I. Pirogov Russian National Research Medical University, Ministry of
Health of the Russian Federation, Moscow, Russia, email@example.com.
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.
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
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 .
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 . However, reperfusion may induce oxidative stress , inflammatory cell infiltration and calcium dysregulation . All these players contribute to the heart damage such as contraction and arrhythmias , generally named myocardial I/R injury. Recently, more and more evolving therapies have been put into use for I/R injury.
Pulsed electromagnetic field (PEMF) is the most widely
tested and investigated technique in the various forms of
electromagnetic stimulations for wound healing , alleviating traumatic pain and neuronal regeneration [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 , and could improve arrhythmia, hypertension and MI . The MI rats were exposed to active PEMF for 4 cycles per day (8 min/cycle, 30±3 Hz, 6 mT) after MI induction. In vitro,
PEMF induced the degree of human umbilical venous endothelial cells
tubulization and increased soluble pro-angiogenic factor secretion [VEGF
and nitric oxide (NO)] .
However, the role of PEMF in ischaemia and reperfusion diseases remains
largely unknown. Our study aimed to investigate the effects of PEMF
preconditioning on myocardial I/R injury and to investigate the involved
In our study, we verified the
cardioprotective effects of PEMF in myocardial I/R rats and the
anti-apoptotic effects of PEMF in neonatal rat cardiac ventricular
myocytes (NRCMs) subjected to hypoxia/reoxygenation (H/R). We
hypothesized that PEMF treatment could alleviate myocardial I/R injury
through elevating the protein expression of B-cell lymphoma 2 (Bcl-2),
phosphorylation of protein kinase B (Akt). Meanwhile, it could decrease
Bax. We emphatically made an effort to investigate the MI/R model and
tried to uncover the underlying mechanisms.
MATERIALS AND METHODS
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
The measurement of blood pressure in SHR rats
At the end of 1 week treatment with
PEMF, the rats were anesthetized with chloral hydrate (350 mg/kg, i.p.),
the right common carotid artery (CCA) was cannulated with polyethylene
tubing for recording of the left ventricle pressures (MFlab 200, AMP
20130830, Image analysis system of physiology and pathology of Fudan
University, Shanghai, China).
Myocardial I/R injury rat model and measurement of infarct size
All the rats were divided into
three groups: (1) Sham: The silk was put under the left anterior
descending (LAD) without ligation; (2) I/R: Hearts were subjected to
ischaemia for 45 min and then reperfusion for 4 h; (3) I/R + PEMF: PEMF
device was provided by Biomobie Regenerative Medicine Technology. The
I/R rats were pre-exposed to active PEMF for 2 cycles per day (8 min per
cycle), whereas other two groups were housed with inactive PEMF
generator. I/R was performed by temporary ligation of the LAD coronary
artery for 45 min through an incision in the fourth intercostal space
under anaesthesia .
Then, the ligature was removed after 45 min of ischaemia, and the
myocardium was reperfused for 4 h. Ischaemia and reperfusion were
confirmed and monitored by electrocardiogram (ECG) observation. The
suture was then tightened again, and rats were intravenously injected
with 2% Evans Blue (Sigma–Aldrich). After explantation of the hearts,
the left ventricles were isolated, divided into 1 mm slices, and
subsequently incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC;
Sigma–Aldrich) in 0.9% saline at 37°C for 25 min, to distinguish
infarcted tissue from viable myocardium. These slices were flushed with
saline and then fixed in 10% paraformaldehyde in PBS (pH 7.4) for 2 h.
Next, the slices were placed on a glass slice and photographed by
digital camera, the ImageJ software (NIH) was used in a blind fashion
for analysis. Infarct size was expressed as a ratio of the infarct area
and the area at risk .
