Effect of Rehabilitation with Extremely Low Frequency Electromagnetic Field on Molecular Mechanism of Apoptosis in Post-Stroke Patients

Apoptosis in acute stroke is associated with a negative prognosis and is correlated with the severity of the neurological deficit. However, there is no evidence that indicates that, in the subacute phase of the stroke, the apoptosis process might activate neuroplasticity. Therefore, in this study, we investigated the effect of an extremely low frequency electromagnetic field (ELF-EMF) on the molecular mechanism of apoptosis, as used in the rehabilitation of post-stroke patients. Patients with moderate stroke severity (n = 48), 3–4 weeks after incident, were enrolled in the analysis and divided into ELF-EMF and non-ELF-EMF group. The rehabilitation program in both groups involves the following: kinesiotherapy—30 min; psychological therapy—15 min; and neurophysiological routines—60 min. Additionally, the ELF-EMF group was exposed to an ELF-EMF (40 Hz, 5 mT). In order to assess the apoptosis gene expression level, we measured the mRNA expression of BAX, BCL-2, CASP8, TNFα, and TP53. We found that ELF-EMF significantly increased the expression of BAX, CASP8, TNFα, and TP53, whereas the BCL-2 mRNA expression after ELF-EMF exposition remained at a comparable level in both groups. Thus, we suggest that increasing the expression of pro-apoptotic genes in post-stroke patients promotes the activation of signaling pathways involved in brain plasticity processes. However, further research is needed to clarify this process.


Introduction
The symptoms of brain damage caused by cerebral ischaemia are the consequence of massive cell death in the ischaemic area. Ischaemic cascades-successive biochemical changes leading to degradation of cell structures and membranes, and ultimately to brain cell death-activated after a few seconds of blockage of the cerebral blood flow [1]. Ischaemia causes cell death as a result of necrosis, apoptosis, and autophagy, the markers of which have been well documented [2][3][4][5].
the effect of ELF-EMF on the crucial mechanisms of apoptosis in order to search for the molecular basis of ELF-EMF activity.

Subjects Presentation and Blood Collection
Forty-eight patients with moderate stroke severity, 3-4 weeks after incident, were enrolled for the analysis. Twenty-six patients were recruited into the ELF-EMF study group and twenty two patients were recruited into the non-ELF-EMF control group. The NIHSS scores were 4.9 ± 3.1 and 5.4 ± 2.9 for the study group and control group, respectively; mean age was 48.8 ± 7.7 and 44.8 ± 8.0, respectively. Exclusion criteria included the following: chronic or significant acute inflammatory factors, dementia, haemorrhagic stroke, neurological illness other than stroke, and/or decreased consciousness in their medical pre-stroke history. Patients with metal and/or electronic implants were excluded from the study group. Neurorehabilitation with neurological and internal examination was conducted for four weeks at the Neurorehabilitation Ward of Dr K. Jonscher Municipal Medical Center in Lodz, Poland. This consisted of the following: kinesiotherapy-30 min; psychological therapy-15 min; and neurophysiological routines-60 min; as well as ELF-EMF treatment.
Blood samples were collected into citrate, phosphate, dextrose, adenine (CDPA1) containing tubes, before and after ten sessions of therapy, at the same time of day (between 07:00 and 09:00), under fasting conditions. They were frozen at −80 • C immediately upon collection. All blood samples were collected and stored using the same protocol. The study was conducted in accordance with the principles of the Helsinki Declaration. The Bioethics Committee of the Faculty of Biology and Environmental Protection of the University of Lodz, Poland, confirmed this study with Resolution No. 13/KBBN-UŁ/II/2016.

