Kidney Stones

Vopr Kurortol Fizioter Lech Fiz Kult. 1994 May-Jun;(3):22-4.

The use of an impulse magnetic field in the combined therapy of patients with stone fragments in the upper urinary tract.

[Article in Russian]

Li AA, Nesterov NI, Malikova SN, Kiiatkin VA.

The paper covers the experiment’s details and the results obtained at stimulation of the kidney projection region and upper ureter with remote-controlled impulse magnetic field (IMF). It was found that IMF activates impulse activity of ureteral smooth muscles in 100% of cases. The technique is detailed of managing ureterolith fragments using IMF combined with iodobromine baths. The results of the proposed treatment are given. A single therapeutic course produced effect in 62.9% of the patients.

Kidney Disease

Bull Exp Biol Med. 2014 Aug;157(4):440-2. doi: 10.1007/s10517-014-2585-y. Epub 2014 Aug 12.

Study of microcirculation of the ocular ciliary body in experimental kidney disease.

Obrubov SA1, Ivanova AO, Klyuchnikov SO, Dreval AA, Boginskaya OA.

Author information

  • 1N. I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow, Russia,


We studied disorders in ciliary body microcirculation in experimental chronic glomerulonephritis with tubulointerstitial nephritis and evaluated the hemodynamic effects of low-frequency pulsed electromagnetic field in this pathology. Laser Doppler flowmetry demonstrated vasospasm with reduced nutrient blood fl ow in the ciliary body of animals with experimental chronic glomerulonephritis with tubulointerstitial nephritis. The exposure to low-frequency pulsed electromagnetic field using developed technology will lead to significant reduction of the vascular tone and improve arterial blood supply to the microcirculatory bed.

Anesteziol Reanimatol. 1993 Sep-Oct;(5):49-51.

The magneto-laser effect on liver functions in the complex treatment of hepatorenal failure.

[Article in Russian]

Shimanko II, Limarev VM.

Complex therapy of acute hepatorenal failure included magnetic field and laser applied to hepatic area and blood extracorporeally. In 26 patients magnetic field and laser were applied to the hepatic area. When compared to a control group of 30 patients a more prompt decrease in blood bilirubin was noted, alongside with a drop in blood fibrinogen, which enhanced a threat of profuse bleeding. There was no decrease in blood fibrinogen or an accelerated drop in bilirubin during extracorporeal application of the technique in 23 patients, as compared to a control group of 20 patients. As the technique has different effects on the liver it should be used with care and only when clinically indicated in patients with acute hepatorenal failure.

Urol Nefrol (Mosk). 1993 Mar-Apr;(2):17-20.

The effect of magnetic and laser therapy on the course of an experimental inflammatory process in the kidneys.

[Article in Russian]

Rodoman VE, Avdoshin VP, Eliseenko VI, Andriukhin MI, Rudyk IaV.

Magnetolaserotherapy (impulse power 3.6-15.2 mW, 10-50 mT) in rabbits with acute pyelonephritis resulted in significant improvement against the responses in the control group. Histologically, this appeared as exudation phase reduction, more rapid proliferation, replacement of the inflammation focus for granulation tissue.

Ischemia – Reperfusion Injury

Logo of bsr

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

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

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

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

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


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

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

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



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

The measurement of blood pressure in SHR rats

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

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

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

Pulsed electromagnetic field treatment

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

Detection of myocardium apoptosis

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

Determination of myocardial enzymes in plasma

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

Myocardium cells morphology via TEM

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

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

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

Measurement of nitric oxide concentration and Western blotting

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

Cell culture

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

Cell treatment (hypoxia/reoxygenation)

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

Cell viability assays

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

Measurement of intracellular reactive oxygen species levels

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

Data analysis

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


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

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

Figure 1

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

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

PEMF treatment could observably improve the abundance of EPCs

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

Figure 2

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

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

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

Figure 3

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

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

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

Figure 4

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

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

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

Figure 5

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

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

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

Figure 6

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

Effect of PEMF on NO releasing via Akt/eNOS pathway

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

Figure 7

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


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

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

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

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

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

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

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


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


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


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


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

[grant number ZDSYS14005]



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Ion Cyclotron Resonance

Electromagn Biol Med. 2009;28(2):124-34.

Electric polarization and the viability of living systems: ion cyclotron resonance-like interactions.

Liboff AR.

Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, Florida 33431, USA.


Wellness can be described in physical terms as a state that is a function of the organism’s electric polarization vector P(r, t). One can alter P by invasive application of electric fields or by non invasive external pulsed magnetic fields (PMF) or ion cyclotron resonance (ICR)-like combinations of static and sinusoidal magnetic fields. Changes in human (total) body bioimpedance are significantly altered during exposure to ICR magnetic field combinations. The conductivities of polar amino acids in solution exhibit sharp discontinuities at ICR magnetic fields tuned to the specific charge to mass ratio of the amino acid. It has been reported that protein peptide bonds are broken by such amino acid ICR fields. Remarkably, some of these effects are only found at ultra-low AC magnetic intensities, on the order of .05 muT. This is approximately 10(3) below accepted levels determined by engineering estimates. Such strikingly low magnetic intensities imply the existence of physically equivalent endogenous weak electric field oscillations. These observations not only make claims related to electromagnetic pollution more credible but also provide a basis for future electromagnetic applications in medicine. They also reinforce the notion that physical factors acting to influence the electric polarization in living organisms play a key role in biology.

Electromagn Biol Med. 2009;28(1):71-9.

Cellular ELF signals as a possible tool in informative medicine.

Foletti A, Lisi A, Ledda M, de Carlo F, Grimaldi S.

BITitalia, Milan, Italy.


According to Quantum Electro-Dynamical Theory by G. Preparata, liquid water can be viewed as an equilibrium between of two components: coherent and incoherent ones. The coherent component is contained within spherical so called “coherence domains” (CDs) where all molecules synchronously oscillate with the same phase. CDs are surrounded by the incoherent component where molecules oscillate with casual phases regarding each other. The existence of coherent domain in water has been demonstrated in a set of experiments on pure water exposed to high voltage, under this condition the electric field concentrates inside the water, arranging the water molecules to form high ordered structure. Recently has been studied the influence of combined static and alternating parallel magnetic fields on the current through the aqueous solution of glutamic acid; outlining the relevance of low frequency electro-magnetic field in interacting with biological target. Additional results demonstrate that at combined static and alternating parallel, magnetic fields matching the ion cyclotron energy resonance of a particular charged molecule into biological tissue an intrinsic weak magnetic field is generated by ion currents in the cell. These results should increase the reliability and the clinical feasibility of the use of electromagnetic field, tuned at ion cyclotron resonance of charged molecules, as a biophysical approach to interfere with biological mechanisms. We demonstrate that Exposure of human epithelial cell to ion cyclotron energy resonance generated by a commercial electromedical device (Vega select 719) tuned to calcium ion at 7 Hz act as a differentiation factor, thus opening up the possibility to use particular extremely low frequency electro magnetic field protocols, in informative medicine.

Electromagn Biol Med. 2008;27(3):230-40.

Calcium ion cyclotron resonance (ICR) transfers information to living systems: effects on human epithelial cell differentiation.

Lisi A, Ledda M, De Carlo F, Foletti A, Giuliani L, D’Emilia E, Grimaldi S.

Istituto di Neurobiologia e Medicina Molecolare CNR, Rome, Italy.


The specific aim of the present work concerns the effectiveness of low-frequency electromagnetic fields treatment to modify biochemical properties of human keratinocytes (HaCaT). Cells exposed to a 7 Hz electromagnetic field, tuned to calcium ion cyclotron resonance (ICR), showed modifications in the cytoskeleton. These modifications were related to different actin distributions as revealed by phalloidin fluorescence analysis. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression, indicating that exposure to electromagnetic field carries keratinocytes to an upper differentiation level. This study confirms our previous observation and supports the hypothesis that a 7 Hz calcium ICR electromagnetic field may modify cell biochemistry and interfere in the differentiation and cellular adhesion of normal keratinocytes, suggesting the possibility to use ICR electromagnetic therapy for the treatment of undifferentiated diseases.

Electromagn Biol Med. 2007;26(4):315-25.

Local and holistic electromagnetic therapies.

Liboff AR.

Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, Florida, USA.


Based on decades of experimental evidence an excellent argument can be made for the existence of a fundamental functional relationship between living systems and electromagnetic fields. We have previously hypothesized that this relationship can be expressed in terms of a field vector whose source is the distribution of electric polarization within the system and which has both a phylogenetic and ontogenetic time dependence. Ion cyclotron resonance (ICR)-like magnetic signals have resulted in physiologic changes in many in vitro and in vivo model systems and have been applied medically with success to bone repair and spinal fusion. This type of local ICR-like therapy has recently been broadened into a holistic application following the remarkable discovery that the whole-body bioimpedance is sharply dependent on ICR signals. We relate this observation to the integrated electric polarization vector, in turn a measure of the double layer charge distribution at the cell membrane. This discovery, already being applied to a number of clinical problems, lends strong support to the concept of an overarching electromagnetic framework for living systems.

Electromagn Biol Med. 2006;25(4):227-43.

Some problems in modern bioelectromagnetics.

Zhadin M, Giuliani L.

Institute of Cell Biophysics of RAS, Pushchino, Moscow Region, Russia.


One of the main problems of bioelectromagnetics – the unbelievable narrow resonance peaks at the cyclotron frequency of the alternating magnetic field – was considered. Modern electrodynamics of condensed matter clearly brings out that the reason of this phenomenon is extremely low viscosity within coherence domains of aqueous electrolytic solutions. The electrochemical model of action of combined static and alternating magnetic fields on aqueous solutions of amino acids is proposed. The possibility of arising a succession of changes in ionic forms in these processes was revealed. The dipole ions (zwitterions) together with water molecules electrostatically forming joint groups in the solution, create favorable conditions for arising mixed coherence domains there. Simultaneously with evolution of the coherent processes in these domains, the amino acid zwitterions are transforming into the usual ionic form, fit for cyclotron resonance. The development of cyclotron resonance under action of combined magnetic fields increases the ion kinetic energy, and the ions leave the domains for the incoherent component of the solution according to Del Giudice pattern (Comisso et al., 2006; Del Giudice et al., 2002), creating the peak current through the solution. Then the ions are transforming little by little into zwitterionic form again; after that, the solution becomes ready to react on exposure of magnetic fields again. The possibilities for formation of coherence domains composed of water molecules together with peptide molecules or protein ones are discussed.

Anal Chem. 2005 Sep 15;77(18):5973-81.

Observation of increased ion cyclotron resonance signal duration through electric field perturbations.

Kaiser NK, Bruce JE.

Department of Chemistry, Washington State University, Pullman Washington 99164-4630, USA.


Ion motion in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is complex and the subject of ongoing theoretical and experimental studies. Two predominant pathways for the loss of ICR signals are thought to include damping of cyclotron motion, in which ions lose kinetic energy and radially damp toward the center of the ICR cell, and dephasing of ion coherence, in which ions of like cyclotron frequency become distributed out of phase at similar cyclotron radii. Both mechanisms result in the loss of induced ion image current in FTICR-MS measurements and are normally inseparable during time-domain signal analysis. For conventional ICR measurements which take advantage of ion ensembles, maximization of the ion population size and density can produce the desired effect of increasing phase coherence of ions during cyclotron motion. However, this approach also presents the risk of coalescence of ion packets of similar frequencies. In general, ICR researchers in the past have lacked the tools necessary to distinguish or independently control dephasing and damping mechanisms for ICR signal loss. Nonetheless, the ability to impart greater phase coherence of ions in ICR measurements will allow significant advances in FTICR-MS research by improving the current understanding of ICR signal loss contributions of dephasing and damping of ion ensembles, increasing overall time-domain signal length, and possibly, resulting in more routine ultrahigh resolution measurements. The results presented here demonstrate the ability to employ a high density electron beam to perturb electric fields within the ICR cell during detection of cyclotron motion, in an approach we call electron-promoted ion coherence (EPIC). As such, EPIC reduces ICR signal degradation through loss of phase coherence, and much longer time-domain signals can be obtained. Our results demonstrate that time-domain signals can be extended by more than a factor of 4 with the implementation of EPIC, as compared to conventional experiments with otherwise identical conditions. The application of EPIC has also been observed to reduce the appearance of peak coalescence. These capabilities are not yet fully optimized nor fully understood in terms of the complex physics that underlies the enhancement. However, the enhanced time-domain signals can result in improved resolution in frequency-domain signals, and as such, this result is important for more efficient utilization of FTICR-MS. High resolution and accurate mass analysis are prime motivating factors in the application of advanced FTICR technology. We believe the approach presented here and derivatives from it may have significant benefit in future applications of advanced FTICR technology.

Int J Neurosci. 2004 Aug;114(8):1035-45.

Lithium ion “cyclotron resonance” magnetic fields decrease seizure onset times in lithium-pilocarpine seized rats.

McKay BE, Persinger MA.

Behavioral Neuroscience Laboratory, Laurentian University, Sudbury, Ontario, Canada.


The cyclotron resonance equation predicts that the frequency of an applied magnetic field that might optimally interact with a single ion species may be computed as a function of the charge-to-mass ratio of the ion and the strength of the background static magnetic field. The present study was undertaken to discern the applicability of this equation for optimizing lithium ion utilization in the rat, as inferred by the predicted magnetic “ion resonance “field-induced shift of lithium’s dose-dependent curve for seizure onset times (SOTs) when combined with the cholinergic agent pilocarpine. Groups of rats were administered 1.5 thru 3 mEq/kg lithium chloride (in 0.5 mEq/kg increments) and exposed to reference conditions or to one of three intensities (70 nanoTesla, 0.8 microTesla, or 25 microTesla) of a 85 Hz magnetic field calculated to resonate with lithium ions given the background static geomagnetic field of approximately 38,000 nanoTesla (0.38 Gauss). A statistically significant quadratic relationship for SOT as a function of magnetic field intensity (irrespective of lithium dose) was noted: this U-shaped function was characterized by equal SOTs for the reference and 25 microTesla groups, with a trend toward shorter SOTs for the 70 nanoTesla and 0.8 microTesla groups. Although not predicted by the equations, this report extends other findings suggestive of discrete intensity windows for which magnetic field frequencies derived from the cyclotron ion resonance equation may affect ion activity.

NeuroRehabilitation. 2002;17(1):9-22.

Physical mechanisms in neuroelectromagnetic therapies.

Liboff AR, Jenrow KA.

Department of Physics, Oakland University, Rochester, MI 48309,


Physical parameters that are used to characterize different types of electromagnetic devices used in neurotherapy can include power, frequency, carrier frequency, current, magnetic field intensity, and whether an application is primarily electric or primarily magnetic. Currents can range from tens of microamperes to hundreds of milliamperes, magnetic fields from tens of microtesla to more than one tesla, and frequencies from a few Hz to more than 50 GHz. A division into three device categories is proposed, based on the current applied and the specificity of the therapeutic signal. Two research areas have great potential for new neuroelectromagnetic strategies. Studies of endogenous neural oscillatory states suggest using external fields to reinforce or inhibit such states. Also, various independent groups have reported that weak magnetic fields, in particular ion cyclotron resonance fields, are capable of sharply altering behavior in rats.

Bioelectromagnetics. 1997;18(1):85-7.

Electric-field ion cyclotron resonance.

Liboff AR.

Department of Physics, Oakland University, Rochester, Michigan 48309, USA.


We consider the possibility that DC magnetic fields can interact in a resonant manner with endogenous AC electric fields in biological systems. Intrinsic electric-field ion cyclotron resonance (ICR) interactions would be more physically credible than models based on external AC magnetic fields and might be expected as an evolutionary response to the long-term constancy of the geomagnetic field.

Bioelectromagnetics. 1993;14(4):299-314.

Theoretical study of the resonant behaviour of an ion confined to a potential well in a combination of AC and DC magnetic fields.

Galt S, Sandblom J, Hamnerius Y.

Department of Applied Electron Physics, Chalmers University of Technology, Göteborg, Sweden.


Numerical solutions are presented to the equation of motion for an ion confined to a region of space by a restoring force and subject to DC and AC magnetic fields. We have expanded on the theoretical work of Durney et al. [1988] by including a potential well as a confining factor. This additional term in the equation of motion, being nondissipative, could allow for the buildup of stored energy within the system to a level necessary for a macroscopic resonant phenomenon. Resonant behaviour has been studied, including calculation of the trajectory and energy (kinetic and potential) of a confined ion, with emphases on the appearance of both amplitude and frequency windows. The results are discussed in relation to ion transport through transmembrane channels exposed to magnetic fields. When realistic values of the frictional and restoring-force coefficients are considered, all predicted resonant behaviour disappears, except at very high field strengths.

Bioelectromagnetics. 1993;14(4):315-27.

Experimental search for combined AC and DC magnetic field effects on ion channels.

Galt S, Sandblom J, Hamnerius Y, Höjevik P, Saalman E, Nordén B.

Department of Applied Electron Physics, Chalmers University of Technology, Göteborg, Sweden.


