Heart Disease

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

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

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

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

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


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

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

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



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

The measurement of blood pressure in SHR rats

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

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

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

Pulsed electromagnetic field treatment

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

Detection of myocardium apoptosis

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

Determination of myocardial enzymes in plasma

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

Myocardium cells morphology via TEM

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

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

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

Measurement of nitric oxide concentration and Western blotting

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

Cell culture

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

Cell treatment (hypoxia/reoxygenation)

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

Cell viability assays

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

Measurement of intracellular reactive oxygen species levels

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

Data analysis

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


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

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

Figure 1

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

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

PEMF treatment could observably improve the abundance of EPCs

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

Figure 2

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

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

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

Figure 3

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

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

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

Figure 4

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

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

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

Figure 5

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

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

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

Figure 6

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

Effect of PEMF on NO releasing via Akt/eNOS pathway

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

Figure 7

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


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

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

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

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

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

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

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


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


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


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


This work was supported by the key laboratory program of the Education Commission of Shanghai Municipality

[grant number ZDSYS14005]



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Zoledronate inhibits ischemia-induced neovascularization by impairing the mobilization and function of endothelial progenitor cells. PLoS ONE. 2012;7:e41065. doi: 10.1371/journal.pone.0041065.[PMC free article] [PubMed] [Cross Ref] 15. Jin S., Pu S.X., Hou C.L., Ma F.F., Li N., Li X.H., Tan B., Tao B.B., Wang M.J., Zhu Y.C. Cardiac H2S generation is reduced in ageing diabetic mice. Oxid. Med. Cell. Longev. 2015;2015:758358.[PMC free article] [PubMed] 16. Cheing G.L., Li X., Huang L., Kwan R.L., Cheung K.K. Pulsed electromagnetic fields (PEMF) promote early wound healing and myofibroblast proliferation in diabetic rats. Bioelectromagnetics. 2014;35:161–169. doi: 10.1002/bem.21832. [PubMed] [Cross Ref] 17. Weintraub M.I., Herrmann D.N., Smith A.G., Backonja M.M., Cole S.P. Pulsed electromagnetic fields to reduce diabetic neuropathic pain and stimulate neuronal repair: a randomized controlled trial. Arch. Phys. Med. Rehabil. 2009;90:1102–1109. doi: 10.1016/j.apmr.2009.01.019. [PubMed] [Cross Ref] 18. Li J., Zhang Y., Li C., Xie J., Liu Y., Zhu W., Zhang X., Jiang S., Liu L., Ding Z. HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism. Cardiovasc. Res. 2013;99:674–684. doi: 10.1093/cvr/cvt139. [PubMed] [Cross Ref] 19. Goto T., Fujioka M., Ishida M., Kuribayashi M., Ueshima K., Kubo T. Noninvasive up-regulation of angiopoietin-2 and fibroblast growth factor-2 in bone marrow by pulsed electromagnetic field therapy. J. Orthop. Sci. 2010;15:661–665. doi: 10.1007/s00776-010-1510-0. [PubMed] [Cross Ref] 20. Asahara T., Masuda H., Takahashi T., Kalka C., Pastore C., Silver M., Kearne M., Magner M., Isner J.M. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ. Res. 1999;85:221–228. doi: 10.1161/01.RES.85.3.221. [PubMed] [Cross Ref] 21. Takahashi T., Kalka C., Masuda H., Chen D., Silver M., Kearney M., Magner M., Isner J.M., Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat. Med. 1999;5:434–438. doi: 10.1038/8462. [PubMed] [Cross Ref] 22. Freude B., Masters T.N., Robicsek F., Fokin A., Kostin S., Zimmermann R., Ullmann C., Lorenz-Meyer S., Schaper J. Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J. Mol. Cell Cardiol. 2000;32:197–208. doi: 10.1006/jmcc.1999.1066. [PubMed] [Cross Ref] 23. Martindale J.J., Fernandez R., Thuerauf D., Whittaker R., Gude N., Sussman M.A., Glembotski C.C. Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6. Circ. Res. 2006;98:1186–1193. doi: 10.1161/01.RES.0000220643.65941.8d. [PubMed] [Cross Ref] 24. Yu L., Lu M., Wang P., Chen X. Trichostatin A ameliorates myocardial ischemia/reperfusion injury through inhibition of endoplasmic reticulum stress-induced apoptosis. Arch. Med. Res. 2012;43:190–196. doi: 10.1016/j.arcmed.2012.04.007. [PubMed] [Cross Ref] 25. Maiuri M.C., Criollo A., Tasdemir E., Vicencio J.M., Tajeddine N., Hickman J.A., Geneste O., Kroemer G. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L) Autophagy. 2007;3:374–376. doi: 10.4161/auto.4237.[PubMed] [Cross Ref] 26. Lindqvist L.M., Heinlein M., Huang D.C., Vaux D.L. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak. Proc. Natl. Acad. Sci. U.S.A. 2014;111:8512–8517. doi: 10.1073/pnas.1406425111. [PMC free article] [PubMed] [Cross Ref] 27. Chandna S., Suman S., Chandna M., Pandey A., Singh V., Kumar A., Dwarakanath B.S., Seth R.K. Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of gamma-radiation. Int. J. Rad. Biol. 2013;89:1017–1027. doi: 10.3109/09553002.2013.825059. [PubMed][Cross Ref] 28. Xu M., Zhou B., Wang G., Wang G., Weng X., Cai J., Li P., Chen H., Jiang X., Zhang Y. miR-15a and miR-16 modulate drug resistance by targeting bcl-2 in human colon cancer cells. Zhonghua Zhong Liu Za Zhi. 2014;36:897–902. [PubMed] 29. Zuo L., Best T.M., Roberts W.J., Diaz P.T., Wagner P.D. Characterization of reactive oxygen species in diaphragm. Acta Physiol. (Oxf.) 2015;213:700–710. doi: 10.1111/apha.12410. [PubMed] [Cross Ref] 30. Kalogeris T., Bao Y., Korthuis R.J. Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning. Redox Biol. 2014;2:702–714. doi: 10.1016/j.redox.2014.05.006.[PMC free article] [PubMed] [Cross Ref] 31. Levraut J., Iwase H., Shao Z.H., Vanden H.T., Schumacker P.T. Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation. Am. J. Physiol. Heart Circ. Physiol. 2003;284:H549–H558. doi: 10.1152/ajpheart.00708.2002. [PubMed] [Cross Ref] 32. Dong R., Chen W., Feng W., Xia C., Hu D., Zhang Y., Yang Y., Wang D.W., Xu X., Tu L. Exogenous bradykinin inhibits tissue factor induction and deep vein thrombosis via activating the eNOS/phosphoinositide 3-kinase/Akt signaling pathway. Cell. Physiol. Biochem. 2015;37:1592–1606. doi: 10.1159/000438526. [PubMed] [Cross Ref] 33. Jin R.C., Loscalzo J. Vascular nitric oxide: formation and function. J. 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Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes. Nitric Oxide. 2015;46:204–212. doi: 10.1016/j.niox.2014.11.006. [PubMed] [Cross Ref] 38. Li Y.D., Ye B.Q., Zheng S.X., Wang J.T., Wang J.G., Chen M., Liu J.G., Pei X.H., Wang L.J., Lin Z.X., et al. NF-kappaB transcription factor p50 critically regulates tissue factor in deep vein thrombosis. J. Biol. Chem. 2009;284:4473–4483. doi: 10.1074/jbc.M806010200. [PMC free article] [PubMed] [Cross Ref] 39. Dong R., Xu X., Li G., Feng W., Zhao G., Zhao J., Wang D.W., Tu L. Bradykinin inhibits oxidative stress-induced cardiomyocytes senescence via regulating redox state. PLoS ONE. 2013;8:e77034. doi: 10.1371/journal.pone.0077034. [PMC free article] [PubMed] [Cross Ref] Klin Med (Mosk). 2003;81(1):24-7. Am J Transl Res. 2014; 6(3): 281–290. Published online May 15, 2014

Clinico-functional efficacy of medicinal and photon stabilization in patients with angina pectoris.

[Article in Russian]

Vasil’ev AP, Senatorov IuN, Strel’tsova NN, Gorbunova TIu.

Modification of erythrocytic membrane and the trend in clinicofunctional indices were studied in 90 patients with angina of effort (FC I-IV) in the course of treatment with a combination of membranoprotective drugs (group 1), magneto-laser radiation (group 2) and imitation of laser radiation (group 3). In patients of groups 1 and 2 the treatment resulted in stabilization of cell membrane accompanied with a hypotensive effect and increased exercise tolerance due to more effective cardiac performance.um chloride baths. Its efficacy was 63%. The effect was due to inhibition of high sympathico-adrenal system.

Klin Med (Mosk). 2000;78(3):23-5.

Characteristics of microcirculation and vascular responsiveness in elderly patients with hypertension and ischemic heart disease.

[Article in Russian]

Abramovich SG.

Microcirculation and vascular responsiveness were studied in 52 patients with arterial hypertension and ischemic heart disease versus 48 healthy elderly persons. The patients were found to have defects of the end blood flow in all links of microcirculation, longer and more severe vasoconstriction of conjunctival and skin vessels in response to norepinephrine and cold stimulation tests.

Crit Rev Biomed Eng. 2000;28(1-2):339-47.

The use of millimeter wavelength electromagnetic waves in cardiology.

Lebedeva AYu.

2nd Department of urgent cardiology at State Clinical Hospital, Russian State Medical University, Moscow.


This paper concerns the problems of the use of millimeter wavelength electromagnetic waves for the treatment of cardiovascular disease. The prospects for this use are considered.

Vopr Kurortol Fizioter Lech Fiz Kult. 1999 Sep-Oct;(5):7-9.

The characteristics of the geroprotective action of magnetotherapy in elderly patients with combined cardiovascular pathology.

[Article in Russian]

Abramovich SG, Fedotchenko AA, Koriakina AV, Pogodin KV, Smirnov SN.

Central hemodynamics, diastolic and pumping functions of the heart, myocardial reactivity, microcirculation and biological age of cardiovascular system were studied in 66 elderly patients suffering from hypertension and ischemic heart disease. The patients received systemic magnetotherapy which produced a geroprotective effect as shown by improved microcirculation, myocardial reactivity, central hemodynamics reducing biological age of cardiovascular system and inhibiting its ageing.

Vopr Kurortol Fizioter Lech Fiz Kult. 1996 Mar-Apr;(2):5-8.

The effect of exposure to magnetics and lasers on the clinical status and the electrophysiological indices of the heart in patients with cardiac arrhythmias.

[Article in Russian]

Budnar’ LN, Antiuf’ev VF, Oranskii IE, Bekhter TV.

Magnetolaser radiation has a considerable influence on electrophysiological condition of the sinus node and sinoatrial zone. There are cases when patients with sick sinus syndrome get rid of arrhythmia. The treatment is safe and promising for further studies.

Vopr Kurortol Fizioter Lech Fiz Kult. 1994 Mar-Apr;(2):18-20.

The effect of a low-frequency magnetic field on erythrocyte membrane function and on the prostanoid level in the blood plasma of children with parasystolic arrhythmia.

[Article in Russian]

Vasil’eva EM, Danilova NV, Smirnov IE, Kupriianova OO, Gordeeva GF.

As shown by clinical and biochemical evidence on 23 parasystolic children, the treatment with low-frequency magnetic field improves humoral and cellular processes participating in cardiac rhythm regulation. There is activation of Ca, Mg-ATPase in the red cells, a reduction of plasma thromboxane levels. Red cell phospholipid composition insignificantly change. Further courses of magnetotherapy may lower the risk of recurrent arrhythmia.

Vopr Kurortol Fizioter Lech Fiz Kult. 1993 Sep-Oct;(5):4-9.

Changes in intracellular regeneration and the indices of endocrine function and cardiac microcirculation in exposure to decimeter waves.

[Article in Russian]

Korolev IuN, Geniatulina MS, Popov VI.


An electron-microscopic study of rabbit heart with experimental myocardial infarction revealed that extracardiac exposure to decimetric waves (DW) activated intracellular regeneration in the myocardium. This was associated with enhanced circulation and endocrine activity in the heart. Most pronounced regeneration was registered in adrenal exposure, the effect of the parietal exposure being somewhat less.

Lik Sprava. 1992 May;(5):40-3.

The effect of combined treatment with the use of magnetotherapy on the systemic hemodynamics of patients with ischemic heart disease and spinal osteochondrosis.

[Article in Russian]

Dudchenko MA, Vesel’skii ISh, Shtompel’ VIu.

The authors examined 66 patients with ischemic heart disease and concomitant cervico-thoracic osteochondrosis and 22 patients without osteochondrosis. Differences were revealed in values of the systemic hemodynamics with prevalence of the hypokinetic type in patients with combined pathology. Inclusion of magnetotherapy in the treatment complex of patients with ischemic heart disease and osteochondrosis favours clinical improvement, normalization of indices of central and regional blood circulation.

Vopr Kurortol Fizioter Lech Fiz Kult. 1992 Sep-Dec;(5-6):13-8.

The effect of decimeter waves on the metabolism of the myocardium and its hormonal regulation in rabbits with experimental ischemia.

[Article in Russian]

Frenkel’ ID, Zubkova SM, Liubimova NN, Popov VI.


Biochemical and morphometric methods were employed to study the effect of decimetric waves (460 MHz, 10 and 120 mW/cm2) in cardiac and thyroid exposure on oxygen metabolism, myocardial microcirculation and contractility, thyroid and adrenal hormonal activity, kallikrein-kinin system activity in rabbits with experimental myocardial ischemia. Hypoxia discontinued in all the treatment regimens, but the exposure of the heart (field density 10 mW/sm2) had the additional effect on lipid peroxidation which reduced in the serum and normalized in the myocardium, on myocardial contractility, kallikrein-kinin system and on the adrenal and thyroid hormones.

Hearing Loss – Hypoacusis – Hyperacusis

Photomed Laser Surg. 2010 Jun;28(3):371-7.

Pain threshold improvement for chronic hyperacusis patients in a prospective clinical study.

Zazzio M.

Audio Laser-Kliniken, Flygeln, Hovmantorp, Sweden. audiolaser@mail.nu


OBJECTIVE: The aim of this study was to investigate if laser therapy in combination with pulsed electromagnetic field therapy/repetitive transcranial magnetic stimulation (rTMS) and the control of reactive oxygen species (ROS) would lead to positive treatment results for hyperacusis patients.

BACKGROUND DATA: Eight of the first ten patients treated for tinnitus, who were also suffering from chronic hyperacusis, claimed their hyperacusis improved. Based upon that, a prospective, unblinded, uncontrolled clinical trial was planned and conducted. ROS and hyperacusis pain thresholds were measured.

