Spreading depression and neuronal damage

执行了两套不同的项目。(1)大鼠全脑缺血后谷胱甘肽的释放和转化将微透析探针植入大脑皮层，连续收集透析液。采用高效液相色谱法分析透析液中的亚硝酸盐（NO2）、硝酸盐（NO3）、氧化型谷胱甘肽（GSH）和还原型谷胱甘肽（GSSG）。通过结扎双侧颈动脉和低血压诱导全脑缺血。在再灌注过程中，氧化型谷胱甘肽（GSH）的释放与GSSG的释放同步增加，并在再灌注100 min后终止。NO_2、NO_3在再灌注过程中减少，其浓度与GSH、GSSG呈镜像关系，说明再灌注早期还原型谷胱甘肽迅速合成并氧化为GSH，这种自由基捕获作用可能有助于防止亚硝酸盐和硝酸盐的增加。(2)培养神经元实验性缺血再氧过程中线粒体膜电位的变化在原代培养的大鼠神经元细胞中诱导无糖无氧的实验性缺血。将电压敏感染料JC-1加载到神经元培养物中，用激光扫描显微镜分析线粒体膜电位。发射光的波长为488 nm，分析530 nm（代表去极化）和580 nm（代表极化）的激活光以确定线粒体膜电位。实验性缺血30 min时线粒体膜超极化，60 min时线粒体膜去极化。总之，线粒体膜电位取决于ATP的产生速率。在ATP产生过程中伴随着质子从内膜的排斥，导致超极化。JC-1在缺血30分钟后表达的超极化清楚地反映了缺血后ATP的快速产生。

Two different sets of projects were performed.(1) Release and coversion of glutathione after global ischemia in ratA probe of microdialysis was implanted in the cerebral cortex and dialysate was collected continuously. Dialysate was analyzed for nitrite (NO2), nitrate (NO3), oxygenated glutathione(GSH) and reduced glutathione (GSSG) by using high performance liquid chromatography. Global ischemia was induced by ligation of both carotid arteries and hypotension. During reperfusion, oxygenated glutathione (GSH) increased simultaneously with GSSG.This release was terminated in 100 min after the start of reperfusion. NO2 and NO3 decreased during reperfusion and its profile was mirror image of GSH and GSSG.In conclusion, during early phase of reperfusion, reduced glutathione is rapidly synthetized and oxidized to GSH.This free radical trapping seems to contribute prevention of increase in nitrite and nitrate.(2) Mitochondrial membrane potential during reoxygenation after experimental ischemia in cultured neuronExperimental ischemia, no glucose and no oxygen, was induced in primary rat neuronal cell culture. Voltage sensitive dye, JC-1, was loaded to the neuronal culture and mitochondrial membrane potential was analysed by using Laser Scanning microscope. Wavelength of emitted light was 488nm and activated light of 530nm(represents depolarization) and 580nm(represents polarization) was analysed to determine mitochondrial membrane potential. After 30 min experimental ischemia, mitochondrial membrane was hyperpolarized, while, after 60 min experimental ischemia, mitochondria was depolarized. In conclusion, Mitochondrial membrane potential depends on rate of ATP production. Accompanying exclusion of proton from inner membrane during ATP production results in hyperpolarization. Hyperpolarization expressed by JC-1 after 30 min ischemia clearly reflects rapid production of ATP after ischemia.