer 106: 307–13 [PMC free article] [PubMed] 6. Repacholi MH, Greenebaum B (1999) Interaction of static and extremely low frequency electric and magnetic fields with living systems: health effects and research needs. Bioelectromagnetics 20: 133–60 [PubMed] 7. World Health Organization: Electromagnetic fields and public health (2007) Exposure to extremely low frequency fields. Available: http://www.who.int/mediacentre/factsheets/fs322/en/index.html Accessed 2012 Nov 12. 8. Crocetti S, Piantelli F, Leonzio C (2011) Selective destabilization of tumor cells with pulsed electric and magnetic sequences: a preliminary report. Electromagn Biol Med 30: 128–35 [PubMed] 9. Ruiz-Gómez MJ, Martínez-Morillo M (2005) Enhancement of the cell-killing effect of ultraviolet-C radiation by short-term exposure to a pulsed magnetic field. Int J Radiat Biol 81: 483–90 [PubMed] 10. Yamaguchi S, Ogiue-Ikeda M, Sekino M, Ueno S (2006) Effects of pulsed magnetic stimulation on tumor development and immune functions in mice. Bioelectromagnetics 27: 64–72 [PubMed] 11. Cheung K, Gawad S, Renaud P (2005) Impedance spectroscopy flow cytometry: on-chip label-free cell differentiation. Cytometry A 65A: 124–32 [PubMed] 12. Cheung K, Di Berardino M, Schade-Kampmann G, Hebeisen M, Pierzchalski A, et al. (2010) Microfluidic impedance-based flow cytometry. Cytometry A 77A: 648–66 [PubMed] 13. David F, Hebeisen M, Schade G, Franco-Lara E, Di Berardino M (2012) Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry. Biotechnol Bioeng 109: 483–92 [PubMed] 14. Pierzchalski A, Hebeisen M, Mittag A, Bocsi J, Di Berardino M, et al. (2012) Label-free hybridoma cell culture quality control by a chip-based impedance flow cytometer. Lab Chip 12: 4533–4543 [PubMed] 15. Schade-Kampmann G, Huwiler A, Hebeisen M, Hessler T, Di Berardino M (2008) On-chip non-invasive and label-free cell discrimination by impedance spectroscopy. Cell Prolif 41: 830–40 [PubMed] 16. Chin S, Hughes MP, Coley HM, Labeed FH (2006) Rapid assessment of early biophysical changes in K562 cells during apoptosis determined using dielectrophoresis. Int J Nanomedicine 1: 333–7 [PMC free article][PubMed] 17. Labeed FH, Coley HM, Hughes MP (2006) Differences in the biophysical properties of membrane and cytoplasm of apoptotic cells revealed using dielectrophoresis. Biochim Biophys Acta 1760: 922–9 [PubMed] 18. Sul AR, Park S, Suh H (2006) Effects of sinusoidal electromagnetic field on structure and function of different kind of cell lines. Yonsei Med J 46: 852–861 [PMC free article] [PubMed] 19. Focke F, Schuermann D, Kuster N, Schär P (2010) DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure. Mutat Res 683: 74–83 [PubMed] 20. Koh EK, Ryu BK, Jeong DY, Bang IS, Nam MH, et al. (2008) A 60-Hz sinusoidal magnetic field induces apoptosis of prostate cancer cells through reactive oxygen species. Int J Radiat Biol 84: 945–55 [PubMed] 21. Radeva M, Berg H (2004) Differences in lethality between cancer cells and human lymphocytes caused by LF-electromagnetic fields. Bioelectromagnetics 25: 503–7 [PubMed] 22. Zhivotosky B, Orrenius S (2001) Assessment of apoptosis and necrosis by DNA fragmentation and morphological criteria. Curr Protoc Cell Biol 18: 18.3–18.3.23 [PubMed] 23. Du Plessis-Stoman D, du Preez J, van de Venter M (2011) Combination treatment with oxaliplatin and mangiferin causes increased apoptosis and downregulation of NF?B in cancer cell lines. Afr J Tradit Complement Altern Med 8: 177–84 [PMC free article] [PubMed] 24. Wang X, Becker FF, Gascoyne PR (2002) Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation. Biochim Biophys Acta1564: 412–20 [PMC free article] [PubMed] 25. Mattson MP, Chan SL (2003) Calcium orchestrates apoptosis. Nat Cell Biol 5(12): 1041–3 [PubMed] 26. Cho Y, Kim HS, Frazier AB, Chen ZG, Shin DM, Han A (2009) Whole Cell Impedance Analysis for Highly and Poorly Metastatic Cancer Cells. J microelectromech 18: 808–817 27. Opp D, Wafula B, Lim J, Huang E, Lo JC, et al. (2009) Use of electric cell-substrate impedance sensing to assess in vitro cytotoxicity. Biosens Bioelectron 24: 2625–9 [PMC free article] [PubMed] 28. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.. J Immunol Methods 184(1): 39–51 [PubMed] 29. Ebrahimi Nigjeh S, Yusoff FM, Mohamed Alitheen NB, Rasoli M, Keong YS, et al. (2013) Cytotoxic effect of ethanol extract of microalga, Chaetoceros calcitrans, and its mechanisms in inducing apoptosis in human breast cancer cell line. Biomed Res Int 2013: 783690. [PMC free article] [PubMed] 30. International Commission for Non-Ionizing Radiation Protection (1998) ICNIRP Guidelines for limiting exposure to time-varying electric, magnetic and electromagnetic fields (up to 300 GHZ). Health Physics 74: 494–522 [PubMed] 31. Ivancsits S, Diem E, Jahn O, Rüdiger HW (2003) Intermittent extremely low frequency electromagnetic fields cause DNA damage in a dose-dependent way. Int Arch Occup Environ Health 76: 431–6 [PubMed] 32. Putney JW, Bird GS (2008) Cytoplasmic calcium oscillations and store-operated calcium influx. J Physiol 586: 3055–9 [PMC free article] [PubMed] 33. Shapovalov G, Lehen’kyi V, Skryma R, Prevarskaya N (2011) TRP channels in cell survival and cell death in normal and transformed cells. Cell Calcium 50: 295–302 [PubMed] 34. Haddad JB, Obolensky AG, Shinnick P (2007) The biologic effects and the therapeutic mechanism of action of electric and electromagnetic field stimulation on bone and cartilage: new findings and a review of earlier work. J Altern Complement Med 13: 485–90 [PubMed] 35. Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ (2007) Calcium and cancer: targeting Ca2+transport. Nat Rev Cancer 7: 519–30 [PubMed] 36. Sergeev IN (2004) Calcium as a mediator of 1,25-dihydroxyvitamin D3-induced apoptosis. J Steroid Biochem Mol Biol 89-90: 419–25 [PubMed] 37. Sergeev IN (2005) Calcium signaling in cancer and vitamin D. . J Steroid Biochem Mol Biol 97: 145–51[PubMed] 38. Khaled AR, Reynolds DA, Young HA, Thompson CB, Muegge K, et al. (2001) Interleukin-3 withdrawal induces an early increase in mitochondrial membrane potential unrelated to the Bcl-2 family. Roles of intracellular pH, ADP transport, and F(0)F(1)-ATPase. J Biol Chem 276: 6453–62 [PubMed] 39. Kim JY, Yu SJ, Oh HJ, Lee JY, Kim Y, et al. (2011) Panaxydol induces apoptosis through an increased intracellular calcium level, activation of JNK and p38 MAPK and NADPH oxidase-dependent generation of reactive oxygen species. Apoptosis 16(4): 347–58 [PubMed] Technol Cancer Res Treat.  2012 Feb;11(1):83-93.

Long term survival of mice with hepatocellular carcinoma after pulse power ablation with nanosecond pulsed electric fields.

Chen X, Zhuang J, Kolb JF, Schoenbach KH, Beebe SJ.

Source

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk Virginia, 4211 Monarch Way, Norfolk, Virginia 23508, USA.

Abstract

Novel therapies are needed for treating hepatocellular carcinoma (HCC) without recurrence in a single procedure. In this work we evaluated anti-neoplastic effects of a pulse power ablation (PPA) with nanosecond pulsed electric fields (nsPEFs), a non-thermal, non-drug, local, regional method and investigated its molecular mechanisms for hepatocellular carcinoma tumor ablation in vivo. An ectopic tumor model was established using C57BL/6 mice with Hepa1-6 hepatocellular carcinoma cells. Pulses with durations of 30 or 100 ns and fast rise times were delivered by a needle or ring electrode with different electric field strengths (33, 50 and 68 kV/cm), and 900 pulses in three treatment sessions (300 pulses each session) or a single 900 pulse treatment. Treated and control tumor volumes were monitored by ultrasound and apoptosis and angiogenesis markers were evaluated by immunohistochemistry. Seventy five percent of primary hepatocellular carcinoma tumors were eradicated with 900 hundred pulses at 100 ns pulses at 68 kV/cm in a single treatment or in three treatment sessions without recurrence within 9 months. Using quantitative analysis, tumors in treated animals showed nsPEF-mediated nuclear condensation (3 h post-pulse), cell shrinkage (1 h), increases in active executioner caspases (caspase-3 > -7 > -6) and terminal deoxynucleotidyl transferase dUTP nick-end-labeling (1 h) with decreases in vascular endothelial growth factor expression (7d) and micro-vessel density (14d). NsPEF ablation eliminated hepatocellular carcinoma tumors by targeting two therapeutic sites, apoptosis induction and inhibition of angiogenesis, both important cancer hallmarks. These data indicate that PPA with nsPEFs is not limited to treating skin cancers and provide a rationale for continuing to investigate pulse power ablation for hepatocellular carcinoma using other models in pre-clinical applications and ultimately in clinical trials. Based on present treatments for specific HCC stages, it is anticipated that nsPEFs could be substituted for or used in combination with ablation therapies using heat, cold or chemicals.

Acc Chem Res.   2012 Apr 30. [Epub ahead of print]

Detecting and Destroying Cancer Cells in More than One Way with Noble Metals and Different Confinement Properties on the Nanoscale.

Dreaden EC, El-Sayed MA.

Source

Laser Dynamics Laboratory, Department of Chemistry and Biochemistry, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.

Abstract

Today, 1 in 2 males and 1 in 3 females in the United States will develop cancer at some point during their lifetimes, and 1 in 4 males and 1 in 5 females in the United States will die from the disease. New methods for detection and treatment have dramatically improved cancer care in the United States. However, as improved detection and increasing exposure to carcinogens has led to higher rates of cancer incidence, clinicians and researchers have not balanced that increase with a similar decrease in cancer mortality rates. This mismatch highlights a clear and urgent need for increasingly potent and selective methods with which to detect and treat cancers at their earliest stages. Nanotechnology, the use of materials with structural features ranging from 1 to 100 nm in size, has dramatically altered the design, use, and delivery of cancer diagnostic and therapeutic agents. The unique and newly discovered properties of these structures can enhance the specificities with which biomedical agents are delivered, complementing their efficacy or diminishing unintended side effects. Gold (and silver) nanotechnologies afford a particularly unique set of physiological and optical properties which can be leveraged in applications ranging from in vitro/vivo therapeutics and drug delivery to imaging and diagnostics, surgical guidance, and treatment monitoring. Nanoscale diagnostic and therapeutic agents have been in use since the development of micellar nanocarriers and polymer-drug nanoconjugates in the mid-1950s, liposomes by Bangham and Watkins in the mid-1960s, and the introduction of polymeric nanoparticles by Langer and Folkman in 1976. Since then, nanoscale constructs such as dendrimers, protein nanoconjugates, and inorganic nanoparticles have been developed for the systemic delivery of agents to specific disease sites. Today, more than 20 FDA-approved diagnostic or therapeutic nanotechnologies are in clinical use with roughly 250 others in clinical development. The global market for nano-enabled medical technologies is expected to grow to $70-160 billion by 2015, rivaling the current market share of biologics worldwide. In this Account, we explore the emerging applications of noble metal nanotechnologies in cancer diagnostics and therapeutics carried out by our group and by others. Many of the novel biomedical properties associated with gold and silver nanoparticles arise from confinement effects: (i) the confinement of photons within the particle which can lead to dramatic electromagnetic scattering and absorption (useful in sensing and heating applications, respectively); (ii) the confinement of molecules around the nanoparticle (useful in drug delivery); and (iii) the cellular/subcellular confinement of particles within malignant cells (such as selective, nuclear-targeted cytotoxic DNA damage by gold nanoparticles). We then describe how these confinement effects relate to specific aspects of diagnosis and treatment such as (i) laser photothermal therapy, optical scattering microscopy, and spectroscopic detection, (ii) drug targeting and delivery, and (iii) the ability of these structures to act as intrinsic therapeutic agents which can selectively perturb/inhibit cellular functions such as division. We intend to provide the reader with a unique physical and chemical perspective on both the design and application of these technologies in cancer diagnostics and therapeutics. We also suggest a framework for approaching future research in the field.

Logo of bmengon

Biomed Eng Online. 2010; 9: 13. Published online 2010 Feb 26. doi:  [10.1186/1475-925X-9-13] PMCID: PMC2839970 PMID: 20187951

A statistical model for multidimensional irreversible electroporation cell death in tissue

Alex Golberg1 and Boris Rubinsky

corresponding author

2 Author information Article notes Copyright and License information Disclaimer 1Center for Bioengineering in the Service of Humanity and Society, School of Computer Science and Engineering, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel 2Department of Mechanical Engineering, Graduate Program in Biophysics, University of California at Berkeley, Berkeley CA 84720, USA

corresponding author

Corresponding author. Alex Golberg: moc.liamg@grebloga; Boris Rubinsky: ude.yelekreb.em@yksnibur Received 2009 Sep 23; Accepted 2010 Feb 26. Copyright ©2010 Golberg and Rubinsky; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article has been cited by other articles in PMC.

Abstract

Background

Irreversible electroporation (IRE) is a minimally invasive tissue ablation technique which utilizes electric pulses delivered by electrodes to a targeted area of tissue to produce high amplitude electric fields, thus inducing irreversible damage to the cell membrane lipid bilayer. An important application of this technique is for cancer tissue ablation. Mathematical modelling is considered important in IRE treatment planning. In the past, IRE mathematical modelling used a deterministic single value for the amplitude of the electric field required for causing cell death. However, tissue, particularly cancerous tissue, is comprised of a population of different cells of different sizes and orientations, which in conventional IRE are exposed to complex electric fields; therefore, using a deterministic single value is overly simplistic.

Methods

We introduce and describe a new methodology for evaluating IRE induced cell death in tissue. Our approach employs a statistical Peleg-Fermi model to correlate probability of cell death in heterogeneous tissue to the parameters of electroporation pulses such as the number of pulses, electric field amplitude and pulse length. For treatment planning, the Peleg-Fermi model is combined with a numerical solution of the multidimensional electric field equation cast in a dimensionless form. This is the first time in which this concept is used for evaluating IRE cell death in multidimensional situations.

Results

We illustrate the methodology using data reported in literature for prostate cancer cell death by IRE. We show how to fit this data to a Fermi function in order to calculate the critical statistic parameters. To illustrate the use of the methodology, we simulated 2-D irreversible electroporation protocols and produced 2-D maps of the statistical distribution of cell death in the treated region. These plots were compared to plots produced using a deterministic model of cell death by IRE and the differences were noted.

Conclusions

In this work we introduce a new methodology for evaluation of tissue ablation by IRE using statistical models of cell death. We believe that the use of a statistical model rather than a deterministic model for IRE cell death will improve the accuracy of treatment planning for cancer treatment with IRE.

Background

Electroporation is the physical phenomenon in which the cell membrane becomes permeabilized when certain electric fields are applied across the cell [1]. When cell membrane permeability increase is only temporary and the resealing happens in the next step, reversible electroporation has occurred [28]. Reversible electroporation has important applications in chemical treatment of tissues for drug delivery and gene therapy [911] If permeability increase is sufficiently long to disrupt intracellular homeostasis, irreversible electroporation has occurred and as a consequence the cell dies [12]. Until recently, the main practical application of irreversible electroporation was microbial inactivation in the food industry [1315]. A summary of much of the current information on the use of IRE in the food industry can be found in a recent book on this topic [15]. The use of irreversible electroporation in a non thermal mode for tissue ablation in the body in vivo is a new minimally invasive molecular selective surgical technique [1621]. Tissue electroporation utilizes electrodes brought into contact with tissues in the body to deliver electric pulses, which in turn induce electroporation in a desired volume of tissue [22,23]. Non-thermal irreversible electroporation (NTIRE) is electroporation delivered in such a way that the Joule heating induced temperature elevation in tissue only reaches levels that are not harmful[24]. Therefore, only the cell membrane in the treated area is affected while other molecular structures in the tissue are spared, effectively making NTIRE molecular surgery[23,25]. One application of NTIRE is the treatment of cancerous tumors [16,17,20,23]. In a typical procedure, electrodes are inserted around the tumor and pulses of specific amplitude and frequency are applied in the hope that they will affect the entire area of the tumor and cause complete cell death [16,17,20,23]. Treatment planning is important for NTIRE treatment success. In the past, mathematical studies on electroporation in tissue used a deterministic model to evaluate the electroporation events, i.e. it was assumed that the event of electroporation is associated with a single value of local electric field current and heat distribution during pulse application[17,21,2433]. Particular attention was paid to the electrode confirmation optimization [34,35] and the impact of tissue histology [36]. Nevertheless, assuming a deterministic effect of electroporation parameters is correct only when the cell population is homogeneous and uniform. In malignant tissues the cell population is at different stages of development and is therefore not homogeneous. It has been known in the field of irreversible electroporation since the 1960’s that in a population of aging cells there is a statistical distribution which correlates cell survival to electroporation parameters [37,38]. The outcome of the application of electric pulses across cells depends on many parameters. These include field amplitude, polarity, number of electric pulses, shape of pulses, length of pulse, interval between pulses, and environmental temperature. Particularly relevant to tissue are the additional parameters of cell type, morphology, age and size [28,26,37,38]. All these parameters determine if the cell membrane will undergo reversible electroporation, irreversible electroporation or no electroporation at all. When treating cancer cells with NITRE, it is obviously important to deliver the electric pulses such that the electric conditions that destroy cells are achieved throughout the entire volume of targeted undesirable tissue. The use of NTIRE for tissue ablation is complicated by the fact that the electric fields which occur in the treated tissue are complex and vary in space as a function of distance from the electrodes, tumor and electrode geometry e.g [17,25]. Therefore, there is evident need for a mathematical methodology of treatment planning which will ensure that the entire volume of undesirable tissue undergoes electric conditions that destroy all the cells.

The food industry, from which some of the first fundamental studies on IRE emerged [37,38] has long recognized that electroporation is a statistical event in a heterogeneous population of cells. In food processing, it is important to completely destroy undesirable cells; as is in treatment of cancer. Therefore, statistical models of cell destruction by irreversible electroporation have been developed in the food industry for processing planning. Our goal in this study is to show how these models can be used in treatment planning for ablation of cancer cells in tissue.

The first mathematical models to describe pulsed electric field induced cell death employed a first order inactivation kinetics model and are given in equation (1), [39]

equation image

(1)

Where S is the survival ratio, k is the kinetic constant which depends of pulse strength and t is the total treatment time.

However, experimental studies show that cell death by pulsed electric fields depends on more parameters than those included in a first order kinetic model. Hülsheger and Niemann proposed a model which is different from first order inactivated models and incorporates more of the relevant pulsed electric field parameters, Equation (2), [40]:

equation image

(2)

Where bis a regression constant, which is bacteria and medium type dependent. E is the applied field and Eis a cell size and pulse length dependent parameter, obtained by extrapolation to 100% survivals. Further model development [14,41,42] have lead to the model in Equation 3, which also includes brings the pulse length as a critical parameter in electric pulse field induced cell death:

equation image

(3)

Where tand Eare microorganism and medium type dependent, E is the applied field and t is the treatment time.

Additional models were developed which take into account the fact that the treated microorganisms population is not homogeneous, hence each individual cell has its own resistance to the applied treatment. Assuming a natural distribution among cells, the survival curve can be described by a distribution function[4345].

Peleg [46]proposed an inactivation model, Equation 4, based on Fermi function:

equation image

(4)

Where, Ec(n) is the field at which 50% of a population of cells are dead and A(n) are function of the number of pulses, n.

Recently, a Weibull distribution, function has been shown to describe effectively several microbial inactivation curves, Equation 5, [44,45]:

equation image

(5)

Where n(E) and b(E) are constants and depend on microbial and media type and treatment parameters (electric field and treatment time).

Several additional models have been reported in the literature [4749]. San Martin et al [50] and Alvarez et al [51] made a comparison study of several proposed statistical models.

The statistical mathematical models used in the food industry deal with one dimensional electric field. These models have practical value in the food industry because the majority of the geometrical configurations in which IRE is used in that industry are one-dimensional. However, when irreversible electroporation is used for medical treatment the electric fields that develop in the treated tissue they are seldom one dimensional[17]. In developing NTIRE mathematical models for medicine it would be beneficial to have a methodology that could predict the outcome of a particular electroporation treatment in tissues made of a variety of cells that experience multidimensional and complex electric fields at complex electroporation protocols.

The goal of this study is to introduce such a methodology, which will lead to the treatment planning according to parameters we previously discussed. Specifically, we suggest combining a mathematical model that calculates the multidimensional electric field in tissue with a statistical and empirical model that predicts cellular damage as a function of the local and temporal values of electric fields and the electroporation protocols. Mathematical models that calculate the multi-dimensional electric fields which occur during tissue electroporation through the solution of the electric field equation have been used successfully in the past for electroporation analysis and research [22,52] as well as for treatment planning in NTIRE [17,20,53]. In the past these mathematical models of electric fields were combined with a deterministic single valued evaluation of the electric field that affects cell viability and the results were expressed as a demarcation line which separates between cells that were electroporated and those that are not. There has been no methodology introduced, up to our knowledge, which evaluates the statistical distribution of electroporated cells. Here we propose a second step after the electric field calculations which consists of inserting the calculated local value of the electric fields into a statistical empirical model of the type derived in the food industry for estimate of local cell damage. This analysis should produce a map of tissue damage in the treated region for a certain electroporation protocol which is the goal of treatment planning. We anticipated that the major difference in the outcome of the analysis between the methodologies proposed in this study and the mathematical methodology used in the past is the occurrence of a domain in which there will be a transition between electroporated and non-electroporated tissue, rather than a discrete demarcation line. Knowing this transition zone is obviously important in treatment of cancer.

