可逆性大脑中动脉阻塞时神经元凋亡与坏死的形态学特点

THE MORPHOLOGICAL CHARACTERISTICS OF APOPTOTIC AND NECRO TIC NEURONS CAUSED BY REVERSIBLE MIDDLE CEREBRAL ARTERY OCCLUSION IN RATS

目的 :观察可逆性大脑中动脉阻塞时神经元凋亡与坏死的形态学特点及其发生过程 ,探讨缺血性神经元损伤的病理机制。方法 :阻塞大鼠大脑中动脉 2小时 ,再灌流 0 .5～ 48小时制成局灶性脑缺血模型 ,HE和TUNEL染色观察凋亡细胞和坏死细胞形态、图象分析并计数。结果 :HE染色可见凋亡细胞有四种形态 :核固缩深染细胞大而肿胀或胞质无明显异常的凋亡细胞、细胞核裂解但胞膜完整的凋亡细胞、凋亡小体 ,出现于再灌流 3小时 ;再灌流 6小时可见坏死细胞。TUNEL染色凋亡细胞着色深呈圆形、新月形和凋亡小体 ,0 .5小时出现 ,1 2小时达高峰 ;坏死细胞着色浅体积较大。 1 2小时纹状体凋亡细胞率 81 .5 6± 6 .2 4%。结论 :本研究结果表明TUNEL染色是辨认凋亡细胞敏感而可靠的方法 ;局灶性脑缺血时凋亡和坏死并存 ,但以细胞凋亡为主 ,因此提示对凋亡机制的干预可能为中风的治疗提供新的思路。

Objective: To investigate the morphological characteristics and development of apoptotic and necrotic neurons caused by reversible middle cerebral artery occlusion in rats and the phathological mechanism of ischemic injury. Methods: The focal cerebral ischemic model in rats was established by blocking middle cerebral artery 2hours and reperfusing 0.5～48hours. The apoptotic and necrotic cells were observed after HE and TUNEL staining. The number of apoptotic and necrotic cells was analyzed by image instrument and was treated statistically. Results: With HE staining, four types of apoptotic cells were observed: apoptotic cells (chromatin condensation) with a normal cytoplasm or a large and swollen cytoplasm; apoptotic bodies; apoptotic cells with nucleus fragments and cytoplasmic membrane intact. The apoptotic cells first presented themselves 3 hours of reperfusion. The necrotic cells were first observed after 6 hours of reperfusion. With TUNEL staining, the apoptotic cells were clearly stained dark red and were round (or oval) and crescent in shape. It began to appear after 0.5 hour and peaked at the 12 hour of reperfusion. The features of necrotic cells showed a diffuse light labeling and with larger body. The ratio of apoptotic cells in striatum at the 24th hour of reperfusion was 81.56±6.24%. Conclusion: These data indicated that TUNEL staining is a reliable and sensitive method to identify the apoptotic cells. In addition, these results also showed that necrosis and apoptosis co-exist in rats subjected to focal cerebral ischemia, and the apoptotic cells predominated over the necrotic cells in number, suggesting that interference with the apoptotic mechanism might provide new insight into stroke treatment.