Detection And Verification Of Abnormal Expression Of Cochlin In The Eyes Of Guinea Pig Myopic Models

Objectives:Myopia has become an epidemic,which is often accompanied by pathological changes such as axial elongation and thinning of the choroid and sclera tissues in the ocular posterior pole.However,the factor mediating choroidal and scleral thinning remains elusive.The purpose of this study was to explore differentially-expressed proteins closely related to experimental myopia of guinea pigs through high-throughput proteomics,in order to reveal key protein molecule in the myopic pathogenesis,providing new ideas for exploring effective targets for myopic intervention.Materials and Methods:Three week-old pigmented guinea pigs were randomly assigned to three groups for 6 weeks.The normal controls were without any treatment.Myopia was induced monocularly by translucent latex facemasks and-10.00 D concave lenses in the right eyes of form-deprived myopia(FDM)group and lens-induced myopia(LIM)group,respectively.The left eyes of the two groups served as self-controls.At 0,2,4 and 6 week after the induction,refraction,axial length,and radius of corneal curvature of guinea pigs among the three groups were measured,respectively.At 6 week after the induction,all animals were deeply anesthetized and sacrificed.All the right eyes were quickly enucleated,some of which were paraffin-sectioned for H&E staining and examining the posterior scleral thickness and morphological changes.Whereas the posterior polar tissues of the remaining right eyes were collected,some of which were subjected to high-throughput proteomics analysis for detection of differentially-expressed proteins among the three groups.The distribution and expression of Cochlin were detected by immunohistochemistry in the posterior polar tissues of the three groups.Subsequently,quantitative real-time PCR and western blot were used to verify the expression of Cochlin at the m RNA and protein levels in the FDM group and the normal control group,respectively.Results:Before the induction,there were no significant differences in the binocular differences(the variation between the right eye and the left eye)of the refraction,axial length and radius of corneal curvature among the three groups(all P > 0.05).Following the induction,the right eyes of both the FDM and the LIM groups showed significant reduction in refraction and elongation in axial length,as compared to the normal control group(all P < 0.05).No significant differences were found in refraction and axial length between the FDM group and the LIM group(all P > 0.05).In addition,there was also no significant difference in radius of corneal curvature among the three groups(all P > 0.05).At 6 week after the induction,the H&E staining result revealed diminished thickness and disordered collagen fibers in the posterior sclera in the FDM and the LIM groups,as compared to the normal control group.The GO functional analysis of proteomics showed that the two myopic groups of differentially-expressed proteins were mainly involved in ion binding,structural molecular activity,and cytoskeletal protein binding function.KEGG enrichment analysis showed that the FDM group was mainly associated with glycolysis pathway,while the LIM group was mainly related to tight junction pathway.Significant upregulation of Cochlin was detected in both myopic model eyes(3.77 fold for FDM vs normal control,1.71 fold for LIM vs normal control).The immunohistochemistry staining revealed that Cochlin was predominantly located in the outer segment of photoreceptor cells,abutting the widespread RPE cells.Furthermore,the results of quantitative real-time PCR and western blot confirmed that the expression of Cochlin at the m RNA and protein levels was significantly increased in the FDM group,compared with the normal control group(both P < 0.05).Conclusions:The induced myopia is mainly caused by axial length elongation,and has nothing to do with the change of corneal curvature,often accompanied by posterior scleral remodeling.FDM is mainly related to the glycolysis pathway,while LIM is mainly related to the tight junction pathway.The upregulation of Cochlin at the m RNA and protein levels was detected in the posterior polar tissues of myopic eyes of guinea pigs,indicating the crucial role of Cochlin in the pathogenesis and progression of myopia.The specific mechanism of action remains to be further explored.