This study describes this mathematical model of electroporation in tissue. Since we want to introduce a general methodology, we will employ dimensionless analysis – which is basic in fundamental engineering analysis. To illustrate the method we will use a Peleg-Fermi type statistical model [46]. Because there is no good experimental data in the literature for IRE in tissue and to nevertheless focus ideas we use and extrapolate limited experimental data obtained for DU 145 prostate cancer cells in a previously published work, based on in vivo experiments, by Canatella et al[54]. The experimental parameters in this specific study. which included field strength from 0.1 to 3.3 kV/cm, pulse length 50 ?sec -20 ms, number of pulses 1-10 [41], fall to the range of parameters used in vivo studies for the successful irreversible electroporation [16,20,22,53]; therefore, we applied these results for demonstration in the current 2D treatment planning model application. In the investigated electroporation study the pulse lengths were significantly longer than the cell membrane charging time which is about 1 ?sec [55] and thus a steady state DC analyses can be implemented. Obviously, for this method to become practical much experimental research is needed to obtain statistical data for cells in tissue.

Methods

To develop the methodology we will employ a statistical empirical model of cell damage by electroporation based on the Peleg-Fermi formulation[46]. The reason for choosing this model over others is related to recent findings in the field of tissue NTIRE. These findings show that the number of pulses is an important treatment parameter[16,26,56]. We chose to use the Peleg-Fermi model since it directly incorporates the dependence of cell death on pulse number and field strength for the given pulse length. Other models, for instance, Weibull function parameters do not incorporate directly the pulse number and pulse length as basic parameters and include only the effect of field amplitude and total treatment time. Obviously the other models can be also used and it is quite likely that new statistical models will be developed in the future for treatment of tissue; however, this study should be viewed primarily as a first attempt at introducing statistical modeling in the analysis of tissue electroporation.

Peleg [46] depicts the dependence of the survival ratio S (S = N/Nor the ratio of living cell count after IRE treatment (N) and before IRE treatment (No)) on the electric field that cells experience, E [V/m] and number of pulses, n, for various electroporation protocols.

The model is based on the Fermi equation of the form described in Equation 4.

The equation incorporates Ec(n) whose typical behavior is

equation image

(6)

Where Eco is the intersect of the curve with the y-axis and is cell type and pulse type specific, n, is the number of pulses and k1 is cell type and pulse type specific. The pulse type specificity relates to all the other parameters of electroporation that are not included explicitly in the equation (i.e. shape of pulse, length of pulse, interval between pulses).

The equation for A(n), whose typical behavior is,

equation image

(7)

The electric field during the electroporative pulses application is obtained from the solution of the Equation 8,

equation image

(8)

where, [S] is the local conductivity and ?[V] is the local potential

To determine the electric potential in the analyzed region Equation (8) is solved subject to the electroporation boundary condition which are:

equation image

(9)

where ?1, ?are the geometrical locations of the electroporation electrode boundaries.

Boundary conditions that do not relate to the electrodes are handled in a standard way, as insulating boundaries. A typical example will be shown later in the results section.

Since we introduce here a general methodology we will employ dimensionless analysis, as commonly done in engineering analysis. We assume that the typical dimension of this problem is the distance L

[m]

, between the centers of gravity of the two electroporation electrodes. We will non-dimensionalize space variables with respect to the dimension, L, and electric field quantities with respect to Eco which is a typical quantity with units of electric field and dependent on the tissue type and electroporation protocol. Specifically:

equation image

(11)

The dimensionless form of Equations (4) and (6-11) becomes,

equation image

(12)

We anticipate that mathematical modeling of IRE will be performed the following way. The experimental data, gathered in preliminary experiments with tissues, will be cast in a statistical model of cell death as a function of various electroporation parameters rather than a deterministic model. It is quite possible that the experimental studies will reveal other parameters of importance for the statistical model; for instance, the effect of the variable polarity, anisotropic properties in relation to the electric fields, heterogeneity to mention a few. From the data gathered in the food industry we have little doubt that in tissue the cell electroporation as a function of electroporation parameters will have a statistical distribution rather than be deterministic. Then the Laplace equation is solved for the particular geometry and electroporation protocol and the statistical model can be used as a survival look-up table with the calculated local electric field to determine the transition region to complete cell death. It should be emphasized that in other tissue ablation techniques such as cryosurgery and thermal ablation this statistically affected transition region has become an important consideration in treatment planning.

Results and Discussion

The goal of this part of the study is to illustrate the methodology with an example. Since there is no experimental statistical data available for tissues we decided to illustrate the concept using some limited data available from experiments with prostate DU 145 cancer cells in the work by Canatella et al[54], which we extrapolate. The goal of this study was to introduce the idea that electroporation effects on tissue should be analyzed as a statistical, probabilistic event rather than as a deterministic event. Tissues are obviously heterogeneous at the microscopic and macroscopic scale and often anisotropic. Others and we have published, studies on the effects of tissue heterogeneity on tissue electroporation and it is substantial [27,30,36,5760]. However, in order to single out the effect of a statistical distribution of electroporation events on the outcome of electroporation, we chose to model the tissue as homogeneous. This approach to the analysis of a newly examined phenomenon is obviously quite standard [22,33].

We could have used data from experiments with micro-organisms from the food industry or just simple parametric studies; however, we thought that although limited, the prostate cancer cell data is somewhat more relevant. Obviously future experimental studies on tissues are needed in this field.

The data of Canatella et al [54]gives the percentage cell survival as a function of applied field intensity for 1, 2, 4 and 10 pulses with pulse lengths of 50 ?sec, 100 ?sec, 1 msec and 10 msec.

We have curve fitted the data of Canatella et al. [54] to the Fermi type model of Peleg, Equation 1 [46], The curve fitted parameters Ec and A as a function of n were calculated from the experimental data and are shown in Figures ?Figures1A1A to ?to1D1D.

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Object name is 1475-925X-9-13-1.jpg

Figure 1

Dependence of Ec and A on the number of pulses as developed from the work of Canatella et al [54]. A. 50 ?sec pulse lenth. B.100 ?sec pulse length, C. 1 msec pulse length and D. 10 msec pulse length.

From the plots in Figures ?Figures1A1A to ?to1D1D we extrapolated to n = 0 to obtain the values of Eco and Ao for each electroporation protocol. The plots in Figures ?Figures1A1A to ?to1D1D were non-dimensionalized as in Equations 16 and 17 and further extrapolated to larger number of pulses than in the experiments of Canatlela et al[54]. These dimensionless representations are shown in Figures (2A, B, C and ?and2D).2D). It should be obvious that what we show is a general methodology and the particular use of the Canatella et al[54] data is to have some basis grounded on experimentation for the description of the methodology.

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Object name is 1475-925X-9-13-2.jpg

Figure 2

Dependance of Ec and A on the number of applied pulses, normalized to Eco. A. 50 ?sec pulse lenth. B.100 ?sec pulse length, C. 1 msec pulse length and D. 10 msec pulse length.

We will further illustrate the methodology by analyzing a configuration that is typical to the NTIRE experiments described previously[61]. Specifically, in those experiments two long 1 mm diameter cylindrical electrodes are placed at a separation of 1 cm between them in a parallel configuration. This situation is primarily two dimensional. For simplicity we will assume that the tissue is isotropic (although the method is obviously not restricted to these conditions) with = 0.42 S/m[62].

The electric field equation is solved using the finite element method with Comsol Multiphysics (version 3.4). The paradigm of the analysis is as follows. The field equation is solved for prescribed voltage boundary conditions on the electrodes and insulating boundary conditions on the outer edges of the domain, and then the curves in Figure ?Figure22 are used to evaluate the cell survival for each value of the local field and the appropriate number of pulses and electroporation protocols. In a typical parametric treatment study we have varied the C values (dimensionless voltage on the electrodes) and treatment parameters (number of pulses and length of pulses) and plotted from the electric field data a spatial depiction of the cell survival. The calculated dimensionless field distribution in the tissue is given in Figures 3(A-C) The cell survival 2D plots are shown in Figures 4(A-H).

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Figure 3

Dimensionless electric field distribution solution in the treated tissue for A. C = 0.5, B. C = 1.5 C. C = 2.5.

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Figure 4

Viability plots for IRE in prostate tissue in 2D for different electroporation protocols that have various number of pulses (n), voltages on the electrodes C, and pulse length, (t). A. n = 10 C = 1.5 t = 100 ?sec B. n = 50 C = 1.5 t = 100 ?sec C. n = 100 C = 1.5 t = 100 ?sec. D. n = 50 pulses, C = 0.5, t = 100 ?sec E. n = 50 pulses c = 1.5 t = 100 ?sec F. n = 50 pulses c = 2.5 t = 100 ?sec. G. n = 50 C = 1.5 t = 100 ?sec H. n = 50 C = 1.5 t = 1 msec.

Figures 4(A-H) show the distribution of cells which survive IRE in relation to the location of the electroporation electrodes for various electroporation protocols. The depiction of the cell damage is obtained from the calculation of the electric fields and the use of the Peleg-Fermi type empirical data. The most important aspect of our findings is that around the treated tissue there is a rim of tissue in which the NTIRE caused damage is partial. The existence and the extent of regions in which only part of the cells are ablated cannot be determined from the deterministic cell death models which have been used before The shape of the treated region is obviously a function of the electrical parameters and the geometry of the probes. From the results it is evident that the damaged region increases as a function of applied voltage, pulse number and pulse length. Both regions of the sub-lethal injured and totally inactivated cells are changing as a function of the applied protocol. The general pattern is interesting: larger numbers of pulses increase the region in which there is complete cell death (blue color) while large field amplitude and longer pulse length increase both the region in which there is complete cell death as well as the transition region of partial cell injury (Figures 4(A-C)). These findings further illustrate the importance of using a statistical distribution model for a precise analysis of the effects of NTIRE. The geometrical form of the treated area changes its shape with the treatment parameters in a form similar to that observed in other studies [33].

In this study we introduce a methodology for evaluating cell death in a volume of tissue treated by IRE using a statistical cell death model rather than the deterministic model for cell death used in the past.

The examples shown in this study illustrate the methodology for mathematical analysis of IRE for multidimensional electroporation protocols from fundamental information on the empirical, statistical relation between cell survival and electroporation protocols in experiments and mathematical solution of the field equation. For a desired region of tissue ablation it is possible to employ this methodology for choosing the desirable electric pulse protocol in terms of pulse amplitude, length, number of pulses and intervals between the pulses. Because non-thermal irreversible electroporation also requires pulses that do not produce thermal damage future studies may also require solving this model of electric fields together with thermal models dealing with temperature distributions as well as thermal damage. While shown for irreversible electroporation this mode of analysis could be employed in a similar form with experimental curves for reversible electroporation. Obviously this is a theoretical study whose goal it is to propose a statistical model for IRE mathematical modeling. It should be empathized that the data used in this work is for illustration purposes only and real curves and parameters should be developed for each specific case. We performed the simulations based on two assumptions. First, we extrapolated data from in vitro experiment performed by Canatella et al. [54] to an in vivo situation in tissue, second we used the Peleg-Fermi model to extrapolate the effect of electric field delivered at a much larger number of pulses than was reported by Canatella et al. [54]. Eventually, in order to use the theoretical methodology introduced in this work in clinical applications experimental studies need to be performed to develop real values for statistical analysis.

The results that were obtained show that when a statistical model is used to predict cell destruction by IRE there is a transition zone between complete cell destruction and complete cell survival. In contrast, previous mathematical models of IRE which employed deterministic models show a sharp transition line. Obviously, knowing precisely the extent of complete tissue ablation is important in treatment of cancer. The mode of analysis and treatment planning design presented in this study may become important in attempts to optimize the use of NTIRE in treatment of cancer.

Conclusion

This study has introduced a new mathematical methodology for analysis of tissue ablation by irreversible electroporation using statistical models of cell death. The methodology was illustrated using data derived from single cell studies. Much experimental work remains to obtain similar data for cells in tissue. However, once the experimental data becomes available, the use of a statistical model rather than a deterministic model for IRE cell death will improve the accuracy of treatment planning for cancer treatment with IRE.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AG performed data collection modeling and drafted the manuscript. BR conceived of the study and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the Israel Science Foundation grant # 403/06.

References

  • Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electrical fields. The EMBO Journal. 1982;1:841–845. [PMC free article][PubMed]
  • Chen C, Smye SW, Robinson MP, Evans JA. Membrane electroporation theories: a review. Med Biol Eng Comput. 2006;44:5–14. doi: 10.1007/s11517-005-0020-2. [PubMed] [CrossRef]
  • Kinosita K, Tsong TY. Formation and resealing of pores of controled sizes in human erythrocyte membrane. Nature. 1977. pp. 438–441. [PubMed] [CrossRef]
  • Kinosita K, Tsong TY. Voltage-induced pore formation and hemolyses of human erythrocytes. Biochim Biophys Acta. 1977;471:227–242. doi: 10.1016/0005-2736(77)90252-8. [PubMed][CrossRef]
  • Saulis G. Pore disappearence in a cell after electroporation. Teoretical simulation and comparison with exeriments. Biophys J. 1997;73:1299–1309. doi: 10.1016/S0006-3495(97)78163-3.[PMC free article] [PubMed] [CrossRef]
  • Saulis G, Venslauskas MS, Naktinis J. Kinetics of pore resealing in cell membrane after electroporation. Bioelectrochemistry Bioenerg. 1991;26:1–13. doi: 10.1016/0302-4598(91)87029-G.[CrossRef]
  • Zimmerman U, Vienken J, Pilwat G. Dielsctric breakdown of cell membranes. Biophys J. 1974;14:881–899. doi: 10.1016/S0006-3495(74)85956-4. [PMC free article] [PubMed] [CrossRef]
  • Weaver JC, Chimadzev YA. Theory of electroporation: A review. Bioelectrochemistry and Bioenergetics. 1996;41:135–160. doi: 10.1016/S0302-4598(96)05062-3. [CrossRef]
  • Miklavcic D, Kotnik T. In: Bioelectromagnetic Medicine. Rosh PJ, Markov MS, editor. Informa HealthCare; 2004. Electroporation for Electrochemotherapy and Gene Therapy; pp. 637–656.
  • Mir LM, Gehl J, Serša G, Collins CG, Garbay JR, Billard V, Geertsen PF, Rudolf Z, O’Sullivan GC, Marty M. Standard operating procedures of the electrochemotherapy: Instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes. European J of Cancer Supplements. 2006;4:14–25. doi: 10.1016/j.ejcsup.2006.08.003. [CrossRef]
  • Neumann E. In: Electrical manipulation of cells. Lynch PT, Davet MR, editor. New York: Chapman and Hall; 1996. Gene delivery by membrane elecroporation; pp. 157–184.
  • Esser AT, Smith KC, Gowrishankar TR, Weaver JC. Towards Solid Tumor Treatment by Irreversible Electroporation: Intrinsic Redistribution of Fields and Currents in Tissue. Technol Cancer Res Treat. 2007;6:261–273. [PubMed]
  • Barbosa-Canovas GV, Pothakamury UR, Palou E, Swanson BG. Nonthermal Preservation of Foods.New York: Marcel Dekker; 1999.
  • FDA. Kinetics of Microbial Inactivation for Alternative Food Processing Technologies. Food and Drug Administration Center for Food Safety and Applied Nutrition; 2000.
  • Lelieved HLM, Notermans S, de Haan SWH. Food Preservation by pulsed electric fields. From Research to Application. Cambridge, England: World Publishing Limited; 2007.
  • Al-Sakere B, Andre F, Bernat C, Connault E, Opolon P, Davalos RV, Rubinsky B, Mir LM. Tumor Ablation with Irreversible Electroporation. PLoS ONE. 2007;2(11):e1135. doi: 10.1371/journal.pone.0001135. [PMC free article] [PubMed] [CrossRef]
  • Davalos RD, Mir LM, Rubinsky B. Tissue ablation with Irreversible Electroporation. Ann Biomed Eng. 2005;33:223–231. doi: 10.1007/s10439-005-8981-8. [PubMed] [CrossRef]
  • Lavee J, Onik G, Mikus P, Rubinsky B. A novel nonthermal energy source for surgical epicardial atrial ablation: irreversible electroporation. Heart Surg Forum. 2007;10:e162–167. doi: 10.1532/HSF98.20061202. [PubMed] [CrossRef]
  • Maor E, Ivorra A, Leor J, Rubinsky B. Irreversible electroporation attenuates neointimal formation after angioplasty. IEEE T Biomed Eng. 2008;59:2268–2274. doi: 10.1109/TBME.2008.923909.[PubMed] [CrossRef]
  • Onik G, Mikus P, Rubinsky B. Irreversible electroporation implications for prostate ablation. Technol Cancer Res Treat. 2007;6:295–300. [PubMed]
  • Rodrigo D, Mart?nez A, Harte F, Barbosa-Canovas GV, Rodrigo M. Study of inactivation of Lactobacillus plantarum in orange-carrot juice by means of pulsed electric fields: Comparison of inactivation kinetics models. J Food Protection. 2001;64:259–263. [PubMed]
  • Edd JF, Horowitz L, Davalos RD, Mir LM, Rubinsky B. In vivo results of a new focal tissue ablation technique: irreversible electroporation. IEEE T Biomed Eng. 2006;153:1409–1415. doi: 10.1109/TBME.2006.873745. [PubMed] [CrossRef]
  • Rubinsky B. Irreversible electroporation in medicine. Technol Cancer Res Treat. 2007;6:255–260.[PubMed]
  • Davalos RD, Rubinsky B. Temperature considerations during irreversible electroporation. Int J Heat Mass Transfer. 2008;51:5617–5622. doi: 10.1016/j.ijheatmasstransfer.2008.04.046. [CrossRef]
  • Edd JF, Davalos RV. Mathematical modeling of irreversible electroporation for treatment planning. Technol Cancer Res Treat. 2007;6:275–286. [PubMed]
  • Miller L, Leor J, Rubinsky B. Cancer Cells Ablation with Irreversible Electroporation. Technol Cancer Res Treat. 2005;4:699–705. [PubMed]
  • Neal RE, Davalos RD. The Feasibility of Irreversible Electroporation for the Treatment of Breast Cancer and Other Heterogeneous Systems. Ann Biomedl Eng. 2009. p. 10439. [PubMed]
  • Pavselj N, Miklavcic D. Numerical modeling in electroporation-based biomedical applications. Radiol Oncol. 2008;42:159–168. doi: 10.2478/v10019-008-0008-2. [CrossRef]
  • Pavselj N, Miklavcic D. In: 14th Nordic-Baltic Conference on Biomedical Engineering and Medical Physics; Riga, Latvia. Katashev A, Dekhtyar Y, Spigulis J, editor. Springer Berlin Heidelberg; 2008. Numerical Models of Skin Conductivity Changes during Electroporation; pp. 307–310. full_text.
  • Miklavcic D, Semrov D, Mekid H, Mir LM. A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for gene therapy. Biochim Biophys Acta. 2000;1523:73–83. [PubMed]
  • Semrov D, Miklavcic D. In: Electrochemotherapy, Electrogenetherapy, and Transdermal Drug Delivery. Jaroszeski MK, Heller R, Gilbert R, editor. NJ: Humana Press; 2000. Numerical modeling for in vivo electroporation; pp. 63–81. full_text.
  • Pliquett U. Joule heating during solid tissue electroporation. Med Biol Eng Comput. 2006;41:215–219. doi: 10.1007/BF02344892. [PubMed] [CrossRef]
  • Davalos RD, Rubinsky B, Mir LM. Theoretical analysis of the thermal effects during in vivo tissue electroporation. Bioelectrochemistry. 2003;61:99–107. doi: 10.1016/j.bioelechem.2003.07.001.[PubMed] [CrossRef]
  • Corovic S, Pavlin M, Miklavcic D. Analytical and numerical quantification and comparison of the local electric field in the tissue for different electrode configurations. Biomed Eng Online. 2007;37:1–14. [PMC free article] [PubMed]
  • Corovic S, Zupanic A, Miklavcic D. Tenth International Conference on Computer Modeling and Simulation. IEEE; 2008. Numerical modeling and optimization of local electric field distribution in anisotropic tissue for in vivo electrochemotherapy and electrogene transfer.
  • Mesojednik S, Pavlin D, Sersa G, Coer A, Kranjc S, Grosel A, Tevz G, Cemazar M. The effect of the histological properties of tumors on transfection efficiency of electrically assisted gene delivery to solid tumors in mice. Gene Ther. 2007;14:1261–1269. doi: 10.1038/sj.gt.3302989. [PubMed][CrossRef]
  • Sale AJ, Hamlton WA. Effect of high electric field on micro-organisms. I. Killing of bacteria and yeast. II Mechanism of action of lethal effect. Biochim Biophys Acta. 1967;148:781–800.
  • Sale AJ, Hamilton WA. Effects of high electric fields on microorganisms. III. Lysis of erythrocytes and protoplasts. Biochim Biophys Acta. 1968;163:37–43. doi: 10.1016/0005-2736(68)90030-8.[PubMed] [CrossRef]
  • San Mart?n MF, Sepúlveda DR, Altunakar B, Góngora-Nieto MM, Swanson BG, Barbosa-Cánovas GV. Evaluation of selected mathematical models to predict the inactivation of Listeria innocua by pulsed electric fields. LWT. 2007;40:1271–1279. doi: 10.1016/j.lwt.2006.08.011. [CrossRef]
  • Hülsheger H, Nieman EG. Lethal effect of high-voltage pulses on E. coli K12. Radiat Environ Biophys. 1980;18:281–288. doi: 10.1007/BF01324271. [PubMed] [CrossRef]
  • Hülsheger H, Potel J, Niemann EG. Killing of bacteria with electric pulses of high field strength. Radiat Environment Biophys. 1981. p. 20. [PubMed]
  • Hülsheger H, Potel J, Niemann EG. Electric field effects on bacteria and yeast cells. Radiat Environ Biophys. 1983;22:149–162. doi: 10.1007/BF01338893. [PubMed] [CrossRef]
  • Cerf O. Tailing of survival curves of bacterial spores. J Appl Bacteriol. 1977;42:1–19. [PubMed]
  • Peleg M, Cole MB. Estimating the survival of Clostridium botulinum spores during heat treatments. J Food Protection. 2000;63:190–195. [PubMed]
  • Peleg M, Penchina CM. Modelling microbial survival during exposure to lethal agent varying intensity. Critical Reviews in Food Science and Nutrition. 2000;40:159–172. doi: 10.1080/10408690091189301. [PubMed] [CrossRef]
  • Peleg M. A model of microbial survaval after exposure to pulse electric field. J Sci Food Agric.1995. pp. 93–99. [CrossRef]
  • Castro AJ, Barbarosa-Canovas GV, Swanson BG. Microbial inactivation of foods by pulsed electric fields. J Food Processing Preserv. 1993. pp. 47–73. [CrossRef]
  • Pruitt K, Kamau DN. Mathematical models of bacteria growth, inhibition and death under combinated stress conditions. J Industr Microbiol. 1993;12:221–231. doi: 10.1007/BF01584194.[CrossRef]
  • Augustin JC, Carlier V, Rozier J. Mathematical modelling of the heat resistance of Listeria monocytogenes. J Appl Microbiol. 1998;84:185–191. doi: 10.1046/j.1365-2672.1998.00317.x.[PubMed] [CrossRef]
  • San Mart?n MF, Sepulveda DR, Altunakar B, Gongora-Nieto M, Swanson BG, Barbosa-Canovas GV. Evaluation of selected mathematical models to predict the inactivation of Listeria innocua by pulsed electric fields. LWT. 2007;40:1271–1279. doi: 10.1016/j.lwt.2006.08.011. [CrossRef]
  • Alvarez I, Virto R, Raso J, Codon S. Comparing predicting models for the Escherichia coli inactivation by pulsed electric fields. Innov Food Sci & Emerging Technol. 2003;4:195–202.
  • Miklavcic D, Beravs K, Semrov D, Cemazar M, Demsar F, Sersa G. The importance of electric field distribution for effective in vivo electroporation of tissues. Biophys J. 1998;74:2152–2158. doi: 10.1016/S0006-3495(98)77924-X. [PMC free article] [PubMed] [CrossRef]
  • Rubinsky B, Onik G, Mikus P. Irreversible Electroporation: A New Ablation Modality – Clinical Implications. Technol Cancer Res Treat. 2007;6:37–48. [PubMed]
  • Canatella PJ, Karr JF, Petros JA, Prausnitz MR. Quantitative Study of Electroporation-Mediated Molecular Uptake and Cell Viability. Biophys J. 2001;80:755–764. doi: 10.1016/S0006-3495(01)76055-9. [PMC free article] [PubMed] [CrossRef]
  • Weaver JC. Electroporation of cells and tissues. IEEE T Plasma Sci. 2000;28:24–33. doi: 10.1109/27.842820. [CrossRef]
  • Rubinsky J, Onik G, Paul Mikus P, Rubinsky B. Optimal Parameters for the Destruction of Prostate Cancer Using Irreversible Electroporation. J Urology. 2008;180:2668–2674. doi: 10.1016/j.juro.2008.08.003. [PubMed] [CrossRef]
  • Daniels C, Rubinsky B. Electrical Field and Temperature Model of Nonthermal Irreversible Electroporation in Heterogeneous Tissues. J Biomech Eng. 2009. p. 131. [PubMed]
  • Esser AT, Smith KC, Gowrishankar TR, Weaver JC. Towards Solid Tumor Treatment by Irreversible Electroporation: Intrinsic Redistribution of Fields and Currents in Tissue. Technol Cancer Res Treat. 2007;6:255–360. [PubMed]
  • Esser AT, Smith KC, Gowrishankar TR, Weaver JC. Towards Solid Tumor Treatment by Nanosecond Pulsed Electric Fields. Technol Cancer Res Treat. 2009;8:249–314. [PubMed]
  • Corovic S, Zupanic A, Miklavcic D. Numerical Modeling and Optimization of Electric Field Distribution in Subcutaneous Tumor Treated With Electrochemotherapy Using Needle Electrodes. IEEE T Plasma Sci. 2008;36:1665–1672. doi: 10.1109/TPS.2008.2000996. [CrossRef]
  • Mir LM, Belehradek M, Domenge C, Orlowski S, Poddevin B, Belehradek J Jr, Schwaab G, Luboinski B, Paoletti C. Electrochemotherapy, a new antitumor treatment: first clinical trial. C R Acad Sci III. 1991;313:613–618. [PubMed]
  • Andreuccetii D, Fossi R, Petrucci C. Dielectric Properties of Body Tissues:Output data. Italian natinal Research Council Institute for Applied Physics IFAC.http://niremf.ifac.cnr.it/tissprop/htmlclie/htmlclie.htm#atsftag>

