The Protective Effects And Underlying Mechanisms Of α-melanocyte-stimulating Hormone In Rodent Models Of Glaucoma

ObjectiveTo investigate the protective effects of α-melanocyte stimulating hormone(α-MSH)on retinal damage in two different glaucoma animal models: N-methyl-D-aspartic acid(NMDA)-induced retinal excitotoxity model in C57 mice and Sprague Dawley(SD)rats,and spontaneous glaucoma DBA mice.The methylated RNA immunoprecipitation sequencing(Me RIP-seq)was used to study the protective mechanism of α-MSH on the retinal damage.MethodsC57 mice were randomly divided into 5 groups: normal control group,2 m M NMDA group,10 m M NMDA group,20 m M NMDA group,and 40 m M NMDA group.SD rats were randomly divided into normal control group,low-dose NMDA group(0.05 μM,0.1 μM,0.15 μM,and 0.2 μM NMDA),and high-dose NMDA group(16.7 m M,33.3 m M,50 m M,and 66.7 m M NMDA).One week after injection,the C57 mice and SD rats were analyzed by the optical coherence tomography(OCT),the hematoxylin and eosin(H&E)staining and the retina whole mount immunofluorescent staining.After determining the injection concentration of NMDA,SD rats were divided into three groups: normal control group,NMDA group and NMDA+α-MSH group.One week later,SD rats were analyzed by H&E staining,retina whole mount immunofluorescent staining and flash visual-evoked potential(F-VEP).In addition,the retinas of the three groups of SD rats were collected for the detection of m5 C m RNA methylation.The intraocular pressure(IOP)of DBA female mice was measured every week,and the anterior segment was observed by slit lamp.When IOP in DBA mice was stable and more than 15 mm Hg,half of the mice were injected with normal saline into the vitreous cavity and the other half were injected with α-MSH.Then all DBA mice were taken slit lamp photography,IOP,H&E staining,and the retina whole mount immunofluorescent staining.ResultsThe total retinal thicknesses of C57 male mice treated with NMDA at different concentrations were significantly smaller than that of the normal control group(all P < 0.01).H&E staining showed that the number of cells in the retinal ganglion cell layer(GCL)treated with NDMA was significantly lower than that in the normal control group(all P < 0.01).In addition,retinal whole mount staining showed that the number of β3-Tubulin-positive cells in retina could be reduced by intravitreal injection of different concentrations of NMDA(all P < 0.001).In the SD rat model,there was significant difference in GCL cell number between the normal control group and some low dose NMDA groups,including 0.05,0.15 and 0.2 μM NMDA groups(all P < 0.01).In addition,compared with the normal control group,high dose NMDA had significant damage effect on GCL cells of retina(all P < 0.001).In particular,50 m M NMDA reduced the number of GCL cells to half of the normal control.The sharp decrease of retinal nerve cells resulted in optic nerve dysfunction,so F-VEP measurement showed 29.57% of latency prolongation and 79.61% of amplitude decrease in NMDA group.However,all these abnormalities were corrected or improved by intravitreal administration of α-MSH.Me RIP-seq results showed that compared with NMDA group,1946 differential m5 C methylation genes were identified in NMDA+α-MSH group,1591 of which were significantly upregulated and 355 significantly downregulated.GO analysis showed that the different methylation genes were involved in some biological processes such as nervous system regulation and cell calcium homeostasis.KEGG analysis revealed that the differentially methylated m RNAs were related to Hedgehog signaling pathway,Adenosine-3’,5’-cyclic monophosphate(c AMP)signaling pathway and calcium signaling pathway.There was no significant difference in the anterior segment of C57 control mice at different weeks,and the IOP remained at about 10 mm Hg.However,in DBA mice,iris atrophy,iris pigment dispersion,and iris light transmission gradually appeared after the age of 20w(about 5 months).The IOP of DBA mice began to rise at 28 weeks and reached 15 mm Hg one week later.There was significant difference in IOP of DBA mice of different weeks(F = 2.450,P = 0.010).Overall comparison,the difference of IOP of DBA mice injected with normal saline and α-MSH was statistically significant(t = 3.479,P = 0.001).H&E staining showed that the number of GCL cells in retina of DBA mice decreased significantly,only 52.29% of C57 group(P < 0.001),while the number of GCL cells in α-MSH group increased significantly to 79.32% of normal control group(P < 0.001,DBA group vs DBA+α-MSH group;P > 0.05,normal control group vs DBA+α-MSH group).The results of retina whole mount staining showed that compared with the DBA group,the number of β3-Tubulin-positive cells in DBA+α-MSH group was significantly increased.ConclusionsThe total thickness of retina,the number of cells in GCL,and the number of β3-Tubulin-positive cells in retina of male C57 mice and SD rats were reduced by intravitreal administration of NMDA in a dose-dependent manner.And α-MSH has protective effects on the RGCs death,retinal morphology and optic nerve function damage induced by 50 m M NMDA in rats.In terms of mechanism,α-MSH may play a protective role by regulating the m5 C methylation of m RNA in Hedgehog signaling pathway,c AMP signaling pathway,and calcium signaling pathway.The IOP of DBA female mice was increased gradually from 28 weeks to 30 weeks and reached the peak value and remained stable and maintained stable.In the meantime,the iris of DBA mice were appeared a series of phenotypes such as pigment dispersion,light transmission,and atrophy.In the DBA mice model of spontaneous glaucoma,α-MSH can improve IOP and significantly decrease the loss of RGCs.