海马局部脑组织血流量和神经元特征性变化与半胱氨酸天冬氨酸蛋白酶8介导癫痫大鼠的关系

Changes of neuron characteristics and blood flow in local brain tissue in rats with epilepsy induced by caspase-8

目的:检测半胱氨酸天冬氨酸蛋白酶8在癫痫大鼠海马神经元的表达及其与癫痫发作大鼠局部脑血流量和神经元变化的关系。方法:实验于2002-05/2003-10在复旦大学神经病研究所完成。选择清洁级SD雄性大鼠60只,随机分为①正常对照组6只:杏仁核注射磷酸盐缓冲液。②空白对照组6只:磷酸盐缓冲液(脑室)+磷酸盐缓冲液(杏仁核内)。③单纯癫痫组36只:杏仁核内注射红藻氨酸诱发癫痫发作,发作1h后经股静脉注射安定(30mg/kg)终止发作,根据处死大鼠的时间点分0,2,4,8,24,72h6组,每组6只大鼠。④试验给药组6只:半胱氨酸天冬氨酸蛋白酶8抑制剂z-IETD-fmk(脑室,0.3μg于红藻氨酸注射前20min,5min及红藻氨酸注射后1h分3次给药)+红藻氨酸(杏仁核内)。⑤实验对照组6只:磷酸盐缓冲液(脑室)+红藻氨酸(杏仁核内);均于安定注射终止发作后24h处死大鼠。结果评估:①激光多普勒血流测定仪记录局部脑血流。②用脱氧核糖核酸末端转移酶介导的缺口末端标记染色和cresylviolet染色观察海马神经元凋亡和存活的变化及半胱氨酸天冬氨酸蛋白酶8抑制剂z-IETD-fmk对海马CA3区的脱氧核糖核酸末端转移酶介导的缺口末端标记阳性细胞和存活细胞的影响。③用免疫荧光和蛋白印迹检测海马半胱氨酸天冬氨酸蛋白酶8的表达。结果:造模过程中有2只大鼠死亡,

AIM:To detect the expression of caspase 8 in neuron, and observe the relation between caspase 8 and the changes of blood flow and neuron in local brain tissue in seizure rats. METHODS:The experiment was finished in the Institute of Neuropathy of Fudan University from May 2002 to October 2003.Totally 60 SD rats were divided randomly into five groups:①Normal control group(n=6)was injected with phosphate buffer solution(PBS) into nucleus amygdalae.②Blank control group(n=6)was injected with PBS(brain ventricle)+PBS(nucleus amygdalae).③Seizure group(n=36)was injected with kainic acid into nucleus amygdalae to induce seizure,one hour later,diazepam(30 mg/kg) was injected into femoral vein to terminate the seizure.The rats were divided averagely into six groups(n=6) according to the time points of death 0,2,4,8,24 and 72 hours.④Administration group(n=6)was injected with caspase 8 inhibitor z IETD fluoromethyl ketone(z IETD fmk)(brain ventricle,0.3 μg was given at 20,5 minutes before and 1 hour after injection of kainic acid)+kainic acid(nucleus amygdalae).⑤Experimental control group(n=6)was injected with PBS(brain ventricle)+kainic acid(nucleus amygdalae),and rats were killed at 24 hours following injection of diazepam.Evaluation of the results:①Regional cerebral blood flow was recorded with Laser Doppler flowmetry.②The survival and apoptosis of neurons in hippocampus,and the effect of z IETD fmk on positive and surviving cells in CA3 of hippocampus were observed by using Terminal deoxynucleotidyl transferrase mediated dUTP nick end labeling(TUNEL and cresyl violet staining.③The expression of caspase 8 in hippocampus was detected with immunofluorescence and western blot. RESULTS:Two rats died during establishing models,and another two were selected to keep 60 rats.①The rate of cerebral perfusion increased lightly 10-20 minutes following the injection of kainic acid,then decreased gradually during 20-60 minutes,after 60 minutes,the rate of cerebral perfusion kept stable,and there was no significant changes between before and after seizure.②Cleavage of caspase 8 was detected at 0 hour,TUNEL positive neurons appeared at 8 hours and reached to the top at 24 hour,and the surviving neurons decreased following seizure cessation. Intracerebroventricular infusion of caspase 8 inhibitor(z IETD fmk) significantly decreased TUNEL positive neurons and increased the numbers of surviving cells.③The results of immunofluorescence and western blot indicated:immunoreactivity of caspase 8 increased in the ipsilateral CA3 region neurons injected with kainic acid. CONCLUSION:The seizures lead to the activation of caspase 8.Caspase 8 may play a role in the mechanism of neuronal damage in CA3 region of hippocampus induced by seizures,but not related with regional cerebral blood flow.