Abstract

Introduction

Methamphetamine (METH) is one of the most frequently abused pharmacologic psychostimulant illicit drugs with strong neurotoxic effects on the central nervous system (CNS). Long-term or high-dose use of METH causes abnormalities in the structure, chemistry, and function of brain, resulting in a number of associated medical complications and fatalities (Zhu et al., 2000Northrop and Yamamoto, 2015Shaerzadeh et al., 2018). Notably, METH abusers are more likely to develop schizophrenia, depression, Parkinson’s disease, and other neuropsychiatric and cognitive disorders; these are mostly attributed to METH-induced neurotoxicity (Schroder et al., 2003Hruba et al., 2010Kuehn, 2011).

The neurotoxic mechanism of METH is complex and involves multiple pathways (Ceccatelli, 2013Shin et al., 2017). METH intake elicits a massive release of dopamine (DA) and excessive glutamate production in the brain, generating a large amount of reactive oxygen species (ROS), and subsequently leading to mitochondrial dysfunction and endoplasmic reticulum (ER) stress (Beauvais et al., 2011Irie et al., 2011Dang et al., 2018Shin et al., 2018). METH has been shown to significantly increase the Bax/Bcl-2 ratio and to disrupt the potential of mitochondrial membranes, triggering a caspase cascade (Jayanthi et al., 2001). METH also increases the detachment of the major chaperone protein from the transmembrane ER signaling protein glucose-related protein 78 (GRP78) and induces apoptosis by escalating the level of the CCAAT/enhancer-binding protein homologous protein (CHOP) (Irie et al., 2011Takeichi et al., 2012). In addition, microglial activation and METH-mediated neuroinflammation also contribute to neurotoxicity by attacking the neuron with inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 (Coelho-Santos et al., 2012). Thus, METH can cause terminal damage and neurodegeneration of the neuronal cells in the hippocampus, eventually resulting in cognitive disorder.

The ever-increasing levels of METH abuse have necessitated the development of effective intervention strategies that protect neural cells against METH-induced neurotoxic consequences (Yang et al., 2018). Molecular hydrogen, which is characterized by its anti-oxidative, anti-inflammatory, and anti-apoptotic properties, has recently gained attention as a potential novel healthcare product for preventive and therapeutic applications in a wide range of pathological conditions (Fujita et al., 2009Ohta, 2012Spulber et al., 2012). Several studies have demonstrated the protective effects of molecular hydrogen in various CNS disease animal models, such as stroke, traumatic brain injury, Alzheimer’s disease, and Parkinson’s disease (Fu et al., 2009Dohi et al., 2014Ono et al., 2017Nishimaki et al., 2018). Our previous study found that hydrogen-rich saline (HRS) injections significantly attenuated naloxone-precipitated morphine withdrawal symptoms and morphine withdrawal-induced anxiety-like behaviors as well as elevated corticosterone levels (Wen et al., 2017). A recent study also showed that consumption of hydrogen-rich water (HRW) prevented stress-induced impairments in learning tasks during chronic physical restraint by buffering the effect of oxidative stress (Nagata et al., 2009). It’s encouraging that a multicenter randomized trial is underway to confirm the efficacy of molecular hydrogen on neurological outcomes in patients with post-cardiac arrest syndrome (Tamura et al., 2017). Some methods have also been used to administer hydrogen to humans, including drinking or bathing with HRW and inhalation of hydrogen gas (HG). HRW drinking is expected to be easily used in daily life, and the results of several clinical studies of HRW were recently released (Kang et al., 2011Iida et al., 2016Korovljev et al., 2019). Herein, we hypothesized that the consumption of HRW may potentially exert a preventive effect on METH-induced neurotoxicity and spatial learning and memory impairments.

In the current study, we have performed the Barnes maze and Morris water maze tests to investigate the effects of ad libitum HRW consumption on high-dose METH exposure-induced spatial learning task impairment and memory loss. The neuronal damage was evaluated by Nissl staining, and the index of mitochondrial dysfunction, ER stress, and neuroinflammation were also measured by the detection of Bax/Bcl-2, caspase 3, GRP78, CHOP and p-NF-κB p65 expression, and IL-6 and TNF-α level in hippocampus.

