锂-匹罗卡品致痫模型海马星形胶质细胞缝隙连接研究

Astrocytic gap junction in the hippocampus of rats with lithium pilocarpine-induced epilepsy

目的应用锂-匹罗卡品致痫动物模型研究急性癫痫持续状态(status epilepticusSE)及继发性慢性自发性发作海马星形胶质细胞的缝隙连接的改变。方法 60只SD大鼠分为实验组与对照组,应用锂-匹罗卡品腹腔注射诱发大鼠急性SE。实验组又分为8个亚组,分别为出现急性SE后2、12、24h,3、7、15、30、60d组,分属急性期(急性SE24h内)、静止期(急性SE后3～15d)和慢性期(急性SE后30～60d),应用尼氏染色观察各期大鼠海马病理改变,用免疫组化检测胶质原纤维酸性蛋白(GFAP),缝隙连接蛋白43(CX43)表达以了解各期大鼠海马各区星形胶质细胞反应及其缝隙连接的改变。结果锂-匹罗卡品腹腔注射后,诱发大鼠急性SE持续6～30h后进入静止期,存活大鼠30～45d后出现慢性自发性发作。尼氏染色显示致痫后12～24h可见明显海马神经元损害,7d后海马各区开始出现反应性胶质细胞增多,以后持续性加重,到观察终点60天最明显。致痫后7dGFAP免疫阳性反应开始增强,30d时较前更明显,60d最明显。CX43在对照组大鼠海马显示散在分布免疫阳性细胞;致痫后2h,CA1区与CA3区的分子层与起层出现散在曲张静脉状阳性突起;12h突起增多增粗,24h最明显(P<0.05),CA1、CA3区比齿状回明显(P<0.05),进入静止期3～7d逐渐下降,15d与对照组无区别,慢性期30～60d未见有明显阳性细胞及突起。结论急性SE星形胶质细胞的缝隙连接增强,有助于维持胞外微环境的稳定;慢性颞叶癫痫动物模型海马星形胶质细胞缝隙连接缺陷,可能是引起慢性自发性发作的因素之一。

Objective To investigate the changes in the gap junction of the hippocampal astrocytes of a rat model of lithium pilocarpine-induced epilepsies. Methods Lithium chloride and pilocarpine were injected intraperitoneally in SD rats to induce status epilepticus （SE）. At 2,12,24 h and 3,7,15,30 and 60 days after SE,the rats were sacrificed to observe the pathological changes in the hippocampus using Nissl staining. Immunohistochemistry for GFAP and connexin43 （CX43） were used to evaluate reactive astrogliosis and the changes in the astrocytic gap junctions. Results SE induced by lithium and pilocarpine lasted for 6-30 h,and after a seizure-free period of about 30-45 days,the rats developed spontaneous recurrent seizures. Nissl staining showed that the most obvious neuronal damage occurred 12-24 h after seizure onset. Reactive gliosis began to be progressively prominent after 7 days till the end of the observation. GFAP expression increased 7 days after SE induction,intensified at 30 days,and became the most prominent at 60 days. In control rats,CX43 immustaining was diffuse in the hippocampus; in the epileptic rats,diffuse CX43-positive varicosities appeared in the molecular layer and stratum oriens of the CA1 and CA3 areas 2 after seizure onset,and the number,length and immunostaining intensity of the varicosities increased at 12 h. These changes became the most prominent at 24 h after seizure onset,followed by gradual decrease of the immunoactivity,which was virtually absent till 30 and 60 days. Conclusion The hippocampal astrocytic gap junction coupling increases in acute SE to maintain the stability of the extracellular microenvironment. The defects in gap junction coupling of the astrocytes in chronic temporal lobe epilepsy may contribute to the development of spontaneous seizures.