Ischemia – Reperfusion Injury

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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.
 .
Abstract

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

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

Keywords: apoptosis, Bax, B-cell lymphoma 2 (Bcl-2), ischaemia/reperfusion (I/R) injury, pulsed electromagnetic field (PEMF)

INTRODUCTION

Hypertension, arrhythmia, myocardial infarction (MI) and myocardial ischaemia/reperfusion (I/R) injury are all the most common cardiac diseases, which are the major causes of mortality in the world []. Among them, myocardial I/R injury is the most important cause of cardiac damage. Its pathological process is closely related to postoperative complications [,] 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 []. However, reperfusion may induce oxidative stress [], inflammatory cell infiltration and calcium dysregulation []. All these players contribute to the heart damage such as contraction and arrhythmias [], 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 [], alleviating traumatic pain and neuronal regeneration [,]. 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 [], and could improve arrhythmia, hypertension and MI []. 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)] []. However, the role of PEMF in ischaemia and reperfusion diseases remains largely unknown. Our study aimed to investigate the effects of PEMF preconditioning on myocardial I/R injury and to investigate the involved mechanisms.

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

MATERIALS AND METHODS

Animals

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

The measurement of blood pressure in SHR rats

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

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

All the rats were divided into three groups: (1) Sham: The silk was put under the left anterior descending (LAD) without ligation; (2) I/R: Hearts were subjected to ischaemia for 45 min and then reperfusion for 4 h; (3) I/R + PEMF: PEMF device was provided by Biomobie Regenerative Medicine Technology. The I/R rats were pre-exposed to active PEMF for 2 cycles per day (8 min per cycle), whereas other two groups were housed with inactive PEMF generator. I/R was performed by temporary ligation of the LAD coronary artery for 45 min through an incision in the fourth intercostal space under anaesthesia []. 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 [].

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) []. 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 []. 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 [].

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 [].

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

Cell viability assays

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

Measurement of intracellular reactive oxygen species levels

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

Data analysis

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

RESULTS

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

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

Figure 1

The 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

The 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

Protective 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

Apoptotic 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

NRCMs 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

PEMF 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

The 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 expression 

DISCUSSION

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

Many previous studies showed that extracorporeal PEMF-treated(5 mT, 25 Hz, 1 h daily) could enhance osteanagenesis, skin rapture healing and neuronal regeneration, suggesting its regenerative potency [,,]. 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 [] system and promoting EPCs mobilized to the ischaemic myocardium [,]. 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 [,,]. 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 []. 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 []. 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 []. 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 [,,,].

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 []. 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 [,,]. 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 [,]. 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 []. NO could also prevent platelet activation and promote vascular smooth muscle cells proliferation []. NO generation from eNOS is considered to be endothelium-derived relaxing and ROS-related factor [,]. Some researchers found that bradykinin limited MI induced by I/R injury via Akt/eNOS signalling pathway in mouse heart []. And bradykinin inhibited oxidative stress-induced cardiomyocytes senescence by acting through BK B2 receptor induced NO release []. Such evidence indicated that Akt phosphorylation could activate eNOS, which lead to NO releasing, and resulted in ROS reducing. In the present study, we found that PEMF decreased ROS via Akt/eNOS pathway.

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

Acknowledgments

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

Abbreviations

Akt protein kinase B
Bax Bcl-2 associated X protein
Bcl-2 B-cell lymphoma 2
CCA common carotid artery
CCK-8 Cell Counting Kit-8
CK creatine kinase
CKMB creatine kinase isoenzyme-MB
DAPI 4,6?-diamidino-2?-phenylindole
DHE dihydroethidium
DMEM/F12 Dulbecco’s modified Eagle’s medium/F-12
dUTP deoxyuridine triphosphate
eNOS endothelial nitric oxide synthase
EPCs endothelial progenitor cells
flk-1 fetal liver kinase-1
GAPDH glyceraldehyde-3-phosphate dehydrogenase
HBDH ?-hydroxybutyrate dehydrogenase
H/R hypoxia/reoxygenation
HRP horseradish peroxidase
I/R ischaemia/reperfusion
LAD left anterior descending
LDH lactate dehydrogenase
MI myocardial infarction
MI/R myocardial infarction/reperfusion
MI/RI myocardial infarction/reperfusion injury
NRCMs neonatal rat cardiac ventricular myocytes
PEMF pulsed electromagnetic field
ROS reactive oxygen species
Sca-1 stem cell antigen-1
SD Sprague Dawley
SHR spontaneously hypertensive rats
TTC 2,3,5-triphenyltetrazolium chloride
TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling
VEGF vascular endothelial growth factor

AUTHOR CONTRIBUTION

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

FUNDING

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

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Nitric Oxide

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Oxidative Medicine and Cellular Longevity
Oxid Med Cell Longev. 2017; 2017: 2181942.
Published online 2017 Sep 12. doi:  10.1155/2017/2181942
PMCID: PMC5613626

Benign Effect of Extremely Low-Frequency Electromagnetic Field on Brain Plasticity Assessed by Nitric Oxide Metabolism during Poststroke Rehabilitation

Natalia Cicho,corresponding author 1 Piotr Czarny, 2 Micha Bijak, 1 Elbieta Miller, 3 , 4 Tomasz liwiski, 5 Janusz Szemraj, 2 and Joanna Saluk-Bijak 1
1Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, Lodz, Poland
2Department of Medical Biochemistry, Medical University of Lodz, Mazowiecka 6/8, Lodz, Poland
3Department of Physical Medicine, Medical University of Lodz, Pl. Hallera 1, Lodz, Poland
4Neurorehabilitation Ward, III General Hospital in Lodz, Milionowa 14, Lodz, Poland
5Department of Molecular Genetics, Laboratory of Medical Genetics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Pomorska 141/143, Lodz, Poland
corresponding authorCorresponding author.
Academic Editor: Tanea T. Reed
Author information Article notes Copyright and License information
Received 2017 May 12; Revised 2017 Jul 2; Accepted 2017 Aug 14.

Abstract

Nitric oxide (NO) is one of the most important signal molecules, involved in both physiological and pathological processes. As a neurotransmitter in the central nervous system, NO regulates cerebral blood flow, neurogenesis, and synaptic plasticity. The aim of our study was to investigate the effect of the extremely low-frequency electromagnetic field (ELF-EMF) on generation and metabolism of NO, as a neurotransmitter, in the rehabilitation of poststroke patients. Forty-eight patients were divided into two groups: ELF-EMF and non-ELF-EMF. Both groups underwent the same 4-week rehabilitation program. Additionally, the ELF-EMF group was exposed to an extremely low-frequency electromagnetic field of 40Hz, 7mT, for 15min/day. Levels of 3-nitrotyrosine, nitrate/nitrite, and TNF? in plasma samples were measured, and NOS2 expression was determined in whole blood samples. Functional status was evaluated before and after a series of treatments, using the Activity Daily Living, Geriatric Depression Scale, and Mini-Mental State Examination. We observed that application of ELF-EMF significantly increased 3-nitrotyrosine and nitrate/nitrite levels, while expression of NOS2 was insignificantly decreased in both groups. The results also show that ELF-EMF treatments improved functional and mental status. We conclude that ELF-EMF therapy is capable of promoting recovery in poststroke patients.

1. Introduction

Cardiovascular diseases, including ischemic stroke (IS), are a serious problem of the modern age, killing 4 million people each year in Europe []. Stroke is caused by ischemia of brain tissue. Brain structure damage occurring during ischemia/reperfusion is due to the generation of significant amounts of reactive oxygen species and inflammatory mediators []. Damage to brain tissue as a result of a stroke cannot be undone. However, the most important part of poststroke therapy is immediate and long-term rehabilitation, considering the enormous plasticity of the brain []. Although extremely low-frequency electromagnetic field (ELF-EMF) therapy is not a standard treatment in the poststroke rehabilitation, some authors suggest its increased positive effect on patients []. ELF-EMF treatment is based on regeneration, osteogenesis, analgesics, and anti-inflammatory action. Its biological effect is related to processes of ion transport, cell proliferation, apoptosis, protein synthesis, and changes in the transmission of cellular signals []. The regenerative and cytoprotective effect of ELF-EMF is based on mechanism associated with nitric oxide induction, collateral blood flow, opioids, and heat shock proteins [].

Nitric oxide (NO) is an unstable, colourless, water-soluble gas with a short half-life (3–6sec). The compound has one unpaired electron, which makes it a highly reactive free radical. It is characterized by the multiplicity of action in the body, in both physiological and pathological conditions []. Synthesis of NO in the organism is catalysed by nitric oxide synthase (NOS), occurring in three isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS), encoded by different genes whose expression is subject to varying regulation. The constituent isoforms of NOS are eNOS and nNOS, whose activity is associated with concentration of calcium ions and the level of calmodulin in a cell, as well as with hypoxia, physical activity, and the level of certain hormones, that is, oestrogens []. In contrast, because it is closely related with the calmodulin, iNOS does not require a high concentration of calcium ions but is regulated by various endogenous and exogenous proinflammatory factors [].

The two-stage synthesis of NO consists of the oxidation of L-arginine to N-hydroxy-L-arginine and, under the influence of NOS and oxygen, formation of L-citrulline and release of NO. All isoforms of NOS require the same cofactors: nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH4), iron protoporphyrin IX (heme), and O2[].

Nitric oxide is one of the most important signal molecules, involved in both physiological and pathological processes. One of the major functions of NO is as a potent vasodilation, increasing the blood flow and regulation of blood pressure, which has been used in clinical practice for many years. Deficiency of this compound is observed in various disorders of many systems: cardiovascular, gastrointestinal, respiratory, and genitourinary []. The beneficial effects of NO lie in its platelet inhibition, macrophage cytotoxicity (antibacterial, antiviral, and antiparasitic), and protection of the mucosal lining of the digestive system. On the other hand, excessive expression of iNOS can be disadvantageous, for example, during sepsis. The adverse action of NO is associated with the production of superoxide anions and subsequent generation of peroxynitrite and hydroxyl radicals, which are highly toxic [].

In the central nervous system, NO as a neurotransmitter regulates cerebral blood flow, as well as neurogenesis and synaptic plasticity. Furthermore, neuronal death is caused by high concentrations of NO by caspase-dependent apoptosis process and promotion of inflammation. Elevated levels of nitric oxide promote necrosis by energy depletion. On the basis of these mechanisms, NO is involved in the etiology of many neurological diseases, such as major depression, schizophrenia, epilepsy, anxiety, and drug addiction [].

Our study was designed to investigate the effect of ELF-EMF on the metabolism of NO, as a signal molecule in the central nervous system, in the rehabilitation of acute poststroke patients.

2. Materials and Methods

2.1. Blood Sample Collection

Blood samples were collected into CPDA1-containing tubes (Sarstedt, Nümbrecht, Germany). Immediately upon doing so, a portion of the sample was frozen at 80°C and the rest of the samples centrifuged to isolate the plasma (15min, 1500g) at 25°C. Blood samples were collected twice, at an interval of 14 days before and after a standard 10 sessions of therapy. For additional analysis of 3-nitrotyrosine levels, the blood samples were collected three times, at an interval of 28 days: before treatment, after 10 treatments, and after 20 treatments. All blood samples were taken in the morning (between 7am and 9am) under patient fasting condition and stored using the same protocol.