Pulsed electromagnetic field treatment
PEMF were generated by a
commercially available healing device (length × width × height: 7 cm ×
5cm × 3cm) purchased from Biomoble Regenerative Medicine Technology. The
adapter input voltage parameter is approximately 100–240 V and output
parameter is 5 V. Fields were asymmetric and consisted of 4.5 ms pulses
at 30±3 Hz, with an adjustable magnetic field strength range (X-axis 0.22±0.05 mT, Y-axis 0.20±0.05 mT, Z-axis
0.06±0.02 mT). The I/R rats were housed in custom designed cages and
exposed to active PEMF for 2 cycles per time (8 min for 1 cycle),
whereas the I/R rats were housed in identical cages with inactive PEMF
generator. For in vitro study, culture dishes were directly exposed to PEMF for 1–2 cycles as indicated (8 min for 1 cycle, 30 Hz, X-axis 0.22 mT, Y-axis 0.20 mT, Z-axis 0.06 mT) . The background magnetic field in the room area of exposure animals/samples and controls is 0 mT.
Detection of myocardium apoptosis
transferase-mediated dUTP nick-end labelling (TUNEL) assay was applied
to analyse cardiomyocyte apoptosis. Heart samples were first fixed in
10% formalin and then paraffin embedded at day 14. Then, the hearts were
cut into 5 ?m sections. TUNEL staining was carried out as described
previously . When apoptosis occurred, cells would look green.
Determination of myocardial enzymes in plasma
Blood samples were collected after haemodynamic measurement and centrifuged at 3000 g for
15 min to get the plasma. Creatine kinase (CK), lactate dehydrogenase
(LDH), creatine kinase isoenzyme-MB (CKMB) and ?-hydroxybutyrate
dehydrogenase (HBDH) were quantified by automatic biochemical analyzer
(Cobas 6000, Roche). All procedures were performed according to the
Myocardium cells morphology via TEM
At the end of the experiment,
sections from myocardial samples of left ventricular were immediately
fixed overnight in glutaraldehyde solution at 4°C and then incubated
while protected from light in 1% osmium tetroxide for 2 h. After washing
with distilled water for three times (5 min each), specimens were
incubated in 2% uranyl acetate for 2 h at room temperature and then
dehydrated in graded ethanol concentrations. Finally, sections were
embedded in molds with fresh resin. The changes in morphology and
ultrastructure of the myocardial tissues were observed and photographed
under a TEM .
Scal-1+/flk-1+ cells counting of endothelial progenitor cells
We applied antibodies to the stem
cell antigen-1 (Sca-1) and fetal liver kinase-1 (flk-1) to sign
endothelial progenitor cells (EPCs) as described before, and used the
isotype specific conjugated anti-IgG as a negative control. The amount
of Scal-1+/flk-1+ cells would be counted by flow cytometry technique .
Measurement of nitric oxide concentration and Western blotting
Plasma concentrations of NO were
measured with Griess assay kit (Beyotime Institute of Biotechnology)
according to the manufacturer’s protocol. The expressions of Bax, Bcl-2,
p-Akt, Akt, p-endothelial nitric oxide synthase (eNOS), eNOS and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed using
Western blot as described recently .
Proteins were measured with Pierce BCA Protein Assay Kit (Thermo).
Hippocampal protein lysates (50 mg/well) were separated using (SDS/PAGE)
under reducing conditions. Following electrophoresis, the separated
proteins were transferred to a PVDF membrane (Millipore). Subsequently,
non-specific proteins were blocked using blocking buffer (5% skim milk
or 5% BSA in T-TBS containing 0.05% Tween 20), followed by overnight
incubation with primary rabbit anti-rat antibodies specific for target
proteins as mentioned before (Cell Signaling Technology) at 4°C. Blots
were rinsed three times (5 min each) with T-TBS and incubated with
horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000,
Proteintech) for 2 h at room temperature. The blots were visualized by
using enhanced chemiluminescence (ECL) method (Thermo). GAPDH was
applied to be the internal control protein. Intensity of the tested
protein bands was quantified by densitometry.
