The efficiency of electromagnetic field treatment in Complex Regional Pain Syndrome Type I.
Durmus A, Cakmak A, Disci R, Muslumanoglu L.
Istanbul Medical Faculty, Department of Physical Medicine and Rehabilitation, Istanbul University, Istanbul Turkey.
INTRODUCTION: Complex Regional Pain Syndrome Type I is a pathological
condition that occurs without evident nerve injury and follows a course
characterized by severe pain.
PURPOSE: The aim of this study is to assess whether or not
electromagnetic field treatment administered with calcitonin and
exercise has positive effects on clinical improvement, scintigraphic
assessment and bone markers compared to calcitonin and exercise
administration.
METHOD: In this randomized double-blind, placebo-controlled study, 40
patients with Complex Regional Pain Syndrome Type I, that developed
after a Colles fracture were included in the assessments and were
administered calcitonin and exercise treatment for 6 weeks. In addition
to this treatment, half the patients received electromagnetic field
treatment, and the other half received placebo treatment. The patients
were evaluated at the beginning and end of treatment with clinical
parameters, scintigraphic assessment and biochemical markers.
RESULTS: Although we found some significant improvements in our
evaluation criteria, we could not find a significant statistical
difference between groups.
CONCLUSIONS: The absence of a significant difference between the two
groups in the assessment parameters has been interpreted as evidence
that electromagnetic field treatment does not provide additional benefit
to calcitonin and exercise treatment.
Effects of a static magnetic field on wound healing: results in experimental rat colon anastomoses.
Nursal TZ, Bal N, Anarat R, Colakoglu T, Noyan T, Moray G, Haberal M.
Department of General Surgery, Adana Teaching and Research Center,
Baskent University, Dadaloglu Mah. 39. Sok. No. 6, 01250 Yuregir Adana,
Turkey. tznursal@baskent-adn.edu.tr
Abstract
BACKGROUND: Research has shown that pulsed electromagnetic fields
(EMFs) promote wound healing in experimental colonic anastomosis;
however, the effects of static EMFs in this setting have not been
investigated to date.
METHODS: Fifty male Wistar rats were used. Ten served as controls for
mechanical strength testing, and the other 40 underwent descending
colon resection and anastomosis. Twenty of these 40 animals (M group)
had NeFeB magnets placed in contact with the anastomosis site (magnetic
field strength at the site 390 to 420 G). The other 20 animals (sham [S]
group) had non-magnetized NeFeB bars of the same dimensions and weight
implanted. Half of the animals in each group were killed and assessed
for healing parameters on postoperative day 3 (M3 and S3 groups) and the
other half on postoperative day 7 (M7 and S7 groups). Four types of
assessment were done: gross healing, mechanical strength, hydroxyproline
deposition, and histopathology.
RESULTS: There were no differences between the M and S animals with
respect to gross healing parameters. The mechanical strength was also
not different between groups (23.8 +/- 12.7 and 24.7 +/- 9.6 mm Hg for
M3 and S3, respectively; P = .863 and 91.3 +/- 65.4 and 94.8 +/- 55.9 mm
Hg for M7 and S7, respectively; P = .902). Similarly, hydroxyproline
deposition was not different between groups on postoperative day 3 or
day 7. On postoperative day 3, the M group had significantly higher
scores than the S group for fibroblast infiltration (2.4 +/- 0.7 vs 1.4
+/- 0.7, respectively; P = .008) and capillary formation (2.5 +/- 0.7 vs
0.9 +/- 0.4, respectively; P <.001). However, these effects were
reversed and did not endure by day 7.
CONCLUSIONS: The study results suggest that static EMF has no effect on experimental colonic wound healing in the rat.
Int J Colorectal Dis. 2003 Mar;18(2):136-41. Epub 2002 Nov 30.
Effect of electromagnetic fields and early postoperative 5-fluorouracil on the healing of colonic anastomoses.
Nayci A, Cakmak M, Renda N, Aksoyek S, Yucesan S.
Department of Pediatric Surgery, Mersin University Medical Faculty, Mersin, Turkey. anayci@mersin.edu.tr
Abstract
BACKGROUND AND AIMS: Studies have indicated a deleterious effect of
perioperative 5-fluorouracil (5-FU) administration on the healing of
intestinal anastomoses. This study examined the effect of early
postoperative 5-FU on the healing of colonic anastomoses and
investigated the effect of electromagnetic fields (EMF) on colonic
anastomotic repair under normal physiological conditions and in the
presence of 5-FU therapy in a rat model.
MATERIALS AND METHODS: Forty male Wistar rats were randomly assigned
into four groups and underwent a standardized left colonic resection and
anastomoses. The animals then served as control or received
intraperitoneal 5-FU (20 mg/kg per day, 5 days), EMF stimulation (10.76
mT, 50 Hz; 2-h on/10-h off cycles, 7 days) or both, starting on the day
of surgery. After 7 days anastomotic healing was assessed by measurement
of hydroxyproline content and breaking strength.
RESULTS: Hydroxyproline content increased in EMF exposed group
(1.53+/-0.11 to 1.92+/-0.11 microg/mg) and in EMF + 5-FU group
(1.53+/-0.11 to 1.89+/-0.12 microg/mg). Breaking strength also increased
in the EMF group (0.23+/-0.02 to 0.27+/-0.01 MPa) and in the EMF + 5-FU
group (0.23+/-0.02 to 0.28+/-0.01 MPa. No differences were found in
hydroxyproline content or breaking strength between the 5-FU group and
controls.
CONCLUSION: Early postoperative 5-FU administration did not impair
the healing of colonic anastomoses in rats. Additionally, EMF
stimulation provided a significant gain in colonic anastomotic strength,
in rat intestines in control animals and in animals exposed to 5-FU.
Dis Colon Rectum. 2001 Aug;44(8):1181-8.
Comparison of electromagnetic field stimulation on the healing of small and large intestinal anastomoses.
Nayci A, Cakmak M, Aksoyek S, Renda N, Yucesan S.
Department of Pediatric Surgery, Mersin University Medical Faculty, PK 33070, Mersin, Turkey.
Abstract
PURPOSE: Magnetic fields have been shown to affect biologic
processes. Accordingly, an experimental study was designed to
investigate the effect of electromagnetic field stimulation on
intestinal healing and to compare small and large intestinal
anastomoses.
METHODS: An ileal or a colonic anastomosis was constructed in rats.
Beginning the day after surgery, randomly assigned groups were exposed
to sinusoidal electromagnetic field stimulation of 10.76-mT intensity
and 50-Hz frequency, with 2-hour-on/10-hour-off cycles. After seven
days, intestinal anastomoses were assessed for hydroxyproline content
and breaking strength. Statistical comparison between each experimental
and control group yielded significance (P < 0.05) in all cases.
RESULTS: Hydroxyproline content increased significantly in ileum from
1.650 +/- 0.11 (mean +/- standard error of the mean) to 2.036 +/- 0.11
microg/mg (P = 0.0249) and in colon from 1.526 +/- 0.11 to 1.922 +/-
0.11 microg/mg (P = 0.0135). Breaking strength also increased
significantly in ileum from 0.213 +/- 0.01 to 0.255 +/- 0.01 MPa (P =
0.001) and in colon from 0.227 +/- 0.01 to 0.270 +/- 0.01 MPa (P =
0.006).
CONCLUSIONS: Electromagnetic field stimulation provided a significant
gain in anastomotic healing in both small and large intestine. There
were no apparent differences detected between the healing of small and
large intestinal anastomoses except for slight differences in the time
sequences of events and magnitude. The study demonstrated a significant
increase in both biochemical and mechanical parameters. Additional
investigations are needed to determine optimal conditions and promote
selective biologic responses.
Dis Colon Rectum. 1996 Sep;39(9):1031-8.
Influence of pulsed electromagnetic fields on healing of experimental colonic anastomosis.
Mente? BB, Ta?cilar O, Tatlicio?lu E, Bor MV, I?man F, Türközkan N, Celebi M.
Department of Surgery, Gazi University Medical School, Ankara, Turkey.
Abstract
PURPOSE: The study investigated the influence of pulsed
electromagnetic fields (PEMFs) on the mechanical strength and collagen
content of uncomplicated colonic anastomosis in rats.
METHODS: A standardized left colonic resection was performed 3 cm
above the peritoneal reflection, and end-to-end anastomosis was
constructed with eight interrupted inverting sutures. Beginning
immediately after surgery, randomly assigned groups were exposed to one
of the following: 1) 100 Hz (frequency), 1 mT (intensity) PEMFs with
16-hour on/8-hour off cycles (n = 8); 2) 100 Hz, 2 mT PEMFs with 16-hour
on/8-hour off cycles (n = 8); 3) 100 Hz, 1 mT PEMFs with 6-hour
on/6-hour off cycles (n = 6), whereas the control group (n = 10)
received no PEMFs. Relaparatomy was performed at 72 hours
postoperatively, and the bursting pressure of the anastomotic segment
was recorded in situ. The hydroxyproline contents of the anastomotic and
adjacent perianastomotic segments of equal lengths were determined.
