Neuroprotective Effect And The Underlying Mechanism Of Hydrogen Sulfide On Retinal Ganglion Cells In An Experimental Glaucoma Model

Purpose:Glaucoma is a neurodegenerative eye disease with the major risk factor of intraocular pressure（IOP）elevation and characterized by progressive loss of retinal ganglion cells（RGCs）.An experimental animal model simulating glaucoma is essential for the basic research on mechanisms underlying the pathogenesis and treatment of glaucoma.Rodents are the most commonly used animals to build experimental glaucoma models,however,there are advantages and disadvantages in the existing glaucoma-modeling methods,like laser photocoagulation of trabecular meshwork around the corneal limbus,cauterization or ligation of episcleral veins,injection of microbeads into anterior chamber and injection of hypertonic saline into episcleral veins et al.Thus,it is necessary to explore a novel rodent glaucoma model which is stable,effective and easy to operate.Hydrogen sulfide（H₂S）is a recently discovered endogenous gasotransmitter,which was reported to exert neuroprotective effect on neurons in neurodegenerative disease in central nervous system.Howerver,it has not been elucitated that whether H2S works to protect retinal ganglion cells in glaucoma.Thus,this study focused on the changes of retinal endogenous H₂S level and expressions of H₂S-associated synthases,cystathionineβ-synthase（CBS）,cystathionineγ-lyase（CSE）and3-mercaptopyruvate sulphurtransferase（3-MST）,in a newly explored rat model of chronic ocular hypertension（COH）.Further more,sodium hydrosulfide（NaHS）,an exogenous H₂S donor,was applied to treat the rats exposed to COH and whether H₂S was able to protect RGCs as well as its underlying mechanism was investigated,which would surely help to understand the mechanisms underlying the pathogenesis of glaucoma and benefit the exploration of therapeutic interventions and strategies for glaucoma.Methods:Primary RGCs were isolated from retinas of Sprague Dawley（SD）rats and co-cultured with a cross-linking hydrogel for 1,3 and 7 days before the cytotoxicity was evaluated by a cell counting kit-8（CCK-8）and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling（TUNEL）method;the cross-linking hydrogel was injected into the anterior chamber of SD rat to induce COH and measurement of IOP was conducted in the following 10 weeks;RGCs density in the area of 1/6,3/6,5/6 retinal radius from optic disc was quantified by retrograde labeling of RGCs with injection of 1,1’-dioctadecyl-3,3,3’,3’-tetramethy-indocarbocyanin perchlorate（DiI）into the superior colliculus,and the thickness of retinal inner plexiform layer,inner nuclear layer,outer nuclear layer and cell density in ganglion cell layer were evaluated by hematoxylin-eosin（HE）staining after 2,4,8 weeks of COH.The H₂S level in blood plasma and retina was measured,and the expressions of retinal CBS,CSE and 3-MST were evaluated by immunohsitochemical staining and Western blot after 2,4,8 weeks of COH.After administration of NaHS injection for 4 weeks following COH induction,retinal H₂S level was measured;RGCs density in the area of 1/6,3/6,5/6 retinal radius from optic disc was quantified by retrograde labeling of RGCs with DiI;the apoptosis of isolated RGCs was evlaluated by TUN EL staining;the expressions of endogenous apoptosis-associated Bcl-2,Bax and cleaved Caspase 3,abnormal glia activation-associated Iba-1 and GFAP in retina were evaluated by immunohistochemical staining and Western blot;the expressions of nulear factor-κB pathway activity-associated IκBαand phospho-IκBα,oxidative stress-associated transmembrane subunit gp91^phox of reduced nicotinamide adenine dinucleotidephosphate（NADPH）oxidase,cellular autophagy-associated Beclin-1 and LC3 in retina were evaluated by Western blot;the level of TNF-αin retina was evaluated by enzyme-linked immunosorbent assay（ELISA）.After receiving treatment of NaHS injection combined with delivery of PD98059,a specific inhibitor of extracellular signal-regulated kinase（ERK）pathway,for 4 weeks following COH induction,RGCs density in the area of 1/6,3/6,5/6 retinal radius from optic disc was quantified by retrograde labeling of RGCs with DiI;the apoptosis of isolated RGCs was evlaluated by TUNEL staining;the expressions of Bcl-2,Bax,cleaved Caspase 3,Iba-1,and GFAP in retina were evaluated by immunohistochemical staining and Western blot;the expressions of IκBα,phospho-IκBα,gp91^phox,Beclin-1 and LC3 in retina were evaluated by Western blot;the level of TNF-αin retina was evaluated by enzyme-linked immunosorbent assay（ELISA）.Results:CCK-8 test and TUNEL staining showed that the cross-linking hydrogel didn’t affect the survival and apoptosis of co-cultured RGCs significantly;IOP was elevated by at least 30%for more than 10weeks with a success rate of 80%after injection of the cross-linking hydrogel into the rat anterior chamber;RGCs density in distinctive retinal areas was decreased in a time-dependent way after COH induction;cell density in ganglion cell layer was progressively decreased after COH induction,along with the reduction of thickness of inner nuclear layer and outer nuclear layer in varying degreees.The H₂S level and expressions of CBS,CSE and 3-MST were decreased in retina in varying degrees after COH induction,however,the H₂S level in blood plasma didn’t change comparably.The RGCs density and rate of TUNEL-positive isolated RGCs were significantly improved by NaHS administration in COH rats;the retinal expressions of cleaved Caspase 3,Iba-1,GFAP,gp91^phox,Beclin-1 and TNF-α,as well as the relative ratio of phospho-IκBα/IκBαand LC3Ⅱ/LC3Ⅰexpressions,were all significantly attenuated by NaHS administration in COH rats;the retinal relative ratio of Bcl-2/Bax expressions was enhanced by NaHS administration in COH rats.The ratio of（phospho-ERK1/2）/（ERK1/2）expressions was significantly downregulated by NaHS administration in COH rats;the RGCs density and rate of TUNEL-positive isolated RGCs were not obviously affected by alone administration of PD98059 in COH rats;the retinal expressions of cleaved Caspase 3,Beclin-1 and relative ratio of Bcl-2/Bax,LC3Ⅱ/LC3Ⅰexpressions were not obviously affected by alone administration of PD98059 in COH rats;the retinal expressions of Iba-1,GFAP,gp91^phox,TNF-αand the relative ratio of phospho-IκBα/IκBαexpressions were significantly attenuated by alone administration of PD98059;the administration of NaHS combined with PD98059induced the similar effects as the alone administration of NaHS did in COH rats,except for a greater reduction of retinal expression of gp91^phox.Conclusions:A novel COH rat model with stable performance and high success rate was built by injection of the cross-linking hydrogel into anterior chamber,which required simple operations;retinal expressions of CBS,CSE and 3-MST were downregulated in COH rats,which led to decrease of retinal H₂S level;H₂S presented a neuroprotective effect on RGCs in COH rats,which might correlate with the downregulations of cellular endogeous apoptosis,abnormal glia activation,NF-κB pathway activity,oxidative stress,TNF-αexpression and cellular autophagy;the ERK1/2 signaling pathway activity was downregulated by H₂S in COH rat;the attenuations of abnormal glia activation,NF-κB pathway activity,oxidative stress and TNF-αexpression in COH rat induced by H₂S supplement might correlate with the downregulation of ERK1/2 signaling pathway activity;the attenuations of cellular endogenous apoptosis and autophagy induced by H₂S supplement might not directly correlate with the ERK1/2 signaling pathway activity.