Brain protection from the neuronal death induced by spreading depression

我们已经完成了两个项目的支持，这笔赠款。首先，我们研究了缺血后再灌注期间自由基的形成和自由基清除。第二，我们检测了缺氧缺糖后复氧过程中线粒体膜电位。第一个项目提出了再灌注早期神经元对自由基毒性作用的生理保护机制。因此，我们转移到线粒体机制参与主动神经元自杀，细胞凋亡，因为主动神经元死亡由神经元本身执行似乎是缺血后神经元损伤的主要原因.谷胱甘肽在脑缺血再灌注过程中的作用本实验观察了大鼠脑缺血再灌注过程中谷胱甘肽氧化和活性氮的动态变化。GSH和GSSG升高并达到峰值;再灌注25分钟时分别为3408 ^^+__-1710%（平均^^+__- SE）（P<0.0001）;再灌注50分钟时分别为329 ^^+__- 104%（p=0.06）。NO_2水平无明显变化。提示再灌注早期GSH的释放及其快速氧化有助于防止活性氮的增加.缺血性神经元死亡的线粒体机制的参与将原代海马培养物接种在用于荧光显微镜检查的35 mm有孔培养皿中，置于密封室中进行厌氧孵育（氧-葡萄糖剥夺（OGD））。在OGD期间，MMP降低至0.72 ^^+__- 0.03（标准化JC-1荧光），然后在OGD 30 min后的60 min复氧期间增加至超极化水平1.99 ^^+__- 0.12。OGD和复氧90分钟后，MMP降低，再也没有恢复。缺氧30 min后再复氧30 min，细胞内ATP含量分别为8.1 ^^+_- 6.6%和3.2 ^^+_- 1.9%。这一观察结果表明，在复氧过程中电子重入内膜的抑制和F_0F_1-ATP合酶的干扰。

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.