| Glutamate is the main excitatory neurotransmitter in the mammalian CNS. To ensure an appropriate signal transmission and to prevent glutamate neurotoxicity resulting from excessive activation of glutamate receptors, extracellular glutamate concentration must be maintained at relatively low levels in physiological conditions.Glutmate transporters, most especially the glutamate transporter-1(GLT-1) and the glutamate-aspartate transporter (GLAST) that are located in astrocytes and microglia, play an important role in the regulation of the glutamate concentration in the synaptic cleft. Glial glutamate transpoters are responsible for clearing more than80%of the synaptic glutamate into glial cells.Glial cells can also express glutamine synthetase to convert glutamate to glutamine, the latter amino acid is then recycled into neurons,thus the glutamate can be used repeatedly.Disfunction of glutamate transporters can lead to accumulation of glutamate in the synaptic cleft, and the glutamate receptor are continued activated then, causing well-known excitotoxicity, leading to neuronal death at last.Previous research have provided compelling evidence that brain trauma and many neurodegenerative disorders are associated with change in localization and/or expression of some of the subtypes of these transporters. This would suggest that therapies directed toward enhancing transporter expression might be beneficial. Considering that GLAST and GLT-1are mainly located in astrocytes and microglia, and they play a predominant role in transporting glutemate, it was necessary to investigate expression changes and regulation of glial glutamate transporters GLAST and GLT-1in different glial cells using Quantitative immunohistochemistry after spinal cord injury in adult rats. The conclusion may provide a theoretical basis for further research which aim at regulating the expression of glutamate transporters, thereby decreasing extracellular glutamate levels, reducing excitotoxicity, reducing the secondary spinal cord injury at last.PURPOUS(1) To investigate the changes of glial glutamate transporters GLAST and GLT-1expression after spinal cord injury in adult rats.(2) To investigate the role ERK1/2signalling pathway on regulation of glial glutamate transporters GLAST and GLT-1expression after spinal cord injury in adult rats.METHODSPart1:Five rats were selected randomly from25adult healthy female S-D rats as the normal control group, the rest rats were divided into4groups after acute contusive spinal cord injury(SCI) was performed, each group contained5rats. At1d,3d,7d and14days after SCI, the neurological function of hind limbs was assessed with the modified Tarlov scale in rats, then the rats were executed and the spinal cord segments which were adjacent to the epicenter of injury were obtaind, five rats were executed at every timepoint. Changes of the GLAST and GLT-1expression was detected semi-quantitativly with immunofluorescence and computer image analysis system after SCI in rats.Part2:Five rats were selected randomly from55adult healthy female S-D rats as the normal control group, the rest rats were divided into two groups after acute contusive injury was performed:experimental group and SCI group, each group contained25rats. Rats of experimental group received U0126(500μg/kg) intraperitoneal injection at30min before SCI and at1h and6h after SCI; U0126was replaced with saline in SCI group. At8h,12h,1d,3d and7days after SCI, the rats were sacrificed and the spinal cord segments which were adjacent to the epicenter of injury were obtaind, five rats of each group were executed at every timepoint in each group. Changes of the GLAST and GLT-1expression was detected semi-quantitativly with immunofluorescence and computer image analysis system after SCI in rats.RESULTSPart1:(1) In the spinal cord cross sections of the normal control group rats, GLAST mainly expressed in the gray matter, some expressed in the white matter, the latter was mainly in superfical parts of the white matter. The expression of GLAST increased slightly on Id after SCI, then it decreased progressively on3d and7d after SCI.The expression of GLAST was the lowest on7d among all the timepoints after SCI, then it increased slightly on14d after SCI, but it remained lower expression than that in the control group, significant difference in GLAST expression was revealed on3d and7d compared to the control group after SCI in rats (P<0.05);(2) In the spinal cord cross sections of the normal control group rats, GLT-1mainly expressed in the gray matter, some expressed in the white matter, the latter was mainly in superfical parts of the white matter. The expression of GLT-1increased slightly on Id after SCI, then it decreased on3d after SCI.The expression of GLT-1was the lowest on3d among all the timepoints after SCI, then it increased slightly on7d and14d progressively after SCI, but it remained lower expression than that in the control group, significant difference in GLT-1expression was revealed on3d,7d and14d compared to contral group after SCI in rats (P<0.05);(3) Double immunofluorescent staining showed that in the spinal cord cross sections of the normal control group rats, there was obvious co-localization of GLAST with GFAP positive astrocytes. The double positive area (DPA) of GLAST and GFAP showed a down-regulation on1d after SCI, then it showed progressive down-regulation on3d and7d after SCI. The double positive area was the lowest on7d among all the timepoints after SCI, then it showed up-regulation slightly on14d after SCI, but it remained lower DPA than that in the control group, significant difference in DPA of GLAST and GFAP was revealed on3d and7d compared to contral group after SCI in rats (P<0.05).