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

本研究旨在探讨选择性凝血酶抑制剂阿加曲班能否预防大鼠局灶性脑缺血后神经细胞死亡，以及超亚低温(35℃)是否能增强选择性凝血酶抑制剂的神经保护作用。SD大鼠采用线栓法制作大脑中动脉闭塞模型，持续2小时。大鼠再灌流24小时，断头取脑进行脑梗塞和脑水肿分析。阿加曲班治疗组在脑缺血后连续注射阿加曲班(3.0 mg/kg)24小时，而赋形剂治疗组给予相同剂量的赋形剂。在缺血期间，常温动物的颞肌和直肠温度监测并维持在37℃，低温动物的温度维持在35℃。动物随机分为4组(每组6只)：(I)37℃(直肠和颞肌温度)的常温组(对照组)；(Ii)阿加曲班…组常温37℃组；(Iii)35℃车用低温组；(4)35℃阿加曲班低温组。缺血时，颞肌和直肠温度分别维持在37±0.2℃(常温组)和35±0.2℃(亚低温组)。阿加曲班(162±28 mm^3)与对照组(205±55 mm^3)相比，皮质缺血损伤明显减轻(p&lt；0.05)。亚低温阿加曲班可显著缩小脑皮质梗死体积(114±28 mm^3)，与I、III组(170±27 mm^3)相比，差异有统计学意义(P<0.05)。此外，亚低温阿加曲班还能显著减少脑皮质水肿体积(29±11 mm^3)，与I、II、III组(68±23 mm^3、52±13 mm^3、61±16 mm^3)比较，差异有统计学意义(p&lt；0.05)。在皮质和纹状体交界区，阿加曲班降低促凋亡蛋白Bax的表达，上调抗凋亡蛋白Bc l的表达。阿加曲班组交界区TUNEL阳性细胞减少，神经症状明显改善(p&lt；0.05)，存活率也明显提高。这些结果表明，超亚低温(35℃)增强了选择性凝血酶抑制剂阿加曲班的神经保护作用，提示这种联合治疗可能是治疗急性卒中的一种新的治疗策略。较少

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 … More -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. Less