Primary neonatal rat cardiac ventricular myocytes (NRCMs) were collected as previously described .
Briefly, the ventricles of new born SD rats (1–3 days old) were minced
and digested with 0.125% trypsin. Isolated cardiomyocytes were cultured
in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12, Life Technologies)
supplemented with 10% (v/v) FBS (Life Technologies), 100 units/ml
penicillin and 100 mg/ml streptomycin. The following experiments used
spontaneously beating cardiomyocytes 48–72 hours after plating. (37°C
with 5% CO2).
Cell treatment (hypoxia/reoxygenation)
NRCMs were prepared according to the methods recently described .
To establish the H/R model, the cells were cultured in DMEM/F-12
without glucose and serum. The cells were exposed to hypoxia (99% N2+5% CO2)
for 8 h, followed by reoxygenation for 16 h. The cells were pretreated
with PEMF for 30 min before the H/R procedure. The control group was
cultured in DMEM/F-12 with low glucose (1000 mg/l) and 2% serum under
normoxic air conditions for the corresponding times.
Cell viability assays
The viability of NRCMs cultured in
96-well plates was measured by using the Cell Counting Kit-8 (CCK-8)
(Dojindo Molecular Technologies) according to the manufacturer’s
instructions. The absorbance of CCK-8 was obtained with a microplate
reader at 450 nm.
Measurement of intracellular reactive oxygen species levels
Reactive oxygen species (ROS)
levels in NRVMs were determined by dihydroethidium (DHE, Sigma–Aldrich)
fluorescence using confocal microscopy (Zeiss, LSM 710). After different
treatments, cells were washed with D-PBS and incubated with DHE
(10 ?mol/l) at 37°C for 30 min in the dark. Then, residual DHE was
removed by PBS-washing. Fluorescent signals were observed (excitation,
488 nm; emission, 610 nm) under a laser confocal microscope (Zeiss).
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
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 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 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 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 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 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 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 expression…Go 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
And some researchers had found that PEMF therapy (8 min/cycle, 30±3 Hz,
6 mT) could improve the myocardial infarct by activating VEGF–Enos  system and promoting EPCs mobilized to the ischaemic myocardium [1,19].
Consistent with the previous work, our present study demonstrated that
PEMF therapy could significantly alleviate cardiac dysfunction in I/R
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 [22–24].
Myocardial apoptosis plays a significant role in the pathogenesis of
myocardial I/R injury. We assumed that PEMF might play its role in
improving cardiac function through inhibiting cell apoptosis. The Bcl-2
family is a group of important apoptosis-regulating proteins that is
expressed on the mitochondrial outer membrane, endoplasmic reticulum
membrane and nuclear membrane. Overexpression of Bcl-2 proteins blocks
the pro-apoptosis signal transduction pathway, thereby preventing
apoptosis caused by the caspase cascade .
The role Bax plays in autophagy is a debatable. Recently, new genetic
and biochemical evidence suggest that Bcl-2/Bcl-xL may affect apoptosis
through its inhibition of Bax .
Overexpression of Bax protein promotes the apoptosis signal pathway. In
the present study, we applied TUNEL staining to find that PEMF has a
perfect effect on cardiac cell apoptosis by regulating apoptosis-related
proteins Bcl-2 and Bax [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 .
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
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 . NO could also prevent platelet activation and promote vascular smooth muscle cells proliferation . NO generation from eNOS is considered to be endothelium-derived relaxing and ROS-related factor [36,37]. Some researchers found that bradykinin limited MI induced by I/R injury via Akt/eNOS signalling pathway in mouse heart .
And bradykinin inhibited oxidative stress-induced cardiomyocytes
senescence by acting through BK B2 receptor induced NO release .
Such evidence indicated that Akt phosphorylation could activate eNOS,
which lead to NO releasing, and resulted in ROS reducing. In the present
study, we found that PEMF decreased ROS via Akt/eNOS pathway.
