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BDNF Regulates GLAST and Glutamine Synthetase in Mouse Retinal Muller Cells

Author: DaiMin
Tutor: XiaXiaoBo
School: Central South University
Course: Ophthalmology
Keywords: BDNF Hypoxia M(u ¨)ller glia cells Glutamate transporter Glutamine synthetase Glutamate
CLC: R774.1
Type: PhD thesis
Year: 2012
Downloads: 39
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Abstract


Chapterl Effect of BDNF on the GLAST and GS in mouse retinal Miiller cells under normal oxygen conditionObjective:To study the effect of brain-derived neurotrophic factor (BDNF) on the expression and function of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) in mouse retinal Miiller cells (RMCs) under normal oxygen conditions.Methods:Retinal Miiller cells (RMCs) of Kunming mouse at postnatal3to7days were cultured by enzymatic digestion. RMCs passaged three times were used in next experiments. RMCs cultures were divided into BDNF group:Cultured retinal Muller cells were maintained with different concentrations of recombinant human BDNF (50ng/ml,75ng/ml,100ng/ml,125ng/ml or150ng/ml)for24h in vitro, and Control group:Cultured retinal Muller cells were maintained without BDNF. The expression of GLAST and GS mRNA was measured by RT-PCR. The expression of GLAST and GS protein was measured by western blotting and immunocytochemical staining. L-[3,4-3H]-glutamic acid uptake was used to quantify glutamate uptake function of RMCs in BDNF group (100ng/ml) and normal control group.Results:Different concentrations of BDNF could up-regulate GLAST and GS expression compared to normal control (p<0.05). The expression of GLAST and GS reached the maximum when the concentration of BDNF was100ng/ml. Immunocytochemistry demonstra-ted that GLAST was predominantly found in the membrane and cytoplasm, while GS was predominantly found in the cytoplasm. Both GLAST and GS immunostaining appeared more intense in BDNF-treated cells. L-[3,4-3H]-glutamic acid uptake of BDNF group (100ng/ml) was significantly higher than normal control group.Conclusion:BDNF may up-regulate GLAST and GS expression to increase extracellular glutamate uptake under normal oxygen conditions. Chapter2Effect of hypoxia on the GLAST and GS in mouse retinal Muller cellsObjective:This study investigated the effect of hypoxia on the expression and function of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) in mouse retinal Muller cells (RMCs)Methods:RMCs of Kunming mouse at postnatal3to7days were cultured by enzymatic digestion. RMCs cultures were treated with CoCl2(125μmol/L) for6h,12h,24h,48h or72h respectively in vitro. RMCs cultures were maintained without CoCl2in normal control group. The expression of GLAST and GS mRNA was determined by RT-PCR. The expression of GLAST and GS protein was determined by western blotting and immunocytochemical staining. L-[3,4-3H]-glutamic acid uptake was used to quantify glutamate uptake function of RMCs. The apoptosis of RMCs was confirmed by annexin V-FITC/PI staining.Results:In early-stage of CoCl2-induced hypoxia (treated with CoCl2for6h,12h,24h or48h), the expression of GLAST was up-regulated (p<0.001) and reached the maximum when RMCs have been treated with CoCl2for12h (p<0.05). After RMCs have been treated with CoCl2for72h, the expression of GLAST had no difference compared to normal control (p>0.05). CoCl2-induced hypoxia (treated with CoCl2for6h,12h,24h,48h or72h) also up-regulated the expression of GS (p<0.001), which reached the maximum when RMCs have been treated with CoCl2for48h (p<0.001). The L-[3,4-3H]-glutamic acid uptake of hypoxia groups were higher than normal control group (p<0.05), and after having treated RMCs with CoCl2for48h, the L-[3,4-3H]-glutamic acid uptake was highest (p<0.005). Treatments with CoCl2did not induce apoptosis in RMCs.Conclusion:In early hypoxia stage, GLAST and GS may be up-regulated and extracellular glutamate uptake may be increased. However, continued hypoxia may cause gradual dysfunction and decreasing of GLAST and GS expression, as well as glutamate uptake. Chapter3Effect of BDNF on the GLAST and GS in mouse retinal Miiller cells under hypoxiaObjective:To study the effect of brain-derived neurotrophic factor (BDNF) on the expression and function of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) in mouse retinal Miiller cells (RMCs) under hypoxia.Method:RMCs of Kunming mouse at postnatal3to7days were cultured by enzymatic digestion. RMCs cultures were treated with CoCl2(125μmol/L) for72h, then combined with BDNF (100ng/ml) for24,48,72, or96h. RMCs cultures were maintained without CoCl2and BDNF in normal controls, RMCs cultures were maintained with CoCl2for72h in hypoxia controls. The expression of GLAST and GS mRNA was determined by RT-PCR. The expression of GLAST and GS protein was determined by western blotting and immunocytochemical staining. L-[3,4-3H]-glutamic acid uptake was used to quantify glutamate uptake function of RMCs. The apoptosis of RMCs was confirmed by annexin V-FITC/PI staining.Result:BDNF treatment increased GLAST and GS mRNA and protein levels compared to nomal controls (p<0.001) and to hypoxia controls (p<0.05). Maximum protection was observed at48h after BDNF treatment (p<0.05), However, when BDNF treatment occurred at24h before CoCl2treatment, GLAST and GS expression had no difference compared to normal controls (p>0.05). Both GLAST and GS immunostaining appeared more intense when BDNF treatment occurred after CoCl2treatment, but not when BDNF treatment occurred at24h before CoCl2treatment. At72h after hypoxia, BDNF treatment also increased L-[3,4-3H]-glutamic acid uptake compared to nomal controls (p <0.001) and hypoxia controls (p<0.005), and this effect was maximal at48h after BNDF treatment. Hypoxia combined with BNDF had not induce death (apoptotic and necrotic) of RMCs compared with controls (p>0.05). Conclusion:BDNF may up-regulate GLAST and GS expression and increase extracellular glutamate uptake in RMCs under hypoxia, but BDNF treatment has no effect at24h before hypoxia.

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CLC: > Medicine, health > Ophthalmology > Retina and optic nerve diseases > Retinal disease
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