Research on the Mechanism of Ischemic Tolerance-Involvement in Caspase-

采用沙鼠短暂性全身缺血模型进行实验研究。用吸入1.5%氟烷麻醉成年雄性蒙古沙鼠。用动脉瘤夹闭塞双侧颈总动脉（BCCAs）引起全身缺血。用加热毯将动物的直肠和脑温度保持在36.5 ~ 37.5℃，直到动物从手术中取出。实验动物分为4组。ⅰ组（缺血耐受组）：缺血5min 48h前，阻断bcca 2min。II组（颏下缺血组）：假手术前48 h，关闭bcca 2 min。III组（Rethal缺血组）：假手术48 h后，关闭bcca 5 min。第四组（假手术组）：假手术48 h后行假手术。在预先设定的再灌注间隔，沙鼠被麻醉并灌注固定在10ml肝素化磷酸盐缓冲液中，然后用100m mol/1磷酸盐（pH 7,4）缓冲60ml 4%多聚甲醛。取下脑，用10%福尔马林和石蜡包埋后固定。在海马中背水平采集脑切片，苏木精和伊红染色。取左右海马体存活的CA1神经细胞计数平均值，并以每毫米CA1计数表示。与缺血组相比，ⅰ组（缺血耐受组）动物的CA1神经存活明显增加。ⅲ组（Rethal缺血组）缺血5 min后2 d，采用TUNEL法观察DNA断裂。此外，采用Velier's方法进行Caspase-3抗体免疫细胞化学（Velier JJ, J Neurosci 19: 5932-5941, 1999）。免疫细胞化学分析未发现诱导缺血前Caspase-3蛋白的表达。缺血3、6、12、24小时（5 min）后，ⅲ组（Rethal缺血组）也未诱导Caspase-3蛋白表达。免疫细胞化学分析I组（缺血耐受组）在缺血5min 2天后显示Caspase-3蛋白，而III组（Rethal缺血组）在相同时间间隔内未显示Caspase-3蛋白。少

Experimental study was done by using transient global ischemic model in gerbil. Adult male Mongolian gerbils were anesthetized with inhalation 1.5 % halothane. Global ischemia was induced by occluding bilateral common carotid arteries (BCCAs) by using aneurysm clip. The rectal and brain temperature was maintained at 36.5 to 37.5 ℃ with a heating blanket until the animals removed from surgery. Animals were divided into 4 groups. : Group I (Ischemic tolerance group) : Before 48 hours of 5 min ischemia, animal were occleded BCCAs for 2 min. Group II (Subrethal ischemic group) : Before 48 hours of sham operation, animal were occleded BCCAs for 2 min. Group III (Rethal ischemic group) : After 48 hours of sham operation, animal were occleded BCCAs for 5 min. Group IV (Sham operation group) : After 48 hours of sham operation, sham operation was done in this group. At the predetrmined reperfusion intervals, gerbils were anethetized and perfusion fixed with 10 ml heparinized phosphate-buffered … More saline followed by 60 ml 4 % paraformaldehyde buffered with 100 m mol/1 phosphate (pH 7, 4). The brains were removed, postfixed in 10 % formalin and paraffin embedded. Brain sections were collected at the level of middorsal hippocampus and stained for hematoxylin and eosin. Surviving CA1 neurinal cell counts from the left and right hipocampi were averaged and expressed as counts per millimeter for CA1. Animals in Group I (Ischemic tolerance group), were showed a significant increase in CA1 neurinal survival compared with those in Group III (Rethal ischemic group). DNA fragmentation by using TUNEL method, was found at 2 day after 5 min ischemia in Group III (Rethal ischemic group). Furthermore, immunocytochemistry with antibody against Caspase-3 was done by using Velier's method (Velier JJ, J Neurosci 19 : 5932-5941, 1999). Immunocytochemical analysis has not revealed Caspase-3 protein before inducing rethal ischemia. After 3, 6, 12, 24 hours of 5 min ischemia, Caspase-3 protein was also not induced in Group III (Rethal ischemic group). Immunocytochemical analysis in Group I (Ischemic tolerance group), revealed Caspase-3 protein after 2 days of 5 min ischemia, however that in Group III (Rethal ischemic group) did not reveal Caspase-3 protein at the same interval. Less