In the present study, we evaluated the neuroprotective effects of hydrogen saline against cerebral ischemia-reperfusion injury. The major findings were that hydrogen saline could reduce cerebral infarction and improve neurological function in the MCAO rat model, which were mediated by the reduction of oxidative stress (8-OHdG, nitrotyrosine and MDA) and inflammatory factors, and subsequent decrease of neuronal apoptosis (TUENL positive cells, expression of Bcl-2 and Bax, and caspase-3 activity). The therapeutic window of hydrogen saline was similar to other prominent neuroprotectants. The protective effects were more pronounced if they were applied immediately after reperfusion, but the protective effects could be achieved to a certain extent when they were applied at 6 h after reperfusion. Our findings were consistent with previous studies in which protective effects of hydrogen gas through scavenging ROS have been confirmed in a cardiac ischemia-reperfusion injury model .
Increasing evidence has demonstrated ROS contribute to ischemia/reperfusion induced brain damage in a 2-phase pattern, an immediately occurring direct cytotoxic damage and a post-ischemia/reperfusion inflammatory injury . ROS is massively produced in the brain after ischemia/reperfusion, and oxidative damage to brain tissues has been regarded as a fundamental mechanism of brain damage after transient or permanent cerebral ischemic injury [19, 20]. All of these species interact with nearby cellular components, such as proteins, lipids, and DNA [4, 5]. Some components in the reactive oxygen species such as superoxide anion and H2O2can be detoxified by antioxidant defense enzymes, while there is no enzyme to detoxify OH and ONOO-, extremely reactive free radicals in cells, until a recent study reported that hydrogen gas could selectively reduce these two harmful free radicals . Hydrogen molecule is electronically neutral and has the ability to penetrate the membranes of cell, nucleus and mitochondria. 8-OHdG is a product of direct oxidation of DNA by hydroxyl radicals and has been used as a marker for oxidative stress . Our results showed reduced number of 8-OHdG positive cells after MCAO by hydrogen saline. Our findings were consistent with a recent study on hydrogen inhalation in which hydrogen inhalation also reduced the oxidative stress following ischemia/reperfusion .
Oxidative stress can also lead to inflammatory response after ischemic stroke, which is characterized by enhanced cytokines production . Among the known cytokines, IL-1β and TNF-α are produced by macrophages, endothelial cells, astrocytes and neurons, and play crucial roles in the ischemic brain injury . Reduction of oxidative stress by hydrogen saline may result in the suppressed production of TNF-α and IL-1β as demonstrated by our study. A possible direct anti-inflammatory effect of hydrogen saline in cerebral ischemia warrants further investigation.
Oxidative stress and inflammation contribute to the activation of program cell death following cerebral ischemia . Oxidative stress can cause changes in the mitochondrial permeability resulting in the release of cytochrome c which then activates caspase-3 executing cell death signals. By reducing oxidative stress and inflammation, hydrogen saline suppressed caspase-3 activity in the ischemic cortex, which might be related to the decreased release of cytochrome c. Two other important mitochondrial apoptotic factors Bcl-2 and Bax were examined in the present study . Consistently, hydrogen saline treatment also up-regulated the Bcl-2 expression and down-regulated the Bax expression.
Of note, although the protective effects were also observed in our previous study, the therapeutic effects of hydrogen saline were more profound than those of hydrogen inhalation. In addition, the effects of intravenous administration of hydrogen were inferior to those of intraperitoneal treatment, which may be explained by rapid elimination of hydrogen through pulmonary gas exchange. But the exact mechanism should be further investigated. Although the intravenous application was more clinical than intraperitoneal administration, intraperitoneal injection was frequently performed in animals. Therefore, in the present study, intraperitoneal administration of hydrogen saline was conducted to observe the neuroprotective effects. Furthermore, in our pilot study on animals and humans, some parameters did not show evident side effects even with several large doses of hydrogen saline were applied.
Taking together, hydrogen has been shown anti-oxidative stress and is beneficial on lipid and glucose metabolism in humans . Hydrogen water also decreased superoxide formation caused by ischemia-reperfusion in the brain slices of mice . For the safety and the convenience of hydrogen administration, hydrogen saline was prepared and protective effects of hydrogen saline confirmed in rat cardiac ischemia/reperfusion and neonatal hypoxia-ischemia models [11, 12]. In the present study, we further demonstrated that intraperitoneal administration of hydrogen saline yielded similar neuroprotective effects comparable to hydrogen inhalation [7, 22]. Therefore, our study for the first time showed hydrogen saline had potentials as an alternative pharmacological strategy in ischemic stroke.