亚低温对缺氧缺血新生大鼠海马星形胶质细胞增殖和凋亡的影响

摘要

目的 观察亚低温对新生大鼠海马星形胶质细胞增殖和凋亡的影响,以探讨亚低温对缺氧缺血脑损伤保护作用的机制.方法 ①体外实验:取3日龄大鼠海马脑片,培养至第4天用氧糖剥夺法制备标本,于33℃(亚低温组)和37℃培养48 h(常温组);对照组脑片不进行氧糖剥夺处理,37℃培养7 d.采用免疫荧光染色方法观察培养脑片星形胶质细胞的活化和增殖.②体内实验:取7日龄大鼠,分手术组和假手术组.手术组永久性结扎左侧颈总动脉,然后置于含8%O2+92%N2中缺氧2 h,制备缺氧缺血模型,分为手术亚低温亚组和手术常温亚组.假手术组仅分离左侧颈总动脉,不结扎,不予缺氧处理,分为假手术常温亚组和假手术亚低温亚组.各组于术后3和7 d处死取材,采用免疫荧光染色方法检测海马星形胶质细胞的活化、增殖和凋亡.结果①体外实验:氧糖剥夺后3 d常温组较对照组胶质纤维酸性蛋白(GFAP)阳性细胞数量明显增多;亚低温组与常温组比较,GFAP阳性细胞数量明显减少.②体内实验:术后3和7 d亚低温组GFAP阳性细胞数量较常温组显著降低,较常温对照组和亚低温对照组显著增高.GFAP与caspase-3免疫荧光双标记结果显示,术后3 d常温组海马区84.5%GFAP阳性细胞表达caspase-3,亚低温组仅32.3%GFAP阳性细胞表达caspase-3,差异有统计学意义.结论 亚低温能减轻新生大鼠缺氧缺血后海马星形胶质细胞的活化增殖和凋亡.%Objective To study the effects of hypothermia on the proliferation and apoptosis of hippocampal astrocytes in hypoxic-ischemic neonatal rat brain.Methods In vitro: hippocampal slices were prepared from 3-day-old neonatal rat pups and cultured using transwell plate for 4 days prior to experimentation.Tissues were randomly divided into normothermia ( 37℃ ) and hypothermia ( 33℃ , 24 h ) groups after oxygen-glucose deprivation ( OGD ).The proliferation of hippocampal astrocytes was detected by immunofluorescence staining.In vivo: 7-day-old rats were subjected to left carotid artery ligation and followed by 8％ O2 +92％ N2 for 2 h.Animals were randomly divided into hypothermia group ( rectal temperature 32-33℃ for 24 h ) and normothermia group ( 36-37 ℃ for 24 h ).The proliferation and apoptosis of hippocampal astrocytes were detected by immunofluorescence staining.Results In vitro: the number of astrocytes significantly increased after OGD.However,hypothermia decreased the number of astrocytes.There was a significant difference between hypothermia group and control group ( P＜0.01 ).In vivo: compared with control group, the number of astrocytes of hypothermia group significantly decreased ( P ＜0.05 ).consistent with in vitro study.GFAP and caspase-3 using double labelling methods, it was found that hypothermia could decrease the apoptosis of astrocytes after hypoxic-ischemic brain injury.Conclusions Hypothermia can decrease the proliferation and apoptosis of hippocampal astrocytes in the neonatal rat brain after hypoxic-ischemic injury.