RESULTS: Mean bursting pressure values of the groups that received
100 Hz, 1 or 2 mT PEMFs with 16-hour on/8-hour off cycles (90.88 +/-
19.13 and 83.88 +/- 7.08 mmHg, respectively) were significantly higher
than those of the control group (61.66 +/- 10.6 mmHg) and the group with
6-hour on/6-hour off cycles (64.83 +/- 7.36 mmHg; P < 0.05 for all
comparisons). Hydroxyproline contents of the anastomotic and
perianastomotic segments were consistently higher in the 16-hour
on/8-hour off PEMF groups, compared with those of the corresponding
segments of the control group.
CONCLUSIONS: PEMFs applied externally to unrestrained rats within a
“window of PEMF parameters” provided a significant gain in the
mechanical strength of the colonic anastomosis, at least 72 hours
post-operatively. Associated relative increases in the hydroxyproline
contents of the (peri)anastomotic colonic segments suggest that an
altered collagen metabolism might contribute to this enhancement of the
anastomotic repair. Further investigations based on these preliminary
data and the definition of the exact measures regarding the effects of
PEMFs on biologic systems, in general, may lead to an efficient and new
adjunctive modality in colorectal surgery.
Tokai J Exp Clin Med. 1993 Jun;18(1-2):49-55.
The effect of electromagnetic field stimulation on corticosteroids-inhibited intestinal wound healing.
Dindar H, Renda N, Barlas M, Akinay A, Yazgan E, Tinçer T, Cakmak M, Konkan R, Gökçora IH, Yücesan S.
Department of Pediatric Surgery, School of Medicine, Ankara University, Türkiye.
Abstract
Electromagnetic field (EMF) stimulation has been used successfully in
the clinical setting to promote healing of ununited fractures. In a few
studies, EMF stimulation enhanced soft tissue healing. To investigate
the effect of EMF stimulation on intestinal wound healing in normal rats
and in those treated with corticosteroids, 80 Wistar rats received
twice-daily injections of either saline dexamethasone (0.1 mg/kg/day for
2 weeks. Animals then underwent creation of single-layer, inverting
small intestine anastomoses. All injections were continued
postoperatively. Animals were grouped as intestinal anastomoses;
intestinal anastomoses plus EMF stimulation; intestinal anastomoses plus
dexamethasone; and intestinal anastomoses plus dexamethasone plus EMF.
On postoperative days 7 and 14, the anastomosed intestines were removed
and the tensile strength (TS) and hydroxyproline (OH-P) contents
measured. EMF stimulation significantly increased intestinal wound
healing in normal animals by the 7th and 14th day. Corticosteroids
significantly impaired the healing of the small intestine anastomoses,
with decreased TS and OH-P contents after the first and second weeks.
However, EMF stimulation significantly reversed this inhibitory effect.
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.
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.
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Articles from Neural Regeneration Research are provided here courtesy of Medknow Publications
Curr Alzheimer Res. 2015;12(5):481-92.
Jeong YJ, Kang GY, Kwon JH, Choi HD, Pack JK, Kim N, Lee YS, Lee HJ1.
Author information
1Division of Radiation Effects, Korea Institute of Radiological &
Medical Sciences, Seoul, 139-706, Korea. hjlee@kirams.re.kr.
. Abstract
The involvement of radiofrequency electromagnetic fields (RF-EMF) in the
neurodegenerative disease, especially Alzheimer’s disease (AD), has
received wide consideration, however, outcomes from several researches
have not shown consistency. In this study, we determined whether RF-EMF
influenced AD pathology in vivo using Tg-5xFAD mice as a model of
AD-like amyloid (Aß) pathology. The transgenic (Tg)-5xFAD and wild type
(WT) mice were chronically exposed to RF-EMF for 8 months (1950 MHz, SAR
5W/kg, 2 hrs/day, 5 days/week). Notably, chronic RFEMF exposure
significantly reduced not only Aß plaques, APP, and APP
carboxyl-terminal fragments (CTFs) in whole brain including hippocampus
and entorhinal cortex but also the ratio of Aß42 and Aß40 peptide in the
hippocampus of Tg-5xFAD mice. We also found that parenchymal expression
of ß-amyloid precursor protein cleaving enzyme 1(BACE1) and
neuroinflammation were inhibited by RF-EMF exposure in Tg-5xFAD. In
addition, RF-EMF was shown to rescue memory impairment in Tg-5xFAD.
Moreover, gene profiling from microarray data using hippocampus of WT
and Tg- 5xFAD following RF-EMF exposure revealed that 5 genes (Tshz2,
Gm12695, St3gal1, Isx and Tll1), which are involved in Aß, are
significantly altered inTg-5xFAD mice, exhibiting different responses to
RF-EMF in WT or Tg-5xFAD mice; RF-EMF exposure in WT mice showed
similar patterns to control Tg-5xFAD mice, however, RF-EMF exposure in
Tg- 5xFAD mice showed opposite expression patterns. These findings
indicate that chronic RF-EMF exposure directly affects Aß pathology in
AD but not in normal brain. Therefore, RF-EMF has preventive effects
against AD-like pathology in advanced AD mice with a high expression of
Aß, which suggests that RF-EMF can have a beneficial influence on AD.
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.
Neurol Sci. 2015 May;36(5):689-700. doi: 10.1007/s10072-015-2120-6. Epub 2015 Feb 27.
Neurostimulation in Alzheimer’s disease: from basic research to clinical applications.
Nardone R1, Höller Y, Tezzon F, Christova M, Schwenker K, Golaszewski S, Trinka E, Brigo F.
Author information
1Department of Neurology, Christian Doppler Klinik,
Paracelsus Medical University and Center for Cognitive Neuroscience,
Salzburg, Austria, raffaele.nardone@asbmeran-o.it.
Abstract
The development of different methods of brain stimulation provides a
promising therapeutic tool with potentially beneficial effects on
subjects with impaired cognitive functions. We performed a systematic
review of the studies published in the field of neurostimulation in
Alzheimer’s disease (AD), from basic research to clinical applications.
The main methods of non-invasive brain stimulation are repetitive
transcranial magnetic stimulation and transcranial direct current
stimulation. Preliminary findings have suggested that both techniques
can enhance performances on several cognitive functions impaired in AD.
Another non-invasive emerging neuromodulatory approach, the transcranial
electromagnetic treatment, was found to reverse cognitive impairment in
AD transgenic mice and even improves cognitive performance in normal
mice. Experimental studies suggest that high-frequency electromagnetic
fields may be critically important in AD prevention and treatment
through their action at mitochondrial level. Finally, the application of
a widely known invasive technique, the deep brain stimulation (DBS),
has increasingly been considered as a therapeutic option also for
patients with AD; it has been demonstrated that DBS of
fornix/hypothalamus and nucleus basalis of Meynert might improve or at
least stabilize cognitive functioning in AD. Initial encouraging results
provide support for continuing to investigate non-invasive and invasive
brain stimulation approaches as an adjuvant treatment for AD patients.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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]:
?=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
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.
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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.
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.
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.
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.
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.
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.
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.
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).
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.
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]:
?=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
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.
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Radiofrequency fields, transthretin, and Alzheimer’s disease.
Söderqvist F, Hardell L, Carlberg M, Mild KH.
Department of Oncology, University Hospital, Orebro, Sweden.
Abstract
Radiofrequency field (RF) exposure provided cognitive benefits in an
animal study. In Alzheimer’s disease (AD) mice, exposure reduced brain
amyloid-beta (Abeta) deposition through decreased aggregation of Abeta
and increase in soluble Abeta levels. Based on our studies on humans on
RF from wireless phones, we propose that transthyretin (TTR) might
explain the findings. In a cross-sectional study on 313 subjects, we
used serum TTR as a marker of cerebrospinal fluid TTR. We found a
statistically significantly positive beta coefficient for TTR for time
since first use of mobile phones and desktop cordless phones combined
(P=0.03). The electromagnetic field parameters were similar for the
phone types. In a provocation study on 41 persons exposed for 30 min to
an 890-MHz GSM signal with specific absorption rate of 1.0 Watt/kg to
the temporal area of the brain, we found statistically significantly
increased serum TTR 60 min after exposure. In our cross-sectional study,
use of oral snuff also yielded statistically significantly increased
serum TTR concentrations and nicotine has been associated with decreased
risk for AD and to upregulate the TTR gene in choroid plexus but not in
the liver, another source of serum TTR. TTR sequesters Abeta, thereby
preventing the formation of Abeta plaques in the brain. Studies have
shown that patients with AD have lowered TTR concentrations in the
cerebrospinal fluid and have attributed the onset of AD to insufficient
sequestering of Abeta by TTR. We propose that TTR might be involved in
the findings of RF exposure benefit in AD mice.
Many-level polysensory stimulation of brain functions by physical therapeutic agents.
[Article in Russian]
Tyshkevich TG, Ponomarenko GN.
A combination of physiotherapeutic methods for neurorehabilitative
treatment has been developed and applied to the treatment of 576
patients with neurosurgical problems including the loss of brain
functions as a sequel to nervous system lesions of traumatic, vascular,
and other origin. Methodologically, this complex is adapted to the level
and extent of the lesion and the character of regeneration of the
nervous tissues. It implies many-level stimulation of neuroregeneration
by syndromically and pathogenetically substantiated application of
physical factors in the early post-injury and postoperative periods. The
proposed approach allows the brain function to be completely restored
by virtue of persistent compensatory changes in the nervous system. A
combination of many-level magnetic, electrical, and laser stimulation is
recommended to manage lesions in the speech, motor, and visual
analyzers. Combined laser and differential electrostimulation may be
prescribed to patients with nerve lesions, extremely high frequency
therapy to those with epileptic syndrome, combined microwave therapy to
cases with impairment of consciousness, and a variant of systemic UV
irradiation with underwater shower-massaging for the treatment of
vegetative and asthenic disturbances. Selected physiological aspects of
the action of the above physical factors are specified. This
physiotherapeutic system is protected by 20 RF patents of invention.
