| Membrane water transport is critically involved in brain volume homeostasis and in the pathogenesis of brain edema, which is of major importance in the progress of stroke. Although the detail has not been definite, we can deduce that the handicap of water transport is the key of brain edema. Discovery of aquaporin-1 answered the long-standing biophysical question of how crosses plasma membranes. Characterization of aquaporins provided molecular insight into fundamental processes of normal water balance and disorders of water balance outside brain. In human aquaporin family, AQP4 is the most abundantly distributed in the astrocytes that are specialized for water transport. Then, it is likely to indicate a potential mechanism of the brain edema to investigate the relationship between brain edema and AQP4. The primary culture of astrocytes is the first step of our study, then replicate the model of hypoxia and reoxygenation to observe the changes of AQP4 by immunocytochemiscal examinations and Western blot. Firstly, cultures of astrocytes was prepared from cerebral cortices of newborn Wistar rats(less than 24 hours). Trypsinized and dissociated cells were plated in culture flakes containing high glucose Dulbecco's modifided Eagle's medium supplemented with 20% fetal bovine serum. After incubation for 7~10 days ,the cells were trypsinized and subcultured. It would take 4 progress of subculture to get the stable cells. Homogeneity of the cell populations was confirmed by immunocytochemical examinations with anti-glia fibrillary acid protein. When the cultured astrocytes became confluent in culture dishes, then they were placed in a controlled atmosphere culture chamber, a humidified airtight apparatus with inflow and outflow valves, into which a mixture of 95% N2 and 5% CO2 was flushed for the appointed time. The chamber was sealed to maintain the gas composition and kept at 37℃. During the exposure to hypoxia and reoxygenation, the cultured cells were collected at the indicated times from 0 to 12 hours for monitoring the lactate dehydrogenase (LDH) and MTT to estimate cellular damage. After exposure to hypoxia for indicated periods (from 0 to 12 hours), the culture dishes were transferred to an ordinary incubator with 95% air and 5% CO2 from the cells reoxygenation. The culture medium was not replaced during hypoxia and reoxygenation. At the same time, the cultured cells were collected for immunocytochemistry and Western blot . Membrane preparations were obtained by the following method. The cells were harvested with phosphate buffered saline at the indicated times and centrifuged for 10mins, yielding pelleted membrane fractions. Samples were separated by 10% sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (PAGE) and then transferred to an Immobilon-P transfer membranes. Then the following steps were operated as the routine. The results showed that hypoxia did not impair the cells seriously during 12 hours, but reoxygenation make a damage obviously and the longer time, the greater damage in the shape of astrocytes. Correspondingly, the LDH changed little in hypoxia and made great transform in reoxyenation. The MTT was looked as the same. So we could deduce that hypoxia and reoxyenation induce the damage of the cultured cells especialy. The expression of AQP4 was decreased by hypoxia in a time-dependent manner. Reoxyenation increased the level of AQP4 as the control after 3 hours, and reach a higher level at 6 and 9 hours. We induced that hypoxia did not do a serious harm to the cultured cells, and at the same time, AQP4 protein have protective impact on astrosytes. When the damage became serious in reoxygenation, AQP4 manifest a harmful influence on astrocytes. We concluded that it can actualize the model of hypoxia and rexoygenation to alter the atmosphere of the cultured astrocytes. The cells do not change greatly in hypoxia and shift in opposition, the expression of AQP4 become less in hypoxia than the control and become more as the...
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