Expression Of Jarid1b And H3K4me3 After Optic Nerve Injury,Primary Culture And Identification Of Retinal Ganglion Cells

ObjectiveThe expression of Jarid1 b and H3K4me3 in the axons after optic nerve injury was studied by establishing a rat model of optic nerve injury,so as to understand whether Jarid1 b plays a certain role in optic nerve injury.The primary culture model of retinal ganglion cells(RGCs)was established to observe the cell morphology under light microscope,and the purity of RGCs was identified by immunocytochemistry,which provided a certain basis for the study of the physiological function or biological characteristics of RGCs.Methods1.Fifteen adult SD rats of clean grade were selected,regardless of gender.Each rat was treated with the left eye as the injured eye,and microvascular clamp was used to clamp the optic nerve vertically at about 2mm behind the eye for about 15 seconds to damage the optic nerve.The right eye was treated as the control eye,the optic nerve was exposed for 15 s without injury.After the injury or exposure of the optic nerve,the eyes of the rats were examined and FVEP was detected,and the results were compared with those before the injury to ensure the success of the optic nerve injury model.At day 3,7 and 14 after optic nerve injury,5 rats were randomly sacrificed.Pathological sections of left eye and right eye optic nerve tissues were made and used as acute injury group and normal control group respectively.After immunohistochemical staining,the positive expression rates of Jarid1 b and H3K4me3 in the two groups were calculated for statistical analysis.2.SD rats born within 7 days were selected,and their eyeballs were removed after anesthesia to detach the retinal tissue.We used papain to digestion the retinal tissue to preparation of RGCs suspension.Then rabbit anti-rat macrophage polyclonal antibody,goat anti-rabbit IgG(H + L),goat anti-mouse IgG(H + L)and Thy-1 antibody were used to screen and purify RGCs.Finally,the cells were cultured in 24-well cell culture plates pretreated with poly-D-lysine and laminin.Until the third day,Thy-1monoclonal antibody was used for immunocytochemical identification.3.SPSS version 22.0 software was used for statistical analysis in this paper.Independent sample t test was used to compare results of the same days in different groups,anova was used for the comparison of different days in the same group,and LSD-t test was used for the pairwise comparison of different days in the acute injury group.The test level was 0.05,and P < 0.05 indicates that the difference was statisticallysignificant.Results1.The expression of Jarid1 b in the axons of the normal control group was moderate,and there was no significant change at different time points.The expression of Jarid1 b in the acute injury group was significantly lower than that in the normal control group at each time point.It decreased on the third day and reached a peak,then gradually increased,and the expressions of Jarid1 b on the seventh and fourteenth days were still lower than that in the normal control group.2.The expression of H3K4me3 in the axons of the normal control group was high,and there was no significant change at different time points.The expression of H3K4me3 in the acute injury group was significantly higher than that in the normal control group at each time point.It increased on the third day,reached a peak on the seventh day,and then gradually decreased.The expression of H3K4me3 in the acute injury group was still higher than that in the normal control group on the fourteenth day.3.Observed under optical microscope,the RGCs were almost completely adherent after 24 hours,the volume increased gradually and most cells could grow axone after 48 hours.At day 3 to 5,the number of protuberances continued to increase.On day 6,some cells died.On day 7,the number of cells decreased significantly and most cells died.4.Under fluorescence microscope,RGCs can be labeled as red by thy-1 antibody with a purity of 87.26%.Conclusion1.In the axons,the expression of Jarid1 b decreased and the expression of H3K4me3 increased after optic nerve injury,suggesting that :(1)in optic nerve injury,the expression of H3K4me3 is correlated with the expression of Jarid1b;(2)the low expression state of Jarid1 b may be one of the mechanisms inhibiting axonal regeneration of RGCs;(3)Jarid1b is expected to be a new target for in-depth study on the repair and regeneration mechanism of optic nerve injury.2.The RGCs cultured by this method grew well,and the survival time in vitro was up to 7 days with high purity,which can be used for further research.