杏仁核内注射KA诱导的边缘叶发作大鼠海马中caspase-9的表达

目的 :红藻氨酸 (kainicacid ,KA)诱导的大鼠边缘叶发作模型 ,检测半胱氨酸天冬酶 9(caspase 9)在癫大鼠海马神经元表达。方法 :立体定位杏仁核注射KA诱导癫发作 ,以持续记录脑电、局部脑血流 (regionalcerebralbloodflow ,r CBF) ,用TUNEL染色和cresylviolet染色观察海马神经元存活和凋亡 ;用免疫荧光和Westernblot检测海马caspase 9的表达。结果 :发作终止 4h时caspase 9出现裂解片段 ,8h时出现TUNEL阳性细胞 ,2 4h时达高峰。脑室内注射caspase 9抑制剂z LEHD fluoromethylketone(z LEHD fmk)可减少TUNEL阳性细胞 ,增加存活神经元 ;发作后在KA注射同侧海马的CA3区神经元caspase 9免疫反应性增强 ;对侧海马未见TUNEL阳性细胞及caspase 9的上述变化。发作前后r CBF无明显变化。结论 :癫发作可诱导caspase 9的激活。caspase 9可能是癫潜在的治疗靶点。

The Expression of Caspase-9 in Hippocampus of Following Limbic Seizure Induced by Kainic Acid Injection into Amygdaloid Nucleus

Aim:To establish the focal limbic seizure model to explore seizure-induced changes in caspase-9 protein expression and the effect of caspase-9 inhibition in vivo.Methods:Animals received intra-amygdaloid injection of kainic acid (KA) to induce seizure under monitoring with continuous electroencephalographic (EEG) and Laser-Doppler Flowmetry for 1 h,after then diazepam (30 mg/kg) was administered to terminate the seizure.The apoptotic and surviving neurons in hippocampus were observed by terminal deoxynucleotidyl transferrase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, caspase-9 was detected with immunofluoresence and Western blot.Results:Cleavage of caspase-9 was detected at 4 h,TUNEL-positive neurons appeared at 8 h and reached maximal at 24 h following seizure cessation within the ipsilateral CA3 subfield of hippocampus.Intracerebroventricular infusion of caspase-9 inhibitor z-LEHD-fluoromethyl ketone (z-LEHD-fmk) significantly decreased TUNEL-positive neurons and increased the numbers of surviving cells. Caspase-9 immunoreactivity increased within the ipsilateral CA3 neurons.TUNEL-positive neurons and the alterations of the expression of caspase-9 within the ipsilateral CA3 were not detected within the contralateral hippocampus.There was little change in r-CBF after the seizure.Conclusion:Seizures lead to the activation of caspase-9,caspase-9 may play a role in the mechanism of brain damage induced by seizures,but not related with r-CBF.Caspase-9 is possibly a potential therapeutic target in the treatment of brain injury associated with seizures.