The Role And Mechanism Of Melatonin In NMDA Induced Retinal Ganglion Cells Injury Mouse Model

Purpose: Glaucoma,one of the most common ocular neurodegenerative diseases worldwide,is characterized by retinal ganglion cells(RGCs)loss.There is a large body of literature that describes the neuroprotective role of melatonin against neurodegenerative diseases by regulating neuroinflammation,although the exact mechanism through which melatonin acts on RGCs is still uncertain.This study aims to explore the role and related mechanisms of melatonin in NMDAinduced RGCs injury mice model.Methods: C57/BL6 J wild-type mice were used to establish NMDAinduced RGCs injury mice model.Melatonin was daily intraperitoneal injection for treatment.Retinal immunofluorescence staining,retinal electrophysiological examination and visual evoked potentials were used to evaluate the protective effect of melatonin on injury RGCs.Terminal deoxynucleotidyl transfer d UTP nick end labeling(TUNEL)and propidium iodide(PI)staining were used to evaluate the effects of melatonin on apoptosis and necrosis of retinal cells.Immunofluorescence staining of frozen section was undertaken to detect the activation of retinal microglia,the expression of inflammatory factors and inflammatory related pathway proteins.The effect of melatonin on the expression of inflammatory factors in NMDA-induced RGCs injury mice model was detected by RT-q PCR and ELISA,and the effect of melatonin on inflammatory related pathways in NMDA-induced RGCs injury model was detected by western blot.PLX3397 was used to ablate microglia.TNFα neutralizing antibody,infliximab,and p38 MAPK pathway inhibitor,SB202190,were used to further investigate the mechanisms of melatonin in NMDA-induced RGCs injury mouse model.Results: Our study found that the numbers of RGCs in the retina of NMDA-induced RGCs injury mice model were decreased,the numbers of apoptosis and necrosis cells in the ganglion cell layer(GCL)and inner nuclear layer(INL)in retina were increased,and the retinal function was decreased.However,melatonin treatment reduced the loss of RGCs,reduced apoptosis and necrosis of retinal cells,and improved retinal function.In addition,in NMDA-induced RGCs injury retinas,the number of retinal microglia,the percentage of activation of CD68+ phagocytic microglia,and the expression of inflammatory factors(TNFα,NOS2 and IL-17)were increased.Melatonin treatment inhibited the activation of microglia and reduced the expression level of these inflammatory factors.After the administration of PLX3397(used to ablate microglia),the expressions of inflammatory factors in NMDA-induced RGCs injury retinas were decreased,and the survival of RGCs was increased.The administration of infliximab,TNFα neutralizing antibody,increased the number of RGCs in NMDA-induced RGCs injury retinas,and suppressed the activation of microglia.Further study has found that the p38 MAPK signaling pathway activated in NMDA-induced RGCs injury retina,and the p-p38 MAPK protein was highly expressed on RGCs.Treatment with melatonin decreased the expression of p-p38 MAPK protein on RGCs,while the administration of SB202190,p38 MAPK pathway inhibitor,promoted RGCs survival of retinas in NMDA-induced RGCs injury mice model.Conclusion: Our results suggest that melatonin protects against NMDA-induced RGCs injury by inhibiting the microglia-TNFα-RGCs p38 MAPK pathway.It should be considered a candidate neuroprotective therapy against retinal neurodegenerative diseases.