Journal of Alzheimer’s Disease J Alzheimers Dis. 2016; 53(3): 753–771. Published online 2016 Aug 3. Prepublished online 2016 May 30. doi:  10.3233/JAD-160165 PMCID: PMC4981900

Review of the Evidence that Transcranial Electromagnetic Treatment will be a Safe and Effective Therapeutic Against Alzheimer’s Disease

Gary W. Arendash^* NeuroEM Therapeutics, Inc., Phoenix, AZ, USA ^*Correspondence to: Gary W. Arendash, PhD, NeuroEM Therapeutics, Inc., 144 E. Boca Raton Rd., Phoenix, AZ 85022, USA. Tel.: +1 480 395 1481; E-mail: moc.meoruen@hsadnera.yrag. Author information ? Article notes ? Copyright and License information ? Accepted 2016 Apr 18. Copyright IOS Press and the authors. All rights reserved This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial (CC BY-NC 4.0) License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

We have demonstrated in multiple studies that daily, long-term electromagnetic field (EMF) treatment in the ultra-high frequency range not only protects Alzheimer’s disease (AD) transgenic mice from cognitive impairment, but also reverses such impairment in aged AD mice. Moreover, these beneficial cognitive effects appear to be through direct actions on the AD process. Based on a large array of pre-clinical data, we have initiated a pilot clinical trial to determine the safety and efficacy of EMF treatment to mild-moderate AD subjects. Since it is important to establish the safety of this new neuromodulatory approach, the main purpose of this review is to provide a comprehensive assessment of evidence supporting the safety of EMFs, particularly through transcranial electromagnetic treatment (TEMT). In addition to our own pre-clinical studies, a rich variety of both animal and cell culture studies performed by others have underscored the anticipated safety of TEMT in clinical AD trials. Moreover, numerous clinical studies have determined that short- or long-term human exposure to EMFs similar to those to be provided clinically by TEMT do not have deleterious effects on general health, cognitive function, or a variety of physiologic measures—to the contrary, beneficial effects on brain function/activity have been reported. Importantly, such EMF exposure has not been shown to increase the risk of any type of cancer in human epidemiologic studies, as well as animal and cell culture studies. In view of all the above, clinical trials of safety/efficacy with TEMT to AD subjects are clearly warranted and now in progress.Keywords: Aßoligomers, Alzheimer’s disease, cognition, electromagnetic treatment, memory, transcranial

INTRODUCTION

There are currently no effective therapeutics to delay or reverse the cognitive impairment of Alzheimer’s disease (AD). Over the past decade, the many pharmacologic interventions against AD have all failed, in part because drugs have difficulty passing the blood-brain barrier and have even less bioavailability inside neurons to affect the AD pathologic process therein [1]. This is critical because intraneuronally-produced amyloid-ß (Aß), a small toxic protein, aggregates into toxic oligomers of up to eight A? molecules within neurons. These A? oligomers appear to be the initiating pathologic agents in AD, as supported by many recent studies [2, 3]. Indeed, changes in CSF levels of A? oligomers are associated with progression of cognitive decline in AD patients [4]. A? oligomers have a high affinity for intraneuronal mitochondria, especially for mitochondrial electron transport proteins on the inner mitochondrial membrane, resulting in suppression of mitochondrial function/ATP production [5, 6]. This A?-induced mitochondrial dysfunction appears not only to be central to AD pathogenesis, but is also an early event therein [6–9]. Thus, we believe that any effective AD therapeutic will need to penetrate not only the blood-brain barrier, but also neuronal cell membranes and then outer mitochondrial membranes in order to address the toxic “intraneuronal” A? oligomerization causative to mitochondrial dysfunction of AD.

Given the many years of unsuccessful drug intervention studies against AD, investigating new and innovative “non-pharmacologic” interventions against AD are now clearly warranted. Neuromodulatory approaches have consequently emerged and are currently being clinically tested against AD. These approaches include transcranial magnetic stimulation (tMS) [10, 11], transcranial direct current stimulation (tDCS) [12], and deep brain stimulation (DBS) [13, 14] via chronically-implanted electrodes. All three of these approaches provide generalized stimulatory/inhibitory effects on neuronal activity, apparently without any direct “disease-modifying” actions against AD. To our knowledge, none of these approaches have been demonstrated to be efficacious against AD endpoints in cell culture or animal models for AD.

The newest neuromodulatory approach against AD is transcranial electromagnetic treatment (TEMT), which we have pioneered in pre-clinical electromagnetic field (EMF) treatment studies [15–19] to AD transgenic mice. Much different from tMS, TEMT (and electromagnetic treatment in general) involves interdigitated magnetic and electric waves that are perpendicular to one another and to the direction which they are propagating. These interwoven magnetic/electric waves leave an antenna source and radiate away, never to return. At the EMF frequencies we have utilized, TEMT easily penetrates the entire human forebrain to impact “intraneuronal” pathologic processes, such as intraneuronal A? oligomer formation. Thus, TEMT is very different technology from the magnetic fields generated by tMS, which involve magnetic energy emitted by and returned to a coil conductor source. TEMT is also superior to other neuromodulatory approaches in being able to directly impact the entire forebrain while the other three neuromodulatory approaches can only affect cortical areas directly (tDCS, tMS) or a limited sub-cortical region directly (DBS). As well, tDCS and tMS require frequent clinical visits, while theneurosurgery required for DBS is both invasive and costly. By contrast, TEMT will be administered in-home by the patient’s caregiver, treat all affected areas of the AD brain, and be available to essentially the entire AD population. Thus, TEMT has distinct advantages over other neuromodulatory approaches, which should enhance the chances for it providing true therapeutic efficacy against AD.

