Damages And Mechanisms Of Retinal Bipolar Cells In N-methyl-D-aspartate Induced Glaucoma Model

Part1 Characteristics of N-Methyl-D-Aspartate induced damage in retina rod bipolar cellsPurpose:To investigate the morphological and functional characteristics of N-methyl-D-aspartate(NMDA)induced damage in retina rod bipolar cells.Methods:C57BL/6J mice(3 weeks)were randomly chosen,3 ?1 of 10 mM NMDA in PBS was injected into the vitreous cavity of the right eye and the same volume of PBS was administered into the fellow eye,which served as a control.The extent of NMDA-induced neurotoxicity in the retina was quantified by electroretinogram(ERG)on the whole animal level.The retinas were also processed by immunofluorescence staining histologically to determine morphological changes under microscope and patch clamp technique to invest the electrophysiological characteristics of different types of bipolar cells.Results:Immunofluorescence staining results revealed a significant decrease of RGCs after 24 h NMDA treatment,and the situation is the same with the RBC.The thickness of inner plexiform layer(IPL)was also decreased.ERG displayed an obvious decrease of b wave amplitude without obvious delay in a wave and b wave.The results of patch clamp displayed that the depolarization current of ON-RBC is completely suppressed after 24 h NMDA treatment.Conclusion:Administration of NMDA can result in excitotoxic damage of RGCs and RBCs,which is similar to glaucomatous degeneration and of important value in experimental research of glaucoma.Part2 Mechanisms of NMDA induced functional damage in retinal rod bipolar cellsPurpose:To investigate the mechanisms of NMD A induced functional damage in retinal rod bipolar cellsMethods:C57BL/6J mice(3 weeks)were randomly divided into three groups,3?1 of 10 mM NMDA?NMDA(10 mM)+KN93(1 nM)and NMDA(10 mM)+MG132(20 ug/ml)in PBS was injected into the vitreous cavity of the right eye correspondingly and the same volume of PBS was administered into the fellow eye,which served as a control.The retinas were then processed by immunofluorescence staining histologically to determine morphological changes under microscop and patch clamp technique to invest the electrophysiological characteristics of RBC after treatment.Injected the 3 ?1 of 10 mM NMDA into the vitreous cavity after received 200nM ONX0912 gavage in a dosage of 30 mg/kg for 5 d,then the retinas were then processed by immunofluorescence staining histologically.We use single cell realtime polymerase chain reaction to detect the RNA quantity of PKC,TRPM1 and Go etc.in rod bipolar cells.After anterior chamber infusion and optic nerve crush to modeling the mice glaucoma model,then processed the retinas by immunofluorescence staining histologically to determine morphological changes.Results:After intravitreal injection of NMDA,a robust decrease in expression of PKCa in the rod bipolar cell body and dendrites was observed as well as functional changes.KN93,the inhibitor of CaMK ?,could reverse the disorder in RBC caused by NMDA,and it was similar with the protease inhibitors MG132 and ONX0912.Single cell RT-PCR found that the RNA quantity of PKC?TRPM1?G?3?RGS7?G?5 decrease in NMDA injected retina,while the RNA quantity of mGluR6?G?o?GPR179?RGS11?GDIA?GDIB?GDIG increase in NMDA injected retina,the differences were statistically significant.In the glaucoma model via anterior chamber infusion and optic nerve crush,we can also observe the damage in RBC.Conclusion:Administration of NMDA can result in excitotoxic damage of RGCs and PKCa trafficking in rod bipolar cells by CaMK ? and proteasome.The injury of bipolar cells caused by NMDA is through increasing or decreasing the protein quantity of the components and the regulators in G protein signal pathway in bipolar cell.The damage of RBC in different glaucoma models has an universality in some degree.