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.
Department 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.
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
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.
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.
Biull Eksp Biol Med. 1992 Nov;114(11):483-4.
The effect of ultrahigh-frequency electromagnetic radiation on learning and memory processes.
Low-intensity electromagnetic field (12.6 cm, 2375 MHz, power density
1 mW/cm2) produced retrograde amnesia in the rat passive avoidance
test. No effect was registered of microwave irradiation on the open
field behavior and the pain sensitivity. Functional activity of the
m-cholinergic receptors decreased, but their number increased in the
brain cortex. It is suggested that cholinergic system plays an important
role in the effects of electromagnetic field on memory processes.
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.
Acupunct Electrother Res. 1992;17(2):107-48.
Common factors contributing to intractable pain and medical problems
with insufficient drug uptake 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)
Electromagn 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.
Clin EEG Neurosci. 2004 Jan;35(1):4-13.
Current status of the utilization of antiepileptic 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.
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.
The paper presents rationale for design of new procedures of
physiotherapy in children with expanded palate and upper lip;
morphofunctional features of prenatal and early postnatal formation of
central nervous system; a multilayer technique of magneto-laser
radiation to the projection of the anterior central gyrus and the
technique of electrostimulation by the system of the wink reflex which
can influence central mechanisms of regulation and adaptation before and
early after uranoplasty and cheiloplasty.
A new pathogenetically based non-medicamentous method for correction
of uteroplacental bloodflow disturbances has been developed on the model
of chronic placental insufficiency in rats. A single 5 min
laser-magnetic exposure on day 21 of normal pregnancy resulted in a
vasodilating effect with reduction of the peripheral resistance in the
uterine horn vessels and with improvement of their blood supply. A new
LAMA laser magneto-therapeutic device was employed. Daily 5 min sessions
of laser magnetic therapy administered to rats with chronic placental
insufficiency from pregnancy days 15-16 to 21 normalized uterine horn
contractility and resulted in positive morphofunctional changes in the
components of the uterine horns and placenta, being associated with a
noticeable improvement of fetal functions. Hence, laser magnetic therapy
may be regarded as an effective non-drug method for therapy of chronic
placental insufficiency.
Correction of immune and mediator characteristics by low-frequency magnetotherapy in children who frequently fall ill.
[Article in Russian]
[No authors listed]
Abstract
The objective of this work was to compare characteristics of clinical
condition and immune status of children with repeated respiratory
diseases of different clinical and nosological forms after standard
treatment and magnetotherapy. It was shown that magnetotherapy produces
well-apparent immunocorrective effects in children with the affected
upper and lower respiratory tracts including patients with bronchial
obstruction syndrome. Positive changes of both cellular and humoral
immunity characteristics were documented coupled to the improvement of
serum cortisol levels. Results of the study give reason to recommend
inclusion of magneotherapy in the combined treatment of children with
repeated respiratory diseases.
Electromagn Biol Med. 2007;26(4):311-3.
Utilization of extremely low frequency (ELF) magnetic fields in chronic disease; five years experience: three case reports.
Mancuso M, Ghezzi V, Di Fede G.
Institute of Biological Medicine, Milano, Italy.
Abstract
We present three examples of the use of ELF magnetic therapy, two
cases of multiple sclerosis and one of chronic pulmonary disease. In
each of the two MS cases the Seqex device was applied as an adjunct to
antioxidant medication two times a week for six weeks. Radiological and
MRI examination indicated improvement in the two MS patients and
stabilization in the patient with obstructive pulmonary disease
following merely five treatments.
Use of nonmedicamental methods of treatment assists to improve the
control of children’s bronchial asthma clinical course. Pulsed
low-frequency electromagnetic field regulates the state of central and
vegetative nervous system and improves psychological status of child.
Inphytotherapy has bronchial spasmolytic and immune correction effects.
Physiotherapy with rotating pulse magnetic field in combined therapy of chronic obstructive pulmonary disease.
[Article in Russian]
Lobanov AIu, Gilinskaia NIu, Chereiskaia NK.
Rationale and technique are proposed and clinical trial has been made
of efficacy of chronic obstructive pulmonary disease (COPD) treatment
with impacts of rotating impulse magnetic field (RIMF) from the device
Polyus-VIEM (two fields, induction 48-72 mTe, reversive rotation, 4-6 s
reversion duration, 20-30 Hz, 7-10 min exposure of each field, 7-10
procedures). The addition of RIMF in combined treatment of COPD improves
treatment and shortens its duration.
Acta Physiol Hung. 2003;90(4):327-34.
The effect of the pulsatile electromagnetic field in children suffering from bronchial asthma.
Sadlonova J, Korpas J, Salat D, Miko L, Kudlicka J.
From the bibliography it is well known that pulsatile electromagnetic
field has an anti-inflammatory and analgesic effect. It causes
vasodilatation, myorelaxation, hyper-production of connective tissue and
activation of the cell membrane. Therefore our aim was to study the
possible therapeutic effect of pulsatile electromagnetic field in
asthmatic children. Forty-two children participating in this study were
divided in two groups. The 1st group consisting of 21 children (11
females, 10 males, aged 11.8 +/- 0.4 yr) was treated by pulsatile
electromagnetic field and pharmacologically. The 2nd group served as
control, consisting also of 21 children (11 females, 10 males, aged 11.7
+/- 0.3 yr) and was treated only pharmacologically. Therapeutic effect
of the pulsatile electromagnetic field was assessed on the basis of
pulmonary tests performed by means of a Spirometer 100 Handi (Germany).
The indexes FVC, IVC, ERV, IRV, FEV1, FEV1/FVC%, MEF75,50,25, PEF, PIF
and the changes of the flow-volume loop were also registered. The
pulsatile electromagnetic field was applied by means of the device MTU
500H, Therapy System (Brno, Czech Republic) for 5 days, two times daily
for 30 minutes (magnetic induction: 3 mT, frequency: 4 Hz as recommended
by the manufacturer). The results in children of the 1st group showed
an improvement of FVC of about 70 ml, IVC of about 110 ml, FEV1 of about
80 ml, MEF75 of about 30 ml, PEF of about 480 ml, PIF of about 550 ml.
The increases of ERV, IRV and FEV1/FVC and decreases of MEF25,50 were
statistically insignificant. The results in the 2nd group were less
clear. The flow-volume loop showed a mild improvement in 14 children.
This improvement in the 2nd group was less significant. The clinical
status of children and their mood became better. We believe that the
pulsatile electro-magnetotherapy in children suffering from asthma is
effective. On the basis of our results we can recommend it as a
complementary therapy.
Bratisl Lek Listy. 2002;103(7-8):260-5.
The effect of the pulsatile electromagnetic field in patients
suffering from chronic obstructive pulmonary disease and bronchial
asthma.
Sadlonova J, Korpas J, Vrabec M, Salat D, Buchancova J, Kudlicka J.
Department of Internal Medicine, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia. sadlonova@jfmed.uniba.sk
Abstract
Pulsatile electromagnetotherapy (PETh) stimulates biological tissues
and processes; it modulates ion exchange across cell membranes and thus
regulates the tone of smooth muscles. On the basis of these effects we
hypothetized that PETh might treat COPD and bronchial asthma. We
examined 117 (61 females, 56 males) adult patients who were decided in 4
groups. The 1st consisted of 16 patients with COPD who were treated by
PETh and pharmacologically. The 2nd group (control) consisted of 24
patients with COPD who were treated only with medicaments. The 3rd group
consisted of 37 asthmatics, treated by PETh and medicaments. The 4th
group (control) consisted of 40 asthmatics treated only with
medicaments. The effectiveness of PETh was assessed by lung function
tests, which were performed using a Spirometer 100 Handi (Germany). We
measured FVCex, FEV1, percentage of FEV1/FVCex, MEF25, 50, 75, PEF and
registered the flow-volume loops. PETh was applied by apparatus MTU 500H
(Therapy System, Czech Republic). It was administered 10 doses; once
daily for 20 min, with a frequency of 4.5 Hz and a magnetic induction 3
T. The initial 3 doses were about 25% lower then the later doses. PETh
was very effective in patients with COPD. The measured indexes improved
about 200-660 ml or ml x s(-1), except FVC. PETh was less effective in
asthmatics. Most indices improved without statistical significance,
about 50-620 ml or ml x s(-1). The indices of FEV1/FVC and MEF25
deteriorated. The changes in controls without PETh were very small.
(Tab. 2, Fig. 1, Ref. 19.)
Bratisl Lek Listy. 2000;101(2):71-7.
The sensitivity of tussiphonography for assessing the effectiveness of treatment.
Korpas J, Salat D, Sadlonova J, Vrabec M, Kudlicka J.
Department of Pathophysiology, Jessenius Medical School Martin, Slovakia.