Cell Biol Int.  2002;26(7):599-603.

Extremely low frequency (ELF) pulsed-gradient magnetic fields inhibit malignant tumour growth at different biological levels.

Zhang X, Zhang H, Zheng C, Li C, Zhang X, Xiong W.

Source

Biomedical Physics Unit, Department of Physics, Wuhan University, Wuhan, 430072, China.

Abstract

Extremely low frequency (ELF) pulsed-gradient magnetic field (with the maximum intensity of 0.6-2.0 T, gradient of 10-100 T.M(-1), pulse width of 20-200 ms and frequency of 0.16-1.34 Hz treatment of mice can inhibit murine malignant tumour growth, as seen from analyses at different hierarchical levels, from organism, organ, to tissue, and down to cell and macromolecules. Such magnetic fields induce apoptosis of cancer cells, and arrest neoangiogenesis, preventing a supply developing to the tumour. The growth of sarcomas might be amenable to such new method of treatment.

Technol Health Care.  2011;19(6):455-67.

Solid Ehrlich tumor growth treatment by magnetic waves.

Ali FM, El Gebaly RH, El Hag MA, Rohaim AM.

Source

Department of Biophysics, Faculty of Science, Cairo University, Giza, Egypt.

Abstract

In this work the retardation of Ehrlich tumor growth implanted in mice was studied by employing 4.5 Hz magnetic field. Eighty female Balb/c mice were used, twenty as normal group; the other sixty mice were inoculated with Ehrlich tumor, then they were divided equally into three groups namely A, B and C. Group A (control group) animals were not exposed to the magnetic field. The tumors in the thigh of the animals of group B were exposed to 4.5 Hz, 2 Gauss square wave magnetic field by using a small solenoid connected to a power square wave generator. Group C animals were whole body exposed inside a large solenoid to 4.5 Hz, 2 Gauss square wave magnetic field. Both groups B and C were exposed for a period of 2 weeks at a rate 2 hours per day. Tumor volume, survival period, histological examination and dielectric relaxation of the tumor were measured to investigate the activity of the tumor of the exposed and the unexposed animals. The results indicated that exposing the tumor tissue to 4.5 Hz square wave magnetic field for 2 weeks at a rate 2 hours/day inhibited tumor growth and increased the survival period of the animals. However, group B showed more improvements than did group C. This was attributed to some distortions in the square waveform in the large solenoid (group C). By comparing data from current and previous work, it was concluded that the use of magnetic waves showed better results over previously published work using amplitude modulated electromagnetic waves with the same frequency.

Expert Opin Investig Drugs.  2011 Aug;20(8):1099-106. doi: 10.1517/13543784.2011.583236. Epub 2011 May 9.

Tumor treating fields: concept, evidence and future.

Pless M, Weinberg U.

Source

Medical Oncology, Department of Internal Medicine, and Tumor Center, Kantonsspital Winterthur, Brauerstrasse, Switzerland. miklos.pless@ksw.ch

Abstract

INTRODUCTION: Local control is fundamental, both for the curative as well as the palliative treatment of cancer. Tumor treating fields (TTFields) are low intensity (1 2 V/cm), intermediate frequency (100 ? 200 kHz) alternating electric fields administered using insulated electrodes placed on the skin surrounding the region of a malignant tumor. TTFields were shown to destroy cells within the process of mitosis via apoptosis, thereby inhibiting tumor growth. TTFields have no effect on non-dividing cells.

AREAS COVERED: This article reviews in vitro and in vivo preclinical studies, demonstrating the activity of TTFields both as a monotherapy as well as in combination with several cytotoxic agents. Furthermore, it summarizes the clinical experience with TTFields, mainly in two indications: one in recurrent glioblastoma multiforme: in a large prospective randomized Phase III trial TTFields was compared with best standard care (including chemotherapy): TTFields significantly improved median overall survival (OS) compared with standard therapy (7.8 vs 6.1 months) for the patients treated per protocol. Importantly, quality of life was also better in the TTFields group. The second indication was a Phase II study in second-line non-small cell lung cancer, where TTFields was administered concomitantly with pemetrexed. This combination resulted in an excellent median OS of 13.8 months. Interestingly, the progression-free survival (PFS) within the area of the TTFields was 28, however, outside the TTFields the PFS was only 22 weeks.

EXPERT OPINION: The proof of concept of TTFields has been well demonstrated in the preclinical setting, and the clinical data seem promising in various tumor types. The side effects of TTFields were minimal and in general consisted of skin reaction to the electrodes. There are a number of ways in which TTFields could be further evaluated, for example, in combination with chemotherapy, as a maintenance treatment, or as a salvage therapy if radiotherapy or surgery is not possible. While more clinical data are clearly needed, TTFields is an emerging and promising novel treatment concept. Br J Cancer. Aug 23, 2011; 105(5): 640–648. Published online Aug 9, 2011. doi:  10.1038/bjc.2011.292 PMCID: PMC3188936

Treatment of advanced hepatocellular carcinoma with very low levels of amplitude-modulated electromagnetic fields

F P Costa,1,* A C de Oliveira,1 R Meirelles,1 M C C Machado,1 T Zanesco,1 R Surjan,1 M C Chammas,2 M de Souza Rocha,2 D Morgan,3 A Cantor,4 J Zimmerman,5 I Brezovich,6 N Kuster,7 A Barbault,8 and B Pasche5,*1Department of Transplantation and Liver Surgery, Hospital das Clínicas da Faculdade de Medicina, University of São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 255, São Paulo 05403-000, Brazil 2Department of Radiology, Hospital das Clínicas, University of São Paulo, São Paulo 05403-000, Brazil 3Department of Radiology, University of Alabama at Birmingham and UAB Comprehensive Cancer Center, Birmingham, AL 35294, USA 4Biostatistics and Bioinformatics Shared Facility, University of Alabama at Birmingham and UAB Comprehensive Cancer Center, Birmingham, AL 35294, USA 5Division of Hematology/Oncology, Department of Medicine, University of Alabama at Birmingham and UAB Comprehensive Cancer Center, 1802 6th Ave South, NP 2566, Birmingham, AL 35294-3300, USA 6Department of Radiation Oncology, The University of Alabama at Birmingham and UAB Comprehensive Cancer Center, Birmingham, AL 35294, USA 7IT’IS Foundation, Swiss Federal Institute of Technology, Zurich, Switzerland 8Rue de Verdun 20, Colmar 68000, France *E-mail: moc.liamg@atsocogerepocirederf*E-mail: ude.bau.ccc@ehcsaP.siroBAuthor information ?Article notes ?Copyright and License information ? Revised July 4, 2011; Accepted July 6, 2011. Copyright © 2011 Cancer Research UK This work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ This article has been cited by other articles in PMC.

Abstract

Background:

Therapeutic options for patients with advanced hepatocellular carcinoma (HCC) are limited. There is emerging evidence that the growth of cancer cells may be altered by very low levels of electromagnetic fields modulated at specific frequencies.

Methods:

[gt-or-equal, slanted]

A single-group, open-label, phase I/II study was performed to assess the safety and effectiveness of the intrabuccal administration of very low levels of electromagnetic fields amplitude modulated at HCC-specific frequencies in 41 patients with advanced HCC and limited therapeutic options. Three-daily 60-min outpatient treatments were administered until disease progression or death. Imaging studies were performed every 8 weeks. The primary efficacy end point was progression-free survival 6 months. Secondary efficacy end points were progression-free survival and overall survival.

Results:

Treatment was well tolerated and there were no NCI grade 2, 3 or 4 toxicities. In all, 14 patients (34.1%) had stable disease for more than 6 months. Median progression-free survival was 4.4 months (95% CI 2.1–5.3) and median overall survival was 6.7 months (95% CI 3.0–10.2). There were three partial and one near complete responses.

Conclusion:

Treatment with intrabuccally administered amplitude-modulated electromagnetic fields is safe, well tolerated, and shows evidence of antitumour effects in patients with advanced HCC.Keywords: hepatocellular carcinoma, phase II study, radiofrequency electromagnetic fields, tumour-specific modulation frequencies, 27.12MHz

Treatment of inoperable or metastatic solid tumours is a major challenge in oncology, which is limited by the small number of therapeutic agents that are both well tolerated and capable of long-term control of tumour growth. Hepatocellular carcinoma (HCC) is the second most common cause of cancer death in men and the sixth in women worldwide (Jemal et al, 2011). Hepatocellular carcinoma is the most common tumour in certain parts of the world, particularly in East Asia, Africa, and certain countries of South America. This tumour is less frequent in Europe and in the United States, but has become the fastest rising cancer in the United States (Jemal et al, 2011). In the United States alone, it is estimated that 24120 new cases were diagnosed and there were 17430 deaths from HCC in 2010 (Jemal et al, 2010), a 27% increase in the number of new cases since 2004 (Jemal et al, 2004). The prognosis of patients suffering from advanced HCC is poor with an average survival of fewer than 6 months (Kassianides and Kew, 1987; Jemal et al, 2011).

Therapies for HCC are limited. Resections of the primary tumour or liver transplantation are the preferred therapeutic approaches in patients who are surgical candidates (Bruix and Sherman, 2005). Although these interventions result in long-term survival for some patients, only a minority benefit from them because of limitations due to tumour size, patient’s overall condition, and presence of hepatic cirrhosis (Cance et al, 2000). Only a small number of randomised trials show a survival benefit in the treatment of HCC. Chemoembolisation has been shown to confer a survival benefit in selected patients with unresectable HCC (Llovet et al, 2002). Data from two phase III randomised placebo-controlled studies demonstrate improved survival in patients with advanced HCC receiving the multikinase inhibitor sorafenib (Llovet et al, 2008b; Cheng et al, 2009). Additional therapies for this disease are sorely needed, especially for the large number of patients with advanced disease who cannot tolerate chemotherapy or intrahepatic interventions because of impaired liver function (Thomas and Zhu, 2005).

The intrabuccal administration of low and safe levels of electromagnetic fields, which are amplitude-modulated at disease-specific frequencies (RF AM EMF) (Figure 1), was originally developed for the treatment of insomnia (Pasche et al, 1990). The highest levels of EMFs encountered during treatment are found at the interface between the tongue and the mouth probe and are compliant with international safety limits (ICNIRP, 1998; Pasche and Barbault, 2003). Tumour-specific modulation frequencies have been identified for several common forms of cancer and one report suggests that this novel therapeutic approach is well tolerated and may be effective in patients with a diagnosis of cancer (Barbault et al, 2009). However, the safety and potential efficacy of this treatment approach in the treatment of advanced HCC are unknown. We designed this single-group, open-label, phase I/II study to assess the feasibility of this treatment in patients with advanced HCC and limited therapeutic options.

Figure 1

Figure 1 Delivery of HCC-specific modulation frequencies. (A) The generator of AM EMFs is a battery-driven RF EMF generator connected to a spoon-shaped mouthpiece. (B) Schematic description of AM EMFs. The carrier frequency (27.12MHz) is sinusoidally Go to:

Patients and methods

Patients

The study was aimed at offering treatment to patients with Child–Pugh A or B advanced HCC and limited therapeutic options. Patients were classified as having advanced disease if they were not eligible for surgical resection or had disease progression after surgical or locoregional therapies or had disease progression after chemotherapy or sorafenib therapy. Patients with measurable, inoperable HCC were eligible for enrolment. Previous local or systemic treatments were allowed as long as they were discontinued at least 4 weeks before enrolment. Inclusion criteria included Eastern Cooperative Oncology Group performance status of 0, 1, or 2 and biopsy-confirmed HCC. Also allowed were patients with no pathological confirmation of HCC with a level of ?-fetoprotein higher than 400ngml?1 and characteristic imaging findings as assessed by multislice computer tomography (CT) scan or intravenous contrast ultrasound (US). As per the University of São Paulo Department of Transplantation and Liver Surgery guidelines, liver biopsies are avoided in patients eligible for transplant or with severely impaired liver function. Exclusion criteria included confirmed or suspected brain metastasis, Child–Pugh C, previous liver transplant, and pregnancy.

Study design

This was an investigator-initiated, single centre, uncontrolled phase I/II trial in patients with advanced HCC. The trial was approved by the local human investigation committee and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient. The protocol was registered: clinicaltrial.gov identifier no. NCT00534664.

Administration of AM EMFs

The generator of AM EMFs consists of a battery-driven radiofrequency (RF) EMF generator connected to a 1.5m long 50? coaxial cable, to the other end of which a stainless-steel spoon-shaped mouthpiece is connected via an impedance transformer (Figure 1A). The RF source of the device corresponds to a class C amplifier operating at 27.12MHz. The carrier frequency is AM (Figure 1B) with a modulation depth of 85±5%, whereas the modulation frequency is generated by a digital direct synthesiser with a resolution of 10?7. The treatment sequence is controlled by a microcontroller (Atmel AT89S8252, Fribourg, Switzerland), that is, duration of session, sequence of modulation frequencies and duration of each sequence can be programmed via PC over a RS232 interface. The RF output is adjusted to 100mW into a 50? load, which results in an emitting power identical to that of the device used for the treatment of insomnia (Pasche et al, 1990; Reite et al, 1994; Pasche et al, 1996). The United States Food and Drug Administration has determined that such a device is not a significant risk device and it has been used in several studies conducted in the United States (Reite et al, 1994; Pasche et al, 1996; Kelly et al, 1997). A long-term follow-up survey of 807 patients who have received this therapy in the United States, Europe and Asia showed that the rate of adverse reactions was low and was not associated with increases in the incidence of malignancy or coronary heart disease (Amato and Pasche, 1993). The maximum specific absorption rate (SAR) of the applied RF averaged over any 10g of tissue has been estimated to be less than 2Wkg?1, and the maximum temperature increase is significantly lower than 1°C anywhere in the body owing to RF absorption. The induced RF field values within the primary and metastatic tumours are significantly lower. In contrast, the RF fields induced during RF ablation of tumours cause hyperthermia and result in SAR in the range of 2.4 × 105Wkg?1 (Chang, 2003), that is, more than 100000 times higher than those delivered by the device used in this study.

We have previously reported the discovery of HCC-specific modulation frequencies in 46 patients with HCC using a patient-based biofeedback approach and shown the feasibility of using AM EMFs for the treatment of patients with cancer (Barbault et al, 2009). The treatment programme used in this study consisted of three-daily outpatient treatments of 1h duration, which contained HCC-specific modulation frequencies ranging between 100Hz and 21kHz administered sequentially, each for 3s (Figure 1C and Supplementary Table S1).

The treatment method consists of the administration of AM EMFs by means of an electrically conducting mouthpiece, which is in direct contact with the oral mucosa (Figure 1D). The patients were instructed on the use of the device and received the first treatment at the medical centre’s outpatient clinic. A device was provided to each patient for the duration of the study. The patients were advised to self-administer treatment three times a day. Treatment was administered until tumour progression was objectively documented. At that time, treatment was discontinued. Treatment compliance was assessed at every return visit by recording the number of treatments delivered in the preceding 2 months.

Efficacy end points and disease assessment

The primary end point of this trial was the proportion of patients progression-free at 6 months. Secondary end points were progression-free survival (PFS) (first day of treatment until progression of disease or death) and overall survival (OS) (first day of receiving treatment to death). Objective response was assessed using the Response Evaluation Criteria in Solid Tumours group classification for patients with disease assessed by either helical multiphasic CT (Therasse et al, 2000). Whenever contrast-enhanced US radiological assessment was used, it was performed and reviewed by the same radiologist specialised in HCC (MCC) as this imaging modality is investigator dependent. Tumour measurements were performed at baseline and every 8 weeks. Only patients with at least one repeat tumour measurement during therapy were considered for response analysis. Throughout the study, lesions measured at baseline were evaluated using the same technique (CT or contrast-enhanced US). Overall tumour response was scored as a complete response (CR), partial response (PR), or stable disease (SD) if the response was confirmed at least 4 weeks later. Alpha-fetoprotein (AFP) levels were measured every 8 weeks in all patients throughout the study, but changes in AFP were not an end point for assessment of response. Pain was assessed according to the NCI-CTCAE v.3.0 (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf).