MATERIALS AND METHODS: Forty-eight patients were treated twice a week with a combination of therapeutic laser, rTMS, and the control and adjustment of ROS. A magnetic field of no more than 100 microT was oriented behind the outer ear, in the area of the mastoid bone. ROS were measured and controlled by administering different antioxidants. At every treatment session, 177-504 J of laser light of two different wavelengths was administered toward the inner ear via meatus acusticus.

RESULTS: The improvements were significantly better in the verum group than in a placebo group, where 40% of the patients were expected to have a positive treatment effect. The patients in the long-term follow-up group received significantly greater improvements than the patients in the short-term follow-up group.

CONCLUSION: The treatment is effective in treating chronic hyperacusis.

Vestn Otorinolaringol. 2002;(1):11-4.

Electrophysical effects in combined treatment of neurosensory hypoacusis.

Article in Russian]

Morenko VM, Enin IP.

The authors consider different methods of electrobiophysical impacts on the body in the treatment of neurosensory hypoacusis: laser beam, laser puncture, electrostimulation, magnetotherapy, magnetolasertherapy, electrophoresis, etc. These methods find more and more intensive application in modern medicine. Further success of physiotherapy for neurosensory hypoacusis depends on adequate knowledge about mechanisms of action of each physical method used and introduction of novel techniques.

Vestn Otorinolaringol. 2001;(4):10-2.

Cerebral hemodynamics in patients with neurosensory hearing loss before and after magnetotherapy.  a prospective clinical study.

Article in Russian]

Morenko VM, Enin IP.

Magnetotherapy effects on cerebral hemodynamics were studied using rheoencephalography (REG). When the treatment results and changes in cerebral hemodynamics were compared it was evident that normalization or improvement of vascular status in vertebrobasilar and carotid territories registered at REG results in better hearing. This confirms the role of vascular factor in pathogenesis of neurosensory hypoacusis of different etiology and effectiveness of magnetotherapy in such patients.

Vestn Otorinolaringol. 1996 Nov-Dec;(6):23-6.

The treatment of hypoacusis in children by using a pulsed low-frequency electromagnetic field.

[Article in Russian]

Bogomil’skii MR, Sapozhnikov IaM, Zaslavskii AIu, Tarutin NP.

The authors provide specifications of the unit INFITA supplied with ELEMAGS attachment of their own design; the technique of treating hypoacusis in children with utilization of impulse low-frequency electromagnetic field; the results of this treatment in 105 hypoacusis children. The method was found highly effective and valuable for wide practice.

Med Tekh. 1995 Mar-Apr;(2):40-1.

ELEMAGS. apparatus and clinical experience in its use in the treatment of children with hypoacusis and otalgia.

[Article in Russian]

Zaslavskii AIu, Sapozhnikov IaM, Markarov GS, Gelis IuS.

To enhance effectiveness of magnetotherapy in the treatment of otic diseases the authors propose to use impulse low-frequency electromagnetic field in combination with constant magnetic field. ELEMAGS equipment based on the above principles is introduced to treat cochlear neuritis and neurosensory hypoacusis in children.

Vopr Kurortol Fizioter Lech Fiz Kult. 1994 Jan-Feb;(1):16-9.

The magnetic amplipulse therapy of vestibular dysfunctions of vascular origin by using the Sedaton apparatus (experimental research).

[Article in Russian]

Mal’tsev AE.

The paper describes the results of combined utilization of magnetic field (MF), sinusoidal modulated current (SMC) and galvanic current (GC) generated by a specially devised unit “Sedaton”. This multimodality physiotherapy was tested in chronic experiments on 25 cats with experimental vascular and vestibular dysfunction. MF in combination with SMC displayed greater efficacy than in monotherapy. Positive physiological reactions were more pronounced.


Headache.  2010 Jul;50(7):1153-63. Epub 2010 Jun 10.
Transcranial magnetic stimulation for migraine: a safety review.
Dodick DW, Schembri CT, Helmuth M, Aurora SK.


Mayo Clinic, Phoenix, AZ, USA.



To review potential and theoretical safety concerns of transcranial magnetic stimulation (TMS), as obtained from studies of single-pulse (sTMS) and repetitive TMS (rTMS) and to discuss safety concerns associated with sTMS in the context of its use as a migraine treatment.


The published literature was reviewed to identify adverse events that have been reported during the use of TMS; to assess its potential effects on brain tissue, the cardiovascular system, hormone levels, cognition and psychomotor tests, and hearing; to identify the risk of seizures associated with TMS; and to identify safety issues associated with its use in patients with attached or implanted electronic equipment or during pregnancy.


Two decades of clinical experience with sTMS have shown it to be a low risk technique with promise in the diagnosis, monitoring, and treatment of neurological and psychiatric disease in adults. Tens of thousands of subjects have undergone TMS for diagnostic, investigative, and therapeutic intervention trial purposes with minimal adverse events or side effects. No discernable evidence exists to suggest that sTMS causes harm to humans. No changes in neurophysiological function have been reported with sTMS use.


The safety of sTMS in clinical practice, including as an acute migraine headache treatment, is supported by biological, empirical, and clinical trial evidence. Single-pulse TMS may offer a safe nonpharmacologic, nonbehavioral therapeutic approach to the currently prescribed drugs for patients who suffer from migraine.

Appl Psychophysiol Biofeedback. 2007 Dec;32(3-4):191-207. Epub 2007 Nov 2.

Headache treatment with pulsing electromagnetic fields: a literature review.

Vincent W, Andrasik F, Sherman R.

Department of Psychology, Georgia State University, PO Box 5010, Atlanta, GA 30302-5010, USA.


Pulsing electromagnetic field (PEMF) therapy may be a viable form of complementary and alternative medicine. Clinical applications include the treatment of fractures, wounds, and heart disease. More recent applications involve treatment of recurrent headache disorders. This paper reviews available studies investigating PEMF for headache management. Possible mechanisms for effects (neurochemical, electrophysical, and cardiovascular) are discussed. The available data suggest that PEMF treatment for headache merits further study. Suggestions for future research are provided.

EEG EMG Z Elektroenzephalogr Elektromyogr Verwandte Geb. 1985 Dec;16(4):227-30.

Cerebral use of a pulsating field in neuropsychiatry patients with long-term headache.

[Article in German]

Grunner O.

A pulsed magnetic field (f = 260 Hz; t = 3 ms; induction B = 1.9 mT; gradient = 0.5 mT/cm) was applied at 40 patients with headaches of various etiology. The change of cephalea intensity was evaluated according the patients statements. These statements were further compared with the changes of the EEG. By means of frequency analysis of the EEG significant changes in percentages of delta and alpha 1 activities (7.5-9.5/s) were stated after the application of the real treatment regarding the sham treatment. Any treatment lasted one half hour. The retreat of subjective difficulties as well as the amelioration of EEG were stated accordingly at headaches, which were bounded with cerebral arteriosclerosis, with states after cerebral concussion, with depressive neurosis, or with tension headache. Pulsed magnetic field could be applied only there, where the visual evaluation stated EEG as physiological.

Headache: The Journal of Head and Face Pain
Volume 39 Issue 8 Page 567  – September 1999
Treatment of Migraine With Pulsing Electromagnetic Fields: A Double-Blind, Placebo-Controlled Study
Richard A. Sherman, PhD; Nancy M. Acosta, BS; Linda Robson, BA
The effect of exposure to pulsing electromagnetic fields on migraine activity was evaluated by having 42 subjects (34 women and 8 men), who met the International Headache Society’s criteria for migraine, participate in a double-blind, placebo-controlled study. Each subject kept a 1-month, pretreatment, baseline log of headache activity prior to being randomized to having either actual or placebo pulsing electromagnetic fields applied to their inner thighs for 1 hour per day, 5 days per week, for 2 weeks. After exposure, all subjects kept the log for at least 1 follow-up month. During the first month of follow-up, 73% of those receiving actual exposure reported decreased headaches (45% good decrease, 14% excellent decrease) compared to half of those receiving the placebo (15% worse, 20% good, 0% excellent). Ten of the 22 subjects who had actual exposure received 2 additional weeks of actual exposure after their initial 1-month follow-up. All showed decreased headache activity (50% good, 38% excellent). Thirteen subjects from the actual exposure group elected not to receive additional exposure. Twelve of them showed decreased headache activity by the second month (29% good, 43% excellent). Eight of the subjects in the placebo group elected to receive 2 weeks of actual exposure after the initial 1-month follow-up with 75% showing decreased headache activity (38% good, 38% excellent). In conclusion, exposure of the inner thighs to pulsing electromagnetic fields for at least 3 weeks is an effective, short-term intervention for migraine, but not tension headaches.
Headache: The Journal of Head and Face Pain
Volume 38 Issue 3 Page 208  – March 1998
Initial Exploration of Pulsing Electromagnetic Fields for Treatment of Migraine
Richard A. Sherman, PhD; Linda Robson, BA; Linda A. Marden, MD
Two studies were conducted during which 23 patients with chronic migraine were exposed to pulsing electromagnetic fields over the inner thigh. In an open study, 11 subjects kept a 2-week headache log before and after 2 to 3 weeks of exposure to pulsing electromagnetic fields for 1 hour per day, 5 days per week. The number of headaches per week decreased from 4.03 during the baseline period to 0.43 during the initial 2-week follow-up period and to 0.14 during the extended follow-up which averaged 8.1 months. In a double-blind study, 9 subjects kept a 3-week log of headache activity and were randomly assigned to receive 2 weeks of real or placebo pulsing elactromagnetic field exposures as described above. They were subsequently switched to 2 weeks of the other mode, after which they kept a final 3-week log. Three additional subjects in the blind study inadvertently received half-power pulsing electromagnetic field exposures. The 6 subjects exposed to the actual device first showed a change in headache activity from 3.32 per week to 0.58 per week. The 3 subjects exposed to only half the dose showed no change in headache activity. Large controlled studies should be performed to determine whether this intervention is actually effective
Curr Rev Pain. 1999;3(5):342-347. Sphenopalatine Ganglion Analgesia. Day M. Texas Tech University Health Sciences Center, Department of Anesthesiology, 3601 4th Street, Room 1C282, Lubbock, TX 79430, USA. The sphenopalatine ganglion and its involvement in the pathogenesis of pain has been the subject of debate for the last 90 years. The ganglion is a complex neural center composed of sensory, motor, and autonomic nerves, which makes it difficult to determine its pathophysiology. Current indications for blockade of the sphenopalatine ganglion include sphenopalatine and trigeminal neuralgia, migraine and cluster headaches, and atypical facial pain. Methods of blockade use local anesthetics, steroids, phenol, and conventional radiofrequency and electromagnetic field- pulsed radiofrequency lesioning. The techniques for blockade range from superficial to highly invasive. Efficacy studies, though few and small, show promise in patients who have failed pharmacologic or surgical therapies. Anesth Pain Control Dent. 1992 Spring;1(2):85-9.

The management of craniofacial pain in a pain relief unit.

Hillman L, Burns MT, Chander A, Tai YM.

Russells Hall Hospital, Dudley, United Kingdom.

This paper reports the results of 34 craniofacial pain sufferers who were treated at the Dudley Pain Relief Unit over a 1-year period. Most of the patients were referred by their general medical practitioners. They were adults representing all age groups, with a female-male ratio of 4:1. The average history of pain was 5.5 years. Neuralgic pain (as distinct from temporomandibular joint dysfunction syndrome, migrainous disorders, and pain of iatrogenic origin) was most frequently seen. Oral drug therapy, local injection of corticosteroids and analgesics, peripheral neurolysis, magnetotherapy, hypnotherapy, and acupuncture were the lines of management available. By the end of this study period, pain had been relieved or eliminated in 30 of the patients (88%).


J Physiol Pharmacol.  2012 Sep;63(4):397-401. Pulsating electromagnetic field stimulation of urothelial cells induces apoptosis and diminishes necrosis: new insight to magnetic therapy in urology. Juszczak K, Kaszuba-Zwoinska J, Thor PJ. Source Department of Pathophysiology, Jagiellonian University, Medical College, Cracow, Poland. kajus13@poczta.onet.pl. Abstract The evidence of electromagnetic therapy (EMT) efficacy in stress and/or urge urinary incontinence, as well as in detrusor overactivity is generally lacking in the literature. The potential EMT action of neuromuscular tissue depolarization has been described. Because there is no data on the influence of pulsating electromagnetic fields (PEMF) on the urothelium, we evaluated the effect of PEMF stimulation on rat urothelial cultured cells (RUCC). In our study 15 Wistar rats were used for RUCC preparation. RUCC were exposed to PEMF (50 Hz, 45±5 mT) three times for 4 hours each with 24-hour intervals. The unexposed RUCC was in the same incubator, but in a distance of 35 cm from the PEMF generator. Annexin V-APC (AnV+) labelled was used to determine the percentage of apoptotic cells and propidium iodide (PI+), as standard flow cytometric viability probe to distinguish necrotic cells from viable ones. The results are presented in percentage values. The flow cytometric analysis was carried out on a FACS calibur flow cytometer using Cell-Quest software. In PEMF-unstimulated RUCC, the percentage of AnV+, PI+, and AnV+PI+ positive cells were 1.24±0.34%, 11.03±1.55%, and 12.43±1.96%, respectively. The percentages of AnV+, PI+, and AnV+PI+ positive cells obtained after PEMF stimulation were 1.45±0.16% (p=0.027), 7.03±1.76% (p<0.001), and 9.48±3.40% (p=0.003), respectively. The PEMF stimulation of RUCC induces apoptosis (increase of AnV+ cells) and inhibits necrosis (decrease of PI+ cells) of urothelial cells. This leads us to the conclusion that a low-frequency pulsating electromagnetic field stimulation induces apoptosis and diminishes necrosis of rat urothelial cells in culture. Vopr Kurortol Fizioter Lech Fiz Kult. 2005 Jan-Feb;(1):26-8.

Efficacy of general magnetotherapy in conservative therapy of uterine myoma in women of reproductive age.

[Article in Russian]

Kulishova TV, Tabashnikova NA, Akker LV.

Sixty women of the reproductive age with uterine myoma were divided into two groups. Thirty patients of the study group received combined therapy plus general magnetotherapy (GMT). Patients of the control group received only combined treatment. Ultrasound investigation registered a reduction in the size of myoma nodes by 16.7% in the study group, while in the controls myoma size did not change (p < 0.05). 1-year follow-up data for the study group demonstrated no cases of the myoma growth while 16.6% of the controls showed growth of myoma nodes, in 6.6% of them supravaginal myoma amputation was made for rapidly growing myoma.