Our previous studies have demonstrated that tussiphonogram is
suitable not only for the detection of pathological condition in the
respiratory tract but also for treatment effectiveness assessment. The
purpose of this study was to evaluate the possibilities of
tussiphonography in detection of already little pathological changes in
the airways and lungs. Therefore the changes of voluntary cough sound
indexes were compared with pulmonary function tests in selected group of
asthmatics before and after a pulsatile electromagnetic therapy in
which the effect of therapy on pulmonary function tests was minimal.
After magnetotherapy in 18 patients with increased expiratory forced
lung capacity by 7.3% and increased peak inspiratory flow by 31.7% in
average the voluntary cough sound intensity decreased by 37.8%, the
sound duration shortened by 11% and the sound pattern showed the
tendency to normalization. The improvement of mentioned cough indexes
was absent in 17 patients who were treated by magnetotherapy too, but at
the same time suffered from respiratory viral infection and in 22
patients treated only with climatotherapy and antiasthmatics. Changes of
flow-volume loops in patients were not in the close relation to other
followed indices. The correlation analysis showed a functional
connection in relative differences of cough sound indices and some
pulmonary function tests. The results confirmed the suitability of
tussiphonography to indicate even mild pathological changes in
respiratory tract. (Fig. 4, Ref. 21.)
The use of a pulsed traveling magnetic field in patients with chronic obstructive bronchitis.
[Article in Russian]
Achkasov VV.
A course treatment with pulse running magnetic field generated by
ALIMP-1 unit of patients with chronic obstructive bronchitis provides a
subjective response, higher exercise tolerance due to improved function
of the external respiration. The inductors-solenoids are fixed
tangentially on the chest by means of the jacket.
The rehabilitative treatment of children with bronchial asthma
[Article in Russian]
Alymkulov DA, To?chieva FM, Saralinova GM, Le?kina LF.
Abstract
Staged regimen of decimetric wave electromagnetic therapy and
microclimate of high altitude salt mines were used in sanatorium
treatment of children with bronchial asthma. Pretreatment with the above
magnetic field induced positive changes in the reflex-segmental zone
which reflected in better adaptation to the high altitude climate. The
latter promoted beneficial rearrangement of respiratory function and
cardiovascular system.
A validation for the combined transcerebral exposure to a UHF
electrical field and to decimeter waves in the area of the splenic
projection in bronchial asthma.
[Article in Russian]
Maliavin AG, Rychkova MA, Nikoda NV.
Abstract
Thirty patients with bronchial asthma of moderate severity in
unstable remission were treated with transcerebral UHF electric field
and decimeter waves on the spleen region. Clinical and laboratory
postexposure findings provided evidence in favour of the regimens used.
Tolerance of the procedures, comparative efficacy regarding the
clinico-pathogenetic variant, probable mechanisms of therapeutic action
are discussed.
Voen Med Zh, 3, 1989, . 35-36.
The Efficacy of the Use of Low-Frequency Fields in Chronic Bronchitis
Iurlov VM, Eksareva TA, Dolodarenko VF.
Low frequency electromagnetic field therapy and treatment with
pulsed electromagnetic fields were effective in patients suffering from
chronic bronchitis in combined treatment with drug therapies in this
double-blind, placebo-controlled study. Magnetic field therapy consisted
of a total of 15, 15 to 20 minute daily exposures.
Vrach Delo. 1989 Mar;(3):55-6.
The use of microwave resonance therapy on patients with chronic nonspecific lung diseases.
[Article in Russian]
Dziublik AA, Mukhin AA, Ugarov BN, Chechel’ LV.
Abstract
The authors investigated the efficacy of using electromagnetic
radiation of extremely high frequency and low intensity (bioresonance
therapy) in the treatment of patients with some forms of chronic
unspecific pulmonary diseases. The object of the investigation were
patients with bronchial asthma, chronic nonobstructive and obstructive
bronchitis. The treatment efficacy was 80%.
Pulsed electromagnetic field (PEMF)
is used to treat bone and joint disorders for over 30 years. Recent
studies demonstrate a significant effect of PEMF on bone and cartilage
proliferation, differentiation, synthesis of extracellular matrix (ECM)
and production of growth factors. The aim of this study is to assess if
PEMF of low frequency, ultralow field strength and short time exposure
have beneficial effects on in-vitro cultured human chondrocytes.
Materials and Methods:
Primary human chondrocytes cultures
were established using articular cartilage obtained from knee joint
during joint replacement surgery. Post characterization, the cells were
exposed to PEMF at frequencies ranging from 0.1 to 10 Hz and field
intensities ranging from 0.65 to 1.95 ?T for 60 min/day for 3
consecutive days to analyze the viability, ECM component synthesis,
proliferation and morphology related changes post exposure. Association
between exposure doses and cellular effects were analyzed with paired’t’
test.
Results:
In-vitro PEMF exposure of
0.1 Hz frequency, 1.95 ?T and duration of 60 min/day for 3 consecutive
days produced the most favorable response on chondrocytes viability (P < 0.001), ECM component production (P<
0.001) and multiplication. Exposure of identical chondrocyte cultures
to PEMFs of 0.65 ?T field intensity at 1 Hz frequency resulted in less
significant response. Exposure to 1.3 ?T PEMFs at 10 Hz frequency does
not show any significant effects in different analytical parameters.
Conclusions:
Short duration PEMF exposure may represent a new therapy for patients with Osteoarthritis (OA).Keywords: Human chondrocytes, osteoarthritis, pulsed electromagnetic field
MeSh terms: Osteoarthritis, cartilage, articular, chondrocytes, electromagnetic fields
Introduction
Pulsed electromagnetic field (PEMF) has been used to treat bone and joint disorders for over 30 years.1Clinical use of PEMF preceded systematic research in its utility for bone and joint healing.2
Later studies identified that PEMF is capable of producing significant
cellular changes in bone and cartilage cells by proliferation,
differentiation, synthesis of extracellular matrix (ECM) and production
of growth factors.3,4,5,7,8,9,10
A systematic review based on 3 clinical studies which assessed effect
of PEMF therapy for osteoarthritis (OA) of knee, incorporating factors
like pain, physical function, patient assessment, joint imaging, health
related quality of life and physician global assessment indicates that
electrical stimulation therapy may be useful in OA of knee, but stresses
the need for confirmation in future studies.11 Proteoglycan (PG) loss occurs in joint cartilage in OA and PEMF therapy has been shown to induce PG synthesis in-vivoand in-vitro.12
PEMF has also demonstrated to have positive effect on cellular
proliferation and DNA synthesis through opening of voltage sensitive
calcium channels.13 Animal models have shown that PEMF therapy retards progression of OA.14,15
Most studies employing PEMF have used
frequencies of 6- 75 Hz and field strengths of 0.4- 2.3 milli Tesla
(mT). We desired to enquire if low frequency (0.1- 10 Hz), low field
strength of 0.65- 1.95 µT and short duration exposure (60 min/day) of
PEMF results in favorable effects on cultured human chondrocytes
(synthesis of ECM; cell viability, proliferation and morphology).
Further need for the study is to arrive at a minimal PEMF exposure
protocol that is expected to decrease the concern related to unfavorable
cellular changes and chromosomal aberrations that may result with high
dose PEMF exposure.16
Materials and Methods
Isolation and characterization of chondrocytes
Articular cartilage samples were
obtained from knee joint during joint replacement surgery after
obtaining informed consent from patients. The study protocol was
approved by Institutional Ethics Committee. Cartilage tissue over the
nonweight bearing portion of the joint was removed and minced in
Dulbecco’s modified eagle medium (DMEM) (Biogene technologies, India)
supplemented with 10% FBS (Biogene technologies, India) and 1 ml
Pen-strep (10000 units of penicillin and 10 mg of streptomycin,
Invitrogen, India). Following this, the tissue was transferred into a
conical flask and initially digested with pronase (1 mg/ml) (Biogene
technologies, India) for 60 min, followed by type II collagenase (1
mg/1ml) (Invitrogen) for 16- 18 hours at 37°C. The following day,
cellular debris and undigested tissue were removed and cells were
separated using a 100 micron cell strainer. Isolated cells were seeded
into 25 cm 2 culture flasks (TPP, India) with DMEM complete medium and maintained at 37°C with 5% CO2
levels. The cells were subcultured on attainment of 80% confluency. The
attached cells were characterized by chondrocyte specific anti-Sox 9
transcription factor antibody staining (Abcam, India.). Chondrocytes
that failed to form monolayer culture were not processed further. Post
characterization, 4 × 105 cells were seeded in each flask and used for PEMF exposure after first passage.
Pulsed electromagnetic field exposure
The PEMF coil system fashioned for
exposure is a four member coil frames, two larger (inner) and two
smaller (outer) coil frames. The coils are mounted coaxially and in a
co-planar fashion to form an enclosure, where it delivers currents in
milliamps at desired waveforms, varying frequencies and magnetic field
strength (Madras Institute of Magnetobiology, Chennai, India). This
system designed according to the parametrical equation of Fansleau and
Brauenbeck and a modified version of the Helmhotz coil. A box is housed
inside the coil in which a 100 W bulb with regulator was used to
maintain the temperature at 37°C and water to maintain humidity. Instead
of 5% CO2, 20 mM HEPES was used as a buffering system. The
chondrocytes were exposed to PEMF while monitoring field strength,
frequency and temperature. The control (unexposed) cells were placed in
the same environment and temperature but not exposed to PEMF.