Statistical analyses and efficacy assessment

[gt-or-equal, slanted]

All eligible patients who began treatment were considered assessable for the primary and secondary end points. A Simon two-stage phase II minimax design was used (Simon, 1989) to evaluate the rate of progression-free survival at 6 months. The interim analysis was performed once enrolment into the first stage was completed. In the first stage, 23 patients were observed. If two or fewer patients had progression-free survival 6 months, the trial would be terminated early for lack of efficacy. If the progression-free survival of 3 or more of the first 23 patients was equal or greater than 6 months, then an additional 18 patients would be enrolled to a maximum of 41 patients. If eight or more of the 41 had PFS of at least 6 months, we would conclude that the treatment was efficacious. This design had a Type I error rate of 5% and a Type II error rate of 10% for the null hypothesis of a 6-month PFS rate of 10% vs the alternative of 27.5%. Kaplan–Meier estimates of survival, PFS, and duration of response were calculated with standard errors based on Greenwood’s formula. These calculations were performed using the Proc Lifetest in SAS 9.2 (SAS Institute Inc., Cary, NC, USA).

Results

Patient recruitment and follow-up

From October 2005 to July 2007, 267 patients were assessed for eligibility (Figure 2). In all, 43 patients with advanced HCC and Child–Pugh A or B were enrolled in this study. The date of last patient follow-up is 9 June 2011. Of these, 20 patients (46.5%) had histological confirmation of HCC; 23 patients (53.5%) were diagnosed based on elevated levels of ?-fetoprotein and characteristic imaging findings such as vascular invasion and characteristic differences in tumour blood flow. One patient was excluded because liver biopsy established the diagnosis of metastatic breast cancer. Another patient was excluded because of severely impaired liver function (Child–Pugh C11). These two patients who did not meet the inclusion criteria were registered as screening failures. Hence, a total of 41 patients were eligible to receive experimental therapy (Figure 2).

Figure 2

Figure 2 CONSORT diagram.

Two patients were lost to follow-up as they did not come back for their scheduled appointments. Repeated efforts were made to reach the patients and their families. The date of death of only one patient is known, and no information on response to treatment is available for either patient. Four patients withdrew consent while receiving therapy after 8.0, 9.3, 20.3, and 21.0 months, respectively (Figure 2). One patient elected to receive chemotherapy, one patient had poor treatment compliance as defined by administration of less than 50% of planned treatments at two consecutive return visits, one patient elected to enrol in another experimental protocol, and one patient requested to be considered for liver transplantation as part of an extended indication, which does not fulfil the Milan criteria (Mazzaferro et al, 1996). This latter patient experienced disease progression and was ultimately not eligible for liver transplantation. Of the 35 patients who discontinued experimental therapy, four died of gastrointestinal bleeding, three of sepsis, three of hepatic failure, one of chronic obstructive pulmonary disease, two of chemotherapy- and chemoembolisation-related complications, and one of myocardial infarction (Figure 2). The remaining 24 patients discontinued because of disease progression assessed by imaging or significant clinical deterioration as assessed by the investigator (Figure 2). Estimated 60-day mortality was 27.8% seven of 10 deaths were directly related to progression of disease. They were caused by liver failure in association with significant hepatic tumour involvement, without other cause of death, other than tumour involvement. Two deaths were secondary to gastrointestinal bleeding. One death was due to liver failure.

A total of 31 patients (75.6%) had radiological evidence of disease progression at the time of enrolment as defined by comparison of baseline imaging studies, with imaging studies obtained within the previous 6 months; 34 (82.9%) patients had received therapy before enrolment, five (14.6%) of them systemic chemotherapy or sorafenib (Table 1). Seven (17.1%) patients had not received therapy before enrolment for the following reasons: (1) severely impaired liver function in five cases; and (2) two patients refused to receive chemotherapy for metastatic disease. As shown in Table 2, the majority of patients had severely impaired liver function as demonstrated by the fact that 22 (53.7%) patients had Child–Pugh B disease and 35 (85.4%) BLCL stage C disease.

Table 1

Table 1Treatments received by patients with advanced HCC before enrolment (n=41)

Table 2

Table 2Patients’ baseline characteristics

Treatment efficacy

[gt-or-equal, slanted]

Six of the first 23 patients (26.1%) had progression-free survival 6 months, which led us to continue enrolling patients up to the preplanned total of 41 patients (Figure 2). In total, 14 patients (34.1%) had SD for more than 6 months, which met our preplanned primary efficacy end point. Median progression-free survival was 4.4 months (95% CI 2.1–5.3) and median OS was 6.7 months (95% CI 3.0–10.2) (Figure 3A and B). One patient, previously enrolled in the SHARP study (Llovet et al, 2008b) and with evidence of disease progression at the time of enrolment, remains on therapy with a near complete response for 58 months (Figure 3C). Estimated survival at 12, 24 and 36 months is 27.9% (s.e.=7.1%), 15.2% (s.e.=5.7%), and 10.1% (s.e.=4.8%), respectively. Subset analyses by Child-Pugh stage and accompanying figures are reported in Supplementary Information.

Figure 3

Figure 3 Progression-free and overall survival. (A) Median progression-free survival was 4.4 months (95% CI 2.1–5.3). (B) Median overall survival was 6.7 months (95% CI 3.0–10.2). (C) Long-term partial response in a patient with

A total of 28 patients were evaluable for tumour response (Figure 2). Four (9.8%) patients had a partial response assessed with CT with or without contrast-enhanced ultrasound (Table 3). All partial responses were independently reviewed by two authors (MSR and DM). Three patients had biopsy-confirmed HCC and three had radiological evidence of disease progression at the time of enrolment (Table 4). Two patients had Child–Pugh A, one Child–Pugh B disease, and one had no cirrhosis. One of these patients without biopsy-proven disease subsequently withdrew consent after 4.9 months to undergo liver transplantation. The patient died of progression of disease 9.4 months later before undergoing liver transplantation. One patient with Child–Pugh B disease had a partial response lasting 11.7 months and died of gastrointestinal bleeding. One patient died of disease progression at 44.6 months. Overall, there were six long-term survivors with an OS greater than 24 months and four long-term survivors with an OS greater than 3 years. Importantly, five of the six (83%) long-term survivors had radiological evidence of disease progression at the time of study enrolment (Table 4). Two of three patients with the longest survival (44.6 and +58 months) had radiological evidence of disease progression at the time of enrolment, BLCL stage C disease, as well as portal vein thrombosis, three predictors of short survival (Llovet et al, 2003). Serial AFP measurements, which predict radiological response and survival in patients with HCC (Chan et al, 2009; Riaz et al, 2009), were available for 23 patients. AFP decreased by 20% or more in four (9.8%) patients following initiation of therapy (Table 5). Figure 3D shows the time course of a 37-fold decrease in AFP in a patient who had a long-lasting (11.7 months) partial response as assessed by CT.

Table 3

Table 3Independently reviewed best response (N=41)

Table 4

Table 4Characteristics of patients with either PR and/or long-term survival in excess of 24 months

Table 5

Table 5Changes in AFP levels

In all, 11 patients reported pain before treatment initiation, 3 patients reported grade 3, 5 patients reported grade 2, and 3 patients grade 1. Five patients reported complete disappearance of pain and two patients reported decreased pain shortly after treatment initiation. Two patients reported no changes and two patients reported increased pain. There were no treatment-related grade 2, 3, or 4 toxicities. The only treatment-related adverse events were grade 1 mucositis (one patient) and grade 1 somnolence (one patient) over a total of 266.8 treatment months.Go to:

Discussion

Treatment with AM EMFs did not show any significant toxicity despite long-term treatment. The lack of toxicity experienced by the 41 patients presented in this report as well as the 28 patients from our previous report (Barbault et al, 2009) can be readily explained by the very low and safe levels of induced RF EMFs, which are more than 100000 times lower than those delivered during RF ablation procedures (Chang, 2003). Hence, the putative mechanism of action of this novel therapeutic approach does not depend on temperature changes within the tumour.

These data are comparable to recent phase II studies evaluating the effectiveness of standard chemotherapy as well as novel targeted therapies in HCC (Abou-Alfa et al, 2006; Boige et al, 2007; Chuah et al, 2007; Cohn et al, 2008; Dollinger et al, 2008; Siegel et al, 2008). In a large phase II study assessing the effects of sorafenib in patients with HCC and Child–Pugh A and B who had not received previous systemic treatment, Abou-Alfa et al (2006) observed partial responses using the WHO criteria in 2.2% of patients. Investigator-assessed median time to progression was 4.2 months, and median OS was 9.2 months. Of note, all 137 patients from that study had evidence of disease progression after 14.8 months (Abou-Alfa et al, 2006), whereas, at the same time point, four (9.8%) of the patients enrolled in this study did not have evidence of disease progression. These findings suggest that RF AM EMF may increase the time to radiological progression in advanced HCC.

The majority of patients enrolled in this study had either failed standard treatment options or had severely impaired liver function that limited their ability to tolerate any form of systemic or intrahepatic therapy. Indeed, 16 patients (39.0%) had Child–Pugh B8 or B9 disease. Among these patients, the median progression-free survival was 4.4 months (95% CI 1.6–7.6 months), which is identical to that of the entire group. Five of these 16 patients (31.3%) received therapy for more than 7.5 months, which indicates that this therapy is well tolerated even in patients with severely impaired liver function.

Previous treatment with standard chemotherapy or sorafenib does not seem to impact the effectiveness of AM EMFs in the treatment of HCC. Indeed, three of the four patients who had a partial response while receiving AM EMFs had received previous systemic therapies (chemotherapy and sorafenib) and one had received intrahepatic therapy with 131I-lipiodol.

[gt-or-equal, slanted]

Tumour shrinkage as assessed by radiological imaging as well as changes in AFP levels were documented in patients with advanced HCC receiving RF EMF modulated at HCC-specific frequencies administered by an intrabuccal probe. Antitumour activity in patients with advanced HCC was exemplified by partial responses observed in four patients (9.8%) and decreases in AFP levels greater than 20% in four patients. A total of 18 patients (43.9%) either had objective response or SD 6 months.

Importantly, this therapeutic approach has long-lasting therapeutic effects in several patients with metastatic cancer. Two of these patients, one with recurrent thyroid cancer metastatic to the lungs (Figure 4) enrolled in our feasibility study (Barbault et al, 2009) and the patient shown in Figure 3C, are still receiving treatment without any evidence of disease progression and without side effects almost 5 years after being enrolled in these studies. These findings suggest that, in some patients, this therapeutic approach may achieve permanent control of advanced cancer with virtually no toxicity.

Figure 4

Figure 4 A 70-year-old man with recurrent thyroid cancer metastatic to the lungs: stable disease at 57.5 months. Long-term stable disease in a 70-year-old man with recurrent biopsy-proven thyroid carcinoma metastatic to the lungs enrolled in the previously published

Our phase I/II study has several limitations. First, only 19 of the 41 patients had biopsy-proven HCC, and the others were diagnosed by clinical criteria, an approach similar to that used in a recently reported phase II trial evaluating the clinical and biological effects of bevacizumab in unresectable HCC (Siegel et al, 2008). Importantly, analysis restricted to these 19 patients shows rates of progression-free survival at 6 months, median progression-free survival and OS that are similar to those without biopsy-proven HCC (Supplementary Figures 1C and D). Furthermore, three of the four partial responses were observed in patients with biopsy-proven HCC. Hence, these findings strongly suggest that treatment with AM EMFs yields similar results in patients with and without biopsy-confirmed HCC. Another potential limitation of our study consists in the use of contrast-enhanced ultrasound for the monitoring of some patients with HCC. It should be pointed out that recent studies indicate that the use of this imaging technique is comparable to that of CT scan with respect to the measurement of HCC tumours (Choi, 2007; Maruyama et al, 2008).

Antitumour response is considered the primary end point for phase II studies to proceed to further investigations. Studies applying Cox proportional hazards analysis indicate that this end point is consistently associated with survival in trials of locoregional therapies for HCC (Llovet et al, 2002) and a recent consensus article suggests that randomised studies are necessary to capture the true efficacy of novel therapies in HCC (Llovet et al, 2008a). In summary, the encouraging findings from this study warrant a randomised study to determine the impact of AM EMFs on OS and time to symptomatic progression.

Acknowledgments

We thank Drs Al B Benson III, Northwestern University and Leonard B Saltz, Memorial Sloan-Kettering Cancer Center for reviewing the manuscript.

Notes

AB and BP have filed a patent related to the use of electromagnetic fields for the diagnosis and treatment of cancer. AB and BP are founding members of TheraBionic LLC.

Footnotes

Supplementary Information accompanies the paper on British Journal of Cancer website (http://www.nature.com/bjc)

Supplementary Material

Supplementary Figure 1

Click here for additional data file.(241K, pdf)

Supplementary Figure 2

Click here for additional data file.(172K, pdf)

Supplementary Information

Click here for additional data file.(73K, doc)

Supplementary Table 1

Click here for additional data file.(29K, xls)Go to:

References

  • Abou-Alfa GK, Schwartz L, Ricci S, Amadori D, Santoro A, Figer A, De GJ, Douillard JY, Lathia C, Schwartz B, Taylor I, Moscovici M, Saltz LB. Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J Clin Oncol. 2006;24 (26:4293–4300. [PubMed]
  • Amato D, Pasche B. An evaluation of the safety of low energy emission therapy (published erratum appears in Compr Ther 1994;20(12):681. Compr Ther. 1993;19:242–247. [PubMed]
  • Barbault A, Costa F, Bottger B, Munden R, Bomholt F, Kuster N, Pasche B. Amplitude-modulated electromagnetic fields for the treatment of cancer: discovery of tumor-specific frequencies and assessment of a novel therapeutic approach. J Exp Clin Cancer Res. 2009;28 (1:51. [PMC free article] [PubMed]
  • Boige V, Raoul JL, Pignon JP, Bouche O, Blanc JF, Dahan L, Jouve JL, Dupouy N, Ducreux M. Multicentre phase II trial of capecitabine plus oxaliplatin (XELOX) in patients with advanced hepatocellular carcinoma: FFCD 03-03 trial. Br J Cancer. 2007;97 (7:862–867. [PMC free article] [PubMed]
  • Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology. 2005;42 (5:1208–1236. [PubMed]
  • Cance WG, Stewart AK, Menck HR. The National Cancer Data Base Report on treatment patterns for hepatocellular carcinomas: improved survival of surgically resected patients, 1985–1996. Cancer. 2000;88 (4:912–920. [PubMed]
  • Chan SL, Mo FK, Johnson PJ, Hui EP, Ma BB, Ho WM, Lam KC, Chan AT, Mok TS, Yeo W. New utility of an old marker: serial alpha-fetoprotein measurement in predicting radiologic response and survival of patients with hepatocellular carcinoma undergoing systemic chemotherapy. J Clin Oncol. 2009;27 (3:446–452. [PubMed]
  • Chang I. Finite element analysis of hepatic radiofrequency ablation probes using temperature-dependent electrical conductivity. Biomed Eng Online. 2003;2:12. [PMC free article] [PubMed]
  • Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, Luo R, Feng J, Ye S, Yang TS, Xu J, Sun Y, Liang H, Liu J, Wang J, Tak WY, Pan H, Burock K, Zou J, Voliotis D, Guan Z. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol. 2009;10 (1:25–34. [PubMed]
  • Choi BI. Doppler and harmonic ultrasound for hepatocellular carcinoma. Hepatol Res. 2007;37 (Suppl 2:S172–S177. [PubMed]
  • Chuah B, Lim R, Boyer M, Ong AB, Wong SW, Kong HL, Millward M, Clarke S, Goh BC. Multi-centre phase II trial of Thalidomide in the treatment of unresectable hepatocellular carcinoma. Acta Oncol. 2007;46 (2:234–238. [PubMed]
  • Cohn AL, Myers JW, Mamus S, Deur C, Nicol S, Hood K, Khan MM, Ilegbodu D, Asmar L. A phase II study of pemetrexed in patients with advanced hepatocellular carcinoma. Invest New Drugs. 2008;26 (4:381–386. [PubMed]
  • Dollinger MM, Behrens CM, Lesske J, Behl S, Behrmann C, Fleig WE. Thymostimulin in advanced hepatocellular carcinoma: a phase II trial. BMC Cancer. 2008;8:72. [PMC free article] [PubMed]
  • ICNIRP Guidelines for limiting exposure to time-varying electric, magnetic and electromagnetic fields (up to 300 GHz) Health Phys. 1998;74:494–522. [PubMed]
  • Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. Cancer J Clin. 2011;61 (2:69–90. [PubMed]
  • Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. Cancer J Clin. 2010;60 (5:277–300. [PubMed]
  • Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, Thun MJ. Cancer statistics, 2004. Cancer J Clin. 2004;54 (1:8–29. [PubMed]
  • Kassianides C, Kew MC. The clinical manifestations and natural history of hepatocellular carcinoma. Gastroenterol Clin North Am. 1987;16 (4:553–562. [PubMed]
  • Kelly TL, Kripke DF, Hayduk R, Ryman D, Pasche B, Barbault A. Bright light and LEET effects on circadian rhythms, sleep and cognitive performance. Stress Med. 1997;13:251–258. [PubMed]
  • Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet. 2003;362 (9399:1907–1917. [PubMed]
  • Llovet JM, Di Bisceglie AM, Bruix J, Kramer BS, Lencioni R, Zhu AX, Sherman M, Schwartz M, Lotze M, Talwalkar J, Gores GJ, for the Panel of Experts in HCC-Design Clinical Trials Design and endpoints of clinical trials in hepatocellular carcinoma. J Natl Cancer Inst. 2008a;100 (10:698–711. [PubMed]
  • Llovet JM, Real MI, Montana X, Planas R, Coll S, Aponte J, Ayuso C, Sala M, Muchart J, Sola R, Rodes J, Bruix J. Arterial embolisation or chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma: a randomised controlled trial. Lancet. 2002;359 (9319:1734–1739. [PubMed]
  • Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Haussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J, the SHARP Investigators Study Group Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008b;359 (4:378–390. [PubMed]
  • Maruyama H, Yoshikawa M, Yokosuka O. Current role of ultrasound for the management of hepatocellular carcinoma. World J Gastroenterol. 2008;14 (11:1710–1719. [PMC free article] [PubMed]
  • Mazzaferro V, Regalia E, Doci R, Andreola S, Pulvirenti A, Bozzetti F, Montalto F, Ammatuna M, Morabito A, Gennari L. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med. 1996;334 (11:693–700. [PubMed]
  • Pasche B, Erman M, Hayduk R, Mitler M, Reite M, Higgs L, Dafni U, Amato D, Rossel C, Kuster N, Barbault A, Lebet JP. Effects of low energy emission therapy in chronic psychophysiological insomnia. Sleep. 1996;19:327–336. [PubMed]
  • Pasche B, Erman M, Mitler M. Diagnosis and management of insomnia. N Engl J Med. 1990;323:486–487. [PubMed]
  • Pasche B, Barbault A. 2003. Low-energy emission therapy: current status and future directions Bioelectromagnetic Medicine,Rosch PJ, Markov MS (eds) pp321–327.327Marcel Dekker Inc.: New York, NY
  • Reite M, Higgs L, Lebet JP, Barbault A, Rossel C, Kuster N, Dafni U, Amato D, Pasche B. Sleep inducing effect of low energy emission therapy. Bioelectromagnetics. 1994;15:67–75. [PubMed]
  • Riaz A, Ryu RK, Kulik LM, Mulcahy MF, Lewandowski RJ, Minocha J, Ibrahim SM, Sato KT, Baker T, Miller FH, Newman S, Omary R, Abecassis M, Benson AB, III, Salem R. Alpha-fetoprotein response after locoregional therapy for hepatocellular carcinoma: oncologic marker of radiologic response, progression, and survival. J Clin Oncol. 2009;27 (34:5734–5742. [PubMed]
  • Siegel AB, Cohen EI, Ocean A, Lehrer D, Goldenberg A, Knox JJ, Chen H, Clark-Garvey S, Weinberg A, Mandeli J, Christos P, Mazumdar M, Popa E, Brown RSJ, Rafii S, Schwartz JD. Phase II trial evaluating the clinical and biologic effects of bevacizumab in unresectable hepatocellular carcinoma. J Clin Oncol. 2008;26 (18:2992–2998. [PMC free article] [PubMed]
  • Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials. 1989;10 (1:1–10. [PubMed]
  • Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG. New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst. 2000;92 (3:205–216. [PubMed]
  • Thomas MB, Zhu AX. Hepatocellular carcinoma: the need for progress. J Clin Oncol. 2005;23 (13:2892–2899. [PubMed]

Electromagn Biol Med.  2010 Dec;29(4):132-43.

Bioelectromagnetic field effects on cancer cells and mice tumors.

Berg H, Günther B, Hilger I, Radeva M, Traitcheva N, Wollweber L.

Source

Laboratory Bioelectrochemistry, Beutenberg Campus, Jena, Germany.

Abstract

We present possibilities and trends of ELF bioelectromagnetic effects in the mT amplitude range on cancer cells and on mice bearing tumors. In contrast to invasive electrochemotherapy and electrogenetherapy, using mostly needle electrodes and single high-amplitude electropulses for treatment, extremely low-frequency (ELF) pulsating electromagnetic fields (PEMF) and sinusoidal electromagnetic fields (SEMF) induce tumor cell apoptosis, inhibit angiogenesis, impede proliferation of neoplastic cells, and cause necrosis non invasively, whereas human lymphocytes are negligibly affected. Our successful results in killing cancer cells-analyzed by trypan blue staining or by flow cytometry-and of the inhibition of MX-1 tumors in mice by 15-20?mT, 50?Hz treatment in a solenoid coil also in the presence of bleomycin are presented in comparison to similar experimental results from the literature. In conclusion, the synergistic combinations of PEMF or SEMF with hyperthermia (41.5°C) and/or cancerostatic agents presented in the tables for cells and mice offer a basis for further development of an adjuvant treatment for patients suffering from malignant tumors and metastases pending the near-term development of suitable solenoids of 45-60?cm in diameter, producing >20?mT in their cores. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi.  2010 Oct;27(5):1128-32.