In 2007, our laboratory, in collaboration with multiple others, began investigating the effects of EMF treatment on cognitive function and brain A? pathology in AD transgenic mice. In a variety of studies, we discovered and confirmed that daily EMF treatment over periods of 1–9 months can prevent and reverse cognitive impairment, as well as reverse A? aggregation/deposition. These benefits apparently occurred through the complementary mechanisms of A? disaggregation (both small/oligomeric and fibrillar/compact forms), mitochondrial enhancement, and enhanced neuronal activity. All of these studies involved EMF treatment within the ISM radiofrequency band (902–928?MHz) reserved for Industry, Science, and Medicine and at Specific Absorption Rate (SAR) power levels below FDA/FCC limits. Although these pre-clinical studies clearly justify the TEMT clinical trial currently in progress, it is important to gauge to the extent currently possible the safety of this neuromodulatory approach for long-term use in humans. As such, the purpose of this article is to review evidence regarding the safety and efficacy of TEMT (and EMF treatment in general) as a new therapeutic intervention against neurodegenerative diseases. The review is divided into three sections, with Section I presenting pre-clinical data/studies supportive of EMF efficacy in AD animal models. Section II then presents pre-clinical data/studies that provide insight into TEMT safety. Finally, Section III describes human studies that relate to TEMT safety and potential physiologic/cognitive benefits.

SECTION I: SUPPORTIVE PRE-CLINICAL STUDIES OF EMF TREATMENT EFFICACY

Since 2010, we have published five peer-reviewed papers showing the utility of EMF treatment in AD transgenic mice (Tg; AD mice) to provide cognitive benefits, anti-aggregation effects on brain A?, mitochondrial enhancement, and enhanced neuronal activity. These transgenic mice overexpress the mutant Swedish form of human APP alone (APPsw) or in combination with a mutant human PS1 gene (APPsw+PS1)—both mutations are causative to the early-onset form of AD via A? production/aggregation. In our initial paper [15], we reported that twice daily whole body EMF treatment (pulsed at 918?MHz, 1.05?W/kg SAR) begun early in adulthood before compact A? plaques and cognitive impairment occur, protected AD mice from otherwise certain cognitive impairment months later; this, in a complex cognitive interference test (Fig. 1A-C). If EMF treatment was delayed until older age (when compact A? plaques were extensive and cognitive impairment present), daily EMF treatment over months reversed both cognitive impairment (Fig. 2A) and A? deposition (Fig. 2B) [16–18].

Fig.1 EMF treatment, begun in young adulthood, protects AD mice (Tg) mice from cognitive impairment and improves basic memory of normal mice. Cognitive interference testing at 4-5 months (A) and 6-7 months (B) into EMF treatment revealed overall [Tg and non-Tg(NT)…

Fig.2 At 8 months into EMF treatment, cognitively-impaired AD mice (Tg) mice exhibited cognitive benefits and reduced brain A? deposition. A) Cognitive interference testing revealed Tg/EMF mice as vastly superior to Tg controls in 3-trial recall and …

Of greater significance than the reductions in “extracellular” compact A? plaques was the preventive effect of TEMT on “intraneuronal” oligomeric A? aggregation following sonication of hippocampal homogenates from aged (14-month-old) AD mice. Over the course of 6 days, the progressive increase in the 80?kD A? oligomer was prevented by twice daily EMF treatment to these homogenates (Fig. 3) [15]; this result indicates that EMF treatment exerts a “direct” anti-aggregating effect on oligomeric A?. In addition to this in vitro prevention of A? oligomeric formation, aged AD transgenic mice given one month of twice-daily EMF treatment exhibited a 5–10-fold increase in “mitochondrial” soluble A? levels within hippocampal synaptosomes (Fig. 4) [19], which is consistent with EMF treatment-induced disaggregation of oligomeric to monomeric A? within these mitochondria. Thus, TEMT penetrates neurons to destabilize/disrupt oligomeric A? therein, possibly through destabilization of hydrogen bonds between individual A? monomers or through disruption of dipole-dipole coupling.

Fig.3In Vitro EMF treatment of hippocampal homogenates from aged Tg mice results in progressively decreased A? oligomerization between 3 and 6 days into treatment. Western blots display the 80?kDa A? oligomer on top and the ?-Actin …

Fig.4 Long-term EMF treatment of aged AD (Tg) mice dramatically increased soluble A?_1–40 levels in mitochondria preparations from both cortex and hippocampus. These 5–10x increases in mitochondrial A? are consistent with an EMF-induced …

Linked to the above A? disaggregation were 50–150% enhancements of mitochondrial function across six established measures evaluated in the same tissue (Fig. 5) [19]. This finding suggests that EMF-induced removal of oligomeric A? from neuronal mitochondria results in a substantial increase in neuronal mitochondrial function—exactly the therapy needed for the mitochondrial dysfunction and hypo-metabolism present in brains of AD subjects. Importantly, EMF-induced mitochondrial enhancement was observed even in hippocampal mitochondria from normal aged mice [19], indicating that EMF treatment-induced increases in mitochondrial function (especially for Complex IV enhancement) do not require removal of oligomeric A? aggregates. Indeed, both young adult and aged “normal” mice exhibit enhanced cognitive function with long-term EMF treatment (Fig. 1D, 2A) [15].

Fig.5 EMF treatment greatly enhances mitochondrial function within both cerebral cortex and hippocampus of aged AD (Tg) mice. Shown are percent changes across six measures of mitochondrial function, wherein 50–150% enhancements were induced by EMF treatment. …

As yet another mechanism of EMF action, we have reported that EMF treatment for 2 months increases “neuronal activity” by 21% within entorhinal cortex of aged AD mice and normal aged mice, while increasing cognitive performance in the same animals (Fig. 6) [16]. This EMF treatment-induced increase in neuronal activity may be at least partially responsible for the minimal 0.1–0.3°C rise in brain temperature sometimes seen during treatment sessions in aged AD mice and normal mice (see Section II).

Fig.6 TEMT increases neuronal activity in entorhinal cortex of aged AD mice, as indicated by the number of cFos-stained neurons. Note increased number of active neurons in AD mice given long-term TEMT (right) compared to control AD mice not given TEMT (left).…

It should be underscored that all of our pre-clinical data (which is comprehensively reviewed in [18]) was attained 2–8?h after EMF administration, indicating lasting benefits of EMF treatment beyond any daily treatment period. As detailed in Section II, all of the benefits of EMF treatment occurred through “non-thermal” mechanisms because we have shown that there are no increases in brain temperature during treatment sessions or in comparison to sham controls [17]. Importantly, the benefits of long-term EMF treatment that we began reporting in 2010 have been confirmed in publications from three independent laboratories that utilized electromagnetic treatment in AD mice [20–22].

