INTRODUCTION
Trimethyltin (TMT) is a potent neurotoxic compound that induces severe damage to the central nervous system (CNS), particularly causing neuronal death and neuroinflammation in the hippocampus (Park et al., 2022). Due to these characteristics, TMT is widely used as a significant animal model to study neurodegenerative disorders, such as Alzheimer disease, as it induces oxidative stress, inflammatory responses, and neurodegeneration (Rostami et al., 2022). The neurotoxic mechanisms of TMT are closely associated with alterations in the expression of proinflammatory cytokines, which contribute to the acceleration of neuroinflammation and neuronal damage.
Interleukin (IL)-1β is a key mediator of the inflammatory response and is secreted by microglia and macrophages activated by TMT (Zeng et al., 2024). IL-1β amplifies inflammatory responses by inducing the activation of various immune cells at the site of inflammation and promotes neuronal death. The upregulation of IL-1β plays a critical role in TMT-induced neuroinflammation and neurotoxic damage, accelerating the progression of CNS injury (Corvino et al., 2013; Mendiola and Cardona, 2018). In contrast, IL-4 is an anti-inflammatory cytokine that regulates the activity of T cells and B cells, suppresses the production of proinflammatory cytokines, and modulates immune responses to exert neuroprotective effects (Gadani et al., 2012; Lazarski et al., 2013). IL-4 maintains the balance between Th1 and Th2 immune responses and suppresses inflammation, offering a mechanism to mitigate inflammatory neuronal damage induced by TMT.
Prostaglandin E2 (PGE2) is a crucial lipid mediator of inflammatory responses, produced via the cyclooxygenase (COX)-2 pathway, which promotes the expression of inflammatory cytokines and exacerbates neuronal damage (Lopez and Ballaz, 2020). In TMT-induced neurotoxicity models, PGE2 amplifies inflammatory responses by inducing the expression of proinflammatory cytokines such as IL-1β, further contributing to inflammation and neuronal injury.
Acetylcholinesterase (AChE) is an enzyme responsible for the breakdown of acetylcholine and plays a vital role in regulating neurotransmission in the CNS (Halder and Lal, 2021). TMT-induced neurotoxicity alters AChE activity, disrupting cholinergic neurotransmission and impairing memory and cognitive function (Ye et al., 2020). Increased AChE activity accelerates the degradation of acetylcholine, reducing neurotransmission efficiency, which is considered one of the mechanisms underlying cognitive dysfunction caused by TMT.
Grounding refers to direct skin contact with the Earth’s surface via bare feet or hands, or the use of grounding systems, and has recently gained attention as a method that offers antioxidant and anti-inflammatory effects (Oschman et al., 2015). Grounding is thought to allow free electrons from the Earth to enter the body, reducing oxidative stress and suppressing inflammation. Moreover, studies suggest that grounding positively influences immune system regulation and mitigates inflammatory responses.
This study aims to investigate the effects of an grounding mat on the expression of inflammatory and anti-inflammatory cytokines, such as IL-1β, IL-4, and PGE2, as well as on AChE activity in a TMT-induced neurotoxicity model. By doing so, this research seeks to evaluate the neuroprotective potential of grounding in mitigating neuroinflammation and neuronal damage caused by TMT.
MATERIALS AND METHODS
Materials
TMT was procured from Sigma-Aldrich (St. Louis, MO, USA). Rat IL-1β and IL-4 assay kits were obtained from Abcam (Cambridge, UK), the PGE2 assay kit was purchased from R&D Systems (Abingdon, UK), and the rat AChE assay kit was acquired from MyBioSource (San Diego, CA, USA).
Experimental animals
Seven-week-old Sprague-Dawley rats were maintained under controlled conditions, including a temperature of 20°C±2°C, humidity of 50%±10%, and a 12-hour light/dark cycle. Rats were divided into five groups: no grounding mat with saline (Nor), no grounding mat with TMT (Con), electric mat for 21 days with TMT (EM), grounding mat for 7 days with TMT (A-7D), and grounding mat for 21 days with TMT (A-21D). Following a one-week acclimatization period, the animals were exposed to an grounding mattress for 24 hours per day over a duration of 7 or 21 days, or to an electric mat for 24 hours per day for 21 days. Subsequent experimental procedures were performed after the designated exposure periods. These experiments were executed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, revised in 1996, and were approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHUAP[SE]-13-041).
Assessment of spatial cognition in the TMT-induced neurotoxicity model
Spatial cognition was evaluated using the Y-maze test, a method for assessing short-term memory-based spontaneous alternation behavior. The Y-maze apparatus consisted of three arms, each measuring 42 cm in length, 8 cm in width, and 21 cm in height, with an angle of 120° between each arm. The apparatus was constructed from black polyvinyl plastic. During the test, the arms were labeled A, B, and C, and a rat was carefully placed in one arm. The movement of the rat was recorded for 8 min, with entries considered valid only when the rat’s entire body, including the tail, entered an arm. Reentries into previously visited arms were also documented. Sequential entries into three different arms were awarded one point, while nonsequential entries did not receive points. Spontaneous alternation behavior was calculated as the number of alternation points divided by the total number of arm entries minus two, expressed as a probability.
