The Effects Of High Glucose On The Content Of Glutamate In Cultured Astrocytes Extracellular Solution Of Rats Stimulated By High K~+

ObjectiveTo investigate the effect of high glucose on the content of glutamate incultured rats astrocytes extracellular solution stimulated by high K+.MethodsThe cells was made into suspension by0.125ï¼…trypsin and mechanicaldissociation from the neonatal rat （aged1³days） cerebral cortex，and it wascultured in3⁴Generation.Then the cultivated astrocytes were identified byimmunohistochemical staining with anti-rabbit GFAP.And the cells were dividedinto four group,randomly:The cultured astrocytes were incubated in extracellularsolution （ES） with different concentrations of glucose (5,10,20and40mmol·L^-1)ï¼›and then the cells were incubated with high KCl(75mmol·L^-1) for0、1、2、3、4and5min，and different concentrations of glucose doesn’t change，and glutamate concentrations were measured.AST morphology were observedunder inverted microscopeï¼›Cell viability were assayed by MTT stainingï¼›Theconcentrations of glutamate at different times were measured by HPLC withprecolumn O-phthaldialdehyde （OPA）and N-lsobutyryl-D-cysteine （IBDC）derivatization.ResultsWith immunohistochemisty appraise, the proportion of astrocyte was>95ï¼….The concentrations of Glu were measured in the culture of AST extracellularsolution.Concentrations of Glu were Determine in extracellular fluid of AST by culture.The linearity of Glu was good in the range of12.5～200μg·L^-1（r=0.9959）；Relative Standard Deviation of Peak area was0.79%ï¼›AverageRecovery was98.5%，it show that detection methods of Glu was reliable.Concentrations of Glu were measured by HPLC in extracellular fluid.Therewas no significant change between Concentrations of Glu in culture supernatantwith different concentration of glucose within3h，and Cell survival was noobvious change with MTT for3h.While after incubation with high KCl(75mmol·L^-1)，compared with culture supernatant by glucose5mmol·L^-1，Glucontents in culture supernatant with glucose10,20,40mmol·L^-1significantlyincreased （P<0.01）.But changes of Glu content were different in thesegroups.The change concentration of Glu in Mannitol group was like5mmol·L^-1group.ConclusionsHigh glucose could effect concentration of Glu in culture supernatant ofAST，which maybe through increasing release and inhibiting the uptake ofGlu.So this will increase Concentrations of Glu in extracellular solution.