半胱氨酸蛋白酶3在局灶性脑缺血再灌注损伤皮质中的动态时空表达

Spatiotemporal expression of caspase-3 in perifocal cortex after focal cerebral ischemia and reperfusion

目的:观察半胱氨酸蛋白酶3在局灶性脑缺血再灌注不同时间、灶周皮质的动态时空变化。方法:实验于2005-05/08在河北医科大学第二医院神经分子影像医学和神经病学实验室完成。①选择清洁级健康新西兰白兔66只,雄性,兔龄4.5～5个月,体质量(2.6±0.2)kg,采用兔大脑中动脉阻断局灶性脑缺血再灌注模型,随机分为假手术组(n=6),模型组(n=60),模型组按再灌注时间又分为6个时间点,即1h、6h、1d、3d、7d、14d,每个时间点10只大白兔,各时间点依方法不同又分为组织学检测组(n=5)和流式细胞术检测组(n=5)。②采用免疫组化SP法检测皮质半胱氨酸蛋白酶3表达:应用MetaMorph显微图像分析系统于400倍光镜下随机观察并测量灶周皮质5个不重复视野神经细胞胞浆半胱氨酸蛋白酶3阳性表达光密度值,计算其平均数。③流式细胞术检测细胞凋亡率和灶周皮质半胱氨酸蛋白酶3的表达:经计算机分析系统,绘制细胞数DNA分布图,以二倍体峰前亚二倍体峰(凋亡峰)判定细胞凋亡,计算DNA断裂的凋亡细胞百分数(细胞凋亡率)。④透射电镜取材和观察:开颅取脑后,立即取梗死灶周1mm×1mm×2mm大小的标本,经过一系列处理后,在光镜下进一步确认已切到梗死灶周后,行超薄切片。醋酸铀、枸橼酸铅双重染色后,日立H-9000型透射电镜进行超微结构观察并照相。⑤结果行单因素方差分析。结果:66只大白兔全部进入结果分析。①免疫组化及流式细胞术分析发现,半胱氨酸蛋白酶3在2h缺血再灌注1h迅速增多,并随再灌注时间延长而加剧,3d达高峰,然后逐渐下降,14d仍高于基线水平。②灶周皮质凋亡率和脱氧核苷酰转移酶法计数TUNEL阳性细胞数在再灌注1h也逐渐增多,1～3d达高峰,然后逐渐下降,14d降至基线水平。③超微结构显示假手术组的细胞结构正常。再灌注3d神经元损伤最重,7d时形态有所改善。结论:半胱氨酸蛋白酶3活性表达在再灌注期先于凋亡发生前,并且其表达时程较长,是检测缺血再灌注凋亡的敏感指标,也是溶栓后治疗的靶点。

AIM： To investigate the spatiotemporal change of caspase-3 at different time in perifocal cortex after focal cerebral ischemia and reperfusion. METHODS： The experiment was carried out in the Laboratory of Molecular Imaging and Neurology, Second Hospital, Hebei Medical University from May to August 2005. ①Sixty-six clean grade healthy male New Zealand rabbits aged 4.5-5 months with body mass of （2.6±0.2） kg were selected. Focal cerebral ischemia reperfusion models were induced by transient occlusion of middle cerebral artery （MCA） and randomly divided into sham operation group （n=6） and model group （n=60）. The model group was subdivided into 1 hour, 6 hours, 1 day, 3 days, 7 days and 14 days groups according to 6 time points for reperfusion with 10 rabbits in each subgroup. Moreover, each subgroup was divided into histological examination group （n=5） and flow cytometry group （n=5）. ②Expression of caspase-3 in cortex was detected with immunohistochemical SP method： The optical density values of the positive expression of caspase-3 in nerve cell kytoplasm of 5 irreplicated fields were observed and measured with the Meta Morph microimage analysis system under 400 fold light microscope and the nean value was calculated. ③Expression of caspase-3 and apoptosis rate in perifocal cortex： The cell population DNA profile was drawn by the computer analysis system; the cell apoptosis was identified by the subdiploid peak （apoptotic peak） prior to the diploid peak, and the DNA ruptured apoptosis cell percentage （cell apoptotic rate） was calculated. ④Sampling and observation by transmission electron microscope： The sample of 1 mm× mm ×2 mm from infarction focus was taken after removed the brain. The ultrathin section was prepared after identification under light microscope. The uhrastructural changes were observed by electron microscopy （Hitachi H-9000） after uranyl acetate and lead citrate staining.⑤The single factor analysis of variance was used for results analysis. RESULTS： All the 66 animals entered the result analysis. ①The immunochemistry and flow cytometry suggested that the caspase-3 was rapidly and strongly activated at 1 hour of reperfusion and become severe with time, reached the active peak at day 3, and gradually decreased but was still higher than basal level at day 14 of reperfusion. ②The apoptotic rate and number of TUNEL-positive neural cells were also increased at 1 hour of reperfusion, reached the peak at days 1-3, and gradually decreased to basal level at day 14 of reperfusion. ③The ultrastructural changes showed the cellular structure of the sham operation group was normal, presented severe neural injury at day 3 of reperfusion and improved at day 7.CONCLUSION： The expression of caspase-3 activation is prior to the apoptosis in the perifocal cortex with a longer expression, which is a sensitive index to detect ischemia reperfusion apoptosis and therapeutic target after thrombolysis.