The hypothesis that specific combinations of DC and low frequency AC magnetic fields at so-called cyclotron-resonance conditions could affect the transport of ions through ion channels, or alter the kinetics of ion channels (opening and closing rates), has been tested. As a model system, the ion channels formed by gramicidin A incorporated in lipid bilayer membranes were studied. No significant changes in channel conductance, average lifetime, or formation rate as a function of applied fields could be detected over a wide range of frequencies and field strengths. Experiments were carried out to measure the time-resolved single-channel events and the average conductances of many-channel events in the presence of K+ and H+ ions. The channel blocking effect of Ca++ was also studied.

Interference with Cardiac Pacemakers

J Am Coll Cardiol. 2005 Mar 15;45(6):896-900.

Clinical study of interference with cardiac pacemakers by a magnetic field at power line frequencies.

Trigano A, Blandeau O, Souques M, Gernez JP, Magne I.

Department of Cardiology, Centre Hospitalier Universitaire Nord, Marseille, France.


OBJECTIVES: This study examined the risk of interference by high magnetic flux density with permanent pacemakers. BACKGROUND: Several forms of electromagnetic energy may interfere with the functions of implanted pacemakers. No clinical study has reported specific and relevant information pertaining to magnetic fields near power lines or electrical appliances. METHODS: A total of 250 consecutive tests were performed in 245 recipients of permanent pacemakers during 12-lead electrocardiographic monitoring. A dedicated exposure system generated a 50-Hz frequency and maximum 100-microT flux density, while the electrical field was kept at values on the order of 0.10 V/m. RESULTS: A switch to the asynchronous mode was recorded in three patients with devices programmed in the unipolar sensing configuration. A sustained mode switch was followed by symptomatic pacing inhibition in one patient. No effect on devices programmed in bipolar sensing was observed, except for a single interaction with a specific capture monitoring algorithm. CONCLUSIONS: The overall incidence of interaction by a magnetic field was low in patients tested with a wide variety of conventionally programmed pacemaker models. A magnetic field pulsed at power frequency can cause a mode switch and pacing inhibition in patients with devices programmed in the unipolar sensing configuration. The risk of interference appears negligible in patients with bipolar sensing programming.

AACN Clin Issues. 2004 Jul-Sep;15(3):391-403.

Electromagnetic interference in cardiac rhythm management devices.

Sweesy MW, Holland JL, Smith KW.

Pacer School, Greenville, SC, USA.


Clinicians caring for cardiac device patients with implanted pacemakers or cardioverter defibrillators (ICDs) are frequently asked questions by their patients concerning electromagnetic interference (EMI) sources and the devices. EMI may be radiated or conducted and may be present in many different forms including (but not limited to) radiofrequency waves, microwaves, ionizing radiation, acoustic radiation, static and pulsed magnetic fields, and electric currents. Manufacturers have done an exemplary job of interference protection with device features such as titanium casing, signal filtering, interference rejection circuits, feedthrough capacitors, noise reversion function, and programmable parameters. Nevertheless, EMI remains a real concern and a potential danger. Many factors influence EMI including those which the patient can regulate (eg, distance from and duration of exposure) and some the patient cannot control (eg, intensity of the EMI field, signal frequency). Potential device responses are many and range from simple temporary oversensing to permanent device damage Several of the more common EMI-generating devices and their likely effects on cardiac devices are considered in the medical, home, and daily living and work environments.

Arch Mal Coeur Vaiss. 2003 Apr;96 Spec No 3:35-41.

Effects of 50 to 60 Hz and of 20 to 50 kHz magnetic fields on the operation of implanted cardiac pacemakers.

[Article in French]

Frank R, Souques M, Himbert C, Hidden-Lucet F, Petitot JC, Fontaine G, Lambrozo J, Magne I, Bailly JM.

Institut de cardiologie, service de rythmologie, hôpital La Pitié-La Salpêtrière, 52-56, bd Vincent Auriol, 75013 Paris.


The effect of 50 Hz and 60 Hz (frequencies of current distribution) and 20 kHz to 50 kHz (frequencies of induction cooktop) magnetic interference on implanted pacemakers have been assessed with the present generation of device technology. Sixty patients implanted in 1998 and 1999 with dual chamber pacemakers from 9 different manufacturers were monitored with telemetry while passing through, and standing between a system of two coils. They generated a 50 Hz or a 60 Hz magnetic field at 50 microT. Then, patients used a cooktop at different power. The recordings were made with the standard setting of “medically correct” sensing parameters chosen for the patients. Then pacemakers were reprogrammed to the unipolar mode, with the highest atrial (A) and ventricular (V) sensitivity that did not induce muscular inhibition while moving. Between each exposure (50 Hz, 60 Hz or 20 kHz to 50 kHz), the pacemaker programmation was controlled. At the end of the tests, pacemakers will be reprogrammed with the standard setting. The medical observer being blind to the existence or not of the magnetic field. No pacemaker was influenced by the vicinity of the magnetic field at medically correct settings. At unipolar high sensitivity, no inhibition nor reprogramming was observed. Transient reversion to interference mode was observed in 6 cases, 3 transient acceleration due to atrial detection of the interference, and one T wave detection by the ventricular lead. All were observed with the 60 Hz, and only 3 with the 50 Hz magnetic field. One device (Biotronik) shifted out of its special program (hysteresis research) during the tests with the induction cooktop, but it maintained its standard program, and the event could not be repeated despite further testing. CONCLUSION: Actual pacemakers do not present any electromagnetic interference with 50 Hz and 60 Hz or induction cooktop frequency working. They are insensitive with medically correct settings. Unusual high sensitivity leads only to noise reversion mode, or transient ventricular tracking.

Arch Mal Coeur Vaiss. 2003 Apr;96 Spec No 3:30-4.

The interference of electronic implants in low frequency electromagnetic fields.

Silny J.

Research Center for Bioelectromagnetic Interaction (femu), University Hospital of Aachen University, Pauwelsstrasse 20, 52074 Aachen, Germany.


Electronic implants such as cardiac pacemakers or nerve stimulators can be impaired in different ways by amplitude-modulated and even continuous electric or magnetic fields of strong field intensities. For the implant bearer, possible consequences of a temporary electromagnetic interference may range from a harmless impairment of his well-being to a perilous predicament. Electromagnetic interferences in all types of implants cannot be covered here due to their various locations in the body and their different sensing systems. Therefore, this presentation focuses exemplarily on the most frequently used implant, the cardiac pacemaker. In case of an electromagnetic interference the cardiac pacemaker reacts by switching to inhibition mode or to fast asynchronous pacing. At a higher disturbance voltage on the input of the pacemaker, a regular asynchronous pacing is likely to arise. In particular, the first-named interference could be highly dangerous for the pacemaker patient. The interference threshold of cardiac pacemakers depends in a complex way on a number of different factors such as: electromagnetic immunity and adjustment of the pacemaker, the composition of the applied low-frequency fields (only electric or magnetic fields or combinations of both), their frequencies and modulations, the type of pacemaker system (bipolar, unipolar) and its location in the body, as well as the body size and orientation in the field, and last but not least, certain physiological conditions of the patient (e.g. inhalation, exhalation). In extensive laboratory studies we have investigated the interference mechanisms in more than 100 cardiac pacemakers (older types as well as current models) and the resulting worst-case conditions for pacemaker patients in low-frequency electric and magnetic fields. The verification of these results in different practical everyday-life situations, e.g. in the fields of high-voltage overhead lines or those of electronic article surveillance systems is currently in progress. In case of the vertically-oriented electric 50 Hz fields preliminary results show that per 1 kV/m unimpaired electrical field strength (rms) an interference voltage of about 400 microVpp as worst-case could occur at the input of a unipolar ventricularly controlled, left-pectorally implanted cardiac pacemaker. Thus, already a field strength above ca. 5 kV/m could cause an interference with an implanted pacemaker. The magnetic fields induces an electric disturbance voltage at the input of the pacemaker. The body and the pacemaker system compose several induction loops, whose induced voltages rates add or subtract. The effective area of one representing inductive loop ranges from 100 to 221 cm2. For the unfavourable left-pectorally implantated and atrially-controlled pacemaker with a low interference threshold, the interference threshold ranges between 552 and 16 microT (rms) for magnetic fields at frequencies between 10 and 250 Hz. On this basis the occurrence of interferences with implanted pacemakers is possible in everyday-life situations. But experiments demonstrate a low probability of interference of cardiac pacemakers in practical situations. This apparent contradiction can be explained by a very small band of inhibition in most pacemakers and, in comparison with the worst-case, deviating conditions.

Pacing Clin Electrophysiol. 2001 Apr;24(4 Pt 1):465-8.

Incidence of electromagnetic interference in implantable cardioverter defibrillators.

Kolb C, Zrenner B, Schmitt C.

Deutsches Herzzentrum München and 1. Med. Klinik, Klinikum rechts, Isar der Technischen Universität München, München, Germany.


Electromagnetic interference (EMI) with ICDs can lead to temporary inhibition of the device or to inappropriate delivery of antitachycardia pacing and shocks. The incidence of interactions between electronic devices and the current generation of ICDs is not known. In a retrospective study of 341 patients (665 patient-years) who underwent a regular follow-up every 3 months, five episodes of EMI were detected in four different patients. The risk for receiving inappropriate shocks due to EMI is < 1% per year and patient. In conclusion, although inappropriate delivery of shocks by ICDs due to EMI rarely occurs, patient information should emphasize the avoidance of situations of possible interference. Further efforts concerning lead technology and detection algorithms are necessary to minimize the risk of EMI.

Pacing Clin Electrophysiol. 1991 Dec;14(12):2114-22.

The influence of elevated 50 Hz electric and magnetic fields on implanted cardiac pacemakers: the role of the lead configuration and programming of the sensitivity.

Toivonen L, Valjus J, Hongisto M, Metso R.

First Department of Medicine, Helsinki University Central Hospital, Finland.


The influence of the electromagnetic interference (EMI) on performance of 15 implanted cardiac pacemakers (12 generator models) was tested during exposure at a high voltage substation. All patients had an adequate spontaneous heart rate during the study. Tests were performed in the ventricular inhibited mode with unipolar sensing in all pacemakers and repeated with bipolar sensing in four pacemakers. The sensitivity was set to a regular, functionally proper level and then to the highest available level. Exposure was done to moderate (1.2-1.7 kV/m) and strong (7.0-8.0 kV/m) electric fields, which correspond to the immediate vicinity of 110 and 400 kV power lines, respectively. In moderate electric fields the output was inhibited in one pacemaker at regular sensitivity (1.7-3.0 mV) and in five pacemakers at the highest sensitivity (0.5-1.25 mV). In strong electric fields the output was inhibited in five pacemakers at regular sensitivity and several pacemakers converted to noise reversion mode at the highest sensitivity. In bipolar mode only one of four pacemakers at high sensitivity (0.5-1.0 mV) was inhibited in the strongest electric field, whereas all four did so in the unipolar mode. One pacemaker with unipolar sensitivity at 0.5 mV was interfered by 63 microT magnetic field. The results confirm that the programmed sensitivity level and the lead configuration markedly influence pacemakers’ vulnerability to EMI. Bipolar sensing mode is rather safe in the presence of EMI, which is encountered in public environments. The programmable features of today’s pacemakers permit individualized, less stringent safety measures to avoid electromagnetic hazards.

Intense Ultrashort Pulsed Fields

Technol Cancer Res Treat.  2012 Feb;11(1):83-93.

Long term survival of mice with hepatocellular carcinoma after pulse power ablation with nanosecond pulsed electric fields.

Chen X, Zhuang J, Kolb JF, Schoenbach KH, Beebe SJ.


Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk Virginia, 4211 Monarch Way, Norfolk, Virginia 23508, USA.


Novel therapies are needed for treating hepatocellular carcinoma (HCC) without recurrence in a single procedure. In this work we evaluated anti-neoplastic effects of a pulse power ablation (PPA) with nanosecond pulsed electric fields (nsPEFs), a non-thermal, non-drug, local, regional method and investigated its molecular mechanisms for hepatocellular carcinoma tumor ablation in vivo. An ectopic tumor model was established using C57BL/6 mice with Hepa1-6 hepatocellular carcinoma cells. Pulses with durations of 30 or 100 ns and fast rise times were delivered by a needle or ring electrode with different electric field strengths (33, 50 and 68 kV/cm), and 900 pulses in three treatment sessions (300 pulses each session) or a single 900 pulse treatment. Treated and control tumor volumes were monitored by ultrasound and apoptosis and angiogenesis markers were evaluated by immunohistochemistry. Seventy five percent of primary hepatocellular carcinoma tumors were eradicated with 900 hundred pulses at 100 ns pulses at 68 kV/cm in a single treatment or in three treatment sessions without recurrence within 9 months. Using quantitative analysis, tumors in treated animals showed nsPEF-mediated nuclear condensation (3 h post-pulse), cell shrinkage (1 h), increases in active executioner caspases (caspase-3 > -7 > -6) and terminal deoxynucleotidyl transferase dUTP nick-end-labeling (1 h) with decreases in vascular endothelial growth factor expression (7d) and micro-vessel density (14d). NsPEF ablation eliminated hepatocellular carcinoma tumors by targeting two therapeutic sites, apoptosis induction and inhibition of angiogenesis, both important cancer hallmarks. These data indicate that PPA with nsPEFs is not limited to treating skin cancers and provide a rationale for continuing to investigate pulse power ablation for hepatocellular carcinoma using other models in pre-clinical applications and ultimately in clinical trials. Based on present treatments for specific HCC stages, it is anticipated that nsPEFs could be substituted for or used in combination with ablation therapies using heat, cold or chemicals. PLos One.  2011;6(12):e28419. Epub 2011 Dec 2.

DNA electrophoretic migration patterns change after exposure of jurkat cells to a single intense nanosecond electric pulse.

Romeo S, Zeni L, Sarti M, Sannino A, Scarfì MR, Vernier PT, Zeni O.


CNR – Institute for Electromagnetic Sensing of Environment, Naples, Italy.


Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi.  2010 Oct;27(5):1128-32.

Focusing properties of picosecond electric pulses in non-invasive cancer treatment.

[Article in Chinese] Long Z, Yao C, Li C, Mi Y, Sun C.


State Key Laboratory of Power Transmission Equipment & System Security and New Technology, Chongqing University, Chongqing 400044, China.


In the light of optical theory, we advanc an ultra-wideband impulse radiating antenna (IRA) which is composed of an ellipsoidal reflector and a cone radiator. The high-intensity ultra-short electric pulses radiated by IRA can be transferred into the deep target in tissue non-invasively and be focused effectively. With the focused picosecond electric pulses, the organelles (mitochondria) transmembrane potential shall change to collapse under which the tumor cells will be targetly induced to apoptosis, so the method of non-invasive treatment of tumors would be achieved. Based on the time-domain electromagnetic field theory, the propagation characteristics of picosecond electric pulses were analyzed with and without the context of biological tissue, respectively. The results show that the impulse characteristics of input pulse were maintained and the picosecond electric pulses can keep high resolution in target areas. Meanwhile, because of the dispersive nature of medium, the pulse amplitude of the pulses will attenuate and the pulse width will be broadened.

Bioelectrochemistry. 2010 Oct;79(2):257-60. Epub 2010 Mar 10.

Electroporation and alternating current cause membrane permeation of photodynamic cytotoxins yielding necrosis and apoptosis of cancer cells.

Traitcheva N, Berg H.

Institute of Plant Physiology “M. Popov,” Bulgarian Acad. of Sciences, Sofia, Bulgaria.


In order to increase the permeability of cell membranes for low doses of cytostatic drugs, two bioelectrochemical methods have been compared: (a) electric pore formation in the plasma membranes by single electric impulses (electroporation), and (b) reordering of membrane structure by alternating currents (capacitively coupled). These treatments were applied to human leukemic K-562 cells and human lymphoma U-937 cells, yielding apoptotic and necrotic effects, determined by flow cytometry. Additional cell death occurs after exposure to light irradiation at wavelengths lambda > 600 nm, of cells which were electroporated and had incorporated actinomycin-C or daunomycin (daunorubicin). It is observed that drug uptake after an exponentially decaying electroporation pulse of the initial field strength Eo=1.4 kV/cm and pulse time constants in the time range 0.5-3 ms is faster than during PEMF-treatment, i.e., application of an alternating current of 16 kHz, voltage U<100 V, I=55 mA, and exposure time 20 min. However, at the low a.c. voltage of this treatment, more apoptotic and necrotic cells are produced as compared to the electroporation treatment with one exponentially decaying voltage pulse. Thus, additional photodynamic action appears to be more effective than solely drugs and electroporation as applied in clinical electrochemotherapy, and more effective than the noninvasive pulsed electromagnetic fields (PEMFs), for cancer cells in general and animals bearing tumors in particular.

Arch Biochem Biophys. 2010 May;497(1-2):82-9. Epub 2010 Mar 24.

Nanosecond pulsed electric fields stimulate apoptosis without release of pro-apoptotic factors from mitochondria in B16f10 melanoma.

Ford WE, Ren W, Blackmore PF, Schoenbach KH, Beebe SJ.

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508, USA.


