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

本研究的目的是确定选择性凝血酶抑制剂阿加曲班是否能预防神经细胞死亡，以及超亚低温（35℃）是否能增强选择性凝血酶抑制剂在大鼠短暂局灶性缺血后的神经保护作用。采用腔内缝合技术对Sprague-Dawley大鼠进行MCAo 2h。大鼠再灌注24h后斩首进行梗死和水肿分析。阿加曲班治疗组在缺血发作后连续注射阿加曲班（3.0mg/kg） 24小时，而载药组则给予相同剂量的载药。在缺血过程中，监测常温动物的颞肌和直肠温度在37℃，低温动物的颞肌和直肠温度在35℃。将动物随机分为4组（每组n = 6）：(1) 37℃（直肠和颞肌温度）常温灌胃组（对照组）；（II）加曲班…More处理常温组，37℃；（三）35℃载药低温组；（IV） 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℃（低温组）。与对照组（205±55 mm^3）相比，阿加曲班（162±28 mm^3）显著改善皮质缺血损伤（p<0.05）。阿加曲班与亚低温组相比，脑皮质梗死面积（114±28 mm^3）明显减少（170±27 mm^3），差异有统计学意义（p<0.05）。阿加曲班与亚亚体温组（68±23 mm^3, 52±13 mm^3, 61±16 mm^3）相比，显著降低脑皮质水肿（29±11 mm^3）体积（p<0.05）。在皮层和纹状体交界区，阿加曲班降低促凋亡蛋白Bax的表达，而增加抗凋亡蛋白Bcl的上调。阿加曲班能显著改善神经系统症状（p<0.05），提高生存率。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，表明这种联合治疗可能是治疗急性卒中的一种新的治疗策略。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