From our collective body of pre-clinical EMF studies, we have identified three mechanisms of action associated with EMF treatment’s ability to protect against or reverse cognitive impairment in AD mice: 1) disaggregation of “intraneuronal” A? oligomers and extracellular A? plaques, 2) mitochondrial enhancement within neurons, and 3) increase in neuronal activity—all three within brain areas importantfor cognitive function such as the cerebral cortex, hippocampus, and entorhinal cortex. Critical to these beneficial effects is the ability of electromagnetic waves (at the parameters utilized) to easily penetrate deep human brain areas and all neurons therein, as we have demonstrated in human phantom FDTD computer simulation studies (Fig. 7).

Fig.7 An FDTD computer simulation showing deep electric field penetration by an excitation element (one of eight elements) positioned on the cranium. Deep brain regions, such as the hippocampus and entorhinal cortex, are easily affected by this single element. …

It is important to note that there are currently no AD therapeutics in clinical trials that have been shown to be capable of Mechanisms 1 or 2, much less both of them. By attacking the AD-initiating processes of “intraneuronal” A? aggregation and suppressed energy production, and in multiple brain areas impacted by AD, TEMT is not based on a single pathogenic hypothesis like most drugs are. Regarding TEMT’s anti-A? aggregation actions (Mech. 1),NeuroEM has identified both direct and indirect processes that are likely involved. As for TEMT’s mitochondrial enhancement actions (Mech. 2), a direct enhancement of Complex IV activity and an indirect enhancement of overall mitochondrial function via removal/disaggregation of A? oligomers within mitochondria are involved. A detailed description of the multiple EMF mechanisms of action against AD will be the subject of a follow-up article.

Collectively, our pre-clinical studies of EMF treatment efficacy exceed the pre-clinical work performed for most potential AD drugs prior to their advancement to clinical trials. As such, clinical trials of safety/efficacy with TEMT administration to AD patients are now clearly warranted and are in progress.

Note, since all of our pre-clinical studies, and essentially all other animal studies, have involved “whole body” EMF treatment and not EMF treatment limited to the head/cranium, these animal studies are being referred to as “EMF treatment studies”. The term “TEMT” is reserved for human clinical studies that have had, or will have, EMF treatment limited to the head/cranium, such as with our TEMT treatment device (see http://www.neuroem.com).

SECTION II: PRE-CLINICAL ANIMAL/CELL CULTURE STUDIES OF EMF TREATMENT SAFETY

Section I presented strong evidence from our EMF treatment studies in AD (transgenic) mice that long-term EMF treatment provides both cognitive and neuropathologic benefits relevant to AD. The studies within this section will evaluate the safety of EMF treatment parameters (around 900?MHz and ?1.6?W/kg SAR) used in our pre-clinical studies and in our currently underway clinical trial – this, from the perspectives of animal and cell culture/in vitro studies. A particular emphasis will be placed on the inability of such EMF treatment to induce, or contribute to the induction of cancer, as demonstrated by a wide breath of basic science and biophysical studies.

Animal studies from our laboratories

All of our pre-clinical studies showing cognitive benefit and neuropathologic reversal in AD mice involved an EMF treatment frequency (918?MHz) within the ISM radiofrequency band (902–928?MHz) reserved for Industry, Science, and Medicine and SAR power levels (1.05?W/kg) below FCC limits for commercial devices: EMF treatment involved two 1-hour sessions every day. Animal studies have concluded time and time again that long-term exposure to radiofrequency waves in/near this ISM band have no negative impact on health, a conclusion that is underscored by safety endpoints evaluated in our own long-term studies. Those studies indicate that far from being deleterious to cognitive function in both AD mice and normal mice, daily long-term EMF treatment for up to 8 months actually improves cognitive function while not affecting sensorimotor function or anxiety levels [15–19]. The reader is referred to these five published papers for details on the cognitive benefits and sensorimotor effects of EMF treat in both normal and AD mice.

The inability of long-term EMF treatment at 918?MHz to deleteriously affect brain homeostasis is indicated by our neurochemical analysis performed in both AD mice and normal mice following daily TEMT for over 7 months [15]. For both AD mice and normal mice, TEMT had essentially no effect on hippocampal DNA repair enzymes (OGG1, PARP), antioxidant enzyme markers (cytosolic and mitochondrial SOD, GSH/GSSH), or protein oxidative damage (protein carbonyl content). Furthermore, histologic evaluation of brains from both AD mice and normal mice in our studies revealed no histologic or cytologic abnormalities, and no cancerous growths [15–17]. As well, major peripheral organs (liver, heart, lungs, kidneys) were all normal in appearance.

Also underscoring the safety of EMF treatment at 918?MHz and below FCC power limits for commercial devices, all of the benefits of EMF treatment that we have reported occurred without any acute or long-term increases in brain temperature; in other words, EMF treatment provided cognitive and neuropathologic benefits through “non-thermal” mechanisms. For example, acute EMF treatment (two 1-hour treatments in a single day) to several types and ages of naïve AD mice and controls revealed no change in brain temperature during or between the two treatments (Fig. 8A) [12]; this was the same brain temperature profile observed in non-treatment mice (Fig. 8B). Note the strong correlation between brain and body temperatures in this study (Fig. 8C), although brain temperature is typically around 0.3–0.4°C cooler than body temperature. Longer term TEMT treatment (daily for 12 days) to AD mice also resulted in no change in brain or body temperature, both in relation to OFF periods and compared to control mice not given EMF administration (Fig. 9) [17]. In yet another study, we attained brain temperature measurements from aged AD mice and normal mice at 1, 3, and 6 weeks into EMF treatment [17, 18]. Throughout this 6-week study period, brain temperature remained stable or was minimally elevated by 0.1–0.3°C during ON periods. Following any such brain temperature elevations, brain temperature always returned to pre-treatment levels during OFF periods. Collectively, these results suggest that clinical use of our human TEMT device will result in either no increase in brain temperature or a minimal increase of no physiologic significance. It is noteworthy that, during moderate exercise in rodents and humans, brain temperature can increase by a much more prominent 1-2°C compared to any incremental elevation induced by 900?MHz EMF exposure/administration [23].