2.2. Subject Presentation

Forty-eight poststroke patients were enrolled in the study. Participants were randomly divided into two groups: ELF-EMF (n = 25) and non-ELF-EMF (n = 23). Patients with metal and/or electronic implants (pacemakers, etc.) were excluded from the ELF-EMF group, for safety reasons. The ELF-EMF group had already undergone ELF-EMF therapy with specific parameters (40Hz frequency, magnetic induction of 5mT (B), rectangular and bipolar waveforms) (Figure 1), which was conducted using a Magnetronic MF10 generator (EiE Elektronika i Elektromedycyna, Otwock, Poland). The parameters were selected on the basis of the fact that low-intensity stimuli improve the vital functions of the body. In addition, rectangular pulses are more intense than sinusoidal and trapezoid, while bipolar pulses show more range of changes than unipolar pulses []. The ELF-EMF and non-ELF-EMF groups were treated for the same amount of time (15minutes). The non-ELF-EMF subjects were given only sham exposure. The pelvic girdle of the patients was exposed to the electromagnetic field, because exposure of the head to ELF-EMF can affect the activation of the epilepsy focus in the brain. The same therapeutic program was used for both subject groups. This consisted of aerobic exercise (30min), neurophysiological routines (60min), and psychological therapy (15min). Poststroke patients with moderate stroke severity according to NIHSS scores of 4.9 ±3.1 in the ELF-EMF group (aged 48.8 ±7.7) and 5.4 ±2.9 (aged 44.8 ±8.0) in the non-ELF-EMF group were enrolled in the study. Table 1 shows the clinical and demographic characteristics. Participants with haemorrhagic stroke, dementia, chronic or significant acute inflammatory factors, decreased consciousness, and/or neurological illness other than stroke in their medical prestroke history were excluded. The subjects had undergone neurorehabilitation for 4 weeks in Neurorehabilitation Ward III of the General Hospital in Lodz, Poland, as well as internal and neurological examinations. The Bioethics Committee of the Faculty of Biology and Environmental Protection of The University of Lodz, Poland, approved the protocol with resolution numbers 28/KBBN-U/II/2015 and 13/KBBN-U/II/2016. All participants provided written informed consent prior to participation. Depression was screened in both groups using the Geriatric Depression Scale (GDS). Cognitive status was estimated in a Mini-Mental State Examination (MMSE), and functional status using the Barthel Index of Activities of Daily Living (ADL). The GDS, ADL, and MMSE were administered either on the same day as the blood sampling or on the afternoon before.

Figure 1

ELF-EMF description. B=5mT; T = 1.3sec.

Table 1

Clinical demographic characteristics.

2.3. Magnetronic MF10 Devices

ELF-EMF therapy was performed by a Magnetronic MF10 generator as per accepted guidelines. This device is able to produce pulses in rectangular, trapezoid, and sinusoidal shapes. The pulses were applied using an AS-550 applicator (EiE, Otwock, Poland), which has the following properties: 550 mm in diameter, 270mm in length, and 5 layers of 187 turns of 1.45mm twin-parallel wires. Magnetic induction was set at 5mT. The electromagnetic field intensity was not uniformed; its distribution is vertical, while the induction coils are set horizontally. Induction of the electromagnetic field of 5mT is present at the geometric center of the applicator, and the value increases in the proximity to the surface about 7mT. Other factors that could affect EMF were eliminated (electronic measuring instruments occurring in rehabilitation room and other electronic equipment).

2.4. Immunodetection of 3-Nitrotyrosine by c-ELISA

Levels of 3-NT-containing proteins in plasma were determined using a modified c-ELISA method, as described by Khan et al. []. 96-well microtiter plates were coated with nitro-fibrinogen (nitro-Fg) (1mg/mL) and kept overnight at 4°C. Concentrations of nitrated proteins inhibiting the binding of anti-nitrotyrosine antibodies were assessed from the standard curve (10–100nM nitro-Fg equivalents) and expressed as nitro-Fg equivalents [].

2.5. Nitrate/Nitrite Estimation

Plasma samples were diluted twice before the measurement of nitrate/nitrite concentration using a Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical Company, USA), based on the two-step Griess method. In the first step, the nitrate is converted to nitrite with nitrate reductase, while in the second step, after addition of the Griess reagent, the nitrite is converted to a deep purple azo compound. The absorbance measurement was performed at 540nm in a 96-well microplate reader (SPECTROstarNano, BMG Labtech, Ortenberg, Germany) [].

2.6. Determination of NOS2 Expression in Whole Blood Samples

RNA was isolated from the frozen whole blood samples (?80°C), in accordance with the manufacturer’s protocol using TRI Reagent® (Sigma-Aldrich, USA). The aqueous phase was purified in accordance with the manufacturer’s protocol using an InviTrap Spin Universal RNA Mini Kit (Stratec Biomedical Systems, Germany). The purity and quantity of isolated RNA were assessed using a Synergy HTX Multi-Mode Microplate Reader equipped with a Take3 Micro-Volume Plate and connected to a PC running Gen5 Software (BioTek Instruments Inc., Winooski, VT, USA). Isolated RNA (20ng/L) was transcribed onto cDNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA, USA). Quantitative assays were executed using a TaqMan Hs01075529_m1 probe for human NOS2 genes and an Hs02786624_g1 for endogenous control, which was GAPDH (Life Technologies). Reactions were carried out using a TaqMan Universal Master Mix II, without UNG (Life Technologies) in a BioRad CFX96 real-time PCR system (BioRad Laboratories, Hercules, CA, USA), all in accordance with the manufacturers’ protocols. Relative expression of NOS2 was obtained using the equation 2Ct, where Ct is the threshold cycle (Ct) value for the target gene minus Ct values obtained for the housekeeping gene GAPDH [].

2.7. Determination of TNF?

Measurements of human tumour necrosis factor alpha (TNF?) in plasma samples were made with a Human TNF? ELISA development kit (MABTECH, Cincinnati, OH, USA), in accordance with the manufacturer’s protocol. The combination of two coating antibodies (TNF3 and TNF4) were used for the analysis. The absorbance was measured at 450nm, and TNF?.

Oxidative Medicine and Cellular Longevity

2.8. Data Analysis

Biochemical and clinical data were expressed as mean ±SD. All measurements were executed in duplicate. Output value (100%) was determined for each measured parameter of each patient before treatment. Data from tests performed on the same study subjects after therapy constituted a percentage of the output value. Percentage values were presented as mean ± SD. Statistical analyses were performed using the Statistica 12 statistical software (StaftSoft Inc.). A Shapiro-Wilk test was used to analyse for normality. A paired Student t-test was used to the calculate differences between the values obtained for subjects before and after therapy, whereas unpaired Student t-test or Mann–Whitney U tests were used to determine differences between the ELF-EMF and non-ELF-EMF groups. p values of 0.05 were accepted as statistically significant for all analyses.

3. Results

Our comparative analysis demonstrated an increased level of 3-nitrotyrosine (3-NT) (p< 0.05) (Figure 2) and an elevated nitrate/nitrite concentration (p < 0.01) (Figure 3) in the plasma of patients from the ELF-EMF group. The gain in the 3-NT level was significantly higher with an increased amount of sessions (Figure 2). In the non-ELF-EMF group, we saw that the effect of rehabilitation on nitrative stress was largely weaker and not statistically significant (p > 0.05) (Figures (Figures22 and and3).3). The 3-NT level increased more in the ELF-EMF group than in the non-ELF-EMF after 10 treatments (68% versus 17%, p < 0.05) (Figure 2). The level of nitrate/nitrite in the non-ELF-EMF group even decreased after 10 treatments (although not statistically significantly) (Figure 3).

Figure 2

The comparison of 3-NT levels in plasma proteins obtained from the ELF-EMF group versus those from the non-ELF-EMF group. Statistical significance between the ELF-EMF and non-ELF-EMF groups: B versus D (p < 0.05).

Figure 3

The comparison of nitrate/nitrite levels in plasma proteins obtained from the ELF-EMF group versus those from the non-ELF-EMF group. Statistical significance between ELF-EMF and non-ELF-EMF groups: B versus D (p < 0.05).

In the next set of experiments, we determined the effect of magnetotherapy on gene expression in the whole blood samples of NOS2 mRNA. Its expression was unmeasurable in 35% of subjects from both the ELF-EMF and non-ELF-EMF groups. We observed a statistically insignificant decrease in the level of NOS2 mRNA expression after treatment in both the ELF-EMF and non-ELF-EMF groups (Figure 4).

Figure 4

The comparison of NOS2 mRNA expression obtained from the ELF-EMF group versus that from the non-ELF-EMF group.

Subsequently, we determined the concentration of proinflammatory cytokine TNF?. We found that the concentration of TNF? was comparable before treatment in both the ELF-EMF and non-ELF-EMF-groups. The cytokine level did not change in either groups after rehabilitation (Figure 5).

Figure 5

The comparison of TNF? levels in plasma proteins obtained from the ELF-EMF group versus those from the non-ELF-EMF group.

The ADL, MMSE, and GDS were used to evaluate the functional and mental status of poststroke patients undergoing rehabilitation. We demonstrated that treatment using ELF-EMF improves their clinical parameters, particularly in cognitive and psychosomatic functions.

Motor abilities estimated by ADL score changed at similar levels in both groups, with the observed improvement being statistically significant in all rehabilitated patients (p < 0.001) (Table 2).

Table 2

Clinical parameters: ADL, MMSE, and GDS measured in the ELF-EMF and non-ELF-EMF groups. Data presented as the delta of a clinimetric scale before and after the standard series of treatments ADL=the increase of ADL; MMSE= the 

The baseline MMSE values before treatment in both groups were comparable, but statistically different (p < 0.05) after rehabilitation. After 2 weeks of rehabilitation, MMSE parameters improved markedly in the ELF-EMF group (p = 0.002), while a small increase in the non-ELF-EMF group was not statistically significant (p = 0.2) (Table 2).

Depression syndrome expressed by GDS improved significantly in both groups after rehabilitation. However, the GDS value reached about a 60% lower result in the ELF-EMF group than in the non-ELF-EMF group (p = 0.018), starting from a similar base level in both groups (p > 0.05) (Table 2).

4. Discussion

In this study, we provide the evidence that application of extremely low-frequency electromagnetic field increases nitric oxide generation and its metabolism, as well as improving the effectiveness of poststroke ischemic patients’ treatments.

Ischemic stroke is one of the major causes of morbidity and mortality in the world’s population and is one of the main causes of long-term disability. The mechanisms of neurological function recovery after brain injury associated with neuroplasticity (cortical reorganization) are still insufficiently understood. Poststroke neurorehabilitation is designed to provide external stimuli, improving the effectiveness of compensatory plasticity [].

In the central nervous system, NO is both a pre- and postsynaptic signal molecule. The activity of NO is associated with a cGMP-mediated signalling cascade. The presynaptic excitatory action of NO is related to the phosphorylation of synaptophysin by the cGMP-dependent protein kinase G (PKG) pathway and the subsequent potentates of glutamatergic neurotransmission []. On the other hand, NO causes a neurotransmission inhibition through gamma-aminobutyric acid- (GABA-) ergic synaptic communication. It is associated with ion exchange and regulation of membrane excitation []. Moreover, NO as an important vasodilation factor mediates neurovascular coupling. The enlargement of vessel diameter is caused by increasing metabolic consumption as a result of neuronal activity. Neurovascular coupling maintains functional and structural brain integrity [].

