Retinal Bipolar Cells Changes In N-Methyl-D-Aspartate Induced Glaucoma Model

Part1 Characteristics of N-Methyl-D-Aspartate induced retinal neurotoxicity in miceObjective:To investigate the morphological and functional characteristics of N-methyl-D-aspartate (NMDA) induced retinal neurotoxicity in mice.Methods:C57BL/6J mice (6 weeks) were randomly divided into low, medium and high three groups to receive a single dose of 1 μL intravitreal injection of 10 nmol,20 nmol and 30 nmol of NMD A per injection respectively.6 h,12 h,24 h,48 h and 7 d after NMD A injection, mice were killed and the eyes were enucleated. The extent of NMDA-induced neurotoxicity in the retina was quantified by fundus photograph and electroretinogram (ERG) on the whole animal level. The retinas and optic nerves were also processed histologically to determine morphological changes under microscope.Results:Cell counting revealed a significant decrease of RGCs after 24 h NMDA treatment in three dose groups. The thickness of inner plexiform layer (IPL) was also decreased.30 nmol NMDA treated retina showed a pale color change caused by vasospasm. ERG displayed an obvious decrease of b wave amplitude without obvious delay in a wave and b wave. Following retinal vessel labeling by Isolectin IB4, we observed no obvious retinal vessel damage. A progressive degeneration of RGCs and optic nerve axons was seen in NMDA treated eyes related to the time NMDA administrated. By using antibodies against Brn-3a on retinal whole mounts, a significant loss of RGCs (approximately 83.97%) was shown in 24 h (P<0.01).Conclusion:Administration of NMDA can result in excitotoxic damage of RGCs, which is similar to glaucomatous degeneration and of important value in experimental research of glaucoma. ERG b wave amplitude may of great value in the diagnosis and assessment of retinal neurotoxicity.Part2 NMDA induced functional PKCa trafficking in retinal rod bipolar cellObjective:To investigate a novel finding in NMDA induced retinal neurotoxicity in mice, linking together NMDA induced functional important PKCa trafficking in Rod bipolar cell.Methods:C57BL/6J mice (6 weeks) received a single dose of 1 uL intravitreal injection of 30 nmol of NMDA.12h,24 h and 48h after NMDA injection, mice were killed and the eyes were enucleated. The retinas were processed histologically to determine apoptosis of retinal neurons, PKCa labeled rod bipolar cells and NMDAR subunits synaptic trafficking under microscope. We also removed the retina and recorded the electrophysiological properties change of retinal bipolar cells by patch clamp whole-cell mode, using single cell polymerase chain reaction to detect the mRNA level of mGluR6-Go-TRPM1 signaling pathway in rod bipolar cells.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, significantly reducing the amplitude of the CPPG-evoked rod bipolar cells responses, measured using patch clamp recording. NR1, NR2B and NR2D synaptic trafficking localized precisely with PKCa trafficking in ONL of retina., In addition, NMDA damage the GPRC signal pathways and induce TRPM1 channel opening through G protein signaling, this may reveal how NMDA injure the function of RBCs.Conclusion:Administration of NMDA can result in excitotoxic damage of RGCs and PKCa trafficking in rod bipolar cells by mGluR6-Go-TRPM1 signaling pathway, the potential novel research on which would contribute to the progression of the related retinal diseases.