咪唑安定对脑缺血再灌注沙土鼠血管内皮生长因子表达的调节作用

Modulatory effects of midazolam on the expression of vascular endothelial growth factor after cerebral ischemia-reperfusion injury in Mongolian gerbil

目的观察咪唑安定对沙土鼠全脑缺血再灌注损伤过程中血管内皮生长因子(VEGF)表达的影响,为咪唑安定临床合理应用提供实验依据。方法健康雄性沙土鼠共72只,随机分为对照组、损伤组和治疗组(n=24)。采用双血管法建立沙土鼠全脑缺血再灌注模型,对照组仅游离双侧颈总动脉但不夹闭;损伤组夹闭双侧颈总动脉10min,放开动脉夹后即刻腹腔注射生理盐水50ml/kg1次,随后每天同一时间点在动物清醒状态下腹腔注射生理盐水50ml/kg,共6d;治疗组夹闭双侧颈总动脉10min,放开动脉夹后即刻腹腔注射0.01%咪唑安定注射液5mg/kg,随后每天同一时间点在动物清醒状态下腹腔注射0.01%咪唑安定5mg/kg,共6d。以双侧动脉夹放开时间为基准点,分别在放开后6h、1d、3d、7d运用正电子发射断层显像(PET)观察脑梗死体积变化,用免疫组织化学方法观察脑组织VEGF的表达,每次的观察时间点均设在腹腔注射前。结果对照组脑内再灌注面积无明显异常;与对照组比较,损伤组与治疗组沙土鼠在再灌注后各时间点的脑内灌注面积明显缩小(P<0.01);与损伤组比较,治疗组在再灌注后6h脑内灌注面积无差异,但在1d、3d、7d时明显增加(P<0.01)。对照组脑内可见少量的VEGF阳性细胞表达。损伤组沙土鼠在再灌注6h即有VEGF阳性表达,随再灌注时间延长,表达逐渐增强,于再灌注3d时表达最强,以后缓慢下降,至7d时仍有部分表达,与再灌注6h时比较,有显著差异(P<0.01)。表达细胞主要为神经胶质细胞和巨噬细胞,其次为神经细胞及血管内皮细胞。治疗组VEGF的表达趋势与损伤组相似,但在再灌注后各时间点的VEGF表达均较损伤组强(P<0.01)。结论咪唑安定5mg/(kg·d)可减少沙土鼠脑缺血再灌注损伤后脑梗死的体积,促进脑缺血再灌注损伤中内源性VEGF的表达,并由此可能对损伤后神经功能中远期的恢复产生影响。

Objective To study the effects of midazolam on expression of vascular endothelial growth factor （VEGF） of gerbils following total cerebral ischemia-reperfusion injury to look for an experimental basis for the rational clinical use of midazolarrL Methods Seventy two mate gerbils （Mongolian gerbil） were randomly assigned into three groups （24 each）： sham injury group, injury group and midazolam treatment group. Total cerebral ischemia was reproduced by blocking the bilateral carotid arteries for 10 minutes with bulldog clamps. When reperfusion began, with release of the clamps, 5mg/kg of midazolam was intraperitoneally injected to the animals in midazo lain group, and 50ml/kg of normal saline was given by the same way in the gerbils in injury group. Then the parameters listed below were observed： positron emission tomography （PET） images at 6h, 1d, 3d and 7d after reperfusion, and the expression of VEGF in cerebral tis sue was immunohistoehemieally assessed. Results No obvious abnormality was found in the cerebral tissue of sham injury group. For the animals in the injury group and midazolam treatment group, the brain reinfusion area enlarged obviously （P〈0.01）. There was no obvious difference in the brain reinfusion area between injury group and midazolam treatment group at 6h after reperfusion, but significant difference was observed on 1d, 3d and 7d after reperfusion （P〈0.01）. A number of VEGF positive cells were seen in sham injury group. In injury group, VEGF began to express at 6h and reached the peak value on 3d after reperfusion. In midazolam treatment group, the tendency of VEGF expression was similar to that of injury group, but the number of VEGF positive cells was larger than that in operated group （P〈0.05）. VEGF expressed in glia cells, macrophages, neurocytes and vascular endothelial cells. Conclusion Endogenous VEGF can be increased by midazolam in the process of ischemia reperfusion injury of the brain, thus significantly promoting the recovery of the brain after of ischemia and reperfusim injury.