Setting and Treatment of ELF-EMF
Magnetronic MF10 generator (EiE Elektronika i Elektromedycyna, Otwock, Poland) was used to generate ELF-EMF with the following parameters: magnetic induction-5 mT, frequency-40 Hz, wave forms-rectangular, bipolar, time of session-30 min, and exposed area-pelvic girdle ( Figure 1). In this research, a coil applicator with five layers of 187 turns of 1.45 mm twin-parallel wires was used with the following parameters: length-270 mm and diameter-550 mm. ELF-EMF intensity inside the solenoid was nonequivalent. The induction coils of the generator were assembled horizontally, while apportionment of ELF-EMF was vertical. The established value of magnetic induction of 5 mT was located in the applicator geometrical center, whereas, with the proximity of the coil surface, the change of ELF-EMF intensity increased by around plus 1.4. The computer system controlled ELF-EMF itself. Post-stroke patients were exposed in 10 standard sessions, five times per week for two weeks. All subjects were placed in applicator, but only patients in the ELF-EMF group were exposed to ELF-EMF, and patients in the non-ELF-EMF group were given sham exposures. The sham exposure was obtained by setting, but not turning on the device.

Isolation of RNA
Reagent ® (Sigma-Aldrich, Saint Louis, MO, USA) was used for lysis of frozen samples, following which phase separation was effectuated. InviTrap Spin Universal RNA Mini Kit (Stratec Biomedical Systems, Birkenfeld, Germany) was used for purification of the RNA-containing aqueous phase. A Synergy HTX Multi-Mode Microplate Reader, equipped with a Take3 Micro-Volume Plate (BioTek Instruments, Inc., Winooski, VT, USA), was used to estimate, purify, and quantify the RNA.

Reverse Transcription
Diluted RNA samples (to 20 ng/µL) were transcribed onto cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA, USA). All procedures were conducted in accordance with the producents' protocol.

Real-Time PCR
Levels of expression of the investigated genes (BAX, BCL-2, CASP8, TNFα, and TP53) were obtained using TaqMan probes: Hs00180269_m1 for human BAX gene; Hs00608023_m1 for human BCL-2 gene; Hs01018151_m1 for human CASP8 gene; Hs00174128_m1 for human TNFα gene; Hs01034249_m1 for human TP53 gene, and Hs99999905_m1 for human GAPDH gene (endogenous control) (Life Technologies, Carlsbad, CA, USA). A TaqMan Universal Master Mix II (Life Technologies, Carlsbad, CA, USA) was used to perform the real-time PCRs, which were executed in a CFX96 real-time PCR system (BioRad Laboratories, Hercules, CA, USA). All steps were conducted according to the producers' recommendations. The equation 2-ΔCt (ΔCt = Ctstudied gene -CtGAPDH) was used to calculate the relative expressions of the analyzed genes.

Data Analysis
All assays were conducted twice and calculated as mean values. For all participants, the values of the expression before treatment were used as the output value (100%). Data from the assays

Isolation of RNA
Reagent ® (Sigma-Aldrich, Saint Louis, MO, USA) was used for lysis of frozen samples, following which phase separation was effectuated. InviTrap Spin Universal RNA Mini Kit (Stratec Biomedical Systems, Birkenfeld, Germany) was used for purification of the RNA-containing aqueous phase. A Synergy HTX Multi-Mode Microplate Reader, equipped with a Take3 Micro-Volume Plate (BioTek Instruments, Inc., Winooski, VT, USA), was used to estimate, purify, and quantify the RNA.

Reverse Transcription
Diluted RNA samples (to 20 ng/µL) were transcribed onto cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA, USA). All procedures were conducted in accordance with the producents' protocol.

Real-Time PCR
Levels of expression of the investigated genes (BAX, BCL-2, CASP8, TNFα, and TP53) were obtained using TaqMan probes: Hs00180269_m1 for human BAX gene; Hs00608023_m1 for human BCL-2 gene; Hs01018151_m1 for human CASP8 gene; Hs00174128_m1 for human TNFα gene; Hs01034249_m1 for human TP53 gene, and Hs99999905_m1 for human GAPDH gene (endogenous control) (Life Technologies, Carlsbad, CA, USA). A TaqMan Universal Master Mix II (Life Technologies, Carlsbad, CA, USA) was used to perform the real-time PCRs, which were executed in a CFX96 real-time PCR system (BioRad Laboratories, Hercules, CA, USA). All steps were conducted according to the producers' recommendations. The equation 2-∆Ct (∆Ct = Ctstudied gene -CtGAPDH) was used to calculate the relative expressions of the analyzed genes.

