大鼠脑缺血再灌注后海马CA1区超微结构及Apaf-1表达的变化

Hippocampal CA1 ultrastructure and expression of Apaf-1 in cerebral ischemia reperfusion rats

目的观察大鼠脑缺血再灌注后海马CA1区超微结构及凋亡相关基因凋亡蛋白激活因子(Apaf)-1表达的变化。方法健康雄性SD大鼠随机分为假手术组和模型组,其中模型组缺血30 min后再灌注,根据再灌注时间不同又分为2、24、72 h 3个亚组。采用Pulsinelli四血管阻断法建立大鼠全脑缺血再灌注模型,透射电镜下观察海马CA1区神经元超微结构变化,免疫组织化学法和Western印迹法检测大鼠海马组织凋亡相关基因Apaf-1蛋白表达的变化。结果透射电镜结果显示:假手术组海马CA1区神经元胞核较大,呈圆形或椭圆形,核膜清晰完整,常染色质呈细颗粒状均匀分布,胞浆内可见丰富的细胞器。模型2 h组海马CA1区神经元核膜凹陷,核染色质密度增高,线粒体轻微肿胀;模型24 h组海马CA1区神经元核膜邹缩严重,核仁物质增多、偏移,线粒体结构松散、空泡状,线粒体嵴断裂,粗面内质网脱颗粒;模型72 h组海马CA1区神经元核固缩,核染色质呈团块状边集于核膜下,线粒体不同程度脱空。免疫组化和Western印迹结果显示:假手术组Apaf-1蛋白仅有少量表达,模型组各时间点Apaf-1蛋白表达均增高(P<0.05)。结论大鼠脑缺血再灌注可导致海马CA1区神经元的凋亡,这可能与凋亡相关基因Apaf-1的激活有关。

Objective To observe hippocampal ultrastructure and expression of Apaf-1 after cerebral ischemia reperfusion in rats.Methods The male SD rats were randomly divided into sham operation and model groups.According to reperfusion time,rats of model were divided into 2,24,72 h subgroups.The global cerebral ischemia-reperfusion models of rats were established by Pulsinelli 4 vessel occlusion.Change of hippocampal neurons ultrastructure was observed by transmission electron microscopy.Immunohistochemistry and Western blot were used to measure the expression of Apaf-1 protein.Results Nucleus of the sham operation group were large,round or oval,clear and complete nuclear membrane.Euchromatin was finely evenly distributed and cytoplasm organelles were rich.Hippocampal CA1 neurons of model 2 h group appeared nuclear membrane introcession and chromatin density increased,as well as mitochondrial slight swelling.Hippocampal CA1 neurons of model 24 h group appeared nuclear membrane serious introcession,nucleoli material was increased and deviated,rarefaction and vacuoles of the mitochondrial structure,break of the mitochondrial cristae,rough endoplasmic reticulum degranulated.Karyopyknosis,mitochondria swelling and vacuolar degeneration chromatin was set of edges the nuclear membrane in the model 72 h group hippocampal CA1 neurons could be seen.Compared with the sham operation group,the expression of Apaf-1 protein in hippocampus every time points were increased obviously in model group by immunohistochemistry and Western blot(P < 0.05).Conclusions Hippocampal neurons apoptosis associated with apoptosis-related gene Apaf-1 activation after global cerebral ischemia and reperfusion in rat.