Fig.8 A, B) There are no changes in brain temperature of AD transgenic mice (both APPsw and APPsw+PS1) and normal mice (NT) during acute EMF treatment (two 1-h treatments during a single day) compared naïve Tg and NT mice of various ages. C) The strong …

Fig.9 Body and brain temperature measurements for AD mice recorded prior to the start of EMF treatment (control), as well as at 5 days and 12 days into EMF treatment. For both control and treatment time points, there were no differences between EMF-treated …

Cancer and radiofrequency exposure: Animal and cell culture studies

Numerous studies have administered radiofrequency (RF) EMF treatment involving ?900?MHz frequency at around 1.6?W/kg SAR to rodents in order to determine any cancer-causing effects that might arise. These full-body exposure studies have determined that such radiofrequency treatment does not initiate, nor does it promote, any type of cancer investigated. With RF treatment at these parameters extending from 5 months to life-long, four studies found no evidence for an induction of brain tumors [24–27], and another study reported no ability of such RF treatment to promote brain tumor growth initiated by a chemical carcinogen [28]. Similarly, 900?MHz RF treatment extending from several weeks to life-long did not promote chemically-induced breast cancer [29–31], nor did it promote UV radiation-induced skin cancer [32]. Indeed, no increases in any type of cancer induced by non-ionizing radiation were observed in rodents exposed to 900?MHz RF treatment for 11/2 years [33]. The National Cancer Institute’s 2015 website summarizes these studies nicely in stating, “It is generally accepted that damage to DNA is necessary for cancer to develop. However, radiofrequency energy, unlike ionizing radiation, does not cause DNA damage in cells, and it has not been found to cause cancer in animals or to enhance the cancer-causing effects of known chemical carcinogens in animals”.

The above animal studies are supported by human/rodent cell culture studies looking at DNA damage (genotoxicity) of the same 900?MHz RF treatment. Although such studies are not particularly relevant to human RF treatment because the vast majority of them are acute (less than 24 hours), they have almost universally reported no effects of 900?MHz RF exposure on indices of genotoxicity/DNA damage [34]. In this regard, RF treatment to cell cultures had no effect on DNA strand breaks [35–39] or micronucleus induction [40–42]. Relatedly, exposing brain suspensions from mice to 900?MHz RF resulted in no effects on DNA stand breaks or chromatin conformation [43]. A number of cell culture studies have measured the activity of ornithine decarboxylase (ODC), an enzymatic marker for increased cell proliferation/cancer, and found ODC activity to be similarly unaffected by RF treatment [44–46]. Krewski [47] presented multiple studies showing that RF exposure to cell cultures does not induce DNA strand breaks, chromosome aberrations, sister chromatid exchanges, or DNA repair synthesis. Verschaeve [48] reviewed the data on alleged RF-induced genetic effects and concluded that the evidence for genotoxic effects of RF exposure (which would be important for demonstrating enhanced cancer risk) is extremely weak.

Consistent with the large body of human, animal, and cell culture studies indicating no association between 900?MHz RF treatment and any type of cancer, extensive research has not established any biologic mechanisms through which such RF treatment could cause cancer, even if an association were present. There is certainly a link between some forms of electromagnetic radiation (e.g., UV radiation, x-rays, and gamma rays) and some cancers. These electromagnetic forms have extremely high frequencies that are many orders of magnitude higher than RF waves. Since the photons of these very high frequency forms of radiation carry a large amount of energy compared to RF, they can break covalent chemical bond; importantly, all carcinogenic agents act by breaking covalent bonds [49]. In sharp contrast, RF-generated photons have a much lower energy level that is insufficient to break, damage, or weaken any covalent bonds. Although RF photons can induce rotational motion of strongly dipolar residues [50] or produce resonance/vibrational effects on some molecules [51, 52], these effects are not deleterious in causing or promoting cancer. The impossibilityof radiofrequency waves, and thus our TEMT device, to induce cancer is supported by the research of none other than Albert Einstein. He won the 1905 Nobel Prize in Physics for establishing that much higher electromagnetic frequencies (UV,x-rays, gamma rays) are required to break covalent bonds in molecules and, thus, to increase cancer risk.

Cognitive function in rodents

We have performed multiple studies investigating the long-term cognitive effects of daily RF treatment to normal and AD mice [15–18]. All of these studies involved pulsed 918?MHz frequency and 1.05?W/kg SAR for two one-hour treatment periods daily, very close to the parameters built into our human TEMT device and the same daily treatment paradigm (two 1-hour periods). In none of these comprehensive studies were any cognitive impairments observed in either normal mice or AD mice in any cognitive task evaluated. Indeed, cognitive enhancement was often seen, and usually in a complex task that is measure-for-measure analogous to a human task of the same name that is used to distinguish AD and pre-AD patients from normal aged individuals—namely, the cognitive interference (CI) task. In an initial study, we found that AD mice started on daily TEMT in young adulthood were protected from otherwise inevitable cognitive impairment in the CI task at 7 months into treatment [15]. In follow-up studies involving the start of EMF treatment at older ages (when AD mice were cognitively impaired), 2–8 months of daily treatment reversed cognitive impairment in the CI task and in the Y-maze task [15–18]. Even normal mice receiving treatment in these studies showed cognitive improvement in both the CI and Y-maze tasks. In all of our studies, beneficial effects lessening brain AD neuropathology [15–18] and/or enhancing brain metabolic function [19] were observed. Although all of these mouse studies involved whole body RF treatment, mouse brains were receiving RF exposure (thus TEMT) very similar to that provided by our human TEMT device.

Other investigators have investigated cognitive endpoints in “normal” rats or mice given 900?MHz RF exposure. All of the well-designed studies involving adult animals have reported no overall effects of 10 days to 19 months RF treatment on a variety of cognitive tasks such as the 8-arm radial maze and Morris water maze [53–58]. Although one of these studies [57] reported transient cognitive impairment midway through 10 weeks of RF treatment, the authors did not find any impairment at earlier or later time points and concluded that rats can adapt to long-term RF exposure. Interestingly, one study involving RF treatment for 5 weeks to “immature” rats reported an enhancement in Morris maze memory retention [59]. Why have all other 900?MHz RF studies involving normal “adult” rodents failed to find the cognitive benefits that we have reported in normal mice? First, most of these prior studies involved shorter-term treatment (30 days or less), which our work shows is usually not sufficient for cognitive benefit in normal animals [15]. Second, in contrast to our cognitive interference task, the cognitive tasks selected have often been tasks that are relatively insensitive to various cognitive domains and not directly relevant to humans. It should be noted that some other rodent studies have actually reported cognitive impairment resulting from RF treatment [60–64]. However, most of these studies were poorly designed. For example, there was often an inexplicable delay of 2–18 months between RF treatment and cognitive testing [60, 63] or RF treatment was compromised by stressful background radio noise that was not controlled for [61]. In one of these studies, animals were given a single treatment lasting only a few seconds, then tested 12 and 18 months thereafter [63]. To summarize, well-designed RF treatment studies involving “normal” rodents have not demonstrated any long-term cognitive impairment resulting from treatment.