Data Analysis
All assays were conducted twice and calculated as mean values. For all participants, the values of the expression before treatment were used as the output value (100%). Data from the assays executed on these same participants after treatment were formulated as a percentage of the output value. Data obtained in this way were expressed as mean ± SD.
Stats Direct statistical software v.2.7.2 was used for all statistical analyses. Paired Student's t-tests (for normal distribution) and Wilcoxon tests (for distribution deviating from normal) were used to analyze significant differences between the data obtained for subjects before and after treatments. Changes in parameters after appropriate treatments for comparison of differences between the ELF-EMF group and the non-ELF-EMF group were calculated. For this analysis, unpaired Student's t-tests or Mann-Whitney U tests were used. Additionally, a correlation analysis between the changes in experimental and clinical parameters was executed using a Spearman's rank correlation. A level of p < 0.05 was accepted as statistically significant for all analyses.

Results
Our comparative analysis shows the effect of ELF-EMF therapy on the expression level of various genes involved in apoptosis. As presented in Figure 2, the expression of the BAX gene in the ELF-EMF group after 10 sessions of rehabilitation was significantly higher as compared with the non-ELF-EMF group (p < 0.001). The increase of the BAX mRNA gene expression level in the ELF-EMF group was about 100% (p < 0.001), while in the non-ELF-EMF group, it did not change (p > 0.05) ( Figure 2). Brain Sci. 2020, 10, x FOR PEER REVIEW 5 of 12 executed on these same participants after treatment were formulated as a percentage of the output value. Data obtained in this way were expressed as mean ± SD. Stats Direct statistical software v.2.7.2 was used for all statistical analyses. Paired Student's ttests (for normal distribution) and Wilcoxon tests (for distribution deviating from normal) were used to analyze significant differences between the data obtained for subjects before and after treatments. Changes in parameters after appropriate treatments for comparison of differences between the ELF-EMF group and the non-ELF-EMF group were calculated. For this analysis, unpaired Student's t-tests or Mann-Whitney U tests were used. Additionally, a correlation analysis between the changes in experimental and clinical parameters was executed using a Spearman's rank correlation. A level of p < 0.05 was accepted as statistically significant for all analyses.

Results
Our comparative analysis shows the effect of ELF-EMF therapy on the expression level of various genes involved in apoptosis. As presented in Figure 2, the expression of the BAX gene in the ELF-EMF group after 10 sessions of rehabilitation was significantly higher as compared with the non-ELF-EMF group (p < 0.001). The increase of the BAX mRNA gene expression level in the ELF-EMF group was about 100% (p < 0.001), while in the non-ELF-EMF group, it did not change (p > 0.05) ( Figure 2). Results formulated as parameter changes before and after therapy (100% expressed for the level of BAX mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.0001.
We also found that the expression of the BCL-2 gene after a series of 10 physical treatments remained at a comparable level in both groups ( Figure 3). Subsequently, we estimated the impact of the ELF-EMF on CASP8 gene expressions in the whole blood samples. We showed that, after ELF-EMF therapy, the expression of CASP8 mRNA in the ELF-EMF group increased by about 50% (p < 0.01), but in the non-ELF-EMF group, it remained at the same level (p > 0.05) (Figure 4). Similarly, as shown in Figure 5, the expression level of TNFα was increased in the ELF-EMF group by about 50% (p < 0.001), whereas in the non-ELF-EMF group, it remained at the same level (p > 0.05). Comparison of the BAX mRNA expression acquired from the study group vs. control. Results formulated as parameter changes before and after therapy (100% expressed for the level of BAX mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.0001.
We also found that the expression of the BCL-2 gene after a series of 10 physical treatments remained at a comparable level in both groups ( Figure 3). Subsequently, we estimated the impact of the ELF-EMF on CASP8 gene expressions in the whole blood samples. We showed that, after ELF-EMF therapy, the expression of CASP8 mRNA in the ELF-EMF group increased by about 50% (p < 0.01), but in the non-ELF-EMF group, it remained at the same level (p > 0.05) (Figure 4). Similarly, as shown in Figure 5, the expression level of TNFα was increased in the ELF-EMF group by about 50% (p < 0.001), whereas in the non-ELF-EMF group, it remained at the same level (p > 0.05).
Brain Sci. 2020, 10, x FOR PEER REVIEW 6 of 12     Results formulated as parameter changes before and after therapy (100% expressed for the level of CASP8 mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.01.
Additionally, we examined the TP53 mRNA expression level. We demonstrated that, after rehabilitation, the mRNA expression of TP53 gene in the study group increased by about 100% (p < 0.001), while in the control group, it remained unchanged (p > 0.05) ( Figure 6).