This study was designed to investigate the impact of ELF-EMF on the metabolism of nitric oxide in the rehabilitation of acute poststroke patients.

In our study, we demonstrate that poststroke rehabilitation increases the level of 3-NT and nitrate/nitrite concentrations. Due to its vasodilating and proangiogenic effects, NO serves as a protective function during cerebral ischemia. Su et al. investigated the role of simvastatin-regulated TRPV1 receptors (transient receptor potential vanilloid type 1) in NO bioavailability, activation of eNOS, and angiogenesis in mice. They demonstrated that simvastatin causes an influx of calcium ions through the TRPV1-TRPA1 (transient receptor potential ankyrin 1) pathway, which then causes activation of CaMKII (Ca2+/calmodulin-dependent protein kinase II). This then enhances the formation of the TRPV1-eNOS complex, which also includes CaMKII, AMPK (5AMP-activated protein kinase), and Akt (protein kinase B), which leads to activation of eNOS, production of NO, and thus the promotion of endothelial angiogenesis []. There have been numerous reports of the protective effects of NO against inflammation and oxidative stress []. Transgenic eNOS-deficient mice demonstrated a more extensive infarct of the middle cerebral artery (MCA), compared to controls []. NO effects on the regulation of endothelial integrity, anti-inflammatory and anti-apoptotic effects, as well as maintenance of cerebral blood flow, inhibition of platelet aggregation, and reduction of leukocyte adhesion []. Khan et al. studied structurally different NO donors as agents of cerebrovascular protection in experimentally induced stroke in rats. They showed that NO donors promote cerebral blood flow through S-nitrosylation and may be an effective drug for acute stroke [].

Furthermore, Greco et al. proved the protective effect of nitroglycerin (donors of NO) on cerebral damage induced by MCA occlusion in Wistar rats. They observed a significant reduction in stroke volume in preinjected rats compared to their control group, which confirms the protective effect of nitroglycerin in vivo. They speculated that the mechanism of action is associated with the generation of a complex chain of phenomena, triggering activation of apoptosis and subsequent activation of antiapoptotic responses [].

The biological action of ELF-EMF is still being investigated. It is suggested that ELF-EMF has an impact on the physicochemical properties of water, the liquid crystal structure generated by cholesterol, and its derivatives []. Changes in ion balance caused by ELF-EMF appeal to the structure of tissue with piezoelectric and magnetostrictive properties, free radicals, diamagnetic molecules, and uncompensated magnetic spins of paramagnetic elements []. Therefore, ELF-EMF causes depolarization of cells having the ability to spontaneously depolarize, predominantly through Ca2+ influx []. In our previous study, we investigated the effect of ELF-EMF on oxidative stress in patients after ischemic stroke. We demonstrated that ELF-EMF causes activation of antioxidant enzymes [], which leads to reduction of the oxidative modification of plasma protein (this is detailed in an article published in Advances in Clinical and Experimental Medicine). As a highly reactive molecule, NO can also regulate the level of oxidative stress. Through the covalent interaction, NO influences the activity of various enzymes. Mechanisms of this modulation can be varied: NO reacts with coenzymes and active centers containing metal ions and interacts with cysteine residues of proteins [].

In the current study, we observed that in the ELF-EMF group, the level of plasma 3-NT was increased (Figure 2). The formation of 3-NT in protein molecules occurs in vivo by the action of nitrating agents on the polypeptide chain. The formation of 3-NT is mainly attributed to NO and superoxide anions (O2??), which react rapidly to form peroxynitrite (ONOO?). This is one of the major oxidizing and nitrating agents produced in vivo in acute and chronic inflammation, as well as in ischemia/reperfusion. Endothelial cells, macrophages, and neutrophils release large amounts of NO and O2?. Thus, increased amounts of NO contribute to the creation of 3-NT [].

To investigate the effect of ELF-EMF on NO metabolism, we determined nitrate/nitrite concentrations in plasma. We showed that in the ELF-EMF group, the level of nitrate/nitrite compounds in plasma increased after treatment (Figure 3), and these results correspond with the data presented by Chung et al. []. The authors investigated the effects of ELF-EMF (60Hz, 2mT) on the level of NO, biogenic amines, and amino acid neurotransmitters in the hippocampus, cortex, thalamus, cerebellum, and striatum in rats. They found a significant increase in NO concentration in the hippocampus, thalamus, and striatum. Moreover, ELF-EMF also caused a change in the level of biogenic amines and amino acid neurotransmitters in the brain. However, the observed effect and range were different, depending on the brain area. Balind et al. determined the effect of ELF-EMF (50Hz, 0.5mT) on oxidative stress in gerbils with induced cerebral ischemia. They measured the level of NO using the Griess reagent and showed an increased level of NO, provoked by electromagnetic fields. Moreover, ELF-EMF reduces oxidative stress generated during cerebral ischemia, thus leading to a decrease in the damaged brain tissue [].

NO is produced from L-arginine with the involvement of nitric oxide synthase. Three NOS isoforms are expressed in different tissues. Although, in the blood, only NOS2 is expressed, in 35% of the subjects in both the ELF-EMF and non-ELF-EMF groups, mRNA expression of NOS2 was under detection. In the remaining patients, the expression of NOS2 had not significantly changed after treatment. The NOS2 gene in fact encodes for iNOS, which is primarily activated during inflammation. In order to exclude deeper inflammation, we measured the concentration of TNF?, one of the main proinflammatory cytokines. TNF? is a pleiotropic cytokine that is involved in nearly all phenomena of inflammatory responses: initiating chemokine synthesis, promoting the expression of adhesion molecules, promoting the maturation of dendritic cells, and inducing the production of inflammatory mediators and other proinflammatory cytokines []. TNF? stimulates collagenase synthesis in synovial fibroblasts and synovial cartilage chondrocytes and activates osteoclasts, leading to joint cartilage damage, hypertrophy, bone resorption and erosion, and angiogenesis. It also activates monocytes and macrophages, enhancing their cytotoxicity and stimulating cytokine production. Chemokines and growth factors are responsible for T cell proliferation, proliferation and differentiation of B lymphocytes, and the release of inflammatory cytokines by the lymphocytes. Moreover, in the hypothalamus, TNF? stimulates prostaglandin E and IL-1 synthesis []. Pena-Philippides et al. investigated the effect of pulsed electromagnetic fields on injury size and neuroinflammation in mice after middle cerebral artery occlusion (MCAO). They found, using magnetic resonance imaging (MRI), that EMF reduced infarct size, as well as changed expression of genes encoding pro- and anti-inflammatory cytokines in the hemisphere with ischemic injury. After EMF exposure, genes encoding IL-1 and TNF superfamily were downregulated, while IL-10 expression was upregulated. Thus, the authors suggested that application of EMF to poststroke patients could have been beneficial through anti-inflammatory effect and reduction of injury size [].

On the basis of our results, we suggest that the observed increase in NO level is associated with nNOS and/or eNOS activities, but not with iNOS expression. Our research is consistent with evidence shown by Cho et al., who established that ELF-EMF (60Hz, 2mT) increased the expression and activation of nNOS in rat brains [].

The activities of nNOS and eNOS depend on calcium ions. There are many reports that the biological effect of ELF-EMF is related to the control of calcium channels []. In view of these findings, the observed mechanism of increased NO generation and metabolism may be associated with calcium-ion flux.

Additionally, we noticed that ELF-EMF treatment enhances the effectiveness of poststroke rehabilitation (Table 2). Some researchers suggest that electromagnetic fields have a beneficial effect on ischemic/reperfusion injury, and in some places, therapeutic programs using ELF-EMF are considered to be standard therapy for poststroke patients []. The beneficial effects of ELF-EMF include the following: improvement in the transport of cellular and mitochondrial membranes; normalization of blood rheological values; counteraction of tissue oxidation; intensification of regenerative processes; stimulation of axon growth in undamaged neurons; intensification of neuronal dissociation and differentiation; reduction of stress-induced emotional reactions and free radicals; acceleration of the return of fibre function in functional disorders; reduction of periapical scarring; and increase of the level of energetic substances in the brain tissue and erythrocytes []. Grant et al. estimated the impact of low-frequency pulsed electromagnetic field on cerebral ischemia in rabbit. They observed using MRI that exposure to electromagnetic field caused extenuation of cortical ischemia oedema and reduction of neuronal injury in cortical area [].

In conclusion, ELF-EMF therapy increases the metabolism and generation of NO, which has both neuroprotective and cytotoxic properties. An increase in NO level is probably associated with nNOS and/or eNOS activities, but not with iNOS expression, which increases mainly during inflammation. We suggested that in poststroke patients, NO demonstrated a protective effect due to significant improvement in patient functional status. Thus, our studies promote the validity of this method in poststroke rehabilitation therapy.

Acknowledgments

This study was supported by the Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz (no. 506/1136), and Laboratory of Medical Genetics, Faculty of Biology and Environmental Protection, University of Lodz (no. B161100000004601), and Grants for Young Scientists and PhD Students, Faculty of Biology and Environmental Protection, University of Lodz (B1611000001145.02).

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

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Measurements of human tumour necrosis factor alpha (TNF?) in plasma samples were made with a Human TNF? ELISA development kit (MABTECH, Cincinnati, OH, USA), in accordance with the manufacturer’s protocol. The combination of two coating antibodies (TNF3 and TNF4) were used for the analysis. The absorbance was measured at 450nm, and TNF? concentration was expressed as pg/mL [].

Glioblastoma

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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.

Abstract

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 []. 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 []. However, in a major breakthrough paper, Kirson et al. [] 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 [,,]. This discovery has been translated into a clinical application termed Tumor Treating Fields (TTFields). Based on the results of a Phase III clinical trial [], 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 []. In late 2015, TTFields were also approved for newly diagnosed GBM patients in combination therapy with temozolomide [,] 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 [,]. Both static magnetic and electric fields have altered the mitotic index and cell cycle progression of a number of cell types in various species []. 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 [,,]. 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 [,]. 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 []. Following these experimental results, two specific mechanisms of action of TTFields have been proposed [,,] 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 [,,] 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 [] 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 [], Pohl et al. [] 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 [,] 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 [,,]. A detailed review of this aspect can be found elsewhere [].

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 []. 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 []. 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 []. Arguments were presented to account for the influence of the membrane conductance, surface admittance and spatial charge effects [], as well as for the geometric and material properties of the cell and the surrounding medium []. 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 []. Later models aimed to study electric polarization effects on oblate and prolate homogeneous and single-shell spheroids have been developed []. They were later extended to arbitrarily oriented cells of the general ellipsoidal shape []. 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 [,,,,]. 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 [,]. 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. [] 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 [,], 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 [].

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 []. The directions of the electrical field near the cell membrane resemble already predicted results [].

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 []. 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 []. 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 [,]. 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 []. 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 [,] and that cell volume is increased in almost all cell lines treated with TTFields []. 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 [].