Nanosecond pulsed electric fields (nsPEFs) eliminates B16f10 melanoma in mice, but cell death mechanisms and kinetics of molecular events of cell death are not fully characterized. Treatment of B16f10 cells in vitro resulted in coordinate increases in active caspases with YO-PRO-1 uptake, calcium mobilization, decreases in mitochondria membrane potential with decreases in forward light scatter (cell size), increases in ADP/ATP ratio, degradation of actin cytoskeleton and membrane blebbing. However, there was no mitochondrial release of cytochrome c, AIF or Smac/DIABLO or generation of reactive oxygen species. Phosphatidylserine externalization was absent and propidium iodide uptake was delayed in small populations of cells. The results indicate that nsPEFs rapidly recruit apoptosis-like mechanisms through the plasma membrane, mimicking the extrinsic apoptosis pathway without mitochondrial amplification yet include activation of initiator and executioner caspases. nsPEFs provide a new cancer therapy that can bypass cancer-associated deregulation of mitochondria-mediated apoptosis in B16f10 melanoma.

Melanoma Res. 2009 Aug 26. [Epub ahead of print]

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment.

Chen X, James Swanson R, Kolb JF, Nuccitelli R, Schoenbach KH.

Department of Hepatobiliary Surgery, the First Affiliated Hospital Zhejiang University School of Medicine, Hangzhou, Zhejiang, China Frank Reidy Research Center for Bioelectrics cDepartment of Biological Sciences, Old Dominion University, Norfolk, Virginia, USA.


Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana-Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.

Anticancer Res. 2008 Jul-Aug;28(4B):2245-51.

Effect of steep pulsed electric field on proliferation, viscoelasticity and adhesion of human hepatoma SMMC-7721 cells.

Song G, Qin J, Yao C, Ju Y.

Department of Bioengineering, College of Bioengineering, Ministry of Education of China, Chongqing University, Chongqing, PR China.


It has been proven that steep pulsed electric field (SPEF) can directly kill tumor cells and plays an important role in anticancer treatment. The biorheological mechanisms, however, that destroy tumor cells are almost unknown. To resolve this issue, here, an SPEF generator was used to assess the effects of high- and low-dose SPEF on the proliferation of human hepatoma SMMC-7721 cells by MTT assay, and on the viscoelasticity, adhesion of SMMC-7721 cells to endothelial cells by micropipette aspiration technique. Viability and proliferation of SPEF-treated SMMC-7721 cells were significantly inhibited. Cell cycle analysis indicated that SPEF arrested the cell cycle progression of SMMC-7721 cells at the G0/G1 transition to the S-phase. Viscoelastic data fitted by a standard linear solid model showed that viscoelasticity of SMMC-7721 cells changed after treatment with SPEF. Moreover, the adhesive force of low-dose SPEF-treated SMMC-7721 cells to endothelial cells markedly decreased compared to that of control cells. These results suggest that the suppressant effects of SPEF on the proliferation of SMMC-7721 cells appeared to be mediated, at least in part, through arresting cell cycle progression and altering the viscoelastic and adhesive properties of the cells, which provides a novel biorheological mechanism for the antitumor therapy of SPEF.

The Effect of Intense Subnanosecond Electrical Pulses on Biological Cells Schoenbach, K.H.   Shu Xiao   Joshi, R.P.   Camp, J.T.   Heeren, T.   Kolb, J.F.   Beebe, S.J.
Old Dominion Univ., Norfolk; This paper appears in: Plasma Science, IEEE Transactions on
Publication Date: April 2008
Volume: 36,  Issue: 2, Part 1
On page(s): 414-422
Location: Eindhoven, Netherlands,
ISSN: 0093-3813
INSPEC Accession Number: 9921271
Digital Object Identifier: 10.1109/TPS.2008.918786
Current Version Published: 2008-04-08 AbstractNanosecond electrical pulses have been successfully used to treat melanoma tumors by using needle arrays as pulse delivery systems. Reducing the pulse duration of intense electric field pulses from nanoseconds into the subnanosecond range will allow us to use wideband antennas to deliver the electromagnetic fields into tissue with a spatial resolution in the centimeter range. To explore the biological effect of intense subnanosecond pulses, we have developed a generator that provides voltage pulses of 160 kV amplitude, 200 ps rise time, and 800 ps pulse width. The pulses are delivered to a cylindrical Teflon chamber with polished flat electrodes at either end. The distance between the electrodes is variable and allows us to generate electric fields of up to 1 MV/cm in cell suspensions. The pulses have been applied to B16 (murine melanoma) cells, and the plasma membrane integrity was studied by means of trypan blue exclusion. For pulse amplitudes of 550 kV/cm, approximately 50% of the cells took up trypan blue right after pulsing, whereas only 20% were taking it up after 1 h. This indicates that the plasma membrane in a majority of the cells affected by the pulses recovers with a time constant of about 1 h. The cells that show trypan blue uptake after this time suffer cell death through apoptosis. Evaluation of the experimental results and molecular dynamics modeling results indicate that with a pulse duration of 800 ps, membrane charging and nanopore formation are the dominant bioelectric effects on B16 cells. This information has been used in a continuum model to estimate the increase in membrane permeability and, consequently, the increase in pore size caused by repetitive pulsing.

Conf Proc IEEE Eng Med Biol Soc. 2008;2008:1044-7.

Experiment and mechanism research of SKOV3 cancer cell apoptosis induced by nanosecond pulsed electric field.

Yao C, Mi Y, Hu X, Li C, Sun C, Tang J, Wu X.

State Key Laboratory of Power Transmission Equipment & System Security and New Technology, Chongqing University, Chongqing 400044, China.


This paper studies the apoptosis of human ovarian carcinoma cell Line (SKOV3) induced by the nanosecond pulsed electric field (10kV/cm, 100ns, 1 Hz) and its effect on intracellular calcium concentration ([Ca2+]i). These cells were doubly marked by Annexin V-FITC/PI, and the apoptosis rate was analyzed with flow cytometry. After AO/EB staining the morphological changes were observed under fluorescent microscope, and their ultrastructural changes were observed under scanning electron microscope (SEM). With Fluo-3/AM as calcium fluorescent marker, laser scanning confocal microscope (LSCM) was used to detect the effect of nsPEF on [Ca2+]i and the source of Ca2+. The results showed that the early apoptosis rate of the treatment group was (22.21+/-2.71)%, significantly higher than that of the control group (3.04+/-0.44)% (P<0.01). The typical features of apoptotic cell have been observed by fluorescent microscope and SEM. It is proved that nsPEF can induce apoptosis of SKOV3 cells and result in distinct increase in [Ca2+]i (P0.01), which was independent of extracellular calcium concentration (P>0.05). Since nsPEF can penetrate cell membrane due to its high frequency components, one of the mechanisms of nsPEF-induced apoptosis may be that activating intracellular calcium stores can increase the [Ca2+]i, and consequently, the apoptotic signal pathway can be induced.

Apoptosis. 2007 Sep;12(9):1721-31.

Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells.

Hall EH, Schoenbach KH, Beebe SJ.

Center for Pediatric Research, Children’s Hospital of the King’s Daughters, Department of Physiological Sciences, Eastern Virginia Medical School, PO Box 1980, Norfolk, VA 23501-1980, USA.


Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53(+/+) and HCT116p53(-/-) colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53(+/+) and HCT116p53(-/-) cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2007 Feb;24(1):230-4.

Biological effects and their applications in medicine of pulsed electric fields.

[Article in Chinese]

Huang H, Song G, Wang G, Sun C.

Key Laboratory for Biomnechanics & Tissue Engineering of the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China.


Pulsed electric fields can induce various kinds of biological effects that are essentially different from the normal electric fields, especially the interactions of Nanosecond Pulsed electric field (nsPEF) with cells. The biological effects of different pulsed electric fields on cell membranes, cytoplasmic matrixes, cell growth are introduced in this paper. Based on these effects, some applications of pulsed electric fields in cancer therapy, gene therapy, and delivery of drugs are reviewed in details.

Biochem Biophys Res Commun. 2006 May 5;343(2):351-60. Epub 2006 Mar 10.

Nanosecond pulsed electric fields cause melanomas to self-destruct.

Nuccitelli R, Pliquett U, Chen X, Ford W, James Swanson R, Beebe SJ, Kolb JF, Schoenbach KH.

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.


We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 micros. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 degrees C, ten degrees lower than the minimum temperature for hyperthermia effects.

Conf Proc IEEE Eng Med Biol Soc. 2006;1:6370-2.

Outlook for the use of focused shock waves and pulsed electric fields in the complex treatment of malignant neoplasms.

Garilevich BA, Andrianov YV, Olefir YV, Zubkov AD, Rotov AE.

Central Air Force Clinical Hosp., Moscow, Russia.


The experimental studies the synchronous action of electric field microsecond range with amplitude within the range of 1-7 kV/sm and shock waves with pressure before 100 MPa on cells membrane permeability of the mouse’s ascitic tumors in vitro have shown the intensification the efficiency of the forming the irreversible pores under synchronous action. Thereby, enabling the electric field in the compression phase of shock wave pulse which can essentially reduce the electric field intensity required for breakdown cell membrane. In usual condition at amplitude of electric field, specified above, electric breakdown membrane carries basically reversible nature. At the same time in the pressure field tension phase of shock-wave pulse reversible pores, created by electric field, can grow before sizes, under which wholeness membrane is not restored. Under simultaneous action on cellular suspension the shock wave and electric field with moderate intensity cells survival is reduced in 5 once in contrast with occuring at different time’s action, and in 10 once in contrast with checking. The most sensitive to influence by under study fields are cells in phase of the syntheses DNA, preparation to fission and in phase of the mitosis. Thereby, continuation of the studies on use synchronous action shock waves and pulsed electric fields in complex treatment of the tumors introduces perspectiv

Clin Cancer Res. 2005 Oct 1;11(19 Pt 2):7093s-7103s.

Application of high amplitude alternating magnetic fields for heat induction of nanoparticles localized in cancer.

Ivkov R, DeNardo SJ, Daum W, Foreman AR, Goldstein RC, Nemkov VS, DeNardo GL.

Triton BioSystems, Inc., Chelmsford, Massachusetts 01824, USA.


OBJECTIVE: Magnetic nanoparticles conjugated to a monoclonal antibody can be i.v. injected to target cancer tissue and will rapidly heat when activated by an external alternating magnetic field (AMF). The result is necrosis of the microenvironment provided the concentration of particles and AMF amplitude are sufficient. High-amplitude AMF causes nonspecific heating in tissues through induced eddy currents, which must be minimized. In this study, application of high-amplitude, confined, pulsed AMF to a mouse model is explored with the goal to provide data for a concomitant efficacy study of heating i.v. injected magnetic nanoparticles.

METHODS: Thirty-seven female BALB/c athymic nude mice (5-8 weeks) were exposed to an AMF with frequency of 153 kHz, and amplitude (400-1,300 Oe), duration (1-20 minutes), duty (15-100%), and pulse ON time (2-1,200 seconds). Mice were placed in a water-cooled four-turn helical induction coil. Two additional mice, used as controls, were placed in the coil but received no AMF exposure. Tissue and core temperatures as the response were measured in situ and recorded at 1-second intervals.

RESULTS: No adverse effects were observed for AMF amplitudes of < or = 700 Oe, even at continuous power application (100% duty) for up to 20 minutes. Mice exposed to AMF amplitudes in excess of 950 Oe experienced morbidity and injury when the duty exceeded 50%.

CONCLUSION: High-amplitude AMF (up to 1,300 Oe) was well tolerated provided the duty was adjusted to dissipate heat. Results presented suggest that further tissue temperature regulation can be achieved with suitable variations of pulse width for a given amplitude and duty combination. These results suggest that it is possible to apply high-amplitude AMF (> 500 Oe) with pulsing for a time sufficient to treat cancer tissue in which magnetic nanoparticles have been embedded.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004 Aug;21(4):546-8.

Effects of steep pulsed electric fields on cancer cell proliferation and cell cycle.

[Article in Chinese]

Yao C, Sun C, Mi Y, Xiong L, Hu L, Hu Y.

Key Lab of High Voltage Engineering and Electrical New Technology, Ministry of Education, Chongqing University, Chongqing 400044, China.


To assess study the cytocidal and inhibitory effects of steep pulsed electric fields (SPEFs) on ovarian cancer cell line SKOV3, the cancer cell suspension was treated by SPEFs with different parameters (frequency, pulse duration, peak value of voltage). Viability rate and growth curves of two test groups (high dosage and low dosage of SPEFs) and one control group were also measured. The DNA contents and cell cycle were analyzed by flow cytometry (FCM). Different dosing levels of SPEFs exerted obviously different effects on cancer cell viability. With the enhancement of each pulse parameter, the viability rate was promoted and the inhibitory effect on the proliferation of treated cells was more evident. The cells exposed to SPEFs grew slower than the control. The ratio of S+G2/M phase cells was decreased, which restrained the DNA synthesis and division, but the ratio of G0/G1 phase cells was increased in the treated groups. It was also indicated that the SPEFs blocked the cell transition from G0/G1 phase to S+G2/M phase. There was a significant difference in cell cycle between treated group and control group (P<0.01). Lethal effects of SPEFs were represented by inhibiting the cancer cell proliferation at the cell level and by influencing the cell cycle at the DNA level.

Physiol Meas. 2004 Aug;25(4):1077-93.

Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms.

Beebe SJ, Blackmore PF, White J, Joshi RP, Schoenbach KH.

Center for Pediatric Research, Eastern Virginia Medical School, Children’s Hospital for The King’s Daughters, Norfolk, VA, USA.

These studies describe the effects of nanosecond (10-300 ns) pulsed electric fields (nsPEF) on mammalian cell structure and function. As the pulse durations decrease, effects on the plasma membrane (PM) decrease and effects on intracellular signal transduction mechanisms increase. When nsPEF-induced PM electroporation effects occur, they are distinct from classical PM electroporation effects, suggesting unique, nsPEF-induced PM modulations. In HL-60 cells, nsPEF that are well below the threshold for PM electroporation and apoptosis induction induce effects that are similar to purinergic agonistmediated calcium release from intracellular stores, which secondarily initiate capacitive calcium influx through store-operated calcium channels in the PM. NsPEF with durations and electric field intensities that do or do not cause PM electroporation, induce apoptosis in mammalian cells with a well-characterized phenotype typified by externalization of phosphatidylserine on the outer PM and activation of caspase proteases. Treatment of mouse fibrosarcoma tumors with nsPEF also results in apoptosis induction. When Jurkat cells were transfected by electroporation and then treated with nsPEF, green fluorescent protein expression was enhanced compared to electroporation alone. The results indicate that nsPEF activate intracellular mechanisms that can determine cell function and fate, providing an important new tool for probing signal transduction mechanisms that modulate cell structure and function and for potential therapeutic applications for cancer and gene therapy.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004 Jun;21(3):433-5.

Effect of steep pulsed electric fields on survival of tumour-bearing mice.

[Article in Chinese]

Yao C, Sun C, Xiong L, Mi Y, Liao R, Hu L, Hu Y.

College of Electrical Engineering, Chongqing University, Chongqing, 400044, China.

To investigate the lethal effect of steep pulsed electric fields (SPEFs) on cancer cells and the life-prolonging effect of SPEFs on the survival of tumour-bearing mice, this study was carried out with the use of SPEFs to treat 40 BALB/C mice inoculated by cervical cancer. The lethal effect on cancer cells and the life-prolonging effect on tumour-bearing mice were examined and compared between the experiment group and control group. The survival periods of the experiment group and control group were 52.05 days and 33.03 days, respectively. There was a significant difference in survival curve between the two groups. The results confirmed he inhibitiory effect and lethal effect of SPEFs on cancer cells. SPEFs can prolong the survival period of tumour-bearing mice.

Technol Cancer Res Treat. 2002 Feb;1(1):71-82.

Enhancing the effectiveness of drug-based cancer therapy by electroporation (electropermeabilization).

Rabussay DP, Nanda GS, Goldfarb PM.

Genetronics, Inc., 11199 Sorrento Valley Road, San Diego CA 92121, USA.


Many conventional chemotherapeutic drugs, as well as DNA for cancer gene therapy, require efficient access to the cell interior to be effective. The cell membrane is a formidable barrier to many of these drugs, including therapeutic DNA constructs. Electropermeabilization (EP, often used synonymously with “electroporation”) has become a useful method to temporarily increase the permeability of the cell membrane, allowing a broad variety of molecules efficient access to the cell interior. EP is achieved by the application of short electrical pulses of relatively high local field strength to the target tissue of choice. In cancer therapy, EP can be applied in vivo directly to the tumor to be treated, in order to enhance intracellular uptake of drugs or DNA. Alternatively, EP can be used to deliver DNA into cells of healthy tissue to achieve longer-lasting expression of cancer-suppressing genes. In addition, EP has been used in ex vivo therapeutic approaches for the transfection of a variety of cells in suspension. In this paper, we communicate results related to the development of a treatment for squamous cell carcinomas of the head and neck, using electropermeabilization to deliver the drug bleomycin in vivo directly into the tumor cells. This drug, which is not particularly effective as a conventional therapeutic, becomes highly potent when the intracellular concentration is enhanced by EP treatment. In animal model experiments we found a drug dose of 1 U/cm(3) tumor tissue (delivered in 0.25 mL of an aqueous solution/cm3 tumor tissue) and an electrical field strength of 750 V/cm or higher to be optimal for the treatment of human squamous cell tumors grown subcutaneously in mice. Within 24-48 hours, the majority of tumor cells are rapidly destroyed by this bleomycin-electroporation therapy (B-EPT). This raises the concern that healthy tissue may be similarly affected. In studies with large animals we showed that normal muscle and skin tissue, normal tissue surrounding major blood vessels and nerves, as well as healthy blood vessels and nerves themselves, are much less affected than tumor tissue. Normal tissues did show acute, focal, and transitory effects after treatment, but these effects are relatively minor under standard treatment conditions. The severity of these effects increases with the number of electric pulse cycles and applied voltage. The observed histological changes resolved 20 to 40 days after treatment or sooner, even after excessive EP treatment. Thus, B-EPT is distinct from other ablative therapies, such as thermal, cryo, or photodynamic ablation, which equally affect healthy and tumor tissue. In comparison to surgical or radiation therapy, B-EPT also has potential as a tissue-sparing and function-preserving therapy. In clinical studies with over 50 late stage head and neck cancer patients, objective tumor response rates of 55-58%, and complete tumor response rates of 19-30% have been achieved.

