Development Of Animal Models For Cataract And Corneal Disorders Using N-Methyl-N-Nitrosourea And Hydrogen Saline Preventing Cataract Related Therapeutics

Purpose: N-methyl-N-nitrosourea(MNU)is an alkylating toxicant with potent mutagenic ability and is sufficient to induce ocular cell apoptosis.MNU has been most extensively utilized for the induction of retinal degeneration.This study was designed to induce lens epithelial cells(LECs)and corneal endothelial cells(CECs)apoptosis via MNU administration.We sought to build two types of ocular disease models(cataract in rats and corneal endothelial decompensation in rabbits)with reliable modeling parameters.Furthermore,this study intended to explore the therapeutic effect of hydrogen-rich saline(HRS)on MNU-induced cataract model and the underlying mechanism.Methods: MNU was delivered into the intraperitoneal cavity of neonatal rats and the anterior chamber of adult rabbits,respectively.Subsequently,the MNU administered animals were subjected to a series of morphological and functional analysis at various time points.Moreover,in MNU-induced cataract model rats were randomly divided into the following groups: control(Group A),MNU plus HRS treated(Group B),MNU plus pirenoxine treated(Group C),MNU plus normal saline(NS)(Group D),MNU induced(Group E).Examinations of slit-lamp,Pentacam and spectrophotometer in vivo and vitro were performed to assess the effects of HRS on protecting lens from MNU-induced cataract at P21(21 days after MNU administration).Subsequently,lens samples in Groups B and D were collected at P21.Using tandem mass tags(TMT)labeling quantitative proteomics technique,high resolution liquid chromatographytandem mass spectrometry(LC-MS/MS)and intensive bioinformatic analysis,the quantitative proteome in HRS and NS treated lens of MNU-induced cataract model were studied.The differentially expressed proteins were confirmed by parallel reaction monitoring(PRM).Results: MNU administration induced pervasive LECs and CECs apoptosis following a doseand time-dependent manner.Mature cataract was found in the neonatal rat 3 weeks after MNU administration.Histology assay showed that MNU toxicity induced swelling,vacuolation,liquefaction of the lens fibers in the lens of MNU administered rats.Pentacam examination showed that the average density of rat lens increased significantly after MNU administration.TUNEL assay showed pervasive apoptotic staining in the lens sections of MNU administered rats.Moreover,the intracameral administration of MNU induced corneal edema in rabbit eyes.Central corneal thickness of the MNU administered rabbits increased significantly and peaked at P14.Morphological and immunochemistry assay showed that CECs were effectively ablated in these MNU administered rabbits.The 8-OHd G expression level in the cornea sections of MNU administered rabbits increased significantly compared with vehicle treated controls.Cataract formation in rats was significantly suppressed by HRS in the terms of lens opacification,density and light transmission.Proteomics indicated that a total of 2017 proteins were quantified,and 393 differentially expressed proteins with quantitative ratio over 1.5 were obtained.These differentially expressed proteins were involved in different biological functions,metabolic pathways and pathophysiological processes.In addition,14 differentially expressed proteins mainly involved in oxidative phosphorylation were confirmed by PRM analyses.Conclusion: Cataract and corneal endothelial decompensation animal models were successfully induced by MNU and evaluated systematically,providing valuable tools for future therapeutic trials of these diseases.HRS can prevent MNU-induced cataract in rats.Proteome analysis provided a scientific basic for understanding of the mechanism related to HRS rescuing MNU-induced cataract.