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Acute hypoxia and programmed cell death in developing CNS: Differential vulnerability of chick optic tectum layers
V M Pozo Devoto1, J C Chavez, S Fiszer de Plazas
1Institute of Cell Biology and Neuroscience "Prof. E. De Robertis," School of Medicine, University of Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina.
Insights
Acute hypoxia transiently increases neuronal death in specific layers of the embryonic chick optic tectum. This programmed cell death is layer-dependent and primarily affects neurons, with effects observed hours after oxygen exposure.
Area of Science:
- Neuroscience
- Developmental Biology
- Cell Biology
Background:
- The developing chick optic tectum has distinct cellular layers.
- Programmed cell death (PCD) is crucial for neural development.
- Understanding hypoxic effects on developing neural tissue is important.
Purpose of the Study:
- To characterize programmed cell death in specific optic tectum layers at embryonic day 12.
- To assess the vulnerability of these layers to acute hypoxia.
- To identify the primary cell type affected by hypoxic cell death.
Main Methods:
- Assessed PCD using Hoechst staining, TUNEL assay, and active caspase-3 immunofluorescence.
- Exposed chick embryos to acute hypoxia (8% O2) for 60 minutes.
- Analyzed cell death rates in different optic tectum layers during normoxic recovery.
Main Results:
- Control embryos showed consistent cell death rates across layers.
- Hypoxic embryos exhibited a significant, delayed increase in cell death in TCC3, C"h-i-j", and SGC layers 6 hours post-hypoxia.
- Neurons (NeuN+) were primarily affected, while the SvZ layer remained unaffected.
Conclusions:
- Programmed cell death in the embryonic chick optic tectum is layer-dependent.
- Acute hypoxia induces a transient, delayed increase in neuronal death in specific tectal layers.
- Hypoxic neuronal death in the optic tectum resembles apoptosis morphologically.
Abstract:
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.