(4) Double immunofluorescent staining showed that in the spinal cord cross sections of the normal control group rats, there was obvious co-localization of GLT-1with GFAP positive astrocytes.The double positive area of GLT-1and GFAP showed a down-regulation on1d after SCI, then it showed progressive down-regulation on3d and7d after SCI.The double positive area was the lowest on7d among all the timepoints after SCI, then it showed up-regulation slightly on14d after SCI, but it remained lower DPA than that in the control group, significant difference in DPA of GLT-1and GFAP was revealed on1d,3d,7d and14d compared to control group after SCI in rats(P<0.05).(5) Double immunofluorescent staining showed that in the spinal cord cross sections of the normal control group rats, there was some co-localization of GLAST with OX-42positive microglia.The double positive area of GLAST and OX-42showed a up-regulation on1d after SCI, then it showed progressive up-regulation on3d,7d and14d after SCI. The DPA was the largest on14d among all the timepoints after SCI, significant difference in DPA of GLAST and OX-42was revealed on3d,7d and14d compared to contral group after SCI in rats(P<0.05).(6)Double immunofluorescent staining showed that in the spinal cord cross sections of the normal control group rats, there was some co-localization of GLT-1with OX-42positive microglia.The double positive area of GLT-1and OX-42showed a up-regulation on Id after SCI, then it showed progressive up-regulation on3d,7d and14d after SCI. The DPA was the largest on14d among all the timepoints after SCI, significant difference in DPA of GLT-1and OX-42was revealed on1d,3d,7d and14d compared to contral group after SCI in rats(P<0.05).Part2(1) In the spinal cord cross sections of the normal control group rats, there was moderate amount expression of GLAST, the expression of GLAST in the gray matter was higher than that in the white matter. The expression of GLAST increased slightly on8h in SCI group after SCI, then it decreased progressively from12h to7d after SCI.The expression of GLAST was the lowest on7d among all the timepoints after SCI, significant difference in GLAST expression was revealed on8h,12h,3d and7d compared to the control group after SCI in rats (P<0.05); Change of the GLAST expression in experimental group had the same trend as that in SCI group, significant difference in GLAST expression was revealed on8h,12h and7d compared to the control group after SCI in rats (P<0.05); The expression of GLAST in experimental group was higher than that in SCI Group on all observed timeponts after SCI, significant difference in GLAST expression in the experimental group was revealed at 8h,12h and24h compared to that in SCI group after SCI in rats(P<0.05).(2)In the spinal cord cross sections of the normal control group rats, there was moderate amount expression of GLT-1, the expression of GLT-1in the gray matter was higher than that in the white matter. The expression of GLT-1increased slightly on8h in SCI group after SCI, then it decreased progressively from12h to3d after SCI.The expression of GLT-1was the lowest on3d among all the timepoints after SCI, then it increased slightly on7d after SCI, but it remained lower expression than that in the control group, significant difference in GLT-1expression was revealed on8h,12h,3d and7d compared to the control group after SCI in rats(P<0.05); Change of the GLT-1expression in experimental group had the same trend as that in SCI group, significant difference in GLT-1expression was revealed on8h,12h and24h compared to the control group after SCI in rats (P<0.05); The expression of GLT-1in the experimental group was higher than that in the SCI Group on all observed timeponts after SCI, significant difference in GLT-1expression in the experimental group was revealed at8h compared to that in SCI group after SCI in rats(0.01<P<0.05).CONCLUSIONS:(1) The expression of GLAST and GLT-1increase shortly after SCI, then they decrease progressively, and restored gradually at last after SCI in rats.(2) The expression of GLAST and GLT-1decrease in astrocytes, and restored gradually at last after SCI in rats;The expression of GLAST and GLT-1increase continiously in microglial cells after SCI in rats.(3) ERK1/2signalling pathway is involved in regulating the expression of GLAST after spinal cord injury in adult rats.(4) These results indicate that the expression of glutamate transporters GLAST and GLT-1are regulated through different signalling pathways following SCI, and astrocytes and microglial cells may play different roles in the course of secondary injury and on recovery of neural function following SCI in adult rats. |
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