局灶性脑缺血及脑缺血再灌注动物模型的制作

Establishment of animal models of focal cerebral ischemia reperfusion

目的:介绍一种简便易行的大鼠局灶性脑缺血再灌注模型的制备方法。方法:实验于2003-03/2005-03在泰山医学院神经生物学实验室完成。选择健康成年清洁级SD大鼠48只,随机数字表法分为假手术组6只,脑缺血组24只,脑缺血再灌注组18只。选择韩国产的3号圆柱形尼龙钓鱼线,直径0.2864mm,剪成长30mm若干段,将其一端粘少许用乙醇稀释合适浓度的指甲油,可形成一薄层保护膜,使插线前端光滑无锐缘,且插线前端无明显膨大,然后垂直倒放,自然凉干,于距处理端18,20mm处做黑色标记备用。自制插线从颈外动脉经颈内动脉插入,栓塞大脑中动脉,形成脑缺血状态,分别缺血30min,24h,2,3d,每个时相点6只。精确控制栓塞时间30min,拔出插线,形成脑缺血再灌注状态,分别再灌注24h,2,3d,每个时相点6只。观察动物模型的成功率,大鼠脑组织溴化2,3,5-氯化三苯基四氮唑红染色结果,光镜下脑组织尼氏小体的显示结果,脑细胞电镜观察结果,凋亡细胞观察结果。结果:①假手术组动物模型成功率为100%,脑缺血组动物模型成功率为91.7%,脑缺血再灌注组动物模型成功率为94.4%。②溴化2,3,5-氯化三苯基四氮唑红染色结果:脑缺血30min肉眼几乎分辨不出梗死灶;脑缺血24h梗死灶主要累及大脑皮质额叶颞侧及其纹状体颞侧靠近大脑皮质下的边缘部分;脑缺血2d梗死灶几乎累及缺血侧大脑半球额顶叶的全部及其纹状体的大部分;脑缺血3d梗死灶累及缺血侧的全部脑组织,甚至累及部分对侧脑组织;脑缺血再灌注显示梗死灶主要累及缺血侧大脑皮质颞侧及纹状体的颞侧部分。③尼氏染色结果显示脑缺血30min,脑组织结构无明显变化;脑缺血24h,脑损伤侧梗死区内广泛细胞坏死,部分细胞自溶;脑缺血30min再灌注24h,脑损伤侧呈现点状坏死灶;脑缺血2d,脑损伤侧呈现较多片状坏死灶;脑缺血30min再灌注2d脑细胞边界呈现毛刺样;脑缺血3d,损伤侧脑组织大片状坏死;脑缺血30min再灌注3d,脑损害趋向稳定。④电镜结果显示:脑缺血30min,神经细胞结构、层次破坏不明显;脑缺血24h,细胞内出现许多高密度中毒颗粒。脑缺血30min再灌注24h,神经细胞胞质内出现少量小的空泡及高密度中毒颗粒;脑缺血2d,很少见到能分辨的细胞结构;脑缺血30min再灌注2d,神经细胞膜性结构继续破坏,结构尚可辨认;脑缺血3d,未见形态可辨的神经细胞;脑缺血30min再灌注3d,能够分辨细胞残存的部分膜性结构。⑤脑缺血组、脑缺血再灌注组凋亡细胞高于假手术组,差异有显著性意义[分别为(75.0±2.3),(47.0±3.7),(8.0±1.2)个,F=12.3,P=0.019<0.05]。结论:该方法简便快捷(手术时间不足15min),结果可靠,稳定性好,是较理想的一种大鼠局灶性脑缺血再灌注动物模型。

AIM： To introduce one reliable and simple preparation for rat models of focal cerebral ischemia reperfusion. METHODS： The experiment was conducted in the Laboratory of Neurobiology, Taishan Medical College between March 2003 and March 2005. Totally 48 clean grade healthy adult SD rats were selected and randomly divided into sham operation group （n=6）, cerebral ischemia group （n=24） and cerebral ischemia reperfusion group （n=18）. A No. 3 cylindrical nylon fishing line made in Korea, 0.286 4 mm in diameter was sheared into several parts with 30 mm of each part, and one end of the line was covered with nail polish diluted by ＂alcohol, which could form protective tissue to make the tip smooth without obvious intumescence. Then the line was posed vertically and marked black at 18 and 20 mm from the tip after dried. The line was inserted into middle cerebral artery beyond the origin of the internat carotis artery to form occlusion for 30 minutes, 24 hours, 2 and 3 days respectively with 6 rats at each time point. After 30 minutes of occlusion, the line was pulled out to produce reperfusion for 24 hours, 2 and 3 days with 6 rats at each time point. The successful rate of models and results of 2, 3, 5-triphenyhetrazoluym chloride （TTC） staining of cerebral tissue were observed. Meanwhile, the results of Nissl body under light microscope, brain cells under electron microscope and apoptotic cells were observed. RESUILTS： ①The successful rate of models of the sham operation group was 100%, the cerebral ischemia group was 91.7% and the cerebral ischemia reperfusion group was 94.4%. ②Results of Trc staining： After 30 minutes of ischemia, the infarction could hardly found by naked eyes; and after 24 hours, the infarction was mainly found in frontal lobe temple of cerebral cortex and its striatal temple near the edge of cortex; after 2 days of ischemia, the infarction distributed over the whole frontoparietal lobe and majority of its striatal temple of the ischemic cerebral hemisphere; after 3 days of ischemia, all the ischemic cerebral tissue was covered with infarction, even part of the other side of cerebral tissue; all above indicated that the infarction was mainly occupied the frontal lobe temple and its striatal temple of cerebral cortex. ③Results of Nissl staining suggested that after 30 minutes of ischemia, the cerebral tissue structure changed little; after 24 hours, many cells necrosis in the infarction area of the injured side were found, part of cells autolysis; after 30 minutes ischemia and 24 hours reperfusion, spotty necrosis infarction was found in the injured side; after 2 days of ischemia, many patchy necrosis infarction appeared in the injured side; after 30 minutes ischemia and 2 days reperfusion, the edge of the brain cells showed spicule： after 3 days ischemia, most of the injured cerebral tissue showed patchy necrosis infarction; after 30 minutes ischemia and 3 days reperfusion, the cerebral injury became stable.④Resuits of electron microscope suggested： After 30 minutes of ischemia, no obvious structural or order damage of neural cells were found; after 24 hours of ischemia, many high density toxic granulation were found in cells; after 30 minutes isehemia and 24 hours reperfusion, few vacuoles and high density toxic granulation were found in cytoplasm of neural cells; after 2 days ischemia, there were few cells with clear structure; after 30 minutes ischemia and 2 days reperfusion, the membranous structure was continuously damaged, but the structure could still be identified; after 3 days of ischemia, there was no clear neural cells; after 30 minutes ischemia and 3 days reperfusion, only part of residual membranous structure was observed. ⑤The apoptotic rates of the cerebral ischemia group and cerebral ischemia reperfusion group were higher than the sham operation group, which had significant differences [（75.0±2.3）, （47.0±3.7）, （8,0±1.215） cells, F=12,3, P=0.019 〈 0.05]. CONCLUSION： This method is simple and convenient （operation time is less than 15 minutes）, reliable and stable. It is an ideal method to study rat models of focal cerebral ischemia reperfusion.