1. IntroductionRepeated immobilization stress is an easy and well-known method to induce chronic physical and emotional stress [1]. The psychological and physiological changes to repeated immobilization stress are initiated by activation of the hypothalamic–pituitary adrenal axis, and these results in the release of catecholamines and stress hormones such as corticotropin-releasing factor (CRF) [2,3]. The CRF system plays a key role in a diversity of behaviors accompanying stress, anxiety and depression [4,5,6]. We previously demonstrated increased CRF expression in the paraventricular nuclei of repeated restraint-stressed rats [6]. However, little is known about the specific interventions for stress-related disorders.Grounding, also called earthing, is a therapeutic technique that involves doing activities that “ground” or electrically reconnect us to the earth. This conductive contact of the human body with the surface of the earth can have intriguing benefits on our physiology. Recently, researchers have studied the physiological effects of grounding from a variety of perspectives [7,8]. For example, electrically conductive contact of the human body with the surface of the Earth (grounding or earthing) produces intriguing effects on physiology and health [9,10]. Chevalier et al. reported that grounding reduces pain and alters the numbers of circulating neutrophils and lymphocytes, and also affects various circulating chemical factors related to inflammation [9]. Some studies reported that environmental medicine generally influences environmental factors with a negative impact on human health [7,9]. Mounting evidence shows that the Earth’s negative potential can produce a stable internal bioelectrical environment for the normal functioning of all body systems [8,9,10]. It is known that electrons from antioxidant molecules normalize reactive oxygen species involved in immune, inflammatory and stress response. Therefore, it is possible that the influx of free electrons absorbed into the body through direct contact with the Earth normalize free radicals and may reduce stress vulnerability. However, no studies investigated the anti-stress effects or mechanism of earthing mat underlying stress responses. The main hypothesis of this study is that connecting the body to the earth through earthing mat may have anti-inflammatory and antioxidant effects and, therefore, exposure with earthing mat has an anti-stress efficacy in animal models of stress.

In this study, we aimed to investigate whether grounding in the rats could change stress-related anxiety and depressive behaviors and the production of corticotrophin-releasing factors in the brain region. To achieve this goal, anxiety and depressive-like behaviors were tested via an elevated plus maze (EPM), tail suspension test (TST) and forced swimming test (FST). Moreover, we further assessed the expression of c-Fos and corticotrophin-releasing factor (CRF) in the paraventricular nucleus of the hypothalamus (PVN).

4. Discussion

The present study showed that in the elevated plus maze (EPM), the Control group decreased spent time in the open arm compared to the Normal group. However, earthing mat groups significantly increased spent time in the open arm compared to the Control group. When resulted in the expression of the CRF, CRF immunoreactive neurons in the Control group were significantly increased compared to the Normal group. However, the expression of CRF immunoreactive neurons in the earthing mat groups was markedly decreased compared to the Control group. The expression of the c-Fos immunoreactive neurons in the seven-day earthing mat group trended to decrease more than in the Control group. However, there were no significant differences among the groups.

Immobilization stress is one of the main and potent sources of stress, inducing a strong hormonal and behavioral reaction [37]. The HPA axis is activated in rodents to a different degree when the stress is mild (e.g., mild handling, needle stick, time in elevated plus maze [38], common symptoms of stress-related behavior [39,40]. A decrease in general exploratory activity in an open arena after restraint stress has been previously described [41]. In the elevated plus-maze, most studies found a decrease in the percentage of open-arm entries and/or time spent in them [41,42]. Consistent with the previous study, the present result showed a decrease in time spent in the open arm after repeated stress. However, an increase in time spent in the open arms was shown in earthing mat groups. Our result also showed that the earthing mat decreased anxiety-like behavior compared to the Control group. Another pilot study showed that grounding improves measurements of mood within 1 h, suggesting a potentially positive effect on health [43,44,45,46,47,48]. Therefore, it can be assumed that earthing mat may be effective against stress-related anxiety.It is well known that repeated stress has an effect on the central nervous system (CNS)-neuroendocrine behavior. The immunohistochemical expression of c-Fos-like proteins in the nervous system is considered a marker of neuronal activation. The logic of this methodology is based on the demonstration that the expression of this protein is increased in neurons after stress exposure [49,50]. The expression of the c-Fos immunoreactive neurons in the seven-day earthing mat group trended to decrease more than in the Control group. However, there were no significant differences among the groups.Corticotropin-releasing factor (CRF) is a key component of stress responsivity, modulating related behaviors, including anxiety and reward. The primary stress response involves the activation of hypothalamic neurons producing CRF, an initial step in the cascade that leads to the synthesis and release of glucocorticoids [51,52,53,54,55,56,57]. Our data also showed the activation of CRF immunoreactive neurons in the PVN after repeated 14 days of restraint stress. However, earthing mat groups decreased the CRF-ir expression in the PVN compared to the Control group. Mounting evidence suggests that the Earth’s negative potential can create a stable internal bioelectrical environment for the normal functioning of all body systems [43,44,45,46]. Moreover, oscillations of the intensity of the Earth’s potential may be important for setting the biological clocks regulating diurnal body rhythms, such as cortisol secretion [51]. The results of the study were consistent with previous studies [18,51].Earthing means reconnecting the conductive body to the Earth’s natural electric charge. Earthing influences the basic bioelectrical function of the body. The present study showed that earthing influenced corticosterone secretion and stress-related behavioral changes such as anxiety and learned helplessness. These results suggest that connecting the whole body to the earth can free electrons and diurnal electrical rhythms to enter the body, setting the biological clocks for hormones that regulate stress responses, consistent with previous studies proving that Earthing stabilizes the physiology, reduces inflammation and pain and improves sleep [43,44,45,46,47,58,59]. Another study reported that the electrodynamics of red blood cells has revealed that earthing significantly reduces blood viscosity and cardiovascular disease [60].

Taken together, these data suggest that earthing mats may be helpful in stress management via the regulation of corticotrophinergic mechanisms. In light of such limitations, our finding provides preliminary evidence of the safety of earthing mats and their potential to decrease stress responses in an animal model. Moreover, we are planning to analyze stress-related neurotransmitter markers such as serotonin, dopamine and GABA synthesis and release in the future study.