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. [] 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 [], 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 [] 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 [,] but protein conductivity has been found to be strongly dependent on the hydration state of proteins []. Electrical properties of cells and their components were promoted by Szent-Györgyi [,], 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 [,]. Owing to the fact that there have been many previous reviews of electromagnetic effects in biology [,,,], 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 [] 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 [,] and have been theorized to support dipolar and/or ionic kink-like soliton waves traveling at speeds in the 2–100 m/s range [,]. 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 [,]. Electric fields generated by MTs have been modeled extensively and reviewed recently [,,], 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 []. 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 []. However, this is also strongly tubulin-isotype dependent, so these numbers vary a lot between various tubulin isotypes from 500 to 4000 debye []. 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 []. 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 []), 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 []. 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 [,,] 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 []. 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 [,]. Since MTs form a cylinder with a hollow inner volume (lumen), MTs have also been theorized to have special conducting properties involving the lumen [] 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 [].

Interestingly, Sahu et al. [,] 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 [,]. 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 [].

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 []. 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. []. 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 []. Impedance spectroscopy enabled the measurements of the dielectric constant of tubulin as ? = 8.41 []. Uppalapati et al. [] 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 [], 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 []. From Priel et al.’s conductance data, the approximate ionic conductivity of MTs is found to be an astonishing 367 S/m []. 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. [] 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 [,] 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 []. 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 [], 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 []. The motion of ions within the filter was intensively studied applying classical molecular dynamics (MD) methods (for a summary see Reference []) and density functional studies (e.g., []). 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 [] and 5 × 10?11 s []. Transition rates (from potential mean force maps and the Kramer transition rate model [] 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) []. 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

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 [], 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 [], the position Xt of such a particle is given as a function of time by []:

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 []:

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 []. 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 []:

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 [].

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. [] 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 [,]. Electric fields in the range of 3 V/cm were applied by Stracke et al. [] 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. [] 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. [] 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. []. 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. [], 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 []. This wave is predicted to travel as a “kink” solitary wave with a phase velocity of vph = 2 nm/ps []. 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

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 [] postulated that polyelectrolytes may have condensed ions in their surroundings if a sufficiently high linear charge density is present on the polymer’s surface []. 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. [] 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 []. Using a Poisson–Boltzmann approach, the capacitance of an elementary ring of an MT consisting of 13 dimers is found as []:

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 ? [,]. 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 [].

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 [].

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 []. 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 []. 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 [] 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. [,] 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 [] 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. [] 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. [] 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 []. They are characterized by a high electrostatic charge density [,] resulting in ionic current conductivity involving the counter-ions surrounding them [], which is very similar to the effects observed for MTs []. The observed wave patterns in electrically-stimulated AFs [] were very similar to the solitary waveforms recorded for electrically-stimulated non-linear transmission lines []. In these experiments [,], 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 [] 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 []. 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 [] 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 [] with an estimated velocity of propagation between 1 and 100 m/s []. This model has been recently updated with a more plausible estimation of model parameters []. Like MTs [], AFs can be manipulated by external electric fields []. 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 [].

5.6. Electric Field Effects on DNA

Anderson and Record [] 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 []. 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 [] 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. [] 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 [] 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 [] 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.

Acknowledgments

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.

Abbreviations

The following abbreviations are used in this manuscript:

DC direct current
AC alternating current
TTFields Tumor Treating Fields
GBM glioblastoma multiforme
EM electromagnetic
MT microtubule
DEP dielectrophoretic
AF actin filament
TT C-terminal tail
MAP microtubule 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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Osteosarcoma

J Orthop Surg Res. 2015; 10: 104.
Published online 2015 Jul 7. doi:  10.1186/s13018-015-0247-z
PMCID: PMC4496869

Nanosecond pulsed electric field inhibits proliferation and induces apoptosis in human osteosarcoma

Xudong Miao,# Shengyong Yin,# Zhou Shao, Yi Zhang, and Xinhua Chencorresponding author
The Department of Orthopedics, the Second Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang Province 310003 China
The Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital, Zhejiang University, Collaborative Innovation Center for Diagnosis Treatment of Infectious Diseases, 79 Qinchun Road, Hangzhou, Zhejiang Province 310003 China
The Department of Gynecology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province 310000 China
Xinhua Chen, Phone: +86-571-87236570, nc.ude.ujz@nehc_auhnix.
corresponding authorCorresponding author.
#Contributed equally.
Author information ? Article notes ? Copyright and License information ?
Received 2015 Jun 11; Accepted 2015 Jun 29.

Abstract

Objective

Recent studies suggest that nanosecond pulsed electric field (nsPEF) is a novel minimal invasive and non-thermal ablation method that can induce apoptosis in different solid tumors. But the efficacy of nsPEF on bone-related tumors or bone metastasis is kept unknown. The current study investigates antitumor effect of nsPEF on osteosarcoma MG-63 cells in vitro.

Method

MG-63 cells were treated with nsPEF with different electric field strengths (0, 10, 20, 30, 40, and 50 kV/cm) and different pulse numbers (0, 6, 12, 18, 24, and 30 pulses). The inhibitory effect of nsPEF on the growth of MG-63 cells was measured by Cell Counting Kit-8 (CCK-8) assay at different time points (0, 3, 12, 24, and 48 h post nsPEF treatment). The apoptosis was analyzed by Hoechst stain, in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), and flow cytometric analysis. The expression of osteoprotegerin (OPG), receptor activator of NF-kB ligand (RANKL), and tumor necrosis factor a (TNF-a) was examined by reverse-transcription polymerase chain reaction (RT-PCR) and western blot.

Results

The CCK-8 assay showed that nsPEF induced a distinct electric field strength- and pulse number-dependent reduction of cell proliferation. For treatment parameter optimizing, the condition 40 kV/cm and 30 pulses at 24 h post nsPEF achieved the most significant apoptotic induction rate. Hoechst, TUNEL, and flow cytometric analysis showed that the cell apoptosis was induced and cells were arrested in the G0/G1 phase. PCR and western blot analysis demonstrated that nsPEF up-regulated OPG expression had no effect on RANKL, increased OPG/RANKL ratio.

Conclusion

NsPEF inhibits osteosarcoma growth, induces apoptosis, and affects bone metabolism by up-regulating OPG, indicating nsPEF-induced apoptosis in osteosarcoma MG-63 cells. NsPEF has potential to treat osteosarcoma or bone metastasis. When nsPEF is applied on metastatic bone tumors, it might be beneficial by inducing osteoblastic differentiation without cancer proliferation. In the future, nsPEF might be one of the treatments of metastatic bone tumor.

Keywords: Osteosarcoma, MG-63 cells, Nanosecond pulsed electric field, Apoptosis

Introduction

Osteosarcoma is a malignant bone tumor with high occurrence in children and young adolescents. Retrospective review showed that in the past 30 years, osteosarcoma had a poor prognosis and there was no significant improvement of disease-free survival and the stagnated situation has not improved even with the aggressive use of neoadjuvant chemotherapy and radiation therapy [1]. Patients did not benefit from overtreatment, and as a result, a high rate of lung metastasis, recurrence, and pathological fracture frequently occur, keeping osteosarcoma still one of the lowest survival rates in pediatric cancers [2]. Thus, new therapeutic strategy needs to be developed.

Nanosecond pulsed electric field (nsPEF) is an innovative electric ablation method based on high-voltage power technology, which came into medical application in the last decade [3]. NsPEF accumulates the electric field energy slowly and releases it into the tumor in ultra-short nanosecond pulses, altering electrical conductivity and permeability of the cell membrane, causing both cell apoptosis and immune reaction [4].Quite different from any other traditional local ablation method, nsPEF accumulate less Joule heating and showed no hyperthermic effects [5], indicating unique advantage over other thermal therapies such as radiofrequency, cryoablation, microwave, and interstitial laser; nsPEF can be used alone and so avoid the side effect caused by chemotherapy or percutaneous ethanol injection [6].

We have used nsPEF to ablate tumor and showed the equal outcome as the radical resection with proper indication [7]. Clinical trials and pre-clinical studies from different groups proved that nsPEF has direct antitumor effects by inhibiting proliferation and causing apoptosis in human basal cell carcinoma [8, 9], cutaneous papilloma, squamous cell carcinoma [10], melanoma [11, 12], hepatocellular tumor [13], pancreatic tumor [14], colon tumor [15, 16], breast cancer [17, 18], salivary adenoid cystic carcinoma [19], oral squamous cell carcinoma [20], et al. Local ablation with nsPEF indicates the noticeable advantage of not only eliminating original tumors but also inducing an immune reaction, e.g., enhance macrophage [21] and T cell infiltration [22] and induce an immune-protective effect against recurrences of the same cancer [23]. The characteristic of electric field on bone metabolism [24] is extremely helpful for osteosarcoma patients with pathological fracture which leads to poor prognosis [25, 26].

Considering osteosarcoma is especially prevalent in children and young adults during quick osteoblastic differentiation [1, 2], unstable RB gene and p53 gene are commonly involved in this malignant transformation process [27]; we hypothesize that nsPEF affects osteosarcoma growth by targeting the Wnt/?-catenin signaling pathway, a key signaling cascade involved in osteosarcoma pathogenesis. Here, we investigate nsPEF-induced changes on human osteosarcoma MG-63 cells to determine (1) the dose-effect relationship and time-effect relationship of nsPEF on osteosarcoma cell growth and apoptosis induction and (2) the nsPEF effect on the osteosarcoma cell; osteoblast specific gene and protein expression (receptor activator of NF-?B ligand (RANKL) and osteoprotegerin (OPG)) were measured along with the production of the pro-inflammatory cytokine tumor necrosis factor a (TNF-a).

Materials and methods

Cell lines and cell culture

MG-63 human osteosarcoma cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS, SAFC Biosciences, Lenexa, KS, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin (Sigma, Aldrich, St. Louis, MO, USA). Cells were kept in a humidified atmosphere of 5 % CO2 at 37 °C.

The nsPEF treatment and dose-effect exam

The nsPEF treatment system was made by Leibniz Institute for Plasma Science and Technology, Germany, and an nsPEF generator with duration of 100 ns was applied. Varied electric fields were released in a cell treatment system from 10 to 60 kV/cm. Waveforms were monitored with a digital phosphor oscilloscope (DPO4054, Tektronix, USA) equipped with a high voltage probe (P6015A, Tektronix, USA). MG-63 human osteosarcoma cells were harvested with trypsin and resuspended in fresh DMEM with 10 % FBS to a concentration of 5.0 × 106 cells/mL. Five hundred microliters of cell suspension were placed into a sterile electroporation cuvette (Bio-Rad, US, 0.1-cm gap). Cells were exposed to 100 pulses at 0, 10, 20, 30, 40, 50, and 60 kV/cm electric field strengths, respectively. Under the 50 kV/cm electric field strength, the different pulse numbers were applied (0, 6, 12, 18, 24, and 30 pulses). The experiments were repeated for three times. After incubation for 24 h, cells were calculated by Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan).

Measurement of apoptosis with TUNEL assay, Hoechst stain, and flow cytometry

At different hours after nsPEF treatment (40 kV/cm, 30 pulses), the treated cells were incubated for 0, 3, 12, 24, and 48 h to determine single-cell apoptosis using the assay of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) with In Situ Cell Death Detection Kit (Millipore, USA) and Hoechst stain kit (Beyotime, Shanghai, China) according to the manufacturer’s instruction, as previously described [14]. Under different electric field strengths and with different pulses, the treated cells were incubated for 24 h to detect cell apoptosis by Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). The cell cycle was also analyzed as previously described [14].

