大鼠局灶性脑缺血再灌注脑组织中活性氧自由基表达的时程变化

目的 采用大鼠大脑中动脉梗死(middle cerebral artery occlusion, MCAO)再灌注模型,研究缺血脑组织再灌注后活性氧自由基(reactive oxygen species, ROS)的时程变化,探讨ROS在脑缺血损伤中的作用。方法 采用数字表法将25只健康雄性SD大鼠随机分为5组:假手术(Sham)组,MCAO组(缺血90 min,再灌注3、6、12、24 h),每组5只大鼠。使用线栓法制作大鼠缺血90 min再灌注模型,术中监测大鼠平均动脉压及肛温,使其保持在正常范围。每组大鼠分别于再灌注后3、6、12、24 h时处死,迅速取脑组织进行TTC染色,检测大鼠脑梗死体积,并制作新鲜脑组织冰冻切片,采用ROS特异性染色方法——H₂DCF-DA荧光染色法,检测再灌注后不同时间点缺血半暗带区ROS的表达。结果 1) 在MCAO手术过程中,Sham组以及MCAO组大鼠的平均动脉压及肛温差异无统计学意义。2)Sham组大鼠脑内无缺血半暗带区,其相应脑区内只可见极少量的H₂DCF-DA阳性染色细胞。而MCAO组大鼠再灌注后3,6,12,24 h 4个时间点,脑组织半暗带区域均可见大量的H₂DCF-DA阳性染色细胞。且从再灌注3 h起,MCAO组大鼠脑组织半暗带区域H₂DCF-DA阳性染色细胞数量持续增加,其中再灌注6 h与3 h相比,差异有统计学意义(P<0.05),再灌注24 h与12 h相比,差异有统计学意义(P<0.05)。3)TTC染色结果显示,Sham组大鼠无脑梗死发生。MCAO组大鼠脑组织随再灌注时间的延长(从3 h 到24 h),相对梗死体积逐渐增大,差异有统计学意义(P<0.05)。4) MCAO组大鼠脑组织H₂DCF-DA阳性细胞数目与脑梗死体积比率呈正相关(r=0.833,P<0.01)。结论 局灶性脑缺血模型大鼠再灌注后缺血侧脑组织中ROS量随再灌注时间延长递增,ROS在脑缺血再灌注损伤中具有重要作用。

The dynamic changes of reactive oxygen species expression in rat brain tissue following focal cerebral ischemia/reperfusion

Objective To investigate the dynamic changes of reactive oxygen species (ROS) during reperfusion time in a rat middle cerebral artery occlusion (MCAO)/reperfusion model and to investigate the role of ROS in cerebral ischemic injury. Methods Twenty-five male Sprague Dawley rats were divided randomly into 5 groups: sham group and MCAO group (90 min ischemia with 3,6,12 or 24 h reperfusion). MCAO operation was performed by using suture method. The rats underwent right MCAO for 90 min. The mean arterial blood pressure (MABP) and rectal temperature were monitored to keep in normal range during the operation. Brains were removed after the animals were euthanized at 3, 6, 12 and 24 h after reperfusion respectively and dyed with 2,3,5-triphenyltetrazolium chloride (TTC) to measure the infarct volume. Cryostat sections were prepared on a freezing microtome. ROS-specific staining and H₂DCF-DA fluorescent staining were used to detect the levels of ROS in the penumbra of the brain. Results 1) There was no statistically significant difference in MABP and rectal temperature between sham and MCAO groups. 2) Sham group rats had no penumbra in the brain where little H₂DCF-DA-positive cells were observed. Lots of H₂DCF-DA-positive staining cells were observed in the penumbra of MCAO group rats at 3, 6, 12 and 24 h after reperfusion. From 3 h after reperfusion, the numbers of H₂DCF-DA-positive staining cells in the penumbra of MCAO group rats showed reperfusion-time-dependent increasing tendency, with significant difference between 3 h and 6 h, as well as 12 h and 24 h (P<0.05). 3) The result of TTC staining showed that no infarction was observed in the brain of sham group rats. The infarct volume of MCAO group rats showed reperfusion-time-dependent increasing tendency (from 3 h to 24 h after reperfusion, P< 0.05). 4) The number of H₂DCF-DA-positively stained cells numbers showed positive correlation with the volume of brain infarction in MCAO group (r=0.833,P<0.01). Conclusion ROS level was increased along with the extension of reperfusion time in a rat focal cerebral ischemia/reperfusion model. ROS might play an important role in cerebral ischemic/reperfusion injury.