Research on the Mechanism of Neuroprotection of Mild Hypothermia (35℃)-Combination Therapy with Neuroprotective Agents-

本研究的目的是确定神经保护剂（选择性凝血酶抑制剂阿加曲班或免疫抑制剂FK 506）是否能防止大鼠短暂局灶性脑缺血后神经细胞死亡，以及超轻度低温（35℃）是否能增强选择性凝血酶抑制剂的神经保护作用。使用管腔内缝合技术使Sprague-Dawley大鼠经受MCAo 2小时。将大鼠重复灌注24小时并断头进行梗死和水肿分析。阿加曲班治疗组在缺血后24小时内连续注射阿加曲班（3.0mg/kg），黄芪治疗组注射相同剂量的黄芪。FK 506处理组动物在缺血诱导后2小时接受单次FK 506（0.3mg/kg）皮下注射，而Vehide处理组接受相同剂量的Vehide。在缺血期间，监测颞肌和直肠温度，并将常温动物的温度维持在37℃， ...更多信息 在低温动物身上。将动物随机分为以下4组（每组，n=6）：（I）37℃（直肠和颞肌温度）的溶剂处理常温组（对照）;（II）37℃的阿加曲班或FK 506处理常温组;（III）35℃的溶剂处理低温组;（IV）35 ℃的阿加曲班或FK 506处理低温组。在缺血期间，颞肌和直肠温度维持在37±0.2℃（常温组）或35±0.2℃（低温组）。阿加曲班组（162±28mm^3）与对照组（205±55mm^3）相比，脑缺血后皮质损伤明显减轻（p<0.05）。阿加曲班联合亚低温治疗组脑梗死体积（114± 28 mm ^3）明显小于I、III组（170± 27 mm ^3）（p<0.05）。阿加曲班联合亚低温治疗组皮质水肿体积（29± 11 mm ^3）明显小于Ⅰ、Ⅱ、Ⅲ组（68± 23 mm ^3、52 ± 13 mm ^3、61 ± 16 mm ^3）（p<0.05）。在皮质和纹状体的交界区，阿加曲班减少促凋亡Bax蛋白的表达，而增加抗凋亡蛋白Bcl的上调。阿加曲班治疗组边缘区TUNEL阳性细胞减少，神经症状明显改善（p<0.05），生存率提高。FK 506和亚低温联合治疗显著减少了梗死体积（皮质，-61%;纹状体，-31%）和水肿体积（皮质，-57%;纹状体，-41%），而亚低温或单独使用FK 506未能改善缺血性脑损伤。这些结果表明，超亚低温（35℃）增强了神经保护剂（选择性凝血酶抑制剂阿加曲班或免疫抑制剂FK 506）的神经保护作用，提示超亚低温（35℃）联合神经保护剂可能是治疗急性脑卒中的一种新的治疗策略。少

The aim of this study is to determine whether neuroprotective agents (a selective thrombin inhibitor, argatroban, or immunosuppressant, FK506), would prevent neuronal cell death and whether extra-mildhypothermia (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 repeifused 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 vehide-treated groups received same dose of vehide. FK506-treated animals received a single injection of FK506 (0.3mg/kg) venously at 2 hours after isehemic induction, while vehide-treated groups received same dose of vehide. During ischemia, temporal muscle and rectal temperatures were monitored and maintained at 37℃ in the normothermic animals and at 35℃ … More 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 or FK506-treated normothermic group at 37℃; (III) vehicle-treated hypothermic group at 35℃; (IV) argatroban or FK506-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±28mm^3) ameliorated the cortical ischemic damage compared with the control (205±55mm^3) significantly (p<0.05). Moreover, argatroban with mild hypothermia decreased the cortical infarct volume (114±28mm^3) significantly compared with those of groups I and III(170±27mm^3) (p<0.05). Furthermore, argatroban with mild hypothermia also decreased the cortical edema (29±11mm^3) volume significantly compared with those of groups I, II and III (68±23mm^3、52±13mm^3、61±16mm^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. The combination of FK506 and mild hypothermia significantly reduced infarct volume (cortex,-61%;striatum,-31%) and edema volume (cortex,-57%;striatum,-41%), while mild hypothermia or FK506 alone failed to improve ischemic brain damage. These results demonstrate that extra-mild hypothermia (35℃) enhances neuroprotective effects of neuroprotective agents (a selective thrombin inhibitor, argatroban or immunosuppressant, FK506), suggesting that this combined therapy, extra-mild hypothermia (35℃) plus neuroprotective agents, may be a new therapeutic strategy for the treatment of acute stroke. Less