Reverse-transcription polymerase chain reaction

Reverse-transcription polymerase chain reaction (RT-PCR) was performed for assessing the expression of OPG, RANKL, and TNF-a. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a house keeping gene, was used as the internal control to calculate the comparative expression. Total RNA was extracted using TRIzol reagent (Sangon, Shanghai, China). The first strand cDNA synthesis from 1 mg of RNA was performed using SuperScript II Reverse Transcriptase (Invitrogen) and Oligo dT primer (Promega, Madison, WI, USA) according to the manufacturer’s instructions. PCR was performed using the oligunucleotides listed as the following. The specific primers were made by Sangon, Shanghai, China, which were listed as the following: RANK: F: CAGGAGACCTAGCTACAGA, R: CAAGGTCAAGAGCATGGA, 95 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min; OPG (264 bp): F: AGTGGGAGCAGAAGACAT, R: TGGA CCTGGTTACCTATC, 95 °C, 1 min; 57 °C, 1 min; 72 °C, 1 min; TNF-a: F: GTGGCAGTCTCAAACTGA, R: TATGGAAAGGGGCACTGA, 94 °C, 40 s; 55 °C, 40 s; 72 °C, 40 s; GAPDH: F: CAG CGACACCCACTCCTC, R: TGAGGTCCA CCACCCTGT, 94 °C, 1 min; 57 °C, 1 min; 72 °C, 1 min.

Western blotting analysis

MG-63 cells (5 × 105) were plated and treated with different doses of nsPEF. Cells were then lysed with a lysis buffer and then quantified. The equal amounts of protein were loaded, and electrophoresis was applied on a 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis mini-gel. Proteins were transferred to a PVDF membrane and blocked with casein PBS and 0.05 % Tween-20 for 1 h at room temperature. Membranes were incubated with mouse monoclonal OPG, anti-OPG (1:500), RANKL (1:200), TNF-a (1:300), GAPDH (1:1000) antibodies which were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibody was purchased from Zhongshan (Zhongshan Golden Bridge, Beijing, China.). The protein expression was visualized with enhanced chemiluminescence reagent (ECL kit, Amersham, UK).

Statistical analysis

Statistical significance was determined using Student’s t test, using SPSS 13.0. P < 0.05 was considered to indicate a statistically significant result.

Results

NsPEF parameter optimizing by CCK-8 and flow cytometry

CCK-8 assay was used to calculate the IC50 values, and flow cytometry was used to detect apoptosis. There were significant growth inhibition and apoptosis induction in a dose-dependent manner following nsPEF treatment for 24 h. MG-63 cell growth was inhibited in an electric field strength- and pulse number-dependent manner. There was significant (P > 0.001) growth inhibition when electric field strength was 40–50 kV/cm (Fig. 1a) and when pulse number was 30 (Fig. 1d) vs control. Cells were treated by nsPEF and then incubated for 24 h. Apoptotic and dead cells were analyzed by flow cytometry using dual staining with propidium iodide (PI) and Annexin V-FITC. NsPEF induced viable apoptotic cells stained with Annexin. The apoptotic cell rate is significantly increased when electric field strength was 40–50 kV/cm (Fig. 1b, c) and when pulse number was 30 (Fig. 1e, f).

Fig. 1

NsPEF treatment parameter optimizing by CCK-8 and flow cytometry. After 24 h post nsPEF, CCK-8 assay was used to calculate the IC50 values under different electric field strengths (a) and different pulse numbers (d). The flow cytometry was used to detect

Apoptosis induction at different times post nsPEF treatment

To determine the effects of nsPEF on the induction of apoptosis in MG-63 cells, the Annexin V assay was performed. After 40 kV/cm and 30 pulses of nsPEF treatment, the control and treated cells were stained with Hoechst 33528 (Fig. 2a upper lane) and TUNEL (Fig. 2a lower lane). The statistical analysis of the positive apoptotic cells were counted and shown in Fig. 2b at different hours (0, 3, 12, 24, and 48 h). Apoptotic cells induced by nsPEF treatment were recognized by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), detecting DNA fragmentation by labeling the terminal end of nucleic acids. The number or percentages of apoptotic cells detected following nsPEF treatment was shown in Fig. 2b. The quantitative analysis showed the percentages of apoptotic cells detected following nsPEF treatment which were 2.6 % (0 h), 8.8 % (3 h), 21 % (12 h), 42 % (24 h), and 15 % (48 h) without nsPEF treatment. The apoptotic induction 12 and 24 h post nsPEF treatment showed significance (P = 0.01243, 0.00081, respectively, vs control). The cell cycle was analyzed by flow cytometry (Fig. 2c) and statistically analyzed in Fig. 2d, which indicates that nsPEF arrest cells in the G0/G1 phase (Fig. 2d).

Fig. 2

Apoptosis induction at different times post nsPEF treatment. After 40 kV/cm and 30 pulses of nsPEF treatment, the control and treated cells were stained with Hoechst 33528 (a upper lane) and TUNEL (a lower lane). The statistical analysis of the positive

The effect of nsPEF on OPG/RANKL, TNF-? gene, and protein expression

With 30 pulses, 24 h post treatment, PCR and western blot were used to determine the different electric field strengths on cell OPG/RANKL, TNF-? gene (Fig. 3a), and the corresponding protein expression (Fig. 3b). NsPEF significantly increased OPG transcription and protein expression at 20–50 kV/cm (Fig. 3a, c). RANKL was almost undetectable both in the control and nsPEF-treated MG-63 cells (Fig. 3a, c). NsPEF slightly down-regulated TNF-a (Fig. 3a, c). The OPG is important in the regulation of bone formation. PCR results showed that the nsPEF-treated cells demonstrated a significantly up-regulation of OPG transcription. Western blot analysis confirmed that nsPEF stimulated osteoprotegerin protein production in the MG-63 cells.

Fig. 3

The nsPEF effect on gene and protein expression. With 30 pulses, 24 h post treatment, PCR and western blot were used to determine the different electric field strengths on cell OPG/RANKL, TNF-a gene (a), and protein expression (b). NsPEF significantly

Discussion

The primary bone malignancy osteosarcoma is still a challenge for orthopedics. For patients who are not suitable for radical resection, the minimal invasive ablation techniques can be used as an alternative to surgery. NsPEF has been proved to be a novel non-thermal ablation method which can activate a protection immune response [2123]. According to the Clinical Practice Guidelines in Oncology of the National Comprehensive Cancer Network (NCCN), local ablation can be used for curative or palliative intent, either alone or in combination with immunotherapy or chemotherapy [11]. The effect of systemic chemotherapy may be enhanced by the physiological changes produced by ablation [11]. Furthermore, ablation can sometimes be used as a complement to surgery [13].

A number of studies have demonstrated that local ablation is effective in osteosarcoma [2830]. To our best knowledge, the application of nsPEF in osteosarcoma has never been reported. The bone-related tumor study is extremely important because many solid tumors tend to have metastasis in bones. The present study applies a new ablation methodology in osteosarcoma and identifies its molecular target. Our data suggest that nsPEF had direct effects on osteosarcoma cells, including the inhibition of tumor cell proliferation and induction of apoptosis. These results are consistent with previous reports. NsPEF inhibits cell proliferation and induces apoptosis in tumor cells [11, 16].

The development of osteoclasts is controlled by cytokine synthesized by osteoblasts like receptor activator of NF-?B ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor ? (TNF-a) [31].The extension of the current study is the investigation of nsPEF’s effect on bone resorption when nsPEF is in its ablation dosage. OPG is a member of the tumor necrosis factor receptor family. It has multiple biological functions such as regulation of bone turnover. OPG can block the interaction between RANKL and the RANK receptor [31]. NsPEF increased OPG expression in MG-63 in in vitro assays. Our data indicate that nsPEF up-regulated the OPG expression. Bone remodeling can be assessed by the relative ratio of OPG to RANKL [32]. NsPEF had no effect on RANKL expression. Defined as a potent bone-resorbing factor, TNF-a is responsible for stimulating bone resorption. TNF-? exerts its osteoclastogenic effect by activating NF-?B with RANKL [33]. Our results show that in osteosarcoma MG-63, in addition to apoptosis induction, nsPEF can regulate bone metabolism through adjusting OPG/RANKL ratio.

TNF-a expression still needs further investigation due to the weak expression. But, it is the key cytokine that we assume which would change the local inflammatory microenvironment in the ablation zone.

The limit of the current study

In this in vitro study, the MG-63 osteosarcoma cell line is used as a model system. Therefore, results obtained from cultured cells only gave hints for the nsPEF treatment of osteosarcoma. The current results need to be tested in an in vivo osteosarcoma model, e.g., MG-63 cell xenografts.

Conclusion

NsPEF can be considered as a potential therapeutic intervention to suppress bone remodeling and osteoclast activity involved in osteosarcoma. Further in vivo studies are required to optimize the dosing regimen of nsPEF to fully study its antitumor potential in the bone microenvironment.

Acknowledgments

All authors acknowledge Dr.Karl H. Shoenbach, Dr. Stephen Beebe, and Mr. Frank Reidy from Old Dominion University for their kind support.

Financial support

This research is supported by National Natural Science Foundation of China (Nos. 81372425 and 81371658), National S & T Major Project (No. 2012ZX10002017), Zhejiang Natural Science Foundation (LY13H180003), and Xinjiang Cooperation Project (2014KL002).

Footnotes

Xudong Miao and Shengyong Yin contributed equally to this work.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

XM and SY carried out the molecular genetic studies and drafted the manuscript. ZS carried out the immunoassays. YZ participated in the design of the study and performed the statistical analysis. XC conceived of the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.

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Indian J Orthop. 2016 Jan-Feb; 50(1): 87–93.
doi:  10.4103/0019-5413.173522
PMCID: PMC4759881

Low dose short duration pulsed electromagnetic field effects on cultured human chondrocytes: An experimental study

Selvam Anbarasan, Ulaganathan Baraneedharan,1 Solomon FD Paul, Harpreet Kaur, Subramoniam Rangaswami,2 andEmmanuel Bhaskar3
Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India
1Department of Biomedical Sciences, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India
2Department of Orthopaedics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India
3Department of General Medicine, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India
Address for correspondence: Mr. Selvam Anbarasan, Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India. E-mail: moc.liamg@ivakbna
Author information ? Copyright and License information ?

Abstract

Background:

Pulsed electromagnetic field (PEMF) is used to treat bone and joint disorders for over 30 years. Recent studies demonstrate a significant effect of PEMF on bone and cartilage proliferation, differentiation, synthesis of extracellular matrix (ECM) and production of growth factors. The aim of this study is to assess if PEMF of low frequency, ultralow field strength and short time exposure have beneficial effects on in-vitro cultured human chondrocytes.

