脑血流监测对大鼠脑缺血模型制备的评价作用

目的 探讨脑血流监测对线栓法制备大鼠大脑中动脉阻塞(MCAO)局灶性脑缺血模型的评价作用。方法 分别将线栓插入30只SPF级Wistar Han大鼠颈内动脉颅内段(16.0±0.5)、(18.0±0.5)和(20.0±0.5)mm,制备3种局灶性脑缺血模型(各10只),然后将所有实验大鼠依据颅底有无血凝块及2,3,5氯化三苯基四氮(TTC)染色后大脑中动脉供血区有无梗死灶分为不全阻塞组、完全阻塞组及过深阻塞组,对阻塞颈内动脉颅内段前后及拔出线栓再灌注后每只大鼠大脑中动脉供血区脑皮质的血流量以激光多普勒法进行监测记录并进行统计学分析。大脑中动脉供血区脑皮质的血流量以相对流量单位PU值表示;阻塞后及再灌注后的脑皮质血流量变化以与阻塞前脑皮质血流量的百分比表示。结果 模型制作过程中,1只大鼠死亡;不全阻塞组9只,完全阻塞组15只,过深阻塞组5只。不全阻塞组8只大鼠线栓插入深度在(16.0±0.5)mm,不能完全阻止大脑前动脉向大脑中动脉的血流,缺血6 h后大鼠Longa评分0~1分;颅底动脉环周围无血凝块,经TTC染色后无梗死灶。完全阻塞组9只大鼠线栓插入深度在(18.0±0.5)mm,大脑前动脉的血流被完全阻断,缺血6 h后大鼠Longa评分2~3分;颅底动脉环周围无血凝块而TTC染色提示存在大脑中动脉供血区的梗死灶。过深阻塞组5只大鼠线栓插入深度在(20.0±0.5)mm,可完全阻断大脑前动脉血流,缺血6 h后大鼠Longa评分3~4分;解剖可见颅底血凝块,TTC染色后可见中动脉供血区梗死灶。插入线栓后,不全阻塞组、完全阻塞组和过深阻塞组大鼠脑皮质血流量均较阻塞前下降(分别为94±17比256±36、43±9比286±44、44±6比294±46,均P0.05),组间差异有统计学意义(F=56.57,P0.01),完全阻塞组和过深阻塞组血流量明显低于不全阻塞组(均P0.05),完全阻塞组与过深阻塞组间差异无统计学意义(P0.05);3组阻塞后与阻塞前脑皮质血流量的百分比分别为(36.93±0.06)%、(15.09±0.02)%、(15.52±0.04)%,组间差异有统计学意义(F=39.14,P0.01)。再灌注后,不全阻塞组、完全阻塞组和过深阻塞组脑皮质血流量(分别为213±31、147±17、96±14)均较阻塞后有明显回升(均P0.05),组间差异有统计学意义(F=50.05,P0.01),过深阻塞组脑皮质血流量明显低于完全阻塞组(P0.05);3组再灌注后与阻塞前脑皮质血流量水平百分比分别为(83.10±0.02)%、(51.83±0.05)%、(33.49±0.09)%,差异有统计学意义(F=93.23,P0.01)。结论 以激光多普勒对脑血流进行监测,可作为判断线栓法制备大鼠MCAO脑缺血模型成功与否的一种实时、便捷、微创、客观可靠的评价手段。

Effects of the monitoring of cerebral blood flow in the preparation of cerebral ischemia model in rats

Objective To investigate whether the monitoring of cerebral blood flow can be used to evaluate the successful preparation of rat models with focal cerebral ischemia.Methods With the line plug inserted into the left internal cerebral artery of 30 SPF Wistar Han rats at (16.0±0.5) mm,(18.0±0.5) mm and (20.0±0.5) mm respectively,three kinds of focal cerebral ischemia models (10 each) were prepared.All the rats were divided into incomplete occlusion group,complete occlusion group and deep occlusion group according to whether the blood clots were found in skull base and the infarction was appeared in the middle cerebral artery territory after the whole brain was stained with 2,3,5-triphenyl tetrazolium chloride.The cerebral blood flow in the middle cerebral artery territory of each animal was monitored by laser Doppler at the time of before and after blocking with the line plug inserted and reperfusion with the line plug pulled out and recorded for statistical analysis.The cerebral blood flow was expressed as the relative flow unit (perfusion unit,PU);The changes of the cerebral blood flow after occlusion and reperfusion were expressed as a percentage of that blood flow and those before occlusion.Results During the process of modeling,1 rats died,and the other in the incomplete occlusion group (n=9),in the complete occlusion group (n=15),and in the deep occlusion group (n=5).The depths of inserting of 8 rats in the incomplete occlusion group were about (16±0.5) mm,and the blood flow from the anterior cerebral artery to middle cerebral artery could not be prevented completely,the Longa score was evaluated 6 h after ischemia and ranged from 0 to 1,there were no blood clots in skull base and no infarction after TTC staining.The depths of inserting of 9 rats in the complete occlusion group were about (18±0.5) mm,and the anterior cerebral artery blood flow was completely blocked,the neurological dysfunction was significant after 6 h ischemia and the Longa score was ranged from 2 to 3,the blood clots in skull base were absent and the infarctions in middle cerebral artery territory were obvious after TTC staining.The depth of inserting of 5 rats in the deep occlusion group were about (20±0.5) mm,and the anterior cerebral artery blood flow was also completely blocked,the neurological dysfunction was serious after 6 h ischemia and the Longa score was ranged from 3 to 4,there existed blood clots in skull base and obvious infarctions in middle cerebral artery territory after TTC staining.The cerebral blood flows after the line plug inserted compared with those before occlusion in the incomplete occlusion group,complete occlusion group and deep occlusion group were all decreased (94±17 vs.256±36,43±9 vs.286±44,44±6 vs.294±46,respectively,all P<0.05) ,and the difference between groups was statistically significant (F=56.57,P<0.01),Those in complete occlusion group and deep occlusion group were lower than that in incomplete occlusion group(all P<0,05),but the difference between deep occlusion group with complete occlusion group was not statistically significant(P>0.05);The percentages of cerebral blood flow after occlusion and before were (36.93±0.06)%,(15.09±0.02)%,(15.52±0.04)% respectively,and the difference between groups was statistically significant (F=39.14,P<0.01).The cerebral blood flow after reperfusion were 213±31,147±17,96±14,respectively,and the difference was also statistically significant (F=50.05,P<0.01),and the cerebral blood flow in deep occlusion group was less than that in complete occlusion group(P<0.05);The percentages of cerebral blood flow after reperfusion and before occlusion were (83.10±0.02)%,(51.83±0.05)%,(33.49±0.09)% respectively,and the difference was also statistically significant (F=93.23,P<0.01).Conclusion The cerebral blood flow monitored by laser Doppler can be used as a real-time,convenient,micro invasive,objective and reliable standard to evaluate the successful preparation of rat MCAO models with line plug method.