In conclusion, this is the first study
suggesting that PEMF treatment could improve cardiac dysfunction through
inhibiting cell apoptosis. Furthermore, in vitro, we first
clarified PEMF still plays a profound effect on improving cell death and
removing excess ROS via regulating apoptosis-related proteins and
Akt/eNOS pathway. All these findings highlight that PEMF would be
applied as a potentially powerful therapy for I/R injury cure.
We thank all of the members of the Laboratory of Pharmacology of Chen Y., Ding Y.J. for their technical assistance.
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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Electric polarization and the viability of living systems: ion cyclotron resonance-like interactions.
Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, Florida 33431, USA. firstname.lastname@example.org
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.
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
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.
Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, Florida, USA. email@example.com
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.
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.
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
Physical mechanisms in neuroelectromagnetic therapies.
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.
Electric-field ion cyclotron resonance.
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.
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.  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
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.
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
AACN Clin Issues. 2004 Jul-Sep;15(3):391-403.
Electromagnetic interference in cardiac rhythm management devices.
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
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.
Research Center for Bioelectromagnetic Interaction (femu), University
Hospital of Aachen University, Pauwelsstrasse 20, 52074 Aachen,
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.
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.
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.
Frank Reidy Research Center for Bioelectrics, Old Dominion
University, Norfolk Virginia, 4211 Monarch Way, Norfolk, Virginia 23508,
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
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. firstname.lastname@example.org
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
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
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.
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
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,
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,
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. email@example.com
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.
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
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.
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
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
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. firstname.lastname@example.org
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
Technol Cancer Res Treat. 2002 Feb;1(1):71-82.
Enhancing the effectiveness of drug-based cancer therapy by electroporation (electropermeabilization).
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. email@example.com
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
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
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. firstname.lastname@example.org
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.
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.
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. email@example.com
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.
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. firstname.lastname@example.org
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
Clin Neurophysiol. 2000 Nov;111(11):1936-41.
Nocturnal magnetic field exposure: gender-specific effects on heart rate variability and sleep.
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.
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.
Human sleep under the influence of pulsed radiofrequency
electromagnetic fields: a polysomnographic study using standardized
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.
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
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
Nocturnal exposure to intermittent 60 Hz magnetic fields alters human cardiac rhythm.
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
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
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
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
Author information ? Article notes ? Copyright and License information ?
Received 2013 Mar 1; Accepted 2013 Apr 27.
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 .
In human joint damages, chondrocytes are able to respond to the
depletion of extracellular matrix and abnormal biomechanical functions
trying to preserve matrix integrity .
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 , .
On the other hand continuous remodeling by bone cells such as
osteoblasts and osteoclasts allows the skeleton to grow, adapt and
repair itself , .
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 .
The number of osteoblasts decreases with age, affecting the balance of
formation and resorption in the bone tissue, and potentially leading to
osteoporosis . Moreover, osteoblasts if adequately stimulated produce increased levels of pro-inflammatory cytokines .
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 .
Moreover, osteoblasts also produce a protein named osteoprotegerin
(OPG) that, preventing the biological effects of RANKL, plays an
important osteoprotective role .
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
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 , .
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
In addition, PEMFs stimulate proteoglycan synthesis without affecting
the degradation suggesting their potential use to preserve cartilage
integrity and function .
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 .
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) . A1 and A3ARs inhibit adenylate cyclase activity and decrease cAMP production whilst A2A and A2BARs exert an increase of cAMP accumulation .
Modulation of ARs has an important role in the regulation of
inflammatory processes suggesting their involvement in different
pathologies based on inflammation –. 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 , . Other studies reported that A2B or A2AAR
stimulation could be implicated in osteoblastic differentiation
revealing their involvement in bone formation and fracture repair , .
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 , . The treatment with PEMFs induced a selective increase in A2AARs expressed in rat cortex membranes and in rat cortical neurons . 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 . A role of ARs and PEMFs in modulating bovine chondrocytes and synoviocytes activity has been previously documented , . Functional studies in human synoviocytes have suggested an anti-inflammatory effect linked to A2AARs that is primarily based on the inhibition of PGE2 production .