Figure 5.
Comparison of the TNFα mRNA expression acquired from the study group vs. control group. Results formulated as parameter changes before and after therapy (100% expressed for the level of TNFα mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.0001.
Additionally, we examined the TP53 mRNA expression level. We demonstrated that, after rehabilitation, the mRNA expression of TP53 gene in the study group increased by about 100% (p < 0.001), while in the control group, it remained unchanged (p > 0.05) ( Figure 6). Figure 6. Comparison of the TP53 mRNA expression acquired from the ELF-EMF group vs. the non-ELF-EMF group. Results formulated as parameter changes before and after therapy (100% expressed for the level of TP53 mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.0001. Results formulated as parameter changes before and after therapy (100% expressed for the level of TNFα mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.0001. Figure 5. Comparison of the TNFα mRNA expression acquired from the study group vs. control group. Results formulated as parameter changes before and after therapy (100% expressed for the level of TNFα mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.0001.
Additionally, we examined the TP53 mRNA expression level. We demonstrated that, after rehabilitation, the mRNA expression of TP53 gene in the study group increased by about 100% (p < 0.001), while in the control group, it remained unchanged (p > 0.05) ( Figure 6). Figure 6. Comparison of the TP53 mRNA expression acquired from the ELF-EMF group vs. the non-ELF-EMF group. Results formulated as parameter changes before and after therapy (100% expressed for the level of TP53 mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.0001. Figure 6. Comparison of the TP53 mRNA expression acquired from the ELF-EMF group vs. the non-ELF-EMF group. Results formulated as parameter changes before and after therapy (100% expressed for the level of TP53 mRNA expression in each patient sample before treatment). Statistical significance between ELF-EMF and non-ELF-EMF groups after 10 sessions amount to p < 0.0001.