Laryngoscope. 2001 Jan;111(1):52-6.

Electroporation therapy for head and neck cancer including carotid artery involvement.

Allegretti JP, Panje WR.

Department of Otolaryngology, Rush-Presbyterian-St Luke’s Medical Center, Rush Medical College, Chicago, Illinois 60612, USA.


OBJECTIVES: Electroporation therapy with intralesional bleomycin (EPT) is a novel, technically simple outpatient technique in which high-voltage electric impulses delivered into a neoplasm transiently increase cell membrane permeability to large molecules, including cytotoxic agents, causing localized progressive necrosis. Unlike many laser ablation methods, EPT can treat bulky tumors (>2 cm) with complete penetration. Our recent publication confirms an excellent response rate in the use of EPT in a clinical trial. STUDY

DESIGN, PATIENTS, AND METHODS: Following our initial prospective study report in 1998, we have followed our entire initial cohort (10 patients) of patients with head and neck cancer beyond 24-months follow-up. Additionally, we have used this approach to treat four additional patients (total: 9 males/5 females) with upper aerodigestive tract squamous cell carcinoma, including three with internal carotid artery (ICA) involvement up to or within the skull base. Two patients underwent preoperative balloon test occlusion with cerebral perfusion studies followed by carotid embolization. EPT was then done safely at least 2 weeks later to avoid the temporary hypercoagulable state.

RESULTS: Within the overall cohort (14 patients) 6 patients had a complete response, 6 had a partial response, and 2 did not respond (overall 85.7% response rate). Both patients with ICA involvement had a partial or complete response to treatment; neither patient had a hemorrhagic or neurologic complication. Overall, 13 of the 14 patients were treated for persistent or recurrent head and neck cancer. Two of the four patients with early recurrent stage tumors had no evidence of recurrence after EPT with an average follow-up of 31.5 months. The overall early stage tumor group had four complete responders out of five (80%). On the contrary, only 2 of 9 patients with advanced recurrent stage tumors were disease-free at 18 months. Morbidity was low for early stage tumors, but higher for advanced tumors with complications, including poor wound healing, dysphagia, and osteomyelitis. There were no treatment-related deaths.

CONCLUSION: We found EPT to be safe and efficacious in patients with head and neck cancer, even with internal carotid artery involvement. Patients with early stage recurrences have the potential for prolonged survival beyond 2 years without the morbidity of surgery and radiation or toxicity of systemic chemotherapy. Because of its superb access qualities even for bulky tumors, EPT is a potential method of delivery for other tumoricidal agents such as in genetic-altering schemes.

J Physiol Biochem. 1999 Jun;55(2):79-83.

Growth modification of human colon adenocarcinoma cells exposed to a low-frequency electromagnetic field.

Ruiz Gómez MJ, Pastor Vega JM, de la Peña L, Gil Carmona L, Martínez Morillo M.

Departamento de Radiología y Medicina Física, Facultad de Medicina, Universidad de Málaga, Teatinos, Spain.


The influence of variable low-intensity, low-frequency electromagnetic fields on culture cells is investigated. Human colon adenocarcinoma cells were exposed to a rectangular and variable magnetic field (1 and 25 Hz; 1.5 mT peak). Cultures were exposed to a dose for 15 and 360 minutes, and after 24 hours incubation, cell viability was measured with neutral red stain. The group treated for 15 minutes showed a statistically significant increase in cell growth with 1 Hz (p < 0.002) and 25 Hz (p < 0.003). In contrast, a significant decrease in cell growth was found in those cultures treated with 1 Hz for 360 minutes (p < 0.02). The effects reported could be influenced by the magnetic field frequency and the exposure time.

In Vivo. 1991 Jan-Feb;5(1):39-40.

Effect of a 9 mT pulsed magnetic field on C3H/Bi female mice with mammary carcinoma.  A comparison between the 12 Hz and 460 Hz frequencies.

Bellossi A, Desplaces A.

Laboratoire de Biophysique, Faculte de Medecine, Rennes, France.

In a previous experiment, the exposure of tumoral C3H/Bi female mice to a 9 mT, 460 Hz pulsed magnetic field led to an increase in the length of survival in the late period of the disease; this might be due to a hampered metastatic process. In the present study 27 controls and 52 exposed mice were treated with the same protocol (a 10-minute exposure, 3 non-consecutive days a week, from 2-3 weeks after the tumors appeared until death) but with a 12 Hz PMF. In this experiment the 12 Hz PMF appeared to increase length of survival times in the early period of the disease.

Insomnia – Sleep

Int J Electron Healthc. 2008;4(3-4):339-49.

Static magnetotherapy for the treatment of insomnia.

Shieh YY, Tsai FY.

Department of Radiological Sciences, School of Medicine, UCIrvine Medical Center, Orange CA 92868, USA.


Magnets have been used for centuries to treat a number of physical disorders. The vast majority of research, however, on static magnet therapy for insomnia has been confined to the auricular type of therapy, with publications limited to Chinese journals. Most of these studies have depended on the subjective self-assessment of participants rather than objective scientific measurements. In this study, the authors report the positive preliminary results of insomnia treatment using pillows with embedded magnets, magnetic insoles and TriPhase bracelets. The analysis is based on objective actigraphic and polysomnographic data. A theory of accelerated transition from wakefulness to sleep is proposed to explain the process of insomnia relief through low-strength static magnetic fields. Analysis by functional Magnetic Resonance Imaging (fMRI) is used to further investigate the theory.

J Sleep Res 16(3) 253-8 (2007)

Pulsed radio-frequency electromagnetic fields: dose-dependent effects on sleep, the sleep EEG and cognitive performance

Regel SJ, Tinguely G, Schuderer J, Adam M, Kuster N, Landolt HP, Achermann P

To establish a dose-response relationship between the strength of electromagnetic fields (EMF) and previously reported effects on the brain, we investigated the influence of EMF exposure by varying the signal intensity in three experimental sessions. The head of 15 healthy male subjects was unilaterally exposed for 30 min prior to sleep to a pulse-modulated EMF (GSM handset like signal) with a 10 g-averaged peak spatial specific absorption rate of (1) 0.2 W kg(-1), (2) 5 W kg(-1), or (3) sham exposed in a double-blind, crossover design. During exposure, subjects performed two series of three computerized cognitive tasks, each presented in a fixed order [simple reaction time task, two-choice reaction time task (CRT), 1-, 2-, 3-back task]. Immediately after exposure, night-time sleep was polysomnographically recorded for 8 h. Sleep architecture was not affected by EMF exposure. Analysis of the sleep electroencephalogram (EEG) revealed a dose-dependent increase of power in the spindle frequency range in non-REM sleep. Reaction speed decelerated with increasing field intensity in the 1-back task, while accuracy in the CRT and N-back task were not affected in a dose-dependent manner. In summary, this study reveals first indications of a dose-response relationship between EMF field intensity and its effects on brain physiology as demonstrated by changes in the sleep EEG and in cognitive performance.

Conf Proc IEEE Eng Med Biol Soc. 2005;6:6214-6.

Influence of time-varying magnetic field on the release of neurotransmitters in raphe nuclei of rats.

Zhang J, Wang X, Wang M.

Mayo Clinic Rochester, MN 55905, USA.


A specially-designed time-varying magnetic field was developed to treat insomnia. Clinical results showed that this method could shorten the time to go to sleep and prolong the sleep duration. However, the mechanism of this method is still not well understood. In this study, the effect of magnetic stimulation on the release of serotonin (5-HT), noradrenaline (NE), dopamine (DA) in raphe nuclei of rats, which are known to play an important role in the sleep-wake regulation, was investigated. It was shown that there was a significant difference in the release of serotonin between control group and experimental group (p<0.01). The release of serotonin of the experimental group increased significantly. No obvious release changes of NE and DA are found (p>0.05). The results indicates that one possible mechanism of inducing sleep using specially designed magnetic field is to change the release of sleep-related neurotransmitters.

Neurosci Behav Physiol. 2005 Feb;35(2):165-70.

Actions of pulsed ultra-broadband electromagnetic irradiation on the EEG and sleep in laboratory animals.

Petrova EV, Gulyaeva NV, Titarov SI, Rozhnov YV, Koval’zon VM.

Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117865 Moscow, Russia.

Irradiation of animals with ultrashort impulses of ultra-broadband magnetic irradiation with an impulse repetition frequency of 6 Hz for 1 h induced changes in the spectral composition of cerebral cortex electrical activity in rats, measured over the 5 min immediately after irradiation, as compared with controls. In particular, there was suppression of frequencies close to the impulse sequence frequency, along with a decrease in interhemisphere coherence. Continuous recording of polygrams for 22 h from rabbits after irradiation revealed a “delayed” effect–a significant increase in paradoxical sleep, starting 16 h after the end of irradiation and persisting to the end of the recording period. It is suggested that irradiation has a direct action both on the mechanisms of generation of the theta rhythm (septohippocampal) and on the system controlling circadian rhythms (the suprachiasmatic nucleus-epiphysis system).

Eur J Neurosci. 2004 Jun;19(11):3099-104.

Intracortical inhibition and facilitation upon awakening from different sleep stages: a transcranial magnetic stimulation study.

De Gennaro L, Bertini M, Ferrara M, Curcio G, Cristiani R, Romei V, Fratello F, Pauri F, Rossini PM.

Dipartimento di Psicologia, Università di Roma La Sapienza, Via dei Marsi 78, 00185 Rome, Italy.


Intracortical facilitation and inhibition, as assessed by the paired-pulse transcranial magnetic stimulation technique with a subthreshold conditioning pulse followed by a suprathreshold test pulse, was studied upon awakening from REM and slow-wave sleep (SWS). Ten normal subjects were studied for four consecutive nights. Intracortical facilitation and inhibition were assessed upon awakening from SWS and REM sleep, and during a presleep baseline. Independently of sleep stage at awakening, intracortical inhibition was found at 1-3-ms interstimulus intervals and facilitation at 7-15-ms interstimulus intervals. Motor thresholds were higher in SWS awakenings, with no differences between REM awakenings and wakefulness, while motor evoked potential amplitude to unconditioned stimuli decreased upon REM awakening as compared to the other conditions. REM sleep awakenings showed a significant increase of intracortical facilitation at 10 and 15 ms, while intracortical inhibition was not affected by sleep stage at awakening. While the dissociation between motor thresholds and motor evoked potential amplitudes could be explained by the different excitability of the corticospinal system during SWS and REM sleep, the heightened cortical facilitation upon awakening from REM sleep points to a cortical motor activation during this stage.

Neurosci Lett. 2004 May 13;362(1):31-4.

Changes of motor cortical excitability in human subjects from wakefulness to early stages of sleep: a combined transcranial magnetic stimulation and electroencephalographic study.

Manganotti P, Fuggetta G, Fiaschi A.

Section of Neurological Rehabilitation, Department of Neurological and Visual Sciences, Giambattista Rossi Hospital, University of Verona, Verona, Italy.


The effect of sleep on human motor cortical excitability was investigated by evaluating the latency and amplitude of motor evoked potentials in ten subjects using transcranial magnetic stimulation. Motor evoked potentials and electroencephalographic data were recorded simultaneously and analyzed. Recordings were performed before, during and after a sleep period. A significant decrease in motor evoked potentials amplitude and a slight change in motor evoked potentials latency were noted in the recordings during the different sleep stages with a return to baseline values on awakening. A decrease in motor cortical excitability is suggested as explaining the effect of sleep.

Ross Fiziol Zh Im I M Sechenova. 2003 Jul;89(7):786-94.

Effect of impulse extrabroad-band electromagnetic radiation on electroencephalogram and sleep in laboratory animals.

[Article in Russian]

Petrova EV, Guliaeva NV, Titarov SI, Rozhnov IuV, Koval’zon VM.

Institute of Higher Nervous Activity and Neurophysiology, Russian Acad. Sci., Russia, 117865, Moscow.


1-hour exposure to ultra-short impulse low-frequency (6 Hz) superbroad band electromagnetic radiation altered cortical EEG in rats just after the exposure and increased the paradoxical sleep in rabbits within 16-22 hours following the radiation.

Advances in Therapy, Volume 18, Number 4 / July, 2001

Impulse magnetic-field therapy for insomnia: A double-blind, placebo-controlled study.

Rainer B. Pelka1 , Christof Jaenicke2 and Joerg Gruenwald2

(1) Universität der Bundeswehr München, Werner-Heisenberg-Weg 39, 85577 Neubiberg/München, Germany. (2) PhytoPharm Consulting Institute for Phytopharmaceuticals, Berlin, Germany


This 4-week double-blind, placebo-controlled study assessed the efficacy of impulse magnetic-field therapy for insomnia. One hundred one patients were randomly assigned to either active treatment (n = 50) or placebo (n = 51) and allocated to one of three diagnostic groups: (1) sleep latency; (2) interrupted sleep; or (3) nightmares. Efficacy endpoints were intensity of sleep latency, frequency of interruptions, sleepiness after rising, daytime sleepiness, difficulty with concentration, and daytime headaches. In the active-treatment group, the values of all criteria were significantly lower at study end (P < .00001). The placebo group also showed significant symptomatic improvement (P < .05), but the differences between groups were highly significant (P < .00001). Seventy percent (n = 34) of the patients given active treatment experienced substantial or even complete relief of their complaints; 24% (n = 12) reported clear improvement; 6% (n = 3) noted a slight improvement. Only one placebo patient (2%) had very clear relief; 49% (n = 23) reported slight or clear improvement; and 49% (n = 23) saw no change in their symptoms. No adverse effects of treatment were reported.

J Sleep Res. 2002 Dec;11(4):289-95.

Electromagnetic fields, such as those from mobile phones, alter regional cerebral blood flow and sleep and waking EEG.

Huber R, Treyer V, Borbély AA, Schuderer J, Gottselig JM, Landolt HP, Werth E, Berthold T, Kuster N, Buck A, Achermann P.

Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.


Usage of mobile phones is rapidly increasing, but there is limited data on the possible effects of electromagnetic field (EMF) exposure on brain physiology. We investigated the effect of EMF vs. sham control exposure on waking regional cerebral blood flow (rCBF) and on waking and sleep electroencephalogram (EEG) in humans. In Experiment 1, positron emission tomography (PET) scans were taken after unilateral head exposure to 30-min pulse-modulated 900 MHz electromagnetic field (pm-EMF). In Experiment 2, night-time sleep was polysomnographically recorded after EMF exposure. Pulse-modulated EMF exposure increased relative rCBF in the dorsolateral prefrontal cortex ipsilateral to exposure. Also, pm-EMF exposure enhanced EEG power in the alpha frequency range prior to sleep onset and in the spindle frequency range during stage 2 sleep. Exposure to EMF without pulse modulation did not enhance power in the waking or sleep EEG. We previously observed EMF effects on the sleep EEG (A. A. Borbély, R. Huber, T. Graf, B. Fuchs, E. Gallmann and P. Achermann. Neurosci. Lett., 1999, 275: 207-210; R. Huber, T. Graf, K. A. Cote, L. Wittmann, E. Gallmann, D. Matter, J. Schuderer, N. Kuster, A. A. Borbély, and P. Achermann. Neuroreport, 2000, 11: 3321-3325), but the basis for these effects was unknown. The present results show for the first time that (1) pm-EMF alters waking rCBF and (2) pulse modulation of EMF is necessary to induce waking and sleep EEG changes. Pulse-modulated EMF exposure may provide a new, non-invasive method for modifying brain function for experimental, diagnostic and therapeutic purposes.

Neurosci Biobehav Rev. 2001 May;25(3):235-60.

A detailed ethological analysis of the mouse open field test: effects of diazepam, chlordiazepoxide and an extremely low frequency pulsed magnetic field.

Choleris E, Thomas AW, Kavaliers M, Prato FS.

Room 9222D, Department of Psychology, Social Science Center, University of Western Ontario, London, Ontario, Canada N6A 5C2.


