脳虚血後の神経細胞傷害と傷害抑制に関する研究 -アポトーシスおよび非アポトーシス性傷害の検討-

2002 Fiscal Year Final Research Report Summary

• Project/Area Number: 12671475
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Anesthesiology/Resuscitation studies
• Research Institution: Yamaguchi University
• Principal Investigator: FUKUDA Shirou Yamaguchi Univ. Hospital, Research Associate, 医学部附属病院, 助手 (70322245)
• Co-Investigator(Kenkyū-buntansha): MATSUMOTO Mishiya Yamaguchi Univ. Hospital, Assistant Professor, 医学部附属病院, 講師 (60243664) ; SAKABE Takefumi Yamaguchi Univ. School of Medicine, Professor, 医学部, 教授 (40035225)
• Project Period (FY): 2000 – 2002
• Keywords: Ras / apoptosis / forebrain / ischemia-reperfusion / immunoblotting / manumycin A / neuron / hippocampus

Research Abstract

It has been noticed that delayed neuronal death after brain ischemia has a certain effect on apoptosis. Ras, one of the factors in intracellular signal transduction, is reported as an inhibitor of apoptosis, intermediating by PI 3-kinase and Akt. In this study, we have investigated the role of Ras in delayed neuronal cell death, based on the presence of apoptosis in hippocampus after ischemia-reperfusion by the model of rat forebrain ischemia.
#Experiment 1.
METHODS; we utilized the forebrain ischemia (15 minutes) model of male Wistar rat. As ischemic condition, temporary occlusion of bilateral carotid arteries was followed by desanguative hypotension. Histochemical study was performed by HE staining and TUNEL staining. In addition, the amounts of Ras, PI 3-kinase, Akt, Bcl-2, and breakdown product of a-fodrin were surveyed by immunoblotting method.
RESULTS; significant decreases of the amounts of Ras, PI 3-kinase, Akt, and Bcl-2 were shown 3 days after ischemia-reperfusion comparing to c ontrol group. Breakdown product of a-fodrin increased significantly. Neuronal cells in hippocampus revealed vacuolization and pyknosis of nucleus in the brain sample by HE staining, stained-positive in the same position by TUNEL staining.
#Experiment 2
METHODS; in the forebrain ischemia (10 minutes) model, manumycin A was injected intracerebroventricularly before ischemia, and sacrificed 3 days after reperfusion. The hippocampi were stained by HE staining and TUNEL method, the quantities their breakdown products of a-fodrin were measured by immunoblotting. As the control group, physiological saline or 2% DMSO were injected intracerebroventricularly before ischemia-repersion.
RESULTS; manumycin A injected group revealed that the hippocampal neurons injured less than the control group in every methods.
#CONCLUSIONS
The degradation of both Ras and Bcl-2 in forebrain ischemia might suggest the regulation of apoptosis in delayed neuronal cell death, the neuronal cell death in hippocampus might be non-apoptotic judging from the result of the experiment 2.