DNA Demethylase TETs In Optic Ganglion Cell Injury

Objective:The aim of this study was to investigate the effect of optic nerve injury on retinal ganglion cell apoptosis,and to further investigate the expression of TET1/2/3 at different times of optic nerve injury and its role in optic ganglion cell injury,with a view to providing possible strategies for the study of protection and repair of optic nerve injury.Methods:Following the currently accepted method of optic nerve crush to construct a simulated optic nerve injury(ONC)model,we applied the immunofluorescence(TF)method to determine the apoptosis rate of RGCs at different times of optic nerve injury by retinal Brn3 a expression at different times after ganglion cell injury.Further,the changes in TET1/2/3 expression and localization characteristics at different times after retinal ganglion cell injury were assessed from transcriptional level(q PCR),protein level(WB)and immunofluorescence(IF)experiments.Results:We found that the retinal ganglion cells began to decrease 1d after ONC,and the arrangement of RGCs began to become disorganized and sparse,with substantial death occurring after 5d,most significantly at 7d and 14 d.After optic nerve injury,the survival of RGCs gradually decreased with time compared with normal mouse retinal tissues.The differences in the relative expression of TET1/2/3 m RNA in the retinal tissues of the right and left eyes of normal mice were not statistically significant(P > 0.05).The expression of TET3 m RNA in the retina of adult mice was higher than that of TET1/2,and there was a statistically significant difference(P <0.001).The relative expression of TET1/2/3 m RNA was not statistically different at1 d and 14 d of optic nerve entrapment injury(P > 0.05),while it decreased significantly at 5d and 7d,with statistically significant differences(P < 0.001),and the expression of TET1/2/3 m RNA was higher at 14 d of optic nerve injury compared to 7d,with statistically significance.There was no statistical difference(P > 0.05)in TET3 protein expression at 1d and 14 d of optic nerve entrapment compared to normal mouse retinal TET3 protein expression,while there was a statistically significant decrease(P < 0.001)at 5d and 7d.Immunofluorescence staining of mouse retinal tissues at 1d,5d,7d and 14 d after ONC modelling revealed that TET1/3 decreased at1 d after ONC modelling,decreased more significantly at 5d and 7d,and gradually increased at 14 d,and was mainly expressed in the ganglion cell layer and the intermediate neuronal layer.Conclusion:The ONC model can successfully induce the death of RGCs and the survival of RGCs gradually decreases at 1d,5d,7d and 14 d after ONC.TET1/2/3 were expressed in the normal left and right retinas of mice,with TET3 expression being the highest,TET1 the second highest and TET2 the lowest,and there was no difference in expression between the left and right eyes.There was a clear temporal trend of TET1/2/3 expression in the retina after optic nerve injury.TET1/2/3 expression gradually decreased within one week after ONC,but showed an increasing trend after two weeks,and the expression was mainly increased in the ganglion cell layer and the intermediate neuronal ganglion layer.Our results suggest that the TET family may be involved not only in the apoptosis of RGCs after optic nerve injury and the maintenance of repair and function after injury,but also in intermediate neuronal damage.