The open field test (OFT) is a widely used procedure for examining the behavioral effects of drugs and anxiety. Detailed ethological assessments of animal behavior are lacking. Here we present a detailed ethological assessment of the effects of acute treatment with the benzodiazepines, diazepam (DZ, 1.5mg/kg) and chlordiazepoxide (CDP, 5.0 and 10.0mg/kg), as well as exposure to a non-pharmacological agent, a specific pulsed extremely low frequency magnetic field (MAG) on open field behavior. We examined the duration, frequency and time course of various behaviors (i.e. exploration, walk, rear, stretch attend, return, groom, sit, spin turn, jump and sleep) exhibited by male mice in different regions of a novel open field. Both DZ and CDP consistently reduced the typical anxiety-like behaviors of stretch attend and wall-following (thigmotaxis), along with that of an additional new measure: ‘returns’, without producing any overall effects on total locomotion. The drugs also differed in their effects. CDP elicited a shift in the locomotor pattern from a ‘high explore’ to a ‘high walk’, while DZ mainly elicited alterations in sit and groom. The MAG treatment was repeated twice with both exposures reducing horizontal and vertical (rearing) activity and increasing grooming and spin turns. However, the anxiety-like behaviors of stretch attend and return were marginally reduced by only the first exposure. We conclude that a detailed ethological analysis of the OFT allows not only the detection of specific effects of drugs and non-pharmacological agents (i.e. pulsed magnetic field) on anxiety-like behaviors, but also permits the examination of non-specific effects, in particular those on general activity.

Clin Neurophysiol. 2000 Nov;111(11):1936-41.

Nocturnal magnetic field exposure: gender-specific effects on heart rate variability and sleep.

Graham C, Sastre A, Cook MR, Gerkovich MM.

Midwest Research Institute, 425 Volker Boulevard, Kansas City, MO 64110, USA.


OBJECTIVE: To determine if controlled exposure to power-frequency magnetic fields alters heart rate variability (HRV) and polysomnographic endpoints in healthy men (n=22) and women (n=24), 40-60 years of age.

METHODS: A randomized, double-blind, crossover design was used. Study endpoints collected during all-night exposure to 60 Hz magnetic fields at an occupational intensity (resultant flux density=28.3 microTesla, microT) were compared to similar endpoints obtained under equivalent, counterbalanced, no-exposure (< or =0.2 microT) control conditions.

RESULTS: Older men, but not women, exposed to the magnetic fields showed power reductions in the LF band of the HRV frequency spectrum, which is associated with sympathetically-mediated blood pressure and thermoregulatory control (P<0.04). Older women, but not men, exposed to the fields showed a pattern of disrupted sleep, with reductions in the duration of REM sleep (P=0.03), and strong trends for reductions in sleep efficiency (P=0.06) and total sleep time (P=0.06).

CONCLUSIONS: The gender-specific effects seen here with older volunteers replicate the results of previous exposure studies with younger men and women.

Neuroreport. 2000 Oct 20;11(15):3321-5.

Exposure to pulsed high-frequency electromagneticf field during waking affects human sleep EEG.

Huber R, Graf T, Cote KA, Wittmann L, Gallmann E, Matter D, Schuderer J, Kuster N, Borbely AA, Achermann P.

Institute of Pharmacology and Toxicology, University of Zurich, Switzerland.

The aim of the study was to investigate whether the electromagnetic field (EMF) emitted by digital radiotelephone handsets affects brain physiology. Healthy, young male subjects were exposed for 30 min to EMF (900 MHz; spatial peak specific absorption rate 1 W/kg) during the waking period preceding sleep. Compared with the control condition with sham exposure, spectral power of the EEG in non-rapid eye movement sleep was increased. The maximum rise occurred in the 9.75-11.25 Hz and 12.5-13.25 Hz band during the initial part of sleep. These changes correspond to those obtained in a previous study where EMF was intermittently applied during sleep. Unilateral exposure induced no hemispheric asymmetry of EEG power. The present results demonstrate that exposure during waking modifies the EEG during subsequent sleep. Thus the changes of brain function induced by pulsed high-frequency EMF outlast the exposure period.

Neurosci Lett. 1999 Nov 19;275(3):207-10.

Pulsed high-frequency electromagnetic field affects human sleep and sleep electroencephalogram.

Borbely AA, Huber R, Graf T, Fuchs B, Gallmann E, Achermann P.

Institute of Pharmacology and Toxicology, University of Zurich, Switzerland.

To investigate whether the electromagnetic field (EMF) emitted by digital radiotelephone handsets affects the brain, healthy, young subjects were exposed during an entire night-time sleep episode to an intermittent radiation schedule (900 MHz; maximum specific absorption rate 1 W/kg) consisting of alternating 15-min on-15-min off intervals. Compared with a control night with sham exposure, the amount of waking after sleep onset was reduced from 18 to 12 min. Spectral power of the electroencephalogram in non-rapid eye movement sleep was increased. The maximum rise occurred in the 10-11 Hz and 13.5-14 Hz bands during the initial part of sleep and then subsided. The results demonstrate that pulsed high-frequency EMF in the range of radiotelephones may promote sleep and modify the sleep EEG.

Bioelectromagnetics. 1998;19(3):199-202.

Human sleep under the influence of pulsed radiofrequency electromagnetic fields: a polysomnographic study using standardized conditions.

Wagner P, Roschke J, Mann K, Hiller W, Frank C.

Department of Psychiatry, University of Mainz,

To investigate the influence of radiofrequency electromagnetic fields (EMFs) of cellular phone GSM signals on human sleep electroencephalographic (EEG) pattern, all-night polysomnographies of 24 healthy male subjects were recorded, both with and without exposure to a circular polarized EMF (900 MHz, pulsed with a frequency of 217 Hz, pulse width 577 micros, power flux density 0.2 W/m2. Suppression of rapid eye movement (REM) sleep as well as a sleep-inducing effect under field exposure did not reach statistical significance, so that previous results indicating alterations of these sleep parameters could not be replicated. Spectral power analysis also did not reveal any alterations of the EEG rhythms during EMF exposure. The failure to confirm our previous results might be due to dose-dependent effects of the EMF on the human sleep profile.

Neuropsychobiology. 1996;33(1):41-7.

Effects of pulsed high-frequency electromagnetic fields on human sleep.

Mann K, Roschke J.

Department of Psychiatry, University of Mainz, Germany.

In the present study we investigated the influence of pulsed high-frequency electromagnetic fields of digital mobile radio telephones on sleep in healthy humans. Besides a hypnotic effect with shortening of sleep onset latency, a REM suppressive effect with reduction of duration and percentage of REM sleep was found. Moreover, spectral analysis revealed qualitative alterations of the EEG signal during REM sleep with an increased spectral power density. Knowing the relevance of REM sleep for adequate information processing in the brain, especially concerning mnestic functions and learning processes, the results emphasize the necessity to carry out further investigations on the interaction of this type of electromagnetic fields and the human organism.

Neuropsychobiology. 1998 Nov;38(4):251-6.

No effects of pulsed high-frequency electromagnetic fields on heart rate variability during human sleep.

Mann K, Roschke J, Connemann B, Beta H.

Department of Psychiatry, University of Mainz, Germany.

The influence of pulsed high-frequency electromagnetic fields emitted by digital mobile radio telephones on heart rate during sleep in healthy humans was investigated. Beside mean RR interval and total variability of RR intervals based on calculation of the standard deviation, heart rate variability was assessed in the frequency domain by spectral power analysis providing information about the balance between the two branches of the autonomic nervous system. For most parameters, significant differences between different sleep stages were found. In particular, slow-wave sleep was characterized by a low ratio of low- and high-frequency components, indicating a predominance of the parasympathetic over the sympathetic tone. In contrast, during REM sleep the autonomic balance was shifted in favor of the sympathetic activity. For all heart rate parameters, no significant effects were detected under exposure to the field compared to placebo condition. Thus, under the given experimental conditions, autonomic control of heart rate was not affected by weak-pulsed high-frequency electromagnetic fields.

Bioelectromagnetics. 1998;19(2):98-106.

Nocturnal exposure to intermittent 60 Hz magnetic fields alters human cardiac rhythm.

Sastre A, Cook MR, Graham C.

Midwest Research Institute, Kansas City, Missouri 64110, USA.


Heart rate variability (HRV) results from the action of neuronal and cardiovascular reflexes, including those involved in the control of temperature, blood pressure and respiration. Quantitative spectral analyses of alterations in HRV using the digital Fourier transform technique provide useful in vivo indicators of beat-to-beat variations in sympathetic and parasympathetic nerve activity. Recently, decreases in HRV have been shown to have clinical value in the prediction of cardiovascular morbidity and mortality. While previous studies have shown that exposure to power-frequency electric and magnetic fields alters mean heart rate, the studies reported here are the first to examine effects of exposure on HRV. This report describes three double-blind studies involving a total of 77 human volunteers. In the first two studies, nocturnal exposure to an intermittent, circularly polarized magnetic field at 200 mG significantly reduced HRV in the spectral band associated with temperature and blood pressure control mechanisms (P = 0.035 and P = 0.02), and increased variability in the spectral band associated with respiration (P = 0.06 and P = 0.008). In the third study the field was presented continuously rather than intermittently, and no significant effects on HRV were found. The changes seen as a function of intermittent magnetic field exposure are similar, but not identical, to those reported as predictive of cardiovascular morbidity and mortality. Furthermore, the changes resemble those reported during stage II sleep. Further research will be required to determine whether exposure to magnetic fields alters stage II sleep and to define further the anatomical structures where field-related interactions between magnetic fields and human physiology should be sought.

Ann Biomed Eng. 1996 May-Jun;24(3):424-9.

Electroencephalographic changes following low energy emission therapy.

Lebet JP, Barbault A, Rossel C, Tomic Z, Reite M, Higgs L, Dafni U, Amato D, Pasche B.

Symtonic SA., Renens, Switzerland.


Low energy emission therapy (LEET) is a novel approach to delivering low levels of amplitude-modulated electromagnetic fields to the human brain. The sleep electroencephalogram (EEG) effects of a 15-min LEET treatment were investigated in a double-find cross-over study to assess sleep induction. Fifty-two healthy volunteers were exposed to both active and inactive LEET treatment sessions, with a minimum interval of 1 week between the two sessions. Baseline EEGs were obtained, and 15-min posttreatment EEGs were recorded and analyzed according to the Loomis classification. A significant increase in the duration of stage B1 sleep (0.58 +/- 2.42 min [mean +/- SD], p = 0.046), decreased latency to the first 10 sec epoch of sleep (-1.23 +/- 5.32 min, p = 0.051) and decreased latency to sleep stage B2 (-1.21 +/- 5.25 min, p = 0.052) were observed after active treatment. Additionally, establishment of slow waves with progression from stages B to C was significantly more pronounced after active LEET treatment (p = 0.040). A combined analysis of these results with those of an identical study performed in Denver showed that LEET had a significant effect on afternoon sleep induction and maintenance with shorter sleep latencies (decreased latency to the first 10 sec epoch of sleep; -1.00 +/- 5.51 min, p = 0.033; decreased latency to sleep stage B2; -1.49 +/- 5.40 min, p = 0.003), an increased duration of stage B2 (0.67 +/- 2.50 min, p = 0.003), an increase in the total duration of sleep (0.69 +/- 4.21 min, p = 0.049), and a more prominent establishment of slow waves with progression to a deeper sleep stage (p = 0.006). It is concluded that the intermittent 42.7 HZ amplitude modulation of 27.12-MHz electromagnetic fields results in EEG changes consistent with shorter sleep latencies, longer sleep duration, and deeper sleep in healthy subjects.

Sleep. 1996 May;19(4):327-36.

Effects of low energy emission therapy in chronic psychophysiological insomnia.

Pasche B, Erman M, Hayduk R, Mitler MM, Reite M, Higgs L, Kuster N, Rossel C, Dafni U, Amato D, Barbault A, Lebet JP.

Symtonic USA, Inc., New York, New York 10162, USA.


The treatment of chronic psychophysiological insomnia presents a challenge that has not been met using currently available pharmacotherapy. Low energy emission therapy (LEET) has been developed as a potential alternative therapy for this disorder. LEET consists of amplitude-modulated electromagnetic fields delivered intrabuccally by means of an electrically conducting mouthpiece in direct contact with the oral mucosa. The effect of LEET on chronic psychophysiological insomnia was assessed with polysomnography (PSG) and sleep rating forms on a total of 106 patients at two different centers. Active or inactive LEET was administered for 20 minutes in late afternoon three times a week for a total of 12 treatments. Primary efficacy endpoints evaluating the results were changes from baseline in PSG-assessed total sleep time (TST) and sleep latency (SL). Secondary endpoints were changes in sleep efficiency (SE), sleep stages, and reports by the subjects of SL and TST. There was a significant increase in TST as assessed by PSG between baseline and post-treatment values for the active treatment group (76.0 +/- 11.1 minutes, p = 0.0001). The increase for the inactive treatment group was not statistically significant. The TST improvement was significantly greater for the active group when compared to the inactive group (adjusted for baseline TST; p = 0.020. R1 = 0.20). There was a significant decrease in SL as assessed by PSG between baseline and post-treatment values for the active treatment group (-21.6 +/- 5.9 minutes, p = 0.0006), whereas the decrease noted for the inactive treatment group was not statistically significant. The difference in SL decrease between the two treatment groups was marginally significant (adjusted for baseline SL and center, p = 0.068, R2 = 0.60). The number of sleep cycles per night increased by 30% after active treatment (p = 0.0001) but was unchanged following inactive treatment. Subjects did not experience rebound insomnia, and there were no significant side effects. The data presented in this report indicate that LEET administered for 20 minutes three times a week increased TST and reduced SL in chronic psychophysiological insomnia. LEET is safe and well tolerated and it effectively improved the sleep of chronic insomniacs given 12 treatments over a 4-week period by increasing the number of sleep cycles without altering the percentage of the various sleep stages during the night. The therapeutic action of LEET differs from that of currently available drug therapies in that the sleep pattern noted in insomniacs following LEET treatment more closely resembles nocturnal physiological sleep. This novel treatment may offer an attractive alternative therapy for chronic insomnia.


J Inflamm Res. 2015 Feb 23;8:59-69. doi: 10.2147/JIR.S78631. eCollection 2015.

Effect of pulsed electromagnetic field treatment on programmed resolution of inflammation pathway markers in human cells in culture.

Kubat NJ1, Moffett J2, Fray LM2.

Author information

  • 1Nicole Kubat Consulting, Pasadena, CA, USA.
  • 2Life Science Department, Regenesis Biomedical, Inc., Scottsdale, AZ, USA.


Inflammation is a complex process involving distinct but overlapping biochemical and molecular events that are highly regulated. Pulsed electromagnetic field (PEMF) therapy is increasingly used to treat pain and edema associated with inflammation following surgery involving soft tissue. However, the molecular and cellular effects of PEMF therapy on pathways involved in the resolution of inflammation are poorly understood. Using cell culture lines relevant to trauma-induced inflammation of the skin (human dermal fibroblasts, human epidermal keratinocytes, and human mononuclear cells), we investigated the effect of PEMF on gene expression involved in the acute and resolution phases of inflammation. We found that PEMF treatment was followed by changes in the relative amount of messenger (m)RNAs encoding enzymes involved in heme catabolism and removal of reactive oxygen species, including an increase in heme oxygenase 1 and superoxide dismutase 3 mRNAs, in all cell types examined 2 hours after PEMF treatment. A relative increase in mRNAs encoding enzymes involved in lipid mediator biosynthesis was also observed, including an increase in arachidonate 12- and 15-lipoxygenase mRNAs in dermal fibroblasts and epidermal keratinocytes, respectively. The relative amount of both of these lipoxygenase mRNAs was elevated in mononuclear cells following PEMF treatment relative to nontreated cells. PEMF treatment was also followed by changes in the mRNA levels of several cytokines. A decrease in the relative amount of interleukin 1 beta mRNA was observed in mononuclear cells, similar to that previously reported for epidermal keratinocytes and dermal fibroblasts. Based on our results, we propose a model in which PEMF therapy may promote chronic inflammation resolution by mediating gene expression changes important for inhibiting and resolving inflammation. PLoS One. 2013; 8(5): e65561. Published online 2013 May 31. doi:  10.1371/journal.pone.0065561 PMCID: PMC3669296

Pulsed Electromagnetic Fields Increased the Anti-Inflammatory Effect of A2Aand A3 Adenosine Receptors in Human T/C-28a2 Chondrocytes and hFOB 1.19 Osteoblasts

Fabrizio Vincenzi,1 Martina Targa,1 Carmen Corciulo,1 Stefania Gessi,1 Stefania Merighi,1 Stefania Setti,2 Ruggero Cadossi,2 Mary B. Goldring,3 Pier Andrea Borea,1 and Katia Varani1,* David M. Ojcius, Editor 1Department of Medical Sciences, Pharmacology Unit, University of Ferrara, Ferrara, Italy 2Igea Biophysics Laboratory, Carpi, Italy 3Laboratory for Cartilage Biology, Hospital for Special Surgery, Weill Cornell Medical College, New York, New York, United States of America University of California Merced, United States of America * E-mail: ti.efinu@krvCompeting Interests: SS is an employee and RC is the president and scientific director of Igea (Carpi, Italy) who provided the PEMF generator system. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Conceived and designed the experiments: FV PAB KV. Performed the experiments: MT CC SG SM SS. Analyzed the data: FV PAB KV. Contributed reagents/materials/analysis tools: SS RC MBG. Wrote the paper: FV RC PAB KV. Author information ? Article notes ? Copyright and License information ? Received 2013 Mar 1; Accepted 2013 Apr 27. Copyright notice This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This article has been cited by other articles in PMC.