Pulsed electromagnetic field treatment
The chondrocytes were seeded in 25 cm 2 culture flasks at concentrations of 6.5 × 105
cells/ml after 20 h being plated the cells were washed with phosphate
buffer saline (PBS), and given fresh medium and exposed to PEMF for the
first three daily trials; media was not changed from this point onwards.
PEMF at a frequency of 0.1, 1 and 10 Hz were applied with flux
densities of 0.65, 1.3 and 1.95 µT (peak-to-peak) for 60 min/day for 3
consecutive days. Whereas exposure to PEMFs at a repetition rate of 0.1
and 1 Hz with 1.95 and 0.65 µT caused a significant increase in
chondrocyte viability that was dependent on PEMF amplitude, PEMFs
applied at a repetition rate of 10 Hz and 1.3 µT did not produce any
noticeable effects over cell viability and were not dealt with further
in this manuscript. To test for effects of different exposure durations,
cells were exposed to PEMFs of 1.95 and 0.65 µT magnitude and at
frequency of 0.1 and 1 Hz for 60 min/day for 3 days. Cells were analyzed
on third day for further experimental studies.
Cell viability assessment
Chondrocytes were cultured in 96 well plates at a density of 5 × 103
cells per well and exposed to PEMF in accordance to the exposure
protocol mentioned. Twenty microliter of 0.5%
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Invitrogen) in phosphate buffered saline was added to each well after
removal of medium and cells were incubated for 3 h at 37°C. Post
incubation, 150 µl dimethyl sulfoxide (Hi-media, India) was added to
each well and absorbance values (optical density value) were noted at
570 nm and 695 nm in spectrophotometer.17
Quantitative measurement of extracellular matrix proteoglycan and glycosaminoglycan synthesis
Chondrocytes were cultured in 48 well plates at densities of 104
cells per well and exposed to PEMF in accordance to the exposure
protocol mentioned. Postexposure, glycosaminoglycan (GAG) synthesis was
quantified by the dimethyl methylene blue (DMMB) assay. The DMMB reagent
(Sigma, India) was prepared as detailed by Panin et al.18 and 200 µL was added to each well after removal of culture medium. Subsequently, absorbance values at 525 nm were noted.
Analysis of cell cycle by flow cytometry
Chondrocytes were cultured in 25 cm 2
culture flasks and exposed to PEMFs as mentioned earlier. After
exposure, the cells were trypsinized, converted to single cell
suspension in PBS and subjected to flow cytometery (FACS calibur, Becton
Dickinson, Germany) according to the manufacturer’s instruction
(Invitrogen, India) as follows: The suspension was spun at 1000 rpm for
10 min and the cell pellet was fixed in 70% ice cold ethanol at 4°C
overnight. The cells were washed with PBS, treated with 500 µl RNAse A
(40 µg/ml) (Sigma, India.) for 30 min at 37°C and stained with 500 µl
propidium iodide (40 µg/ml) for 15 min incubation at room temperature.
Postincubation, cell distributions at distinct phases of the cell cycle
were analyzed by flow cytometery.
Analysis of cell architecture and morphology
Cell architecture and morphology
were analyzed by staining of actin filaments in chondrocytes.
Chondrocytes were cultured on cover slips in 6 well culture plates and
exposed to PEMFs as described earlier. Processing of cells was done
according to the manufacturer’s instructions (Invitrogen, India.).
Briefly, the cells were fixed in 3.7% formaldehyde solution for 10 min
after washing the slide with PBS and permeabilized in 0.1% Triton X-100
for 5 min. After washing with PBS, the cells were stained with 0.05
mg/ml Phalloidin solution at room temperature for 20-30 min, followed by
counterstaining with 300 µl Propidium Iodide (500 nM). The coverslips
were then rinsed in PBS, placed on a glass slide and cellular
architecture and stress fiber formation was qualitatively analyzed by
fluorescent confocal microscopy (LSM 510 META, Carl Zeiss, Germany).
Statistical analysis
Discrete variables were expressed
as number (%) and continuous variables expressed as mean ± Standard
Deviation. Association between field strengths (0.65, 1.3, and 1.95 µT)
in variable frequencies (0.1, 1, and 10 Hz) and cellular effects (cell
viability and ECM production,) was analyzed with paired ‘t’ test. A P
< 0.05 was considered as statistically significant. Analysis was
done with Statistical Package for the social sciences (SPSS) software
version 21.0. This software was released in 2012 and used to solve
business and research problems by means of ad-hoc analysis, hypothesis
testing and predictive analysis.
Results
Isolation of chondrocytes
Healthy chondrocytes were observed in
cultures by 3 days and these monolayers were 80% confluent by a week.
The chondrocytes were spherical prior to attachment and later appeared
polygonal in shape [Figure 1].
Figure 1
Primary human chondrocytes displaying typical polygonal conformation after attachment
Cell viability assessment
Viability of chondrocytes after PEMF
exposure was quantified by the MTT assay to ascertain the effects PEMFs
on chondrocytes which were exposed to PEMFs of field intensities between
1.95 and 0.65 µT at frequencies of 0.1 and 1 Hz for 60 min/day for 3
days. Following the third day exposure, samples were treated with MTT to
quantify the cell viability and compared to control (unexposed)
cultures. A highly significant viability of chondrocyte was observed in
following field intensities and frequencies (1.95 µT-0.1Hz [P <
0.001], 1.95 µT -1Hz [P < 0.001] and 0.65 µT-0.1 Hz [P < 0.001]).
Moderate favourable response was observed in other field intensities and
frequencies [Table 1].
After 3 days of 60 min daily exposure to 1.95 µT PEMFs at a frequency
of 0.1 Hz, the total number of cells in the culture increased,
indicating heightened viability in response to PEMFs.
Table 1
MTT assay for detection of viable cells after exposure to PEMFs for 3 consecutive days
Quantitative measurement of proteoglycan glycosaminoglycan synthesis
Our spectrophotometric quantification of
the ECM components such as GAG and PGs were assayed with identical PEMF
parameters (field strengths, frequencies, and days of exposure and
duration of exposure) as those used for MTT assay of cell viability with
identical results. As compared with previously observed results,
favorable responses to the production of ECM components were seen in
following field strengths and frequencies (1.95 µT-0.1 Hz [P <
0.001], 1.95 µT -1 Hz [P < 0.001], 0.65 µT-0.1 Hz [P < 0.001],
0.65 µT-1 Hz [P < 0.001], 1.95 µT-10 Hz [P = 0.001] and 0.65 µT-10 Hz
[P = 0.001]. Moderate favorable response was observed in other field
intensities and frequencies [Table 2].
Our spectrophotometric quantification thus corroborates and strengthen
our MTT assay results, indicating that exposure with 1.95 µT field
intensity at frequency of 0.1 Hz for 60 min/day was most effective in
production of GAG and PG of chondrocytes.
Table 2
DMMB assay for detection of ECM components after exposure to PEMFs for 3 consecutive days
Cell cycle analysis
Cells were analyzed to assess their
distribution at different phases of the cell cycle by flow cytometry
after staining of DNA with propidium iodide and recording of 106
events for each exposure parameter. The cells distribution in four
distinct phases could be recognized in a proliferating cell population: G1, S (DNA synthesis Phase), G2 and M (Mitosis). As both G2
and M phase have an identical DNA content, they could not be
discriminated based on their differences in their DNA content. The
percentage values were assigned to each population and also dot plot
[Figure ?[Figure2a2a and ?andb]b] and histogram [Figure ?[Figure2c2c and ?andd]d]
were used to denote the distribution of cells in distinct phases. PEMF
at different field strengths and frequencies was found to promote cell
cycle progression from the G1 phase to the S and G2-M phases.
Cells present in G2-M phase are in dividing state and show increased
rate of proliferation. A shift to top of cell population (G2-M) in dot plot shows great proliferation [Figure ?[Figure2a2a and ?andb].b].
Based on the percentage of cells distribution in G2-M phase,
proliferation effect was determined at different exposure parameters.
Histogram indicates, cells exposed at 0.1 Hz frequency with 1.95 µT of
PEMFs show 20.24% of their significant presence in G2-M phase compared
to other filed strengths such as 0.65 (18.9%) and 1.3 µT (17.54%) [Figure 2c].
The cells exposed to 1.95 µT of PEMFs at 0.1 Hz frequency shows 20.24%
of their significant presence in G2-M phase compared to other
frequencies such as 1 Hz (19.46%) and 10 Hz (17.83%) [Figure 2d].
Figure 2
Cell cycle analysis by flow cytometer to determine the
proliferative effect of chondrocytes in distinct cell cycle phases.
Percentage of chondrocytes distribution in G2-M phase indicates cell
proliferation effects as it has all mitotic cells. Significant …
Analysis of cell architecture and morphology
Actin filaments of the cytoplasm stained
by Phalloidin and nucleus was counterstained with propidium iodide
observed by confocal fluorescent microscopy showed a significant
difference in morphological structure and formation of stress fibers
between exposed chondrocytes at varying frequencies (0.1, 1 and, 10 Hz)
with specific field strength 1.95 µT and unexposed cells. Stress fiber
formation was increased in chondrocytes exposed at frequency of 0.1 Hz
with 1.95 µT compared to unexposed [Figure 3]. Stress fiber formation indicates that the cells stability, strength and their healthy attachment.