Focusing properties of picosecond electric pulses in non-invasive cancer treatment.

[Article in Chinese] Long Z, Yao C, Li C, Mi Y, Sun C.

Source

State Key Laboratory of Power Transmission Equipment & System Security and New Technology, Chongqing University, Chongqing 400044, China. longzaiquan@foxmail.com

Abstract

In the light of optical theory, we advanc an ultra-wideband impulse radiating antenna (IRA) which is composed of an ellipsoidal reflector and a cone radiator. The high-intensity ultra-short electric pulses radiated by IRA can be transferred into the deep target in tissue non-invasively and be focused effectively. With the focused picosecond electric pulses, the organelles (mitochondria) transmembrane potential shall change to collapse under which the tumor cells will be targetly induced to apoptosis, so the method of non-invasive treatment of tumors would be achieved. Based on the time-domain electromagnetic field theory, the propagation characteristics of picosecond electric pulses were analyzed with and without the context of biological tissue, respectively. The results show that the impulse characteristics of input pulse were maintained and the picosecond electric pulses can keep high resolution in target areas. Meanwhile, because of the dispersive nature of medium, the pulse amplitude of the pulses will attenuate and the pulse width will be broadened.

BMC Cancer. 2010 Apr 24;10:159.

Anti-proliferative effect of extremely low frequency electromagnetic field on preneoplastic lesions formation in the rat liver.

Jiménez-García MN, Arellanes-Robledo J, Aparicio-Bautista DI, Rodríguez-Segura MA, Villa-Treviño S, Godina-Nava JJ.

Department of Physics Center of Research and Advanced Studies of the National Polytechnic Institute, Mexico City, Mexico. jj@fis.cinvestav.mx

Abstract

BACKGROUND: Recently, extremely low frequency electromagnetic fields (ELF-EMF) have been studied with great interest due to their possible effects on human health. In this study, we evaluated the effect of 4.5 mT-120 Hz ELF-EMF on the development of preneoplastic lesions in experimental hepatocarcinogenesis.

METHODS: Male Fischer-344 rats were subjected to the modified resistant hepatocyte model and were exposed to 4.5 mT – 120 Hz ELF-EMF. The effects of the ELF-EMF on hepatocarcinogenesis, apoptosis, proliferation and cell cycle progression were evaluated by histochemical, TUNEL assay, caspase 3 levels, immunohistochemical and western blot analyses.

RESULTS: The application of the ELF-EMF resulted in a decrease of more than 50% of the number and the area of gamma-glutamyl transpeptidase-positive preneoplastic lesions (P = 0.01 and P = 0.03, respectively) and glutathione S-transferase placental expression (P = 0.01). The number of TUNEL-positive cells and the cleaved caspase 3 levels were unaffected; however, the proliferating cell nuclear antigen, Ki-67, and cyclin D1 expression decreased significantly (P < or = 0.03), as compared to the sham-exposure group.

CONCLUSION: The application of 4.5 mT-120 Hz ELF-EMF inhibits preneoplastic lesions chemically induced in the rat liver through the reduction of cell proliferation, without altering the apoptosis process.

Cell Biochem Biophys. 2009;55(1):25-32. Epub 2009 Jun 18.

Evaluation of the potential in vitro antiproliferative effects of millimeter waves at some therapeutic frequencies on RPMI 7932 human skin malignant melanoma cells.

Beneduci A.

Department of Chemistry, University of Calabria, Via P. Bucci, Cubo 17/D, Arcavacata di Rende (CS), Italy.beneduci@unical.it

Abstract

The potential antiproliferative effects of low power millimeter waves (MMWs) at 42.20 and 53.57 GHz on RPMI 7932 human skin melanoma cells were evaluated in vitro in order to ascertain if these two frequencies, comprised in the range of frequency used in millimeter wave therapy, would have a similar effect when applied in vivo to malignant melanoma tumours. Cells were exposed for 1 h exposure/day and to repeated exposure up to a total of four treatments. Plane wave incident power densities <1 mW/cm(2) were used in the MMWs-exposure experiments so that the radiations did not cause significant thermal effects. Numerical simulations of Petri dish reflectivity were made using the equations for the reflection coefficient of a multilayered system. Such analysis showed that the power densities transmitted into the aqueous samples were < or = 0.3 mW/cm(2). Two very important and general biological endpoints were evaluated in order to study the response of melanoma cells to these radiations, i.e. cell proliferation and cell cycle. Herein, we show that neither cell doubling time nor the cell cycle of RPMI 7932 cells was affected by the frequency of the GHz radiation and duration of the exposure, in the conditions above reported.

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Bioelectrochemistry. 2010 Oct;79(2):257-60. Epub 2010 Mar 10.

Electroporation and alternating current cause membrane permeation of photodynamic cytotoxins yielding necrosis and apoptosis of cancer cells.

Traitcheva N, Berg H.

Institute of Plant Physiology “M. Popov,” Bulgarian Acad. of Sciences, Sofia, Bulgaria.

Abstract

In order to increase the permeability of cell membranes for low doses of cytostatic drugs, two bioelectrochemical methods have been compared: (a) electric pore formation in the plasma membranes by single electric impulses (electroporation), and (b) reordering of membrane structure by alternating currents (capacitively coupled). These treatments were applied to human leukemic K-562 cells and human lymphoma U-937 cells, yielding apoptotic and necrotic effects, determined by flow cytometry. Additional cell death occurs after exposure to light irradiation at wavelengths lambda > 600 nm, of cells which were electroporated and had incorporated actinomycin-C or daunomycin (daunorubicin). It is observed that drug uptake after an exponentially decaying electroporation pulse of the initial field strength Eo=1.4 kV/cm and pulse time constants in the time range 0.5-3 ms is faster than during PEMF-treatment, i.e., application of an alternating current of 16 kHz, voltage U<100 V, I=55 mA, and exposure time 20 min. However, at the low a.c. voltage of this treatment, more apoptotic and necrotic cells are produced as compared to the electroporation treatment with one exponentially decaying voltage pulse. Thus, additional photodynamic action appears to be more effective than solely drugs and electroporation as applied in clinical electrochemotherapy, and more effective than the noninvasive pulsed electromagnetic fields (PEMFs), for cancer cells in general and animals bearing tumors in particular.

Arch Biochem Biophys. 2010 May;497(1-2):82-9. Epub 2010 Mar 24.

Nanosecond pulsed electric fields stimulate apoptosis without release of pro-apoptotic factors from mitochondria in B16f10 melanoma.

Ford WE, Ren W, Blackmore PF, Schoenbach KH, Beebe SJ.

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508, USA.

Abstract

Nanosecond pulsed electric fields (nsPEFs) eliminates B16f10 melanoma in mice, but cell death mechanisms and kinetics of molecular events of cell death are not fully characterized. Treatment of B16f10 cells in vitro resulted in coordinate increases in active caspases with YO-PRO-1 uptake, calcium mobilization, decreases in mitochondria membrane potential with decreases in forward light scatter (cell size), increases in ADP/ATP ratio, degradation of actin cytoskeleton and membrane blebbing. However, there was no mitochondrial release of cytochrome c, AIF or Smac/DIABLO or generation of reactive oxygen species. Phosphatidylserine externalization was absent and propidium iodide uptake was delayed in small populations of cells. The results indicate that nsPEFs rapidly recruit apoptosis-like mechanisms through the plasma membrane, mimicking the extrinsic apoptosis pathway without mitochondrial amplification yet include activation of initiator and executioner caspases. nsPEFs provide a new cancer therapy that can bypass cancer-associated deregulation of mitochondria-mediated apoptosis in B16f10 melanoma.

J Physiol Pharmacol. 2010 Apr;61(2):201-5.

Pulsating electromagnetic field stimulation prevents cell death of puromycin treated U937 cell line.

Kaszuba-Zwoinska J, Wojcik K, Bereta M, Ziomber A, Pierzchalski P, Rokita E, Marcinkiewicz J, Zaraska W, Thor P.

Department of Pathophysiology, Jagiellonian University Medical College, Cracow, Poland. jkaszuba@cm-uj.krakow.pl

Abstract

Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1 x 10(6) cells/ml to 0.0625 x 10(6) cells/ml, were exposed to a pulsating magnetic field 50 Hz, 45+/-5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. Viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1 x 10(6)cells/ml and 0.5 x 10(6) cells/ml density. To eliminate density effect on cell death, for further studies density 0.25 x 10(6) cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 microg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin.

Int J Radiat Biol. 2010 Feb;86(2):79-88.

Growth of injected melanoma cells is suppressed by whole body exposure to specific spatial-temporal configurations of weak intensity magnetic fields.

Hu JH, St-Pierre LS, Buckner CA, Lafrenie RM, Persinger MA.

Department of Biology, Laurentian University, Sudbury, Ontario, Canada.

Abstract

PURPOSE: To measure the effect of exposure to a specific spatial-temporal, hysiologically-patterned electromagnetic field presented using different geometric configurations on the growth of experimental tumours in mice.

METHODS: C57b male mice were inoculated subcutaneously with B16-BL6 melanoma cells in two blocks of experiments separated by six months (to control for the effects of geomagnetic field). The mice were exposed to the same time-varying electromagnetic field nightly for 3 h in one of six spatial configurations or two control conditions and tumour growth assessed.

RESULTS: Mice exposed to the field that was rotated through the three spatial dimensions and through all three planes every 2 sec did not grow tumours after 38 days. However, the mice in the sham-field and reference controls showed massive tumours after 38 days. Tumour growth was also affected by the intensity of the field, with mice exposed to a weak intensity field (1-5 nT) forming smaller tumours than mice exposed to sham or stronger, high intensity (2-5 microT) fields. Immunochemistry of tumours from those mice exposed to the different intensity fields suggested that alterations in leukocyte infiltration or vascularisation could contribute to the differences in tumour growth.

CONCLUSIONS: Exposure to specific spatial-temporal regulated electromagnetic field configurations had potent effects on the growth of experimental tumours in mice.

Melanoma Res. 2009 Aug 26. [Epub ahead of print]

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment.

Chen X, James Swanson R, Kolb JF, Nuccitelli R, Schoenbach KH.

Department of Hepatobiliary Surgery, the First Affiliated Hospital Zhejiang University School of Medicine, Hangzhou, Zhejiang, China bFrank Reidy Research Center for Bioelectrics cDepartment of Biological Sciences, Old Dominion University, Norfolk, Virginia, USA.

Abstract

Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana-Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.

Cancer Biol Ther. 2009 Sep;8(18):1756-62. Epub 2009 Sep 17.

Static magnetic fields impair angiogenesis and growth of solid tumors in vivo.

Strelczyk D, Eichhorn ME, Luedemann S, Brix G, Dellian M, Berghaus A, Strieth S.

Walter-Brendel-Center for Experimental Medicine (WBex), Campus Grosshadern, University of Munich (LMU), Munich, Germany.

Abstract

Exposure to static magnetic fields (SMFs) results in a reduced blood flow in tumor vessels as well as in activation and adherence of platelets. Whether this phenomenon may have a significant functional impact on tumors has not been investigated as yet. The aim of our study was to evaluate the effects of prolonged exposure to SMFs on tumor angiogenesis and growth. Experiments were performed in dorsal skinfold chamber preparations of Syrian Golden hamsters bearing syngenic A-Mel-3 melanomas. On 3 d following tumor cell implantation one group of animals was immobilized and exposed to a SMF of 586 mT for three h. Control animals were immobilized for the same duration without SMF exposure. Using in vivo-fluorescence microscopy the field effects on tumor angiogenesis and microcirculation were analyzed for seven days. Tumor growth was assessed by repeated planimetry of the tumor area during the observation period. Exposure to SMFs resulted in a significant retardation of tumor growth ( approximately 30%). Furthermore, histological analysis showed an increased peri- and intratumoral edema in tumors exposed to SMFs. Analysis of microcirculatory parameters revealed a significant reduction of functional vessel density, vessel diameters and red blood cell velocity in tumors after exposure to SMFs compared to control tumors. These changes reflect retarded vessel maturation by antiangiogenesis. The increased edema after SMF exposure indicates an increased tumor microvessel leakiness possibly enhancing drug-uptake. Hence, SMF therapy appears as a promising new anticancer strategy-as an inhibitor of tumor growth and angiogenesis and as a potential sensitizer to

J Exp Clin Cancer Res. 2009 Apr 14;28:51.

Amplitude-modulated electromagnetic fields for the treatment of cancer: discovery of tumor-specific frequencies and assessment of a novel therapeutic approach.

Barbault A, Costa FP, Bottger B, Munden RF, Bomholt F, Kuster N, Pasche B. Cabinet Médical, Avenue de la Gare 6, Lausanne, Switzerland. alexandre.barbault@gmail.com

Abstract PURPOSE: Because in vitro studies suggest that low levels of electromagnetic fields may modify cancer cell growth, we hypothesized that systemic delivery of a combination of tumor-specific frequencies may have a therapeutic effect. We undertook this study to identify tumor-specific frequencies and test the feasibility of administering such frequencies to patients with advanced cancer. PATIENTS AND METHODS: We examined patients with various types of cancer using a noninvasive biofeedback method to identify tumor-specific frequencies. We offered compassionate treatment to some patients with advanced cancer and limited therapeutic options. RESULTS: We examined a total of 163 patients with a diagnosis of cancer and identified a total of 1524 frequencies ranging from 0.1 Hz to 114 kHz. Most frequencies (57-92%) were specific for a single tumor type. Compassionate treatment with tumor-specific frequencies was offered to 28 patients. Three patients experienced grade 1 fatigue during or immediately after treatment. There were no NCI grade 2, 3 or 4 toxicities. Thirteen patients were evaluable for response. One patient with hormone-refractory breast cancer metastatic to the adrenal gland and bones had a complete response lasting 11 months. One patient with hormone-refractory breast cancer metastatic to liver and bones had a partial response lasting 13.5 months. Four patients had stable disease lasting for +34.1 months (thyroid cancer metastatic to lung), 5.1 months (non-small cell lung cancer), 4.1 months (pancreatic cancer metastatic to liver) and 4.0 months (leiomyosarcoma metastatic to liver). CONCLUSION: Cancer-related frequencies appear to be tumor-specific and treatment with tumor-specific frequencies is feasible, well tolerated and may have biological efficacy in patients with advanced cancer. J Ethnopharmacol. 2009 Jun 22;123(2):293-301. Epub 2009 Mar 24.

Induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by flavonoids from Astragalus complanatus.

Hu YW, Liu CY, Du CM, Zhang J, Wu WQ, Gu ZL.

Department of Pharmacology, Medical College of Soochow University, 199 RenAi Road, Suzhou 215123, PR China.

Abstract

AIM OF THE STUDY: Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.

MATERIALS AND METHODS: Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.

RESULTS: The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.

CONCLUSIONS: These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways. J Exp Clin Cancer Res. 2009; 28(1): 51. Published online Apr 14, 2009. doi:  10.1186/1756-9966-28-51 PMCID: PMC2672058

Amplitude-modulated electromagnetic fields for the treatment of cancer: Discovery of tumor-specific frequencies and assessment of a novel therapeutic approach

Alexandre Barbault,1,2 Frederico P Costa,3 Brad Bottger,4 Reginald F Munden,5 Fin Bomholt,6 Niels Kuster,7 and Boris Pasche

corresponding author

1,81Cabinet Médical, Avenue de la Gare 6, Lausanne, Switzerland 2Rue de Verdun 20, Colmar, France 3Sirio-Libanes Hospital, Oncology Center, São Paulo, Brazil 4Radiology Associates, Danbury Hospital, Danbury, CT, USA 5Department of Radiology, The University of Alabama at Birmingham and UAB Comprehensive Cancer Center, Birmingham, AL, USA 6SPEAG AG, Zurich, Switzerland 7IT’IS, Swiss Federal Institute of Technology, Zurich, Switzerland 8Division of Hematology/Oncology, Department of Medicine, The University of Alabama at Birmingham and UAB Comprehensive Cancer Center, Birmingham, AL, USA

corresponding author

Corresponding author. Alexandre Barbault: moc.liamg@tluabrab.erdnaxela; Frederico P Costa: moc.liamg@atsocogerepocirederf; Brad Bottger: ten.enilnotpo@regttob; Reginald F Munden: ude.bau@nednum; Fin Bomholt: moc.gaeps@tlohmob; Niels Kuster: hc.zhte.siti@retsuk; Boris Pasche: ude.bau.ccc@ehcsap.sirob Author information ? Article notes ? Copyright and License information ? Received January 8, 2009; Accepted April 14, 2009. Copyright © 2009 Barbault et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article has been cited by other articles in PMC.

Abstract

Purpose

Because in vitro studies suggest that low levels of electromagnetic fields may modify cancer cell growth, we hypothesized that systemic delivery of a combination of tumor-specific frequencies may have a therapeutic effect. We undertook this study to identify tumor-specific frequencies and test the feasibility of administering such frequencies to patients with advanced cancer.

Patients and methods

We examined patients with various types of cancer using a noninvasive biofeedback method to identify tumor-specific frequencies. We offered compassionate treatment to some patients with advanced cancer and limited therapeutic options.

Results

We examined a total of 163 patients with a diagnosis of cancer and identified a total of 1524 frequencies ranging from 0.1 Hz to 114 kHz. Most frequencies (57–92%) were specific for a single tumor type. Compassionate treatment with tumor-specific frequencies was offered to 28 patients. Three patients experienced grade 1 fatigue during or immediately after treatment. There were no NCI grade 2, 3 or 4 toxicities. Thirteen patients were evaluable for response. One patient with hormone-refractory breast cancer metastatic to the adrenal gland and bones had a complete response lasting 11 months. One patient with hormone-refractory breast cancer metastatic to liver and bones had a partial response lasting 13.5 months. Four patients had stable disease lasting for +34.1 months (thyroid cancer metastatic to lung), 5.1 months (non-small cell lung cancer), 4.1 months (pancreatic cancer metastatic to liver) and 4.0 months (leiomyosarcoma metastatic to liver).

Conclusion

Cancer-related frequencies appear to be tumor-specific and treatment with tumor-specific frequencies is feasible, well tolerated and may have biological efficacy in patients with advanced cancer.

Trial registration

clinicaltrials.gov identifier NCT00805337

Background

We have previously shown that the intrabuccal administration of low and safe levels of electromagnetic fields, amplitude-modulated at a frequency of 42.7 Hz by means of a battery-powered portable device modifies the electroencephalographic activity of healthy subjects [1,2], and is associated with subjective and objective relaxation effects [3]. We have also shown that sequential administration of four insomnia-specific frequencies, including 42.7 Hz, results in a significant decrease in sleep latency and a significant increase in total sleep time in patients suffering from chronic insomnia [4,5]. This approach has been termed Low Energy Emission Therapy (LEET)[4]. Dosimetric studies have shown that the amount of electromagnetic fields delivered to the brain with this approach is 100 to 1000 times lower than the amount of electromagnetic fields delivered by handheld cellular phones and does not result in any heating effect within the brain [6]. The U.S. FDA has determined that such a device is not a significant risk device. A long-term follow-up survey of 807 patients who have received this therapy in the U.S., Europe and Asia revealed that the rate of adverse reactions were low and were not associated with increases in the incidence of malignancy or coronary heart disease [7].

While many discoveries in medicine have evolved from a scientific rationale based on in vitro and in vivo findings, several seminal discoveries are the results of biological effects first observed in humans. For example, the development of modern cancer chemotherapy can be traced directly to the clinical observation that individuals exposed to mustard gas, a chemical warfare agent, had profound lymphoid and myeloid suppression. These observations led Goodman and Gilman to use this agent to treat cancer[8]. Given the advantageous safety profile of athermal, non-ionizing radiofrequency electromagnetic fields[7] and the emerging evidence that low levels of electromagnetic or electric fields may modify the growth of tumor cells [911], we hypothesized that the growth of human tumors might be sensitive to different but specific modulation frequencies. We tested this hypothesis through examination of a large number of patients with biopsy-proven cancer. Using a patient-based biofeedback approach we identified strikingly similar frequencies among patients with the same type of cancer and observed that patients with a different type of cancer had biofeedback responses to different frequencies. These findings provided strong support for our initial hypothesis. Following identification of tumor-specific frequencies in 163 patients with a diagnosis of cancer, we offered compassionate treatment to 28 patients with advanced cancer and limited palliative therapeutic options. We are reporting the results of our frequency discovery studies as well as the results of a feasibility study making use of Low Energy Emission Therapy in the treatment of cancer.

Methods

Frequency discovery consists in the measurement of variations in skin electrical resistance, pulse amplitude and blood pressure. These measurements are conducted while individuals are exposed to low and safe levels of amplitude-modulated frequencies emitted by handheld devices. Exposure to these frequencies results in minimal absorption by the human body, which is well below international electromagnetic safety limits [12,13]. Patients are lying on their back and are exposed to modulation frequencies generated by a frequency synthesizer as described below. Variations in the amplitude of the radial pulse were used as the primary method for frequency detection. They were defined as an increase in the amplitude of the pulse for one or more beats during scanning of frequencies from 0.1 to 114,000 Hz using increments of 100 Hz. Whenever a change in the amplitude of the pulse is observed, scanning is repeated using increasingly smaller steps, down to 10-3 Hz. Frequencies eliciting the best biofeedback responses, defined by the magnitude of increased amplitude and/or the number of beats with increased amplitude, were selected as tumor-specific frequencies.

During our initial search for frequencies in patients with a diagnosis of cancer, we identified frequencies in the 1,000 to 15,000 Hz range. The range of these frequencies was higher than the frequencies previously identified in patients with insomnia (< 300 Hz). To enable the administration of well defined signals at these higher frequencies, the signal synthesizer used in the insomnia studies was redesigned and its accuracy verified at the laboratories of the Foundation for Research on Information Technology in Society (IT’IS, Zurich, Switzerland). The Direct Digital Synthesis (DDS) based synthesizer AD9835 (Analog Devices, Norwood, MA) with a frequency precision of 10-7 was used for frequency detection in patients with a diagnosis of cancer. Subsequently, the same frequency synthesizer was used for treatment administration. The concept of this novel device is depicted in Figure ?Figure11.

