大鼠局灶性脑缺血再灌注早期DNA单链断裂损伤及神经元形态变化

Single-strand DNA breaks and morphological change of neurons at early stage of rats focal cerebral ischemia/reperfusion

目的 探讨大鼠大脑中动脉阻塞再灌注 (MCAO/ R)早期 DNA断裂损伤的情况及其意义。方法 使用尼氏染色方法观察再灌注早期损伤区域神经元数量和形态的变化 ,使用 DNA聚合酶 - Klenow片段原位检测 DNA的断裂损伤。结果 再灌注 6 h损伤区域部分神经元出现形态变化时 ,可见少量 Klenow阳性细胞 ;再灌注2 4 h损伤区域大量神经元出现形态变化时 ,Klenow阳性细胞数量陡然显著增加 (P<0 .0 1) ;再灌注 72 h损伤区域神经元数量明显减少时 ,Klenow阳性细胞数量较 2 4 h时有所下降 (P<0 .0 1)。结论 (1)局灶性脑缺血再灌注 2 4 h时 ,数量达到峰值的 Klenow阳性细胞中多数应为单链 DNA断裂的细胞 ,并推测这一损伤形式可能是由自由基引发的一系列 DNA损伤过程中的一个重要环节 ;(2 )单链 DNA断裂大量产生的同时 ,损伤区域大量神经元出现病理形态变化 ,说明神经元的不可逆损伤过程在单链 DNA断裂时可能就已经发生。

Objective To discuss the DNA strand breaks at early stage of middle cerebral artery occlusion/reperfusion(MCAO/R). Methods Neurons number and morphologic change were observed through Nissl staining method,and DNA strand breaks were in situ detected by DNA polymerase ⅠKlenow fragment mediated nick end labeling method. Results At 6h after reperfusion,a few neurons in damaged regions appeared morphologic changes while a few Klenow positive cells were detected (P<0.01). At 24h after reperfusion,lots of neurons showed morphologic change while the number of Klenow positive cells immediately and remarkably increased (P<0.01). At 72h after reperfusion,the number of neurons decreased significantly and Klenow positive cells were also less than those at 24h (P<0.01). Conclusion (1)24h after reperfusion when the total number of Klenow positive cells reaches the peak,DNA single strand breaks (SSBs) take place in many Klenow positive cells,and DNA SSBs are presumed to be an important step in DNA damage procession which might be induced by free radicals. (2)At the same time when lots of DNA SSBs are produced,many neurons in the damaged regions show morphological change,which indicates that lots of neurons have been irreversibly damaged when DNA SSBs take place.