内毒素预处理对大鼠急性局灶脑缺血的影响

Influence of endotoxin preconditioning on acute focal cerebral ischemic injury in rats

目的:观察经内毒素预处理后急性局灶脑缺血大鼠脑梗死体积、凋亡细胞数和凋亡抑制基因Bcl-2蛋白表达的变化。探讨内毒素预处理对急性局灶脑缺血损伤大鼠脑功能的保护作用。方法:实验于2004-11/2005-02在沈阳市第五人民医院动物室完成。采用HaruoNagasawa法制作大鼠局灶脑缺血模型。将48只健康成年Wistar大鼠随机分为3组:生理盐水对照组、内毒素预处理组、缺血预处理组,每组16只。内毒素预处理组大鼠在局灶脑缺血模型制备前3d,皮下注射内毒素0.05mg/kg;缺血预处理组大鼠在第一次缺血预处理再灌注3d后再次麻醉大鼠,把插线再次推进到第一次插线深度,停留60min后,将线退出;生理盐水对照组大鼠在局灶脑缺血模型制备前3d,皮下注射生理盐水0.2mL。各组大鼠在局灶脑缺血1h再灌注23h后断头取脑,红四氯氮唑染色观察大鼠脑梗死的体积;多聚甲醛固定,石蜡包埋,连续切片,原位细胞凋亡检测法观察神经细胞凋亡情况,免疫组化染色观察Bcl-2蛋白表达的变化。结果:各组大鼠在实验过程中无死亡,均进入结果分析。①红四氯氮唑染色显示缺血预处理组和内毒素预处理组的梗死体积较生理盐水对照组明显减小,差异有显著性意义[(64±14.74),(71.3±10.35),(159±9.79)mm3,P<0.01]。②缺血预处理组和内毒素预处理组仅可见散在的凋亡细胞出现于皮质、胼胝体及基底节区。生理盐水对照组梗死灶周围的半暗带内可见大量神经元胶质细胞及部分血管内皮细胞发生凋亡。缺血预处理组和内毒素预处理组梗死灶周围的凋亡细胞数较生理盐水对照组明显减少[(33.5±7.45),(35.6±4.18),(57.88±0.96)个/切片,P<0.01]。③缺血预处理组和内毒素预处理组缺血侧大脑半球Bcl-2阳性细胞数明显增加,生理盐水对照组在缺血侧梗死周边区Bcl-2蛋白表达也增加。但缺血预处理组和内毒素预处理组缺血侧大脑半球Bcl-2阳性细胞数比生理盐水对照组增加明显[(111.38±13.59),(105.88±9.99)(47.5±11.43),P<0.01]。结论:小剂量内毒素预处理可以通过调节细胞内凋亡抑制基因Bcl-2蛋白的表达诱导抗凋亡,启动内源性保护机制,产生缺血耐受,从而减少梗死面积。

AIM： To investigate the change of infarct volume, the number of neuronal apoptosis and the expression of apoptosis-suppressor gene Bcl-2 protein after preconditioning with endotoxin in rats with acute focal cerebral ischemia, and explore the protective effect of endotoxin preconditioning on brain function of rats with acute focal cerebral injury. METHODS： The experiment was carried out in the Animal laboratory of Fifth People＇s Hospital of Shenyang from November 2004 to February 2005. Rat focal brain ischemia model was made by modified Haruo Nagasawa method. Totally 48 healthy adult Wistar rats were randomly divided into three groups： saline control group, endotoxin preconditioning group and iachemic preconditioning group with 16 rats in each group. The rats in endotoxin preconditioning group received 0.05 mg/kg endotoxin by hypodermical injection at 3 days before the preparation of focal cerebral ischemic models. The rats in the ischemic preconditioning group were drugged once again after 3-day ischemic preconditioning reperfusion. The plug wire was inserted into the same depth as the first time for 60 minutes, and then withdrew it. The rats in the saline control group were treated with 0.2 mL saline by hypodermical injection at 3 days before the preparation of focal cerebral ischemic models. After 1-hour focal ischemia followed by 23 hours reperfusion, the rats were all killed to gain brains. Cerebral infarction volumes were observed by TI＇C staining; post_fixed with paraformaldehyde, and then embedded in pafaffin. The rat brains were cut into sections. Neuronal apoptosis was observed with in situ apoptosis determination. Expression of Bcl-2 protein was observed with immunohistochemical staining. .RESULTS： All the rats were involved in the result analysis, without dropout. ①The infarct volume reduced significantly in the ischemic preconditioning group and endotoxin preconditioning group than the saline control group, which had significant difference [（64±14.74）, （71.3±10.35）, （159 ±9.79） mm^3,P 〈 0.01]. ②There were sporadic apoptosis appeared in cortex, corpus callosum and basal ganglia in the ischemic preconditioning group and endotoxin preconditioning group. Apoptosis of a mass of neuronal glial cells and partial vascular endothelial cells occurred at semidarkness region around infarcted focus of the saline control group. Number of apoptosis around infarcted focus in the ischemic preconditioning group and endotoxin preconditioning group significantly reduced as compared with that in the saline control group [（33.5±7.45）, （35.6±4.18）, （57.88±0.96） in each section,P 〈 0.01]. ③Number of Bcl-2 positive cells increased at the cerebral hemisphere of ischemic side in the ischemic preconditioning group and endotoxin preconditioning group, so did that around infarcted region of ischemic side in the saline control group. Number of Bcl-2 positive cells at the cerebral hemisphere of ischemic side in the ischemic preconditioning group and endotoxin preconditioning group increased significantly as compared with that in the saline control group [（ 111.38±13.59）, （105.88±9.99）, （47.5±11.43）, P 〈 0.01]. CONCLUSION： Small dose of endotoxin preconditioning may induce anti-apoptosis by regulating the expression of apoptosis-related gene Bcl-2 protein, and then activate endogenously neuroproteetive mechanism, produce isehemic tolerance so as to reduce infarct size.