Discussion
An increased apoptotic process in acute phase of stroke is proven, and associated with the severity of neurological deficit [26,27]. There is still little information about apoptosis in the subacute/chronic phase of stroke. Within several minutes after ischaemia, neuronal cells in the infract core died [28], however, in penumbra, neuronal cells died slowly within a couple of hours after the incident [29].
Deng et al. studied the dynamics of apoptotic processes in mice that had experienced an induced stroke. They showed that markers of apoptosis had grown 2-5 h after ischemic, peaking after 5 h. At that time, the stroke volume and neurological deficit expended slowly, implying that apoptosis activation might inhibit ampliation of the ischeamic core and promote neuronal survival. Moreover, after this time, apoptosis was normalized [30].
Apoptosis, as programmed cell death, aims to eliminate damaged cells and, therefore, also those resulting from hypoxia/reperfusion. Magavi et al. investigated the neurogenesis of the adult cerebral cortex. They observed that induced apoptosis promoted the formation of new neurons [12]. Similarly, Chen et al. found the activation of neurogenesis in mice with induced apoptosis in corticospinal motor neurons [11].
Our innovative research concerned ELF-EMF activity in vivo in post-stroke patients. Most of the available studies were conducted on cell lines or animal models. The results obtained from research carried out using other research models cannot be precisely related to human rehabilitation. Thus, we relate current results only to our previous research that showed that ELF-EMF therapy improved neuroregeneration processes at the molecular level. In our previous papers, we found that rehabilitation treatments with the use of ELF-EMF intensification processes of neuroplasticity in post-acute stroke patients [21][22][23]. We found that, after exposition to ELF-EMF, the brain-derived neurotrophic factor (BDNF) expression at both the mRNA and protein level was increased, as well as plasma cytokine levels-hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), stem cell factor (SCF), and interleukin 1β (IL-1β)-were elevated [22,25]. Moreover, we observed that ELF-EMF affected the synthesis of nitric oxide (NO), which participates in plasticity processes [23].
In this study, we demonstrated that ELF-EMF increased BAX mRNA expression in vivo in post-acute stroke patients (Figure 2). Our results concur with the research shown by Wang et al. [31]. They observed that 3 mT EMF promotes osteoclast apoptosis by up-regulation of RANK, NFATc1, TRAP, CTSK, BAX, and BAX/BCL-2 expression, thereby enhancing osteoclast formation and maturation [31]. On the other hand, Tenuzzo et al. [32] assessed the effect of exposition of 6 mT EMF on expression of BAX, p53, HSP70, and BCL-2 in human lymphocytes. They found that, after exposition, the BAX and p53 gene expression rose, while the hsp70 and BCL-2 gene expression decreased.
We observed that, after ELF-EMF rehabilitation, the mRNA expression of BCL-2 remained at a comparable level in both groups. Our results concur with a study shown by Ding et al. [33]. They estimated the effect of ELF-EMF on H 2 O 2 -induced apoptosis and necrosis in human leukaemia HL-60 cells. They observed that the level of BCL-2 in H 2 O 2 -treated cells was comparable to its level in cells treated with both H 2 O 2 and ELF-EMF. They also suggested that EFF-EMF itself cannot induce apoptosis and necrosis [33].
We demonstrated that the CASP8 and TNFα mRNA expression were elevated in the ELF-EMF group (Figure 4; Figure 5). Xie et al. estimated the impact of EMF therapy on chondrocyte morphology and apoptosis, and the expression of apoptosis-related proteins in rabbits with anterior cruciate ligament transection. They found after, exposition on ELF-EMF, the expression level of CASP8 was higher in comparison with the healthy group, but that there was an insignificant difference in comparison with the untreated group with anterior cruciate ligament transection. The EMF group was also characterized by better clinical parameters [34].
In our current study, we found that expression of TP53 mRNA increased by about 100% (Figure 6) in the ELF-EMF group. Vincenzi et al. [35] evaluated the impact of pulsed electromagnetic fields (PEMF) on p53 activation and the stimulation of A3 adenosine receptors in NF-kB, cytotoxicity, apoptosis, and cell proliferation. They found that PEMF treatment alone was not enough to affect regulation of p53 expression. However, the coinstantaneous treatment of tumour cells with PEMF and agonist of A(3) adenosine receptors (A3AR)-2-chloro-N 6-(3-iodobenzyl)adenosine-5 -N-methyl-uronamide (Cl-IB-MECA)-caused a further significant elevation of protein levels of p53, compared with Cl-IB-MECA alone [35]. Similarly, Ma et al. [36] evaluated the impact of ELF-EMF on the proliferation and differentiation of neural stem cells. They observed that, after exposure to ELF-EMF, cell cycle analyses do not significantly change in the studied group compared with the control. Additionally, they found no significant difference in the expression level of the p53 gene [36].