Figure 3
Human chondrocytes morphological structure was studied by staining
with phalloidin and propidium iodide for visualizing stress fibers
(green) and nuclear staining (red). (a) No stress fiber formation in
chondrocytes unexposed to pulsed electromagnetic …
Discussion
Our study observed that short term in-vitro
chondrocyte exposure to PEMFs at frequency of 0.1 Hz and field strength
of 1.95 µT for 60 min/day for 3 consecutive days have shown highly
significant effects in different experimental parameters such as cell
viability, ECM production, cell cycle progression and stress fiber
formation. By contrast, exposure of identical chondrocyte cultures to
PEMFs of 0.65 µT field intensity at 1 Hz frequency resulted in less
significant levels of different parameters. On the other hand, exposure
to 1.3 µT PEMFs at 10 Hz frequency does not shown any significant
effects in different analytical parameters. These findings, apart from
observing benefits of certain range of field strengths, also bring to
light the ability of PEMF to inhibit cellular effects when used at
certain field strengths and frequencies, a fact which has been observed
earlier.
In our study design, we limited our experiments to within 3
days of exposure to PEMF to stay within the realm of better clinical
applicability. For our analysis, we have chosen 3 days as an appropriate
end point as it avoided the over confluence of chondrocytes and also it
would minimize the contact inhibition that can induce changes in
biochemical status and cause dedifferentiation. As the number of days of
exposure to PEMFs increases, it may enhance the proliferative effects
to the chondrocytes. The design of longer day exposure to PEMFs will be
taken into future study. PEMF parameters used in this study such as
frequency, field strength and duration of exposure could translate into
the clinical application and will be innocuous to the target tissue and
their surrounding tissues which are exposed to PEMF during clinical
therapy.
Our study observed correlation between critical cell
characteristics (cell viability and promotion in cell multiplication) of
exposed samples and induction of extracellular components which include
GAG and PG. This raises the question on the validity of using changes
in ECM components as a marker of chondrocyte healing in studies using in-vitro models.
The earliest in-vitro study with bovine articular
chondrocytes exposed using Helmholtz coils found no significant effect
of PEMF on ECM component synthesis.19
Sakai and colleagues studied the effect of 0.4 mT field strength at 6.4
Hz delivered over a period of 5 days on rabbit growth cartilage and
human articular cartilage and observed that PEMF stimulated cell
proliferation and GAG synthesis in growth cartilage cells but resulted
in only cell proliferation with no increase in GAG content in articular
cartilage cells.20
The latter finding of our observation on extracellular components (GAG
and PG) synthesis is comparable with earlier studies observation.
De Mattei et al. exposed chondrocytes from
healthy patients to PEMF to varying duration of exposure (1- 18 h and 1-
6 days) using a field strength of 2.3 mT at 75 Hz. The study observed
that short duration of exposure (1 and 6 h) did not result in increased
DNA synthesis, while longer duration of exposure (9 and 18 h) increased
DNA synthesis.21 Chang et al.,
exposed porcine chondrocytes to a field of 1.8- 3 mT at a frequency of
75 Hz for 2 h/day for 3 weeks and observed that long term 3 weeks PEMF
exposure was beneficial over the short term 1 week exposure.22
However, our observations contradict these findings and reports the
better efficacy of even short term PEMF exposures. Though our study
observed the efficacy of a daily PEMF exposure of 60 min for only 3
days, benefits of exposure should be expected to enhance with daily
exposures exceeding 3 days. We could not observe the benefits beyond day
3, since confluent chondrocyte cultures de-differentiated due to
contact inhibition beyond this period in two-dimensional cultures.
Our observation on promotion of cell cycle from G1 phase
to G2-M phase with certain field strengths is comparable with the
findings of Nicolin et al. which observed similar results with field strength of 2 mT at 75 Hz with an exposure time of 4 h or 12 h/day.23
The striking observation of similar findings in our study with much
lower field strength for exposure duration of 60 min has better clinical
applicability.
A recent in-vivo animal study exposed rabbits
with experimental osteochondral defect to PEMF for a period of 60
min/day for 6 weeks and observed a better total histological score in
the study group to conclude that PEMF is beneficial for hyaline
cartilage formation.24 The only in-vitro
study on human chondrocytes harvested from OA knee reports no effect on
PG production using field strength of 2mT at 50 Hz for 14 days.25 However both studies did not evaluate fine cellular effects (cell viability and cell cycle promotion).
Based on our data, the study informs that the future in-vitro
studies on the topic should probably use exposure duration not more
than 60 min/day but we can increase more number of days to PEMFs at 0.1
and 1 Hz frequencies and 1.95 and 0.65 µT field intensities. However,
future studies should aim to utilize collagen matrix in
three-dimensional (3D) cultures and focus more on exposure for more
number of days to overcome the limitation of dedifferentiation and
contact inhibition due to over confluent in 3D model and also focus on
the effect of PEMF on chondrocyte cytoskeleton (observed as stress
fibers in Phalloidin staining). It would of interest to investigate the
strength of the chondrocyte cytoskeleton between exposed and control
cells. Though it may be argued that occurrence of stress fiber formation
observed with PEMF exposure is a result of heating effect due to
Helmholtz system, the low dose of PEMF is less likely to have produced a
heating effect which may happen with higher doses.
To conclude, our study observed that short
duration (60 min/day) low frequency (0.1 Hz) low field strength (1.95
µT) PEMFs have beneficial effects on chondrocyte viability, ECM
production, multiplication and probably cytoskeleton even for a short
period of 3 days. Short duration PEMF exposure for patients with OA has
the potential to produce favorable clinical effects. However, the
results of the study have to be confirmed with a methodology
incorporating assessment of both mass and strength of PEMF exposed
chondrocytes.
Financial support and sponsorship
Defence Institute of Physiology and
Allied Sciences (DIPAS), Defence Research and Development Organisation
(DRDO), Ministry of Defence, Government of India.
Conflicts of interest
There are no conflicts of interest.
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Alimentary hyperlipemia of rabbits is affected by exposure to low-intensity pulsed magnetic fields.
Luo E, Shen G, Xie K, Wu X, Xu Q, Lu L, Jing X.
Department of Military Medical Equipment & Metrology, Faculty of
Biomedical Engineering, The Fourth Military Medical University, Xi’an,
China. tocooper@hotmail.com
Abstract
An experimental study was carried out in rabbits to investigate the
effects of exposing rabbits to low-intensity pulsed magnetic fields
(PMFs) on alimentary hyperlipemia. Thirty female white big ear rabbits
were randomly divided into three groups. The normal group was fed with a
standard chow diet and the other two groups (hyperlipid and magnetic)
were fed with the chow diet supplemented with cholesterol, yolk powder
and lard. The magnetic group was exposed to 15 Hz pulsed magnetic
fields. After 8 weeks, levels of blood lipid and indices of hemorheology
were examined. In addition, histomorphologic changes of hepatic and
myocardial tissues were compared across the groups respectively.
Compared with the hyperlipid group, hemorheology indices of the magnetic
group reduced significantly from 12.80% to 38.05% (P < 0.01)
indicating lower blood viscosity. Similarly, compared with the
hyperlipid group, the levels of total cholesterol and triglycerides in
the magnetic group decreased 40.52% and 52.42% (P < 0.01). On the
contrary, high density lipoprotein (HDL) value obviously increased
66.67% (P < 0.01). Furthermore, compared with the control group, the
values of triglycerides and HDL of the magnetic group did not show
statistical differences (P > 0.05). The deposit of fatty material on
the inner lining of thoracic aorta wall of the magnetic group was
significantly lighter than that of the hyperlipid group. Numerous
aggregation of lipoids emerged among myocardial myofibrils in the
hyperlipid group, while no notable change was found in both the magnetic
and control group. The results indicate that low-intensity PMFs could
be helpful for the treatment of alimentary hyperlipemia.
Lipids Health Dis. 2007 Nov 16;6:31.
Effects of whole body exposure to extremely low frequency
electromagnetic fields (ELF-EMF) on serum and liver lipid levels, in the
rat.
Torres-Duran PV, Ferreira-Hermosillo A, Juarez-Oropeza MA, Elias-Viñas D, Verdugo-Diaz L.
Department of Physiology, School of Medicine, UNAM, P.O. Box 70250, Mexico, D.F. 04510, Mexico. pavito@correo.unam.mx
Abstract
BACKGROUND: [corrected] The effects of extremely low-frequency
electromagnetic fields (ELF-EMF) on the blood serum and liver lipid
concentrations of male Wistar rats were assessed.
METHODS: Animals were exposed to a single stimulation (2 h) of
ELF-EMF (60 Hz, 2.4 mT) or sham-stimulated and thereafter sacrificed at
different times (24, 48 or 96 h after beginning the exposure).
RESULTS: Blood lipids showed, at 48 h stimulated animals, a
significant increase of cholesterol associated to high density
lipoproteins (HDL-C) than those observed at any other studied time. Free
fatty acid serum presented at 24 h significant increases in comparison
with control group. The other serum lipids, triacylglycerols and total
cholesterol did not show differences between groups, at any time
evaluated. No statistical differences were shown on total lipids of the
liver but total cholesterol was elevated at 24 h with a significant
decrease at 96 h (p = 0.026). The ELF-EMF stimulation increased the
liver content of lipoperoxides at 24 h.
