Characterization Apoptosis Neurogensis Induced Bu Transient Hypoxia In Postnatal Rat Brain

ObjectiveA rat model mimicking neonatal hypoxic was established to investigate the effects of transient hypoxia in brain on the expression level of NeuroD, apoptosis and neurogensis. Importantly, this study may result in novel interventions for management of hypoxic-ischemic encephalopathy.Methods1. The model was established as previously described by Grojean. Briefly, newborn rats were transiently exposed to 100% N2, and reliability of the model was analyzed by behavior analysis, HE staining and transmission electron microscope.2. Neonatal rats were randomly divided into 3 groups of normal control, 10min hypoxia and 20min hypoxia. The expression level of NeuroD in rat brain was determined with Western blot and immunofluorescence staining.3. In other experiments, rats exposed to N2 for 20 min were sacrificed at day 13 , 20 and 27 post-hypoxia, and the expression levels of NeuroD and BrdU in brain were analyzed by immunofluorescence staining and immunochemistry. Alternatively, the rats were sacrificed at day 6 and 20 post-hypoxia, and apoptosis in the brain was analysed with TUNEL-staining.Results1. Transient hypoxia in neonatal rats resulted in systemic cyanosis and abnormal neurological behaviors such as unconsciousness. Histological analysis showed that hypoxia 10 min resulted in the neuronal cells swell swelling which became more severe at hypoxia 20 min. Ultrastructural analysis of the neuronal cells revealed that chromatin margined and secondary lysosomes appeared. Microvascular endothelial cells and pericyte cells appeared vacuolar degeneration. Morphological changes became worse at hypoxia 20 min with that the neuronal cells necrotized, nuclear dissolved and organelles disappeared. The baseboard and cell processes of astrocyte also swelled.2. Immunofluorescence analysis showed that NeuroD was expressed in cerebral cortex and dentate gyrus. Western blot analysis demonstrated a significant up-regulation in the NeuroD expression with the time after hypoxia (P <0.01).3. Immunofluorescence /immunohistochemisty analysis showed that the expression of NeuroD and BrdU significantly increased at day 13 and 27 in group of 20 min hypoxia compared to that in control group (P<0.05), with the highest expression level seen at day 20 (P <0.01). TUNEL-positive cells also significantly increased at day 6 in group of 20 min hypoxia, while no difference was seen between the hypoxia and the control group.Conclusions1. It is feasible to establish a transient HIE model by exposing the newborn rat to 100% N2, that we designed to mimic events of neonatal hypoxic-ischemic brain injury occur in human.2. Transient hypoxia induces the up-regulation of NeuroD , which was found to express mainly in neural stem /progenitor cells rich areas, including cerebral cortex, hippocampus and SVZ.3. The transient neuronal loss induced by birth hypoxia may lead to cell proliferation, neuron differentiation and also may trigger neurogenesis, which in turn could participate in brain repair and functional restoration.