亚低温对大鼠缺血性脑水肿及水通道蛋白AQP4表达的影响

背景:亚低温(28～35℃)正成为一种治疗急性缺血性卒中较有前景的方法,低温可有效地减轻脑水肿是其神经保护作用之一。目的:观察亚低温对大鼠脑缺血再灌注后脑含水量和水通道蛋白4(AQP4)表达的影响,探讨亚低温脑保护的作用机制。设计:随机对照实验。单位:徐州医学院神经生物实验室。材料:选择健康雄性SD大鼠110只,体质量250～300g,由徐州医学院实验动物中心提供No.SYNK(苏)2002-0079]。应用SPSS11.0统计软件将大鼠随机分3组:①假手术组(n=10);②常温组(n=50);③亚低温组(33℃,n=50)。常温组及亚低温组又分为缺血后再灌注6h,1d,2d,3d,7d各亚组,每亚组各10只大鼠。每组中5只用于脑含水量测定,5只用于苏木精-伊红染色及免疫组织化学染色。方法:参考Pulsinelli方法对常温组及亚低温组大鼠制备四动脉结扎全脑缺血模型,缺血时间为15min。假手术组大鼠仅电凝双侧椎动脉及分离颈总动脉,不作结扎,手术后24h断头取脑。对常温组及亚低温组大鼠脑组织切片,HE染色及免疫组化染色,分别于再灌注后6h,1d,2d,3d,7d观察脑组织病理学和AQP4表达水平的动态变化;干湿重法测定各组大鼠各时间点脑含水量。主要观察指标:①常温组及亚低温组大鼠脑组织病理学变化。②所有大鼠各时间点脑含水量及AQP4表达水平。结果:①常温组大鼠在缺血再灌注后6h可见血管周围间隙增宽、细胞外间隙扩大、脑组织变疏松等脑组织水肿表现,以缺血再灌注后2d最明显;亚低温组大鼠各时间点与相应的常温组比较,脑组织水肿表现相对减轻。②常温组及亚低温组大鼠缺血再灌注后6h内即出现脑含水量增高,2d达高峰,7d时脑含水量明显减少,但仍高于假手术组。亚低温组脑含水量均较相应时间点的常温组少(6h,7d组P<0.01,余各组P<0.05)。③常温组及亚低温组大鼠AQP4表达水平在再灌注后6h增高,2d达最高水平,7d时明显降低,但仍较假手术组高。亚低温组AQP4表达水平均较相应时间点的常温组降低(P<0.01)。结论:脑缺血再灌注后AQP4表达水平的变化趋势与脑含水量变化趋势在时间上一致,表明AQP4表达上调可能是缺血性脑水肿形成的分子机制之一。亚低温可减轻缺血性脑水肿,而通过抑制AQP4表达可能是亚低温减轻缺血性脑水肿的作用机制之一。

Effect of mild hypothermia on ischemic brain edema and expression of aquaporin-4 in rats

BACKGROUND: Mild hypothermia (28-35 ℃) is becoming one of the promising methods in treating acute ischemic stroke. Hypothermia can effectively lessen brain edema, which is one of its neuroprotective roles.OBJECTIVE: To investigate the effect of mild hypothermia on brain water content and aquaporin-4 (AQP4) expression level following global cerebral ischemia-reperfusion injury in rats, so as to study the neuroprotective mechanisms of mild hypothermia.DESIGN: A randomized controlled trial.SETTING: Neurobiological Laboratory of Xuzhou Medical College.MATERIALS: 110 healthy male SD rats with body mass 250-300 g, provided by the Animal Center of Xuzhou Medical College, No. SYNK (Jiangsu) 2002-0079, were selected and randomly divided into 3 groups by SPSS 11.0software: ①sham-operated group (n=10);②normothermiagroup (n=50); ③mild hypothermia group (33 ℃, n=50). Normothermia group and mild hypothermia group were subdivided into five reperfusion subgroups for 6 hours, 1, 2, 3 and 7 days, respectively with 10 rats in each subgroup,in which 5 rats were used for measurement of brain water content, and others for HE staining and immunohistochemistry staining.METHODS: The models of global cerebral ischemia were established in the normothermia group and mild hypothermia group by four-vessel occlusion (4-VO) method with ischemia for 15 minutes as Pulsinelli described.The rats in the sham-operated group were only underwent the electrocauterization of bilateral vertebral arteries and the isolation of common carotid arteries except for occlusion of common carotid arteries. Twenty-four hours later, the rats were decapitated to take out the brains. The brains of rats in the normothermia group and mild hypothermia group were taken out to make sections for HE staining and immunohistochemistry staining, and the dynamic change of pathology of the brain tissue and AQP4 expression level were observed after reperfusion for 6 hours, 1, 2, 3 and 7 days, respectively. The brain wet-to-dry weight measurement was used to measure the brain water content of the rats at each time point of each group.MAIN OUTCOME MEASURES: ①The pathologic changes of brain tissues of rats in the normothermia group and mild hypothermia group. ②The brain water content and the AQP4 expression level of all rats at each time point.RESULTS: ①After 6 hours of reperfusion, brain edema appeared in the normothermia group including amplification of periyascular spaces and intercellular spaces, rarefaction of brain tissues, etc, which got worst after 2 days of reperfusion; the phenomenon of brain edema of the rats in the mild hypothermia group at each time point was relatively lighter than the normothermia group. ②Brain water content of the normothermia group and mild hypothermia group was increased after 6 hours of reperfusion and reached peak at the 2nd day; Although decreased at the 7th day, it was still higher than the sham-operated group. The brain water content of the mild hypothermia group at each time point was less than the normothermia group (values after 6 hour and 7 day, P ＜ 0.01, the rest groups P ＜ 0.05).③AQP4 expression level of the normothermia group and mild hypothermia group was increased after 6 hours of reperfusion and reached peak at the 2nd day. Although decreased at the 7th day, it was still higher than the sham-operated group.The AQP4 expression level of the mild hypothermia group at each time point was lower than the normothermia group (P ＜ 0.01).CONCLUSION:The change tendency of AQP4 level is parallel to that of brain water content after ischemia reperfusion, which indicates that the upregulation of AQP4 expression is one of molecular mechanisms for the for mation of ischemicbrain edema. Mild hypothermia can release ischemic brain edema by inhibiting AQP4 expression, which is one of its mechanisms.