Materials and Methods:

Primary human chondrocytes cultures were established using articular cartilage obtained from knee joint during joint replacement surgery. Post characterization, the cells were exposed to PEMF at frequencies ranging from 0.1 to 10 Hz and field intensities ranging from 0.65 to 1.95 ?T for 60 min/day for 3 consecutive days to analyze the viability, ECM component synthesis, proliferation and morphology related changes post exposure. Association between exposure doses and cellular effects were analyzed with paired’t’ test.

Results:

In-vitro PEMF exposure of 0.1 Hz frequency, 1.95 ?T and duration of 60 min/day for 3 consecutive days produced the most favorable response on chondrocytes viability (P < 0.001), ECM component production (P< 0.001) and multiplication. Exposure of identical chondrocyte cultures to PEMFs of 0.65 ?T field intensity at 1 Hz frequency resulted in less significant response. Exposure to 1.3 ?T PEMFs at 10 Hz frequency does not show any significant effects in different analytical parameters.

Conclusions:

Short duration PEMF exposure may represent a new therapy for patients with Osteoarthritis (OA).

Keywords: Human chondrocytes, osteoarthritis, pulsed electromagnetic field
MeSh terms: Osteoarthritis, cartilage, articular, chondrocytes, electromagnetic fields

Introduction

Pulsed electromagnetic field (PEMF) has been used to treat bone and joint disorders for over 30 years.1Clinical use of PEMF preceded systematic research in its utility for bone and joint healing.2 Later studies identified that PEMF is capable of producing significant cellular changes in bone and cartilage cells by proliferation, differentiation, synthesis of extracellular matrix (ECM) and production of growth factors.3,4,5,7,8,9,10 A systematic review based on 3 clinical studies which assessed effect of PEMF therapy for osteoarthritis (OA) of knee, incorporating factors like pain, physical function, patient assessment, joint imaging, health related quality of life and physician global assessment indicates that electrical stimulation therapy may be useful in OA of knee, but stresses the need for confirmation in future studies.11 Proteoglycan (PG) loss occurs in joint cartilage in OA and PEMF therapy has been shown to induce PG synthesis in-vivoand in-vitro.12 PEMF has also demonstrated to have positive effect on cellular proliferation and DNA synthesis through opening of voltage sensitive calcium channels.13 Animal models have shown that PEMF therapy retards progression of OA.14,15

Most studies employing PEMF have used frequencies of 6- 75 Hz and field strengths of 0.4- 2.3 milli Tesla (mT). We desired to enquire if low frequency (0.1- 10 Hz), low field strength of 0.65- 1.95 µT and short duration exposure (60 min/day) of PEMF results in favorable effects on cultured human chondrocytes (synthesis of ECM; cell viability, proliferation and morphology). Further need for the study is to arrive at a minimal PEMF exposure protocol that is expected to decrease the concern related to unfavorable cellular changes and chromosomal aberrations that may result with high dose PEMF exposure.16

Materials and Methods

Isolation and characterization of chondrocytes

Articular cartilage samples were obtained from knee joint during joint replacement surgery after obtaining informed consent from patients. The study protocol was approved by Institutional Ethics Committee. Cartilage tissue over the nonweight bearing portion of the joint was removed and minced in Dulbecco’s modified eagle medium (DMEM) (Biogene technologies, India) supplemented with 10% FBS (Biogene technologies, India) and 1 ml Pen-strep (10000 units of penicillin and 10 mg of streptomycin, Invitrogen, India). Following this, the tissue was transferred into a conical flask and initially digested with pronase (1 mg/ml) (Biogene technologies, India) for 60 min, followed by type II collagenase (1 mg/1ml) (Invitrogen) for 16- 18 hours at 37°C. The following day, cellular debris and undigested tissue were removed and cells were separated using a 100 micron cell strainer. Isolated cells were seeded into 25 cm 2 culture flasks (TPP, India) with DMEM complete medium and maintained at 37°C with 5% CO2 levels. The cells were subcultured on attainment of 80% confluency. The attached cells were characterized by chondrocyte specific anti-Sox 9 transcription factor antibody staining (Abcam, India.). Chondrocytes that failed to form monolayer culture were not processed further. Post characterization, 4 × 105 cells were seeded in each flask and used for PEMF exposure after first passage.

Pulsed electromagnetic field exposure

The PEMF coil system fashioned for exposure is a four member coil frames, two larger (inner) and two smaller (outer) coil frames. The coils are mounted coaxially and in a co-planar fashion to form an enclosure, where it delivers currents in milliamps at desired waveforms, varying frequencies and magnetic field strength (Madras Institute of Magnetobiology, Chennai, India). This system designed according to the parametrical equation of Fansleau and Brauenbeck and a modified version of the Helmhotz coil. A box is housed inside the coil in which a 100 W bulb with regulator was used to maintain the temperature at 37°C and water to maintain humidity. Instead of 5% CO2, 20 mM HEPES was used as a buffering system. The chondrocytes were exposed to PEMF while monitoring field strength, frequency and temperature. The control (unexposed) cells were placed in the same environment and temperature but not exposed to PEMF.

Pulsed electromagnetic field treatment

The chondrocytes were seeded in 25 cm 2 culture flasks at concentrations of 6.5 × 105 cells/ml after 20 h being plated the cells were washed with phosphate buffer saline (PBS), and given fresh medium and exposed to PEMF for the first three daily trials; media was not changed from this point onwards. PEMF at a frequency of 0.1, 1 and 10 Hz were applied with flux densities of 0.65, 1.3 and 1.95 µT (peak-to-peak) for 60 min/day for 3 consecutive days. Whereas exposure to PEMFs at a repetition rate of 0.1 and 1 Hz with 1.95 and 0.65 µT caused a significant increase in chondrocyte viability that was dependent on PEMF amplitude, PEMFs applied at a repetition rate of 10 Hz and 1.3 µT did not produce any noticeable effects over cell viability and were not dealt with further in this manuscript. To test for effects of different exposure durations, cells were exposed to PEMFs of 1.95 and 0.65 µT magnitude and at frequency of 0.1 and 1 Hz for 60 min/day for 3 days. Cells were analyzed on third day for further experimental studies.

Cell viability assessment

Chondrocytes were cultured in 96 well plates at a density of 5 × 103 cells per well and exposed to PEMF in accordance to the exposure protocol mentioned. Twenty microliter of 0.5% 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Invitrogen) in phosphate buffered saline was added to each well after removal of medium and cells were incubated for 3 h at 37°C. Post incubation, 150 µl dimethyl sulfoxide (Hi-media, India) was added to each well and absorbance values (optical density value) were noted at 570 nm and 695 nm in spectrophotometer.17

Quantitative measurement of extracellular matrix proteoglycan and glycosaminoglycan synthesis

Chondrocytes were cultured in 48 well plates at densities of 104 cells per well and exposed to PEMF in accordance to the exposure protocol mentioned. Postexposure, glycosaminoglycan (GAG) synthesis was quantified by the dimethyl methylene blue (DMMB) assay. The DMMB reagent (Sigma, India) was prepared as detailed by Panin et al.18 and 200 µL was added to each well after removal of culture medium. Subsequently, absorbance values at 525 nm were noted.

Analysis of cell cycle by flow cytometry

Chondrocytes were cultured in 25 cm 2 culture flasks and exposed to PEMFs as mentioned earlier. After exposure, the cells were trypsinized, converted to single cell suspension in PBS and subjected to flow cytometery (FACS calibur, Becton Dickinson, Germany) according to the manufacturer’s instruction (Invitrogen, India) as follows: The suspension was spun at 1000 rpm for 10 min and the cell pellet was fixed in 70% ice cold ethanol at 4°C overnight. The cells were washed with PBS, treated with 500 µl RNAse A (40 µg/ml) (Sigma, India.) for 30 min at 37°C and stained with 500 µl propidium iodide (40 µg/ml) for 15 min incubation at room temperature. Postincubation, cell distributions at distinct phases of the cell cycle were analyzed by flow cytometery.

Analysis of cell architecture and morphology

Cell architecture and morphology were analyzed by staining of actin filaments in chondrocytes. Chondrocytes were cultured on cover slips in 6 well culture plates and exposed to PEMFs as described earlier. Processing of cells was done according to the manufacturer’s instructions (Invitrogen, India.). Briefly, the cells were fixed in 3.7% formaldehyde solution for 10 min after washing the slide with PBS and permeabilized in 0.1% Triton X-100 for 5 min. After washing with PBS, the cells were stained with 0.05 mg/ml Phalloidin solution at room temperature for 20-30 min, followed by counterstaining with 300 µl Propidium Iodide (500 nM). The coverslips were then rinsed in PBS, placed on a glass slide and cellular architecture and stress fiber formation was qualitatively analyzed by fluorescent confocal microscopy (LSM 510 META, Carl Zeiss, Germany).

Statistical analysis

Discrete variables were expressed as number (%) and continuous variables expressed as mean ± Standard Deviation. Association between field strengths (0.65, 1.3, and 1.95 µT) in variable frequencies (0.1, 1, and 10 Hz) and cellular effects (cell viability and ECM production,) was analyzed with paired ‘t’ test. A P < 0.05 was considered as statistically significant. Analysis was done with Statistical Package for the social sciences (SPSS) software version 21.0. This software was released in 2012 and used to solve business and research problems by means of ad-hoc analysis, hypothesis testing and predictive analysis.

Results

Isolation of chondrocytes

Healthy chondrocytes were observed in cultures by 3 days and these monolayers were 80% confluent by a week. The chondrocytes were spherical prior to attachment and later appeared polygonal in shape [Figure 1].

Figure 1

Primary human chondrocytes displaying typical polygonal conformation after attachment

Cell viability assessment

Viability of chondrocytes after PEMF exposure was quantified by the MTT assay to ascertain the effects PEMFs on chondrocytes which were exposed to PEMFs of field intensities between 1.95 and 0.65 µT at frequencies of 0.1 and 1 Hz for 60 min/day for 3 days. Following the third day exposure, samples were treated with MTT to quantify the cell viability and compared to control (unexposed) cultures. A highly significant viability of chondrocyte was observed in following field intensities and frequencies (1.95 µT-0.1Hz [P < 0.001], 1.95 µT -1Hz [P < 0.001] and 0.65 µT-0.1 Hz [P < 0.001]). Moderate favourable response was observed in other field intensities and frequencies [Table 1]. After 3 days of 60 min daily exposure to 1.95 µT PEMFs at a frequency of 0.1 Hz, the total number of cells in the culture increased, indicating heightened viability in response to PEMFs.

Table 1

MTT assay for detection of viable cells after exposure to PEMFs for 3 consecutive days

Quantitative measurement of proteoglycan glycosaminoglycan synthesis

Our spectrophotometric quantification of the ECM components such as GAG and PGs were assayed with identical PEMF parameters (field strengths, frequencies, and days of exposure and duration of exposure) as those used for MTT assay of cell viability with identical results. As compared with previously observed results, favorable responses to the production of ECM components were seen in following field strengths and frequencies (1.95 µT-0.1 Hz [P < 0.001], 1.95 µT -1 Hz [P < 0.001], 0.65 µT-0.1 Hz [P < 0.001], 0.65 µT-1 Hz [P < 0.001], 1.95 µT-10 Hz [P = 0.001] and 0.65 µT-10 Hz [P = 0.001]. Moderate favorable response was observed in other field intensities and frequencies [Table 2]. Our spectrophotometric quantification thus corroborates and strengthen our MTT assay results, indicating that exposure with 1.95 µT field intensity at frequency of 0.1 Hz for 60 min/day was most effective in production of GAG and PG of chondrocytes.