The potential mechanism of intensification of apoptosis processes by ELF-ELM could be related to its effect on calcium channels. The effect of ELF-EMF on Ca 2+ flux is well documented [37][38][39][40]. The increase of Ca 2+ ions is an alarm signal that may present differently according to the physiological details. It has been shown that apoptosis can be dependent on an increase in the level of mitochondrial calcium, which in turn induces the release of mitochondrial cytochrome c and activation of proteases [41]. In addition to intrinsic and extrinsic pathways of apoptosis, there are also reticular pathways. The reticulum pathway is caused by disturbance of the ion balance (especially Ca 2+ ions), and the accumulation of improperly folded or modified proteins in the cell. In the caspase-independent pathway, calpain plays an important role in apoptosis. Calpain is a cysteine protease activated by Ca 2+ ions. After stimulation, calcium is released from the endoplasmic reticulum and is bound with many factors, including with calpain [42].
Morabito et al. [43] evaluated the impact of ELF-EMF on morphology, proliferation, and differentiation in pheochromocytoma cells (PC12), as well as on the induction of oxidative stress dependent on Ca 2+ . Oxidative stress causes overproduction of reactive oxygen species (ROS), which leads to apoptotic cell death. Cell growth and viability were examined after ELF-EMF exposure (50 Hz, 01,1 mT) using morphological and colorimetric analysis. Assay immediately after exposure demonstrated an increased level of ROS and a decreased catalase (CAT) activity, without affecting the Ca 2+ level. On the other hand, seven days' exposure caused an increased CAT activity, which may suggest the cell adaptation on ELF-EMF. Furthermore, long-term exposure caused an intracellular Ca 2 level. Calcium activates voltage-gated (L-type) Ca 2 channels, which, via cell pathways (extracellular signal-regulated kinases, c-Jun N-terminal protein kinase/stress-activated protein kinase, and p38), could lead to the activation of gene expression regulating apoptosis, cell survival, and differentiation [43]. Our previous research is compatible with Morabito studies. We observed that, two weeks post-stroke, rehabilitation with ELF-EMF exposition caused an increase in antioxidant enzymes activity: CAT and superoxide dismutase (SOD) [24], as well as elevation of the expression of antioxidant enzymes genes: CAT, SOD (SOD1 and SOD2), and glutathione peroxidase (GPx1 and GPx4) [21]. Moreover, we demonstrated that 10 sessions of ELF-EMF therapy decreased the level of parameters of oxidative stress in patients after stroke [24].
What is particularly important in our previous works is that we reported that, after ELF-EMF treatment, the improvement of biochemical markers was accompanied by the enhancement of clinical parameters. ELF-EMF improves motor condition expressed in the ADL scale, as well as mental efficiency assessed in the MMSE and GDS scales [21][22][23][24][25]. Our research to date clearly shows that ELF-EMF significantly boosts the effectiveness of rehabilitation. In our current study, we established that ELF-EMF additionally increases the expression of pro-apoptotic genes. Enhancement of apoptosis in post-stroke rehabilitated patients can significantly contribute to improving repair processes and increasing neuroplasticity owing to the removal of redundant or damaged cells.
On the basis of all of our research, we suggest that increasing the expression of these genes in actively rehabilitated post-stroke patients promotes the induction and/or intensification of signaling pathways involved in brain plasticity. However, subsequent study is needed to elucidate the exact mechanism of this process, which can include a simultaneous action of a variety of repair systems. Nevertheless, we recommended that the inclusion of ELF-EMF therapy could intensify the efficacy of the treatment after stroke. Funding: This research received no external funding. This study was approved by the Bioethics Committee of the University of Lodz, Poland with Resolution No. 13/KBBN-UŁ/II/2016. All participants gave their written informed consent prior to participation. The study was executed according to the principles of the Helsinki Declaration.

Conflicts of Interest:
The authors declare no conflict of interest.