CONCLUSION: Single exposures to ELF-EMF increases the serum values of
HDL-C, the liver content of lipoperoxides and decreases total
cholesterol of the liver. The mechanisms for the effects of ELF-EMF on
lipid metabolism are not well understand yet, but could be associated to
the nitric oxide synthase EMF-stimulation.
Panminerva Med. 1998 Dec;40(4):276-9
Effect of pulsed magnetic fields on triglyceride and cholesterol levels in plasma of rats.
Bellossi A, Pouvreau-Quilien V, Rocher C, Ruelloux M.
Laboratoire de Biophysique Faculte de Medecine, Rennes, France.
BACKGROUND: Liver is a crucial organ in metabolism. For instance liver is the main source of circulating lipoproteins.
METHODS: In this paper cholesterol and triglyceride plasma levels
were measured in male rats previously exposed to pulsed magnetic fields
(PMF) used in therapy. Rats underwent a one-hour exposure to a 6 mT 12
Hz PMF.
RESULTS: Twenty-four hours after the end of the exposure to the PMF
the rats’ livers were heavier, cholesterol and triglyceride plasma
levels decreased. All these variations were significantly different
according to a variance ratio test as was a rebound in triglyceride
level 48 hours after the end of the exposure. Normal values were
observed 48 and 96 hours after the end of exposure respectively for
cholesterol and triglycerides.
CONCLUSIONS: These alterations may be due to a reversible
accumulation of either triglycerides or of their precursors in liver
following acute exposure to a 12 Hz PMF.
Biofizika. 1985 Mar-Apr;30(2):313-6.
Correction of lipid metabolism in rats with limited mobility by an alternating magnetic field of infra-low frequency.
[Article in Russian]
Temur’iants NA, Evstaf’eva EV, Makeev VB.
Abstract
The effect of changing magnetic field at the frequency of 8 Hz and
intensity at 4.1 A/M on rats lipid metabolism state in the exposition of
continuous hypokinesia was investigated. It was found that changing
magnetic field of such parameters greatly restricted the development of
hyperlipidemia of rats with a low level of mobile activity.
Analysis of parameters of reproductive tract mucosal immunity in
women with chlamydial infection before and after local
magnetolaserotherapy.
[Article in Russian]
Gizinger OA, Dolgushin II, Letiaeva OI.
Abstract
The objective of the present study was to evaluate the influence of
combined treatment with low-intensity laser radiation and magnetic
field on neutrophil function in women presenting with Chlamydial
infection. Dysfunction of neutrophil granulocytes in these patients was
manifest in the first place as the decreased number of phagocytes and
the low rate of phagocytosis. It was shown that the concentration of
active oxygen species in neutrophils in the patients with Chlamydial
infection was significantly smaller than in healthy women. The
concurrent application of low-intensity laser radiation and a magnetic
field not only stimulated phagocytosis but also increased intracellular
production of active oxygen species especially under in vitro
conditions. It is concluded that combined treatment with low-intensity
laser radiation and magnetic field has beneficial effect on the
parameters of mucosal immunity in the reproductive tract of women with
Chlamydial infection.
Best Pract Res Clin Rheumatol. 2007 Feb;21(1):93-108.
Strategies for prevention and management of musculoskeletal conditions. Neck pain.
Jensen I, Harms-Ringdahl K.
Department of Clinical Neuroscience, Section of Personal Injury
Prevention, Karolinska Institutet, and Department of Physical
Therapy, Karolinska University Hospital, Stockholm, Sweden. irene.jensen@ki.se
The aim of this article was to summarise the existing
evidence concerning interventions for non-specific neck pain.
Neck-and-shoulder pain is commonly experienced by both adolescents
and adults. Although the prevalence appears to vary among different
nations, the situation is essentially the same, at least in the
industrialised nations. Explanations for the wide variation in
incidence and prevalence include various methodological issues. Back
and neck disorders represent one of the most common causes for both
short- and long-term sick leave and disability pension. Evidenced
risk factors for the onset and maintenance of non-specific neck and
back pain include both individual and work-related psychosocial
factors. Based on the existing evidence different forms of exercise
can be strongly recommended for at-risk populations, as well as for
the acute and chronic non-specific neck pain patient. Furthermore,
for symptom relief this condition can be treated with transcutaneous
electric nerve stimulation, low level laser therapy, pulse
electromagnetic treatment or radiofrequency denervation.
Pain. 2007 Jan;127(1-2):173-82. Epub 2006 Oct 18.
Pulsed radiofrequency adjacent to the cervical dorsal
root ganglion in chronic cervical radicular pain: a double blind sham
controlled randomized clinical trial.
Van Zundert J, Patijn J, Kessels A, Lamé I, van Suijlekom H, van Kleef M.
Department of Anesthesiology, Pain Management and Research Centre, University Hospital Maastricht, The Netherlands. janvanzundert@pandora.be
Abstract
Cervical radicular pain affects approximately 1 on 1000
adults per year. Although many treatment modalities are described in the
literature, the available evidence for efficacy is not sufficient to
allow definitive conclusions on the optimal therapy to be made. The
effect of pulsed radiofrequency treatment for this type of patients was
evaluated in a prospective audit that showed satisfactory pain relief
for a mean period of 9.2 months, justifying a randomized sham controlled
trial. Twenty-three patients, out of 256 screened, met the inclusion
criteria and were randomly assigned in a double blind fashion to receive
either pulsed radiofrequency or sham intervention. The evaluation was
done by an independent observer. At 3 months the pulsed radiofrequency
group showed a significantly better outcome with regard to the global
perceived effect (>50% improvement) and visual analogue scale (20
point pain reduction). The quality of life scales also showed a positive
trend in favor of the pulsed radiofrequency group, but significance was
only reached in the SF-36 domain vitality at 3 months. The need for
pain medication was significantly reduced in the pulsed radiofrequency
group after six months. No complications were observed during the study
period. These study results are in agreement with the findings of our
previous clinical audit that pulsed radiofrequency treatment of the
cervical dorsal root ganglion may provide pain relief for a limited
number of carefully selected patients with chronic cervical radicular
pain as assessed by clinical and neurological examination.
Rheumatol Int. 2005 Jun 29; [Epub ahead of print]
The effect of pulsed electromagnetic fields in the
treatment of cervical osteoarthritis: a randomized, double-blind,
sham-controlled trial.
Sutbeyaz ST, Sezer N, Koseoglu BF.
Ankara Physical Medicine and Rehabilitation Education and Research Hospital, Turk ocagi S No: 3 Sihhiye, Ankara, Turkey.
The purpose of this study was to evaluate the effect of
electromagnetic field therapy (PEMF) on pain, range of motion (ROM) and
functional status in patients with cervical osteoarthritis (COA).
Thirty-four patients with COA were included in a randomized,
double-blind study. PEMF was administrated to the whole body using a mat
1.8×0.6 m in size. During the treatment, the patients lay on the mat
for 30 min per session, twice a day for 3 weeks. Pain levels in the PEMF
group decreased significantly after therapy (p<0.001), but no change
was observed in the placebo group. The active ROM, paravertebral muscle
spasm and neck pain and disability scale (NPDS) scores improved
significantly after PEMF therapy (p<0.001) but no change was observed
in the sham group. The results of this study are promising, in that
PEMF treatment may offer a potential therapeutic adjunct to current COA
therapies in the future.
J Med Eng Technol. 2002 Nov-Dec;26(6):253-8.
Comparison between the analgesic and therapeutic
effects of a musically modulated electromagnetic field (TAMMEF) and
those of a 100 Hz electromagnetic field: blind experiment on patients
suffering from cervical spondylosis or cervical periarthritis.
Rigato M, Battisti E, Fortunato M, Giordano N.
Department of Physics, Section of Medical Physics University of Sienna, Italy. rigato@unisi.it
The analgesic-therapeutic efficacy and tolerability of a
low-frequency electromagnetic field (ELF), modulated at a frequency of
100 Hz with a sinusoidal waveform and mean induction of a few gauss, has
been demonstrated by the authors in numerous previous studies of
various hyperalgic pathologies, particularly of the locomotor apparatus.
In the present study, the authors tested a new type of all-inclusive
field, denoted TAMMEF, whose parameters (frequency, intensity, waveform)
are modified in time, randomly varying within the respective ranges, so
that all the possible codes can occur during a single application. For
the comparison, 150 subjects (118 women and 32 men, between 37 and 66
years of age) were enrolled. They were affected by cervical spondylosis
(101 cases) or shoulder periarthritis (49 cases). Unbeknownst to them,
they were randomly divided into three groups of 50 subjects. One group
was exposed to the new TAMMEF, another group to the usual ELF, and the
third group to simulated treatment. The results show that the effects of
the new TAMMEF therapy are equivalent to those obtained with the ELF.
Cochrane Database Syst Rev. 2002;(1):CD003523.
Electromagnetic fields for the treatment of osteoarthritis.
Hulme J, Robinson V, DeBie R, Wells G, Judd M, Tugwell P.
