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

利用沙鼠短暂性全身缺血模型进行实验研究。成年雄性沙鼠吸入1.5%氟烷麻醉。使用动脉瘤夹闭塞双侧颈总动脉（BCCA）诱导全身缺血。用加热毯将直肠和脑温度维持在36.5至37.5℃直至动物从手术中取出。将动物分为4组。 ：I组（缺血耐受组）：在5分钟缺血的48小时之前，将动物闭塞BCCA 2分钟。 II组(视网膜下缺血组)：在假手术48小时之前，将动物的BCCA闭塞2分钟。第三组(Rethal缺血组)：假手术48小时后，将动物闭塞BCCA 5分钟。第四组(假手术组)：本组在假手术48小时后进行假手术。在预定的再灌注间隔，将沙鼠麻醉并用 10 ml 肝素化磷酸盐缓冲盐水固定灌注，然后用 60 ml 4% 多聚甲醛（用 100 m mol/1 磷酸盐缓冲）（pH 7, 4）。取出大脑，后固定在 10% 福尔马林中并包埋石蜡。在中背海马水平收集脑切片并进行苏木精和伊红染色。对左海马和右海马的存活 CA1 神经元细胞计数进行平均并表示为 CA1 每毫米的计数。与第 III 组（Rethal 缺血组）相比，第 I 组（缺血耐受组）的动物 CA1 神经元存活率显着增加。第III组（Rethal缺血组）在缺血5分钟后第2天使用TUNEL方法发现DNA片段化。此外，通过使用Velier法(Velier JJ，J Neurosci 19：5932-5941，1999)进行使用针对Caspase-3的抗体的免疫细胞化学。免疫细胞化学分析未发现诱导视网膜缺血前有 Caspase-3 蛋白。 5分钟缺血3、6、12、24小时后，第III组(Rethal缺血组)也没有诱导Caspase-3蛋白。第 I 组（缺血耐受组）中的免疫细胞化学分析在 5 分钟缺血 2 天后显示出 Caspase-3 蛋白，但在第 III 组（Rethal 缺血组）中，在相同时间间隔内未显示出 Caspase-3 蛋白。较少的