Other functions in rodents (immune function, oxidative markers, BBB)

Although several endpoints (immune function, oxidative markers, and blood-brain barrier [BBB] integrity) have not been analyzed to our knowledge in human RF exposure studies, studies in normal rodent studies have investigated the effects of full-body 900?MHz RF treatment on these endpoints. Regarding immune function, Johansson [65] reviewed the literature involving RF effects on the immune system (both T- and B-cell compartments) and found no effects of 900?MHz RF treatment, although effects at harmful “microwave” frequencies (e.g., 2450?MHz) were reported. With 900?MHz RF treatment for 1 month to mice, Gatta [66] reported that neither T- norB-cell compartments were affected and that a clinically relevant effect of RF treatment on the immune system was unlikely. Similarly, Nasta [67] found that the same one-month RF treatment protocol did not affect the B-cell peripheral compartment (T1 and T2 cells, mature follicular and marginal zone B-cells) or antibody (IgM and IgG) production. Most recently, Rosado [68] found no effects of 900?MHz-exposed bone marrow cells on their long-term (3-month) ability to reconstitute peripheral T and B cells, and no differences in thymocyte number, frequency, or proliferation. Collectively, these rodent studies suggest that the immunosystem will not be impacted by TEMT in humans, especially since only the head will be exposed to RF treatment.

Animal and cell culture studies have evaluated oxidative markers for evidence of oxidative stress/damage induced by 900?MHz RF treatment and have largely found little evidence for oxidative stress/damage. Seven days of 900?MHz RF exposure to rabbits resulted in no effects on all brain oxidative markers evaluated, including SOD, GSH-peroxidase, MDA, and NO [69]. Similarly, 900?MHz RF treatment to mouse cell cultures did not affect reactive oxygen species (ROS) production [70], while levels of oxidants/antioxidants (GSSH, SOD, catalase, glutathione peroxidase activity), oxidative damage/toxicity (trypan blue dye exclusion assay), and NO production were unaffected [71]. Results from these animal studies are consistent with our results showing no effects of daily RF treatment for 8 months on oxidative measures [15]. Regarding 900?MHz RF effects on the BBB, Finnie [72] reported that BBB integrity was maintained in mice after two years of daily treatment and Grafstom [73] found no evidence of BBB breakdown in rats treated once weekly for one year. By contrast, Tang [64] found damaged BBB after more acute treatment of 14–28 days. Collectively, these studies suggest that, although temporary effects of EMF on BBB integrity are possible, no long-term effects have beendemonstrated.

Thus, from the standpoints of immune function, oxidative stress, and BBB integrity, there is essentially no evidence from animal studies that 900?MHz RF treatment induces deleteriously effects.

SECTION III: HUMAN STUDIES RELATED TO TEMT SAFETY AND EFFICACY

General health studies

Particularly since 2005, many studies in normal adults have investigated the safety of cell phone use (especially GSM 900 phones) on indices of general human health such as sleeplessness, fatigue, dizziness, digestive disturbances, concentration difficulties, blood cell profiles, blood pressure, or cognitive function. The single antenna of these commercially available devices is held close to the human head during use and their electromagnetic frequency of around 900?MHz and SAR levels of <1.6?W/kg are close to those for any given antenna of the TEMT device that we have in clinical trials. In that only one antenna of the TEMT device is ON/active at any given time, the results of human studies investigating health effects of both short- and long-term GSM 900 cell phone use are especially pertinent to determining safety of our TEMT device. General health aspects of cell phone use will be considered first, followed by an analysis of the purported association between cell phones and brain cancers. It is important to note that this evaluation of human health effects of cell phones largely involves electromagnetic (RF) exposure from GSM 900?MHz cell phones, although some studies also included other cell phone technologies (e.g., GSM 1800/1900?MHz, UMTS). Obviously, GSM 900 cell phones are the closest in electromagnetic parameters to the TEMTdevice.

Valberg [74] summarized findings of the World Health Organization’s workshop on health issues potentially related to cell phone use and concluded that there is little support for adverse health effects from cell phones at or below levels established by international standards. Valberg [74] underscored that the more recent, better-designed human studies are universally negative, particularly regarding cancer development. In a very comprehensive review, Krewski [47] stated that, “All of the authoritative reviews completed within the last 2 years have concluded that there is no clear evidence of adverse health effects associated with radiofrequency fields”. In an update of their original report, Krewski and colleagues [75] again found there was no clear evidence of adverse health effects associated with radiofrequency fields/cell phones. For the period 2000–2011, Moussa [76] evaluated epidemiologic, systemic, and meta-analysis studies, and also found no consistent pattern for exposure to mobile phones being detrimental to health.

The aforementioned studies, and others, have lead prominent health organizations in the U.S. to conclude that there is no clear evidence of adverse health effects associated with radiofrequency fields. The National Institute of Environmental Health Sciences (NIEHS) states that, “The weight of the current scientific evidence has not conclusively linked cell phone use with any adverse health problems.” The FDA states that, “Studies reporting biological changes associated with radiofrequency energy have failed to be replicated and the majority of human epidemiologic studies have failed to show a relationship between RF exposure from cell phones and health problems.” The Centers for Disease Control and Prevention (CDC) states that scientific research as a whole does not support a statistically significant association between cell phone use and healtheffects.