Urologiia. 2004 Mar-Apr;(2):20-2.

Combined therapy of interstitial cystitis using the “Aeltis-Synchro-02-Iarilo” device.

[Article in Russian]

Kalinina SN, Molchanov AV, Rutskaia NS.

Multiple modality therapy of interstitial cystitis (IC)–the disease characterized by nicturia, pelvic pains, imperative pollakiuria–is considered. As IC nature is not well known, its treatment remains empiric. Among the underlying causes, most probable are autoimmune, allergic, infectious, neurological, vascular. Therefore, the treatment should be multi-modality. Most usable now is combined chemotherapy. Perspective is also IC treatment with medicines in combination with physiotherapy (electromagnetolaser AELTIS-SYNCHRO-02-YARILO”). Endovesical electrophoresis can be also applied.

Vopr Kurortol Fizioter Lech Fiz Kult. 1996 Sep-Oct;(5):22-5.

The effect of a low-frequency magnetic field on the clinico-immunological indices of patients with chronic inflammatory diseases of the organs of the female genital system.

[Article in Russian]

Markina LP, Iarustovskaia OV, Alisultanova LS, Derevnina NA, Gontar’ EV.

Low-frequency magnetic field generated by the vaginal inductor used in 120 females with chronic genital inflammation promoted a decrease in leukocytosis, elevation of total population of T-lymphocytes, inhibition of high proliferative activity in PHA test. However, marked immunocorrection was not reached.

Eur J Surg Suppl. 1994;(574):83-6.

Electrochemical therapy of pelvic pain: effects of pulsed electromagnetic fields (PEMF) on tissue trauma.

Jorgensen WA, Frome BM, Wallach C.

International Pain Research Institute, Los Angeles, California.

Unusually effective and long-lasting relief of pelvic pain of gynaecological origin has been obtained consistently by short exposures of affected areas to the application of a magnetic induction device producing short, sharp, magnetic-field pulses of a minimal amplitude to initiate the electrochemical phenomenon of electroporation within a 25 cm2 focal area. Treatments are short, fasting-acting, economical and in many instances have obviated surgery. This report describes typical cases such as dysmenorrhoea, endometriosis, ruptured ovarian cyst, acute lower urinary tract infection, post-operative haematoma, and persistent dyspareunia in which pulsed magnetic field treatment has not, in most cases, been supplemented by analgesic medication. Of 17 female patients presenting with a total of 20 episodes of pelvic pain, of which 11 episodes were acute, seven chronic and two acute as well as chronic, 16 patients representing 18 episodes (90%) experienced marked, even dramatic relief, while two patients representing two episodes reported less than complete pain relief.

Urol Nefrol (Mosk). 1996 Sep-Oct;(5):10-4.

Magnetic-laser therapy in inflammatory and postraumatic lesions of the urinary system.

[Article in Russian]

Loran OB, Kaprin AD, Gazimagomedov GA.

The authors discuss disputable problem of renal and ureteral tissue after trauma or inflammation. These cause irreversible morphological changes in the tissue. Poor results of the standard therapy urged the authors to try magnetic-laser therapy in urological clinic. The technique has been developed on experimental animal models. The resultant morphological characteristics of ureteral wall and parenchyma support the validity of magnetic-laser therapy in urological practice.

Vopr Kurortol Fizioter Lech Fiz Kult. 1996 Nov-Dec;(6):21-4.

A permanent magnetic field in the combined treatment of acute endometritis after an artificial abortion.

[Article in Russian]

Strugatskii VM, Strizhakov AN, Kovalenko MV, Istratov VG, Iakubovich DV.

117 patients with acute endometritis after induced abortion were examined using markers of wound process phases and treated according to the original method. This consists in combination of constant magnetic field with other modalities. Application of the constant magnetic field produced a significant clinical response and reduced the hospital stay through positive effect on healing of the endometrial wound.

Guillian – Barre Syndrome

Zh Nevropatol Psikhiatr Im S S Korsakova. 1989;89(4):41-4.

Current methods of pathogenetic therapy of infectious-allergic polyradiculoneuritis.

[Article in Russian]

Neretin VIa, Ki’riakov VA, Sapfirova VA, Agafonov BV.

This is a survey of the experience in using corticosteroids, plasmapheresis, immunodepressants, hyperbaric oxygenation, laser and magnetotherapy in treating the infectious-allergic Guillain-Barre polyradiculoneuritis. The indications and counter-indications to individual techniques are presented as related to the character and course of the disease. The principles of interrelation of these techniques with other drug and physical therapies are discussed. The authors infer that combination of plasmapheresis with corticosteroids is the best for acute polyradiculoneuritis and prolonged use of maintenance doses of corticosteroids and immunodepressants, physical methods and gymnastics are recommended for chronic polyradiculoneuritis.


Sci Rep. 2017 Nov 6;7(1):14544. doi: 10.1038/s41598-017-14983-9.

Extremely low frequency pulsed electromagnetic fields cause antioxidative defense mechanisms in human osteoblasts via induction of •O2- and H2O2.

Ehnert S1, Fentz AK2, Schreiner A3, Birk J3, Wilbrand B3, Ziegler P3, Reumann MK3, Wang H4, Falldorf K2, Nussler AK3.

Author information

1 Siegfried Weller Institute for Trauma Research, Eberhard-Karls-Universität Tübingen, Schnarrenbergstr. 95, D-72076, Tübingen, Germany. sabrina.ehnert@med.uni-tuebingen.de. 2 Sachtleben GmbH, Hamburg, Spectrum UKE, Martinistraße 64, D-20251, Hamburg, Germany. 3 Siegfried Weller Institute for Trauma Research, Eberhard-Karls-Universität Tübingen, Schnarrenbergstr. 95, D-72076, Tübingen, Germany. 4 Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Dadao 1277#, 430022, Wuhan, China.


Recently, we identified a specific extremely low-frequency pulsed electromagnetic field (ELF-PEMF) that supports human osteoblast (hOBs) function in an ERK1/2-dependent manner, suggesting reactive oxygen species (ROS) being key regulators in this process. Thus, this study aimed at investigating how ELF-PEMF exposure can modulate hOBs function via ROS. Our results show that single exposure to ELF-PEMF induced ROS production in hOBs, without reducing intracellular glutathione. Repetitive exposure (>3) to ELF-PEMF however reduced ROS-levels, suggesting alterations in the cells antioxidative stress response. The main ROS induced by ELF-PEMF were •O2 and H2O2, therefore expression/activity of antioxidative enzymes related to these ROS were further investigated. ELF-PEMF exposure induced expression of GPX3, SOD2, CAT and GSR on mRNA, protein and enzyme activity level. Scavenging •O2 and H2O2 diminished the ELF-PEMF effect on hOBs function (AP activity and mineralization). Challenging the hOBs with low amounts of H2O2 on the other hand improved hOBs function. In summary, our data show that ELF-PEMF treatment favors differentiation of hOBs by producing non-toxic amounts of ROS, which induces antioxidative defense mechanisms in these cells. Thus, ELF-PEMF treatment might represent an interesting adjunct to conventional therapy supporting bone formation under oxidative stress conditions, e.g. during fracture healing. Med Pr. 2014;65(3):343-9.

Effect of extremely low frequency magnetic field on glutathione in rat muscles.

[Article in Polish] Ciejka E, Jakubowska E, Zelechowska P, Huk-Kolega H, Kowalczyk A, Goraca A.



Free radicals (FR) are atoms, molecules or their fragments. Their excess leads to the development of oxidizing stress, the cause of many neoplastic, neurodegenerative and inflammatory diseases, and aging of the organism. Industrial pollution, tobacco smoke, ionizing radiation, ultrasound and magnetic field are the major FR exogenous sources. The low frequency magnetic field is still more commonly applied in the physical therapy. The aim of the presented study was to evaluate the effect of extremely low frequency magnetic field used in the magnetotherapy on the level of total glutathione, oxidized and reduced, and the redox state of the skeletal muscle cells, depending on the duration of exposure to magnetic field.


The male rats, weight of 280-300 g, were randomly devided into 3 experimental groups: controls (group I) and treatment groups exposed to extremely low frequency magnetic field (ELF-MF) (group II exposed to 40 Hz, 7 mT for 0.5 h/day for 14 days and group III exposed to 40 Hz, 7 mT for 1 h/day for 14 days). Control rats were kept in a separate room not exposed to extremely low frequency magnetic field. Immediately after the last exposure, part of muscles was taken under pentobarbital anesthesia. Total glutathione, oxidized and reduced, and the redox state in the muscle tissue of animals were determined after exposure to magnetic fields.


Exposure to low magnetic field: 40 Hz, 7 mT for 30 min/day and 60 min/day for 2 weeks significantly increased the total glutathione levels in the skeletal muscle compared to the control group (p < 0.001).


Exposure to magnetic fields used in the magnetic therapy plays an important role in the development of adaptive mechanisms responsible for maintaining the oxidation-reduction balance in the body and depends on exposure duration.


Logo of ijerph

Int J Environ Res Public Health. 2016 Nov; 13(11): 1128. Published online 2016 Nov 12. doi:  10.3390/ijerph13111128 PMCID: PMC5129338

An Overview of Sub-Cellular Mechanisms Involved in the Action of TTFields

Jack A. Tuszynski,1,2,* Cornelia Wenger,3 Douglas E. Friesen,1 and Jordane Preto1 Mats-Olof Mattsson, Academic Editor 1Department of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada; ac.atreblau@neseirfed (D.E.F.); moc.liamg@oterp.enadroj (J.P.) 2Department of Physics, University of Alberta, Edmonton, AB T6G 2E1, Canada 3The Institute of Biophysics and Biomedical Engineering, Faculdade de Ciências, Universidade de Lisboa, Lisboa 1749-016, Portugal; tp.lu.cf@regnewc*Correspondence: ac.atreblau@tkcaj; Tel.: +1-780-432-8906 Received 2016 Jun 26; Accepted 2016 Nov 7. Copyright © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).


Long-standing research on electric and electromagnetic field interactions with biological cells and their subcellular structures has mainly focused on the low- and high-frequency regimes. Biological effects at intermediate frequencies between 100 and 300 kHz have been recently discovered and applied to cancer cells as a therapeutic modality called Tumor Treating Fields (TTFields). TTFields are clinically applied to disrupt cell division, primarily for the treatment of glioblastoma multiforme (GBM). In this review, we provide an assessment of possible physical interactions between 100 kHz range alternating electric fields and biological cells in general and their nano-scale subcellular structures in particular. This is intended to mechanistically elucidate the observed strong disruptive effects in cancer cells. Computational models of isolated cells subject to TTFields predict that for intermediate frequencies the intracellular electric field strength significantly increases and that peak dielectrophoretic forces develop in dividing cells. These findings are in agreement with in vitro observations of TTFields’ disruptive effects on cellular function. We conclude that the most likely candidates to provide a quantitative explanation of these effects are ionic condensation waves around microtubules as well as dielectrophoretic effects on the dipole moments of microtubules. A less likely possibility is the involvement of actin filaments or ion channels.Keywords: electric fields, biological cells, cancer cells, microtubules, ions, TTFields

1. Introduction

The effects of external electric fields on biological cells have been extensively studied both in the direct current (DC) and alternating current (AC) cases [1]. In order to elucidate possible impact of electric fields on cells, various experimental assays as well as analytical and computational models have been developed in the past. Experimentally obtained findings were further translated into biomedical applications. While DC or low-frequency AC fields are used to induce stimulation of excitable cells through membrane depolarization or to promote wound healing, high-frequency AC fields are associated with tissue heating and membrane rupture, thus finding its application in diathermy or ablation techniques.

Intermediate-frequency AC electric fields in the kHz to MHz range were commonly assumed to lead to no significant biological effects [1]. However, in a major breakthrough paper, Kirson et al. [2] reported the discovery that low-intensity (1–3 V/cm), intermediate frequency (100–300 kHz) electric fields have a profoundly inhibitory effect on the growth rate of various mammalian tumor cell lines [2,3,4]. This discovery has been translated into a clinical application termed Tumor Treating Fields (TTFields). Based on the results of a Phase III clinical trial [5], TTFields have been approved by the United States Food and Drug Administration (FDA) in 2011 for the treatment of recurrent glioblastoma multiforme (GBM) and their efficacy in treating other solid tumor types is currently being investigated clinically [6]. In late 2015, TTFields were also approved for newly diagnosed GBM patients in combination therapy with temozolomide [7,8] due to significantly increased survival times.

It should be noted that electromagnetic (EM) fields may affect the regulation of cellular growth and differentiation, including the growth of tumors [9,10]. Both static magnetic and electric fields have altered the mitotic index and cell cycle progression of a number of cell types in various species [10]. EM low-frequency fields in the range of 50–75 Hz cause perturbations in the mitotic activity of plant and animal cells and a significant inhibitory effect on mitotic activity occurs early during exposure [11,12,13]. While the field amplitudes used are consistent with those of interest to this report, the frequencies are orders of magnitude lower.

The reduction in the cell number due to an application of TTFields was studied by in vitro experiments with various cancer cell lines. A significant prolongation of mitosis was predicted, where treated cells remain stationary at metaphase for several hours, which was accompanied by abnormal mitotic figures as well as membrane rupture and blebbing leading to apoptosis [2,3]. Furthermore, these experiments showed that the inhibitory effect increases with an increasing electric field intensity, resulting in a complete proliferation arrest of rat glioma cells after 24 h exposure to a field intensity of 2.25 V/cm. Additionally, the effects of TTFields have been shown to be frequency-dependent, with a cancer cell line-specific peak frequency of the maximal inhibitory effect, e.g., 200 kHz for glioma cells [3]. Following these experimental results, two specific mechanisms of action of TTFields have been proposed [2,3,4] which we describe below.

Firstly, the applied field is expected to interfere with proper microtubule (MT) formation preventing a functioning mitotic spindle, due to the force of interactions with the large intrinsic dipole moments of the tubulin dimers [14,15,16] that make up MTs. It has been hypothesized that the tubulin dimers might align parallel to the direction of the applied electric field, rather than along the MT axis. Secondly, the cellular morphology during cytokinesis gives rise to a non-uniform intracellular electric field, with a high density at the cleavage furrow between the dividing cells. This non-uniform field leads to the development of dielectrophoretic (DEP) forces [17] acting on polarizable macromolecules such as MTs, organelles and all charged structures present in the cell, such as ions, proteins or DNA.

