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

实验研究是通过在Gerbil中使用瞬态全球缺血模型完成的。成年男性蒙古沙鼠用吸入1.5％的氟烷麻醉。全局缺血是通过使用动脉瘤夹阻塞双侧普通颈动脉（BCCA）来诱导的。直肠和大脑温度保持在36.5至37.5℃，并用加热毯子保持在手术中。动物分为4组。 ：I组（缺血性耐受性组）：在48小时缺血48小时之前，将动物遮住了2分钟。 II组（下缺血组）：在48小时操作之前，将动物遮住2分钟。 III组（复发性缺血组）：在手术48小时后，将动物遮住了5分钟。第四组（假手术组）：48小时的假手术后，该组进行了假手术。在预处理的再灌注间隔中，逐渐消除沙鼠并用10 ml甲基化磷酸盐缓冲固定……更多的盐水，然后用100 m mol/1 mol/1磷酸盐缓冲（pH 7，4）60 ml 4％多聚甲醛。将大脑移除，以10％的福尔马林和石蜡嵌入后固定。在中间海马的水平上收集脑部切片，并染色为苏木精和曙红。将从左右Hipocampi的CA1神经元细胞计数进行平均，并表示为CA1的每毫米计数。与III组（复发性缺血组）相比，I组（缺血性耐受性组）中的动物（缺血性耐受性组）显着增加。使用TUNEL方法在第III组5分钟缺血后2天发现了DNA碎片（复发性缺血组）。此外，使用Velier的方法（Velier JJ，J Neurosci 19：5932-5941，1999）进行了抗Caspase-3的免疫细胞化学化学。免疫细胞化学分析尚未揭示caspase-3蛋白在诱导再现缺血之前。在3、6、12、24小时的缺血5分钟后，在第三组（复位缺血组）中也未诱导caspase-3蛋白。第I组（缺血性）耐受性组的免疫细胞化学分析显示，在5分钟缺血2天后，CASPASE-3蛋白是caspase-3蛋白，但是在III组（复发性缺血组）中，并未在相同的间隔内揭示caspase-3蛋白。较少的