Cognitive/physiologic studies

Regarding subjective symptoms and cognitive function, Kwon [77] conducted an extensive review of studies evaluating behavioral and neurophysiological effects of cell phone use. They found no evidence that any subjective symptoms (sleeplessness, headache, dizziness, fatigue, etc.) were induced by cell phone use; such symptoms reported in supposed hypersensitive individuals are thus psychosomatic in nature. Moreover, in over 30 published papers (most of which involved GSM 900 phones), Kwon [77] found no evidence that cell phone use resulted in any deleterious effects on cognitive function. Similarly, a meta-analysis performed by Barth et al. [78] involving 17 studies found no significant effects of GSM 900 phone exposure on cognitive abilities, a conclusion echoed by an additional meta-analysis by Valentini [79] involving 24 studies. To date, most controlled human studies reporting no deleterious cognitive effects of 900?MHz cell phone exposure have been “acute”, single exposure (3–120?min) studies [80–86], with the exception of three studies involving daily exposure for 6–27 days [87–89]. All of these studies showing no deleterious cognitive effects were exclusively in normal individuals (no AD or other neurologically-diseased subjects) and all of them involved unilateral RF exposure to only one hemisphere via a cell phone held next to the head.

No controlled human studies have investigated the cognitive effects of “long-term” and “bilateral” GSM 900 EMF treatment in normal subjects over months or years. However, two epidemiologic-based human studies have already provided indirect evidence that continued RF exposure via cell phone use could be associated with enhanced cognitive performance (executive function) in normal subjects [90] and a much reduced risk of hospitalization due to AD and vascular dementia for long-term cell phone users of 10 years or more [91]. Although involvinga very high 10,500?MHz frequency and extremely low power levels, a recent pilot study administered EMF clinically to AD patients three times a week for 5 weeks, resulting in significant improvement in a variety of cognitive measures [92]. However, the known inability of such a high EMF frequency to penetrate brain tissue, especially at the extremely low EMF utilized, suggest an unconventional mechanism may be involved in these cognitive benefits.

A number of physiologic effects have been reported with “acute” 900?MHz cell phone exposure in normal humans. First, cortical excitability is enhanced, as measured by evoked potentials [93]. Second, numerous studies have reported that acute 900?MHz cell phone exposure enhances alpha wave activity (important for basic cognitive processing) in awake cortical EEG [84, 94–96]. All of these studies suggest that neuronal activity could be beneficially enhanced by 900?MHz exposure. Since neuronal activity is coupled to glucose utilization, it is not surprising that an increase in brain glucose utilization (indexed by FDG-PET scanning) was observed in brain areas closest to the cell phone antenna [97]. In view of these diverse physiologic studies, electromagnetic waves from cell phones could actually be providing beneficial physiologic effects on brain function in normal humans.

Importantly, Wessapan [98] showed that the electromagnetic parameters we are utilizing in our clinical studies (around 900?MHz and 1.6?W/kg SAR) result in a very minimal 0.1-0.2°C increase in brain temperature in their human head FDTD simulation study. Wang [99], as well as Van Leeuwen [100], also calculated brain temperature in their FDTD simulation studies involving 900?MHz exposure and found no more than a 0.1°C rise in brain temperature. Since any potential health problems due to EMF exposure are linked to temperature increases of at least 2-3°C [19], the very minimal increase in brain temperature calculated in the FDTD studies of Wessapan [98], Wang [99], and Van Leeuwen [100] clearly indicate that the frequency (around 900?MHz) and power level (1.6?W/kg) of our clinical TEMT device is highly unlikely to have any thermally-induced health hazards associated with its use.

Thus, in terms of general health, subjective symptoms, cognitive function, and physiologic measures evaluated in humans, 900?MHz RF exposure has not been associated with any deleterious effects. In the case of cognitive function and physiologic endpoints, there is evidence that such exposure may actually be beneficial.

Brain cancer studies

The notion that GSM 900?MHz or 1800?MHz cell phones can increase the risk of brain cancer originated with a single group of Swedish researchers around 2004 and became prominent around 2008 [101,102]. Investigating the Swedish population, these researchers have repeatedly published epidemiologic studies since then concluding that GSM cell phone exposure doubles the risk of brain glioma and acoustic neuroma after 10 or more years of cell phone use [103–105]. Their most recent epidemiologic study [106] pooled two case-control studies involving Swedish patients diagnosed during 1997–2003 and 2007–2009. With cell phone exposure assessed by a self-administered questionnaire, Hardell [106] reported a 1.8x increased risk of glioma overall through 20 years. It is important to recognize that the current life-long risk of developing any form of brain cancer is about 0.5%. So even if the risk of brain cancer was doubled by long-term cell phone use (which overwhelming evidence says is not the case), the life-long risk of brain cancer would still only be a small 1% ! If NeuroEM’s TEMT device is shown to be an effective therapeutic against AD in clinical trials, the vast majority of AD patients and their families would gladly accept this claimed doubling of brain cancer risk to 1%.

Based in part on the above results reported by Swedish investigators, a working group from the World Health Organization’s International Agency for Research on Cancer (IARC) in 2011 classified radiofrequency fields emitted from mobile phones as “possibly carcinogenic to humans”. The IARC put RF fields into Category 2B, based on “limited” evidence suggesting an association between exposure from mobile phones and two types of brain cancer (glioma and acoustic neuroma) [107]. This report puts mobile phone exposure in the same potential risk Category (2B) as coffee. Any listing of carcinogenic agents by the IARC that suggests coffee is potentially carcinogenic has questionable credibility or is hopelessly out-of-date (the inclusion of coffee in Category 2B has apparently not been updated since 1991). Indeed, over the past 10 years, there has been mounting scientific evidence that coffee reduces risk of many forms of cancer, including liver cancer, rectal cancer, breast cancer, and prostate cancer [108, 109]. Following the 2011 IARC report classifying mobile phones in Category 2B, a number of investigators condemned the report as scientifically invalid and misleading. Vigayalaxmi [110] did a meta-analysis investigating the purported correlation between increased genetic damage and carcinogenesis and found that the Category 2B classification for mobile phones was not supported by genotoxicity-based evidence. Moreover, Wiedemann [111] reported that the IARC’s 2011 study was flawed because characterization of the probability of carcinogenicity was misunderstood by study participants and the respondents greatly overestimated the magnitude of the potential risk from cell phone radiofrequency exposure. In their study reporting no significant effect of intensive cell phone usage on incidence of brain cancers in Taiwan, Hsu [112] even suggested that the IARC should publish more conscientious reports to spare the public unnecessary worries.