Thus, TTFields are considered to be suitable as a novel anti-mitotic cancer treatment modality. In fact, it has been suggested by numerous researchers that endogenous electric fields may play a key role during mitosis. Similar to Cooper [18], Pohl et al. [19] proposed that the onset of mitosis is associated with a ferroelectric phase transition, which establishes an axis of oscillation for the cellular polarization wave. The mitotic spindle apparatus would delineate the polarization field with MTs lined up along the electric field lines. The poles are expected to experience the highest field intensities while the equatorial plane is likely to provide a nodal manifold for the fields. Consequently, the chromosome condensation during this transformation was predicted to be induced by the static dielectric polarization of the chromatin complex as a result of the cellular ferroelectric phase transition. These conclusions have been supported by experimental evidence for peak EM activity during mitosis [20,21] and by physical modeling of the electrostatic forces generated by MTs which generate mechanical force required for chromosome segregation during mitosis and influence chromosomal motion [15,16,22]. A detailed review of this aspect can be found elsewhere [1].

Put together, there is reasonable evidence that especially during mitosis, electric field effects are relevant for the functioning of a dividing cell, especially in the creation of the mitotic spindle. However, to date a rigorous quantitative analysis of the magnitude of these effects within cells exposed to TTFields has not been performed. Furthermore, an analysis of how TTFields might interact with subcellular structures has also never been reported. In a quantitative model, which attempts to explain these effects, an energetic constraint, both from below and above, must be kept in mind. Firstly, for an effect to be of significance at a molecular level, its interaction energy must exceed thermal energy, i.e., kT per degree of freedom (i.e., 4 × 10?21 J). Otherwise, thermal fluctuations will disrupt the action of electric fields. Secondly, it must not produce so much thermal energy as to seriously increase the temperature of the cell. In terms of practical comparisons, a cell generates approximately 3 × 10?12 W of power (3 × 10?12 J/s), much of which is used to maintain a constant physiological temperature. This is found from a simple estimate of energy production by the human body which is 100 W divided by the number of cells in the body which is approximately 3 × 1013 [23]. In terms of subcellular forces at work, a minimal amount of useful force at a nanometer scale is 1 pN. Motor proteins generate forces on the order of several pN. A force of 1 pN applied to a tip of a microtubule may be used to bend it by as much as 1 µm [24]. Below, we review electric conduction effects for subcellular structures of interest.

The paper is structured as follows. In Section 2, we review what is known about the shape and intensity of the electric field within cells exposed to externally applied electric fields, focusing on cells during mitosis. As a preparation for following sections, Section 3 offers a general introduction to subcellular electrical conduction and electrostatics. Section 4 and Section 5 are devoted to a comprehensive review of the literature concerned with the effects of electric fields on biopolymers, and with the identification of additional mechanisms by which TTFields might interact with cells. Section 4 covers electric field interactions with the cell membrane and the cytosol, whereas the focus of Section 5 penetrates deeper into the cell, shedding light on the electric field effects on subcellular structures of interest, i.e., microtubules (MTs), actin filaments (AFs), ionic charges and DNA. Finally, in Section 6, we present a discussion about the significance of our findings and about future directions of research that should be undertaken in this area. We hope this paper will set a solid theoretical foundation for future studies into the biophysics of TTFields. 2. Induced Electric Fields within Biological Cells in Mitosis

The topic of induced electric fields in and around biological cells subject to DC or AC fields has been investigated for decades. The preliminary and most popular studies on the analytical description of steady-state trans-membrane potential induced on spherical cells go back to the work of H.P. Schwan and colleagues [25]. Arguments were presented to account for the influence of the membrane conductance, surface admittance and spatial charge effects [25], as well as for the geometric and material properties of the cell and the surrounding medium [26]. The impact of external electric fields on a living cell significantly depends on the cell’s shape. Concerning analytical solutions for non-spherical cell shapes, many authors proposed appropriate adaption of the governing equations going back to the work presented in Reference [27]. Later models aimed to study electric polarization effects on oblate and prolate homogeneous and single-shell spheroids have been developed [28]. They were later extended to arbitrarily oriented cells of the general ellipsoidal shape [29]. Importantly, the induced electric field inside a spherical cell is uniform, whereas increasing non-uniformities are predicted for deviations of the regular shape.

Another important aspect is the frequency-dependency of the induced trans-membrane voltage and thus also the intracellular field strength, as predicted by the above-mentioned studies and additional research reported elsewhere [30,31,32,33,34]. For low frequencies, the intracellular space is shielded to a large degree from extracellular electric fields. For example, the electric field strength inside a typical spherical cell is approximately five orders of magnitude lower than that outside the cell [35,36]. However, as the frequency of the field increases, the high membrane field gain diminishes, allowing for higher field intensities to penetrate into the cell.

Recently, Wenger et al. [37] developed a computational model to study the application of TTFields to isolated cells during mitosis, specifically during metaphase and at different stages of cytokinesis. Comsol Multiphysics (www.comsol.com) was used to solve for the scalar electric potential V for frequency ranges between 60 Hz and 10 GHz. With voltages of opposite signs set as boundary conditions, a uniform field of 1 V/cm was induced in the model domain. Following 3D confocal microscopy findings [38,39], the metaphase cell was represented by a sphere with a 10 µm radius and three different stages of cytokinesis were modeled with increased distance between the elliptical mother and daughter cell (see Figure 1, left panel). Three model domains, the extracellular space, the cytoplasm and the membrane, were assigned typical dielectric properties, electrical conductivity and relative permittivity [37].

Figure 1

Figure 1 (Left) Schematic diagrams of the cell geometries for metaphase and three stages of cytokinesis. Black lines indicate the electric field contours. (Right) The maximum intracellular electric field strength in V/cm plotted as a function of field frequency 

For a spherical cell during metaphase, the modeling results predict that for frequencies lower than 10 kHz only small changes of the field are detected and the intracellular field strength, Ei, almost equals zero. A first significant increase of Ei is observed at approximately 200 kHz, and Ei increases rapidly as the frequencies increase above this value. This can be seen in the inset of the right panel in Figure 1, which shows a zoomed view of the blue M-phase cell. This transition region depends on the dielectric properties of the cell and its membrane. Nonetheless, above 1 MHz electric current is shunted across the membrane and the impedance is dominated by the cytoplasm. Thus, for an increasing frequency, the electric field inside the cell is augmented and at 1 GHz the cellular structure becomes ‘‘electrically invisible’’ as previously reported [33]. The directions of the electrical field near the cell membrane resemble already predicted results [40].

The model further showed that within the dividing cell the intracellular electric field distribution is non-uniform with highest field intensities at the cleavage plane (black lines in the left panel of Figure 1). These maximum intensities are much higher than the applied field and appear for frequencies in the range 100–500 kHz depending on the stage of cytokinesis, i.e., how far the cell division has already progressed. The corresponding curves are plotted in the right panel of Figure 1, where the highest maximum intracellular field strength of ~22 V/cm is observed for the cell in late cytokinesis.

Due to the inhomogeneity in the electric field distribution, significant dielectrophoretic (DEP) forces are expected to develop within the cell and these DEP forces are thought to be important factors in the mechanism of action of TTFields [41]. This DEP force causes the motion of polarizable particles as a result of the interaction of a non-uniform electric field with their induced dipole moment F=p??E [42]. The DEP force is proportional to the volume of the particle v, its effective polarizability ? and the square of the gradient of the electric field according to: ?F DEP?=1/2?v?Re[?(E˜??)E˜?] using complex phasor notation [42,43]. Thus, the magnitude of the DEP force component is proportional to the magnitude of the gradient of the squared electric field, |F|????|E|2?? in (V2/m3). The DEP force component showed well-defined peak frequencies at 500, 200, and 100 kHz, respectively, for the three stages considered, from the earliest to the latest stage [37]. This coincides with the peaks of the maximum electric field inside the cell, which are presented in the right panel of Figure 1.

Apart form testing different field intesities, the computational study tested another aspect of TTFields. Namely, it has been shown that the optimal frequency for the inhibitory effect of TTFields is inversely related to cell size [2,44] and that cell volume is increased in almost all cell lines treated with TTFields [45]. The simulation results predicted that the above-mentioned peak frequencies decrease and converge as a function of an increasing cell radius. The corresponding maximum values of the DEP force component also decrease with an increasing cell size with equal decay rates for all cytokinesis stages [37].

In summary, these results obtained by computational modeling confirmed several predicted outcomes of the application of TTFields to biological cells. During metaphase a uniform non-zero Ei is induced. Depending on cell properties, the frequency window of the predicted transition range might be shifted. During cytokinesis, a non-uniform Eiis induced with a substantially increased strength at the cleavage furrow. Frequency-, cell size-, and field-intensity dependences were confirmed.

Experimental validation of the predicted induced field strength values would be of great interest. Electric field strengths have typically only been able to be measured inside membranes with voltage dye and patch-clamp techniques. A promising technique by Tyner et al. [46] reported the generation of a nanovoltmeter that can report local electric fields in the cell and its use would be ideal to calibrate the strength and local distribution of electric fields in the presence of externally applied AC electric fields.

3. Subcellular Electrical Conduction and Electrostatics

3.1. Protein Conduction

Biological polymers are made up of various proteins, such as actin and tubulin, or nucleic acids as is the case of DNA and RNA. These structures have uncompensated electrical charges when immersed in water but ionic solutions such as the cytoplasm provide a bath of counter-ions that at least partially neutralize the net electric charge. This, however, results in dipolar and higher-moment electric field distributions complicating the situation greatly. Biological water is also believed to create structures with ordered dipole moments and complex dynamics at multiple scales [47], which adds to the complexity of subcellular electric field effects. Additionally, free ions endow the cell with conducting properties along well-defined polymeric pathways as well as in a diffusive way. Membranes support strong electric fields (on the order of 105 V/cm), which, due to counter-ion attraction to charged surfaces in solution, result in Debye screening. This causes an exponential decay of these electric fields on a nm scale [48] but not their complete disappearance when measured in the cell interior (hence a field strength of 105 V/cm decreases to approximately 0.01 V/cm over 100 nm).

The idea that proteins in organisms may have semiconducting properties dates back many decades [49,50] but protein conductivity has been found to be strongly dependent on the hydration state of proteins [51]. Electrical properties of cells and their components were promoted by Szent-Györgyi [52,53], but significant experimental challenges of measuring electric fields and currents at a sub-cellular level impeded progress in this field. The development of more precise experimental tools in the area of nanotechnology holds great promise for rapid progress in the near future [54,55]. Owing to the fact that there have been many previous reviews of electromagnetic effects in biology [1,56,57,58], here we mainly focus on the electrical properties of MTs, actin AFs, ion channels, cytoplasmic ions and DNA with special interest into dynamical electrical properties involving AC fields in the range of 100 kHz. A crucial role of water in the transmission of electrical pulses due to the structure imparted by hydrophilic surfaces [59] is also worth noting. Charge carriers related to protein semi-conduction have largely been electrons, protons as well as ions surrounding proteins in physiological solution. AFs and MTs have been implicated in facilitating numerous electrical processes involving ionic and electronic conduction [60,61] and have been theorized to support dipolar and/or ionic kink-like soliton waves traveling at speeds in the 2–100 m/s range [14,62]. Due to strong coupling between electrical and mechanical degrees of freedom in proteins, mechano-electric vibrations of MTs have been modeled both analytically and computationally [63,64]. Electric fields generated by MTs have been modeled extensively and reviewed recently [65,66,67], although experimental measurement of these fields remains extremely difficult, especially in a live cellular environment.

3.2. Electrostatic Interactions Involving Charges and Dipoles of Tubulin

The net charge on a tubulin dimer depends on pH and changes from +5 at pH 4.5 to 0 at pH 5 and drops to ?30 at pH 8 [68]. However, in the cytoplasm, a vast majority of electrostatic charges are screened over the distances greater than the Debye length (which varies between 0.6 and 1.5 nm depending on the ionic strength). Therefore, calculating the force due to an electric field of a static electric field with a strength of 1 V/cm acting on a 10 µm-long microtubule, we find from F = qE, with q = 10?13 C for unscreened charges, that results in F = 10 pN assuming the field is largely undiminished when penetrating a cell, which is in general a major oversimplification. This latter issue will be addressed at the end of this review. Even if the force is essentially unchanged, the Debye screening of electrostatic charges means that less than 5% of the charge remains exposed to the field resulting in a net force of at most 0.5 pN, most likely insufficient to exert a major influence on the cytoskeleton. If the field oscillates rapidly, the net force would cancel out over the period of these oscillations, i.e., on a time scale of microseconds or less.

The next aspect of MT electrostatics is the effect on the dipole moments of tubulin dimers and of entire MTs. The dipole moment of tubulin (excluding the very flexible and dynamic C-termini which we discuss separately below) has been estimated to be between 566 debye for the ?-monomer and 1714 for the ?-monomer [69]. However, this is also strongly tubulin-isotype dependent, so these numbers vary a lot between various tubulin isotypes from 500 to 4000 debye [70]. Note that 1 debye is a unit of electric polarization and is equal to 3.33 × 10?30 Cm. Therefore, taking the dipole moment of a free tubulin dimer as p = 3000 debye as a representative number, we find the interaction energy U with an electric field of E = 1 V/cm, and obtain U = ?pE, and hence U = 10?24 J. This is clearly too small (4000 times smaller than thermal energy kT) to affect the dynamics of an individual tubulin dimer. However, a single MT contains 1625 dimers per 1 µm of its length, so it could eventually accumulate enough net dipole strength to be significantly affected by the field. Unfortunately, this is very unlikely because of the almost perfect radial symmetry of tubulin dipole arrangements in an MT, which has been predicted by a computer simulation [70]. The individual dipole moments of constituent dimers will almost perfectly cancel out in the radial arrangement of an MT cylinder. There is a small non-cancellation effect along the MT axis but this amounts to less than 10% of the next dipole moment, hence it is doubtful that an entire MT can be aligned in electric fields with intensities lower than 10 V/cm. Unless one uses time-dependent fields (e.g., those used in Reference [71]), much stronger fields are needed for static effects. To put it another way, the torque ? between a dipole moment of an MT, p, and an external electric field, E, is proportional to their vector product: ? = pxE. For the force to have a meaningful effect on a microtubule, it should exceed 1 pN for lever arm on the order of 1 µm giving a torque of 10?18 Nm. With fields on the order of 1 V/cm, and a dipole moment of 3000 debye per tubulin dimer, even if these dipoles were perfectly aligned, it would result in a 1 µm MT only experiencing a torque of 10?21 Nm, which is approximately 1000 times too low to be of relevance.

