Identification Of Mechanisms Underlying Retinal Ganglion Cell Survival In Zebrafish After Optic Nerve Injury

Purpose: The mechanism of optic nerve injury as well as selective and progressive retinal ganglion cells(RGCs)death during the progression of glaucoma is still not very clear,and currently there is no effective treatment for neuronal protection in clinical practice.In this study,one of the animal models in glaucoma research-zebrafish optic nerve transection injury model,was used to explore the mechanism of retinal ganglion cell survival after optic nerve injury.Meanwhile,inflammatory response and immune regulation-related molecular signaling pathways on retinal ganglion cell survival and optic nerve repair was studied.Overall,we aimed to provide new references for neuronal protection in the process of glaucoma diagnosis and treatment.Methods: isl2b:GFP+ zebrafish,in which a subset of RGCs are labeled with GFP,were utilized at ～ 3 months(equal number of male and female animals).Animals underwent optic nerve transection surgery on the left eye,while the right eye of the same fish was used as the control.To quantify RGCs survival after transection,retinal flat mounts were made and imaged using confocal microscopy at 1,3,7&14 days post injury(dpi)and RGCs survival over time was quantified using Fiji Image J software.Retinae were dissociated at 6,12 and 24 hours post transection and RGCs were sorted and collected by fluorescence-activated cell sorting(FACS).Bulk RNA-Seq was performed in biological triplicates,and gene expression changes after optic nerve transection were quantified.After analysing RNA-seq data,several genes and pathways were identified to be involved in RGCs survival.Based on RNA-seq results,pharmacological experiments were performed by intravitreal injection of JAK/STAT inhibitor P6 and dexamethasone,which suppressed inflammation.The results on RGCs survival were quantified.Moreover,transgenic line mpeg1:m Cherry+,in which macrophages/microglia populations were labeled,and macrophages/microglia depletion drug PLX3397 were used to assess the interaction of immune responses and RGCs survival after optic nerve transection.Results: After optic nerve transection,RGCs survival was: 94.44 ±5.45%(1dpi),92.52 ± 3.54%(3dpi)and 76.35 ± 2.58%(7dpi),with levels returning to 98.71 ± 4.82% at 14 dpi.Analysis of RNA-seq data indicate JAK/STAT signaling pathway and immune responses were activated after optic nerve transection,amongst other pathways.Immunostaining of p STAT3 showed increasing expression of it after optic nerve transection at 1dpi and 7dpi,which were expressed mostly cytoplasmically.Local inhibition of the JAK/STAT pathway leads to the reduction of RGCs survival,while local suppression of immune responses by dexamethasone rescued RGCs after optic nerve transection.Furthermore,depletion of macrophages/microglia by PLX3397 increased RGCs survival after optic nerve transection even with additional inhibition of JAK/STAT signaling.Conclusions: Zebrafish maintain most RGCs after optic nerve transection.JAK/STAT signaling pathway regulates RGCs survival after optic nerve transection.Immune responses play a critical role in RGCs survival after optic nerve transection.