Experimental Studies On Apoptotic Injury And Its Relations With Thrombin In Intracerebral Hemorrhage In Rats

Background and Purpose: The appropriate therapy for intracerebral hemorrhage( ICH) to reduce disability rate and mortality rate remains a subject of debate.In the past, It was believed that the primary mechanisms of neural injury in ICH were due to the massive effects of hematoma and secondary brain edema.So,the main therapy for ICH was clot evacuation and desiccation to maintain intracranial pressure. However,the curative effect is disappointing. Other mechanisms of neural injury must be invovled in ICH. Yet, in comparison with ischemia, relatetively few experimental investigations have been devoted to studying the pathophysiology of ICH. Apoptosis was involved in the occurence and development of many diseases. Previous studies have demonstrated that in ICH there was a "penumbra" around hematoma similar to that existed in cerebral ischemia and that components in hematoma such as thrombin and hemoglobin could induce apoptosis in cell culture. However, it is poorly understood whether apoptosis was involved in ICH,and it has not been reported whether thrombin released after ICH induced neural cells apoptosis, although thrombin can act as a neurotoxicitic medium.So,the purpose of this study is: ① to investigate whether apoptotic injury exists in perihemotoma in ICH in rats and to explore mechanisms of neural injury else in ICH; ② to analyze the rule of apoptosis change and to search "time window" for anti-apoptotic injury therapy after ICH,if apoptoticinjury exists in ICH;③ to investigate the relationship between thrombin and apoptosis in ICH and to find new therapy target for ICH. Methods:It was devided into four parts:Ⅰ.ICH model of rats was established with 50μl tail blood injected into caudoputamen.Rats were killed respectively at 6h,12h,1d,3d,1w,2w,3w,4w after injection,and neurologic impairment score of each rat was measured before killing. TUNEL,flow cytometry, transmission electron microscopy,and immunohistochemistry technology to probe protein Caspase-3 or protein Bax were performed to detect apoptosis around hematoma at different time points,and the rule of apoptosis change after ICH was analysed. Ⅱ. At 1d,3d,and 7d after 10U hirudin following 50μl tail blood was infused in caudate nucleus,rats were killed. TUNEL positive cell and apoptosis associated protein Caspase-3 and Bax were detected at different time points. Ⅲ.Three groups of animals were studied.In the first group ,each rat received an infusion of 10U thrombin; in the second group,10U hirudin following 10U thrombin was infused into caudate nucleus;Control rats received injection of saline only.rats of each group were killed at 12h,1d,3d,1w after injection;then,TUNEL positive cells,protein Caspase-3 and protein Bax were assessed for each rat. Ⅳ.Primary cultures of neuron were prepared from the brain cortex of 1-to 2-day-old rats. Survival neurons were detected by using toluidine blue staining 3 days after neurons were cultured in normal,thrombin containing,or thrombin and hirudin containing culture medium,the expression of protein Caspase-3 and TUNEL positive cells in survival neurons was also detected. Results:1. Neurologic impairment in various degree appeared in each rat after ICH;At 12h,TUNEL positive cells appeared in ICH model and were also present for more than 4 weeks,peaking at 3d; Protein Caspase-3 and Bax were expressed at 6h after ICH,peaking at 1d andlasting for over 2 weeks;The rate of apoptosis in perihematoma was higher than that in control group examined by FCM;Apoptotic morphology change such as karyopyknosis and chromatin margining was found under TEM. 2.After interference by 10U hirudin, neurologic impairment of each group was lessen and the expression of TUNEL positive,protein Caspase-3 and Bax decreased in groups 1d,3d,and 7d after ICH(P(0.01 or P(0.05)3.When 10U thrombin was injected into caudate nucleus,partial rats showed neurologic impairment symptom;TUNEL positive cells appeared at 12h,peaking at 3d,and there were still many TUNEL positive cells at 1w,the amount of TUNEL pos...