Exposure to the grounding mattress
The efficacy of grounding was confirmed using a Walking Test Set EFM 023 (BGT UAC-110), which measured body potential. A reduction in potential from 229 V to 0 V upon contact with the grounding mattress indicated successful grounding. The rats were then exposed to the grounding mattress for specified durations as detailed above.
Measurement of cytokine expression in serum
On the final day of behavioral experiments, the rats were anesthetized with pentobarbital, and blood was collected via cardiac puncture. The blood samples were left at 4°C for 30 min and centrifuged at 3,000 rpm for 15 min (FLETA-5, Hanil Science Inc., Seoul, Korea) to isolate serum. The serum was stored at −70°C until analysis. Enzyme-linked immunosorbent assay (ELISA) was performed using kits for IL-1β and IL-4 (Abcam, Cambridge, UK) and PGE2 (R&D Systems). Absorbance was measured at 450 nm and 540 nm.
Measurement of AChE activity in brain tissue
Brain tissues were extracted, and 10 volumes of lysis buffer were added to the tissues, which were then homogenized using a glass homogenizer. The homogenates were centrifuged at 12,000 rpm for 30 min (FLETA-5, Hanil Science Inc., Seoul, Korea) at 4°C. The supernatants were used for enzyme activity assays. Protein concentration was measured using the Quant-iTProtein Assay Kit (Invitrogen Co., Carlsbad, CA, USA). For the AChE activity assay, 5 μL of enzyme extract was pre-incubated with 65 μL of 50 mM sodium phosphate buffer at 37°C for 15 min. Subsequently, 70 μL of Ellman’s reaction mixture was added to the reaction mixture. Absorbance was measured at 405 nm every 2 min for a duration of 10 min. AChE activity in brain tissues was expressed as a percentage of activity per milligram of protein relative to the control group.
Statistical analysis
Statistical analyses were conducted using IBM SPSS ver. 18.0 (IBM Co., Armonk, NY, USA). Data were expressed as mean±standard error of the mean. Group comparisons were analyzed using one-way analysis of variance, followed by post hoc tests, including Tukey test and t-tests. A P-value of <0.05 was considered statistically significant.
RESULTS
Cognitive effects of grounding mat exposure in a TMT-induced neurotoxicity model evaluated via Y-maze test
The Y-maze test was conducted to evaluate the memory-protective effects of grounding mat exposure in a TMT-induced neurotoxicity model. Spontaneous alternation behavior, a measure of cognitive function, was significantly decreased in the TMT-treated control group compared to the normal group (P<0.05). However, the A-7D group exhibited a significant increase in spontaneous alternation behavior compared to the control group (P<0.05) (Fig. 1). These findings indicate that exposure to the grounding mat improves cognitive function impaired by TMT-induced neurotoxicity.
Effects of grounding mat exposure on cytokine expression in a TMT-induced neurotoxicity model
To investigate the anti-inflammatory effects of grounding, a neurotoxicity model was exposed to the grounding mat, and cytokine levels, including IL-1β, IL-4, and PGE2, were measured in serum using ELISA. IL-1β levels were significantly elevated in the control group compared to the normal group (P<0.05). However, the A-21D group exhibited a significant reduction in IL-1β levels compared to the control group (P<0.01) (Fig. 2A). For IL-4, the control group showed a significant decrease in levels compared to the normal group (P<0.001), while the A-21D group exhibited a significant increase in IL-4 level s compared to the control group (P<0.05) (Fig. 2B). PGE2 levels were significantly increased in the control group compared to the normal group (P<0.001). In contrast, the A-21D group showed a significant reduction in PGE2 levels compared to the control group (P<0.05) (Fig. 2C). These results demonstrate that exposure to the grounding mat suppresses inflammation induced by TMT, suggesting its potential efficacy in mitigating inflammatory responses and neurotoxicity.
Effects of grounding mat exposure on hippocampal AChE activity in a TMT-induced neurotoxicity model
To assess the effects of grounding mat exposure on AChE activity, hippocampal tissue from TMT-induced neurotoxicity models was analyzed using ELISA. The control group exhibited significantly elevated AChE activity in the hippocampus compared to the normal group (P<0.05). However, the A-21D group demonstrated a significant reduction in hippocampal AChE activity compared to the control group (P<0.01) (Fig. 3). These findings suggest that the grounding mat alleviates TMT-induced neurotoxicity and may contribute to the restoration of cholinergic neurotransmission in the hippocampus.
