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

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 12670624
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Neurology
• Research Institution: Nippon Medical School
• Principal Investigator: KATAYAMA Yasuo Nippon Medical School, Department of Internal Medicine, Professor and Chairman, 医学部, 教授 (70152692)
• Co-Investigator(Kenkyū-buntansha): KAMIYA Tatsushi Nippon Medical School, Department of Internal Medicine, Assistant Professor, 医学部, 講師 (70233955)
• Project Period (FY): 2000 – 2001
• Keywords: Ischemic tolerance / Gerbil / Grobal ischemia / Caspase-3 / In situ hybridization / Immunocytochemistry

Research Abstract

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 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.