Table 2

DMMB assay for detection of ECM components after exposure to PEMFs for 3 consecutive days

Cell cycle analysis

Cells were analyzed to assess their distribution at different phases of the cell cycle by flow cytometry after staining of DNA with propidium iodide and recording of 106 events for each exposure parameter. The cells distribution in four distinct phases could be recognized in a proliferating cell population: G1, S (DNA synthesis Phase), G2 and M (Mitosis). As both G2 and M phase have an identical DNA content, they could not be discriminated based on their differences in their DNA content. The percentage values were assigned to each population and also dot plot [Figure ?[Figure2a2a and ?andb]b] and histogram [Figure ?[Figure2c2c and ?andd]d] were used to denote the distribution of cells in distinct phases. PEMF at different field strengths and frequencies was found to promote cell cycle progression from the G1 phase to the S and G2-M phases. Cells present in G2-M phase are in dividing state and show increased rate of proliferation. A shift to top of cell population (G2-M) in dot plot shows great proliferation [Figure ?[Figure2a2a and ?andb].b]. Based on the percentage of cells distribution in G2-M phase, proliferation effect was determined at different exposure parameters. Histogram indicates, cells exposed at 0.1 Hz frequency with 1.95 µT of PEMFs show 20.24% of their significant presence in G2-M phase compared to other filed strengths such as 0.65 (18.9%) and 1.3 µT (17.54%) [Figure 2c]. The cells exposed to 1.95 µT of PEMFs at 0.1 Hz frequency shows 20.24% of their significant presence in G2-M phase compared to other frequencies such as 1 Hz (19.46%) and 10 Hz (17.83%) [Figure 2d].

Figure 2

Cell cycle analysis by flow cytometer to determine the proliferative effect of chondrocytes in distinct cell cycle phases. Percentage of chondrocytes distribution in G2-M phase indicates cell proliferation effects as it has all mitotic cells. Significant

Analysis of cell architecture and morphology

Actin filaments of the cytoplasm stained by Phalloidin and nucleus was counterstained with propidium iodide observed by confocal fluorescent microscopy showed a significant difference in morphological structure and formation of stress fibers between exposed chondrocytes at varying frequencies (0.1, 1 and, 10 Hz) with specific field strength 1.95 µT and unexposed cells. Stress fiber formation was increased in chondrocytes exposed at frequency of 0.1 Hz with 1.95 µT compared to unexposed [Figure 3]. Stress fiber formation indicates that the cells stability, strength and their healthy attachment.

Figure 3

Human chondrocytes morphological structure was studied by staining with phalloidin and propidium iodide for visualizing stress fibers (green) and nuclear staining (red). (a) No stress fiber formation in chondrocytes unexposed to pulsed electromagnetic

Discussion

Our study observed that short term in-vitro chondrocyte exposure to PEMFs at frequency of 0.1 Hz and field strength of 1.95 µT for 60 min/day for 3 consecutive days have shown highly significant effects in different experimental parameters such as cell viability, ECM production, cell cycle progression and stress fiber formation. By contrast, exposure of identical chondrocyte cultures to PEMFs of 0.65 µT field intensity at 1 Hz frequency resulted in less significant levels of different parameters. On the other hand, exposure to 1.3 µT PEMFs at 10 Hz frequency does not shown any significant effects in different analytical parameters. These findings, apart from observing benefits of certain range of field strengths, also bring to light the ability of PEMF to inhibit cellular effects when used at certain field strengths and frequencies, a fact which has been observed earlier.

In our study design, we limited our experiments to within 3 days of exposure to PEMF to stay within the realm of better clinical applicability. For our analysis, we have chosen 3 days as an appropriate end point as it avoided the over confluence of chondrocytes and also it would minimize the contact inhibition that can induce changes in biochemical status and cause dedifferentiation. As the number of days of exposure to PEMFs increases, it may enhance the proliferative effects to the chondrocytes. The design of longer day exposure to PEMFs will be taken into future study. PEMF parameters used in this study such as frequency, field strength and duration of exposure could translate into the clinical application and will be innocuous to the target tissue and their surrounding tissues which are exposed to PEMF during clinical therapy.

Our study observed correlation between critical cell characteristics (cell viability and promotion in cell multiplication) of exposed samples and induction of extracellular components which include GAG and PG. This raises the question on the validity of using changes in ECM components as a marker of chondrocyte healing in studies using in-vitro models.

The earliest in-vitro study with bovine articular chondrocytes exposed using Helmholtz coils found no significant effect of PEMF on ECM component synthesis.19 Sakai and colleagues studied the effect of 0.4 mT field strength at 6.4 Hz delivered over a period of 5 days on rabbit growth cartilage and human articular cartilage and observed that PEMF stimulated cell proliferation and GAG synthesis in growth cartilage cells but resulted in only cell proliferation with no increase in GAG content in articular cartilage cells.20 The latter finding of our observation on extracellular components (GAG and PG) synthesis is comparable with earlier studies observation.

De Mattei et al. exposed chondrocytes from healthy patients to PEMF to varying duration of exposure (1- 18 h and 1- 6 days) using a field strength of 2.3 mT at 75 Hz. The study observed that short duration of exposure (1 and 6 h) did not result in increased DNA synthesis, while longer duration of exposure (9 and 18 h) increased DNA synthesis.21 Chang et al., exposed porcine chondrocytes to a field of 1.8- 3 mT at a frequency of 75 Hz for 2 h/day for 3 weeks and observed that long term 3 weeks PEMF exposure was beneficial over the short term 1 week exposure.22 However, our observations contradict these findings and reports the better efficacy of even short term PEMF exposures. Though our study observed the efficacy of a daily PEMF exposure of 60 min for only 3 days, benefits of exposure should be expected to enhance with daily exposures exceeding 3 days. We could not observe the benefits beyond day 3, since confluent chondrocyte cultures de-differentiated due to contact inhibition beyond this period in two-dimensional cultures.

Our observation on promotion of cell cycle from G1 phase to G2-M phase with certain field strengths is comparable with the findings of Nicolin et al. which observed similar results with field strength of 2 mT at 75 Hz with an exposure time of 4 h or 12 h/day.23 The striking observation of similar findings in our study with much lower field strength for exposure duration of 60 min has better clinical applicability.

A recent in-vivo animal study exposed rabbits with experimental osteochondral defect to PEMF for a period of 60 min/day for 6 weeks and observed a better total histological score in the study group to conclude that PEMF is beneficial for hyaline cartilage formation.24 The only in-vitro study on human chondrocytes harvested from OA knee reports no effect on PG production using field strength of 2mT at 50 Hz for 14 days.25 However both studies did not evaluate fine cellular effects (cell viability and cell cycle promotion).

Based on our data, the study informs that the future in-vitro studies on the topic should probably use exposure duration not more than 60 min/day but we can increase more number of days to PEMFs at 0.1 and 1 Hz frequencies and 1.95 and 0.65 µT field intensities. However, future studies should aim to utilize collagen matrix in three-dimensional (3D) cultures and focus more on exposure for more number of days to overcome the limitation of dedifferentiation and contact inhibition due to over confluent in 3D model and also focus on the effect of PEMF on chondrocyte cytoskeleton (observed as stress fibers in Phalloidin staining). It would of interest to investigate the strength of the chondrocyte cytoskeleton between exposed and control cells. Though it may be argued that occurrence of stress fiber formation observed with PEMF exposure is a result of heating effect due to Helmholtz system, the low dose of PEMF is less likely to have produced a heating effect which may happen with higher doses.

To conclude, our study observed that short duration (60 min/day) low frequency (0.1 Hz) low field strength (1.95 µT) PEMFs have beneficial effects on chondrocyte viability, ECM production, multiplication and probably cytoskeleton even for a short period of 3 days. Short duration PEMF exposure for patients with OA has the potential to produce favorable clinical effects. However, the results of the study have to be confirmed with a methodology incorporating assessment of both mass and strength of PEMF exposed chondrocytes.

Financial support and sponsorship

Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organisation (DRDO), Ministry of Defence, Government of India.

Conflicts of interest

There are no conflicts of interest.

References

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PEMF vs / or combined with Laser Therapy

J Maxillofac Oral Surg. 2014 Dec;13(4):451-7. doi: 10.1007/s12663-013-0551-2. Epub 2013 Jul 26.

Radiodensitometric Assessment of the Effect of Pulsed Electromagnetic Field Stimulation Versus Low Intensity Laser Irradiation on Mandibular Fracture Repair: A Preliminary Clinical Trial.

Refai H1, Radwan D1, Hassanien N1.

Author information

  • 1Department of Oral and Maxillofacial Surgery, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt.

Abstract

PURPOSE:

Closed reduction of mandibular fractures usually entails a relatively long period of immobilization, with the subsequent delay of rehabilitation. Therefore, shorter immobilization period with various approaches to protect or enhance bone healing have been investigated. The aim of this study was to analyze the effects of pulsed electromagnetic field (PEMF) and low intensity laser irradiation (LILI) on the fracture healing process, through radiodensitometric assessment of the bone callus.

PATIENTS AND METHODS:

Eighteen patients with mandibular fractures at the tooth bearing area participated in this prospective study. They were treated by closed reduction using maxillo-mandibular fixation (MMF) and were consecutively assigned into 1 of 3 groups. In group A, the fracture sites were exposed to PEMF for 2 h daily for 12 days. In group B, the fracture sites were exposed to LILI on the tenth and twelfth postoperative days (2 sessions of 6 min per day 2 h apart). The fracture sites in group C acted as controls. MMF was maintained for 2 weeks in group A and 4 weeks in groups B and C. The bone fracture healing was evaluated clinically by investigating the union of the fractured segments and radiographically using computerized densitometry. The union of the fractured segments was tested by manual manipulation and the occlusion was assessed upon removal of MMF. Standardized digital panoramic radiographs were performed for each patient, immediately postoperatively as well as at 2 and 4 weeks. The digital images were manipulated using the IDRISI software. A rectangular area of 10 × 15 mm was drawn along the center of the fracture line. The obtained densitometry values were expressed in gray levels from 0 to 256. The collected data were then tabulated and statistically analyzed.

RESULTS:

After releasing the MMF, the bimanual mobility test of the fractured segments in all patients showed stability of the segments. The preinjury occlusion was maintained in all patients. The postoperative radiographs of all patients revealed good bony alignment of the bony segments. In all groups, comparison between the study intervals with respect to both means and changes percentages of the bone density values showed insignificant differences. At 2nd postoperative week, the mean bone density at the fracture sites decreased by 4.74, 6.6 and 27.89 % in groups A, B and C respectively. The period from the 2nd to the 4th postoperative weeks showed increase in the bone density by 1.49, 1.95 and 14.12 % in groups A, B and C respectively. Insignificant difference was found between the means of bone densities of group A and B throughout the study intervals. On the other hand, both groups showed insignificant difference with group C immediately postoperative and significant increase in bone density at the 2nd and 4th postoperative weeks.

CONCLUSIONS:

Short period immobilization of mandibular fractures for 2 weeks supplemented with PEMF is recommended. Further studies are needed to evaluate the efficacy of LILI as a supplement to reduce the mandibular fracture immobilization period.

