急性脑缺血/再灌注后小鼠皮层和海马部位神经细胞损伤的动态表征

Dynamic characterization of neuronal injury in cortex and hippocampus of mice after acute cerebral ischemia/reperfusion

目的动态表征急性脑缺血/再灌注(ischemia/reperfusion,I/R)后小鼠皮层和海马部位神经细胞的损伤情况。方法取体质量为25~28 g的雄性C57BL/6J小鼠,线栓法阻断小鼠的大脑中动脉,1 h后进行再灌注,制备急性I/R损伤小鼠模型。实验为Sham组、I/R-6 h组、I/R-24 h组和I/R-72 h组。采用Longa神经功能评分评估各时间点小鼠的神经功能;TTC染色检测脑梗死体积;苏木精-伊红染色观察脑组织病理损伤;尼氏染色检测神经细胞损伤;免疫荧光组织化学染色检测星形胶质细胞和小胶质细胞的活化情况,以及成熟神经元的损伤情况;透射电镜检测海马神经元的线粒体损伤;Western blot检测线粒体分裂-融合相关蛋白p-Drp1/Drp1、Mff、Fis1和OPA1的表达情况。结果随着脑I/R时间的延长,小鼠的神经功能损伤、脑梗死体积,皮层和海马部位的神经细胞损伤、胶质细胞活化、神经元缺失、线粒体损伤逐渐加重;线粒体分裂相关蛋白表达逐渐增加,而线粒体融合相关蛋白表达逐渐降低。结论随着脑I/R时间的延长,胶质细胞活化、神经元缺失、线粒体损伤等神经病理损伤逐渐加重,这可能与线粒体动力学失衡有关。

Aim To dynamically characterize neuronal damage in the cortex and hippocampus of mice following acute cerebral ischemia/reperfusion(I/R).Methods Male C57BL/6J mice weighing 25-28 g underwent middle cerebral artery occlusion using the filament method,followed by 1 hour of reperfusion to establish the acute cerebral I/R injury mouse model.The experiment comprised a sham surgery group,I/R-6 h group,I/R-24 h group,and I/R-72 h group.Longa neurological function score was used to assess the neurological function.Triphenyltetrazolium chloride(TTC)staining was conducted to detect cerebral infarct volume.Hematoxylin and eosin(HE)staining was utilized to observe brain tissue pathological damage.Nissl staining was performed to evaluate neuronal damage.Immunofluorescence histochemistry staining was employed to assess the activation of astrocytes and microglia,as well as neuronal loss.Transmission electron microscopy was used to examine mitochondrial damage in hippocampal neurons.Western blot analysis was conducted to detect the expression levels of mitochondrial fission-fusion-related proteins p-Drp1/Drp1,Mff,Fis1,and OPA1.Results With prolonged cerebral I/R time,neurological functional impairment,cerebral infarct volume,neuronal damage in the cortex and hippocampus,glial cell activation,neuronal loss,and mitochondrial damage gradually worsened in mice.The expression of mitochondrial fission-related proteins increased gradually,while the expression of mitochondrial fusion-related proteins decreased gradually.Conclusions Neuronal pathological damage,such as glial cell activation,neuronal loss,and mitochondrial damage,is gradually aggravated with prolonged cerebral I/R time,which may be associated with mitochondrial dynamics imbalance.