Figure 1

Figure 1Block diagram of the novel emitting device making use of the Direct Digital Synthesis (DDS) technology http://www.analog.com/library/analogdialogue/archives/38-08/dds.html. This applicator was used for both the detection and administration of amplitude-modulated

Generation of amplitude-modulated electromagnetic fields: the device consists of a battery-driven radiofrequency (RF) electromagnetic field generator connected to a 1.5 meter long 50 Ohm coaxial cable, to the other end of which a spoon-shaped mouthpiece made of steel is connected with the inner conductor. The RF source of the device corresponds to a high-level amplitude-modulated class C amplifier operating at 27.12 MHz. The modulation frequency can be varied between 0.01 Hz and 150 kHz with a modulation depth of 85 ± 5%. The output signal is controlled by a microcontroller AT89S8252 (Atmel, Fribourg, Switzerland), i.e. duration of a session, sequence of modulation frequencies, and duration of each sequence are programmed prior to the treatment with a PC connected to the panel of the device. The RF output is adjusted to 100 mW into a 50 Ohm load using a sinusoidal modulated test signal, which results in an emitting power identical to that of the device used in the treatment of insomnia [4,5].

Compassionate treatment

Following a period of search and discovery of novel tumor-specific frequencies, outpatient treatment of patients with advanced cancer was initiated in Switzerland and Brazil on a compassionate basis, free of charge. Patients self-administered treatment for 60 min, three times a day. Oral informed consent was provided by seven patients. All other patients signed a written informed consent approved by a local human subject committee in compliance with the Helsinki declaration and the protocol was registered, clinicaltrials.gov identifier # NCT00805337. All patients had histologically confirmed diagnosis of cancer. Except for patients with a diagnosis of ovarian cancer, measurable disease was required. For patients with ovarian cancer, CA 125 level was used as a surrogate marker of measurable disease and a 50% increase in baseline level considered evidence of disease progression. All anticancer therapies had to be discontinued for at least one month prior to treatment initiation. Other eligibility criteria included an Eastern Cooperative Group performance status (PS) of 0 to 2 and an estimated life expectancy of at least 3 months.

Disease assessment

Objective response in patients with measurable disease was assessed using the Response Evaluation Criteria in Solid Tumors group classification [14]. Two of us (B.B. and R.F.M.) independently reviewed all imaging studies.

Toxicity assessment

Patients were evaluated for treatment-related toxicity at a minimum every two months as per the National Cancer Institute Common Toxicity Criteria version 2.0. The worst grade of toxicity per patient was recorded.

Results

Patients characteristics

A total of 115 patients were examined in Switzerland, 48 in Brazil (Table ?(Table1).1). There were 76 females and 87 males. The median age was 59 years (range 19 – 84). The most common tumor types were hepatocellular carcinoma (46), breast cancer (32), colorectal cancer (19), and prostate cancer (17).

Table 1

Table 1 Frequency discovery in 163 patients with a diagnosis of cancer

Compassionate treatment with tumor-specific frequencies was offered to 28 patients (Table ?(Table2).2). Twenty six patients were treated in Switzerland and two patients were treated in Brazil. All patients were white, and 63% (n = 17) were female. Patients ranged in age from 30 to 82 years (median, 61 years) and 75% (n = 21) had PS of 1 (vs 0 or 2). Seventy-nine percent (n = 22) of patients had received at least one prior systemic therapy, 57% (n = 17) had received at least two prior systemic therapies (Table ?(Table22).

Table 2

Table 2 Characteristics of patients treated with amplitude-modulated electromagnetic fields

Once disease progression was observed, most patients elected to resume or initiate chemotherapy and/or targeted therapy. Seven (25%) patients requested to continue experimental treatment in combination with chemotherapy. Continuation of experimental treatment was allowed if desired by the patient and approved by the patient’s oncologist.

Discovery of tumor-specific frequencies

The exact duration of each examination was not recorded but lasted on average three hours. Each patient was examined an average of 3.3 ± 3.4 times (range 1 – 26). Frequency discovery was performed in patients with disease progression, stable disease or partial response. In general, we found more frequencies in patients with evidence of disease progression and large tumor bulk than in patients with stable disease, small tumor bulk or evidence of response. When we restrict the analysis of patients examined in 2006 and 2007, i.e. at a time when we had gathered more than 80% of the common tumor frequencies, we found that patients with evidence of disease progression had positive biofeedback responses to 70% or more of the frequencies previously discovered in patients with the same disease. Conversely, patients with evidence of response to their current therapy had biofeedback responses to 20% or less of the frequencies previously discovered in patients with the same disease. We also observed that patients examined on repeated occasions developed biofeedback responses to an increasing number of tumor-specific frequencies over time whenever there was evidence of disease progression. Whenever feasible, all frequencies were individually retested at each frequency detection session. These findings suggest that a larger number of frequencies are identified at the time of disease progression.

A total of 1524 frequencies ranging from 0.1 to 114 kHz were identified during a total of 467 frequency detection sessions (Table ?(Table1).1). The number of frequencies identified in each tumor type ranges from two for thymoma to 278 for ovarian cancer. Overall, 1183 (77.6%) of these frequencies were tumor-specific, i.e. they were only identified in patients with the same tumor type. The proportion of tumor-specific frequencies ranged from 56.7% for neuroendocrine tumors to 91.7% for renal cell cancer. A total of 341 (22.4%) frequencies were common to at least two different tumor types. The number of frequencies identified was not proportional to either the total number of patients studied or the number of frequency detection sessions (Table ?(Table11).

Treatment with tumor-specific amplitude-modulated electromagnetic fields

Twenty eight patients received a total of 278.4 months of experimental treatment. Median treatment duration was 4.1 months per patient; range 1 to +50.5. Patients treated in Switzerland were re-examined on average every other month for frequency detection; patients treated in Brazil were only examined once. Novel frequencies discovered upon re-examination were added to the treatment program of patients receiving experimental treatment. The first treatment programs consisted of combinations of less than ten frequencies while the most recent treatment programs exceed 280 frequencies (Figure ?(Figure22).

Figure 2

Figure 2Compassionate treatment of a 51 year old patient with ovarian cancer FIGO IIIC with extensive peritoneal carcinomatosis since October 1997. The patient received paclitaxel and cisplatin from March 97, then docetaxel and carboplatin, doxorubicin, and gemcitabine.

The evolution of treatment programs through incremental addition of tumor-specific frequencies is illustrated by the case of a 51 year old woman with ovarian cancer. This patient was diagnosed with FIGO stage III (G2–G3) ovarian cancer in October 1997 and had received multiple courses of palliative chemotherapy until 2005. As seen on Figure ?Figure2,2, the initial treatment consisting of 15 frequencies did not yield any response. Upon re-examination, 11 additional frequencies (total of 26) were added to the treatment program in August 05. Because of disease progression, treatment with single agent bevacizumab was initiated in November 05. Interestingly, the CA 125 level had decreased by 200 units between October and November 2005, prior to the initiation of bevacizumab. Combined treatment with amplitude-modulated electromagnetic fields and bevacizumab resulted in a decrease in CA 125 level from 2140 to 540 in May 06. Treatment was supplemented with cyclophosphamide from March to September 07. The patient was hospitalized with pneumonia and elected to only receive amplitude-modulated electromagnetic fields since September 07. As of April 1, 2009 the patient has stable disease and is asymptomatic. She has been receiving experimental treatment without interruption for a total of +50.5 months.

This case provides empirical evidence that adding tumor-specific frequencies may yield disease stabilization in patients with evidence of disease progression. However, addition of frequencies over time does not appear to be a requirement for therapeutic efficacy. This is illustrated by the case of a 59 yo postmenopausal female with ER/PR positive, ERBB2 negative breast cancer with biopsy confirmed metastasis to the left ischium and right adrenal gland (Figure ?(Figure3A,3A, Figure ?Figure3C,3C, Figure ?Figure3D).3D). She had been previously treated with radiation therapy to the left ischium, had received five different hormonal manipulations (tamoxifen, anastrozole, exemestane, fulvestran and megestrol). She had also received capecitabine, which had been discontinued because of gastrointestinal side effects. The patient was examined only once. In June 2006, at the time of treatment initiation, the patient complained of severe left hip pain, which was limiting her mobility despite the intake of opioids. Within two weeks of experimental treatment initiation with breast cancer-specific frequencies, the patient reported complete disappearance of her pain and discontinued the use of pain medications. She also reported a significant improvement in her overall condition. As seen on Figure ?Figure3B3B and ?and3E,3E, PET-CT obtained three months after treatment initiation showed complete disappearance of the right adrenal and left ischium lesions. The complete response lasted 11 months. Intriguingly, the patient had developed intermittent vaginal spotting in the months preceding experimental treatment initiation. A minimally enhancing uterine lesion was observed on PET-CT prior to treatment initiation. Upon follow-up, FDG uptake increased significantly (Figure ?(Figure3B)3B) and the patient was diagnosed with uterine cancer by hysteroscopy. The patient underwent hysterectomy, which revealed endometrial adenocarcinoma. Hence, while treatment with breast cancer specific frequencies resulted in a complete response, it did not affect the growth of endometrial adenocarcinoma. This observation suggests that breast cancer frequencies are tumor-specific as a response of the metastatic breast cancer was observed while a uterine tumor progressed.

Figure 3

Figure 359 yo postmenopausal female with ER/PR positive, ERBB2 negative breast cancer with biopsy confirmed metastasis to the left ischium and right adrenal gland. A) Baseline PET MIP image demonstrates metastatic disease of the right adrenal gland (small arrow)

As seen in Table ?Table3,3, sixteen patients were evaluable for response by RECIST criteria. A complete response was observed in a patient with hormone-refractory breast cancer metastatic to the adrenal gland and bone (Figure ?(Figure3),3), which lasted 11 months. A partial response was observed in a patient with hormone-refractory breast cancer metastatic to bone and liver, which lasted 13.5 months. Five patients had stable disease for +34.1 months (thyroid cancer with biopsy-proven lung metastases), 6.0 months (mesothelioma metastatic to the abdomen), 5.1 months (non-small cell lung cancer), and 4.1 months (pancreatic cancer with biopsy-proven liver metastases). As of April 1, 2009 two patients are still receiving experimental treatment and four patients are alive.

Table 3

Table 3 Independent review of best response (N = 16) according to RECIST criteria

Adverse and beneficial reactions

No patients receiving experimental therapy reported any side effect of significance and no patient discontinued treatment because of adverse effects. Three patients (10.7%) reported grade I fatigue after receiving treatment. One patient (3.6%) reported grade I mucositis after long-term use (26 months) of the experimental device and concomitant chemotherapy. Two patients with severe bony pain prior to initiation of experimental treatment reported significant symptomatic improvement. Both patients had breast cancer metastatic to the skeleton.

Discussion

In this report we describe the discovery of tumor-specific amplitude modulation frequencies in patients with a diagnosis of cancer using noninvasive biodfeedback methods. Our approach represents a significant departure from the development of novel forms of chemotherapy and targeted therapy, which commonly rely on in vitro and animal experiments, followed by phase I studies to assess tolerability. Given the absence of theoretical health risks related to the administration of very low level of electromagnetic fields and the excellent safety profile observed in patients suffering from insomnia treated for up to several years [7], our approach was entirely patient-based. This allowed us to examine a large number of patients with tumor types commonly encountered in Switzerland and Brazil. It also allowed us to examine the same patients on multiple occasions, which decreased the variability inherent to a single frequency detection session.

Examination of patients with cancer led to the identification of frequencies that were either specific for a given tumor type or common to two or more tumor types. We observed that most frequencies were tumor-specific. Indeed, when the analysis of frequencies is restricted to tumor types analyzed following a minimum of 60 frequency detection sessions (breast cancer, hepatocellular carcinoma, ovarian cancer and prostate cancer), at least 75% of frequencies appear to be tumor-specific. Some frequencies such as 1873.477 Hz, 2221.323 Hz, 6350.333 Hz and 10456.383 Hz are common to the majority of patients with a diagnosis of breast cancer, hepatocellular carcinoma, prostate cancer and pancreatic cancer. The small number of frequency detection sessions conducted in patients with thymoma, leiomyosarcoma, and bladder cancer constitutes a limitation of our study and an accurate estimate of tumor-specific versus nonspecific frequencies cannot yet be provided for these tumor types. Only one patient with thyroid cancer metastatic to the lung was examined 14 times over the course of the past three years and this led to the discovery of 112 frequencies, 79.5% of which were thyroid cancer-specific. These combined findings strongly suggest that many tumor types have a proportion of tumor-specific frequencies of more than 55%. The high number of frequencies observed in patients with ovarian cancer may be due to the various histologies associated with this tumor type.

We observed excellent compliance with this novel treatment as patients were willing to self-administer experimental treatment several times a day. The only observed adverse effects in patients treated with tumor-specific frequencies were grade I fatigue after treatment (10.6%) and grade I mucositis (3.6%). Fatigue was short-lived and no patient reported persistent somnolence. Of note, mucositis only occurred concomitantly with the administration of chemotherapy. The frequency and severity of adverse effects is analogous to what was observed in patients treated with insomnia-specific frequencies [5] and confirm the feasibility of this therapeutic approach and its excellent tolerability.

We did not observe any untoward reaction in patient receiving either chemotherapy or targeted therapy in combination with amplitude-modulated electromagnetic fields. While these latter findings are limited to 7 patients, they are consistent with the lack of theoretical interaction between very low level of electromagnetic fields and anticancer therapy. Furthermore, one patient received palliative radiation therapy concomitantly with experimental therapy without any adverse effects. These findings provide preliminary data suggesting that amplitude-modulated electromagnetic fields may be added to existing anticancer therapeutic regimens.

The objective responses observed suggest that electromagnetic fields amplitude-modulated at tumor-specific frequencies may have a therapeutic effect. Of the seven patients with metastatic breast cancer, one had a complete response lasting 11 months, another one a partial response lasting 13.5 months. These data provide a strong rationale to further study this novel therapy in breast cancer. The increased knowledge of tumor-specific frequencies and the preliminary evidence that additional tumor-specific frequencies may yield a therapeutic benefit (Figure ?(Figure2)2) provides a strong rationale for the novel concept that administration of a large number of tumor-specific frequencies obtained through the follow-up of numerous patients may result in long-term disease control. This hypothesis is partially supported by two long-term survivors reported in this study, a patient with thyroid cancer metastatic to the lung with stable disease for +34.1 months and a heavily pretreated patient with ovarian carcinoma and peritoneal carcinomatosis with stable disease for +50.5 months. Additional support for this hypothesis stems from the observation that four patients with advanced hepatocellular carcinoma in a follow-up phase II study by Costa et al had a partial response, two of them lasting more than 35 months[15]. These exciting results provide hope that this novel therapeutic approach may yield long-term disease control of advanced cancer.

Kirson et al have recently reported the use of continuous wave (CW) electric fields between 100 KHz to 1 MHz [10,11]. These fields were CW, applied at relative high field strengths but lower frequencies than the fields used in our study. These frequencies were found to be effective when applied by insulating external electrodes to animal cancer models and patients with recurrent glioblastoma. In contrast to our approach, the electric fields applied to cancer cells and patients did not include any amplitude modulation. Hence, it is likely that these two different therapeutic modalities have different mechanisms of action.

Computer simulation studies have shown that the specific absorption rate (SAR) in the head resulting from the use of intrabuccally-administered amplitude-modulated electromagnetic fields is in the range of 0.1–100 mW/kg[1]. Hence, the SAR outside the head is substantially below 0.1 mW/kg. We had previously hypothesized that the mechanism of action of electromagnetic fields amplitude-modulated at insomnia-specific frequencies was due to modification in ions and neurotransmitters[6], as demonstrated in animal models[16], as such biological effects had been reported at comparable SARs. However, this hypothesis does not provide a satisfactory explanation for the clinical results observed in patients with advanced cancer. First, the levels of electromagnetic fields delivered to organs such as the liver, adrenal gland, prostate and hip bones, are substantially lower than the levels delivered to the head. Second, there is currently no acceptable rationale for a systemic anti-tumor effect that would involve subtle changes in neurotransmitters and ions within the central nervous system. Consequently, we hypothesize that the systemic changes (pulse amplitude, blood pressure, skin resistivity) observed while patients are exposed to tumor-specific frequencies are the reflection of a systemic effect generated by these frequencies. These observations suggest that electromagnetic fields, which are amplitude-modulated at tumor-specific frequencies, do not act solely on tumors but may have wide-ranging effects on tumor host interactions, e.g. immune modulation. The exciting results from this study provide a strong rationale to study the mechanism of action of tumor-specific frequencies in vitro and in animal models, which may lead to the discovery of novel pathways controlling cancer growth.

Competing interests

AB and BP have filed a patent related to the use of electromagnetic fields for the diagnosis and treatment of cancer. AB and BP are founding members of TheraBionic LLC.

Authors’ contributions

BP and AB conceived and designed the study. FB and NK designed the device and performed the EM dosimetry. AB, BP and FC collected and assembled the data. BB and RF independently reviewed the imaging studies. AB, BP and FC analyzed and interpreted the data. BP wrote the manuscript. All co-authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Al B. Benson, III, Northwestern University and Leonard B. Saltz, Memorial Sloan-Kettering Cancer Center for providing insightful comments and reviewing the manuscript. Neither of them received any compensation for their work. Presented in part: abstract (ID 14072) ASCO 2007; oral presentation (29th Annual Meeting of the Bioelectromagnetics Society, Kanazawa, Japan, 2007). Funding: study funded by AB and BP. The costs associated with the design and engineering of the devices used in this study were paid by AB and BP. BB and RM did not receive any compensation for their independent review of the imaging studies.

References

  • Reite M, Higgs L, Lebet JP, Barbault A, Rossel C, Kuster N, Dafni U, Amato D, Pasche B. Sleep Inducing Effect of Low Energy Emission Therapy. Bioelectromagnetics. 1994;15:67–75. doi: 10.1002/bem.2250150110. [PubMed] [Cross Ref]
  • Lebet JP, Barbault A, Rossel C, Tomic Z, Reite M, Higgs L, Dafni U, Amato D, Pasche B. Electroencephalographic changes following low energy emission therapy. Ann Biomed Eng. 1996;24:424–429. doi: 10.1007/BF02660891. [PubMed] [Cross Ref]
  • Higgs L, Reite M, Barbault A, Lebet JP, Rossel C, Amato D, Dafni U, Pasche B. Subjective and Objective Relaxation Effects of Low Energy Emission Therapy. Stress Medicine. 1994;10:5–13. doi: 10.1002/smi.2460100103. [Cross Ref]
  • Pasche B, Erman M, Mitler M. Diagnosis and Management of Insomnia. N Engl J Med. 1990;323:486–487. [PubMed]
  • Pasche B, Erman M, Hayduk R, Mitler M, Reite M, Higgs L, Dafni U, Rossel C, Kuster N, Barbault A, Lebet J-P. Effects of Low Energy Emission Therapy in chronic psychophysiological insomnia. Sleep. 1996;19:327–336. [PubMed]
  • Pasche B, Barbault A. Low-Energy Emission Therapy: Current Status and Future Directions. In: Rosch PJ, Markov MS, editor. Bioelectromagnetic Medicine. New York: Marcel Dekker, Inc; 2003. pp. 321–327.
  • Amato D, Pasche B. An evaluation of the safety of low energy emission therapy [published erratum appears in Compr Ther 1994;20(12):681] Compr Ther. 1993;19:242–247. [PubMed]
  • Goodman LS, Wintrobe MM, Dameshek W, Goodman MJ, Gilman A, McLennan MT. Landmark article Sept. 21, 1946: Nitrogen mustard therapy. Use of methyl-bis(beta-chloroethyl)amine hydrochloride and tris(beta-chloroethyl)amine hydrochloride for Hodgkin’s disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. By Louis S. Goodman, Maxwell M. Wintrobe, William Dameshek, Morton J. Goodman, Alfred Gilman and Margaret T. McLennan. JAMA: The Journal of the American Medical Association. 1984;251:2255–2261. doi: 10.1001/jama.251.17.2255. [PubMed] [Cross Ref]
  • Kavet R. EMF and current cancer concepts. Bioelectromagnetics. 1996;17:339–357. doi: 10.1002/(SICI)1521-186X(1996)17:5<339::AID-BEM1>3.0.CO;2-4. [PubMed] [Cross Ref]
  • Kirson ED, Gurvich Z, Schneiderman R, Dekel E, Itzhaki A, Wasserman Y, Schatzberger R, Palti Y. Disruption of Cancer Cell Replication by Alternating Electric Fields. Cancer Res. 2004;64:3288–3295. doi: 10.1158/0008-5472.CAN-04-0083. [PubMed] [Cross Ref]
  • Kirson ED, Dbaly V, Tovarys F, Vymazal J, Soustiel JF, Itzhaki A, Mordechovich D, Steinberg-Shapira S, Gurvich Z, Schneiderman R, Wasserman Y, Salzberg M, Ryffel B, Goldsher D, Dekel E, Palti Y. Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors. PNAS. 2007;104:10152–10157. doi: 10.1073/pnas.0702916104. [PMC free article] [PubMed] [Cross Ref]
  • ICNIRP Guidelines for limiting exposure to time-varying electric, magnetic and electromagnetic fields (up to 300 GHz) Health Physics. 1998;74:494–522. [PubMed]
  • Institute of Electrical and Electronics Engineers . Safety Levels with Respect to Human Exposure to Radio Frequency Electromagnetic Fields, 3 kHz to 300 GHz, IEEE C95.1-2005. New York, Institute of Electrical and Electronics Engineers; 2005.
  • Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom A, Christian MC, Gwyther SG. New Guidelines to Evaluate the Response to Treatment in Solid Tumors. J Natl Cancer Inst. 2000;92:205–216. doi: 10.1093/jnci/92.3.205. [PubMed] [Cross Ref]
  • Costa F, de Oliveira AC, Meirelles R, Zanesco T, Surjan R, Chammas M, Barbault A, Pasche B. A phase II study of amplitude-modulated electromagnetic fields in the treatment of advanced hepatocellular carcinoma (HCC) J Clin Oncol (Meeting Abstracts) 2007;25:15155.
  • Adey WR. Biological effects of electromagnetic fields. J Cell Biochem. 1993;51:410–416. [PubMed]

Anticancer Res. 2008 Jul-Aug;28(4B):2245-51.

