运用原位末端标记技术检测大鼠缺氧缺血性脑损伤中III型细胞死亡

Discrimination of the type III cell death in hypoxic-ischemic brain damage in rats by in situ end labeling

目的 探讨新生大鼠脑缺氧缺血 (HI)后迟发性神经元死亡的形式。方法 建立新生大鼠脑HI损伤标准动物模型 ,运用HE染色光镜观察及原位末端标记 (ISEL)技术对HI后不同时间点实验侧大鼠大脑皮质、海马回死亡细胞进行观察与比较。结果 HI迟发性脑损伤中 ,存在一种既不同于细胞凋亡又有别于细胞坏死的一种特殊的细胞死亡———III型细胞死亡。光镜下 ,III型细胞既表现为核固缩、核染色质边聚、细胞体积缩小等I型 (凋亡 )细胞的特征 ,又呈现为胞浆丰富、胞膜完整性丧失等Ⅱ型 (坏死 )细胞的特征。ISEL检测III型细胞无核周空晕、无凋亡小体形成。I型细胞在脑HI后6h开始明显增多 ,平均为 (2 1.3± 3.5 ) / 10个高倍视野 (10hpf) ;2 4h达高峰 ,为 (6 3.7± 3.2 ) / 10hpf,与对照组比较差异有显著意义 (P <0 .0 0 1)。脑HI后 2 4h可检测到III型细胞 ,平均为 (5 0 .6± 6 .3) / 10hpf;48h阳性率最高 ,为 (75 .6± 10 .2 ) / 10hpf,明显高于I型细胞 [(42 .3± 4.5 ) / 10hpf,P <0 .0 1]。III型细胞主要分布在坏死区或其周围。结论 新生大鼠HI迟发性神经元死亡中 ,不仅存在细胞坏死和细胞凋亡 ,而且还存在III型细胞死亡。III型细胞死亡是与凋亡和坏死相关的一种特殊类型的细胞死亡。

Objective To investigate the type of delayed neuron death following cerebral hypoxia ischemia (HI) in neonatal rats. Methods Using HE staining and in situ end labeling (ISEL) methods, the authors observed the histological features of the cell death in the cerebral cortex and hippocampus of experimental hemispherium at different time points in the HI model of neonatal rats. Results As a special type of cell death, the type III cell death was observed in the delayed cerebral damage of HI in neonatal rat brains. Under the light microscope, the type III cell showed the condensed nuclear chromatin and the shrunken cell volume which shared the characteristics with the type I (apoptosis) cells; meanwhile, the type III cell showed the cell rich in cytoplasm and lost integrity of the plasma membrane which shared the characteristics with the type II (necrosis) cells. The type III cells were revealed by ISEL without the perinuclear halo and the apoptotic bodies. The type I (apoptosis) cells increased significantly in the experimental hemisphere 6 hours after HI [ (21.3±3.5)/10 hpf], and reached the peak level at 24 hours [(63.7±3.2)/10 hpf], when compared with the control [(7.3±2.3)/10 hpf, P <0.001]. There was no type III cell in the normal brain tissue, while the type III cells could be observed in the experimental hemisphere 24 hours after HI [(50.6±6.3)/10 hpf], and reached the peak level at 48 hours [(75.6±10.2)/10 hpf], which was higher than the type I cells [(42.3±4.5)/10 hpf] at the same time point ( P <0.01). The type III cells were mainly located in the necrotic area or around the necrotic area. Conclusions The type III cell death was mainly involved in the delayed cell death following HI injury in addition to the necrosis and apoptosis. The type III cell death was a special type of cell death, which related to apoptosis and necrosis.