Corneal Crosslinking Induced Endothelium Dysfunction In Vivo And Its Protection By Topical Ripasudil Treatment

Purpuse:To establish a standardized corneal crosslinking model in vivo for investigations of the changes of corneal endothelium under different crosslinking conditions and the effect of ripasudil on endothelial cells in corneal crosslinking.Methods:The present study used 58 SPF male C57BL/6 mice(6-8-week-old).The corneal crosslinking groups were both infiltrated with riboflavin and then irradiated with 0.54 J/cm2 UVA or 1.08 J/cm2 UVA,while the non-crosslinking groups included the group with neither UVA irradiation nor riboflavin treatment,only 1.08 J/cm2 UVA irradiation group and only riboflavin treatment group.36 hours,4 days,7 days and 14 days after the corneal crosslinking,the corneal opacity was observed by slit lamp,the change of corneal endothelial cells was observed by in vivo confocal and the change of corneal thickness was observed by optical coherence tomography.ZO-1,F-actin,Na+/K+-ATPase staining and scanning electron microscopy to study the structure and function changes of corneal endothelium,and to investigate the DNA changes of corneal endothelial cells by immunofluorescence staining of 8-OHdG andy-H2AX as DNA damage markers.The mice with a corneal crosslinking dose of 1.08 J/cm2 were instilled with ripasudil to explore the protective effect of ripasudil on the corneal endothelium.Results:Slit lamp and optical coherence tomography showed that the combined treatment of UVA and riboflavin could cause the increase of corneal opacity and corneal thickness.In vivo confocal and scanning electron microscopy,it was found that the combined treatment of UVA and riboflavin increased corneal endothelial atypia,damaged the connection between cells,and decreased the cell density.Frozen section and the wholemount of cornea immunofluorescence staining further found that the combined use of UVA and riboflavin caused endothelial cell DNA damage and destroyed the tight junction and pump fuction of corneal endothelium while riboflavin alone or the same dose of UVA did not affect corneal endothelium.Ripasudil could reduce the DNA damage of endothelial cells,increase the density of endothelial cells and protect the structure and function of corneal endothelium in the endothelial damage caused by corneal crosslinking.Conclusion:Riboflavin combined with UVA can damage the structure and function of the corneal endothelium,the wound repair of the corneal endothelial structure and function is dose-dependent,and the ROCK inhibitor ripasudil protects the pump and barrier functions in the endothelial injury induced by corneal crosslinking.