高血糖对大鼠局灶性脑缺血再灌注时小胶质细胞活化的影响

Effects of hyperglycemia on the activation of microglia in focal cerebral ischemia and reperfusion

目的观察高血糖对大鼠局灶性脑缺血再灌注时小胶质细胞活化的影响。方法健康雄性SD大鼠80只,随机分为正常血糖假手术组(NG sham组,n=5)、高血糖假手术组(HG sham组,n=5)、正常血糖脑缺血再灌注组(NG手术组,n=35)和高血糖脑缺血再灌注组(HG手术组,n=35)。采用链脲佐菌素(STZ)腹腔注射法制作SD大鼠1型糖尿病模型,大脑中动脉阻塞法(MCAO)建立大鼠脑缺血再灌注模型,应用小胶质细胞特异性标志蛋白Iba-1免疫组化标记小胶质细胞,观察NG组和HG组大脑中动脉阻塞30min,再灌注0.5、3、6h以及1、3、7、14d大鼠(每时间点5只)室周带和尾状核区小胶质细胞的变化;采用Iba-1和核增殖抗原(PCNA)免疫荧光双标法检测小胶质细胞的增殖变化。结果脑缺血再灌注后,可见大鼠脑组织明显水肿,呈网格状,HE染色变淡,神经元肿胀,胞质空泡状,胞核固缩,并可见炎细胞浸润,HE染色结果显示HG手术组与NG手术组比较脑损伤更为明显。脑缺血再灌注后第3天,免疫组化染色可见梗死周边区、梨状皮质和躯体感觉皮质的小胶质细胞明显活化,于第7天达到峰值,且其活化状态可以持续到再灌注第14天。免疫荧光双标染色结果显示脑缺血再灌注后小胶质细胞数量增加与其增殖有关,其增殖程度同样是在缺血再灌注后第3天增加,第7天时达高峰。与NG手术组比较,HG手术组小胶质细胞的活化及增殖较弱(P<0.05),但均明显高于各自的假手术组(P<0.05)。结论高血糖导致的缺血后脑组织小胶质细胞活化、增殖抑制可能参与了高血糖加重缺血性脑损伤的过程。

Objective To observe the changes of microglia in diabetic cerebral ischemia and reperfusion, and further explore the role of microglia in diabetic rats with cerebral ischemic injury. Methods Eighty healthy male SD rats were randomly divided into 4 groups： normal blood glucose sham operation group （NG sham group, n=5）, diabetic hyperglycemia sham operation group （HG sham group, n=5）, normal blood glucose with cerebral ischemia-reperfusion group （NG MCAO group, n=35） and diabetic hyperglycemia with cerebral ischemia-reperfusion group [HG middle cerebral artery occlusion （MCAO） group, n=35]. The diabetic rats models were established by intraperitoneal injection of streptozotocin （STZ）. The cerebral ischemia reperfusion models were made with MCAO, the specific marker protein Iba-1 was used to immunohistochemically label the microglia. The changes of microglia in the periventricular zone and caudate putamen region of the rats in HG MCAO group and NG MCAO group were observed at ischemia 30min and reperfusion 30min, 3h, 6h, 1d, 3d, 7d and 14d （each time point contains 5 rats）. Iba-1 and proliferating cell nuclear antigen （PCNA） immunofluorescence double labeling method were performed to detect the proliferation of microglia. Results After ischemia-reperfusion, the brain tissue appeared as obvious edema, mesh-like, HE staining faded, neurons swelled, cytoplasm vacuolization, nuclear pyknosis, and inflammatory cell infiltration. All these symptoms of brain injury were more obvious in HG group than in NG group. On the 3rd day after ischemia reperfusion, microglial cells were markedly activated in the infarct peripheral zone, piriform cortex and somatic sensory cortex, the activation reached the peak value atthe 7th day, and the activated state continued to the 14th day of reperfusion. It was found with Iba-1 and PCNA immunofluorescence double labeling that, after cerebral ischemia-reperfusion, the increase of microglia number was related to its proliferation. The microglia proliferation also increased at the 3rd day after ischemia-reperfusion, and reached the peak value at the 7th day. The degree ofmicroglia activation and proliferation was weaker in NG group than in HG group （P〈0.05）, but higher obviously when compared with their each sham group （P〈0.05）. Conclusion Hyperglycemia induced ischemia brain tissue microglia activation and proliferation inhibition may be involved in the hyperglycemia induced ischemic brain damage.