Extremely low-frequency electric field suppresses not only induced stress response but also stress-related tissue damage in mice

Although extremely low-frequency electric fields (ELF-EF) have been utilised for therapeutic purposes, the biological effect and the underlying mechanism of ELF-EF have not been elucidated. Here, we developed a mouse model of immobilisation-induced increase in glucocorticoid (GC) to evaluate the effect of ELF-EF. Mice were exposed to 50-Hz 10 kV/m EF via a parallel plate electrode and immobilised as needed. The ELF-EF suppressed the immobilisation-induced increase in blood GC level. Here, the results of 32 tests using the model were pooled and analysed. The suppressive effect of ELF-EF on immobilisation-induced increase in GC was reproduced, and the GC level was slightly higher in the ELF-EF-treated mice than in the sham-controlled mice, a novel observation. The immobilisation-induced increase in lactate dehydrogenase, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase, markers of tissue damage, was suppressed by co-treatment with EF in the biochemical tests using the same plasma sample. In the metabolome analysis, the changes in corticosterones, leukotrienes, and hydroxyeicosatetraenoic acids, markers of inflammation, showed a pattern similar to that of the plasma GC level. Thus, ELF-EF suppresses the stress response that causes an increase in the GC level and slightly promotes GC production in the absence of stress. Moreover, the suppressive effect of ELF-EF on induced stress response might be involved in stress-induced tissue damage or inflammation in immobilised mice. Overall, the model and the data help explore the biological effect of ELF-EF and explain the stress-relieving effect of EF. They would be useful in determining the medical applications of EF in humans and animals.


Metabolome analysis.
A metabolome analysis was performed using pooled plasma from one experiment (Table 1, note d). The changes in corticosterones showed a pattern similar to those of GC, but the cholesterol level did not differ among the four treatments ( Fig. 4a-c). In addition, the changes in the plasma level of leukotrienes and hydroxyeicosatetraenoic acids (HETEs) (Fig. 4f,g), and progesterones (Fig. 4d,e) showed a pattern similar to those of GC.

