大鼠脑缺血再灌注后大脑皮质小胶质细胞活化及炎症因子的表达

Activation of microglia and expressions of inflammatory cytokines in rat cerebral cortex after ischemia-reperfusion injury

目的检测大鼠缺血再灌注(I/R)后不同时间点大脑皮质小胶质细胞活化情况,同时探究小胶质细胞中的炎症因子白细胞介素6(IL-6)、IL-1β和肿瘤坏死因子α(TNF-α)的表达变化规律。方法通过线栓法制备SD大鼠大脑中动脉栓塞(MCAO)的脑缺血再灌注模型,实验动物随机分为假手术组(SHAM组)、MCAO再灌3 h组(MCAO 3 h组)、MCAO 6 h组、MCAO 12 h组、MCAO 18 h组、MCAO 24 h组和MCAO 72 h组。苏木精-伊红(HE)染色法检测大鼠大脑皮质神经细胞形态改变。Western印迹检测各组大鼠大脑皮质Iba-1的表达。免疫荧光双标记法检测小胶质细胞中炎症因子IL-6、IL-1β和TNF-α的表达水平。结果与SHAM组比较,MCAO各组大鼠皮质的神经细胞损伤严重,Iba-1蛋白表达显著升高(P<0.05)。除IL-1β的表达在MCAO术后3 h与SHAM组比较无显著差异外,其他各MCAO组炎症因子IL-1β、IL-6和TNF-α在小胶质细胞中的表达均显著提高(P<0.05)。此外,3种炎症因子的表达均在MCAO术后24 h达到高峰后开始下降,与MCAO 72 h组相比,差异有统计学意义(P<0.05)。结论 I/R可导致大鼠患侧皮质神经细胞的损伤和小胶质细胞的激活,并导致激活的小胶质细胞中的炎症因子释放增加,且表达高峰存在一致性。

Objective To detect the activation of microglia at different time points in the cortex of rats after ischemiareperfusion injury and investigate the expression pattern of inflammatory factors(IL-6,IL-1β and tumor necrosis factor-α)in activated microglia. Methods The focal cerebral ischemia-reperfusion model was induced by an intraluminal filament embolism. The SD rats were randomly divided into the sham-operated group(SHAM group),the middle cerebral artery occlusion-reperfusion 3 h group(MCAO 3 h group),MCAO 6 h group,MCAO 12 h group,MCAO 18 h group,MCAO 24 h group and MCAO 72 h group. HE staining was employed to detect the damage to the cortex of rats. Western blotting was used to detect the Iba-1 expression in the cerebral cortex of each group. The expression levels of IL-1β,IL-6 and TNF-α in activated microglia were detected by double immunofluorescence labeling. Results Compared with SHAM group,the cerebral cortex neurons in MCAO groups were severely damaged. The expression of Iba-1 protein was increased notably in each group of MCAO compared with the SHAM group. Except the IL-1β in MCAO 3 h group,the expressions of inflammatory factors IL-1β,IL-6 and TNF-α in microglia were significantly augmented in other MCAO groups(P<0.05). In addition,the expression of the three inflammatory factors began to decrease after reaching the peak at 24 h after MCAO,and the difference was statistically significant compared with the MCAO 72 h group(P<0.05). Conclusion Ischemia-reperfusion could cause neuronal damage,activation of microglia in the ipsilateral cortex and lead to increased release of inflammatory factors in activated microglia. The time that the expressions of the three inflammatory factors reach the peak is consistent.