Chronic inflammation represent an important factor in the pathophysiology of several joint diseases [1]. In human joint damages, chondrocytes are able to respond to the depletion of extracellular matrix and abnormal biomechanical functions trying to preserve matrix integrity [2]. The degradation of the cartilage matrix is mediated by a number of different factors including pro-inflammatory cytokines, matrix degrading enzymes, nitric oxide (NO), oxygen derived free radicals and prostaglandins [3], [4].

On the other hand continuous remodeling by bone cells such as osteoblasts and osteoclasts allows the skeleton to grow, adapt and repair itself [5], [6]. In healthy adults bone remodeling, an important homeostatic function, is well balanced and abnormalities in this process can result in a variety of skeletal disorders [7]. The number of osteoblasts decreases with age, affecting the balance of formation and resorption in the bone tissue, and potentially leading to osteoporosis [8]. Moreover, osteoblasts if adequately stimulated produce increased levels of pro-inflammatory cytokines [9]. It is well accepted that osteoblasts release the receptor activator of nuclear factor kB (NF-kB) ligand (RANKL) modulating signaling pathways that promote osteoclast differentiation and survival [10]. Moreover, osteoblasts also produce a protein named osteoprotegerin (OPG) that, preventing the biological effects of RANKL, plays an important osteoprotective role [11].

Previous papers have reported that PEMF exposure could act modulating cartilage and bone metabolism, stimulating chondrocyte and/or osteoblast cell proliferation and the synthesis of extracellular matrix components [12]. The stimulation of chondrocyte and/or osteoblast cell proliferation induced by PEMFs has been shown to have a positive effect in the treatment of fracture healing [13], [14]. In particular, a well observed beneficial effect on osteogenesis has been reported based on the observation that PEMFs stimulate cell proliferation, induce osteoblastogenesis and differentiation of osteoblasts [15]. In addition, PEMFs stimulate proteoglycan synthesis without affecting the degradation suggesting their potential use to preserve cartilage integrity and function [16]. A clinical study has shown that PEMF treatment after arthroscopic surgery results in faster and complete functional recovery compared to controls in the short term, that is maintained at 3 year follow-up [17].

Adenosine, an endogenous modulator of a wide range of biological functions, interacts with four cell surface subtypes classified as A1, A2A, A2B and A3 adenosine receptors (ARs) [18]. A1 and A3ARs inhibit adenylate cyclase activity and decrease cAMP production whilst A2A and A2BARs exert an increase of cAMP accumulation [19]. Modulation of ARs has an important role in the regulation of inflammatory processes suggesting their involvement in different pathologies based on inflammation [20][23]. Recently, it has been well documented that the adenosine and its receptors are involved in bone remodeling. Adenosine A1AR-knockout mice are protected from bone loss suggesting that these receptor subtypes may be a useful target in treating diseases characterized by excessive bone turnover [24], [25]. Other studies reported that A2B or A2AAR stimulation could be implicated in osteoblastic differentiation revealing their involvement in bone formation and fracture repair [26], [27].

It has been reported that different physiological systems seem to be influenced by PEMF exposure as revealed by in vitro experiments. The effect of PEMFs on ARs in various cells and tissues such as in human neutrophils has been investigated [28], [29]. The treatment with PEMFs induced a selective increase in A2AARs expressed in rat cortex membranes and in rat cortical neurons [30]. A potentiated anti-tumoral effect of A3ARs by PEMFs was found in different cell lines such as rat adrenal pheochromocytoma (PC12) and human glioblastoma (U87MG) cell lines [31]. A role of ARs and PEMFs in modulating bovine chondrocytes and synoviocytes activity has been previously documented [32], [33]. Functional studies in human synoviocytes have suggested an anti-inflammatory effect linked to A2AARs that is primarily based on the inhibition of PGE2 production [34]. No papers are present in literature studying the effect of PEMFs and ARs in human chondrocytes and osteoblasts despite their co-presence could be very interesting in the cell functionality.

In the present study we have investigated whether PEMFs modulate the expression of ARs in T/C-28a2 chondrocytes and in hFOB 1.19 osteoblasts. The effect of one of the most important pro-inflammatory stimuli such as IL-1β on A2A and A3ARs in the absence or in the presence of PEMFs has been examined. The stimulation of A2A or A3ARs has been investigated on cAMP production and cell proliferation as well as on the release of PGE2 and some of the most relevant pro-inflammatory cytokines such as IL-6 or IL-8. Moreover, the effects of A2A or A3AR agonists on vascular endothelial growth factor (VEGF) release in T/C-28a2 chondrocytes and on OPG production in hFOB 1.19 osteoblasts have been explored. In addition, the activation of A2A or A3ARs has been examined on NF-kB activation. All the experiments were carried out in the absence or in the presence of PEMF exposure. These results could indicate the possibility of novel therapeutic approaches based on the interaction of AR modulation and a non-invasive stimulus represented by PEMFs.

Materials and Methods

Cell culture


Human immortalized chondrocyte cells (T/C-28a2) were kindly provided by Professor Mary B. Goldring, from Cornell Medical College, NY, USA [35], [36]. Cells were cultured in complete medium DMEM F12 (11), containing 10% FBS. Cells were grown at 37°C, in 5% CO2. T/C-28a2 cells represent an appropriate chondrocyte model as suggested by the significant similarities between human primary chondrocytes and T/C-28a2 cells in the induction of IL-6 synthesis in response to chemical and shear stimulation [37].

Human fetal osteoblast cells (hFOB 1.19) were obtained from ATCC (Manassas, VA, USA). Cells were cultured in Ham’s F12 without phenol red (Gibco, Invitrogen, Carlsbad, CA), containing 10% fetal bovine serum, 0.3 mg/ml G418. Cells were grown at a permissive temperature of 34°C for a rapid cell division and the medium was renewed every 2 to 3 days [38].

Field Exposure System

T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts were exposed to PEMFs generated by a pair of rectangular horizontal coils (14 cm×23 cm), each made of 1400 turns of copper wire placed opposite to each other. The culture was placed between this pair of coils so that the plane of the coils was perpendicular to the culture flasks. The coils were powered by the PEMF generator system (IGEA, Carpi, Italy) used in previous studies[30][32], which produced a pulsed signal with the following parameters: pulse duration of 1.3 ms and frequency of 75 Hz.

The peak intensity of the magnetic field was 1.5±0.1 mT for T/C-28a2 chondrocytes or 2.5±0.2 mT for hFOB 1.19 osteoblasts. It was detected in air between two coils from one side to the other, at the level of the culture flasks, using the Hall probe (HTD61-0608-05-T, F.W. Bell, Sypris Solutions, Louisville, KY, USA) of a gaussmeter (DG500, Laboratorio Elettrofisico, Milan, Italy) with a reading sensitivity of 0.2%. The peak values measured between two coils in air had a maximum variation of 1% in the whole area in which the culture flasks were placed.

RT-PCR analysis of ARs

Total cytoplasmic RNA was extracted by the acid guanidiniumthiocyanate-phenol method from T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts untreated or treated with PEMFs for 24 hours. Quantitative real-time RT-PCR assay of mRNAs was carried out using a gene-specific, fluorescently labeled TaqMan minor groove binder (MGB) probe in an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Warrington Cheshire, UK). For the real-time RT-PCR of A1, A2A, A2B and A3ARs the Assays-on-Demand gene expression products (Applied Biosystems) were used. For the real-time RT-PCR of the reference gene, the endogenous control human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) kit was used, and the probe was labeled with the fluorescent dye VIC (Applied Biosystems). In the negative control, sterile distilled water was added instead of template [39].

Western blot assay for ARs


T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts untreated or treated with PEMFs for 24 hours were lysed in Triton lysis buffer and aliquots of total protein samples (50 µg) were analysed using specific A1, A2A, A2B and A3AR antibody (Alpha Diagnostic, San Antonio, TX, USA). Filters were washed and incubated for 1 hour at room temperature with peroxidase-conjugated secondary antibody (12000 dilution). Specific reaction were revealed with enhanced chemiluminescence Western blotting detection reagent (GE Healthcare). Western blotting assays were also normalized against the housekeeping protein β-actin [20].

Saturation binding experiments to ARs

Saturation binding experiments to A1ARs were performed according to the method described previously using [3H]-1,3-dipropyl-8-cyclopentyl-xanthine ([3H]-DPCPX, specific activity 120 Ci/mmol; Perkin-Elmer, Boston, MA, USA) as radioligand. The membranes derived from PEMFs-treated or untreated T/C-28a2 or hFOB 1.19 cells (100 µg of protein/assay) with different concentrations of the radioligand [3H]-DPCPX (0.1–30 nM) were incubated in Tris–HCl 50 mM, pH 7.4, for 90 min at 4°C. Nonspecific binding was determined in the presence of DPCPX 1 µM [40]. Saturation binding experiments to A2AARs were performed according to the method described previously using [3H]-4-(2-[7-amino-2-(2-furyl)[1,2,4] triazolo [2,3-a] [1,3,5] triazin-5-yl-amino]ethyl ([3H]-ZM 241385, specific activity 27.4 Ci/mmol; American Radiolabeled Chemicals Inc, Saint Louis, MO, USA) as radioligand. The membranes derived from PEMFs-treated or untreated T/C-28a2 or hFOB 1.19 cells (100 µg of protein/assay) were incubated for 60 min at 4°C with various concentrations of the radioligand [3H]-ZM 241385 (0.1–30 nM) and Tris–HCl 50 mM, MgCl210 mM, pH 7.4. Nonspecific binding was determined in the presence of ZM 241385 1 µM [39]. Saturation binding experiments to A2BARs were performed using [3H]-N-benzo [1,3[dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl-oxy]-acetamide ([3H]-MRE 2029F20, specific activity 123 Ci/mmol; GE Healthcare, Little Chalfont, UK) as radioligand. The membranes obtained as previously described (100 µg of protein/assay) with [3H]-MRE 2029F20 in the range 0.1–30 nM were incubated in Tris–HCl 50 mM, MgCl2 10 mM, EDTA 1 mM, pH 7.4 at 4°C for 60 min. Nonspecific binding was determined in the presence of MRE 2029F20 1 µM [41]. Saturation binding experiments to A3ARs were performed using [3H]-5N-(4-methoxyphenylcarbamoyl) amino-8-propyl-2-(2-furyl) pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine ([3H]-MRE 3008F20, specific activity 67 Ci/mmol; GE Healthcare) as radioligand. The membranes treated as above mentioned (100 µg of protein/assay) with [3H]-MRE 3008F20 (0.1–50 nM) were incubated in Tris–HCl 50 mM, MgCl2 10 mM, EDTA 1 mM, pH 7.4, at 4°C for 150 min. Nonspecific binding was determined in the presence of MRE 3008F20 1 µM [42]. At the end of the incubation time, bound and free radioactivity was separated by filtering the assay mixture through Whatman GF/B glass fiber filters by using a Brandel cell harvester. The filter bound radioactivity was counted by Scintillation Counter Perkin Elmer Tri Carb 2810 TR with an efficiency of 62% (Perkin-Elmer).

To verify the effect of pro-inflammatory stimuli on ARs, T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts were also treated with IL-1β at 1 ng/ml for 48 hours, in the absence or in the presence of PEMFs for the last 24 hours. At the end of the treatment the cells were used in saturation binding experiments for A2A or A3ARs or in functional experiments.

Immunoflourescence staining of A2A and A3ARs


T/C-28a2 chondrocyte or hFOB 1.19 osteoblast cells grown on coverslips and incubated in polylysine-treated chambers were fixed with 4% formalin in PBS, pH 7.4 for 15 min at room temperature. After two or five min washes with ice cold PBS, potential sites for nonspecific antibody binding were blocked by 30 min incubation with 1% BSA in PBST pH 7.4. The cells were then incubated with specific A2A or A3ARs polyclonal primary antibody (150 dilution) overnight at 4°C (Alpha Diagnostics Inc). Subsequently, they were incubated with secondary antibody (180) conjugated to fluorescein isothiocyanate (FITC) goat anti-rabbit IgG for 1 hour at room temperature and nuclear stain with 4′,6-diamidino-2- henylindole (DAPI, Sigma) 1 µg/ml for 20 min. After washing with PBS, pH 7.4, the cells were mounted for microscopy with DABCO (1,4-Diazabicyclo (2.2.2) octane, Sigma) and visualized by a microscopy Nikon Eclipse 50i [43].

Measurement of cyclic AMP levels

T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts (106 cells per sample) were suspended in 0.5 ml incubation mixture Krebs Ringer phosphate buffer, containing 1.0 IU/ml adenosine deaminase (Sigma) and preincubated for 10 min in a shaking bath at 37°C with 0.5 mM of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as phosphodiesterase inhibitor. Then the effects of the A2AAR agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680, Sigma) or of the A3AR agonist 2-chloro-N6-(3-iodobenzyl) adenosine-5′-N-methyl-uronamide (Cl-IB-MECA, Tocris, Bristol, UK) at 100 nM concentration were studied. To better investigate the inhibitory effect of Cl-IB-MECA, the cells were also incubated with forskolin (1 µM). A2A or A3ARs selected adenosine antagonists, such as 2-(2-Furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo [4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH 442416, Sigma) or 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl] ester (MRS 1334, Tocris) at the 1 µM concentration, were also used to verify the specific involvement of these subtypes in cAMP production. The final aqueous solution was tested to evaluate cAMP levels by using a competition binding protein assay with [3H]-cAMP, trizma base 0.1 mM, aminophylline 8.0 mM, mercaptoethanol 6.0 mM, pH 7.4 [39]. At the end of the incubation time (150 min at 4°C), and after the addition of charcoal, the samples were centrifuged at 2000× g for 10 min and the clear supernatant was counted in a liquid Scintillation Counter Tri Carb Perkin-Elmer 2810 TR.

Cell Proliferation Assays

T/C-28a2 or hFOB 1.19 cells were seeded in fresh medium with 1 µCi/ml [3H]-Thymidine for 24 hours and simultaneously treated with well-known adenosine agonists such as CGS 21680 or Cl-IB-MECA (100 nM) in the absence or presence of SCH 442416 or MRS 1334 (1 µM). Proliferation assays under the same experimental conditions were also carried out in the presence of PEMF exposure. After 24 hours of labeling, cells were trypsinized, dispensed in four wells of a 96-well plate, and filtered through Whatman GF/C glass fiber filters using a Micro-Mate 196 cell harvester (Perkin-Elmer). The filter-bound radioactivity was counted on Top Count Microplate Scintillation Counter with Micro Scint 20 [43].

IL-6, IL-8, PGE2, VEGF and OPG release

T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts were seeded into 24-well plates and incubated in the absence or in the presence of IL-1β (1 ng/ml) for 48 hours. Some of the cells were also incubated in the absence or in the presence of CGS 21680 or Cl-IB-MECA (1 µM). Selective A2A or A3AR antagonists such as SCH 442416 or MRS 1334 (1 µM) were used to verify the specific involvement of these receptors in cytokine release. In order to examine the effect of PEMFs the cells were also treated for the last 24 hours in comparison with untreated cells. At the end of incubation, the cell suspension was collected and centrifuged at 1000× g for 10 min at 4°C. The pro-inflammatory cytokines IL-6 and IL-8, the lipid mediator PGE2 and the angiogenic factor VEGF levels were determined with specific quantitative sandwich ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer instructions [33], [39], [44]. The production of OPG from hFOB 1.19 osteoblasts was determined by ELISA kit (Abcam, Cambridge, UK) following the manufacturer instructions. Briefly, the reaction was developed with streptavidin-horseradish peroxidase and optical density was read at 450 nm wavelength.

NF-kB activation

Nuclear extracts from T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts were obtained by using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer instructions. The NF-kB activation was evaluated by detecting phosphorylated p65 proteins in nuclear extracts by using the TransAM NF-kB kit (Active Motif, Carlsbad, USA). Phosphorylated NF-kB subunits specifically binds to the immobilized oligonucleotides containing the NF-kB consensus site (5′-GGGACTTTCC-3′). The primary antibody used to detect NF-kB recognized an epitope in the subunits that is accessible only when it is activated and bound to its DNA target. A horseradish peroxidase-conjugated secondary antibody provided a sensitive colorimetric readout that was quantified by spectrophotometry at 450 nm wavelength [21].

Statistical Analysis

Dissociation equilibrium constants for saturation binding, affinity or KD values, as well as the maximum densities of specific binding sites, Bmax were calculated for a system of one or two-binding site populations by non-linear curve fitting using the program Ligand purchased from Kell Biosoft, Ferguson, MO, USA[31], [39]. All data are reported as mean ± SEM of different independent experiments as indicated in Result section or in Figure legend. Analysis of data was performed by one-way analysis of variance (ANOVA) followed by Dunnett’s test or unpaired two-sided Student’s t-test for comparison of two samples and were considered statistically significant with a p value less than 0.05 (Graph Pad Software, San Diego, CA, USA):


mRNA and protein expression of ARs in T/C-28a2 and hFOB 1.19 cells

Figure 1A shows the relative mRNA levels of A1, A2A, A2B and A3ARs in T/C-28a2 chondrocytes obtained by real-time quantitative RT-PCR. The treatment of the cells with PEMFs for 24 hours elicited a statistical significant increase of A2A and A3ARs mRNA levels (p<0.01 vs control conditions). The upregulation of A2A and A3ARs protein expression in T/C-28a2 chondrocytes following PEMF exposure was confirmed by Western blot assays (Figure 1B). In particular, densitometric analysis revealed an increase of 2.1 fold and 2.3 fold for A2A and A3ARs, respectively (Figure 1C, p<0.01 vs control conditions). PEMF treatment did not determined any changes in mRNA or protein expression for A1 and A2BARs in T/C-28a2 cells (Figure 1).