Discussion
In the present study, we elucidated the potential application of ELF-EF in medical field by performing a pooled analysis of previously published results obtained in an immobilisation-induced stress mouse model. Stress response was induced by the immobilisation of mice in a centrifuge tube for 30 min. The plasma GC level in the immobilised mice, that is, the stress(+)/EF(−) group, was approximately threefold higher than that in the control group, suggesting that immobilisation activated the endocrine system of the pituitary-adrenocortical axis and/ or the sympathetic-adrenomedullary system 17,23,24 . www.nature.com/scientificreports/ The plasma GC level increased in male BALB/c mice subjected to both immobilisation stress and exposure to 50 Hz EF, applied via the upper electrode of a parallel plate electrode system, and the GC level was lower than that in the immobilisation-alone group. The suppressive effect of EF on the induced stress response was dependent on both intensity (kV/m) and exposure time 18 . Furthermore, the effect was considerable at 20 min after the initiation of immobilisation 19 and possibly depended on EF intensity distribution in the exposed regions of varied sizes on the body surface, even when the EF strength remained unchanged 21 . Similar effects were observed between EF of 50 and 60 Hz, which are the standard power frequencies in most regions, despite varied environmental brightness levels during EF exposure 21 , and sex and age differences in the mice 5 . As the suppressive effect of  www.nature.com/scientificreports/ ELF-EF on immobilisation-induced increase in the GC level was clearly reproduced, these results suggest the robustness of this animal model to assess the efficacy of biological mechanisms induced by EF exposure (Fig. 1). In contrast, the GC level in mice treated with ELF-EF alone was approximately 1.25-fold higher than that in the sham-controlled mice. This novel finding suggests that ELF-EF may act as a potential stressor. In a study by de www.nature.com/scientificreports/ Bruyn and de Jager 25 , the plasma level of corticosterone (41.9 ± 22.8 ng/mL) in 6-month-old male mice increased by approximately 3.26 times following exposure to an EF of 10 kV/m for 22 h 25 . Compared with the results of de Bruyn and de Jager, the magnitude of increase in the GC level in the current study ( Fig. 1) was considerably small. It has been reported that electric shock from a short circuit between a charged mouse and the exposure system could provoke a stress response in the animal 26 , but this phenomenon was not observed in our study. EF may not act as a stressor but instead may function as an activating stimulus, such as plasma GC level increase after waking up in the morning in humans, which follows a circadian rhythm triggered by light stimulus. Further research is necessary to determine the role of EF-alone treatment in GC level increase. The present study confirmed that 50 Hz EF of 10 kV/m can exert an anti-stress effect in mice. The results of this study suggest that the experimental approach adopted here, with respect to both technical and experimental design, may have a wider application in other studies on the biological effects of EF in different organisms. For example, a 0.1-mm thick polypropylene sheet placed between the lower electrode and the animal completely annihilated the suppressive effect of the EF on stress-induced GC level increase, and the effect reappeared as the sheet thickened 20 . Further development of the current method is necessary for extrapolation to humans.
Our experimental model had a few limitations. In all experiments, an EF intensity of 10 kV/m was selected based on a previous study that assessed EF intensities ranging from 2.5 to 200 kV/m, and 10 kV/m EF showed the highest anti-stress effect 18 . Another reason for selecting this range was EF of intensity greater than 50 kV/m generated vibration and/or noise 18 . The effects of vibrations and/or noise were difficult to distinguish from those of the EF, thereby deterring the exclusion of a possible artefact at higher intensities. Therefore, this issue should be addressed and resolved to ensure that higher EF intensities can be further investigated.
The activities of LDH, GOT, and GPT differed between the immobilisation-alone (stress(+)/EF(−)) and cotreatment (stress(+)/EF(+)) groups (Fig. 3a). In each case, the increase caused by immobilisation was suppressed by the EF, similar to its effect on GC. There was a significant correlation among these three indexes in all combinations (Fig. S1a). Generally, a common feature of these three indexes is an increase in their blood levels during tissue or organ injury with inflammation. In addition, it is well known that GC reduces inflammation [27][28][29][30][31] . These results suggest the effect of ELF-EF on the association between stress and tissue damage involving inflammatory events, but there were no correlations between the three enzymes and GC level (Fig. S1b). Further investigation is necessary to confirm whether inflammation induced by GC can be alleviated by a reduction in the GC level due to ELF-EF exposure.
The purpose of the metabolome analysis of the pooled plasma samples was to understand the GC-related pathways influenced by ELF-EF (Fig. 4). It was expected that the change trend of corticosterone might be similar to that of GC (Fig. 5b,c). The level of cholesterol, which is a source of corticosterone, did not change following immobilisation or EF exposure (Fig. 4a). In addition, ELF-EF did not affect the level of pregnenolone, a corticosterone precursor, although an increase was observed because of immobilisation (data not shown). These data suggest that ELF-EF does not inhibit the synthesis of corticosterone, but instead inhibits its release from the adrenal gland. A pattern similar to that of GC was observed for the female sex hormone progesterone (Fig. 4d,e). Progesterone is biosynthesised from cholesterol, and although it is important for the synthesis of corticosterone, the biological significance of the inhibition of progesterone synthesis must be evaluated in future studies. Furthermore, the levels of HETE and leukotrienes (Fig. 4f,g), which are indicators of inflammation 32,33 , increased owing to immobilisation. Their suppression by ELF-EF supports the possibility that the suppressive effect of ELF-EF on induced stress response may be involved in stress-induced inflammation. Because the metabolome analysis was conducted with one sample, analyses with more samples are necessary to confirm the trend. Nevertheless, further research is necessary to elucidate the hypothesis.

Conclusions
Our results indicated that ELF-EF suppresses the plasma GC level under stress and slightly increases the GC level in a steady state condition. In addition, the plasma level of enzymes, which indicate tissue damage, was suppressed by ELF-EF. Therefore, the suppressive effect of ELF-EF exposure on the induced stress response appears to be involved in stress-induced inflammation in immobilised mice. These results demonstrate the treatment potential of ELF-EF.
The suppressive effect of ELF-EF on stress-induced elevation in the GC level showed high reproducibility (sensitivity (low error)). The animal model can be effectively used in future research on the biological effects of ELF-EF, even if the effects are not associated with stress response. We found that the blood level of markers of tissue damage or inflammation were elevated similar to endogenous steroids during immobilization, and that ELF-EF exerted an inhibitory effect on some of these markers. As stress causes several diseases, the results of this study will increase the use of ELF-EF for preventive or curative treatment. In addition, an increase in the blood level of GC by treatment with ELF-EF alone, although slightly, suggests that the biological effect of ELF-EF should be carefully interpreted from the perspective of not only biohazard but also medical application.  Table 1) were pooled for each treatment (n = 1) and analysed.

