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

缺血耐受机制研究-Caspase参与-

• 批准号: 12670624
• 负责人: KATAYAMA Yasuo
• 金额: $ 1.86万
• 依托单位: Nippon Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670624/
• 关键词: Ischemic tolerance; Gerbil; Grobal ischemia; Caspase-3; In situ hybridization; Immunocytochemistry; In situ hybridizaion

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

采用沙土鼠短暂性全脑缺血模型进行实验研究。成年雄性长爪沙鼠吸入1.5%氟烷麻醉.采用动脉瘤夹夹闭双侧颈总动脉（BCCAs）造成全脑缺血模型。使用加热毯将直肠和脑温维持在36.5 - 37.5 ℃，直至动物从手术中取出。动物分为4组。第一组（缺血耐受组）：缺血5 min前48 h，阻断BCCAs 2 min（亚临界缺血组）：假手术前48小时，将动物的BCCAs封闭2分钟。（视网膜缺血组）：假手术48小时后，阻断BCCAs 5分钟。（假手术组）：假手术48 h后，该组再行假手术。在预定的再灌注时间间隔，沙鼠被麻醉，并用10 ml肝素化磷酸盐缓冲液灌注固定。 ...更多信息 生理盐水，然后加入60 ml用100 mmol/l磷酸盐缓冲的4%多聚甲醛（pH 7.4）。取出脑，在10%福尔马林中后固定并石蜡包埋。在背中海马水平收集脑切片，并用苏木精和伊红染色。对左右海马存活的CA 1神经元细胞计数取平均值，并表示为CA 1每毫米的计数。I组（缺血耐受组）动物的CA 1神经元存活率明显高于III组（缺血再灌注组）。缺血5 min后第2天，TUNEL法检测到缺血组（Ⅲ组）脑组织DNA断裂。此外，通过使用Velier的方法（Velier JJ，J Neurosci 19：5932-5941，1999）用抗Caspase-3的抗体进行免疫细胞化学。免疫细胞化学分析未发现Caspase-3蛋白在诱导大鼠肾缺血前表达。缺血5 min后3、6、12、24 h，再灌注组（Ⅲ组）Caspase-3蛋白也无表达。免疫细胞化学分析显示，缺血耐受组（I组）在缺血2d后5 min有Caspase-3蛋白表达，而缺血再灌注组（III组）在缺血2d后5 min无Caspase-3蛋白表达。少