Figure 1

Figure 1mRNA levels and protein expression of ARs in T/C-28a2 chondrocytes.

Analogous results were obtained in hFOB 1.19 cells following PEMF treatment (Figure 2). A2A and A3AR mRNA levels (Figure 2A) as well as their protein expression (Figure 2B and C) were augmented by PEMFs while A1 and A2BARs were not affected by PEMF exposure.

Figure 2

Figure 2mRNA levels and protein expression of ARs in hFOB 1.19 osteoblasts.

ARs saturation binding experiments in T/C-28a2 and hFOB 1.19 cells

Saturation binding experiments were performed to evaluate the affinity (KD) and density (Bmax) of ARs in T/C-28a2 and hFOB 1.19 cells and to better quantify the upregulation of A2A and A3ARs determined by PEMF exposure. Table 1 shows that the KD and Bmax values for A1 and A2BARs were not affected by PEMF exposure, while it determined an increase of A2A and A3AR density in both the cell line examined. In T/C-28a2 chondrocytes the affinity of the radioligand [3H]-ZM241385 for A2AARs did not change after 24 hours PEMF treatment whereas the Bmax value increased from 126±10 to 297±22* fmol/mg protein (*, p<0.01, Figure 3A, B). In the same cells, saturation binding experiments with the radioligand [3H]-MRE3008F20 revealed a 2.2 fold of increase in A3AR density following PEMF treatment (Figure 3C, D). For A2A and A3ARs, the linearity of the Scatchard plots (Figure 3B and D, respectively) indicated the presence of a single class of high affinity binding site.

Figure 3

Figure 3PEMF exposure up-regulated A2A and A3ARs in T/C-28a2 chondrocytes.

Table 1

Table 1Affinity and density of A1, A2A, A2B and A3ARs in untreated or PEMF treated T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts.

In hFOB 1.19 cells PEMF exposure determined an increase from 110±11 to 275±23* fmol/mg protein and from 135±12 fmol/mg protein to 328±29* fmol/mg protein for A2A and A3AR Bmax values, respectively (*, p<0.01, Figure 4). PEMF exposure did not induced any changes in the affinity values for the A2A and A3ARs in hFOB 1.19 cells. In particular, the KD values of the radioligand [3H]-ZM241385 for A2AARs were 2.52±0.21 nM and 2.47±0.22 nM in the absence or in the presence of PEMFs, respectively (Figure 4A, B). Similarly, the KD values of the radioligand [3H]-MRE3008F20 for A3ARs were 3.02±0.26 nM and 3.21±0.27 nM in the absence or in the presence of PEMFs, respectively (Figure 4C, D). These data suggested that PEMF exposure did not influenced the ligand-receptor interaction but increased the receptor expression in the membrane surface.

Figure 4

Figure 4PEMF exposure up-regulated A2A and A3ARs in hFOB 1.19 osteoblasts.

Since most of the functional experiments were performed in the presence of the pro-inflammatory cytokine IL-1β, we evaluated its effect on A2A and A3AR expression. The treatment of T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts with IL-1β for 48 hours mediated a significant increase of A2A and A3ARs (Table 2). Moreover, when T/C-28a2 and hFOB 1.19 cells were exposed to PEMFs for the last 24 hours during IL-1β treatment, we found a further increase of A2A and A3AR density (Table 2).

Table 2

Table 2Affinity and density of A2A and A3ARs in untreated or PEMF treated T/C-28a2 chondrocytes or hFOB 1.19 osteoblasts before and after IL-1β treatment.

Immunofluorescence staining confirmed the PEMF-induced overexpression of A2A and A3ARs in T/C-28a2 and hFOB 1.19 cells

Immunoflourescence analysis demonstrate the presence of A2A and A3ARs in T/C-28a2 chondrocytes (Figure 5, panel A and C, respectively). Interestingly, PEMF treatment determined an overexpression of these receptor subtypes as shown by the increase of the fluorescent signal (Figure 5B and D). The presence of A2Aand A3ARs in hFOB 1.19 was demonstrated by immunofluorescence analysis and, analogously to T/C-28a2 cells, PEMF exposure for 24 hours determined their upregulation (Figure 5E–H).

Figure 5

Figure 5A2A and A3AR immunofluorescence staining in T/C-28a2 and hFOB 1.19 cells.

PEMF exposure enhanced the differential effects of A2A and A3AR stimulation on cAMP production and cell proliferation in T/C-28a2 and hFOB 1.19

To evaluate if the upregulation of A2A and A3ARs determined by PEMF in the examined cells was accompanied by an increase of their functional responses, we studied the cAMP production induced by A2Aand A3AR stimulation before and after 24 hours of PEMF exposure. As expected, in T/C-28a2 cells the well-known A2AAR agonist CGS 21680 (100 nM) elicited an increase of cAMP from a basal condition of 16±2 to 85±8 pmol/106 cells. The treatment with PEMF enhanced the stimulatory effect of CGS 21680 that reached a cAMP production of 165±11 pmol/106 cells (p<0.01 vs CGS 21680 in control conditions). The selective A2AAR antagonist SCH 442416 (1 µM) was able to abrogate the effect of CGS 21680 (Figure 6A). The inhibitory effect of the A3AR agonist Cl-IB-MECA was studied in the presence of the adenylate cyclase direct activator Forskolin (1 µM). While Cl-IB-MECA at the 100 nM concentration was able to inhibit the forskolin-stimulated cAMP levels by 45%, the pre-treatment of T/C-28a2 chondrocytes with PEMFs increased the effect of the A3AR agonist that was able to reduce the cAMP levels by 76% (p<0.01 vs Cl-IB-MECA in control conditions, Figure 6A). The A3AR antagonist MRS 1334 blocked the effect of Cl-IB-MECA in the presence or in the absence of PEMF exposure. Analogous results were obtained in hFOB 1.19 cells, suggesting the capability of PEMF exposure to increase the specific effects of A2A or A3AR agonists on cAMP production (Figure 6B).

Figure 6

Figure 6Modulation of cAMP production and cell proliferation in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts.

Chondrocyte and osteoblast cell proliferation represent an important issue in the cartilage and bone metabolism. For this reason we have evaluated the effect of A2A and A3AR agonists on T/C-28a2 and hFOB 1.19 cell proliferation in the absence or in the presence of PEMF exposure. CGS 21680 (100 nM) was able to significantly increase cell proliferation in both the cell lines examined (Figure 6C and D). This effect was potentiated by PEMF exposure that determined a further increase of [3H]-Thymidine incorporation of 45% and 36% in T/C-28a2 and hFOB 1.19 cells, respectively (p<0.01 vs CGS 21680 in control conditions). The use of the selective A2AAR antagonist SCH 442416 (1 µM) that completely abrogated the CGS 21680-induced proliferation increase in the absence or in the presence of PEMFs, confirmed that this effect was determined by A2AAR activation. The A3AR agonist Cl-IB-MECA did not influenced the proliferation rate of T/C-28a2 chondrocytes or hFOB 1.19 osteoblast neither in the absence nor in the presence of PEMF exposure (Figure 6C and D).

The anti-inflammatory effects of A2A and A3AR activation are enhanced by PEMF exposure

The A2A and A3AR agonists were both able to inhibit the IL-1β-stimulated release of pro-inflammatory cytokine IL-6 and IL-8 in T/C-28a2 and hFOB 1.19 cells. In particular, in T/C-28a2 chondrocytes the A2AAR agonist CGS 21680 (100 nM) elicited a reduction of IL-6 and IL-8 levels of 43% and 52%, respectively (Figure 7A and B). In the same cells, the A3AR agonist Cl-IB-MECA (100 nM) mediated an inhibition of IL-6 and IL-8 levels of 40% and 65%, respectively. Interestingly, the simultaneous treatment with A2A and A3AR agonists and PEMFs resulted in a major effect on the inhibition of these pro-inflammatory cytokines. As expected, the use of selective A2A and A3AR antagonists (SCH 442416 and MRS 1334, respectively) abrogated the effect of the agonists either in the absence or in the presence of PEMFs (Figure 7A and B).

Figure 7

Figure 7Inhibition of IL-6, IL-8, PGE2 and VEGF by A2A or A3AR agonists and PEMFs in T/C-28a2 chondrocytes.

In T/C-28a2 chondrocytes, the lipid mediator PGE2 levels stimulated by IL-1β were significantly reduced in the presence of CGS 21680 or Cl-IB-MECA and their inhibitory effects were potentiated by PEMF exposure that elicited a further reduction of 34% and 26%, respectively (p<0.05 vs CGS 21680 or Cl-IB-MECA in control condition, Figure 7C). We next investigated the release of VEGF in the same experimental conditions. PEMF exposure potentiated the inhibitory effect of A2A and A3AR activation on IL-1β-stimulated VEGF production in T/C-28a2 cells (Figure 7D). SCH 442416 and MRS 1334 were able to counteract the responses mediated by A2A and A3AR, respectively, suggesting the specific involvement of these receptor subtypes.

IL-6, IL-8 and PGE2 release was also investigated in hFOB 1.19 osteoblasts obtaining similar results to those found in T/C-28a2 chondrocytes (Figure 8A, B and C). To investigate if the co-treatment with both A2A and A3AR agonists determined an even further inhibition of IL-6 as an example of inflammatory mediator we incubated T/C-28a2 or hFOB 1.19 cells with CGS 21680 (100 nM) and Cl-IB-MECA (100 nM). The simultaneous treatment with the two agonists inhibited IL-1β-stimulated IL-6 release of 61% and 57% in T/C-28a2 and hFOB 1.19 cells, respectively, resulting in a higher inhibition than the single agonists.

Figure 8

Figure 8Modulation of IL-6, IL-8, PGE2 and OPG by A2A or A3AR agonists and PEMFs in hFOB 1.19 osteoblasts.

Then, in hFOB 1.19 cells we have examined the effect of A2A and A3AR agonists in the absence or in the presence of PEMFs on the production of OPG, a protein involved in bone metabolism. Neither CGS 21680 nor Cl-IB-MECA at the 100 nM concentration were able to modify the basal release of OPG. Interestingly, PEMF exposure significantly augmented the production of this protein eliciting an increase of 48% respect to basal conditions (p<0.01, Figure 8D). The release of osteoprotegerin following the treatment with CGS 21680 or Cl-IB-MECA in the presence of PEMFs was not different from that obtained with PEMF exposure alone suggesting an effect not related to the capability of PEMFs to modulate ARs signaling (Figure 8D).

Effect of A2A and A3AR agonists and PEMFs on NF-kB activation

The transcription factor NF-kB is a key regulator of inflammatory responses and plays a critical role also in cartilage and bone metabolism. For this reason we have evaluated the effect of CGS 21680 and Cl-IB-MECA in the absence or in the presence of PEMFs on NF-kB p65 subunit activation. Both A2A and A3AR activation resulted in the inhibition of NF-kB stimulated with IL-1β in T/C-28a2 chondrocytes (Figure 9A) and hFOB 1.19 osteoblasts (Figure 9B). This effect was potentiated by the presence of PEMFs that were able to further inhibit the NF-kB p65 subunit activation. In both cell line, the use of the selective antagonists SCH 442416 and MRS 1334 counteracted the effect of the A2A and A3AR agonists, respectively (Figure 9).

Figure 9

Figure 9Inhibition of NF-kB activation by A2A or A3AR agonists and PEMFs in T/C-28a2 and hFOB 1.19 cells.


In this study we demonstrated that PEMF exposure mediates a specific overexpression of A2A and A3AR in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. This effect was confirmed both at a transcriptional level, as shown by RT-PCR assays, as well as in the increase of protein expression, as revealed by Western blotting, immunofluorescence and saturation binding experiments. On the other hand, PEMF treatment did not affected A1 and A2BAR mRNA or protein expression. These data are consistent with those previously reported by our group showing that PEMF exposure are able to induce the overexpression of A2A and A3ARs in different cell types such as human neutrophils [28], [29], bovine chondrocytes and synoviocytes[32] and neural cancer cells [31]. Given the well-recognized anti-inflammatory effects of A2A and A3AR activation, the increase of their density following IL-1β stimulation could be interpreted as a compensatory mechanism to counteract excessive inflammation. This is in agreement with data previously reported showing the up-regulation of A2A and A3ARs in cells or tissues from patients affected by different inflammation-based pathologies such as chronic obstructive pulmonary disease and rheumatoid arthritis [20], [21], [23]. The further increase of A2A and A3AR density elicited by PEMFs could indicate a potentiation of this compensatory mechanism, suggesting the possibility to exploit the PEMF-induced A2A and A3AR upregulation to reduce the inflammatory status. Taken together, these results suggest that at least some of the effect elicited by PEMFs in biological systems could be attributed to the modulation of these AR subtypes. The mechanism by which PEMFs determined an up-regulation of A2A and A3ARs is not yet understood. Several evidence report that PEMF could act either at a membrane level or at a transcriptional level [15],[16], [28][33]. Nevertheless, further studied are necessary to elucidate the exact mode of action of PEMFs. To verify if the PEMF-induced up-regulation of A2A and A3ARs was accompanied by altered receptor functional responses, we have performed cAMP experiments before and after PEMF treatment. The capability of PEMFs to potentiate the typical responses of A2A and A3AR agonists on cAMP production suggested the synergistic use of biophysical stimulation to enhance the well-known anti-inflammatory effect of A2A and A3AR activation.

Articular cartilage may undergo repeated damage involving a degenerative process that includes focal and progressive cartilage loss [45]. Moreover, clinical observation has shown that trauma, cancer, osteoporosis and osteoarthritis can lead to loss of mechanical bone competence and to bone resorption [46]. The main issue in the treatment of these diseases is how to increase chondrocyte proliferation to promote cartilage repair or how to generate more osteoblasts to promote ossification and accelerate osteogenesis. Our data showed that the A2AAR agonist CGS 21680 had a positive effect on both chondrocytes and osteoblasts proliferation, and the simultaneous presence of PEMF exposure potentiated this proliferative action. Thus, the two agents combined could represent a potential alternative strategy for the treatment of pathological conditions characterized by an excessive cartilage degradation or bone resorption. Previous reports in the literature have documented that PEMF exposure may have a proliferative effect on both chondrocytes and osteoblast, although after a longer culture period [47][49]. This may suggest that the presence of the A2AAR agonist could accelerate this process. On the other hand, the A3AR agonist Cl-IB-MECA did not affected cell proliferation nor in the presence or in the absence of PEMF exposure.

Inflammatory mediators play crucial roles in cartilage degenerative conditions as well as in bone metabolism. In this study, we have evaluated the effect of A2A and A3AR stimulation in the absence or in the presence of PEMFs on pro-inflammatory cytokine release from T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. The release of inflammatory mediators were stimulated by using Il-1β, a pro-inflammatory cytokines that interacts with most cell type and is one of the most important mediator of the inflammatory response, especially in cartilage and bone pathologies. In both the cell line examined, CGS 21680 and Cl-IB-MECA were able to significantly decrease the IL-1β-stimulated production of the inflammatory mediators IL-6, IL-8 and PGE2. In T/C-28a2, a similar effect was observed on the production of VEGF, an important mediator of angiogenesis. The data are consistent with those previously found in chondrocytes and/or osteoblasts. In particular, it has been recently shown that A2AAR activation reduced inflammatory mediators in mouse articular chondrocytes stimulated with IL-1β [50]. Moreover, in the osteoblastic cell line MG-63, the treatment with adenosine inhibited IL-6 production via A2AAR activation [51]. The capability of both A2Aand A3AR agonists to mediate anti-inflammatory effect suggests the involvement of different downstream signaling pathways. As a matter of fact, in a previous work we demonstrated that the anti-inflammatory effects of A2AARs were mediated by the modulation of cAMP. In contrast, the inhibitory effect of A3AR activation on pro-inflammatory mediators was completely abrogated by using the PI3K inhibitor LY294002 but not by the Gi inactivator pertussis toxin [40]. Interestingly, the present study highlighted the capability of PEMF exposure to potentiate the anti-inflammatory effects mediated by A2A and A3AR agonists. Numerous evidence have suggested that PEMFs possess a potential anti-inflammatory effect [15][17], [52] and our new data indicate that the up-regulation of A2A and A3ARs could be considered one of the mechanism by which PEMFs exerted their effects (Figure 10).

Figure 10

Figure 10Proposed mechanism of anti-inflammatory effect of PEMFs through the up-regulation of A2A and A3ARs in T/C-28a2 and hFOB 1.19 cells.

Osteoprotegerin is a competitive protein for receptor activator of nuclear factor kappa-B ligand (RANKL) and has been shown to prevent bone resorption by blocking the binding of RANKL with the receptor RANK, thereby inhibiting osteoclast differentiation and activation [53]. Our results obtained from hFOB 1.19 osteoblasts revealed that, although A2A and A3AR stimulation had no effect on osteoprotegerin production, PEMF exposure resulted in a significant increased release of this bone protective factor. These data are consistent with various papers previously reported where PEMF exposure increased osteoprotegerin secretion and mRNA expression in osteoblast-like cells [54], [55]. Moreover, it has been shown that PEMFs stimulated osteoprotegerin in rats preventing ovariectomy-induced bone loss [56].

