大鼠局灶性脑缺血再灌注致多器官功能障碍综合征模型Caspase-3的表达

Expression of Caspase-3 in rats with multiple organ dysfunction syndrome caused by focal cerebral ischemia-reperfusion

目的:建立大鼠局灶性脑缺血再灌注致多器官功能障碍综合征模型,观察Caspase-3的表达。方法:实验于2004-10/12在徐州市中心医院神经内科实验室完成。选择健康雄性Wistar大鼠40只,随机分为正常对照组(5只)、假手术组(5只)及手术组(30只),手术组分2,12,24,48,72h,7d6个时相点,每个时相点5只。采用线栓法制作大鼠局灶性脑缺血再灌注致多器官功能障碍综合征模型。观察并检测大鼠生命体征、血生化指标及主要官病理形态学改变。免疫组织化学方法检测脑、肺及肾脏Caspase-3表达的阳性神经元细胞与数目。结果:纳入动物40只,均进入结果分析。①生命体征观察及血生化检测:手术组大鼠的体温升高,呼吸、心率增快,血生化各项指标亦明显增高,与正常对照组、假手术组相比差异显著。②各组大鼠脏器的病理改变:正常对照组各脏器组织细胞超微结构正常;假手术组病理学改变轻微;手术组主要表现为功能障碍器官以炎症为主的非特异性改变。脑:细胞核固缩,核仁消失,胞浆疏松,染色变浅,间质水肿,细胞密度减少。肺:可见渗出、间质增宽,肺泡内有带状、片状或广泛水肿、血管周围水肿套、部分包膜下水肿;间质大量炎细胞浸润,分类以单核细胞浸润为主;局灶性肺不张,个别小支气管内可见少量渗出物。肝:肝细胞高度肿胀和炎性细胞浸润。肾:肾小球充血肿胀,白细胞浸润或肾小管管腔可见均质蛋白管型,间质明显淤血。③免疫组织化学检测Caspase-3的表达:正常对照组和假手术组脑、肺和肾脏有微量Caspase-3表达;手术组再灌注2h后亦有少量Caspase-3阳性表达,但与正常对照组、假手术组相比差异无显著性,12h明显增多,24h达到高峰,然后逐渐下降,至7d时仍有少量表达。12,24,48h与正常对照组及手术组相比差异显著,以肾脏Caspase-3表达增高最为明显。结论:局灶性脑缺血再灌注致多器官功能障碍综合征后Caspase-3表达上调,脑缺血后细胞凋亡可能与脑源性多器官功能障碍综合征有关。

AIM： To establish model of multiple organ dysfunction syndrome （MODS） caused by focal ischemia-reperfusion in rats, and observe the expression of Caspase-3. METHODS： The experiment was conducted in the Department of Neurology, Xuzhou Municipal Center from October to December 2004. Forty male healthy Wistar rats were selected and randomly divided into normal control group （n=5）, sham-operation group （n=5） and operation group （n=30）. Rats in the operation group were divided into subgroups according to the 6 time-points, i.e. 2 hours, 12 hours, 24 hours, 48 hours, 72 hours and 7 days with 5 rats in each time point. The models of MODS induced by focal cerebral isehemia were made by occluding the middle cerebral artery with nylon line in rats. The vital signs, blood biochemical indicator and the main pathological changes of primary viscera were detected and observed. The immunohistochemieal method was adopted to examine the positive neurons and number in expression of Caspase-3 in brain, lung and kidney. RESULTS： A total .of 40 enrolled animals were involved in the analysis of results. （1）Observation on vital signs and blood biochemical detection： the temperature, breath, heart rate of rats in the operation group were accelerated, and all blood biochemical indicators were obviously increased. The differences between normal control group and sham-operation group were remarkable. （2）The pathological changes in internal organs of rats in all groups： the ultrastraeture of all internal organs in rats of the normal control group was normal, and there were slight pathological changes in rats of the sham-operation group. There were non-specific changes in rats of the operation group, which is mainly represented as organ dysfunction inflammations. Brain： the earyon condensed, entoblast disappeared, endochylema loosened, dying faded, there was interstitial edema and the cell density decreased. Lung： The interstitial substance was widened with bendiformis, lamellar and extensive dropsy inside the alveoli pulmonis. There were edema in the periphery of vessels and edema could be found under partial amieula. There were large amount of inflammatory cell infiltrate in the interstitial substance, and the classification mainly accorded to the infiltration of mononuelear cells. There was focal atelectasis, and a small quantity of effusion could be seen inside individual bronehium. Liver：. the liver cells greatly swelled and there was inflammatory cell infiltrate. Kidney： the renal corpuscle engorged and swelled, there were leukocytie infiltrate ,and homogen protein east could be seen in the lumina of urinary east. There was obvious congestion in the interstitial substance. （3）Determination of the expression of Caspase：3 with immunohistochemieal methods： there were micro-amount in brain, lung and kidney of normal control group and sham-operation group. There were little positive expression in the operation group at 2 hours after perfusion, which was not significantly different from the normal control group and sham-operation group. The expression of Caspase-3 was remarkably increased on the 12^th hour and reached the peak onthe 24^th hour, and then gradually descended, and there was still little on the 7^th day. Differences on the 12^th, 24^th and 48^th hour between normal control group and operation group were marked, and the.increase of expression of Caspase-3 in kidney was the most remarkable. CONCLUSION： The expression of Caspase-3 is up regulated after MODS eaused by focal isehemia-reperfusion, and the apoptosis after eerebral ischemia may be related with MODS eaused by stroke,