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
Materials and Methods
Human immortalized chondrocyte cells (T/C-28a2) were kindly
provided by Professor Mary B. Goldring, from Cornell Medical College,
NY, USA , . 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 .
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 .
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–, 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
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 .
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 .
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 . 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 . Saturation binding experiments to A2BARs were performed using [3H]-N-benzo
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 . 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 .
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
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
[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH 442416, Sigma) or
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 .
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 .
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 , , .
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
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 .
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, .
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 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 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 3PEMF exposure up-regulated A2A and A3ARs in T/C-28a2 chondrocytes.
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
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 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 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 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 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,
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 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 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 , , bovine chondrocytes and synoviocytes and neural cancer cells . 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 , , . 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 ,, –.
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 .
Moreover, clinical observation has shown that trauma, cancer,
osteoporosis and osteoarthritis can lead to loss of mechanical bone
competence and to bone resorption .
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 –. 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 . Moreover, in the osteoblastic cell line MG-63, the treatment with adenosine inhibited IL-6 production via A2AAR activation . 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 .
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 –,  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 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 . 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 , . Moreover, it has been shown that PEMFs stimulated osteoprotegerin in rats preventing ovariectomy-induced bone loss .
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 .
In addition, NF-kB controls the differentiation or activity of the
major skeletal cell types such as osteoclasts, osteoblasts, osteocytes
and chondrocytes . 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 , . 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.
The authors have no support or funding to report.
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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
Adv Ther. 2002 Jan-Feb;19(1):53-60
Impulse magnetic-field therapy for erectile dysfunction: a double-blind, placebo-controlled trial.
Pelka R. Et.al. 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.
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.
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.
Department of Neuroscience at the Institute for Biomedical
Engineering and Rehabilitation Services, Touro College, Bay Shore, NY
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]
An effect was studied of appliances for magnetotherapy, such as
<<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
[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.
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.
Analysis of parameters of reproductive tract mucosal immunity in
women with chlamydial infection before and after local
[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
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.
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.
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.
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
[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
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.
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.
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.
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.
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
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
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.
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.
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
BMC Musculoskelet Disord. 2012 Jun 6;13(1):88. [Epub ahead of print]
I-ONE therapy in patients undergoing total knee arthroplasty: a prospective, randomized and controlled study.
Moretti B, Notarnicola A, Moretti L, Setti S, De Terlizzi F, Pesce V, Patella V.
Total knee arthroplasty (TKA) is often associated with a severe local
inflammatory reaction which, unless controlled, leads to persistent
pain up to one year after surgery. Standard and accelerated
rehabilitation protocols are currently being implemented after TKA, but
no consensus exists regarding the long-term effects. Biophysical
stimulation with pulsed electromagnetic fields (PEMFs) has been
demonstrated to exert an anti-inflammatory effect, to promote early
functional recovery and to maintain a positive long-term effect in
patients undergoing joint arthroscopy. The aim of this study was to
evaluate whether PEMFs can be used to limit the pain and enhance patient
recovery after TKA.
A prospective, randomized, controlled study in 30 patients undergoing
TKA was conducted. Patients were randomized into experimental PEMFs or a
control group. Patients in the experimental group were instructed to
use I-ONE stimulator 4hours/day for 60days. Postoperatively, all
patients received the same rehabilitation program. Treatment outcome was
assessed using the Knee Society Score, SF-36 Health-Survey and VAS.
Patients were evaluated pre-operatively and one, two, six and 12 months
after TKA. Joint swelling and Non Steroidal Anti Inflammatory Drug
(NSAID) consumption were recorded. Comparisons between the two groups
were carried out using a two-tail heteroschedastic Student’s t-test.