Effect of steep pulsed electric field on proliferation, viscoelasticity and adhesion of human hepatoma SMMC-7721 cells.

Song G, Qin J, Yao C, Ju Y.

Department of Bioengineering, College of Bioengineering, Ministry of Education of China, Chongqing University, Chongqing, PR China.

song@cqu.edu.cn

Abstract

It has been proven that steep pulsed electric field (SPEF) can directly kill tumor cells and plays an important role in anticancer treatment. The biorheological mechanisms, however, that destroy tumor cells are almost unknown. To resolve this issue, here, an SPEF generator was used to assess the effects of high- and low-dose SPEF on the proliferation of human hepatoma SMMC-7721 cells by MTT assay, and on the viscoelasticity, adhesion of SMMC-7721 cells to endothelial cells by micropipette aspiration technique. Viability and proliferation of SPEF-treated SMMC-7721 cells were significantly inhibited. Cell cycle analysis indicated that SPEF arrested the cell cycle progression of SMMC-7721 cells at the G0/G1 transition to the S-phase. Viscoelastic data fitted by a standard linear solid model showed that viscoelasticity of SMMC-7721 cells changed after treatment with SPEF. Moreover, the adhesive force of low-dose SPEF-treated SMMC-7721 cells to endothelial cells markedly decreased compared to that of control cells. These results suggest that the suppressant effects of SPEF on the proliferation of SMMC-7721 cells appeared to be mediated, at least in part, through arresting cell cycle progression and altering the viscoelastic and adhesive properties of the cells, which provides a novel biorheological mechanism for the antitumor therapy of SPEF.

The Effect of Intense Subnanosecond Electrical Pulses on Biological Cells Schoenbach, K.H.   Shu Xiao   Joshi, R.P.   Camp, J.T.   Heeren, T.   Kolb, J.F.   Beebe, S.J.
Old Dominion Univ., Norfolk; This paper appears in: Plasma Science, IEEE Transactions on
Publication Date: April 2008
Volume: 36,  Issue: 2, Part 1
On page(s): 414-422
Location: Eindhoven, Netherlands,
ISSN: 0093-3813
INSPEC Accession Number: 9921271
Digital Object Identifier: 10.1109/TPS.2008.918786
Current Version Published: 2008-04-08 AbstractNanosecond electrical pulses have been successfully used to treat melanoma tumors by using needle arrays as pulse delivery systems. Reducing the pulse duration of intense electric field pulses from nanoseconds into the subnanosecond range will allow us to use wideband antennas to deliver the electromagnetic fields into tissue with a spatial resolution in the centimeter range. To explore the biological effect of intense subnanosecond pulses, we have developed a generator that provides voltage pulses of 160 kV amplitude, 200 ps rise time, and 800 ps pulse width. The pulses are delivered to a cylindrical Teflon chamber with polished flat electrodes at either end. The distance between the electrodes is variable and allows us to generate electric fields of up to 1 MV/cm in cell suspensions. The pulses have been applied to B16 (murine melanoma) cells, and the plasma membrane integrity was studied by means of trypan blue exclusion. For pulse amplitudes of 550 kV/cm, approximately 50% of the cells took up trypan blue right after pulsing, whereas only 20% were taking it up after 1 h. This indicates that the plasma membrane in a majority of the cells affected by the pulses recovers with a time constant of about 1 h. The cells that show trypan blue uptake after this time suffer cell death through apoptosis. Evaluation of the experimental results and molecular dynamics modeling results indicate that with a pulse duration of 800 ps, membrane charging and nanopore formation are the dominant bioelectric effects on B16 cells. This information has been used in a continuum model to estimate the increase in membrane permeability and, consequently, the increase in pore size caused by repetitive pulsing.

Conf Proc IEEE Eng Med Biol Soc. 2008;2008:1044-7.

Experiment and mechanism research of SKOV3 cancer cell apoptosis induced by nanosecond pulsed electric field.

Yao C, Mi Y, Hu X, Li C, Sun C, Tang J, Wu X.

State Key Laboratory of Power Transmission Equipment & System Security and New Technology, Chongqing University, Chongqing 400044, China.

Abstract

This paper studies the apoptosis of human ovarian carcinoma cell Line (SKOV3) induced by the nanosecond pulsed electric field (10kV/cm, 100ns, 1 Hz) and its effect on intracellular calcium concentration ([Ca2+]i). These cells were doubly marked by Annexin V-FITC/PI, and the apoptosis rate was analyzed with flow cytometry. After AO/EB staining the morphological changes were observed under fluorescent microscope, and their ultrastructural changes were observed under scanning electron microscope (SEM). With Fluo-3/AM as calcium fluorescent marker, laser scanning confocal microscope (LSCM) was used to detect the effect of nsPEF on [Ca2+]i and the source of Ca2+. The results showed that the early apoptosis rate of the treatment group was (22.21+/-2.71)%, significantly higher than that of the control group (3.04+/-0.44)% (P<0.01). The typical features of apoptotic cell have been observed by fluorescent microscope and SEM. It is proved that nsPEF can induce apoptosis of SKOV3 cells and result in distinct increase in [Ca2+]i (P0.01), which was independent of extracellular calcium concentration (P>0.05). Since nsPEF can penetrate cell membrane due to its high frequency components, one of the mechanisms of nsPEF-induced apoptosis may be that activating intracellular calcium stores can increase the [Ca2+]i, and consequently, the apoptotic signal pathway can be induced.

Apoptosis. 2007 Sep;12(9):1721-31.

Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells.

Hall EH, Schoenbach KH, Beebe SJ.

Center for Pediatric Research, Children’s Hospital of the King’s Daughters, Department of Physiological Sciences, Eastern Virginia Medical School, PO Box 1980, Norfolk, VA 23501-1980, USA.

Abstract

Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53(+/+) and HCT116p53(-/-) colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53(+/+) and HCT116p53(-/-) cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax. Hell J Nucl Med.  2007 May-Aug;10(2):95-101.

Anticancer effects on leiomyosarcoma-bearing Wistar rats after electromagnetic radiation of resonant radiofrequencies.

Avdikos A, Karkabounas S, Metsios A, Kostoula O, Havelas K, Binolis J, Verginadis I, Hatziaivazis G, Simos I, Evangelou A.

Source

Laboratory of Physiology, Unit of Environmental Physiology, Faculty of Medicine, University of Ioannina, Greece.

Abstract

In the present study, the effects of a resonant low intensity static electromagnetic field (EMF), causing no thermal effects, on Wistar rats have been investigated. Sarcoma cell lines were isolated from leiomyosarcoma tumors induced in Wistar rats by the subcutaneous (s.c) injection of 3,4-benzopyrene. Furthermore, smooth muscle cells (SMC) were isolated from the aorta of Wistar rats and cultivated. Either leiomyosarcoma cells (LSC) or SMC were used to record a number of characteristic resonant radiofrequencies, in order to determine the specific electromagnetic fingerprint spectrum for each cell line. These spectra were used to compose an appropriate algorithm, which transforms the recorded radiofrequencies to emitted ones. The isolated LSC were cultured and then exposed to a resonant low intensity radiofrequency EMF (RF-EMF), at frequencies between 10 kHz to 120 kHz of the radiowave spectrum. The exposure lasted 45 consecutive minutes daily, for two consecutive days. Three months old female Wistar rats were inoculated with exposed and non-exposed to EMF LSC (4 x 10(6) LCS for animal). Inoculated with non-exposed to EMF cells animals were then randomly separated into three Groups. The first Group was sham exposed to the resonant EMF (control Group-CG), the second Group after the inoculation of LSC and appearance of a palpable tumor mass, was exposed to a non-resonant EMF radiation pattern, for 5 h per day till death of all animals (experimental control Group-ECG). The third Group of animals after inoculation of LSC and the appearance of a palpable tumor mass, was exposed to the resonant EMF radiation for 5 h per day, for a maximum of 60 days (experimental Group-I, EG-I). A fourth Group of animals was inoculated with LSC exposed to EMF irradiation and were not further exposed to irradiation (experimental Group-II, EG-II). Tumor induction was 100% in all Groups studied and all tumors were histologically identified as leiomyosarcomas. In the case of the EG-I, a number of tumors were completely regretted (final tumor induction: 66%). Both Groups of animals inoculated with exposed or non-exposed to the EMF LSC, (EG-I and EG-II, respectively) demonstrated a significant prolongation of the survival time and a lower tumor growth rate, in comparison to the control Group (CG) and the experimental control Group (ECG). However, the survival time of EG-I animals was found to be significantly longer and tumor growth rate significantly lower compared to EG-II animals. In conclusion, our results indicate a specific anticancer effect of resonant EMF irradiation. These results may possibly be attributed to (a) the duration of exposure of LSC and (b) the exposure of the entire animal to this irradiation.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2007 Feb;24(1):230-4.

Biological effects and their applications in medicine of pulsed electric fields.

[Article in Chinese]

Huang H, Song G, Wang G, Sun C.

Key Laboratory for Biomnechanics & Tissue Engineering of the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China.

Abstract

Pulsed electric fields can induce various kinds of biological effects that are essentially different from the normal electric fields, especially the interactions of Nanosecond Pulsed electric field (nsPEF) with cells. The biological effects of different pulsed electric fields on cell membranes, cytoplasmic matrixes, cell growth are introduced in this paper. Based on these effects, some applications of pulsed electric fields in cancer therapy, gene therapy, and delivery of drugs are reviewed in details.

Biochem Biophys Res Commun. 2006 May 5;343(2):351-60. Epub 2006 Mar 10.

Nanosecond pulsed electric fields cause melanomas to self-destruct.

Nuccitelli R, Pliquett U, Chen X, Ford W, James Swanson R, Beebe SJ, Kolb JF, Schoenbach KH.

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. yaochenguo@cqu.edu.cn

Abstract

We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 micros. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 degrees C, ten degrees lower than the minimum temperature for hyperthermia effects.

Bioelectromagnetics. 2006 May;27(4):258-64.

Effect of millimeter wave irradiation on tumor metastasis.

Logani MK, Szabo I, Makar V, Bhanushali A, Alekseev S, Ziskin MC.

Richard J. Fox Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, PA 19140, USA. mlogani@temple.edu

Abstract

One of the major side effects of chemotherapy in cancer treatment is that it can enhance tumor metastasis due to suppression of natural killer (NK) cell activity. The present study was undertaken to examine whether millimeter electromagnetic waves (MMWs) irradiation (42.2 GHz) can inhibit tumor metastasis enhanced by cyclophosphamide (CPA), an anticancer drug. MMWs were produced with a Russian-made YAV-1 generator. Peak SAR and incident power density were measured as 730 +/- 100 W/kg and 36.5 +/- 5 mW/cm(2), respectively. Tumor metastasis was evaluated in C57BL/6 mice, an experimental murine model commonly used for metastatic melanoma. The animals were divided into 5 groups, 10 animals per group. The first group was not given any treatment. The second group was irradiated on the nasal area with MMWs for 30 min. The third group served as a sham control for group 2. The fourth group was given CPA (150 mg/kg body weight, ip) before irradiation. The fifth group served as a sham control for group 4. On day 2, all animals were injected, through a tail vein, with B16F10 melanoma cells, a tumor cell line syngeneic to C57BL/6 mice. Tumor colonies in lungs were counted 2 weeks following inoculation. CPA caused a marked enhancement in tumor metastases (fivefold), which was significantly reduced when CPA-treated animals were irradiated with MMWs. Millimeter waves also increased NK cell activity suppressed by CPA, suggesting that a reduction in tumor metastasis by MMWs is mediated through activation of NK cells.

Bioelectromagnetics. 2006 Apr;27(3):226-32.

Effect of extremely low frequency electromagnetic fields (ELF-EMF) on Kaposi’s sarcoma-associated herpes virus in BCBL-1 cells.

Pica F, Serafino A, Divizia M, Donia D, Fraschetti M, Sinibaldi-Salimei P, Giganti MG, Volpi A.

Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy.

pica@uniroma2.it

Abstract

Association between extremely low frequency electromagnetic fields (ELF-EMF) and human cancers is controversial, and few studies have been conducted on their influence on oncogenic viruses. We studied the effects of 1 mT, 50 Hz sine waves, applied for 24-72 h, on Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV or HHV-8) in BCBL-1, a latently infected primary effusion lymphoma (PEL) cell line. ELF-EMF exposure did not affect the growth and viability of BCBL-1 cells, either stimulated or not with TPA. The total amount of KSHV DNA detected in ELF-EMF exposed cultures not stimulated with TPA did not differ from that of the unexposed controls (P = ns). However, in the presence of TPA stimulation, total KSHV DNA content was found higher in ELF-EMF exposed than in control BCBL-1 cultures (P = .024) at 72 h exposure, but not earlier. Viral DNA increase significantly correlated with increased mean fluorescence intensity/cell for the lytic antigen gp K8.1A/B (P < .01), but not with percentage of gp K8.1A/B-positive cells or of cells containing virions. Viral progeny produced under ELF-EMF exposure consisted mainly of defective viral particles.

Conf Proc IEEE Eng Med Biol Soc. 2006;1:6370-2.

Outlook for the use of focused shock waves and pulsed electric fields in the complex treatment of malignant neoplasms.

Garilevich BA, Andrianov YV, Olefir YV, Zubkov AD, Rotov AE.

Central Air Force Clinical Hosp., Moscow, Russia.

medic-air@mtu-net.ru

Abstract

The experimental studies the synchronous action of electric field microsecond range with amplitude within the range of 1-7 kV/sm and shock waves with pressure before 100 MPa on cells membrane permeability of the mouse’s ascitic tumors in vitro have shown the intensification the efficiency of the forming the irreversible pores under synchronous action. Thereby, enabling the electric field in the compression phase of shock wave pulse which can essentially reduce the electric field intensity required for breakdown cell membrane. In usual condition at amplitude of electric field, specified above, electric breakdown membrane carries basically reversible nature. At the same time in the pressure field tension phase of shock-wave pulse reversible pores, created by electric field, can grow before sizes, under which wholeness membrane is not restored. Under simultaneous action on cellular suspension the shock wave and electric field with moderate intensity cells survival is reduced in 5 once in contrast with occuring at different time’s action, and in 10 once in contrast with checking. The most sensitive to influence by under study fields are cells in phase of the syntheses DNA, preparation to fission and in phase of the mitosis. Thereby, continuation of the studies on use synchronous action shock waves and pulsed electric fields in complex treatment of the tumors introduces perspectiv

Bioelectromagnetics. 2006 Jan;27(1):64-72.

Effects of pulsed magnetic stimulation on tumor development and immune functions in mice.

Yamaguchi S, Ogiue-Ikeda M, Sekino M, Ueno S.

Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Japan.

chikko@medes.m.u-tokyo.ac.jp

Abstract

We investigated the effects of pulsed magnetic stimulation on tumor development processes and immune functions in mice. A circular coil (inner diameter = 15 mm, outer diameter = 75 mm) was used in the experiments. Stimulus conditions were pulse width = 238 micros, peak magnetic field = 0.25 T (at the center of the coil), frequency = 25 pulses/s, 1,000 pulses/sample/day and magnetically induced eddy currents in mice = 0.79-1.54 A/m(2). In an animal study, B16-BL6 melanoma model mice were exposed to the pulsed magnetic stimulation for 16 days from the day of injection of cancer cells. A tumor growth study revealed a significant tumor weight decrease in the stimulated group (54% of the sham group). In a cellular study, B16-BL6 cells were also exposed to the magnetic field (1,000 pulses/sample, and eddy currents at the bottom of the dish = 2.36-2.90 A/m(2)); however, the magnetically induced eddy currents had no effect on cell viabilities. Cytokine production in mouse spleens was measured to analyze the immunomodulatory effect after the pulsed magnetic stimulation. tumor necrosis factor (TNF-alpha) production in mouse spleens was significantly activated after the exposure of the stimulus condition described above. These results showed the first evidence of the anti-tumor effect and immunomodulatory effects brought about by the application of repetitive magnetic stimulation and also suggested the possible relationship between anti-tumor effects and the increase of TNF-alpha levels caused by pulsed magnetic stimulation.

Clin Cancer Res. 2005 Oct 1;11(19 Pt 2):7093s-7103s.

Application of high amplitude alternating magnetic fields for heat induction of nanoparticles localized in cancer.

Ivkov R, DeNardo SJ, Daum W, Foreman AR, Goldstein RC, Nemkov VS, DeNardo GL.

Triton BioSystems, Inc., Chelmsford, Massachusetts 01824, USA. rivkov@tritonbiosystems.com

Abstract

OBJECTIVE: Magnetic nanoparticles conjugated to a monoclonal antibody can be i.v. injected to target cancer tissue and will rapidly heat when activated by an external alternating magnetic field (AMF). The result is necrosis of the microenvironment provided the concentration of particles and AMF amplitude are sufficient. High-amplitude AMF causes nonspecific heating in tissues through induced eddy currents, which must be minimized. In this study, application of high-amplitude, confined, pulsed AMF to a mouse model is explored with the goal to provide data for a concomitant efficacy study of heating i.v. injected magnetic nanoparticles.

METHODS: Thirty-seven female BALB/c athymic nude mice (5-8 weeks) were exposed to an AMF with frequency of 153 kHz, and amplitude (400-1,300 Oe), duration (1-20 minutes), duty (15-100%), and pulse ON time (2-1,200 seconds). Mice were placed in a water-cooled four-turn helical induction coil. Two additional mice, used as controls, were placed in the coil but received no AMF exposure. Tissue and core temperatures as the response were measured in situ and recorded at 1-second intervals.

RESULTS: No adverse effects were observed for AMF amplitudes of < or = 700 Oe, even at continuous power application (100% duty) for up to 20 minutes. Mice exposed to AMF amplitudes in excess of 950 Oe experienced morbidity and injury when the duty exceeded 50%.

CONCLUSION: High-amplitude AMF (up to 1,300 Oe) was well tolerated provided the duty was adjusted to dissipate heat. Results presented suggest that further tissue temperature regulation can be achieved with suitable variations of pulse width for a given amplitude and duty combination. These results suggest that it is possible to apply high-amplitude AMF (> 500 Oe) with pulsing for a time sufficient to treat cancer tissue in which magnetic nanoparticles have been embedded.

Anticancer Res. 2005 Mar-Apr;25(2A):1023-8.

Frequency and irradiation time-dependant antiproliferative effect of low-power millimeter waves on RPMI 7932 human melanoma cell line.

Beneduci A, Chidichimo G, De Rose R, Filippelli L, Straface SV, Venuta S.

Department of Chemistry, University of Calabria, 87036 Arcavacata di Rende (CS), Italy. beneduci@unical.it

Abstract

The biological effects produced by low power millimeter waves (MMW) were studied on the RPMI 7932 human melanoma cell line. Three different frequency-type irradiation modes were used: the 53.57-78.33 GHz wide-band frequency range, the 51.05 GHz and the 65.00 GHz monochromatic frequencies. In all three irradiation conditions, the radiation energy was low enough not to increase the temperature of the cellular samples. Three hours of radiation treatment, applied every day to the melanoma cell samples, were performed at each frequency exposure condition. The wide-band irradiation treatment effectively inhibited cell growth, while both the monochromatic irradiation treatments did not affect the growth trend of RPMI 7932 cells. A light microscopy analysis revealed that the low-intensity wide-band millimeter radiation induced significant morphological alterations on these cells. Furthermore, a histochemical study revealed the low proliferative state of the irradiated cells. This work provides further evidence of the antiproliferative effects on tumor cells induced by low power MMW in the 50-80 GHz frequency range of the electromagnetic spectrum.

Bioelectromagnetics. 2005 Jan;26(1):10-9.

Effect of millimeter waves on natural killer cell activation.

Makar VR, Logani MK, Bhanushali A, Kataoka M, Ziskin MC.

Richard J Fox Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, PA 19140, USA.

Abstract

Millimeter wave therapy (MMWT) is being widely used for the treatment of many diseases in Russia and other East European countries. MMWT has been reported to reduce the toxic effects of chemotherapy on the immune system. The present study was undertaken to investigate whether millimeter waves (MMWs) can modulate the effect of cyclophosphamide (CPA), an anticancer drug, on natural killer (NK) cell activity. NK cells play an important role in the antitumor response. MMWs were produced with a Russian-made YAV-1 generator. The device produced modulated 42.2 +/- 0.2 GHz radiation through a 10 x 20 mm rectangular output horn. Mice, restrained in plastic tubes, were irradiated on the nasal area. Peak SAR at the skin surface and peak incident power density were measured as 622 +/- 100 W/kg and 31 +/- 5 mW/cm2, respectively. The maximum temperature elevation, measured at the end of 30 min, was 1 degrees C. The animals, restrained in plastic tubes, were irradiated on the nasal area. CPA injection (100 mg/kg) was given intraperitoneally on the second day of 3-days exposure to MMWs. All the irradiation procedures were performed in a blinded manner. NK cell activation and cytotoxicity were measured after 2, 5, and 7 days following CPA injection. Flow cytometry of NK cells showed that CPA treatment caused a marked enhancement in NK cell activation. The level of CD69 expression, which represents a functional triggering molecule on activated NK cells, was increased in the CPA group at all the time points tested as compared to untreated mice. However, the most enhancement in CD69 expression was observed on day 7. A significant increase in TNF-alpha level was also observed on day 7 following CPA administration. On the other hand, CPA caused a suppression of the cytolytic activity of NK cells. MMW irradiation of the CPA treated groups resulted in further enhancement of CD69 expression on NK cells, as well as in production of TNF-alpha. Furthermore, MMW irradiation restored CPA induced suppression of the cytolytic activity of NK cells. Our results show that MMW irradiation at 42.2 GHz can up-regulate NK cell functions.