Materials and Methods

Results

Discussion

This study aimed to check if consumption of HRW may potentially exert a preventive effect on METH-induced neurotoxicity and spatial learning and memory impairments. Herein, we present the first set of findings demonstrating a significant inhibitory effect of molecular hydrogen delivered by ad libitum HRW consumption on the METH-induced spatial learning impairment and memory loss noted in the Barnes maze and Morris water maze tests. We also found that ad libitum HRW consumption significantly restrained neuronal damage in the hippocampus after high-dose METH exposure. Treatment with ad libitum HRW consumption also had an inhibitory effect on METH-induced mitochondrial dysfunction, ER stress, and neuroinflammation. Therefore, our study may pave the way for a novel and effective neuroprotective strategy using molecular hydrogen for METH-induced neurotoxicity.

Illicit drug abuse is a serious global problem. Among the current illicit drugs, METH is the most prevalent illicit drug in numerous countries, including China (Bramness et al., 2015Kwon and Han, 2018Bannwarth et al., 2019). METH abuse has been shown to result in cognitive deficits in attention, learning, and memory in a variety of experimental and clinical studies (Potvin et al., 2018Seyedhosseini Tamijani et al., 2018). Several dose regimens of METH administration have been evaluated in different studies, such as a single high dose (40 mg/kg) or repeated (4 × 10 mg/kg, 2–3-h intervals) or escalating (1–10 mg/kg, twice a day, at 5-h intervals, for 10 days) doses of METH and chronic voluntary oral METH intake (Yang et al., 2018). Since some studies have suggested that exposure to nontoxic amounts of METH can afford protection against the toxic effects of subsequent larger doses, a phenomenon referred to as METH preconditioning, a regimen based on 4 × 10 m/kg METH injections spaced 3 h apart, was used in mice in the present study. Stereotypic behavior and hyperthermia were obviously induced after drug administration. After METH exposure, two different behavioral tests were utilized to analyze and confirm the efficiency of molecular hydrogen. The Barnes maze and Morris water maze tests are both common behavioral models that are valuable for evaluating spatial learning and memory ability of rodents. It’s worthy to note that behavioral tasks involving high levels of stress can influence the animal’s performance on the task. Strong aversive stimulus induced by swimming in the Morris water maze might be a factor account for the different learning curves in this study. Consistent with the results of previous studies (Vorhees, 1997Gutierrez et al., 2018), the findings in both behavioral tests revealed spatial learning task impairment and memory loss at 7 days after drug exposure in the METH-treated mice. Moreover, METH exposure caused damage to the pyramidal neurons in the CA1 and CA3 regions of the hippocampus, which play crucial roles in cognition. In fact, we also tested METH-induced neuronal damage in dentate gyrus (DG). However, the inconsistent result was revealed, and the damage was only observed in one animal. The reason might be complicated, and we could not regard it as a negative region. Observations in more samples were needed in the following study.

Considering the primary mechanisms underlying the effects of METH, reducing oxidative damage or neuroinflammation may be feasible approaches to prevent METH-induced neurotoxicity. Molecular hydrogen was assumed to be effective in relieving the pathologic changes after METH exposure. In this study, we found that molecular hydrogen significantly inhibited the METH-induced spatial learning impairment and memory loss and hippocampal neuronal damage. Injections of HRS and inhalation of HG are common strategies to rapidly deliver molecular hydrogen into experimental animals and the human body. However, these approaches are not practical or suitable for continuous consumption in daily life. Drinking HRW is clearly much more convenient. In our experiments, ad libitum HRW consumption, a method that fits well within the normal lifestyle of humans, was applied to administer hydrogen to animals, and it had significant neuroprotective effects on METH-induced neurotoxicity. As for the concentration of hydrogen, we attempted to measure the hydrogen concentrations in the blood and brain homogenate after intraperitoneal injection of HRS by performing cyclic voltammetry analysis using a hydrogen electrode. We could detect a fivefold and threefold change in the hydrogen concentrations in the blood and brain homogenate, respectively, at 5 min after HRS injection (unpublished data not shown). However, since this test could not be reliably conducted on free-moving animals in the present condition of our lab, we did not test the hydrogen concentration in the blood and brain of mice that consumed HRW ad libitum. Nevertheless, one recent study has reported that the blood concentration of hydrogen incorporated from the stomach of animals consuming HRW was approximately 5 µM (the control data was 1 µM) (Nagata et al., 2009). Since hydrogen can quickly reach the maximal serum concentration and easily cross the blood–brain barrier, we considered that hydrogen had been incorporated into brain tissues after ad libitum HRW consumption.