Laser vs / or combined with PEMF

Lasers Med Sci. 2015 Aug 26. [Epub ahead of print]

LASER versus electromagnetic field in treatment of hemarthrosis in children with hemophilia.

Eid MA1, Aly SM.

Author information

  • 1Department of Physical Therapy, College of Applied Medical Sciences, Najran University, Najran, KSA, Saudi Arabia, mohamed.eid27@yahoo.com.

Abstract

Children with hemophilia usually have recurrent joint bleeding that leads to joint damage, loss of range of motion, and restriction of mobility, therefore affecting the quality of life in these children. The purpose of this study was to compare the effects of low-level laser therapy (LLLT) to that of pulsed electromagnetic field (PEMF) in treatment of hemarthrosis in children with hemophilia. Thirty boys with hemophilia A with ages ranging from 9 to 13 years were selected and assigned randomly, using sealed envelopes, into two equal intervention groups. The study group I received the traditional physical therapy program in addition to LLLT, whereas the study group II received the same physical therapy program given to the study group I in addition to PEMF. Both groups received the treatment sessions three times per week for three successive months. Pain, laboratory investigations, swelling, and range of motion (ROM) of the affected knee joint, in addition to physical fitness were evaluated before, at the end of the sixth week and at 12 weeks of the treatment program. Laser group showed significant improvement in all measured variables after the sixth week of treatment when compared with PEMF. By 12 weeks of treatment, there was a significant improvement in pain, ROM, ESR and leucocytes levels in laser group compared with PEMF, while there was no significant difference in knee circumferences and the 6-min walk test (6MWT) between both groups. Both groups showed significant improvement at 12 weeks of treatment compared with that at 6 weeks. Both LLLT and PEMF are effective modalities in reducing pain, swelling, increasing ROM and improving physical fitness. Twelve weeks of treatment of both modalities demonstrated significant improvement than 6 weeks of treatment. Laser therapy induced significant improvement than electromagnetic therapy in treatment of hemarthrosis-related problems in children with hemophilia.

 
J Maxillofac Oral Surg. 2014 Dec;13(4):451-7. doi: 10.1007/s12663-013-0551-2. Epub 2013 Jul 26.

Radiodensitometric Assessment of the Effect of Pulsed Electromagnetic Field Stimulation Versus Low Intensity Laser Irradiation on Mandibular Fracture Repair: A Preliminary Clinical Trial.

Refai H1, Radwan D1, Hassanien N1.

Author information

  • 1Department of Oral and Maxillofacial Surgery, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt.

Abstract

PURPOSE:

Closed reduction of mandibular fractures usually entails a relatively long period of immobilization, with the subsequent delay of rehabilitation. Therefore, shorter immobilization period with various approaches to protect or enhance bone healing have been investigated. The aim of this study was to analyze the effects of pulsed electromagnetic field (PEMF) and low intensity laser irradiation (LILI) on the fracture healing process, through radiodensitometric assessment of the bone callus.

PATIENTS AND METHODS:

Eighteen patients with mandibular fractures at the tooth bearing area participated in this prospective study. They were treated by closed reduction using maxillo-mandibular fixation (MMF) and were consecutively assigned into 1 of 3 groups. In group A, the fracture sites were exposed to PEMF for 2 h daily for 12 days. In group B, the fracture sites were exposed to LILI on the tenth and twelfth postoperative days (2 sessions of 6 min per day 2 h apart). The fracture sites in group C acted as controls. MMF was maintained for 2 weeks in group A and 4 weeks in groups B and C. The bone fracture healing was evaluated clinically by investigating the union of the fractured segments and radiographically using computerized densitometry. The union of the fractured segments was tested by manual manipulation and the occlusion was assessed upon removal of MMF. Standardized digital panoramic radiographs were performed for each patient, immediately postoperatively as well as at 2 and 4 weeks. The digital images were manipulated using the IDRISI software. A rectangular area of 10 × 15 mm was drawn along the center of the fracture line. The obtained densitometry values were expressed in gray levels from 0 to 256. The collected data were then tabulated and statistically analyzed.

RESULTS:

After releasing the MMF, the bimanual mobility test of the fractured segments in all patients showed stability of the segments. The preinjury occlusion was maintained in all patients. The postoperative radiographs of all patients revealed good bony alignment of the bony segments. In all groups, comparison between the study intervals with respect to both means and changes percentages of the bone density values showed insignificant differences. At 2nd postoperative week, the mean bone density at the fracture sites decreased by 4.74, 6.6 and 27.89 % in groups A, B and C respectively. The period from the 2nd to the 4th postoperative weeks showed increase in the bone density by 1.49, 1.95 and 14.12 % in groups A, B and C respectively. Insignificant difference was found between the means of bone densities of group A and B throughout the study intervals. On the other hand, both groups showed insignificant difference with group C immediately postoperative and significant increase in bone density at the 2nd and 4th postoperative weeks.

CONCLUSIONS:

Short period immobilization of mandibular fractures for 2 weeks supplemented with PEMF is recommended. Further studies are needed to evaluate the efficacy of LILI as a supplement to reduce the mandibular fracture immobilization period.

 

Ankylosing Spondylitis

Rheumatol Int. 2014 Mar;34(3):357-65. doi: 10.1007/s00296-013-2941-7. Epub 2014 Jan 8.

Is magnetotherapy applied to bilateral hips effective in ankylosing spondylitis patients? A randomized, double-blind, controlled study.

Turan Y1, Bayraktar K, Kahvecioglu F, Tastaban E, Aydin E, Kurt Omurlu I, Berkit IK.

Author information

  • 1Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Adnan Menderes University, Ayd?n, Turkey, dryaseminturan@gmail.com.

Abstract

This double-blind, randomized controlled study was conducted with the aim to investigate the effect of magnetic field therapy applied to the hip region on clinical and functional status in ankylosing spondylitis (AS) patients. Patients with AS (n = 66) who were diagnosed according to modified New York criteria were enrolled in this study. Patients were randomly divided in two groups. Participants were randomly assigned to receive magnetic field therapy (2 Hz) (n = 35), or placebo magnetic field therapy (n = 31) each hip region for 20 min. Patients in each group were given heat pack and short-wave treatments applied to bilateral hip regions. Both groups had articular range of motion and stretching exercises and strengthening exercises for surrounding muscles for the hip region as well as breathing and postural exercises by the same physical therapist. These treatment protocols were continued for a total of 15 sessions (1 session per day), and patients were examined by the same physician at months 1, 3 and 6. Visual analogue scale (VAS) pain, VAS fatigue, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrologic Index (BASMI), DFI, Harris hip assessment index and Ankylosing Spondylitis Quality of Life scale (ASQOL) were obtained at the beginning of therapy and at month 1, month 3 and month 6 for each patient. There were no significant differences between groups in the VAS pain, VAS fatigue, morning stiffness, BASDAI, BASFI, BASMI, DFI, Harris hip assessment index and ASQoL at baseline, month 1, month 3 or month 6 (p > 0.05). Further randomized, double-blind controlled studies are needed in order to establish the evidence level for the efficacy of modalities with known analgesic and anti-inflammatory action such as magnetotherapy, particularly in rheumatic disorders associated with chronic pain.

Ankylosing Spondylitis

Rheumatol Int. 2014 Mar;34(3):357-65. doi: 10.1007/s00296-013-2941-7. Epub 2014 Jan 8.

Is magnetotherapy applied to bilateral hips effective in ankylosing spondylitis patients? A randomized, double-blind, controlled study.

Author information

  • 1Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Adnan Menderes University, Ayd?n, Turkey, dryaseminturan@gmail.com.

Abstract

This double-blind, randomized controlled study was conducted with the aim to investigate the effect of magnetic field therapy applied to the hip region on clinical and functional status in ankylosing spondylitis (AS) patients. Patients with AS (n = 66) who were diagnosed according to modified New York criteria were enrolled in this study. Patients were randomly divided in two groups. Participants were randomly assigned to receive magnetic field therapy (2 Hz) (n = 35), or placebo magnetic field therapy (n = 31) each hip region for 20 min. Patients in each group were given heat pack and short-wave treatments applied to bilateral hip regions. Both groups had articular range of motion and stretching exercises and strengthening exercises for surrounding muscles for the hip region as well as breathing and postural exercises by the same physical therapist. These treatment protocols were continued for a total of 15 sessions (1 session per day), and patients were examined by the same physician at months 1, 3 and 6. Visual analogue scale (VAS) pain, VAS fatigue, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrologic Index (BASMI), DFI, Harris hip assessment index and Ankylosing Spondylitis Quality of Life scale (ASQOL) were obtained at the beginning of therapy and at month 1, month 3 and month 6 for each patient. There were no significant differences between groups in the VAS pain, VAS fatigue, morning stiffness, BASDAI, BASFI, BASMI, DFI, Harris hip assessment index and ASQoL at baseline, month 1, month 3 or month 6 (p > 0.05). Further randomized, double-blind controlled studies are needed in order to establish the evidence level for the efficacy of modalities with known analgesic and anti-inflammatory action such as magnetotherapy, particularly in rheumatic disorders associated with chronic pain.

Ankylosing Spondylitis

Rheumatol Int. 2014 Mar;34(3):357-65. doi: 10.1007/s00296-013-2941-7. Epub 2014 Jan 8.

Is magnetotherapy applied to bilateral hips effective in ankylosing spondylitis patients? A randomized, double-blind, controlled study.

Turan Y1, Bayraktar K, Kahvecioglu F, Tastaban E, Aydin E, Kurt Omurlu I, Berkit IK.

Author information

  • 1Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Adnan Menderes University, Ayd?n, Turkey, dryaseminturan@gmail.com.

Abstract

This double-blind, randomized controlled study was conducted with the aim to investigate the effect of magnetic field therapy applied to the hip region on clinical and functional status in ankylosing spondylitis (AS) patients. Patients with AS (n = 66) who were diagnosed according to modified New York criteria were enrolled in this study. Patients were randomly divided in two groups. Participants were randomly assigned to receive magnetic field therapy (2 Hz) (n = 35), or placebo magnetic field therapy (n = 31) each hip region for 20 min. Patients in each group were given heat pack and short-wave treatments applied to bilateral hip regions. Both groups had articular range of motion and stretching exercises and strengthening exercises for surrounding muscles for the hip region as well as breathing and postural exercises by the same physical therapist. These treatment protocols were continued for a total of 15 sessions (1 session per day), and patients were examined by the same physician at months 1, 3 and 6. Visual analogue scale (VAS) pain, VAS fatigue, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrologic Index (BASMI), DFI, Harris hip assessment index and Ankylosing Spondylitis Quality of Life scale (ASQOL) were obtained at the beginning of therapy and at month 1, month 3 and month 6 for each patient. There were no significant differences between groups in the VAS pain, VAS fatigue, morning stiffness, BASDAI, BASFI, BASMI, DFI, Harris hip assessment index and ASQoL at baseline, month 1, month 3 or month 6 (p > 0.05). Further randomized, double-blind controlled studies are needed in order to establish the evidence level for the efficacy of modalities with known analgesic and anti-inflammatory action such as magnetotherapy, particularly in rheumatic disorders associated with chronic pain.