Materials and methods
Pooled data. Table 1 shows the date and a summary of information, including mean, standard deviation, and sample number for each pooled experiment conducted between 2011 and 2019, and the sample that was used for each analysis in this study, indicated as a-d.
EF exposure system. The EF exposure system consisted of the following three major parts: a high-voltage transformer unit (maximum output voltage, 30 kV; Hakuju, Tokyo, Japan), a constant-voltage unit (CVFT1-200H; Tokyo Seiden, Tokyo, Japan) to avoid unexpected interferences from electrical noise originating from the commercial power supply, and an EF exposure cage [18][19][20][21][22] . The exposure system consisted of a cylindrical plastic cage (diameter, 200 mm; height, 100 mm) and two stainless steel electrodes (1,000 mm × 600 mm) that were placed above and under the cylindrical cage (Fig. 5a,b). The cylindrical cage had slits (length, 100 mm; width, 5 mm) throughout at intervals of 5 mm (Fig. 5b) to prevent smudges (from faeces or saliva) from disturbing the formation of a stable EF. To generate an EF of 10 kV/m in the cage, 50 Hz/1 kV was applied to the upper electrode, whereas the lower electrode was grounded. Separate cages and tubes were used for each animal; these were reused after being washed with a neutral detergent and completely dried. Using a digital thermometer placed on the lower electrode, the temperature was measured before EF exposure and at 10, 20, 30, 40, 50, and 60 min after EF exposure. The temperature inside the cage was 25 °C ± 3 °C during the period of EF or sham exposure. The temperature within the cylindrical cage did not change during either period. The humidity was maintained between 45 and 55%. Mice in the EF-treated groups were exposed to an EF of 10 kV/m for 60 min. To generate an EF of 10 kV/m in the cage, 50 Hz/1 kV was applied to the upper electrode, whereas the lower electrode was grounded. To measure www.nature.com/scientificreports/ field intensity and verify the system's operation, an optical fibre voltmeter, which measures EF intensity using the Pockels effect, and an electro-optic voltage sensor attached with a two-cored Bi 12 SiO 20 fibre (FOVM 03; Sumitomo Electric, Osaka, Japan) and a digital multimeter (Fluke 87; Fluke, Everett, WA) were used. The EF intensity was measured at 273 arbitrary points (21 × 13) on each cage floor. The 10 kV/m EF intensity applied to the cage had an error margin of ± 4% outside the exposure cage and ± 0.1% inside the cage. A portable alternating current magnetic field metre (TMM-1; Electric Power Engineering Systems, Kanagawa, Japan) was used to measure the magnetic field intensity in the area where each mouse was exposed to the EF. The magnetic field intensity was approximately 0.12 ± 0.04 mG when 50-Hz, 10 kV/m EF was generated in the space. Table 1, 8-week-old male BALB/c mice were purchased from Charles River Japan (Kanagawa, Japan) and maintained in a pathogen-free environment at 24 °C ± 1 °C and 50% ± 10% humidity with daily artificial illumination (12-:12-h light/dark cycle with lights on from 07:00 to 19:00 h). The mice had free access to standard laboratory chow (CE-2; CLEA, Tokyo, Japan) and water, except during EF exposure and immobilisation.

Animals. For all experiments shown in
To examine the effect of EF exposure, with or without immobilisation stress, on plasma GC level, male mice were divided into the following four groups (n = 8 each): control, stress(−)/EF(−); EF alone, stress(−)/EF(+); immobilisation alone, stress(+)/EF(−); and co-treatment, stress(+)/EF(+). Mice in the EF-treatment groups were exposed to the same EF (50 Hz, 10 kV/m for 60 min), and those in the co-treatment groups were immobilised during the second half (30 min) of the EF exposure period (Fig. 6). Mice in the control group were housed in the EF cage for 60 min without exposure to an EF (i.e., 0 V/m).
GC elevation as a stress response started immediately after animal immobilisation and peaked approximately 30 min later. When immobilisation was continued even after the peak in the GC level, the GC level showed a gradual decrease. Therefore, to use the changes in the GC level as an indicator of stress, we reasoned that the GC level should be measured 30 min after the start of the stress treatment.
All animal experiments were carried out in accordance with the Guiding Principles for the Care and Use of Research Animals of Obihiro University of Agriculture and Veterinary Medicine, Japan. The protocol was approved by the Committee on the Ethics of Animal Experiments of Obihiro University of Agriculture and Veterinary Medicine (Permit number 25-86, 25-155, 26-119, 27-132, 28-152, 29-164, 30-95, 19-162).

