Research on the Mechanism of Thrombin-induced Apoptotic Neuronal Cell Death hi Ischemia

2002 Fiscal Year Final Research Report Summary

• Project/Area Number: 13670672
• 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: KAMIYA Tatsushi Nippon Medical School, Department of Internal Medicine, Assistant Professor, 医学部, 講師 (70233955)
• Co-Investigator(Kenkyū-buntansha): KATAYAMA Yasuo Nippon Medical School, Department of Internal Medicine, Professor and Chairmann, 医学部, 教授 (70152692)
• Project Period (FY): 2001 – 2002
• Keywords: Thrombin / Rat / Focal Ischemia / Apoptosis / Neuroprotection / Immunocytochemistry / In situ hybridization

Research Abstract

The aim of this study is to determine whether a selective thrombin inhibitor, Argatroban, would prevent neuronal cell death and whether extra-mild hypothermia (35℃) would enhance the neuroprotecive effect of a selective thrombin inhibitor following transient focal ischemia in rats. Sprague-Dawley rats were subjected to MCAo using an intraluminal suture technique for 2hrs. The rats were reperfused for 24h and decapitated for infarct and edema analysis. Argatroban-treated animals received a continuous injection of argatroban (3.0mg/kg) for 24 hrs by after the onset of ischemia, while vehicle-treated groups received same dose of vehicle. During ischemia, temporal muscle and rectal temperatures were monitored and maintained at 37 ℃ in the normothermic animals and at 35 ℃ in the hypothermic animals. Animals were randomly divided into the following four groups (each, n = 6): (I) vehicle-treated normothermic group (control) at 37 ℃ (rectal and temporalis muscle temperatures) ; (II) argatroban -treated normothermic group at 37 ℃; (III) vehicle-treated hypothermic group at 35 ℃; (IV) argatroban-treated hypothermic group at 35 ℃. Temporal muscle and rectal temperatures were maintained during ischemia at 37 ± 0.2 ℃ (normothermic groups) or 35 ± 0.2 ℃ (hypothermic groups). Argatroban (162±28 mm^3) ameliorated the cortical ischemic damage compared with the control (205±55 mm^3) significantly (p<0.05). Moreover , argatroban with mild hypothermia decreased the cortical infarct volume (114 ±28 mm^3) significantly compared with those of groups I and III (170 ±27 mm^3) (p<0.05). Furthermore, argatroban with mild hypothermia also decreased the cortical edema (29 ±11 mm^3) volume significantly compared with those of groups I, II and III (68 ±23 mm^3, 52± 13 mm^3, 61 ± 16 mm^3) (p<0.05). In the borderzone of cortex and striatum, argatroban reduced expression of the proapoptotic Bax protein, whereas increased upregulation of antiapoptotic protein Bcl. Moreover, TUNEL positive cells in the borderzone were decreased in the groups treated by argatroban Argatroban improved neurological symptoms significantly (p<0.05) and also improved survival rate. These results demonstrate that extra-mild hypothermia (35℃) enhances neuroprotective effects of a selective thrombin inhibitor, argatroban, suggesting that this combined therapy may be a new therapeutic strategy for the treatment of acute stroke.