| Objective The mechanisms underlying hypoxic ischemic brain damage are only partially understood but are commonly suggested to include excitotoxicity, and induction of secondary apoptosis and inflammation. Previous studies have shown that hypoxia-ischemia (HI) results in a cascade of biochemical events and pathological reactions that lead to neuronal death in the hippocampus.Astrocytes are the most numerous cell types in the central nervous system. They provide structural, metabolic and trophic support to neurons and modulate synaptic activity. There is considerable evidence that astrocyte-conditioned media support the survival of neurons. Hypothermia is effective in reducing brain injury and improving behavioral recovery in animal experiments and is also safe and effective in neonatal clinical settings. Previous studies have shown that mild hypothermia preserves neurons and reduces astrocyte proliferation in adult animal models. Nevertheless, it is still unclear if mild hypothermia can attenuate astrocyte loss in the hippocampus of HI injured immature rat brain, and the corresponding changes of astrocyte proliferation and its apotosis response.Method(1)in vivo:Neonatal HI was induced in rats at postnatal day 7, according to the method described by Rice.The left common carotid artery was isolated and ligated, placed in a chamber perfused with a humidified gas mixture (8% oxygen in nitrogen) for 2h. The operated pups were randomly divided into the normo-group(36-37℃) and hypothermia groups(32-33℃),respectively. Control animals were anesthetized but not subjected to HI. They were then randomized to either normothermia or hypothermia.animals were killed by decapitation at at 0,3 and 7 days after HI. The brains were removed and immersion-fixed, coronal sections were cut on a freezing microtome at a thickness of 30μm for glial fibrillary acidic protein count(GFAP) and caspase-3 immunohistochemical staining. Cells were counted in the CA1 region of ipsilateral hippocampus detected the apoptosis of astrocytes.(B) In vitro:All cultured AST originated from 1 day old rat hippocampus, using oxygen and glucose deprivation (oxygen-glucose deprivation, OGD)method to simulate the HI injury, using CCK-8, Tunel and DAPI staining to assess the activation, proliferation and apoptosis of astrocyte.Results In vivo, GFAP-positive cells of hippocampus was significant decreased at 72 hour and 7 days after HI in hypothermia treatment group (P<0.05), There are significant differences between normothermia and hypothermia groups at 72hours and 7days after HI.84.5% co-expression of GFAP and Caspase-3 cells showed in hippocampus with immunofluorescence staining in HI group, but 32.3% in hypothermia treatment group. In vitro, cell activation and proliferation were observed at 24-72 hours following 1-4 hours OGD, which was significantly decreased in the hypothermia treatment group. Significant increase in cell death occurred 24-48 hours after 1-4 hours OGD, and cell death measured at 24 hours,48 hours was significantly reduced in hypothermia treatment group. Experiments showed that the Tunel positive cells decreased significantly (P<0.05).Conclusion Mild hypothermia can reduce the activation, proliferation,and inhibit apoptosis of the AST in neonatal rat hippocampus injured by HI.
|
PDF Full Text Request