In contrast to the above studies from a single Swedish group and the IARC’s classification, large and well-designed human epidemiologic studies performed since 2010 have concluded time and time again that long-term exposure to RF fields of around 900?MHz (typifying cell phones in the U.S.) have no negative impact on health, particularly on incidence of brain tumors. The large INTERPHONE Study [113], performed by a subsidiary of the WHO, involved 13 nations (including Sweden) with the goal of determining if RF waves from long-term cell phone use of over 10 years increased risk of brain cancers (glioma, acoustic neuroma, meningioma). This huge cased-controlled and recall-based study found no elevated risk of brain cancer with 10 or more years of cell phone use. Also, no relationship was found between lifetime number of phone calls (higher amounts of cell phone use) and brain cancer. A 2011 review of the INTERPHONE Study by the National Institute of Environmental Health and Safety (NIEHS) firmly agreed with the study’s conclusion and underscored that the INTERPHONE Study actually found an overall reduced risk of brain cancer with regular mobile phone use versus non-users [114]. Moreover, a recent extension from the INTERPHONE Study reported no relationship between location of brain tumors and regions of the brain that were exposed to the highest level of RF energy from cell phones [115].

In another huge epidemiologic study [116] with no selection bias and no recall bias, 358,000 cell phone subscribers in Denmark were followed for 17 years (1990–2007). Irrespective of whether subscribers had used cell phones for 10–13 years or more than 13 years, the incidence of brain cancers (glioma, acoustic neuroma, meningioma) was not increased. In the prospective Million Women Study (UK) involving 791,000 women, there was no increased risk of glioma, acoustic neuroma, or meningioma during 7 years of follow-up through 2011 [117, 118]. Barchana [119] actually found a decreased risk of gliomas in the Asian Pacific region after cell phones became available around 1995. Finally, Lagorio [120] recently performed a meta-analysis of 29 studies investigating cell phone use and brain cancer. In long-term cell phone users (more than 10 years), the relative risks of glioma, acoustic neuroma, and meningioma were non-significant.

Because of the aforementioned large and well-designed clinical studies, major health organizations have conclude there are no health problems (including cancer) that have been linked to radiofrequency/cell phone exposure. For example, the National Cancer Institute’s 2015 website states, “To date, there is no evidence from studies of cells, animals, or humans that radiofrequency energy can cause cancer”. Indeed, NCI’s Surveillance, Epidemiology, and End Results (SEER) Program, which tracks cancer incidence in the U.S. over time, found no increase in brain cancer incidence between 1987 and 2007, despite the dramatic increase in cell phone use in the U.S. during that time [121, 122]. Even in Sweden’s national cancer statistics, the incidence rates for glioma have not risen since 1970 [123], and glioma rates in Nordic countries from 1979 through 2008 have not increased [124], despite much increased use of cell phones in these countries. Furthermore, the U.S. FCC states that there is no scientific evidence that shows that wireless phone use can lead to cancer or to other health problems. Similar conclusions have been reached by the National Institute of Environmental Health Sciences (NIEHS), the FDA, and the Centers for Disease Control and Prevention (CDC). These organizations and the multitude of scientific studies since 2010 firmly revoke the Hardell group’s studies in Sweden, which formed the basis for the IARC’s erroneous categorization of mobile phone exposure as “possibly carcinogenic to humans”.

Thus, regarding around 900?MHz RF exposure to humans via long-term cell phone use (i.e., essentially at the same parameters as our TEMT device), many epidemiologic studies from numerous laboratories have strongly affirmed that there is no enhanced risk of brain cancers or any other cancer. Although not at the 900?MHz frequency focused on in this review, in-home RF treatment at 27?MHz to patients with various cancers was not only safe, but appeared to induce anti-tumor effects [125, 126]. Particularly for liver cancer [125], it was concluded that daily RF treatment may increase the time to radiological progression of the disease. Such studies suggest that, far from causing cancer, RF treatment may actually be therapeutic against it

CONCLUSIONS

Since pharmacologic interventions against AD have thus far been unsuccessful in slowing or reversing the AD process, non-pharmacologic therapeutics against the disease must now be seriously considered. Based on a diversity of pre-clinical studies from our laboratory in collaboration with others, the neuromodulatory approach of TEMT appears to offer unique, disease-modifying potential that could limit or reverse AD memory loss. In reviewing the evidence from animal, cell culture, and human clinical studies, this article concludes that TEMT should be a safe therapeutic against AD and other neurodegenerative diseases, even with long-term utility. Our just-initiated Phase I clinical trial involving TEMT administration to AD subjects will provide an even more definitive assessment of TEMT’s safety and potential efficacy against AD.

ACKNOWLEDGMENTS

Funds for the research and writing of this paper have been provided by NeuroEM Therapeutics, Inc. (Phoenix, AZ). We thank our primary collaborators in this work, Drs. Chuanhai Cao and Patrick Bradshaw from the University of South Florida, as well as Dr. Takashi Mori of Saitama Medical University in Japan. We also thank David Kirk (Phoenix, AZ) for his graphic design expertise in the figures.

Authors’ disclosures available online (http://j-alz.com/manuscript-disclosures/16-0165r1).

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

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

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

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

Abstract

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

Introduction

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

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

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

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

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

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

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

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

Electromagnetic Brain Stimulation and Immunomodulation in Neurodegenerative Diseases

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

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

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

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

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

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

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

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

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

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

Low-frequency Electromagnetic Fields Stimulation and Autoimmunity

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

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

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

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

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

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

Low-frequency Electromagnetic Fields Stimulation and Immunomodulation

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

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

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

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

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

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

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

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

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

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

ELF-EMF stimulation and oxidative stress

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

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

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

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

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

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

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

ELF-EMF effects on pro-inflammatory chemokines

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

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

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

Studies have shown the anti-inflammatory effects of ELF-EMF in vivo; for instance, Selvam used a coil system emitting a 5 Hz frequency to treat rats with rheumatoid arthritis for 90 minutes, producing significant anti-exhudative effects and resulting in the restoration of normal functional parameters (Vianale et al., 2008). This anti-inflammatory effect was reported to be partially mediated through the stabilizing action of ELF-EMF on cell membranes, reflected the restoration of intracellular C^a2+ 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.