Measurement of plasma GC and haematological analysis.
Immediately after EF treatment (between 10:00 and 12:30 h), 800 µL of blood was collected from each mouse under 3% isoflurane anaesthesia (Mylan, Tokyo, Japan) and centrifuged at 1500×g for 10 min at 4 °C, and then the plasma was collected and stored at − 80 °C until use.
The stocked plasma from 19 tests was utilised for the pooled analysis of plasma GC (Test Nos. 14-32, Table 1). To derivatise GC for analysis, 200 μL of plasma was mixed with 900 µL of isooctane (2,2,4-trimethylpentane; Wako, Osaka, Japan) followed by vortexing and centrifugation at 380×g for 5 min at 24 °C. The upper layer was discarded, and then 900 µL of chloroform (Wako) was added to the lower layer. The sample was vortexed and centrifuged at 380×g for 5 min at 24 °C. The upper and white membranous layers were removed, and the lower layer was retained for analysis. Eight hundred microlitres of the lower layer was transferred to a new tube and mixed with 320 µL of solution containing 65% concentrated sulphuric acid and 35% ethanol (both from Wako), and then vortexed. The solution was incubated in dark for 3.5 h, and the fluorescence intensity of the sample was measured at 519 nm with excitation at 475 nm using a spectrofluorophotometer (RF-5300PC; Shimadzu, Kyoto, Japan). This method that utilises sulphuric acid-induced fluorescence of GC could be used to measure total GC 24 .

Metabolome analysis.
As 500 µL of plasma was necessary for metabolome analysis, the plasma samples collected from eight mice (Test No. 27 in Table 1) were pooled for each group. Five hundred microlitres of the pooled plasma sample was added to 1500 µL of 1% formic acid/acetonitrile containing an internal standard solution (Solution ID: H3304-1002; Human Metabolome Technologies, Inc., Tsuruoka, Japan) at 0 °C to inactivate enzymes. The solution was thoroughly mixed and centrifuged at 2300×g for 5 min at 4 °C. The supernatant was filtered using Hybrid SPE phospholipid 55,261-U (Supelco, Bellefonte, PA) to remove phospholipids. The filtrate was desiccated and dissolved in 100 µL of isopropanol/Milli-Q for liquid chromatography mass spectrometry. Metabolome analysis was carried out at a facility of Human Metabolome Technologies Inc. (Tsuruoka, Japan). Liquid chromatography time-of-flight mass spectrometry analysis was carried out using an Agilent LC System (Agilent 1200 series RRLC system SL) equipped with an Agilent 6230 Time-of-Flight mass spectrometer (Agilent Technologies, Waldbronn, Germany). The systems were controlled using Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies). The cationic and anionic compounds were analysed using an ODS column (2 mm × 50 mm, 2 μm) according to a previously described method 34 .
Statistical analysis. Normality of data distribution was evaluated using the normality test. The differences among the groups were evaluated using the one-way analysis of variance (non-parametric method) and those between two groups were evaluated using Dunn's multiple comparison test. The correlations and regressions Scientific Reports | (2020) 10:20930 | https://doi.org/10.1038/s41598-020-76106-1 www.nature.com/scientificreports/ were assessed using Pearson correlation analysis and slope regression analysis, respectively. Significance was defined at P < 0.05. All statistical analyses were conducted using Prism Version 8 (GraphPad Software, La Jolla, CA).

Figure 6.
Experimental design employed to assess the influence of immobilisation and electric field (EF). After an acclimation period of 2 weeks, mice aged 10 weeks were divided into the following groups (n = 8 per group): control, stress(−)/EF(−); EF alone, stress(−)/EF(+); immobilisation alone, stress(+)/EF(−); and co-treatment stress(+)/EF(+). Mice in the co-treatment group were exposed to EF for 60 min and were immobilised during the second half (30 min) of the EF exposure period. In the control group, mice were handled in an identical manner, except that the EF condition was 0 V/m, and they were not immobilised. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.