Brain protection from the neuronal death induced by spreading depression

我们已经完成了这一资助下的两个项目。首先，我们检测了缺血后再灌注期间自由基的形成和清除。其次，我们检测了氧-葡萄糖剥夺后再氧化过程中的线粒体膜电位。第一个项目提出了再灌注早期自由基对神经元毒性的生理保护机制。因此，我们转向线粒体机制参与神经元的活动性自杀、凋亡，因为神经元自身执行的活动性神经元死亡似乎是神经元缺血后损伤的主要原因。研究了大鼠脑缺血后再灌注过程中谷胱甘肽氧化和活性氮的时间分布。GSH和GSSG增加并达到峰值；再灌注25 min时3408 ^^+__-1710%（平均^^+__- SE） （P<0.0001）；再灌注50 min时为329 ^^+__- 104% （p=0.06）。NO_2水平无明显变化。这些数据表明，谷胱甘肽在再灌注早期的释放及其快速氧化有助于防止活性氮种类的增加。将原代海马培养物接种于35mm开孔荧光显微镜培养皿中，置于密封室中进行厌氧培养（氧-葡萄糖剥夺（OGD））。在OGD期间，MMP下降到0.72 ^^+__- 0.03（归一化JC-1荧光），然后在OGD 30min后60min再氧化时增加到超极化水平1.99 ^^+__- 0.12。在OGD和再氧化90分钟后，MMP减少且从未恢复。OGD 30mm后细胞内ATP含量为8.1 ^^+__- 6.6%，OGD 30min后再氧30min后细胞内ATP含量为3.2 ^^+__- 1.9%。这一观察结果表明，在再氧化过程中，电子再进入内膜受到抑制，F_0F_1-ATP合成酶受到干扰。

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.