Various special situations involving electrostatic effects on MTs were calculated earlier [68]. Note that a force between a charge and an electric field is given by F = qE(x) where E(x) is screened exponentially over the Debye length, which is approximately 1 nm. Hence, a test charge of +5e a distance of 5 nm from the MT surface for a 10-µm MT, experiences a force of 12 pN in water and 1 pN in ionic solution. A tubulin dimer with a dipole of 3000 debye in the vicinity of a microtubule experiences an electrostatic energy of 3 meV. MT-MT interactions due to their net charges with Debye screening accounted for lead to a net force of 9 pN when separated by 40 nm resulting in net repulsion between them. However, at longer distances attractive forces prevail and the corresponding dipole-dipole attraction at 90 nm is only 0.08 pN. The authors of the references [15,16,22] estimated the maximum electrostatic force in the mitotic plate, which was given as F = 6n2 pN per MT where n is the number of elementary charges on each protofilament. Since F is estimated to be 1–74 pN for a typical MT, the estimate is 0.4–3.5 uncompensated elementary charges per protofilament. The range of values of the forces involved is certainly within the realm of possible force requirements for chromosome segregation (about 700 pN per chromosome).

3.3. MT Conductivity

The building block of an MT is a tubulin dimer, containing approximately 900 amino acid residues with a combined mass of 110 kDa (1 Da is the atomic unit of mass, 1 Da = 1.7 × 10?27 kg). Each tubulin dimer in an MT has a length of 8 nm, along the MT cylinder axis, a width of about 6.5 nm and the radial dimension of 4.6 nm. The inner core of the cylinder, known as the lumen, is approximately 15 nm in diameter. MTs have been predicted to exhibit intrinsic electronic conductivity as well as ionic conductivity along their length [72]. MTs have a highly electro-negatively charged outer surface as well as C-terminal tails (TTs), resulting in a cloud of counter-ions surrounding them. Experiment and theory demonstrate that ionic waves are amplified along MTs [72,73]. Since MTs form a cylinder with a hollow inner volume (lumen), MTs have also been theorized to have special conducting properties involving the lumen [55] but there has been no direct experimental determination of the electric properties of the MT lumen. Many diverse experiments were performed to date in order to measure the various conductivities of MTs, with a range of results largely dependent on the experimental method, and this has been reviewed elsewhere [74].

Interestingly, Sahu et al. [75,76] measured conductivity along the periphery of MTs, where the DC intrinsic conductivities of MTs, from a 200 nm gap, were found to be approximately 10?1 to 102 S/m. Unexpectedly, MTs at certain specific AC frequencies (in several frequency ranges) were found to be approximately 1000 times more conductive, exhibiting astonishing values for the MT conductivities in the range of 103to 105 S/m [55,76]. Some resonance peaks for solubilized tubulin dimers were reported as: 37, 46, 91, 137, 176, 281, and 430 MHz; 9, 19, 78, 160, and 224 GHz; and 28, 88, 127, and 340 THz. However, for MTs, the corresponding resonance peaks were given as: 120, 240, and 320 kHz; 12, 20, 22, 30, 101, 113, 185, and 204 MHz; and 3, 7, 13, and 18 GHz. Therefore, for MTs there is some overlap with the 100 kHz range indicating a possible independent confirmation of the sensitivity of MT AC conductivity to this electric field frequency range. These authors showed experimental evidence that the high conductivity of the MT at specific AC frequencies only occurred when the water channel inside the lumen of the MT remained intact [55].

Electro-orientation experiments involving MTs have shown an increased ionic conductivity (0.150 S/m) compared to the buffer solution free of tubulin by as much as 15-fold [77]. MTs exposed to low frequency AC fields (f < 10 kHz) exhibit a flow motion due to ionic convection. However, for frequencies above 10 kHz this convection effect is absent. Electric fields with intensities above 500 V/cm and frequencies in the range of 10 kHz–5 MHz, are able to orient MTs in solution. As a point of interest, this frequency range overlaps with the range used by Kirson et al. [2]. However, the intensities of the electric fields used are substantially higher. For instance, a 900 V/cm field with f = 1 MHz was able to align MTs within several seconds [77]. Impedance spectroscopy enabled the measurements of the dielectric constant of tubulin as ? = 8.41 [78]. Uppalapati et al. [79] exposed taxol-stabilized MT’s in solution to an AC field, which exhibited electro-osmotic and electro-thermal flow, in addition to MT dielectrophoresis effects. Interestingly above f = 5 MHz, electro-hydrodynamic flows were virtually eliminated, and the conductivity of MTs was estimated at 0.25 S/m.

Priel et al. demonstrated MTs’ ability to amplify ionic charge conductivity, with current transmission increasing by 69% along MTs [60], which was explained by the highly negative surface charge density of MTs that creates a counter-ionic cloud subjected to amplification along the MT axis [60]. From Priel et al.’s conductance data, the approximate ionic conductivity of MTs is found to be an astonishing 367 S/m [74]. Below, in the second part of this review, we quantitatively assess AC electric fields on these ionic conductivity experiments, which are expected to be sensitive to the electric field frequencies in the 100 kHz to 1 MHz range.

The multiple mechanisms of MT conductance provide ample possibility to explain the varied reports on MT conductivity in the literature. Ionic conductivity along the outer edge of the MT, intrinsic conductivity through the MT itself, and possible proton jump conduction and conductivity through the inner MT lumen have all been suggested. It is conceivable that TTFields may affect ionic conductivities along MTs as is argued below.

4. Collective Effects in the Membrane and Cytoplasm

4.1. Membrane Depolymerization Effects

The electric field across the membrane is on the order of 105 V/cm (0.1 V over 8–10 nm), which is 4–5 orders of magnitude greater than TTFields’ amplitude. Therefore, a direct effect of TTFields on cancer cells’ membrane potential is expected to be very minor.

4.2. Ion Channel Conduction Effects

Liu et al. [80] reported activation of a Na+ pumping mode with an oscillating electric field with a strength of 20 V/cm, which is comparable to the fields of interest in this review, but at a much higher frequency (1.0 MHz) than those of interest. Moreover, neither K+ efflux nor Na+ influx was stimulated by the applied field in the frequency range from 1 Hz to 10 MHz. These results indicate that only those transport modes that require ATP splitting under the physiological condition were affected by the applied electric fields, although the field-stimulated K+ influx and Na+ efflux did not depend on the cellular ATP concentration in the range 5 to 800 µM. Computer simulation of a four-state enzyme electro-conformationally coupled to an alternating electric field [81,82] reproduced the main features of the above results.

Channel densities strongly vary among different neuronal phenotypes reflecting different stabilities of resting potentials and signal reliabilities. In model cell types such as in mammalian medial enthorinal cortex cells, modeled and experimental results match best for an average of 5 × 105 fast conductance Na+ and delayed rectifier K+ channels per neuron [83]. In unmyelinated squid axons counts can reach up to 108 channels per cell. In model channels such as the bacterial KcsA channel one K+ ion crosses the channel per 10–20 ns under physiological conductances of roughly 80–100 pS [84], which is consistent with the frequencies of external electric stimulation mentioned above. This allows for a maximum conduction rate of about 108 ions/s. Estimating the distances between the center of the channel pore and the membrane surface to scale along 5 nm and assuming the simplest watery-hole and continuum electro-diffusion model of channels, this would provide an average speed of 0.5 m/s per ion. Ion transition occurs through a sequence of stable multi-ion configurations through the filter region of the channels, which allows rapid and ion-selective conduction [85]. The motion of ions within the filter was intensively studied applying classical molecular dynamics (MD) methods (for a summary see Reference [86]) and density functional studies (e.g., [87]). MD methods used in these simulations solve Newton’s equations of motion for the trajectory of ions.

Time scales for the processes in ion channels can be estimated by the time for translocations (ttr) between two filter sites separated by ~0.3 nm, i.e., 5 × 10?10 s [88] and 5 × 10?11 s [87]. Transition rates (from potential mean force maps and the Kramer transition rate model [89] are consistent with these numbers. Changes between a non-conductive and a conductive state in the KcsA occur at a rate of 7.1 × 103 s?1, giving a life-time of the non-conducting state of 0.14 ms (~10?4 s) [89]. As the duration of the rather (stable) non-conducting state scales in the range 10?3–10?4 s and the within filter translocation time is on the order of 10?11 s, we can expect about 107 filter state changes during a non-conducting state and about 1010 switches per second (10 GHz). Consequently, these time scales are incompatible with those resulting from the effects of 100 kHz electric fields (10 ?s).

4.3. Electric Field Effects on Cytoplasmic Ions

The cytoplasm provides a medium in which fundamental biophysical processes, e.g., cellular respiration, take place. Most biological cells maintain a neutral pH (7.25–7.35) and their dry matter is composed of at least 50% of protein). The remaining dry material is composed of nucleic acids, trace ions, lipids, and carbohydrates. Most of the trace ions are positively charged. A few metallic ions are found which are required for incorporation into metallo-proteins, e.g., Fe2+, typically at nanomolar concentrations. In Table 1, we summarize the composition of the cytoplasm regarding the most abundant and important components.

Table 1

Table 1 Composition of the cytoplasm.

Based on the above, we can estimate the net force on the total charge in the cytoplasm as F = qE, q = 4 × 1011 e and E = 1 V/cm, so the total force is approximately 6 µN, which is sufficient to cause major perturbations in the cell interior. As discussed above, this is strongly depended on the ability of the electric field to penetrate into the cell’s interior, which is easier in the case of non-spherical cells. The net outcome of these ionic oscillations away and towards attractively interacting protein surfaces inside the cytoplasm can be a concomitant series of oscillations of the structures affected by the ionic clouds as schematically shown below.

The viscosity of cytoplasm is approximately ? = 0.002 Pa·s [90], hence we can estimate the friction coefficient for an ion in solution as ? = 6??r where r is the ionic radius (hydration shell radius) and find ? = 2 × 10?12 Pa·s·m. In an oscillating electric field of amplitude 1 V/cm and a frequency f = 200 kHz, an ion’s position will follow periodic motion given by: x(t) = 0.1·A·sin(2?ft), i.e., will execute harmonic motion out of phase with the field, with the same frequency and an amplitude A approximately 10% of the radius. However, these ions are simultaneously subjected to the Brownian motion due to their collisions with the molecules of the solvent.

To estimate the effect of an oscillating external electric field on the diffusion of a single biomolecular particle (protein, DNA, simple ion, etc.), the Langevin equation can be written down and solved. In the Ito interpretation [91], the position Xt of such a particle is given as a function of time by [92]: dXt= F(Xt)?dt+2kBT???????dWt (1)

where ? is the friction coefficient of the particle, T=310 ? is the temperature and kB is the Boltzmann constant. The first term on the RHS of Equation (1) accounts for the influence of deterministic forces F(Xt). Assuming there is no interaction other than the coupling with an external electric field E(Xt), we can write F(Xt)=qE(Xt) where q is the net charge of the particle. At intermediate frequencies, i.e., around 100–200 kHz, the wavelength is around 1000 m, which is obviously much larger than the size of a typical cell. Thus, assuming no important changes due to the dissipation of the field, E can be considered almost constant in a cellular environment: F(Xt)=qE(t). The second term on the RHS represents the random motion, which is due to the many kicks with the surrounding water molecules. Hence, dWt is usually given by [91]: dWt~dt1/2 ?(t) (2)

where ?(t) is a random number, which follows a normal distribution with a mean equal to 0 and a variance equal to 1. Since the Brownian motion is proportional to dt1/2, an estimate of dt is needed to evaluate the influence of the external electric field over the thermal noise. The time step dt can be estimated by the time interval between two series of collisions with water molecules, each series being the sum of enough collisions so that the outcome is approximately Gaussian. In other words, one can assume dt=dx/vH2O, where dx is the typical separation between two water molecules, i.e., dx=mH2O/?H2O??????????3 where mH2O is the mass of one water molecule and ?H2O is the mass density of water. Here, vH2O is the velocity of water molecules given by vH2O=3kBT/mH2O???????????. The use of the above parameters leads to a typical time step of dt~5.0×10?13 s.

The two terms in the RHS of Equation (1) above can be compared to estimate the effect of an electric field over the thermal noise. In the case of a spherical particle, we can assume ?=6??r, where the hydrodynamic radius is r=1.8 ? and the viscosity of the cytoplasm is ?=0.002 Pa·s [90]. By taking q=1 e (a single ion) and E=E0cos2?ft with E0=1 V/cm, it turns out that the amplitude of the coupling term associated with the electric field is qE0/?=2.36 × 10?6 m/s. On the other hand, the noise coefficient is 2kBT/???????? (dt)?1/2=50.2 m/s when the estimate obtained above is used: dt~5.0×10?13 s, which is much larger than the deterministic term. Even in the case of less frequent Brownian collisions, e.g., dt~10?6 s, the noise coefficient is 0.035 m/s which is still much larger than the coupling with the electric field, meaning that an electric field of amplitude 1 V/cmhas an exceedingly small probability to influence the diffusion of a single Brownian particle even if the net charge q is 100–1000 times larger as in the case of a protein.

Alternatively, it can be shown that an oscillating electric field at intermediate frequencies with an amplitude of 1 V/cm has no direct sizable effect on the diffusion of biomolecules by considering an ensemble of molecules instead of a single Brownian particle. Assuming a constant electric field E, the distribution of particles as a function of time is given by [93]: P(x,t)= 12Dt????exp?????(x –x0?qEt?)22Dt???? (3)

Here, D=kBT/? is the diffusion coefficient for one particle. From the above equation, a typical time when the particles start to be drifted away because of the electric field is t=2(kBT)?/(qE)2. For a single ion (q=1 e, r=1.8 ?), t=226.1 s, whereas for a typical globular protein (q~100 e, r~1.0 nm), t=0.13 s, which is much larger than the period of an electric field oscillating at hundreds of kHz.

For the sake of simplicity, we have not discussed here how an electric field could induce conformational changes in biomolecular structures, which would affect their charge distributions and dipolar spectra, which, in turn, could modify their diffusion by inducing new interactions with the surrounding molecules. An estimate of such indirect effects would require careful investigations of the studied system based on realistic MD simulations. In this case, the external electric field can be either computationally modeled by initializing the system with added kinetic energy in the directions of the normal modes or by adding an extra coupling term to the force field [94].