Oxidative stress, the most significant factor contributing to METH-induced neurotoxicity, leads to mitochondrial dysfunction and ER stress. It eventually results in devastating neuropathological effects and neuronal death in the brain due to the subsequent Bax/Bcl-2 changes in the mitochondrial fractions and caspase activation, as well as the accumulation of misfolded proteins and elevation of GRP78 and CHOP expression (Shah and Kumar, 2016Qie et al., 2017). Similar results were obtained in METH-treated mice in the present study. Furthermore, METH activates NF-kB signaling pathway and promoting the transcription of pro-inflammatory cytokines in the brain after 3 to 7 days of METH treatment (Goncalves et al., 2010), which corroborate our data showing a late increase in the levels of markers characteristic of neuroinflammation, such as p-NF-kB p65, and the pro-inflammatory cytokines IL-6 and TNF-α. In addition, HRW significantly inhibited the increase in Bax/Bcl-2, cleaved caspase-3, GRP78, CHOP, and p-NF-kB p65 levels and decreased the amounts of IL-6 and TNF-α in the hippocampus of METH-treated mice. This indicates that molecular hydrogen attenuated the METH-induced mitochondrial dysfunction, ER stress, and neuroinflammation in the hippocampus.

However, the lack of animal general condition record such as change of body weight after large dose of METH treatment was a limitation of this study. Moreover, METH abuse leads to dysfunction of several brain regions, and most of the studies have been extensible exploring the toxic effects of METH in the striatum (Gou et al., 2015Xie et al., 2018Sabrini et al., 2019). In view of this point, we planned to observe the effects of hydrogen on the DA transporter activity and striatal dopaminergic terminal damage in the animal model by using chronic repeated METH treatment regimens in a following study. Although we confirmed the efficiency of HRW consumption on METH-induced neurotoxicity, the main mechanisms underlying the inhibitory effect of molecular hydrogen have not been clearly clarified. The effects of hydrogen on early upregulation of markers characteristic of oxidative damage after METH exposure, such as ROS and malondialdehyde (MDA), and the roles of hydrogen over glia cells need to be explored in future studies.

Conclusion

In summary, the present investigation showed that ad libitum HRW consumption significantly attenuated METH-induced spatial learning impairment and memory loss as well as the neuronal damage in hippocampus. Since reversing or preventing the oxidative damage and neuroinflammation induced by METH exposure might be useful in protecting against neuronal damage, molecular hydrogen may have a potential application in reducing the risk of neurodegeneration frequently observed in METH abusers.

Data Availability

The datasets analyzed in this manuscript are not publicly available. Requests to access the datasets should be directed to moc.621@52110idnew.

Ethics Statement

All experiments were conducted according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Local Committee on Animal Care and Use and Protection of the Hebei Medical University approved the experimental procedures.

Author Contributions

CM, BC, and DW conceived this work. DW wrote the main manuscript text. DW and RH performed the lab experiments. RH, JW, and XS performed the animal experiments. MG performed the Nissl staining. BX and FY analyzed and interpreted the data. All authors read and commented on the manuscript.

Funding

This study was financially supported by the Science and Technology Research Projects for University in Hebei Province (No. BJ2017015).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

References