Journal of Rehabilitation Medicine 51-11 | Page 42

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N. Cichon et al.
ELF-EMF treatment (15 min) was performed using a Mag-
netronic MF10 generator (EiE Elektronika i Elektromedycyna,
Otwock, Poland) (Fig. 1), with specific parameters (magnetic
induction of frequency of 40 Hz, magnetic induction of 5 mT.
The pulses were applied using an AS-550 applicator (EiE
Elektronika i Elektromedycyna, Otwock, Poland), which has
the following properties: 550 mm diameter, 270 mm length,
and 5 layers of 187 turns of 1.45-mm twin-parallel wires. Both
groups were treated for the same time (15 min), although the
non-ELF-EMF group were only given sham exposures. Patients
with metal and/or electronic implants (pacemakers, etc.), were
excluded from the study group.
Blood samples were taken before and after a standard 10 ses-
sions of treatment (with an interval of 14 days). Samples were
collected into anticoagulant solution, containing citric acid,
sodium citrate, monobasic sodium phosphate, dextrose, and
adenine (CPDA1) (Sarstedt, Nümbrecht, Germany), between
7 am and 9 am, under fasting conditions. Immediately upon
collection, a portion of the sample was frozen at –80°C, for
evaluation of mRNA expression. For isolation of plasma, the
remaining samples were centrifuged (1,500 g, 15 min, 25°C).
All blood samples were stored using the same protocol.
The study protocol was approved by the ethics committee
of the Faculty of Biology and Environmental Protection of
University of Lodz, Poland (number 28/2015). All participants
provided written informed consent.
Cytokine level analysis in plasma
Measurements of interleukin 1β (IL-1β), interleukin 2 (IL-2),
interferon-γ (INF-γ) and transforming growth factor β (TGF-β)
in plasma samples were performed with Human IL-1β de-
velopment kit, Human IL-2 development kit, Human INFγ
development kit, and Human TGF-β development kit, respec-
tively (MABTECH, Cincinnati, OH, USA), according to the
manufacturer’s protocol. All measurements were made using
MaxiSorp plates (Nunc, Roskilde, Denmark). The absorbance
was measured at 450 nm.
Isolation of mRNA and reverse transcription
Isolation of RNA from frozen whole-blood samples was ex-
ecuted using TRI Reagent® (Sigma-Aldrich, Saint Louis, MO,
USA). InviTrap Spin Universal RNA Mini Kit (Stratec Biome-
dical Systems, Birkenfeld, Germany) was used for purification
of the RNA-containing aqueous phase. Estimation of purity and
quantity of isolated RNA was performed using a Synergy HTX
Multi-Mode Microplate Reader, equipped with a Take3 Micro-
Volume Plate (BioTek Instruments Inc., Winooski, VT, USA).
Then, RNA samples (diluted to 10 ng/µl) were transcribed onto
cDNA using a High-Capacity cDNA Reverse Transcription Kit
(Applied Biosystems™, Waltham, MA, USA). All steps were
performed in accordance with the manufacturers’ protocol.
Real-time PCR analysis of gene for IL-1β expression
Expression levels of the analysed gene were determined using
the following TaqMan probes: Hs01555410_m1 for human IL-
1β gene, and Hs99999905_m1 for human GAPDH gene as an
endogenous control (Life Technologies, Carlsbad, CA, USA).
Real-time PCRs were performed in a CFX96 Real-Time PCR
system (BioRad Laboratories, Hercules, CA, USA), with used
a TaqMan™ Gene Expression Master Mix (Life Technologies,
Carlsbad, CA, USA). All procedures were conducted according
to the manufacturers’ instructions. The calculation of relative
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expressions of the analysed gene was performed using the equa-
tion 2 –ΔCt , where ΔCt=Ct(target gene) – Ct(GAPDH).
Statistical analysis
For comparison of differences in obtained parameter changes
between the ELF-EMF group and non-ELF-EMF group changes
in these parameters we calculated after appropriate treatments.
For all subjects the values of parameters before treatments were
used as the output value (100%). The raw data are shown in
Table II. Data from experiments performed on these same sub-
jects after appropriate treatments were expressed as percentages
of output value. All results were expressed as means (standard
deviations (SD)). All statistical analyses were performed using
StatsDirect statistical software V. 2.7.2. The results were ana-
lysed for normal distribution with the Shapiro–Wilk test. The
significance of differences between the values obtained for
patients before and after treatments were analysed using a test
for normal distribution (paired Student’s t-test), whereas the
significance of differences between the ELF-EMF group and
non-ELF-EMF group was determined using unpaired Student’s
t-test or Mann–Whitney U test. For all analysis a level of p < 0.05
was considered statistically significant.
RESULTS
This comparative analysis demonstrated the effect
of ELF-EMF treatment on the expression of various
pro-inflammatory cytokines. The plasma level of
IL-1β in the ELF-EMF group after 10 sessions of
rehabilitation was significantly higher than in the
non-ELF-EMF group (p < 0.05). The increase in IL-
1β level in the ELF-EMF group was approximately
100% (p < 0.05), while in the non-ELF-EMF group it
remained at a comparable level (p > 0.05) (Fig. 2). The
effect of ELF-EMF on gene expression in the whole-
blood samples of IL-1β was also investigated. After
ELF-EMF treatment, the expression of IL-1β mRNA
increased approximately 70% (p < 0.001), while in the
non-ELF-EMF group it did not change (Fig. 2).
Moreover, after a standard series of 10 physical
treatments, the IL-2 plasma concentration in the ELF-
EMF group increased approximately 15% (p < 0.05),
but in the non-ELF-EMF group it remained unchanged
(p > 0.05) (Fig. 3). An increase in plasma concentra-
tion, almost identical in value, was measured during
Table II. Raw data for results obtained in this study (before
transformation to % values)
ELF-EMF group
Non-ELF-EMF group
Before
After
Before
After
treatment treatment treatment treatment
IL-1β level [µmol/ml]
IL-1β mRNA expression [2 -ΔCt ]
IL-2 level [µmol/ml]
IFN-γ level [µmol/ml]
TGF-β level [µmol/ml]
18.259 30.802 18.635 19.753
0.03545
73.204
363.918
199.822 0.0588
80.722
387.595
206.830 0.03317
67.909
348.101
204.451 0.03814
65.298
349.703
236.640
ELF-EMF: extremely low frequency electromagnetic field; INFγ: interferon γ;
IL-2: interleukin 2; IL-1β: interleukin 1β; TGF-β: transforming growth factor β.