新生鼠缺氧缺血性脑损伤内源性纤维蛋白溶酶原激活剂变化及其意义

Changes and significance of endogenous tissue plasminogen activators in cerebral hypoxia-ischemia in neonatal rats

目的新生儿缺氧缺血性脑损伤发病机制尚不十分清楚,目前缺乏有效的治疗手段。近年来,越来越多的研究显示组织纤维蛋白溶酶原激活剂(tissue-type plasminogen activator,tPA)在神经系统中起到很重要的作用。该研究欲探讨纤维蛋白溶酶原激活剂(tPA)在新生鼠缺氧缺血脑损伤中的作用。方法选用Wistar大鼠7日龄的新生鼠,运用Rice-Vannucci动物模型造成新生鼠脑缺血缺氧,缺血缺氧后不同时间取新生鼠大脑检测。异硫氰酸荧光素-右旋糖酐左心室灌注分析脑缺血缺氧后新生鼠脑的再灌注障碍。用逆转录酶-聚合酶链反应(RT-PCR),酶谱法检测损伤后不同时间tPA的表达与活性;并采用双重免疫荧光,TUNEL和DAPI染色法检测血小板聚集指标(Integrin GPIIb)与纤维蛋白(fibrin)在缺血缺氧后不同时间的表达及神经细胞凋亡。结果新生鼠脑缺氧缺血后1h,异硫氰酸荧光素-右旋糖酐心室灌注提示新生鼠大脑有明显的缺血梗塞区,而Integrin GPIIb与fibrin的表达也较对照侧明显增加。但4h后缺血梗塞区面积明显缩小,伴有Integrin GPIIb与fibrin的表达减少。tPA的表达与活性在缺血缺氧后明显增加。随着缺氧缺血后恢复的时间延长,大脑凋亡细胞增加。结论tPA表达的增加在脑缺氧缺血的急性期可有助于血栓溶解,但在脑缺氧缺血的亚急性期却会导致神经细胞凋亡而加重脑损伤。

Objective The mechanism of neonatal hypoxic-ischemic brain damage （HIBD） remains unclear and effective treatment approach is limited for this disorder. Many studies have shown that tissue-type plasminogen activator （tPA） plays an important role in nervous system. This study investigated the effect of tPA in HIBD in neonatal rats. Methods Seven-day-old Wistar rat pups were used for the Rice-Vannucci model of neonatal hypoxia-ischemia （HI）. Brain samples were collected 1, 4, and 24 hrs after HI. FITC-Dextran was injected into the left ventricle of pups after HI to observe reperfusion defects of the neonatal brain. RT-PCR and tPA zymogram were used to detect the expression and activity of tPA. Double immunostaining, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL） and DAPI staining were used to detect the expression of integrin GPIIb and fibrin and neuronal apoptosis. Results FITC- Dextran perfusion analysis indicated there was obvious infarct area in the neonatal brain and the expression of integrin GPIIb and fibrin increased significantly 1 hr after HI compared with the contralateral side. The infarct area decreased and the expression of integrin GPIIb and fibrin were reduced 4 hrs after HI. The expression and activity of tPA increased significantly in neonatal rats after HI, and peaked at 4 hrs after HI. The number of apoptotic neural cells increased progressively with the prolonged reperfusion time following HI. Conclusions The increase of tPA in the acute phase after HIBD may be helpful to clot dissolving, but it induces neuronal apoptosis and aggravates brain injury.