急性脑缺血/再灌注损伤后神经元细胞骨架蛋白的动态变化

Dynamic changes in cytoskeletal elements following acute cerebral ischemia and reperfusion in rats

目的探讨急性脑缺血/再灌注大鼠神经元细胞骨架蛋白时空动态变化。方法线栓法阻断大鼠大脑中动脉90 min后实现再灌注,在不同再灌注时间点观察及取材。尼氏染色观察神经细胞损伤,采用神经功能缺损评分和前肢放置实验评估神经功能;免疫组化染色、免疫印迹法观察细胞骨架成分微管相关蛋白2(microtubule associated protein 2,MAP2)、神经丝重链(neurofilament heavy chain,NF-H)的变化;透射电镜观察轴突、树突和神经丝亚显微结构。结果随着再灌注时间的延长,脑损伤和神经行为功能损害逐渐加重。纹状体损伤比皮层出现的更早、更严重。缺血区域的MAP2相关免疫反应强度降低,NF-H相关免疫反应强度升高。超微结构观察显示细胞骨架排列受损,密度降低。结论不同脑区对缺血/再灌注损伤的耐受性不同。神经元细胞骨架的主要成分对缺血和再灌注表现出动态反应,这可能进一步促进脑损伤和神经功能缺损。

Aim To investigate the dynamic time-course changes in neuronal cytoskeleton after acute ischemia and reperfusion in rats.Methods Reperfusion was performedin rats by blocking the middle cerebralarteryfor 90 min,then therats wereobserved and collected at different time points.The brain damage was observed by Nissl staining,and neurobehavioural function was evaluated with neurological deficit score and forelimb placement test.The cellular changes in the alternations of cytoskeletal elements including microtubule associated protein 2(MAP2)and neurofilament heavy chain(NF-H)were observed by immunohistochemistry staining and Western blot.Impaired axons,dendrites and cytoskeletal alternations were detected by electron microscope.Results Brain damage and neurobehavioural function were gradually aggravated with the prolongation of reperfusion.Brain damage appeared earlier and more severe in striatum than in cortex.Moreover,decreased MAP2-related and increased NF-H-related immunoreactive intensities were found in the ischemic areas.Impaired cytoskeletal arrangement and reduced dense were indicated.Damaged cytoskeletal components such as microtubules and neurofilament arrangement,decreased axonal filament density,and swelled dendrites were observed after cerebral ischemia reperfusion by ultrastructural observations.Conclusions Different brain regions have diverse tolerance to ischemia-reperfusion injury.Major elements of neuronal cytoskeleton show dynamic responses to ischemia and reperfusion,which may further contribute to brain damage and neurological impairment following MCAO and reperfusion.