5. AC Electric Field Effects on Subcellular Structures

5.1. Electric Field Effects on MTs

Several experimental efforts were made aimed at measuring the electric field around MTs. Vassilev et al. [71] observed alignment of MTs in parallel arrays due to the application of electric fields with intensities of 0.025 V/cm and of pulsed shape. In cell division, coherent polarization waves have been implicated as playing the key role in chromosome alignment and their subsequent separation [18,19]. Electric fields in the range of 3 V/cm were applied by Stracke et al. [75] to suspended MTs, which moved at pH 6.8 from the negative electrode to the positive one indicating a negative net charge, and an electrophoretic mobility of about 2.6 × 10?4 cm2·V?1·s?1. The work of Uppalapati et al. [79] covers the range of frequencies overlapping with TTFields, although the amplitudes are much larger due to the voltage bias of 40 V across a 20-µm gap giving an electric field of 2 × 104 V/cm as opposed to 1 V/cm). Below 500 kHz, MTs flow toward the centerline of electrodes. The electro-osmotic force causes the movement of the fluid in a vortex-like manner. This represents the Coulomb force experienced by the ionic fluid due to the applied voltage. The fluid flow velocity ? is proportional to the tangential component of the electric field Et, surface charge density ?, the solution’s viscosity ? and the inverse Debye length ? such that: ? = Et ?/??. At lower frequencies, flow velocity is larger. On the other hand, due to strong heating effects of the AC field, the electro-thermal force causes motion of MTs along the length of the electrodes. Above 500 kHz MTs flow toward the gap between the electrodes due to dielectrophoresis. The DEP force experienced by MTs in a non-uniform electric field is given by: ?FDEP?=14??m[?2?m(?p??m)+?m(?p??m)?2?2m+?2m]?|E|2 (4)

where the symbols with subscript “m” refer to the medium and “p” to the particle. Hence, this process is largely driven by the difference between the conductivities and permittivities of the MTs and the medium, (?p ? ?m) and (?p ? ?m), respectively. We predict that lowering the pH of the solution to the isoelectric point of MTs around pH 5 should substantially reduce this effect and additionally lowering the frequency will reduce it further due to the dependence of the first term on the square of the frequency. At ~5 MHz, the electro-osmotic and electro-thermal flow balance each other out with the flow of MTs being solely due to dielectrophoresis. It is important to compare the dielectrophoretic force to Brownian motion in order to determine whether or not electric fields are sufficiently strong to overcome random motion, i.e., to find out if the dielectric potential exceeds the thermal energy, i.e., ?r3?m[(?p??m)(?p+2?m)]E2>kT (5)

where ?m is the dielectric constant of the medium and ?p is the dielectric constant of the particle. E is the electric field strength and r the radius of the particle. Taking as an example a tubulin dimer in solution and the corresponding values of the dielectric constants, one finds that E must exceed 0.25 V/cm for the field to be effective in orienting polarizable tubulin dimers. Similarly, for a 10-µm long MT we replace the factor ?r3 with ?r2 L, where r is the radius of a MT (12.5 nm) and L its length, to obtain a condition that E > 0.01 V/cm. Clearly, the electric field values of 1 V/cm (even if they are screened by a large factor inside the cell) are sufficient to exert electrophoretic effects on tubulin and MTs. The longer the MT, the more pronounced the dielectrophoretic effect is predicted to occur.

Recently, Isozaki et al. [95] used MTs labeled with dsDNA to manipulate the amount of net charge and observe the mobility of these hybrid structures compared to control where MTs where only labeled fluorescently with two different tags. It was found for control MTs that the electrophoretic mobility is approximately: 2 × 10?8 m2·V?1·s?1which is consistent with Stracke et al. [75]. For field strengths of approximately 1 V/cm, one can estimate the average velocity of MT translocations as 2 µm/s. They also stated ?D = 0.74 nm as the Debye length, ? = 8.90 × 10?4 kg·m?1·s?1 and ? = 6.93 × 10?10C·V?1·m?1 as the viscosity and dielectric constant of the buffer, respectively. Importantly, they estimated the effective charges of the TAMRA- and AlexaFluor 488-tagged tubulin dimer as 10 e? and 9.7 e?, which obviously is only a fraction (approximately 20%–30%) of the vacuum values but much larger than earlier experimental estimates. Electrophoresis experiments were also performed by van den Heuvel et al. [96], with electric field strengths of 40 V/cm, yielding MT electrophoretic mobility in the range of 2.6 × 10?8 m2·V?1·s?1, in line with previous reports. They found the effective charge of a tubulin dimer to be approximately 23 e?.

5.2. Tubulin’s C-Termini Dynamics and AC Electric Fields

Computer simulations demonstrate that ionic waves can trigger C-termini to change from upright to downward conformations initiating propagation of a travelling wave [97]. This wave is predicted to travel as a “kink” solitary wave with a phase velocity of vph = 2 nm/ps [97]. A typical time scale for C-termini motion is 100 ps, which is too fast for the 100 kHz frequency range of TTFields. However, C-termini being very flexible and highly charged (with approximately 40% of the tubulin’s charge located there) are likely to dynamically respond to electric fields as local changes of pH are correlated with positive and negative electric field’s polarities, respectively. This effect can cause MT instability as well as interference with motor protein transport as discussed below. A stable dimer conformation is predicted to have C-termini cross-linked between the monomers as shown in Figure 2.

Figure 2

Figure 2 A cross-linked conformation of C-termini stabilizes a straight orientation of a tubulin dimer. A disruption of this conformation can cause MT instability.

5.3. Ionic Waves along MTs and AC Electric Fields

Manning [98] postulated that polyelectrolytes may have condensed ions in their surroundings if a sufficiently high linear charge density is present on the polymer’s surface [99]. The Bjerrum length, ?B, is defined as the distance at which thermal fluctuations are equally strong as the electrostatic interactions between charges in solution whose dielectric constant is ? at a given temperature T in Kelvin. Here, ?0denotes the permittivity of the vacuum and kB is the Boltzmann constant. Counter-ion condensation occurs when the average distance between charges, b, is such that ?B/b = S> 1. In this case, the cylindrical volume of space depleted of ions outside the counter-ion cloud surrounding the polymer functions as an electrical shield. The “cable-like” electro-conducting behavior of such a structure is supported by the polymer itself and the “adsorbed” counter-ions, which are “bound” to the polymer in the form of an ionic cloud (IC). Tuszynski et al. [68] calculated an electrostatic potential around tubulin and extended this to an MT, which demonstrated non-uniformity of the potential along the MT radius with periodically repeating peaks and troughs along the MT axis. Consequently, MTs have been viewed as “conducting cables” composed of 13 parallel currents of ionic flux (corresponding to 13 protofilaments of MTs) and attracting an IC of positive counter-ions close to its surface and along tubulin C-terminal tails (TT), while negative ions of the cytosol are repelled away from the MT surface. The thickness of the negative ion depleted area corresponds to the Bjerrum length. An estimate of the respective condensate thickness ? of the counter-ion sheath for the tubulin dimer (?TD) and C-termini (?TT) is ?TD = 2.5 nm and ?TT = 1.1 nm, as analyzed in [61]. Using a Poisson–Boltzmann approach, the capacitance of an elementary ring of an MT consisting of 13 dimers is found as [100]: C0=2??0?lln(1+lBRIC) (6)

where l stands for the length of a polymer unit and RIC = ?TD + ?TT for the outer radius of an IC. For a tubulin dimer: CTD = 1.4 × 10?16 F and for an extended TT: CTT = 0.26 × 10?16 F. Hence: C0=C0+2×C0=1.92×10?16 F (7)

Estimating the electrical resistance for a complete tubulin ring gives R0 = 6.2 × 107 ? [60,100]. Including the conductance of both nanopores through an MT surface accounts for the leakage of IC cations into the lumen area and gives a conductance G0, of a ring as G0 = ?1 + ?2 = (2.93 + 7.8) nS = 10.7 nS and the corresponding resistivity as R = 1/G0 = 93 M?.

A simple equivalent periodic electric circuit simulating one protofilament of an MT consists of a long ladder network composed of elementary circuit units as shown in Figure 3 [61].

Figure 3

Figure 3 An effective circuit diagram for the n-th unit with characteristic elements for Kirchhoff’s laws applied to a microtubule as an ionic cable [61].

The longitudinal ionic current encounters a series of Ohmic resistors R0 for each ionic conduction unit (an MT ring). The nonlinear capacity with the charge Qn for the n-th site of the ladder is in parallel with the total conductance G0 of the two TTs of a dimer. Then using Kirchhoff’s law: in?in+1=?Qn?t+G0?n, (8) ?n?1??n=R0in, (9)

we find the equations for the voltage propagation: ?Qn?t=C0??n?t?C0?0??n?C0?0?(t?t0)??n?t?2b0C0?n??n?t (10)

Introducing an auxiliary function u(xt) unifying the voltage and its accompanying IC current as: un=Z1/2in=Z?1/2?n (11)

with the characteristic impedance defined as: Z=1?C0, (12)

leads in the continuum limit to the electric signal propagation equation: ?2?u?x?l23?3u?x3?ZC0l?u?t+ZC0?0?l(t?t0)?u?t+2Z3/2b0C0lu?u?t?1l(ZG0+Z?1R0?ZC0?0?)u=0 (13)

The characteristic charging (discharging) time of an elementary unit capacitor C0through the resistance R0 is given by T0 = R0C0 with an estimate for T0 = 1.2 × 10?8 s and the characteristic propagation velocity of the ionic wave: v=l/T0 as v0 = 0.67 m/s. A standard travelling-wave with speed v, for the normalized function u(xt), can be used as a solution of the propagation equation, which is a soliton that preserves its width but its amplitude decays over the length of about 400 units corresponding to 3.2 µm, which is of the order of the MT length. Interestingly, a characteristic time for this excitation can readily be estimated as 1.2 × 10?5 s whose inverse, the frequency, f, is very close to the TTField value, i.e., 90 kHz. The maximum frequency allowed in this model is 68 MHz.

To summarize, ionic conduction along and away from charged protein filaments such as MTs involves cable equations resulting from equivalent RLC circuits surrounding each protein unit in the network. Conduction along the filaments experiences resistance due to viscosity in the ionic fluid. Capacitance is caused by charge separation forming a double layer between the MT surface and ions with a distance separating them comparable to the Bjerrum length. Inductance is caused by helical nature of the MT surface and consequently, solenoidal flows of the ionic fluid along and around the MT. The key numerical estimates of the RLC circuit components are as follows [60]. For a single dimer: C = 6.6 × 10?16 F, R1 = 6 × 106 ? (along the MT), R2 = 1.2 × 106 ? (perpendicular to the MT) and L = 2 × 10?12 H. These numbers can be used to estimate characteristic time scales for the oscillations (LC) and exponential decay (RC) taking place in this equivalent circuit. We obtain for decay times (? = RC) the following values: (a) ?1 = 10?8 s along the MT length and (b) ?2 = 10?9 s away from the MT surface. However, due a low value of inductance L, the corresponding time for electromagnetic oscillations is found using ?0 = (LC)1/2 as ?0 = 0.2 × 10?12 s = 0.2 ps. Clearly, the oscillation times are too short for potential effects with 100 kHz-range fields (the time of TTFields oscillations is on the order of 5–10 µs). The decay times are much closer so we will focus on these parameters. Repeating these calculations for a microtubule of length l, we note that R1 scales with length of a microtubule, while R2 is length independent. The corresponding capacitance in both cases scales with length, therefore ?1 scales with length squared (l2) while ?2 scales with length. To obtain actual values, we need to multiply the values for a single ring by the number of rings in an MT. We use the values found for a single ring, i.e., ?1 = 10?8 s and ?2 = 2 × 10?9 s and scale them accordingly to estimate the length of MTs that could experience resonant effects in terms of ionic currents along and away from their surface. This way we find the scaling factor that leads to the characteristic times on the order of 10 µs. Therefore, for longitudinal effects, on the order of 50 rings, MTs only 400 nm long would respond to 100 kHz stimulation. On the other hand, for ionic flows pulsating radially around an MT, a 20-µm long MT would be required. These results are very sensitive regarding the choice of parameter values, especially the resistivity where diverse estimates can be found in the literature. In general, there is strong overlap between the time scales of ionic wave propagation and electric field stimulation. It is conceivable that both effects play a role depending on the orientation of the field vis a vis the geometry of mitotic spindles and the MTs forming them. It appears that short MTs would be more sensitive to the longitudinal wave generation by TTFields while long MTs should lead to perpendicular wave generation.

Current densities should also be briefly discussed in relation to previously reported endogenous current densities, j, in cells, which range from 0.2 to 60 µA/ cm2 [101]. This translates into 0.002 < j < 0.6 A/m2. Since j = ?E where E = 1 V/cm and ? of the cytoplasm has a large range of values reported between 0.1 and 100, we see that even taking the lower limit of 0.1 would result in ionic currents along MTs that would overwhelm the intrinsic ion flows in a dividing cell. It is possible that these externally stimulated currents cause a major disruption of the process of mitosis and associated intra-cellular effects.

It is also worth mentioning that recently metabolic oscillations in cells with a period of approximately 10 to 12 s, were measured in vivo [102] which is many orders of magnitude slower than any AC electric field effects discussed here. Hence, it is safe to assume that there is a very unlikely possibility of electric field effects in the 100 kHz range to interfere with cellular metabolism.

Finally, it is interesting to address the issue of the power dissipated due to a current flowing along an MT. Again, we take as an example a 10 µm-long MT, and we estimate the average power drain as: ?P?=(1/2)V20[R/(R2+X2c)], (14)

where Xc= 1/?C is the capacitive resistance. Substituting the relevant numbers we obtain the power dissipated to be in the 10?11 W range which is comparable to the power generated by the cell in metabolic processes (100 W of power generation in the body/3 × 1013 cells in the body). Consequently, additional heat generated by these processes may be disruptive to living cells although there is no experimentally detected thermal effect of TTFields.

5.4. Resonance Effects on MTs

Cosic et al. [103,104] reported EM resonances in biological molecules (proteins, DNA and RNA) in THz, GHz, MHz and kHz ranges. They proposed the so-called resonant recognition model (RRM) based on the distribution of energy of delocalized proteins in a biological system and charge transfer under resonance with a velocity of 7.87 × 105m/s and covering distances of 3.8 Å between amino acids, giving a characteristic frequency between 1013 and 1015 Hz. Then they state a variety of charge transfer velocities yielding different resonant frequencies. Of particular interest to this review is the velocity v = 0.0005 m/s which produces EMF in the range of 108–325 kHz for TERT, TERT mRNA and Telomere. This velocity corresponds the propagation of solitons on ?-helices. For tubulin and MTs, three specific ranges of resonant frequencies have been predicted by the RRM approach: 97–101 THz, 340–350 THz and 445–470 THz, none of which overlaps with TTField frequencies.

