Pozo Devoto V M, Chavez J C, Fiszer de Plazas S
Institute of Cell Biology and Neuroscience "Prof. E. De Robertis," School of Medicine, University of Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina.
Neuroscience. 2006 Oct 27;142(3):645-53. doi: 10.1016/j.neuroscience.2006.06.054. Epub 2006 Aug 14.
The chick optic tectum displays an alternating pattern of cellular and plexiform layers and at embryonic day (ED) 12 there are mainly four cellular layers: transient cell compartment 3 (TCC3), compartment "h-i-j"(C"h-i-j"), stratum griseum centrale (SGC) and subventricular zone (SvZ). In the present work we characterized the programmed cell death (PCD) of these layers and their vulnerability to acute hypoxia at ED12, and also identified the main cellular type involved in hypoxic cell death. The colocalization of three independent markers of cell degeneration: pyknotic nuclei by Hoechst staining, fragmented DNA by TdT-mediated dUTP nick-end labeling (TUNEL), and presence of active caspase-3 by immunofluorescence, was analyzed in embryos that developed in normoxic conditions (control embryos) and embryos that were subjected to hypoxia (8% O(2)/92% N(2)) for 60 min (hypoxic embryos), followed by 0-12 h of normoxic recovery. In control embryos cell death rate within each layer was constant through time, but there were significant differences (P<0.01) in cell death rates among the different layers. In contrast, in hypoxic embryos, a significant increase (P<0.01) in cell death rate was observed in layers TCC3, C"h-i-j" and SGC. This change was evident only at 6 h post-hypoxia, and at later time points cell death rate was similar to control values. Each of these layers had a different vulnerability to the hypoxic event while the SvZ layer was not affected. In addition, the significant colocalization between the neuron specific nuclear protein (NeuN) and TUNEL signal showed that hypoxia affected primarily neurons. In conclusion, our findings demonstrate that in the chick optic tectum at ED12, PCD is layer dependent and that acute hypoxia causes a transient increase in neuronal death in a delayed fashion, which is also layer dependent. The morphological features of the neuronal death process at the light microscope level resembled apoptosis.
鸡的视顶盖呈现出细胞层和神经纤维层交替的模式，在胚胎第12天（ED12）主要有四层细胞：瞬态细胞区室3（TCC3）、“h - i - j”区室（C“h - i - j”）、中央灰质层（SGC）和室下区（SvZ）。在本研究中，我们对这些层的程序性细胞死亡（PCD）及其在ED12时对急性缺氧的易损性进行了表征，还确定了参与缺氧性细胞死亡的主要细胞类型。在常氧条件下发育的胚胎（对照胚胎）和经历缺氧（8% O₂/92% N₂）60分钟的胚胎（缺氧胚胎）中，分析了三种独立的细胞变性标记物的共定位情况：通过Hoechst染色检测的固缩核、通过TdT介导的dUTP缺口末端标记（TUNEL）检测的DNA片段化以及通过免疫荧光检测的活性半胱天冬酶 - 3的存在，之后进行0 - 12小时的常氧恢复。在对照胚胎中，各层内的细胞死亡率随时间保持恒定，但不同层之间的细胞死亡率存在显著差异（P<0.01）。相比之下，在缺氧胚胎中，观察到TCC3、C“h - i - j”和SGC层的细胞死亡率显著增加（P<0.01）。这种变化仅在缺氧后6小时明显，在随后的时间点细胞死亡率与对照值相似。这些层中的每一层对缺氧事件都有不同的易损性，而SvZ层未受影响。此外，神经元特异性核蛋白（NeuN）与TUNEL信号之间的显著共定位表明缺氧主要影响神经元。总之，我们的研究结果表明，在ED12的鸡视顶盖中，PCD是层依赖性的，急性缺氧会以延迟的方式导致神经元死亡短暂增加，这也是层依赖性的。光镜水平下神经元死亡过程的形态学特征类似于凋亡。
