Brain protection from the neuronal death induced by spreading depression

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We have accomplished two projects supported by this grant. First, we examined radical formation and radical scavenging during reperfusion period after ischemia. Second, we examined mitochondrial membrane potential during reoxygenation after oxygen-glucose deprivation. The first project suggested physiological protective mechanism of neuron from toxic effect of free radical during early reperfusion phase. Therefore, we moved to mitochondrial mechanism involved in active neuronal suicide, apoptosis because active neuronal death executed by neuron itself seems to be a main cause of neuronal damage after ischemia.1. The role of glutathione during reperfusionA timed profile of glutathione oxidation and reactive nitrogen species during reperfusion after cerebral ischemia in rat was obtained. GSH and GSSG increased and reached a peak; 3408 ^^+__-1710% (mean ^^+__- SE) at 25 min of reperfusion (P<0.0001); and 329 ^^+__- 104% at 50 min of reperfusion (p=0.06), respectively. NO_2 levels did not significantly change. These data suggest that GSH releases during early phase of reperfusion and its rapid oxidation contributes to prevent increase in reactive nitrogen species.2. Involvement of mitochondrial mechanism for ischemic neuronal deathA primary hippocampal culture seeded in a 35mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation (oxygen-glucose deprivation (OGD)). During OGD, MMP decreased to 0.72 ^^+__- 0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99 ^^+__- 0.12 during 60min reoxygenation after 30 min OGD. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1 ^^+__- 6.6% and 3.2 ^^+__- 1.9% after 30 mm OGD and 30 min reoxygenation following 30 min OGD, respectively. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of F_0F_1-ATP synthase.