全脑缺血再灌注后神经元凋亡的时相和分布特征

目的探讨大鼠全脑缺血再灌注后神经元凋亡的时相和分布特征,为神经元损伤的早期干预和继发性脑损害的防治提供实验依据.方法建立大鼠全脑缺血模型,采用TTC染色、原位细胞凋亡检测(原位末端标记法,TUNEL)及AO/EB荧光比色法观察脑缺血再灌后海马、皮层凋亡发生的数量和分布,用荧光指示剂Fura-2 AM标记,检测脑海马细胞内游离钙的浓度.结果 TUNEL显示再灌注3 h海马部位即出现散在凋亡细胞,并向皮层区域扩展,24～48 h达高峰;坏死细胞迟于凋亡出现,弥散分布于凋亡细胞周围,再灌注48 h后坏死细胞增多,72 h最多.AO/EB法显示海马、额叶和顶叶凋亡细胞总数分布在再灌注后24 h和72 h达高峰,并显著高于对照组(P＜0.05).TTC染色证实全脑缺血再灌注后不出现梗死灶,而是在海马及大脑皮层有弥散性坏死.胞内[Ca2 ]i各时间点与对照相比匀显著升高(P＜0.01).结论全脑缺血再灌后受累神经元经历了由凋亡到死亡的过程,对缺血敏感的海马神经元首先受损,并向顶叶和额叶皮层扩展,细胞质内[Ca2 ]i增加是主要原因.利用凋亡时间窗进行早期干预可能有益于保护和挽救凋亡前期神经元,减小继发性损害的严重程度.

Time and distribution characteristics of neuron apoptosis in rat after ischemic insult

Objective To investigate the time and distribution characteristics of neuron apoptosis occurred after global cerebral ischemia in rats,so as to provide some helpful experimental basis both for the early interference of injured neuron and the prevention and treatment of brain secondary damage.Methods In the global cerebral ischemia model with modified 4-vessel occlusion method,the amount and distribution of apoptotic neuron in hippocampus and cortex after ischemia and reperfusion were detected with TTC staining,TUNEL labeling and AO/EB fluorescence examination techniques.Intracellular free calcium concentration was measured with Fura-2 AM fluorescent indicator.Results TUNEL showed that apoptotic neurons scattered in hippocampus 3 h after reperfusion,then spread around to cortex.The amount of apoptotic cell reached to the peak at 24-48 h.The died cells appeared later and mainly dispersed around the apoptotic cells.There was a significant increase of the amount of died cells 72 h after ischemia and reperfusion.AO/EB fluorescence examination showed that the total amount of the apoptotic neurons in hippocampus,frontal cortex and parietal cortex reached their peak at 24 h and 72 h respectively,and they were significantly higher than that in control group(P<0.05).The infarct focuses were not found after reperfusion by TTC staining,which expanded around hippocampus and cortex.Intracellular free calcium concentration was significantly higher than that in control group(P<0.01).(Conclusion) The injured neuron underwent a process from apoptosis to death after global cerebral ischemia and reperfusion.Hippocampal neurons,which are quite sensitive to ischemia,were injured first,and then the apoptotic process extended to frontal and parietal cortex.The early interferences in the apoptotic time window were of help both in the protection of neuron in pre-apoptosis stage and reduce of secondary damage severity.