Institute of Cell Biology and Neuroscience, Prof. E. De Robertis, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.
Neuroscience. 2013 Nov 12;252:190-200. doi: 10.1016/j.neuroscience.2013.07.065. Epub 2013 Aug 8.
Hypoxic insults during the perinatal period lead to motor and cognitive impairments that later appear during childhood. In the adult brain, hypoxic events often lead to necrotic neuronal death, depending on the region and intensity of the event. During development an active apoptotic cell death occurs and could be an important variable affecting the hypoxic insult outcome. In the present work we performed a comparative study, in a chick embryo model, of the phenotypes and molecular markers exhibited during developmental and hypoxic cell death (HxCD). Ultrastructural analysis of optic tectum cells of embryos subjected to hypoxia (8% O2, 60 min) revealed a clear apoptotic morphology that did not differ from the one exhibited during developmental cell death. Integrity of plasma membrane, condensation of chromatin in round well-defined bodies, and gradual shrinkage of the cell are all hallmarks of the apoptotic process and were present in both control and hypoxic cells. To elucidate if hypoxic and developmental cell deaths share a common mechanism we evaluated the activation of both intrinsic and extrinsic apoptotic pathways. A basal cleavage of caspase-9 and cytochrome c release was observed by co-immunofluorescence in control embryos, but hypoxic insult significantly increased the incidence of this colocalization. Caspase-8 cleavage remained unchanged after the hypoxic insult, suggesting that the extrinsic pathway would not be involved in hypoxic death. We also observed a significant decrease of Akt activation immediately after hypoxia, possibly facilitating the later release of cytochrome c. In addition we analyzed the influence of retinal ganglion cells (RGC) in neuronal survival. Transection of RGC fibers at embryonic day (ED) 3 did not induce any change in developmental and HxCD at ED12. In conclusion, our findings demonstrate that a hypoxic insult in the developing brain triggers the same apoptotic pathway as the active developmental death.
围产期缺氧损伤可导致儿童时期出现运动和认知障碍。在成人大脑中，缺氧事件通常导致坏死性神经元死亡，这取决于事件的区域和强度。在发育过程中，会发生活跃的细胞凋亡，这可能是影响缺氧损伤结果的一个重要变量。在本研究中，我们在鸡胚模型中进行了一项比较研究，研究了发育过程中和缺氧细胞死亡（HxCD）中表现出的表型和分子标志物。对暴露于缺氧（8% O2，60 分钟）的胚胎视顶盖细胞的超微结构分析显示，凋亡形态清晰，与发育性细胞死亡中观察到的形态无差异。质膜的完整性、染色质在圆形清晰定义的小体中的浓缩以及细胞的逐渐收缩都是凋亡过程的特征，在对照和缺氧细胞中均存在。为了阐明缺氧和发育性细胞死亡是否共享共同的机制，我们评估了内在和外在凋亡途径的激活。在对照胚胎中通过共免疫荧光观察到 caspase-9 和细胞色素 c 释放的基础切割，但缺氧刺激显著增加了这种共定位的发生率。缺氧刺激后 caspase-8 的切割保持不变，这表明外在途径不会参与缺氧死亡。我们还观察到缺氧后 Akt 激活的显著下降，这可能有利于随后细胞色素 c 的释放。此外，我们还分析了视网膜神经节细胞（RGC）对神经元存活的影响。在胚胎期 3 天（ED3）切断 RGC 纤维不会导致 ED12 时发育性和 HxCD 发生任何变化。总之，我们的研究结果表明，发育中的大脑缺氧刺激触发与主动发育性死亡相同的凋亡途径。