Analysis of variance for each individual subject during the study was
performed using ANOVA for multiple comparisons, applied on each group,
and a Dunnet post hoc test. A p value<0.05 was considered
Pre-operatively, no differences were observed between groups in terms
of age, sex, weight, height, Knee-Score, VAS, SF-36 and joint swelling,
with the exception of the Functional Score. The Knee-Score, SF-36 and
VAS demonstrated significantly positive outcomes in the I-ONE stimulated
group compared with the controls at follow-ups. In the I-ONE group,
NSAID use was reduced and joint swelling resolution was more rapid than
in controls. The effect of I-ONE therapy was maintained after use of the
device was discontinued.
The results of the study show early functional recovery in the I-ONE
group. I-ONE therapy should be considered after TKA to prevent the
inflammatory reaction elicited by surgery, for pain relief and to speed
Effects of biophysical stimulation in patients undergoing
arthroscopic reconstruction of anterior cruciate ligament: prospective,
randomized and double blind study.
Benazzo F, Zanon G, Pederzini L, Modonesi F, Cardile C, Falez F,
Ciolli L, La Cava F, Giannini S, Buda R, Setti S, Caruso G, Massari L.
IRCCS Foundation, Orthopaedic and Traumatology Department, S. Matteo Hospital Institute, University of Pavia, Pavia, Italy.
Pre-clinical studies have shown that treatment by pulsed
electromagnetic fields (PEMFs) can limit the catabolic effects of
pro-inflammatory cytokines on articular cartilage and favour the
anabolic activity of the chondrocytes. Anterior cruciate ligament (ACL)
reconstruction is usually performed by arthroscopic procedure that, even
if minimally invasive, may elicit an inflammatory joint reaction
detrimental to articular cartilage. In this study the effect of I-ONE
PEMFs treatment in patients undergoing ACL reconstruction was
investigated. The study end-points were (1) evaluation of patients’
functional recovery by International Knee Documentation Committee (IKDC)
Form; (2) use of non-steroidal anti-inflammatory drugs (NSAIDs),
necessary to control joint pain and inflammation. The study design was
prospective, randomized and double blind. Sixty-nine patients were
included in the study at baseline. Follow-up visits were scheduled at
30, 60 and 180 days, followed by 2-year follow-up interview. Patients
were evaluated by IKDC Form and were asked to report on the use of
NSAIDs. Patients were randomized to active or placebo treatments; active
device generated a magnetic field of 1.5 mT at 75 Hz. Patients were
instructed to use the stimulator (I-ONE) for 4 h per day for 60 days.
All patients underwent ACL reconstruction with use of quadruple
hamstrings semitendinosus and gracilis technique. At baseline there were
no differences in the IKDC scores between the two groups. At follow-up
visits the SF-36 Health Survey score showed a statistically significant
faster recovery in the group of patients treated with I-ONE stimulator
(P < 0.05). NSAIDs use was less frequent among active patients than
controls (P < 0.05). Joint swelling resolution and return to normal
range of motion occurred faster in the active treated group (P <
0.05) too. The 2-year follow-up did not shown statistically significant
difference between the two groups. Furthermore for longitudinal analysis
the generalized linear mixed effects model was applied to calculate the
group x time interaction coefficient; this interaction showed a
significant difference (P < 0.0001) between the active and placebo
groups for all investigated variables: SF-36 Health Survey, IKDC
Subjective Knee Evaluation and VAS. Twenty-nine patients (15 in the
active group; 14 in the placebo group) underwent both ACL reconstruction
and meniscectomy; when they were analysed separately the differences in
SF-36 Health Survey scores between the two groups were larger then what
observed in the whole study group (P < 0.05). The results of this
study show that patient’s functional recovery occurs earlier in the
active group. No side effects were observed and the treatment was well
tolerated. The use of I-ONE should always be considered after ACL
reconstruction, particularly in professional athletes, to shorten the
recovery time, to limit joint inflammatory reaction and its catabolic
effects on articular cartilage and ultimately for joint preservation.