1950 MHz Electromagnetic Fields Ameliorate Aß Pathology in Alzheimer’s Disease Mice.

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.

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

Abstract

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

Introduction

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

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

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

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

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

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

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

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

Results

Behavioral assessment during long-term EMF treatment

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

Table 1

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 (2^nd block) of testing, irrespective of whether they were receiving EMF treatment or not (Fig. 1B). Furthermore, EMF treatment did not improve the poor performance (e.g, high escape latencies) of both Tg and NT mice in this task.

Figure 1

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

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

Figure 2

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

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

Body/brain temperature recording during long-term EMF treatment

Study I

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

Figure 3

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

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

Study II

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

Figure 4

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

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

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

Figure 5

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

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

AB immunohistochemistry

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

Figure 6

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

Discussion

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

Cognitive and AB deposition effects of EMF treatment

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 7

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 5^th day and 12^th 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]:

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 4^th trial and the 5^th 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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• Long-term electromagnetic field treatment enhances brain mitochondrial function of both Alzheimer’s transgenic mice and normal mice: a mechanism for electromagnetic field-induced cognitive benefit? [Neuroscience. 2011]
• Electromagnetic field treatment protects against and reverses cognitive impairment in Alzheimer’s disease mice. [J Alzheimers Dis. 2010]
• Protection against cognitive deficits and markers of neurodegeneration by long-term oral administration of melatonin in a transgenic model of Alzheimer disease. [J Pineal Res. 2009]
• Caffeine reverses cognitive impairment and decreases brain amyloid-beta levels in aged Alzheimer’s disease mice. [J Alzheimers Dis. 2009]
• Caffeine suppresses amyloid-beta levels in plasma and brain of Alzheimer’s disease transgenic mice. [J Alzheimers Dis. 2009.

J Alzheimers Dis. 2010;20(2):599-606.

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.

J Alzheimers Dis. 2010 Jan;19(1):191-210.

Electromagnetic field treatment protects against and reverses cognitive impairment in Alzheimer’s disease mice.

Arendash GW, Sanchez-Ramos J, Mori T, Mamcarz M, Lin X, Runfeldt M, Wang L, Zhang G, Sava V, Tan J, Cao C.

The Florida Alzheimer’s Disease Research Center, Tampa, FL, USA. arendash@cas.usf.edu

Abstract

Despite numerous studies, there is no definitive evidence that high-frequency electromagnetic field (EMF) exposure is a risk to human health. To the contrary, this report presents the first evidence that long-term EMF exposure directly associated with cell phone use (918 MHz; 0.25 w/kg) provides cognitive benefits. Both cognitive-protective and cognitive-enhancing effects of EMF exposure were discovered for both normal mice and transgenic mice destined to develop Alzheimer’s-like cognitive impairment. The cognitive interference task utilized in this study was designed from, and measure-for-measure analogous to, a human cognitive interference task. In Alzheimer’s disease mice, long-term EMF exposure reduced brain amyloid-beta (Abeta) deposition through Abeta anti-aggregation actions and increased brain temperature during exposure periods. Several inter-related mechanisms of EMF action are proposed, including increased Abeta clearance from the brains of Alzheimer’s disease mice, increased neuronal activity, and increased cerebral blood flow. Although caution should be taken in extrapolating these mouse studies to humans, we conclude that EMF exposure may represent a non-invasive, non-pharmacologic therapeutic against Alzheimer’s disease and an effective memory-enhancing approach in general.

QJM. 2010 Jun 16. [Epub ahead of print]

Bioelectromagnetics, complex behaviour and psychotherapeutic potential.

Pooley DT.

From the Institute of Medical Engineering and Medical Physics, Cardiff School of Engineering, Cardiff University, Queen’s Buildings, The Parade, CARDIFF CF24 3AA, Wales, UK.

Abstract

The brain is a complex non-linear dynamical system that is associated with a wide repertoire of behaviours. There is an ongoing debate as to whether low-intensity radio frequency (RF) bioelectromagnetic interactions induce a biological response. If they do, it is reasonable to expect that the interaction is non-linear. Contradictory reports are found in the literature and attempts to reproduce the subtle effects have often proved difficult. Researchers have already speculated that low-intensity RF radiation may offer therapeutic potential and millimetre-wave therapy is established in the countries of the former Soviet Union. A recent study using transgenic mice that exhibit Alzheimer’s-like cognitive impairment shows that microwave radiation may possibly have therapeutic application. By using a highly dynamic stimulus and feedback it may be possible to augment the small effects that have been reported using static parameters. If a firm connection between low-intensity RF radiation and biological effects is established then the possibility arises for its psychotherapeutic application. Low intensity millimetre-wave and peripheral nervous system interactions also merit further investigation. Controlled RF exposure could be associated with quite novel characteristics and dynamics when compared to those associated with pharmacotherapy.

Neurosci Lett. 2007 May 11;418(1):9-12. Epub 2007 Mar 1.

Fifty Hertz electromagnetic field exposure stimulates secretion of beta-amyloid peptide in cultured human neuroglioma.

Del Giudice E, Facchinetti F, Nofrate V, Boccaccio P, Minelli T, Dam M, Leon A, Moschini G.

Research & Innovation Company, Padova, Italy.

Abstract

Recent epidemiological studies raise the possibility that individuals with occupational exposure to low frequency (50-60 Hz) electromagnetic fields (LF-EMF), are at increased risk of Alzheimer’s disease (AD). However, the mechanisms through which LF-EMF may affect AD pathology are unknown. We here tested the hypothesis that the exposure to LF-EMF may affect amyloidogenic processes. We examined the effect of exposure to 3.1 mT 50 Hz LF-EMF on Abeta secretion in H4 neuroglioma cells stably overexpressing human mutant amyloid precursor protein. We found that overnight exposure to LF-EMF induces a significant increase of amyloid-beta peptide (Abeta) secretion, including the isoform Abeta 1-42, without affecting cell survival. These findings show for the first time that exposure to LF-EMF stimulates Abeta secretion in vitro, thus alluding to a potential link between LF-EMF exposure and APP processing in the brain.

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)