DISCUSSION
Grounding, which involves direct connection of the body to the earth’s surface, facilitates the transfer of free electrons into the body. Research has demonstrated that this process effectively reduces oxidative stress and inflammation (Oschman et al., 2015). Grounding has also been reported to regulate the secretion of stress hormones, stabilize circadian rhythms, restore autonomic nervous system balance, and improve stress-related biomarkers (Chevalier et al., 2012). Moreover, the absorption of free electrons through direct contact with the Earth’s surface neutralizes reactive oxygen species, contributing to the reduction of both acute and chronic inflammation (Oschman et al., 2015).
In this study, the effects of grounding mat exposure on inflammatory responses and cognitive function in a TMT-induced neurotoxicity model were evaluated. The Y-maze test demonstrated significant improvement in cognitive function in the A-21D group. Furthermore, the proinflammatory cytokines IL-1β and PGE2, which were elevated in response to neurotoxicity, were significantly reduced in the A-21D group, while the expression of the anti-inflammatory cytokine IL-4 increased. Additionally, AChE activity in the hippocampus was enhanced in the A-21D group compared to the control group, indicating improvements in cholinergic neurotransmission. These findings suggest that exposure to the grounding mat alleviates TMT-induced neurotoxicity, mitigates inflammation, and improves cognitive deficits.
TMT is a neurotoxic compound known to cause extensive damage to the CNS, particularly in the hippocampus, a brain region essential for memory and learning (Thong-Asa et al., 2020). Hippocampal damage is strongly associated with cognitive decline. Previous studies have reported that TMT administration impairs spatial memory and learning abilities in rodents, as evidenced by reduced performance in tasks such as passive avoidance learning and the Morris water maze, which indicate disruptions in memory and learning processes (Ye et al., 2020). Furthermore, TMT-treated animals exhibit decreased spontaneous alternation behavior in the Y-maze test, reflecting impairments in working memory and exploratory behavior. Consistent with these findings, the present study observed reduced spontaneous alternation rates following TMT administration. However, the A-21D group demonstrated a significant increase in spontaneous alternation compared to the control group, indicating that grounding mat exposure effectively promotes cognitive recovery.
IL-1β and IL-4 play opposing roles in the regulation of inflammatory responses (Kaneko et al., 2019; Pyrillou et al., 2020). IL-1β, a potent proinflammatory cytokine, promotes inflammation, induces neuronal damage, and exacerbates neuroinflammation. Conversely, IL-4 is an anti-inflammatory cytokine that suppresses the production of proinflammatory cytokines, mitigates inflammation, and maintains immune homeostasis. Previous studies have shown that TMT administration increases IL-1β expression while reducing IL-4 expression, resulting in an imbalance in the inflammatory response. Similarly, this study confirmed increased IL-1β levels and decreased IL-4 levels in the control group. In contrast, the A-21D group exhibited a reduction in IL-1β expression and an increase in IL-4 expression, indicating that grounding mat exposure regulates inflammatory responses by suppressing proinflammatory cytokines and enhancing anti-inflammatory cytokines. These changes contribute to the restoration of immune balance and support neuroprotection.
PGE2 is a critical mediator of pain and inflammation. As a lipid mediator produced through the COX-2 pathway, PGE2 amplifies inflammatory responses and exacerbates neuronal damage (Nango et al., 2023). Studies have demonstrated that TMT administration increases PGE2 expression, thereby intensifying inflammatory responses and accelerating neuronal injury (Park et al., 2022). This study similarly observed elevated PGE2 levels in the control group. However, the A-21D group exhibited a significant reduction in PGE2 expression, suggesting that grounding mat exposure suppresses PGE2 production or modulates COX-2 pathway activity, thereby alleviating inflammation. The reduction in PGE2 levels underscores the potential of grounding mats to regulate inflammation and protect against inflammation-related neurotoxicity.
AChE is an enzyme essential for the breakdown of acetylcholine and the regulation of cholinergic neurotransmission. Following TMT administration, AChE activity is typically reduced, leading to impaired acetylcholine metabolism and disrupted cholinergic signaling. These changes contribute to memory impairments and cognitive dysfunction. TMT-induced neurotoxicity damages cholinergic pathways, thereby reducing AChE activity and impairing neurotransmission efficiency (Moss, 2020; Ye et al., 2020). This study also observed reduced AChE activity in the hippocampus following TMT administration. In contrast, the A-21D group exhibited significantly increased AChE activity compared to the control group, indicating that grounding mat exposure contributes to the recovery of cholinergic neurotransmission and maintains appropriate acetylcholine levels. These findings suggest that grounding mats positively influence cognitive function and neuronal integrity.
While this study provides evidence supporting the beneficial effects of grounding mats on TMT-induced neurotoxicity, certain limitations should be considered. The sample size was limited, and the findings may not be directly generalizable to humans. Further studies with larger sample sizes, extended exposure periods, and clinical trials are required to confirm these effects. Additionally, the molecular mechanisms underlying the observed effects of grounding should be further elucidated to enhance understanding.
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