Background and Purpose— Normobaric oxygenation (NBO) and ethanol both provide neuroprotection in stroke. We evaluated the enhanced neuroprotective effect of combining these 2 treatments in a rat stroke model. Methods— Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 2 hours. Reperfusion was then established and followed by treatment with either (1) an intraperitoneal injection of ethanol (1.0 g/kg), (2) NBO treatment (2-hour duration), or (3) NBO plus ethanol. The extent of brain injury was determined by infarct volume and motor performance. Oxidative metabolism was determined by ADP/ATP ratios, reactive oxygen species levels, nicotinamide adenine dinucleotide phosphate oxidase activity, and pyruvate dehydrogenase activity. Protein expression of major nicotinamide adenine dinucleotide phosphate oxidase subunits (p47 phox , gp91 phox , and p67 phox ) and the enzyme pyruvate dehydrogenase was evaluated through Western immunoblotting. Results— NBO and ethanol monotherapies each demonstrated reductions as compared to stroke without treatment in infarct volume (36.7% and 37.9% vs 48.4%) and neurological deficits (score of 6.4 and 6.5 vs 8.4); however, the greatest neuroprotection (18.8% of infarct volume and 4.4 neurological deficit) was found in animals treated with combination therapy. This neuroprotection was associated with the largest reductions in ADP/ATP ratios, reactive oxygen species levels, and nicotinamide adenine dinucleotide phosphate oxidase activity, and the largest increase in pyruvate dehydrogenase activity. Conclusions— Combination therapy with NBO and ethanol enhances the neuroprotective effect produced by each therapy alone. The mechanism behind this synergistic action is related to changes in cellular metabolism after ischemia reperfusion. NBO plus ethanol is attractive for clinical study because of its ease of use, tolerability, and tremendous neuroprotective potential in stroke.

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Oxidative stress plays a role in many neurological diseases. Hypoxic preconditioning (HPC) has been proposed as an intervention that protects neurons from damage by altering their response to oxidative stress. The aim of this study was to investigate the mechanisms by which HPC results in neuroprotection in cultured SH-SY5Y cells subjected to oxidative stress to provide a guide for future investigation and targeted interventions. SH-SY5Y cells were subjected to HPC protocols or control conditions. Oxidative stress was induced by HO. Cell viability was determined via adenosine triphosphate assay. Rapamycin and 3-methyxanthine (3-MA) were used to induce and inhibit autophagy, respectively. Monodansylcadaverine staining was used to observe the formation of autophagosomes. Levels of Microtubule-associated protein light chain 3 B (LC3B), Beclin 1, and p53 were measured by Western blot. Reactive oxygen species (ROS) were also determined. Cell viability in the HPC group following 24-h exposure to 600 μM HO was 65.04 ± 12.91% versus 33.14 ± 5.55% in the control group. LC3B, Beclin 1, and autophagosomes were increased in the HPC group compared with controls. Rapamycin mimicked the protection and 3-MA decreased the protection. There was a moderate increase in ROS after HPC, but rapamycin can abolish the increase and 3-MA can enhance the increase. p53 accumulated in a manner consistent with cell death, and HPC-treated cells showed reduced accumulation of p53 as compared with controls. Treatment with rapamycin decreased p53 accumulation, and 3-MA inhibited the decrease in p53 induced by HPC. HPC protects against oxidative stress in SH-SY5Y cells. Mechanisms of protection may involve the activation of autophagy induced by ROS generated from HPC and the following decline in p53 level caused by activated autophagy in oxidative stress state. This is in line with recent findings in nonneuronal cell populations and may represent an important advance in understanding how HPC protects neurons from oxidative stress.