The activation of the transcription factor NF-kB is known to be central for the regulation of the synthesis and activity of inflammatory cytokines, including TNF-α and IL-1β, and also several other mediators involved in the pathogenesis of inflammatory joint and bone diseases [57]. In addition, NF-kB controls the differentiation or activity of the major skeletal cell types such as osteoclasts, osteoblasts, osteocytes and chondrocytes [58]. It is well-known that many of the anti-inflammatory effects of A2A and A3AR stimulation are mediated by the inhibition of NF-kB signaling pathway [59], [60]. In the present study, we provide evidence that A2Aand A3AR agonists were able to reduce the IL-1β-induced NF-kB p65 subunit activation also in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Interestingly, the simultaneous exposure of the cells with PEMFs enhanced this inhibitory effect, suggesting the potential utilization of PEMFs and A2A and A3AR agonists to reduce overactivation of NF-kB.

In conclusion, our data revealed that PEMFs mediated an up-regulation of A2A and A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. The PEMF-induced overexpression is accompanied by a potentiation of the typical anti-inflammatory responses elicited by A2A and A3AR activation. In addition, PEMFs regulated the production of the bone-protective factor osteoprotegerin from hFOB 1.19 cells. Taken together, these results suggest the potential for a combined use of two different approaches represented by pharmacological tools such as A2A and A3AR agonists and a biophysical stimuli such as PEMFs in order to modulate cartilage and bone activity, especially in inflammatory conditions.

Funding Statement

The authors have no support or funding to report.


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Urologiia. 2009 Nov-Dec;(6):17-21.

Nonpharmacological treatment of erectile dysfunction in obese patients.

[Article in Russian]

glybochko PV, Shaplygin LV, Ra?gorodski? IuM, Spirin PV, Alise?ko SV, Tverdokhleb SA.


An original combined treatment of obese patients with erectile dysfunction including transcranial magnetotherapy and transabdominal electrostimulation in the region of fat deposit for 6 months reduces body weight by up to 17% and elevates testosterone by up to 29%. Erectile function improved to normal in 31.8% patients. This method is pathogenetically sound in minimal use of medicines and low risk of cardiovascular disorders.

Adv Ther. 2002 Jan-Feb;19(1):53-60

Impulse magnetic-field therapy for erectile dysfunction: a double-blind, placebo-controlled trial.

Pelka R. Universitat der Bundeswehr Munchen, Neubiberg/Munich, Germany.

This double-blind, placebo-controlled study assessed the efficacy of 3 weeks of pulsing magnetic-field therapy for erectile dysfunction (ED). In the active-treatment group, all efficacy endpoints were significantly improved at study end (P < or = .01), with 80% reporting increases in intensity and duration of erection, frequency of genital warmth, and general well-being. Only 30% of the placebo group noted some improvement in their sexual activity; 70% had no change. No side effects were reported.

Vopr Kurortol Fizioter Lech Fiz Kult. 1999 Mar-Apr;(2):25-7.

Physical factors in the treatment and rehabilitation of patients with chronic prostatitis complicated by impotence.

[Article in Russian]

Karpukhin IV, Bogomol’nyi VA.

103 patients with chronic prostatitis complicated by erectile impotence were given combined treatment including shock-wave massage, mud applications, local vacuum magnetotherapy. This combination was found to stimulate copulative function, urodynamics of the lower urinary tracts, to produce an antiinflammatory effect. These benefits allow to recommend the above physical factors for management of chronic prostatitis patients with copulative dysfunction.

Vopr Kurortol Fizioter Lech Fiz Kult. 1997 Jul-Aug;(4):24-5.

The sequential use of local vacuum magnetotherapy and papaverine electrophoresis with sinusoidal modulated currents in impotence.

[Article in Russian]

Karpukhin IV, Bogomol’nyi VA.

105 patients with chronic nonspecific prostatitis were examined and treated with papaverin electrophoresis using sinusoidal modulated currents (SMC) and local vacuum magnetotherapy (LVMT). Papaverin SMC electrophoresis and LVMT stimulated cavernous circulation. The highest stimulation was achieved at successive use of LVMT and the electrophoresis. LVMT followed by the electrophoresis maintained good cavernous circulation for 5-6 hours after the procedure in the course of which several spontaneous erections were observed.

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

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

Sandyk R.

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

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

Lik Sprava. 1995 Mar-Apr;(3-4):95-7.

The use of magnetic devices in treating sexual disorders in men.

[Article in Russian]

Gorpinchenko II.

An effect was studied of appliances for magnetotherapy, such as <<Biopotenzor>>, <<Eros>>, <<Bioskan-1>>, on sexual function of 105 men presenting with sexual problems. A total of 96 sexological patients were examined according to a general programme, to study placebo-effect. The magnetic field beneficial effect was recordable in 70-80 % of the patients, that of placebo in 33 % men. It is suggested that augmentation of sexual activity is associated with an increase in cavernous bloodflow.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2001 Dec;18(4):658-60.

The development of clinical application of the rejuvenator and a study of its mechanism for the treatment of functional erectile dysfunction.

[Article in Chinese]

Meng Z, Zou Y, Luo E, Zou L.

Andrology Laboratory, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038.

In this paper, we describe the development and clinical application of the Rejuvenator and report the result of our study on its mechanism for the treatment of functional erectile dysfunction (FED). The Rejuvenator, which can be used both at home and in hospitals to treat patients with FED, was developed on the basis of our clinical practice in the light of the modern theory of traditional Chinese medicine and by integrating multiple techniques of engineering science. It works by means of the paraoral use of the special herbal medicine, electro-magnetic effects, thermal moxibustion and drug-ingression. 2250 patients with FED received the treatment. Using combined electro-neurophysiological techniques, pulsed ultrasound Doppler and microcomputer image-scanning, we further studied the mechanism of the Rejuvenator for the treatment of FED. The total effective rate was 92%. The clinical data and result of study indicate that the Rejuvenator for the patients with functional erectile dysfunction is a safe, effective and scientific new method.

Vopr Kurortol Fizioter Lech Fiz Kult. 1999 Mar-Apr;(2):25-7.

Physical factors in the treatment and rehabilitation of patients with chronic prostatitis complicated by impotence.

[Article in Russian]

Karpukhin IV, Bogomol’nyi VA.

103 patients with chronic prostatitis complicated by erectile impotence were given combined treatment including shock-wave massage, mud applications, local vacuum magnetotherapy. This combination was found to stimulate copulative function, urodynamics of the lower urinary tracts, to produce an antiinflammatory effect. These benefits allow to recommend the above physical factors for management of chronic prostatitis patients with copulative dysfunction.

Immune Effects

Vopr Kurortol Fizioter Lech.  2010 Sep-Oct;(5):30-3.

Analysis of parameters of reproductive tract mucosal immunity in women with chlamydial infection before and after local magnetolaserotherapy.

[Article in Russian]

Gizinger OA, Dolgushin II, Letiaeva OI.


The objective of the present study was to evaluate the influence of combined treatment with low-intensity laser radiation and magnetic field on neutrophil function in women presenting with Chlamydial infection. Dysfunction of neutrophil granulocytes in these patients was manifest in the first place as the decreased number of phagocytes and the low rate of phagocytosis. It was shown that the concentration of active oxygen species in neutrophils in the patients with Chlamydial infection was significantly smaller than in healthy women. The concurrent application of low-intensity laser radiation and a magnetic field not only stimulated phagocytosis but also increased intracellular production of active oxygen species especially under in vitro conditions. It is concluded that combined treatment with low-intensity laser radiation and magnetic field has beneficial effect on the parameters of mucosal immunity in the reproductive tract of women with Chlamydial infection.

Vopr Kurortol Fizioter Lech Fiz Kult. 2010 May-Jun;(3):32-5.

Correction of immune and mediator characteristics by low-frequency magnetotherapy in children who frequently fall ill.

[Article in Russian]

[No authors listed]


The objective of this work was to compare characteristics of clinical condition and immune status of children with repeated respiratory diseases of different clinical and nosological forms after standard treatment and magnetotherapy. It was shown that magnetotherapy produces well-apparent immunocorrective effects in children with the affected upper and lower respiratory tracts including patients with bronchial obstruction syndrome. Positive changes of both cellular and humoral immunity characteristics were documented coupled to the improvement of serum cortisol levels. Results of the study give reason to recommend inclusion of magneotherapy in the combined treatment of children with repeated respiratory diseases.

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

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

[Article in Russian]

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


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

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

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

[Article in Russian]

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


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

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

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

[Article in Russian]

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


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

Probl Tuberk Bolezn Legk. 2007;(4):8-10.

Impact of various millimeter-range electromagnetic radiation schedules on immunological parameters in patients with respiratory sarcoidosis.

[Article in Russian]

Borisov SB, Shpykov AS, Terent’eva NA.


The paper analyzes the impact of various millimeter-range electromagnetic radiation schedules on immunological parameters in 152 patients with new-onset respiratory sarcoidosis. It shows that the immunomodulatory effect of millimeter-range therapy depends on the treatment regimen chosen. There is evidence for the advantages of millimeter-range noise electromagnetic radiation.

Bioelectromagnetics. 2006 Sep;27(6):458-66.

Effect of cyclophosphamide and 61.22 GHz millimeter waves on T-cell, B-cell, and macrophage functions.

Makar VR, Logani MK, Bhanushali A, Alekseev SI, Ziskin MC.

Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.


The present study was undertaken to investigate whether millimeter waves (MMWs) at 61.22 GHz can modulate the effect of cyclophosphamide (CPA), an anti-cancer drug, on the immune functions of mice. During the exposure each mouse’s nose was placed in front of the center of the antenna aperture (1.5 x 1.5 cm) of MMW generator. The device produced 61.22 +/- 0.2 GHz wave radiation. Spatial peak Specific Absorption Rate (SAR) at the skin surface and spatial peak incident power density were measured as 885 +/- 100 W/kg and 31 +/- 5 mW/cm(2), respectively. Duration of the exposure was 30 min each day for 3 consecutive days. The maximum temperature elevation at the tip of the nose, measured at the end of 30 min, was 1 degrees C. CPA injection (100 mg/kg) was given intraperitoneally on the second day of exposure to MMWs. The animals were sacrificed 2, 5, and 7 days after CPA administration. MMW exposure caused upregulation in tumor necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages suppressed by CPA administration. MMWs also caused a significant increase in interferon-gamma (IFN-gamma) production by splenocytes and enhanced proliferative activity of T-cells. Conversely, no changes were observed in interleukin-10 (IL-10) level and B-cell proliferation. These results suggest that MMWs accelerate the recovery process selectively through a T-cell-mediated immune response.

Electromagn Biol Med. 2006;25(4):307-23.

Review of in vivo static and ELF electric fields studies performed at Gazi Biophysics Department.

Seyhan N, Güler G.

Department of Biophysics, Gazi University, Ankara, Turkey.


In vivo effects of Static Electric and ELF Magnetic and Electric fields have been carried out for more than 20 years in the Bioelectromagnetic Laboratory at the Biophysics Department of the Medical Faculty of Gazi University. In this article, the results of in vivo ELF Electric field studies are presented as a review. Static and 50 Hz ELF (Extremely Low Frequency) Electric (E) fields effects on free radical synthesis, antioxidant enzyme level, and collagen synthesis were analyzed on tissues of guinea pigs, such as brain, liver, lung, kidney, spleen, testis, and plasma. Animals were exposed to static and ELF electric fields with intensities ranging from 0.3 kV/m to 1.9 kV/m in vertical and horizontal directions. Exposure periods were 1, 3, 5, 7, and 10 days. Electric fields were generated from a specially designed parallel plate capacitor system. The results indicate that the effects of electric fields on the tissues studied depend significantly on the type and magnitude of electric field and exposure period.

Bioelectromagnetics. 2005 Jan;26(1):10-9.

Effect of millimeter waves on natural killer cell activation.

Makar VR, Logani MK, Bhanushali A, Kataoka M, Ziskin MC.

Richard J Fox Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, PA 19140, USA.


Millimeter wave therapy (MMWT) is being widely used for the treatment of many diseases in Russia and other East European countries. MMWT has been reported to reduce the toxic effects of chemotherapy on the immune system. The present study was undertaken to investigate whether millimeter waves (MMWs) can modulate the effect of cyclophosphamide (CPA), an anticancer drug, on natural killer (NK) cell activity. NK cells play an important role in the antitumor response. MMWs were produced with a Russian-made YAV-1 generator. The device produced modulated 42.2 +/- 0.2 GHz radiation through a 10 x 20 mm rectangular output horn. Mice, restrained in plastic tubes, were irradiated on the nasal area. Peak SAR at the skin surface and peak incident power density were measured as 622 +/- 100 W/kg and 31 +/- 5 mW/cm2, respectively. The maximum temperature elevation, measured at the end of 30 min, was 1 degrees C. The animals, restrained in plastic tubes, were irradiated on the nasal area. CPA injection (100 mg/kg) was given intraperitoneally on the second day of 3-days exposure to MMWs. All the irradiation procedures were performed in a blinded manner. NK cell activation and cytotoxicity were measured after 2, 5, and 7 days following CPA injection. Flow cytometry of NK cells showed that CPA treatment caused a marked enhancement in NK cell activation. The level of CD69 expression, which represents a functional triggering molecule on activated NK cells, was increased in the CPA group at all the time points tested as compared to untreated mice. However, the most enhancement in CD69 expression was observed on day 7. A significant increase in TNF-alpha level was also observed on day 7 following CPA administration. On the other hand, CPA caused a suppression of the cytolytic activity of NK cells. MMW irradiation of the CPA treated groups resulted in further enhancement of CD69 expression on NK cells, as well as in production of TNF-alpha. Furthermore, MMW irradiation restored CPA induced suppression of the cytolytic activity of NK cells. Our results show that MMW irradiation at 42.2 GHz can up-regulate NK cell functions.

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

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

[Article in Russian]

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


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

Bioelectromagnetics. 2003 Jul;24(5):356-65.

Effect of millimeter waves on cyclophosphamide induced suppression of T cell functions.

Makar V, Logani M, Szabo I, Ziskin M.

Richard J. Fox Center for Biomedical Physics, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.


The effects of low power electromagnetic millimeter waves (MWs) on T cell activation, proliferation, and effector functions were studied in BALB/c mice. These functions are important in T-lymphocyte mediated immune responses. The MW exposure characteristics were: frequency = 42.2 GHz; peak incident power density = 31 +/- 5 mW/cm(2), peak specific absorption rate (SAR) at the skin surface = 622 +/- 100 W/kg; duration 30 min daily for 3 days. MW treatment was applied to the nasal area. The mice were additionally treated with cyclophosphamide (CPA), 100 mg/kg, a commonly used immunosuppressant and anticancer drug. Four groups of animals were used in each experiment: naive control (Naive), CPA treated (CPA), CPA treated and sham exposed (CPA + Sham), and CPA treated and MW exposed (CPA + MW). MW irradiation of CPA treated mice significantly augmented the proliferation recovery process of T cells (splenocytes). A statistically significant difference (P <.05) between CPA and CPA + MW groups was observed when cells were stimulated with an antigen. On the other hand, no statistically significant difference between CPA and CPA-Sham groups was observed. Based on flow cytometry of CD4(+) and CD8(+) T cells, two major classes of T cells, we show that CD4(+) T cells play an important role in the proliferation recovery process. MW exposure restored the CD25 surface activation marker expression in CD4(+) T cells. We next examined the effector function of purified CD4(+) T cells by measuring their cytokine profile. No changes were observed after MW irradiation in interleukin-10 (IL-10) level, a Th2 type cytokine, while the level of interferon-gamma (IFN-gamma), a Th1 type cytokine was increased twofold. Our results indicate that MWs enhance the effector function of CD4(+) T cells preferentially, through initiating a Th1 type of immune response. This was further supported by our observation of a significant enhancement of tumor necrosis factor-alpha (TNF-alpha) production by peritoneal macrophage’s in CPA treated mice. The present study shows MWs ameliorate the immunosuppressive effects of CPA by augmenting the proliferation of splenocytes, and altering the activation and effector functions of CD4(+) T cells.

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

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

[Article in Russian]

Oga? VB, Novoselova EG, Fesenko EE.

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


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

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

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

[Article in Russian]

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

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


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

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

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

[Article in Russian]

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

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


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

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

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

[Article in Russian]

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

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


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

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

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

[Article in Russian]

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

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


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

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

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

[Article in Russian]

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

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


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

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

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

[Article in Russian]

Novoselova ET, Fesenko EE.


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

Vopr Kurortol Fizioter Lech Fiz Kult. 1998 Sep-Oct;(5):30-2.

The action of low-frequency alternating magnetic field on the indices of hemodynamics and temperature homeostasis in women.

[Article in Russian]

Gerasimov IG, Samokhina EV, Tedeeva TA.

Magnetic field in physiotherapeutic doses has no negative effects either on hemodynamics or thermoregulation in females. Magnetic field’s positive action on inflammation can be explained by stimulation of nonspecific resistance of immune system. Hemodynamic and thermoregulation parameters may serve for control and regulation of the exposure to magnetic field.

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

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

[Article in Russian]

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


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