ABSTRACT
OBJECTIVE: To elucidate the role of aquaporin-4(AQP4) in cerebral edema formation, we studied the expression and subcellular localization of AQP4 in astrocytes after focal cerebral ischemia. METHODS: Cerebral ischemia were induced by permanent middle cerebral artery(MCA) occlusion in rats and estimated by the discoloration after triphenyltetrazolium chloride(TTC) immersion. Change of AQP4 expression were evaluated using western blot. Localization of AQP4 was assessed by confocal microscopy and its interaction with alpha-syntrophin was analyzed by immunoprecipitation. RESULTS: After right MCA occlusion, the size of infarct and number of apoptotic cells increased with time. The ratio of GluR1/GluR2 expression also increased during ischemia. The polarized localization of AQP4 in the endfeet of astrocytes contacting with ventricles, vessels and pia mater was changed into the diffuse distribution in cytoplasm. The interactions of AQP4 and Kir with alpha-syntrophin, an adaptor of dystrophin complex, were disrupted by cerebral ischemia. CONCLUSION: The deranged spatial buffering function of astrocytes due to mislocalized AQP4/Kir4.1 channel as well as increased assembly of Ca2+ permeable AMPA receptors might contribute to the development of edema formation and the excitotoxic neuronal cell death during ischemia.
Subject(s)
Animals , Rats , Apoptosis , Aquaporin 4 , Astrocytes , Blotting, Western , Brain Edema , Brain Ischemia , Cell Death , Cerebral Infarction , Cytoplasm , Dystrophin , Edema , Immersion , Immunoprecipitation , Ischemia , Microscopy, Confocal , Neurons , Pia Mater , Receptors, AMPA , Receptors, KIRABSTRACT
OBJECTIVE: Activated endothelial cells mediate the cascade of reactions in response to hypoxia for adaptation to the stress. It has been suggested that hypoxia, by itself, without reperfusion, can activate the endothelial cells and initiate complex responses. In this study, we investigated whether hypoxia-induced endothelial products alter the endothelial permeability and have a direct cytotoxic effect on nerve cells. METHODS: Hypoxic condition of primary human umbilical vein endothelial cells(HUVEC) was induced by CoCl2 treatment in culture medium. Cell growth was evaluated by 3,4,5-dimethyl thiazole-3,5-diphenyl tetrazolium bromide (MTT) assay. Hypoxia-induced products (IL-1beta, TGF-beta1, IFN-gamma, TNF-alpha, IL-10, IL-6, IL-8, MCP-1 and VEGF) were assessed by enzyme-linked immunosorbent assay. Endothelial permeability was evaluated by Western blotting. RESULTS: Prolonged hypoxia caused endothelial cells to secrete IL-6, IL-8, MCP-1 and VEGF. However, the levels of IL-1, IL-10, TNF-alpha, TGF-beta, IFN-gamma and nitric oxide remained unchanged over 48 h hypoxia. Hypoxic exposure to endothelial cells induced the time-dependent down regulation of the expression of cadherin and catenin protein. The conditioned medium taken from hypoxic HUVECs had the cytotoxic effect selectively on neuroblastoma cells, but not on astroglioma cells. CONCLUSION: These results suggest the possibility that endothelial cell derived cytokines or other secreted products with the increased endothelial permeability might directly contribute to nerve cell injury followed by hypoxia.