Glutamate And Its Transporter In Neonatal Rats After Hyperoxia

Objective To observe the change of glutmate and its transporters in the brain of neonatal rat exposed to hyperoxia for 24 hours,and to explore the mechanism of glutamate in hyperoxic brain injury.Methods Neonatal rats were divided into hyperoxia group and air control group.In order to make hyperoxic animal model,hyperoxia group was exposed 80% high oxygen at postnatal 6d(P6)for 24 hours.Air control group was exposed to normal air for 24 hours at the same time.All neonatal rats were decapitated to get brain tissue at P7.The change of glutamate(Glu)in the brain tissue and gamma aminobutyric acid(GABA)was detected by enzyme linked immunosorbent assay(ELISA).Western Blot method was used for the detection of high affinity glutamate transporter EAAT1,EAAT2,EAAT3,glucose transporter GLUT1,GLUT3 and vesicular glutamate transporter VGLUT1 and VGLUT2 protein expression.Reverse transcriptase polymerase chain reaction(RT-PCR)was used to detect the gene expression of high affinity glutamate transporter EAAT1 and brain type glucose transporter GLUT1 in the newborn rat brain.The statistical analysis and relevant statistical charts was used by SPSS 19.0 statistical software.Measurement data was caculated by mean ± standard deviation(),the two groups were compared by independent “t”test.Skewness data was caculated by median and four percentile of [M(P25,P75)],the two groups were compared by Mann-Whitney U test.P < 0.05 was considered as statistically significant.sx ±Results 1.It was found that the glutamate in the brain tissue of neonatal rats in the hyperoxia group was significantly higher than that in the control group [27.80(22.68,42.05)vs 17.46(14.75,20.16)]by ELISA test.The difference between the two groups was statistically significant(Z=-2.205,P=0.032).The Glu/GABA ratio in the brain tissue of neonatal rats at P7 in the hyperoxia group was 2.69(2.43,3.08)by ELISA test.The ratio of Glu/GABA in the brain tissue of newborn rats at P7 in the control group was 1.06(0.57,2.03).The difference between the two groups was statistically significant(Z=-2.205,P=0.032).2.The protein expression of glutamate transporter EAAT2,EAAT3,GLUT1 and VGLUT2 tested by Western Blot method in the neonatal rat brain at P7 in the hyperoxia group was significantly lower than that in the control group,there was significant difference between two groups(EAAT2:t=5.206,P=0.008;EAAT3:t= 2.785,P=0.024;GLUT1:t=2.892,P=0.020;VGLUT1: t=2.611,P=0.031).And the protein expression of glutamate transporter EAAT1,GLUT3 and VGLUT1 in the neonatal rat brain at P7 in the hyperoxia group decreased compared with that in the control group,but there was no statistically significance between hyperoxia group and control group(P>0.05).3.High affinity glutamate transporter EAAT1 m RNA expression tested by RT-PCT in the neonatal rat brain at P7 in the hyperoxia group increased compared with that in the control group(2.15±0.51 vs 1.31±0.20),but the difference between the two groups was not statistically significant(P=0.142),glucose transporter GLUT1 m RNA in the neonatal rat brain at P7 in the hyperoxia group reduced compared with that in control group(0.63±0.12 vs 1.09±0.15),there was significant difference between control group and hyperoxia group(t=2.443,P=0.025).The rusult was consistent with the Western Blot,indicating that the high oxygen decreased the expression of glucose transporter GLUT1 in gene level.Conclusion Hyperoxia resulted increase of glutamate,glutamate transporter EAAT2,EAAT3,GLUT1 and VGLUT2 decreased expression of Glu/GABA in brain tissue,the ratio increased,breaking the balance between brain excitatory amino acid and inhibitory amino acid,is one of the causes of brain injury caused by hyperoxia.