Bioelectromagnetics. 2004 Oct;25(7):516-23.

Combined millimeter wave and cyclophosphamide therapy of an experimental murine melanoma.

Logani MK, Bhanushali A, Anga A, Majmundar A, Szabo I, Ziskin MC.

Richard J. Fox Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

mlogani@temple.edu

Abstract

The objective of the present studies was to investigate whether millimeter wave (MMW) therapy can increase the efficacy of cyclophosphamide (CPA), a commonly used anti-cancer drug. The effect of combined MMW-CPA treatment on melanoma growth was compared to CPA treatment alone in a murine model. MMWs were produced with a Russian made YAV-1 generator. The device produced 42.2 +/- 0.2 GHz modulated wave radiation through a 10 x 20 mm rectangular output horn. The animals, SKH-1 hairless female mice, were irradiated on the nasal area. Peak SAR and incident power density were measured as 730 +/- 100 W/kg and 36.5 +/- 5 mW/cm2, respectively. The maximum skin surface temperature elevation measured at the end of 30 min irradiation was 1.5 degrees C. B16F10 melanoma cells (0.2 x 10(6)) were implanted subcutaneously into the left flank of mice on day 1 of the experiment. On days 4-8, CPA was administered intraperitoneally (30 mg/kg/day). MMW irradiation was applied concurrently with, prior to or following CPA administration. A significant reduction (P < .05) in tumor growth was observed with CPA treatment, but MMW irradiation did not provide additional therapeutic benefit as compared to CPA alone. Similar results were obtained when MMW irradiation was applied both prior to and following CPA treatment.

Biofizika. 2004 May-Jun;49(3):545-50.

A comparison of the effects of millimeter and centimeter waves on tumor necrosis factor production in mouse cells.

[Article in Russian]

Sinotova OA, Novoselova EG, Glushkova OV, Fesenko EE.

Abstract

The effects of millimeter (40 GHz) and centimeter (8.15-18.00 GHz) low-intensity waves on the production of tumor necrosis factor (TNE) in macrophages and lymphocytes from exposed mice as well as in exposed isolated cells were compared. It was found that the dynamics of TNF secretory activity of cells varies depending on the frequency and duration of exposure. The application of millimeter waves induced a nonmonotonous course of the dose-effect curve for TNF changes in macrophages and splenocytes. Alternately, a stimulation and a decrease in TNF production were observed following the application of millimeter waves. On the contrary, centimeter waves provoked an activation in cytokine production. It is proposed that, in contrast to millimeter waves, the single application of centimeter waves to animals (within 2 to 96 h) or isolated cells (within 0.5 to 2.5 h) induced a much more substantial stimulation of immunity.

Bioelectromagnetics. 2004 Oct;25(7):503-7.

Differences in lethality between cancer cells and human lymphocytes caused by LF-electromagnetic fields.

Radeva M, Berg H.

Labor Bioelectrochemistry (Campus Beutenberg, Jena) of the Saxonian Academy of Sciences, Leipzig, Germany.

Abstract

The lethal response of cultured cancer cells lines K-562, U-937, DG-75, and HL-60 were measured directly after a 4 h exposure to a pulsating electromagnetic field (PEMF, sinusoidal wave form, 35 mT peak, 50 Hz) [Traitcheva et al. (2003): Bioelectromagnetics 24:148-158] and 24 h later, to determine the post-exposure effect. The results were found to depend on the medium, pH value, conductivity, and temperature. From these experiments, suitable conditions were chosen to compare the vitality between K-562 cells and normal human lymphocytes after PEMF treatment and photodynamic action. Both agents enhance necrosis synergistically for diseased as well as for healthy cells, but the lymphocytes are more resistant. The efficacy of PEMF on the destruction of cancer cells is further increased by heating (hyperthermia) of the suspension up to 44 degrees C or by lowering the pH-value (hyperacidity) to pH 6.4. Similar apoptosis and necrosis can be obtained using moderate magnetic fields (B < or = 15 mT 50/60 Hz), but this requires longer treatment of at least over a week. PEMF application combined with anticancer drugs and photodynamic therapy will be very effective.

Bioelectromagnetics. 2004 Sep;25(6):466-73.

Millimeter wave-induced suppression of B16 F10 melanoma growth in mice: involvement of endogenous opioids.

Radzievsky AA, Gordiienko OV, Szabo I, Alekseev SI, Ziskin MC.

Center for Biomedical Physics, Temple University Medical School, Philadelphia, Pennsylvania 19140, USA. aradziev@temple.edu

Abstract

Millimeter wave treatment (MMWT) is widely used in Eastern European countries, but is virtually unknown in Western medicine. Among reported MMWT effects is suppression of tumor growth. The main aim of the present “blind” and dosimetrically controlled experiments was to evaluate quantitatively the ability of MMWT to influence tumor growth and to assess whether endogenous opioids are involved. The murine experimental model of B16 F10 melanoma subcutaneous growth was used. MMWT characteristics were: frequency, 61.22 GHz; average incident power density, 13.3 x 10(-3) W/cm2; single exposure duration, 15 min; and exposure area, nose. Naloxone (1 mg/kg, intraperitoneally, 30 min prior to MMWT) was used as a nonspecific blocker of opioid receptors. Five daily MMW exposures, if applied starting at the fifth day following B16 melanoma cell injection, suppressed subcutaneous tumor growth. Pretreatment with naloxone completely abolished the MMWT-induced suppression of melanoma growth. The same course of 5 MMW treatments, if started on day 1 or day 10 following tumor inoculations, was ineffective. We concluded that MMWT has an anticancer therapeutic potential and that endogenous opioids are involved in MMWT-induced suppression of melanoma growth in mice. However, appropriate indications and contraindications have to be developed experimentally before recommending MMWT for clinical usage.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004 Aug;21(4):546-8.

Effects of steep pulsed electric fields on cancer cell proliferation and cell cycle.

[Article in Chinese]

Yao C, Sun C, Mi Y, Xiong L, Hu L, Hu Y.

Key Lab of High Voltage Engineering and Electrical New Technology, Ministry of Education, Chongqing University, Chongqing 400044, China.

Abstract

To assess study the cytocidal and inhibitory effects of steep pulsed electric fields (SPEFs) on ovarian cancer cell line SKOV3, the cancer cell suspension was treated by SPEFs with different parameters (frequency, pulse duration, peak value of voltage). Viability rate and growth curves of two test groups (high dosage and low dosage of SPEFs) and one control group were also measured. The DNA contents and cell cycle were analyzed by flow cytometry (FCM). Different dosing levels of SPEFs exerted obviously different effects on cancer cell viability. With the enhancement of each pulse parameter, the viability rate was promoted and the inhibitory effect on the proliferation of treated cells was more evident. The cells exposed to SPEFs grew slower than the control. The ratio of S+G2/M phase cells was decreased, which restrained the DNA synthesis and division, but the ratio of G0/G1 phase cells was increased in the treated groups. It was also indicated that the SPEFs blocked the cell transition from G0/G1 phase to S+G2/M phase. There was a significant difference in cell cycle between treated group and control group (P<0.01). Lethal effects of SPEFs were represented by inhibiting the cancer cell proliferation at the cell level and by influencing the cell cycle at the DNA level.

Physiol Meas. 2004 Aug;25(4):1077-93.

Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms.

Beebe SJ, Blackmore PF, White J, Joshi RP, Schoenbach KH.

Center for Pediatric Research, Eastern Virginia Medical School, Children’s Hospital for The King’s Daughters, Norfolk, VA, USA. sbeebe@chkd.com

These studies describe the effects of nanosecond (10-300 ns) pulsed electric fields (nsPEF) on mammalian cell structure and function. As the pulse durations decrease, effects on the plasma membrane (PM) decrease and effects on intracellular signal transduction mechanisms increase. When nsPEF-induced PM electroporation effects occur, they are distinct from classical PM electroporation effects, suggesting unique, nsPEF-induced PM modulations. In HL-60 cells, nsPEF that are well below the threshold for PM electroporation and apoptosis induction induce effects that are similar to purinergic agonistmediated calcium release from intracellular stores, which secondarily initiate capacitive calcium influx through store-operated calcium channels in the PM. NsPEF with durations and electric field intensities that do or do not cause PM electroporation, induce apoptosis in mammalian cells with a well-characterized phenotype typified by externalization of phosphatidylserine on the outer PM and activation of caspase proteases. Treatment of mouse fibrosarcoma tumors with nsPEF also results in apoptosis induction. When Jurkat cells were transfected by electroporation and then treated with nsPEF, green fluorescent protein expression was enhanced compared to electroporation alone. The results indicate that nsPEF activate intracellular mechanisms that can determine cell function and fate, providing an important new tool for probing signal transduction mechanisms that modulate cell structure and function and for potential therapeutic applications for cancer and gene therapy.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004 Jun;21(3):433-5.

Effect of steep pulsed electric fields on survival of tumour-bearing mice.

[Article in Chinese]

Yao C, Sun C, Xiong L, Mi Y, Liao R, Hu L, Hu Y.

College of Electrical Engineering, Chongqing University, Chongqing, 400044, China.

To investigate the lethal effect of steep pulsed electric fields (SPEFs) on cancer cells and the life-prolonging effect of SPEFs on the survival of tumour-bearing mice, this study was carried out with the use of SPEFs to treat 40 BALB/C mice inoculated by cervical cancer. The lethal effect on cancer cells and the life-prolonging effect on tumour-bearing mice were examined and compared between the experiment group and control group. The survival periods of the experiment group and control group were 52.05 days and 33.03 days, respectively. There was a significant difference in survival curve between the two groups. The results confirmed the inhibitiory effect and lethal effect of SPEFs on cancer cells. SPEFs can prolong the survival period of tumour-bearing mice.

Ann Biomed Eng. 2003 Jan;31(1):80-90.

Viability of cancer cells exposed to pulsed electric fields: the role of pulse charge.

Krassowska W, Nanda GS, Austin MB, Dev SB, Rabussay DP.

Department of Biomedical Engineering, Duke University, Durham, NC 27708-0281, USA. wanda.krassowska@duke.edu

The goal of this study was to collect a comprehensive set of data that related lethal effects of electric fields to the duration of the pulse. Electric pulses of different strengths and durations were applied to a suspension of HEp-2 cells (epidermoid carcinoma of the human larynx) using a six-needle electrode array connected through an autoswitcher to a square wave generator. Pulse durations varied from 50 micros to 16 ms and the ranges of electric field were adjusted for each duration to capture cell viabilities between 0% and 100%. After pulsation, cells were incubated for 44 h at 37 degrees C, and their viability was measured spectrophotometrically using an XTT assay. For each pulse duration (d), viability data were used to determine the electric field that killed half of the cells (E50). When plotted on logarithmic axes, E50 vs. d was a straight line, leading to a hyperbolic relationship: E50=const/d. This relationship suggests that the total charge delivered by the pulse is the decisive factor in killing HEp-2 cells.

Vopr Onkol. 2003;49(6):748-51.

Experience with turbulent magnetic field as a component of breast cancer therapy.

[Article in Russian]

Letiagin VP, Protchenko NV, Rybakov IuL, Dobrynin IaV.

N.N. Blokhin Center for Oncology Research, Russian Academy of Medical Sciences, Zdorovje Research Center, Moscow.

No adverse side-effects were reported in an investigation of the antitumor effect of turbulent magnetic field (TMF) carried out as a component of preoperative chemoradiotherapy for breast cancer at the Center’s Clinic. The study group included 114 patients with locally advanced tumors(T3, N1-N3, M0). According to the clinical, roentgenological and histological evidence on the end-results, the procedure was highly effective. Also, it was followed by shorter and less extensive postoperative lymphorrhea.

Bioelectromagnetics. 2003 Feb;24(2):148-50.

ELF fields and photooxidation yielding lethal effects on cancer cells.

Traitcheva N, Angelova P, Radeva M, Berg H.

Laboratory of Bioelectrochemistry, Institute of Virology, FSU, Jena, Germany.

Abstract

The lethal effect on human cancer cells was studied under three types of treatment: A) an ELF pulsed sinusoidal of 50 Hz electromagnetic field (PEMF) with amplitudes between 10 and 55 mT; B) the field and a cytostatic agent (actinomycin-C); and C) the field, the cytostatic agent, which has a photodynamic effect, and exposure to a halogen lamp. The results show a decreasing vitality of human K-562 and U-937 cancer cells in suspension with each additional treatment. Combination with other parameters as hyperthermia and/or hyperacidity could yield high killing rates by this noninvasive method.

Technol Cancer Res Treat. 2002 Feb;1(1):71-82.

Enhancing the effectiveness of drug-based cancer therapy by electroporation (electropermeabilization).

Rabussay DP, Nanda GS, Goldfarb PM.

Genetronics, Inc., 11199 Sorrento Valley Road, San Diego CA 92121, USA. dietmarr@genetronics.com

Abstract

Many conventional chemotherapeutic drugs, as well as DNA for cancer gene therapy, require efficient access to the cell interior to be effective. The cell membrane is a formidable barrier to many of these drugs, including therapeutic DNA constructs. Electropermeabilization (EP, often used synonymously with “electroporation”) has become a useful method to temporarily increase the permeability of the cell membrane, allowing a broad variety of molecules efficient access to the cell interior. EP is achieved by the application of short electrical pulses of relatively high local field strength to the target tissue of choice. In cancer therapy, EP can be applied in vivo directly to the tumor to be treated, in order to enhance intracellular uptake of drugs or DNA. Alternatively, EP can be used to deliver DNA into cells of healthy tissue to achieve longer-lasting expression of cancer-suppressing genes. In addition, EP has been used in ex vivo therapeutic approaches for the transfection of a variety of cells in suspension. In this paper, we communicate results related to the development of a treatment for squamous cell carcinomas of the head and neck, using electropermeabilization to deliver the drug bleomycin in vivo directly into the tumor cells. This drug, which is not particularly effective as a conventional therapeutic, becomes highly potent when the intracellular concentration is enhanced by EP treatment. In animal model experiments we found a drug dose of 1 U/cm(3) tumor tissue (delivered in 0.25 mL of an aqueous solution/cm3 tumor tissue) and an electrical field strength of 750 V/cm or higher to be optimal for the treatment of human squamous cell tumors grown subcutaneously in mice. Within 24-48 hours, the majority of tumor cells are rapidly destroyed by this bleomycin-electroporation therapy (B-EPT). This raises the concern that healthy tissue may be similarly affected. In studies with large animals we showed that normal muscle and skin tissue, normal tissue surrounding major blood vessels and nerves, as well as healthy blood vessels and nerves themselves, are much less affected than tumor tissue. Normal tissues did show acute, focal, and transitory effects after treatment, but these effects are relatively minor under standard treatment conditions. The severity of these effects increases with the number of electric pulse cycles and applied voltage. The observed histological changes resolved 20 to 40 days after treatment or sooner, even after excessive EP treatment. Thus, B-EPT is distinct from other ablative therapies, such as thermal, cryo, or photodynamic ablation, which equally affect healthy and tumor tissue. In comparison to surgical or radiation therapy, B-EPT also has potential as a tissue-sparing and function-preserving therapy. In clinical studies with over 50 late stage head and neck cancer patients, objective tumor response rates of 55-58%, and complete tumor response rates of 19-30% have been achieved.

Bioelectromagnetics. 2002 Dec;23(8):578-85.

Influence of 1 and 25 Hz, 1.5 mT magnetic fields on antitumor drug potency in a human adenocarcinoma cell line.

Ruiz-Gómez MJ, de la Peña L, Prieto-Barcia MI, Pastor JM, Gil L, Martínez-Morillo M.

Laboratory of Radiobiology, Department of Radiology and Physical Medicine, Faculty of Medicine, University of Málaga, Teatinos, Málaga, Spain.

Abstract

The resistance of tumor cells to antineoplastic agents is a major obstacle during cancer chemotherapy. Many authors have observed that some exposure protocols to pulsed electromagnetic fields (PEMF) can alter the efficacy of anticancer drugs; nevertheless, the observations are not clear. We have evaluated whether a group of PEMF pulses (1.5 mT peak, repeated at 1 and 25 Hz) produces alterations of drug potency on a multidrug resistant human colon adenocarcinoma (HCA) cell line, HCA-2/1(cch). The experiments were performed including (a) exposures to drug and PEMF exposure for 1 h at the same time, (b) drug exposure for 1 h, and then exposure to PEMF for the next 2 days (2 h/day). Drugs used were vincristine (VCR), mitomycin C (MMC), and cisplatin. Cell viability was measured by the neutral red stain cytotoxicity test. The results obtained were: (a) The 1 Hz PEMF increased VCR cytotoxicity (P < 0.01), exhibiting 6.1% of survival at 47.5 microg/ml, the highest dose for which sham exposed groups showed a 19.8% of survival. For MMC at 47.5 microg/ml, the % of survival changed significantly from 19.2% in sham exposed groups to 5.3% using 25 Hz (P < 0.001). Cisplatin showed a significant reduction in the % of survival (44.2-39.1%, P < 0.05) at 25 Hz and 47.5 microg/ml, and (b) Minor significant alterations were observed after nonsimultaneous exposure of cells to PEMF and drug. The data indicate that PEMF can induce modulation of cytostatic agents in HCA-2/1(cch), with an increased effect when PEMF was applied at the same time as the drug. The type of drug, dose, frequency, and duration of PEMF exposure could influence this modulation.

Biofizika. 2002 Mar-Apr;47(2):376-81.

Immunomodulating effect of electromagnetic waves on production of tumor necrosis factor in mice with various rates of neoplasm growth.

[Article in Russian]

Glushkova OV, Novoselova EG, Sinotova OA, Vrublevskaia VV, Fesenko EE.

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

Abstract

The effects of low-density centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 14 days; MW) on tumor necrosis factor production in macrophages of mice with different growth rate of a cancer solid model produced after hypodermic injection of Ehrlich carcinoma ascites cells into hind legs were studied. After irradiation, an increase in the concentration of tumor necrosis factor in immunocompetent cells of healthy and, specially, of tumor-bearing animals was observed; and the effect of stimulation was higher upon exposure of mice carrying rapidly growing tumors. We suggest that the significant immunomodulating effect of low-density microwaves can be utilized for tumor growth suppression.

Biofizika. 2001 Jan-Feb;46(1):131-5.

Effect of centimeter m

Cell Biol. Int. 2002;26(7):599-603.

Extremely low frequency (ELF) pulsed-gradient magnetic fields inhibit malignant tumour growth at different biological levels.

Zhang X, Zhang H, Zheng C, Li C, Zhang X, Xiong W.

Source

Biomedical Physics Unit, Department of Physics, Wuhan University, Wuhan, 430072, China.

Abstract

Extremely low frequency (ELF) pulsed-gradient magnetic field (with the maximum intensity of 0.6-2.0 T, gradient of 10-100 T.M(-1), pulse width of 20-200 ms and frequency of 0.16-1.34 Hz treatment of mice can inhibit murine malignant tumour growth, as seen from analyses at different hierarchical levels, from organism, organ, to tissue, and down to cell and macromolecules. Such magnetic fields induce apoptosis of cancer cells, and arrest neoangiogenesis, preventing a supply developing to the tumour. The growth of sarcomas might be amenable to such new method of treatment.

icrowaves and the combined magnetic field on the tumor necrosis factor production in cells of mice with experimental tumors.

[Article in Russian]

Novoselova EG, Oga? VB, Sorokina OV, Novikov VV, Fesenko EE.

Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia.

Abstract

The effect of fractionated exposure to low-intensity microwaves (8.15-18 GHz, 1 microW/cm2, 1.5 h daily for 7 days) and combined weak magnetic field (constant 65 1 microT; alternating–100 nT, 3-10 Hz) on the production of tumor necrosis factor in macrophages of mice with experimental solid tumors produced by transplantation of Ehrlich ascites carcinoma was studied. It was found that exposure of mice to both microwaves and magnetic field enhanced the adaptive response of the organism to the onset of tumor growth: the production of tumor necrosis factor in peritoneal macrophages of tumor-bearing mice was higher than in unexposed mice.

J Photochem Photobiol B. 2001 Nov 1;64(1):21-6.

Photodynamic effect on cancer cells influenced by electromagnetic fields.

Pang L, Baciu C, Traitcheva N, Berg H.

Institute of Physics, Nankai University, Nankai, PR China.

The synergism of low-frequency electromagnetic field treatment and photodynamic effect on killing of human cancer cells is presented. The weak pulsating electromagnetic field (PEMF) generated by Helmholtz coils in the mT range influences the permeability of cell membranes for photosensitizers. Several types of sensitizers were excited by visible light during incorporation without and with two kinds of PEMF treatment. In the first part suitable photosensitizers were selected in the absorption range between 400 and 700 nm against human myeloid leukaemia K562 and human histiocytic lymphoma U937 cells by treatment of PEMF consisting of rectangular pulse groups. In the second part amplitude and frequency dependencies were measured using sinuous PEMF and white light with the result that after 12 min the PEMF treatment enhanced photodynamic effectivity by more than 40% over the control value. Taking into account the influence of many parameters, an additional optimization will be possible by photodynamic PEMF synergism for an increased drug delivery in general.

Bioelectromagnetics. 2001 Oct;22(7):503-10.

Pulsed electromagnetic fields affect the intracellular calcium concentrati