| ObjectiveTo isolate, purify and identify astrocytes from Sprague-Dawley rats, build the models of Oxygen glucose deprivation/reoxygenation (OGD/R), and investigate the expression and its significance of adenosine preconditioning induced astrocyte glutamate transporter protein GLT-1 and chemokine CX3CL1, lay the foundation for clinical treatment of ischemic cerebrovascular diseases.Methods1. Astrocytes of cerebral cortex in Sprague-Dawley rats were cultured in vitro; Astrocytes were cultured in vitro of neonatal SD rat according to McCarthy and Yu aiqing’s method. With the cortical distribution of astrocytes, cortexes of Sparague-Dawley rats at postonal 24 hours were collected. Vessels and meninges were dissected. Cells were treated with mechanical blow and beat method, adherence method with different speed and cell passage. Cell morphology was observed under inverted phase contrast microscope during culturing.2. The third or forth generation of cells were identified by glial fibrillary acidic protein (GFAP) with immunofluorescence.3.Cultured third or fourth generation of astrocytes were passaged at 96 or 24 well plate, fter attaching to the wall, astrocytes were randomly divided into 3 groups, the normal group, OGD/R group and ADO/R group. Astrocytes in the normal group did not receive OGD and were cultured in normal glucose medium as usual during oxygen-glucose deprivation and reoxygenation. Astrocytes in OGD/R group were cultured in medium without glucose during oxygen-glucose deprivation. And ADO/R preconditioning group were cultured in complete DMEM culture medium with 100 μmol/L adenosine at 24 hours before oxygen-glucose deprivation. And then put OGD/R and ADO/R groups into the chambers which were flushed with a gas mixture of 95% N2 and 5% CO2 for 5 hours at room temperature at 1L/min. After 5 hours, astrocytes at the two groups were taken out and were changed medium with glucose respectively, and then they were put back to cell culture incubator for 24 hours. Cellular morphological changes, cell viability and concentration of CX3CL1 were observed after oxygen-glucose deprivation and reoxygenation. The expression of CX3CL1 and GLT-1 in the astrocytes were checked using Elisa、immunofluorescence and Western blotting All comparisons were performed by one-way ANOVA followed by post hoc analysis with correction using spass 17.0. All values are given as mean ± s.d. The critical P level set for significance is less than 0.05.Results1. The astrocytes were isolated, purified and identified successfully.2. The morphological observation was taken at 24 hours after reoxygenation.Astrocytes in the normal group grew well as usual. The cells in ADO/R group reserved the astrocyte-like characteristics, but the body of them had a slighter edema and their cellular process became thinner and shorter; while the cells in OGD/R group became round because of serious edema and their cellular process completely disappeared. The edema in ADO/R group was less.3. The cell viability was quantified by MTT assay. Compared with the normal group, the cell viability in the OGD/R group and ADO/R group produced a significant decrease (P<0.05), which indicated that the cell viability declined because astrocytes were injured or dead; the viability in ADO/R group were higher than that of OGD/R group, which showed us that ADO/R could improve the viability of the cells. There were significant differences between each two groups (all P<0.05).4. Detection of CX3CL1 vitality in cell culture fluid:compared with the normal group, the CX3CL1 concentration in OGD/R group was significantly increased (P<0.05), which meant that astrocytes suffered seriously; compared with OGD/R group, CX3CL1 concentration in ADO/R group obviously decreased (P<0.05), which showed us that the ADO protected astrocytes from being injured. There were significant differences in CX3CL1 concentration between each two groups (all P<0.05).5.The result of GLT-1 immunofluorescence:the fluorescence signal in cytoplasmic and membrane of the normal group was stronggest; fluorescence signal in membrane was weakened after oxygen-glucose deprivation; fluorescence expression in cytoplasmic was weak and no significant change. Compared with normal group, the fluorescence intensity in membrane of adenosine group decreases, but higher than the OGD/R group.6.Detection of expression of GLT-1 by Westernblot:astrocytes in the normal group have the highest relative protein expression of GLT-1. ADO/R group followed, OGD/ R group lowest.There were significant differences between each two groups (all P<0.05).ConclusionsAstrocytes are injured seriously after oxygen-glucose deprivation;Adenosine can reduce the injury and apoptosis of oxygen-glucose deprivation, A large amount of CX3CL1 were released after OGD/R, adenosine can block this effect. Measurements of injury severity of astrocytes are changed in the correspondence with cell viability and CX3CL1 concentration. Adenosine can up regulation GLT-1 protein and increase the expression GLT-1 resulting in brain ischemic tolerance. |
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