H-bond strength in MTs has been recently computationally estimated [105] as ranging from 11.9 k/mol for the weakest bond to 42.2 kJ/mol for the strongest one and a total of 462 kJ/mol for the ?-tubulin/?-tubulin interactions and 472 kJ/mol for the ?-tubulin/?-tubulin interactions, which based on the Planck relationship between frequency and energy translates into a range of frequency values between 0.3 × 1014 Hz and 1.3 × 1015Hz. Again, these frequencies are much too high to be affected by TTFields. Therefore, we do not expect TTFields to be capable of disrupting the MT structure.

Furthermore, Pizzi et al. [106] measured microwave resonance effects in MTs and found a resonant frequency at 1.510 GHz. This may not correspond to bond-breaking between tubulin dimers but simply to some specific electro-mechanical oscillations. Finally, Preto et al. [92] re-evaluated the Froehlich mechanism for long-range interactions and concluded that classical electromagnetic dipole-dipole interactions at high enough frequencies can lead to attraction between oscillating dipoles over distances comparable to the size of the cell. However, even including a coherently coupled layer of water molecules around a protein, this would require frequencies in the THz range or higher. Consequently, almost all of the resonant frequencies listed above fall well outside the range of potential overlap with the 100 kHz frequencies of TTFields.

5.5. Ionic Wave Conductivity along Actin Filaments and AC Fields

AFs are approximately 7 nm in diameter, with a periodic helical structure repeating every 37 nm. Actin filaments are arranged from actin monomers resulting in an alternating distribution of electric dipole moments along the length of each filament [107]. They are characterized by a high electrostatic charge density [108,109] resulting in ionic current conductivity involving the counter-ions surrounding them [109], which is very similar to the effects observed for MTs [60]. The observed wave patterns in electrically-stimulated AFs [30] were very similar to the solitary waveforms recorded for electrically-stimulated non-linear transmission lines [110]. In these experiments [30,42], an input voltage pulse was applied with an amplitude of 200 mV for a duration of 800 ms. Electrical signals were measured at the opposite end of the AF demonstrating that AFs support axial non-linear ionic currents. Since AFs produce a spatially-dependent electric field arranged in peaks and troughs [111] with an average pitch ~35–40 nm, they can be modeled as an electrical circuit with the following non-linear components: (a) a non-linear capacitor associated with the spatial charge distribution between the ions located in the outer and inner areas of the polymer; (b) an inductor; and (c) a resistor, similar to the model described above developed for MTs. A helical distribution of ions winding around the filament at an approximate distance of one Bjerrum length to the filament corresponds to a solenoid in which an ionic current flows due to the voltage gradient between the two ends. For an AF with n monomers, its effective resistance, inductance, and capacitance are, respectively: Reff=(?ni=11R2,1)?1+?ni=1R1,i, (15) Leff=?ni=1Li, (16) Ceff=?ni=1C0,i, (17)

where R1,i = 6.11 × 106 ?, and R2,i = 0.9 × 106 ?, such that R1,i = 7R2,i [112]. Hence, for a 1-µm length of an AF we find that Reff = 1.2 × 109 ?, Leff = 340 × 10?12 H and Ceff = 0.02 × 10?12 F. The electrical model of an AF is an application of Kirchhoff’s laws to one section of the effective electrical circuit that is coupled to neighboring monomers. In the continuum limit [112] the following equation describes the spatio-temporal behavior of the electric potential propagating along the actin filament: LC0?2V?t2=a2(?xxV)+ R2C0??t(a2(?xxV))? R1C0?V?t+R1C02bV?V?t. (18)

Solitary ionic waves have been described as the solutions of the above nonlinear partial differential equation [112] with an estimated velocity of propagation between 1 and 100 m/s [72]. This model has been recently updated with a more plausible estimation of model parameters [100]. Like MTs [96], AFs can be manipulated by external electric fields [113]. In a similar manner to our analysis of the time scales for MTs as ionic conduction cables with RLC components, we estimate similar time scales for actin and AFs. We readily find for a single actin monomer, that the time scale for LC oscillations is very fast, namely ?0 = (LC)1/2 and ?0 = 6 × 10?14 s. Secondly, the decay time for longitudinal ionic waves is ?1 = R1C = 6 × 10?10 s while the corresponding time for radial waves is ?2 = R2C = 0.9 × 10?10 s. All of the above time scales are not compatible with interactions involving electric fields in the 100 kHz range. However, the situation changes drastically for AFs where there is a similar scaling with the length of the filament as described above for MTs. Taking as an example a 1-µm AF, we find ?0 = 10?11 s, which is still too short but ?1 = R1C = 2.4 × 10?5 s which is in the correct range of time for interactions with AC electric fields in the 100 kHz range. It should be noted that AFs have been found sensitive to AC fields under experimental conditions [114].

5.6. Electric Field Effects on DNA

Anderson and Record [115] described ionic distribution around DNA in great detail. During interphase, DNA contents present in the nucleus are expected to be protected from external fields due to being enclosed in the nearly spherical nuclear membrane [78]. In addition to the screening effects of being shielded both by the cell membrane and the nuclear wall, the irregular geometry of the DNA strands and their short persistence length indicate that while highly charged, DNA is unlikely to participate in ionic conduction effects shown either for AFs or MTs, both of which have very large persistence lengths.

However, at the beginning of mitosis, the nuclear membrane breaks down, thus potentially not shielding the DNA any longer which would allow for the action of electric fields on chromosomes.

5.7. Electric Field Effects on Motor Proteins

Kinesin participates in mass transport along MTs and propagates at a maximum speed of 10?6 m/s. This value depends on the concentration of ATP and the ionic concentrations in the medium. In the case of MTs, kinesin transports various crucial cargo and for actin filaments, dynein does the same at similar speeds. Hence each step of a motor protein takes place over the period of a few ms, which is much longer than the period of AC field oscillations. However, kinesin binds to MTs through C-termini, which are very sensitive to electric field fluctuations and hence it is possible that kinesin transport would be very strongly disrupted by these rapid oscillations of C-termini. This aspect merits careful experimental verification.

Another potential member of the cytoskeleton that has been found affected by TTFields [2] is the protein called septin, which are GTP-binding like tubulin but form oligomeric hetero-complexes including rings and filaments. There is no information at the present time that could shed light on the mechanism of TTField effects with septin-based structures.

6. Discussion

The cytoskeleton and especially, MTs, may participate in numerous interactions with electromagnetic forces due to the complex charge distribution in and around these protein filaments surrounded by poly-ionic solutions. First of all, there are large net charges on tubulin, which are largely but not completely screened by counter-ions. Secondly, some of the charges are localized on C-termini, which are very flexible leading to oscillating charge configurations. Then, there are ions surrounding the protein that can be partially condensed and susceptible to collective oscillations. Moreover, there are large dipole moments on tubulin and microtubules whose geometric organization importantly affects their response to external fields. Finally, there can be induced dipole moments especially in the presence of electric field gradients. Disentangling the relative importance of the various effects under different conditions is not trivial and requires careful examination.

Depending on the orientation of the electric fields with the cell axis and in particular with the MT axis (however, they fan out from centrosomes in mitotic cells, so there will be at different angles to any field), there could in general be three types of ionic waves generated:

  1. Longitudinal waves propagating along the MT surface. In this case each protofilament of a microtubule acts like a cable with its inherent resistance r, so the resistance of an entire microtubule would be R = r/13 since all these cables are in parallel to each other.
  2. Helical waves propagating around and along each microtubule, there could be three or five such waves propagating simultaneously mimicking the three-start or five-start geometry of a microtubule. The effective resistance of such cables would be the individual resistance divided by the number of cables in parallel.
  3. Radial waves propagating perpendicularly to the microtubule surface.

If a field is oriented at an angle to the MT axis, it is expected that all these wave types may be generated simultaneously. Once AC fields generate oscillating ionic flows, these can in turn:

  1. Interfere with ion flows in the cleavage area of dividing cells.
  2. Interfere with motor protein motion and MAP-MT interactions.
  3. May to a lesser degree affect ion channel dynamics.
  4. May in general affect the net charge of the cytoplasm.

Finally, Kirson et al. [2] mention intracellular charged and polar entities such as cytoplasmic organelles as being potentially most directly affected by TTFields. This is not specifically addressed in this paper due to size and scope limitations as well as the scarcity of data in this regard. It has been argued [2] that inhomogeneity in field intensity may exert a uni-directional electric force on all intracellular charged and polar entities, pulling them toward the furrow (regardless of field polarity). It was determined that cytoplasmic organelles are electrically polarized by the field within dividing cells. As a consequence, the TTField-generated forces acting on these organelles may reach values up to 60 pN resulting in their movement toward the cleavage furrow. These organelles can move at velocities up to 30 ?m/s and, as a result, they could pile up at the cleavage furrow within a few minutes, interfering with cytokinesis, which may lead to cell destruction. This aspect needs detailed experimental investigation.

Some measurable heating effects in the cytoplasm might also be expected. These fields are not expected to affect permanent dipoles of proteins such as tubulin and actin. Although TTField effects have not been specifically assessed for AFs, an earlier paper [114] investigated exposure of cells to AC electric fields in a low frequency range of 1–120 Hz and found significant induced alterations in the AF structure, which were both frequency- and amplitude dependent. An application of 1–10 Hz AC fields caused AF reorganization from continuous, aligned cable structures to discontinuous globular patches. Cells exposed to 20–120 Hz electric fields were not visibly affected. The extent of AF reorganization increased nonlinearly with the electric field strength. The characteristic time for AF reorganization in cells exposed to a 1 Hz, 20 V/cm electric field was approximately 5 min. Importantly, applied AC electric fields were shown to initiate signal transduction cascades, which in turn cause reorganization of cytoskeletal structures. Therefore, in addition to direct effects of TTFields, there may be indirect, down-stream interactions.

7. Conclusions

Based on the extensive analysis of the various possible effects AC electric fields can have on living cells, we conclude the following. Electric field gradients, especially in dividing cells, cause substantial DEP forces on tubulin dimers and MTs. The longer the MT, the more pronounced the effect. Additionally, another likely scenario is that ionic current flows along and perpendicular to MT surfaces (as well as actin filaments, but less likely) take place, which can be generated by AC field oscillations in the 100–300 kHz range. The specific frequency selection depends critically on the length of each filament.

Identification of the strength, cause, and function of intracellular electric fields has only recently been experimentally accessible, although speculations in this area have existed for over a decade. These insights may also assist in devising and optimizing ways and means of affecting cells, especially cancer cells, by the application of external electric fields. With the advent of nanoprobe technology, which has shown promise in measuring these fields at a subcellular level, it is very timely to explore the various physical properties of the cytoplasmic environment including the cytoskeleton and the ionic contents of the cytoplasm. This research promises to contribute to our understanding of the cytoplasm in live cells and the role of microtubules and mitochondria in creating dynamic and structural order in healthy functioning cells. It will also be of help to identify biophysical differences in cancer cells that lead to increased metastatic behavior. Such an understanding may lead to optimized therapies and the identification of specific targets to halt metastatic transformation, as well as insights into the mechanism of action of current electromagnetic cancer therapies that are FDA approved and are in development.


Cornelia Wenger was supported by Novocure. Douglas E. Friesen was supported by Novocure. Douglas E. Friesen also gratefully acknowledges support from Alberta Innovates Health Solutions and the Alberta Cancer Foundation. The funding for J.A.T.’s research comes from the Natural Sciences and Engineering Research Council of Canada.


The following abbreviations are used in this manuscript:

DCdirect current
ACalternating current
TTFieldsTumor Treating Fields
GBMglioblastoma multiforme
AFactin filament
TTC-terminal tail
MAPmicrotubule associated protein

Author Contributions

Jack A. Tuszynski produced the first draft of the manuscript. Cornelia Wenger performed the computational studies and contributed to editing the paper. Douglas E. Friesen helped conceive the ideas presented in the paper and contributed to editing the paper. Jordane Preto contributed the analysis of ion motion in electric fields.

Conflicts of Interest

Novocure had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.


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Vestn Oftalmol. 2000 Jul-Aug;116(4):3-5. Differentiated approaches to the treatment of nonstabilized primary open-angle glaucoma with normalized intraocular pressure considering its pathogenic features. [Article in Russian]
Egorov VV, Sorokin EL, Smoliakova GP. Clinical efficiency of dedystrophic treatments for nonstabilized primary open-angle glaucoma (POAG) in the presence of normalized intraocular pressure is compared in 168 patients (246 eyes). In one group of patients ischemic angiopathy and hyperreactivity of optic vessel adrenoreceptors associated with hypokinetic central hemodynamics and constitutional metabolic status of the organism was corrected by cinnarisin and riboxin. Patients with predominating congestive angiopathy symptoms, hyper- or eukinetic circulation and “slow” acetylation were treated by pantothenic acid, endotelon, and hyperbaric oxygenation. In both groups epithalamine, eiconol, and magnetic laser therapy were used, if indicated. This helped stabilize the process in 91% patients with initial POAG, in 87.5% with well-developed condition vs. 66.1% and 38.2% patients treated by traditional therapy (period of observation 3 years). Vestn Oftalmol. 1996 Jan-Mar;112(1):6-8. Possibilities of magnetotherapy in stabilization of visual function in patients with glaucoma. [Article in Russian] Bisvas Shutanto Kumar, Listopadova NA. Courses of magnetotherapy (MT) using ATOS device with 33 mT magnetic field induction were administered to 31 patients (43 eyes) with primary open-angle glaucoma with compensated intraocular pressure. The operation mode was intermittent, with 1.0 to 1.5 Hz field rotation frequency by 6 radii. The procedure is administered to a patient in a sitting posture with magnetic inductor held before the eye. The duration of a session is 10 min, a course consists of 10 sessions. Untreated eyes (n = 15) of the same patients were examined for control. The patients were examined before and 4 to 5 months after MT course. Vision acuity improved by 0.16 diopters, on an average, in 29 eyes (96.7%) out of 30 with vision acuity below 1.0 before treatment. Visocontrastometry was carried out using Visokontrastometer-DT device with spatial frequency range from 0.4 to 19 cycle/degree (12 frequencies) and 125 x 125 monitor. The orientation of lattices was horizontal and vertical. The contrasts ranged from 0.03 to 0.9 (12 levels). MT brought about an improvement of spatial contrast sensitivity by at least 7 values of 12 levels in 22 (84.6%) out of 26 eyes and was unchanged in 4 eyes. Visual field was examined using Humphry automated analyzer. A 120-point threshold test was used. After a course of MT, visual field deficit decreased by at least 10% in 31 (72%) out of 43 eyes, increased in 3, and was unchanged in 9 eyes; on an average, visual field deficit decreased by 22.4% vs. the initial value. After 4 to 5 months the changes in the vision acuity and visual field deficit were negligible. In controls these parameters did not appreciably change over the entire follow-up period. Oftalmol Zh. 1990;(3):154-7. The effect of a pulsed electromagnetic field on the hemodynamics of eyes with glaucoma. [Article in Russian] Tsisel’skii IuV, Kashintseva LT, Skrinnik AV. The influence of pulse electromagnetic field (PEMF) on hemodynamics of the eye in open-angle glaucoma has been studied by means of a method and a device proposed at the Filatov Institute. The PEMF characteristics are: impulse frequency–50 Hz, exposition–0,02 sec., impulse shape–square, rate of impulse rise–4.10(4) c rate of magnetic induction rise–2.10(4) mT/c, amplitude value of magnetic induction at the impulse height–9.0–8.5 mT, duration of the procedure–7 min., a course–10 sessions. Observations over 150 patients (283 eyes) with latent, initial and advanced glaucoma have shown a positive influence of PEMF on hemodynamics of a glaucomatous eye: a rise of rheographic coefficient and relative volume pulse in 87,99 and 81,63%, respectively. The degree of the rise and restoration frequency of rheographic values of the glaucomatous eye under the influence of PEMF to the age norm was more expressed at initial stages of the glaucomatous process (latent and initial glaucoma).
Oftalmol Zh. 1990;(3):154-7.

