Effects of Molecular Hydrogen on Methamphetamine-Induced Neurotoxicity and Spatial Memory Impairment

Abstract

Methamphetamine (METH) is a highly addictive stimulant, and METH exposure can induce irreversible neuronal damage and cause neuropsychiatric and cognitive disorders. The ever-increasing levels of METH abuse worldwide have necessitated the identification of effective intervention strategies to protect the brain against METH-induced neurotoxicity. The protective effects of molecular hydrogen on oxidative stress and related neurodegenerative diseases have been recently elucidated. Herein, we investigated whether treatment with molecular hydrogen ameliorated the METH-induced neurotoxicity and spatial learning and memory impairments. Male C57BL/6 mice received four intraperitoneal METH injections (10 mg/kg, 3-h interval), and stereotypic behaviors and hyperthermia were observed. After METH treatment and behavioral observation, the mice were returned to their home cages, where they received water or hydrogen-rich water (HRW) ad libitum for 7 days. We found that the molecular hydrogen delivered by ad libitum HRW consumption significantly inhibited the METH-induced spatial learning impairment and memory loss evidenced in the Barnes maze and Morris water maze tests. Furthermore, molecular hydrogen significantly restrained the neuronal damage in the hippocampus after high-dose METH exposure. Ad libitum HRW consumption also had an inhibitory effect on the METH-induced increase in the expression of Bax/Bcl-2, cleaved caspase-3, glucose-related protein 78 (GRP 78), CCAAT/enhancer-binding protein homologous protein (CHOP), and p-NF-kB p65 expression and elevation of interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels in the hippocampus. These are the first findings to indicate that hydrogen might ameliorate METH-induced neurotoxicity and has a potential application in reducing the risk of neurodegeneration frequently observed in METH abusers.

Keywords: endoplasmic reticulum stress; methamphetamine; mitochondrial dysfunction; molecular hydrogen; neuroinflammation; spatial learning and memory impairment.

Figure 1

The experimental procedure and drug treatment. The mice were treated with methamphetamine (METH; 10 mg/kg × 4-, 3-h interval), and the naive controls were treated with an equal number of saline injections. Stereotypic behavior and body temperature were observed immediately after the last METH injection. When the mice were returned to their home cages, they were allowed to consume water or hydrogen-rich water (HRW) ad libitum for 7 days (days 2–8). On days 9–14 and 15–20, a subgroup of mice from each group performed the Barnes maze and Morris water maze tests. The remaining mice in each group were decapitated, and their brains were harvested for Nissl staining, Western blot, and enzyme-linked immunosorbent assay (ELISA) on day 9.

Figure 2

METH-induced stereotypic behavior and hypothermia in mice. Stereotypic behavior (A) and body temperature (B) were tested after the last injection of METH. One animal in control group (subsequent HRW group) and one in METH group (subsequent METH group) were excluded because of low mobility in behavioral test, and three mice in METH group were died after the last METH injection. Data are expressed as the mean ± SEM; n = 35 for the control group, and n = 32 for the METH group; and ***p < 0.001, compared to the control group.

Figure 3

Effects of ad libitum HRW consumption on METH-induced spatial learning task impairment and memory loss in the Barnes maze test. (A) Experimental procedure for the Barnes maze test. (B) The escape latencies of mice in the spatial learning task on four training days. (C) The exploration time (percent of total time) spent in the four quadrants (target, opposite, adjacent 1, and adjacent 2) during the probe test. (D) Motion speed of mice in the probe test. Data are expressed as the mean ± SEM; n = 10, 8, 8, and 9, respectively; *p < 0.05, **p < 0.01, ***p < 0.001, compared to the control group; and ^# p < 0.05, ^## p < 0.01 compared to the METH group.

Figure 4

Effects of ad libitum HRW consumption on METH-induced spatial learning task impairment and memory loss in the Morris water maze test. (A) Experimental procedure for the Morris water maze test. (B) The average escape latencies of four trials for each mouse in the spatial learning task on four training days. (C) The swimming time (percent of total time) spent in the four quadrants (target, opposite, adjacent 1, and adjacent 2) during the probe test. (D) Swimming speed of mice in the probe test. Data are expressed as the mean ± SEM; n = 10, 8, 8, and 9, respectively; *p < 0.05, **p < 0.01 compared to the control group; and ^# p < 0.05, ^## p < 0.01 compared to the METH group.

Figure 5

Effects of ad libitum HRW consumption on METH-induced neuronal damage in the hippocampus. (A) Representative photomicrograph of Nissl staining. Bars = 50 mm. (B) Numbers of Nissl-positive dead cells in the CA1 (a) and CA3 (b) regions of the hippocampus. Data are expressed as the mean ± SEM; n = 4 for each group; ***p < 0.001 compared to the control group; and ^## p < 0.01, ^### p < 0.001 compared to the METH group.

Figure 6

Effects of ad libitum HRW consumption on METH-induced changes in the indicators of mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and neuroinflammation. (A) Western blot analysis of Bax, Bcl-2, cleaved caspase 3, caspase 3, GRP78, CHOP, and p-NF-κB p65 expression in the hippocampus. (B) Levels of IL-6 (a) and TNF-α (b) in the hippocampus determined by ELISA. Data are expressed as the mean ± SEM; n = 4 for each group; **p < 0.01, ***p < 0.001 compared to the control group; and ^# p < 0.05, ^## p < 0.01 compared to the METH group.