Cochrane Collaborating Center, Center for Global Health, Institute of
Population Health – University of Ottawa, 1 Stewart Street, Ottawa,
Ontario, Canada, K1N 6N5. jhulme@uottawa.ca
BACKGROUND: As the focus for osteoarthritis (OA)
treatment shifts away from drug therapy, we consider the effectiveness
of pulsed electric stimulation which is proven to stimulate cartilage
growth on the cellular level.
OBJECTIVES: 1)To assess the effectiveness of pulsed
electric stimulation for the treatment of osteoarthritis (OA). 2) To
assess the most effective and efficient method of applying an
electromagnetic field, through pulsed electromagnetic fields (PEMF) or
electric stimulation, as well as the consideration of length of
treatment, dosage, and the frequency of the applications.
SEARCH STRATEGY: We searched PREMEDLINE, MEDLINE,
HealthSTAR, CINAHL, PEDro, and the Cochrane Controlled Trials Register
(CCTR) up to and including 2001. This included searches through the
coordinating offices of the trials registries of the Cochrane Field of
Physical and Related Therapies and the Cochrane Musculoskeletal Group
for further published and unpublished articles. The electronic search
was complemented by hand searches and experts in the area.
SELECTION CRITERIA: Randomized controlled trials and
controlled clinical trials that compared PEMF or direct electric
stimulation against placebo in patients with OA.
DATA COLLECTION AND ANALYSIS: Two reviewers determined
the studies to be included in the review based on inclusion and
exclusion criteria (JH,VR) and extracted the data using pre-developed
extraction forms for the Cochrane Musculoskeletal Group. The
methodological quality of the trials was assessed by the same reviewers
using a validated scale (Jadad 1996). Osteoarthritis outcome measures
were extracted from the publications according to OMERACT guidelines
(Bellamy 1997) and additional secondary outcomes considered.
MAIN RESULTS: Only three studies with a total of 259 OA
patients were included in the review. Electrical stimulation therapy had
a small to moderate effect on outcomes for knee OA, all statistically
significant with clinical benefit ranging from 13-23% greater with
active treatment than with placebo. Only 2 outcomes for cervical OA were
significantly different with PEMF treatment and no clinical benefit can
be reported with changes of 12% or less.
REVIEWER’S CONCLUSIONS: Current evidence suggests that
electrical stimulation therapy may provide significant improvements for
knee OA, but further studies are required to confirm whether the
statistically significant results shown in these trials confer to
important benefits.
Experience in using saprogel mud in combination with a magnetic field in treating cervical osteochondrosis.
[Article in Russian]
Samutin NM.
Patients with cervical osteochondrosis were successfully
treated with Deshembinskoe Lake [correction of Deshembinskaya] sapropel
mud in combination with exposure to magnetic field. The details of this
treatment regimen are described. Combination of pelotherapy with
effects of the magnetic field proved beneficial for patients with
cervical osteochondrosis.
J Rheumatol. 1994 Oct;21(10):1903-11.
The effect of pulsed electromagnetic fields in the
treatment of osteoarthritis of the knee and cervical spine. Report of
randomized, double blind, placebo controlled trials.
Trock DH, Bollet AJ, Markoll R.
Department of Medicine, Danbury Hospital, CT.
Abstract
OBJECTIVE: We conducted a randomized, double blind clinical trial to
determine the effectiveness of pulsed electromagnetic fields (PEMF) in
the treatment of osteoarthritis (OA) of the knee and cervical spine.
METHODS: A controlled trial of 18 half-hour active or placebo
treatments was conducted in 86 patients with OA of the knee and 81
patients with OA of the cervical spine, in which pain was evaluated
using a 10 cm visual analog scale, activities of daily living using a
series of questions (answered by the patient as never, sometimes, most
of the time, or always), pain on passive motion (recorded as none,
slight, moderate, or severe), and joint tenderness (recorded using a
modified Ritchie scale). Global evaluations of improvement were made by
the patient and examining physician. Evaluations were made at baseline,
midway, end of treatment, and one month after completion of treatment.
RESULTS: Matched pair t tests showed extremely significant changes
from baseline for the treated patients in both knee and cervical spine
studies at the end of treatment and the one month followup observations,
whereas the changes in the placebo patients showed lesser degrees of
significance at the end of treatment, and had lost significance for most
variables at the one month followup. Means of the treated group of
patients with OA of the knee showed greater improvement from baseline
values than the placebo group by the end of treatment and at the one
month followup observation. Using the 2-tailed t test, at the end of
treatment the differences in the means of the 2 groups reached
statistical significance for pain, pain on motion, and both the patient
overall assessment and the physician global assessment. The means of the
treated patients with OA of the cervical spine showed greater
improvement from baseline than the placebo group for most variables at
the end of treatment and one month followup observations; these
differences reached statistical significance at one or more observation
points for pain, pain on motion, and tenderness.
CONCLUSION: PEMF has therapeutic benefit in painful OA of the knee or cervical spine.
J Rheumatol. 1993 Mar;20(3):456-60.
A double-blind trial of the clinical effects of pulsed electromagnetic fields in osteoarthritis.
Department of Medicine (Rheumatology), Danbury Hospital, CT 06810.
Abstract
OBJECTIVE: Further evaluation of pulsed electromagnetic
fields (PEMF), which have been observed to produce numerous biological
effects, and have been used to treat delayed union fractures for over a
decade.
METHODS: In a pilot, double-blind randomized trial, 27
patients with osteoarthritis (OA), primarily of the knee, were treated
with PEMF. Treatment consisted of 18 half-hour periods of exposure over
about 1 month in a specially designed noncontact, air-coil device.
Observations were made on 6 clinical variables at baseline, midpoint of
therapy, end of treatment and one month later; 25 patients completed
treatment.
RESULTS: An average improvement of 23-61% occurred in
the clinical variables observed with active treatment, while 2 to 18%
improvement was observed in these variables in placebo treated control
patients. No toxicity was observed.
CONCLUSION: The decreased pain and improved functional
performance of treated patients suggests that this configuration of PEMF
has potential as an effective method of improving symptoms in patients
with OA. This method warrants further clinical investigation.
J Rheumatol. 1993 Mar;20(3):456-60.
The Effect of Pulsed Electromagnetic Fields in the
Treatment of Osteoarthritis of the Knee and Cervical Spine. Report of
Randomized, Double-Blind, Placebo Controlled Trials
Trock D. et.al.
Department of Medicine, Danbury Hospital, CT. J. of Rheumatology
From http://www.curatronic.com/scientific.html#1
OBJECTIVE. We conducted a randomized,
double blind clinical trial to determine the effectiveness of pulsed
electromagnetic fields (PEMF) in the treatment of osteoarthritis (OA) of
the knee and cervical spine.
METHODS. A controlled trial of 18
half-hour active or placebo treatments was conducted in 86 patients with
OA of the knee and 81 patients with OA of the cervical spine, in which
pain was evaluated using a 10 cm visual analog scale, activities of
daily living using a series of questions (answered by the patient as
never, sometimes, most of the time, or always), pain on passive motion
(recorded as none, slight, moderate, or severe), and joint tenderness
(recorded using a modified Ritchie scale). Global evaluations of
improvement were made by the patient and examining physician.
Evaluations were made at baseline, midway, end of treatment, and one
month after completion of treatment.
RESULTS. Matched pair t tests showed
extremely significant changes from baseline for the treated patients in
both knee and cervical spine studies at the end of treatment and the one
month follow-up observations, whereas the changes in the placebo
patients showed lesser degrees of significance at the end of treatment,
and had lost significance for most variables at the one month follow-up.
Means of the treated group of patients with OA of the knee showed
greater improvement from baseline values than the placebo group by the
end of treatment and at the one month follow-up observation. Using the
2-tailed t test, at the end of treatment the differences in the means of
the 2 groups reached statistical significance for pain, pain on motion,
and both the patient overall assessment and the physician global
assessment. The means of the treated patients with OA of the cervical
spine showed greater improvement from baseline than the placebo group
for most variables at the end of treatment and one month follow-up
observations; these differences reached statistical significance at one
or more observation points for pain, pain on motion, and tenderness.
CONCLUSION. PEMF has therapeutic benefit in painful OA of the knee or cervical
Scand J Rehabil Med. 1992;24(1):51-9.
Low energy high frequency pulsed electromagnetic
therapy for acute whiplash injuries. A double blind randomized
controlled study.
Foley-Nolan D. et.al.
Mater Hospital, Dublin, Ireland.
The standard treatment of acute whiplash injuries (soft
collar and analgesia) is frequently unsuccessful. Pulsed electromagnetic
therapy PEMT has been shown to have pro-healing and anti-inflammatory
effects. This study examines the effect of PEMT on the acute whiplash
syndrome. PEMT as described is safe for domiciliary use and this study
suggests that PEMT has a beneficial effect in the management of the
acute whiplash injury.
Minerva Anestesiol. 1989 Jul-Aug;55(7-8):295-9.
Pulsed magnetic fields. Observations in 353 patients suffering from chronic pain.
[Article in Italian]
Di Massa A, Misuriello I, Olivieri MC, Rigato M.
Three hundred-fifty-three patients with chronic pain have been
treated with pulsed electromagnetic fields. In this work the Authors
show the result obtained in the unsteady follow-up (2-60 months). The
eventual progressive reduction of benefits is valued by Spearman’s test.
We noted the better results in the group of patients with post-herpetic
pain (deafferentation) and in patients simultaneously suffering from
neck and low back pain.