The effect of a pulsed electromagnetic field on the hemodynamics of eyes with glaucoma.

[Article in Russian]

Tsisel’skii IuV, Kashintseva LT, Skrinnik AV.

The influence of pulse electromagnetic field (PEMF) on hemodynamics of the eye in open-angle glaucoma has been studied by means of a method and a device proposed at the Filatov Institute. The PEMF characteristics are: impulse frequency–50 Hz, exposition–0,02 sec., impulse shape–square, rate of impulse rise–4.10(4) c rate of magnetic induction rise–2.10(4) mT/c, amplitude value of magnetic induction at the impulse height–9.0–8.5 mT, duration of the procedure–7 min., a course–10 sessions. Observations over 150 patients (283 eyes) with latent, initial and advanced glaucoma have shown a positive influence of PEMF on hemodynamics of a glaucomatous eye: a rise of rheographic coefficient and relative volume pulse in 87,99 and 81,63%, respectively. The degree of the rise and restoration frequency of rheographic values of the glaucomatous eye under the influence of PEMF to the age norm was more expressed at initial stages of the glaucomatous process (latent and initial glaucoma).

Oftalmol Zh. 1990;(2):89-92.

The effect of a pulsed electromagnetic field on ocular hydrodynamics in open-angle glaucoma.

[Article in Russian]

Tsisel’skii IuV.

The influence of pulse electromagnetic field on the hydrodynamics of the eye in open-angle glaucoma has been studied using the method and the device suggested at the Filatov Institute. The characteristics of the action were: impulse frequency–50 Hz, duration–0.02 sec., pulse form–rectangular, rate of pulse rise–4/10(-4) sec., rate of magnetic induction rise–2/10(-4) mT/sec., amplitude value of magnetic induction at the pulse level–8.0-8.5 mT, duration of the procedure–7 min. Ten session in a total. Observations over 150 patients (283 eyes) with latent, initial and advanced glaucoma have shown that the usage of pulse electromagnetic field exerts influence on the hydrodynamics of the eye in open-angle glaucoma; stimulates the rise of aqueous outflow and production, the reduction of the Becker’s coefficient. At the latent stage of the disease, normalization of outflow was recorded in 25% of cases, at the initial and advanced stages–in 17.8% and 16.0% of cases, respectively. The investigations carried out allow to recommend the mentioned method for a complex treatment of open-angle glaucoma.

Vestn Oftalmol. 1994 Apr-Jun;110(2):5-7.

The effect of noninvasive electrostimulation of the optic nerve and retina on visual functions in patients with primary open-angle glaucoma.

[Article in Russian]

Kumar BSh, Nesterov AP.

Electrostimulation courses with OEC-2 Ophthalmologic Electrostimulator were administered to 30 patients (36 eyes) with primary open-angle glaucoma and normal intraocular pressure. An active electrode was placed on the upper lid, an indifferent one on the forearm. Electric pulses (150-900 mcA) were grouped in several sessions, 30 sec each, divided by 30-45 sec intervals. Total duration of a procedure was 16 min, the course consisting of 10 procedures. Control group included 24 eyes of the same patients. The patients were examined before, immediately, and 4-5 months after the treatment. Noticeable changes in vision acuity and visual field were detected. Visual field was examined using Humphrey Field Analyzer and 120-point threshold related test. The treatment resulted in reduction of visual field deficit by 10% or more in 28 (78%) of 36 eyes, in its increase in 2 eyes, and in no changes in 2 cases. Visual field deficit decreased by 25% on an average as against the initial value. Four to five months after the treatment the changes in this parameter were negligible. Vision acuity increased after the treatment in 31 of 36 eyes by 0.17 diopters on an average; 4 to 5 months later no changes occurred. In control eyes no changes were detected either in vision acuity or visual field during and after the treatment.


Clin J Pain. 2009 Oct;25(8):722-8.

Low-frequency pulsed electromagnetic field therapy in fibromyalgia: a randomized, double-blind, sham-controlled clinical study.

Sutbeyaz ST, Sezer N, Koseoglu F, Kibar S.

Fourth Physical Medicine and Rehabilitation Clinic, Ankara Physical Medicine and Rehabilitation Education and Research Hospital, Ankara, Turkey. ssutbeyaz@yahoo.com


OBJECTIVE: To evaluate the clinical effectiveness of low-frequency pulsed electromagnetic field (PEMF) therapy for women with fibromyalgia (FM).

METHODS: Fifty-six women with FM, aged 18 to 60 years, were randomly assigned to either PEMF or sham therapy. Both the PEMF group (n=28) and the sham group (n=28) participated in therapy, 30 minutes per session, twice a day for 3 weeks. Treatment outcomes were assessed by the fibromyalgia Impact questionnaire (FIQ), visual analog scale (VAS), patient global assessment of response to therapy, Beck Depression Inventory (BDI), and Short-Form 36 health survey (SF-36), after treatment (at 4 wk) and follow-up (at 12 wk).

RESULTS: The PEMF group showed significant improvements in FIQ, VAS pain, BDI score, and SF-36 scale in all domains at the end of therapy. These improvements in FIQ, VAS pain, and SF-36 pain score during follow-up. The sham group also showed improvement were maintained on all outcome measures except total FIQ scores after treatment. At 12 weeks follow-up, only improvements in the BDI and SF-36 scores were present in the sham group.

CONCLUSION: Low-frequency PEMF therapy might improve function, pain, fatigue, and global status in FM patients.

Aesthetic Plast Surg. 2008 Jul;32(4):660-6. Epub 2008 May 28.

Effects of pulsed electromagnetic fields on postoperative pain: a double-blind randomized pilot study in breast augmentation patients.

Hedén P, Pilla AA.

Department of Plastic Surgery, Akademikliniken, Storängsvägen 10, 115 42, Stockholm, Sweden. per.heden@ak.se


BACKGROUND: Postoperative pain may be experienced after breast augmentation surgery despite advances in surgical techniques which minimize trauma. The use of pharmacologic analgesics and narcotics may have undesirable side effects that can add to patient morbidity. This study reports the use of a portable and disposable noninvasive pulsed electromagnetic field (PEMF) device in a double-blind, randomized, placebo-controlled pilot study. This study was undertaken to determine if PEMF could provide pain control after breast augmentation.

METHODS: Forty-two healthy females undergoing breast augmentation for aesthetic reasons entered the study. They were separated into three cohorts, one group (n = 14) received bilateral PEMF treatment, the second group (n = 14) received bilateral sham devices, and in the third group (n = 14) one of the breasts had an active device and the other a sham device. A total of 80 breasts were available for final analysis. Postoperative pain data were obtained using a visual analog scale (VAS) and pain recordings were obtained twice daily through postoperative day (POD) 7. Postoperative analgesic medication use was also followed.

RESULTS: VAS data showed that pain had decreased in the active cohort by nearly a factor of three times that for the sham cohort by POD 3 (p < 0.001), and persisted at this level to POD 7. Patient use of postoperative pain medication correspondingly also decreased nearly three times faster in the active versus the sham cohorts by POD 3 (p < 0.001).

CONCLUSION: Pulsed electromagnetic field therapy, adjunctive to standard of care, can provide pain control with a noninvasive modality and reduce morbidity due to pain medication after breast augmentation surgery.

Pain Res Manag. 2007 Winter;12(4):249-58.

A randomized, double-blind, placebo-controlled clinical trial using a low-frequency magnetic field in the treatment of musculoskeletal chronic pain.

Thomas AW, Graham K, Prato FS, McKay J, Forster PM, Moulin DE, Chari S.

Bioelectromagnetics, Imaging Program, Lawson Health Research Institute, Department of Medical Biophysics, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Canada. athomas@lawsonimaging.ca


Exposure to a specific pulsed electromagnetic field (PEMF) has been shown to produce analgesic (antinociceptive) effects in many organisms. In a randomized, double-blind, sham-controlled clinical trial, patients with either chronic generalized pain from fibromyalgia (FM) or chronic localized musculoskeletal or inflammatory pain were exposed to a PEMF (400 microT) through a portable device fitted to their head during twice-daily 40 min treatments over seven days. The effect of this PEMF on pain reduction was recorded using a visual analogue scale. A differential effect of PEMF over sham treatment was noticed in patients with FM, which approached statistical significance (P=0.06) despite low numbers (n=17); this effect was not evident in those without FM (P=0.93; n=15). PEMF may be a novel, safe and effective therapeutic tool for use in at least certain subsets of patients with chronic, nonmalignant pain. Clearly, however, a larger randomized, double-blind clinical trial with just FM patients is warranted.

Pain Res Manag. 2006 Summer;11(2):85-90.

Exposure to a specific pulsed low-frequency magnetic field: a double-blind placebo-controlled study of effects on pain ratings in rheumatoid arthritis and fibromyalgia patients.

Shupak NM, McKay JC, Nielson WR, Rollman GB, Prato FS, Thomas AW.

Lawson Health Research Institute, St. Joseph’s Health Care, London, Ontario N6A 4V2.


BACKGROUND: Specific pulsed electromagnetic fields (PEMFs) have been shown to induce analgesia (antinociception) in snails, rodents and healthy human volunteers.

OBJECTIVE: The effect of specific PEMF exposure on pain and anxiety ratings was investigated in two patient populations.

DESIGN: A double-blind, randomized, placebo-controlled parallel design was used.

METHOD: The present study investigated the effects of an acute 30 min magnetic field exposure (less than or equal to 400 microTpk; less than 3 kHz) on pain (McGill Pain Questionnaire [MPQ], visual analogue scale [VAS]) and anxiety (VAS) ratings in female rheumatoid arthritis (RA) (n=13; mean age 52 years) and fibromyalgia (FM) patients (n=18; mean age 51 years) who received either the PEMF or sham exposure treatment.

RESULTS: A repeated measures analysis revealed a significant pre-post-testing by condition interaction for the MPQ Pain Rating Index total for the RA patients, F(1,11)=5.09, P<0.05, estimate of effect size = 0.32, power = 0.54. A significant pre-post-effect for the same variable was present for the FM patients, F(1,15)=16.2, P<0.01, estimate of effect size = 0.52, power =0.96. Similar findings were found for MPQ subcomponents and the VAS (pain). There was no significant reduction in VAS anxiety ratings pre- to post-exposure for either the RA or FM patients.

CONCLUSION: These findings provide some initial support for the use of PEMF exposure in reducing pain in chronic pain populations and warrants continued investigation into the use of PEMF exposure for short-term pain relief.

Neurosci Lett. 2001 Aug 17;309(1):17-20.

A comparison of rheumatoid arthritis and fibromyalgia patients and health controls exposed to a pulsed (200 microT) magnetic field: effects on normal standing balance.

Thomas AW, White KP, Drost DJ, Cook CM, Prato FS.

The Lawson Health Research Institute, Department of Nuclear Medicine & MR, St. Joseph’s Health Care, 268 Grosvenor Street, London, N6A 4V2, Ontario, Canada. athomas@lawsonimaging.ca

Specific weak time varying pulsed magnetic fields (MF) have been shown to alter animal and human behaviors, including pain perception and postural sway. Here we demonstrate an objective assessment of exposure to pulsed MF’s on Rheumatoid Arthritis (RA) and Fibromyalgia (FM) patients and healthy controls using standing balance. 15 RA and 15 FM patients were recruited from a university hospital outpatient Rheumatology Clinic and 15 healthy controls from university students and personnel. Each subject stood on the center of a 3-D forceplate to record postural sway within three square orthogonal coil pairs (2 m, 1.75 m, 1.5 m) which generated a spatially uniform MF centered at head level. Four 2-min exposure conditions (eyes open/eyes closed, sham/MF) were applied in a random order. With eyes open and during sham exposure, FM patients and controls appeared to have similar standing balance, with RA patients worse. With eyes closed, postural sway worsened for all three groups, but more for RA and FM patients than controls. The Romberg Quotient (eyes closed/eyes open) was highest among FM patients. Mixed design analysis of variance on the center of pressure (COP) movements showed a significant interaction of eyes open/closed and sham/MF conditions [F=8.78(1,42), P<0.006]. Romberg Quotients of COP movements improved significantly with MF exposure [F=9.5(1,42), P<0.005] and COP path length showed an interaction approaching significance with clinical diagnosis [F=3.2(1,28), P<0.09]. Therefore RA and FM patients, and healthy controls, have significantly different postural sway in response to a specific pulsed MF.

Fibroids – Uterine Myoma

Vopr Kurortol Fizioter Lech Fiz Kult. 2005 Jan-Feb;(1):26-8.

Efficacy of general magnetotherapy in conservative therapy of uterine myoma in women of reproductive age.

[Article in Russian]

Kulishova TV, Tabashnikova NA, Akker LV.

Sixty women of the reproductive age with uterine myoma were divided into two groups. Thirty patients of the study group received combined therapy plus general magnetotherapy (GMT). Patients of the control group received only combined treatment. Ultrasound investigation registered a reduction in the size of myoma nodes by 16.7% in the study group, while in the controls myoma size did not change (p < 0.05). 1-year follow-up data for the study group demonstrated no cases of the myoma growth while 16.6% of the controls showed growth of myoma nodes, in 6.6% of them supravaginal myoma amputation was made for rapidly growing myoma.