Establishment Of Two Inherited Corneal Disease Mouse Models And Their Characteristics Of Corneal Disease

N-ethyl-N-nitrosourea(ENU)is a synthetic high-efficiency alkylating agent which is currently recognized as one of the most effective mouse gene mutagens.Following an ENU-mutagenesis screen,a large number of mouse models of human disease were recovered for the study of the occurrences,development mechanisms and gene functions.In this study,we obtained 7 heritable mutant mouse models by ENU mutagenesis:1 Two inherited corneal disease mouse models obtained by ENU mutagenesisIn this study,15 C57BL/6J(B6)male mice about 8-10 weeks old were injected intraperitoneally with ENU.After recovering its reproduction capacity,these injected mice were bred with wild-type B6 female mice.A total of 36 mutant mice were obtained(including white spot,abnormal pupil,anophthalmia,corneal opacity,etc.).According to genetic confirmation testing,7 heritable mutant mouse models have been identified.Two corneal disease mouse models of them were named Coa1(Corneal disease 1)and Cod2(Corneal disease 2),and the follow-up work was carried out.2 HE staining analysis of corneal tissue in Coa1 and Cod2 miceHE staining showed that:(1)wild-type mice have clear corneal structure,arranged neatly epithelial cells,smooth basal layer of epithelium;parallel arranged collagenous fibrils and no blood vessels in the stroma;obvious endothelial cells.(2)collagen fibrils were loose at the bottom of the corneal stromal layer and some endothelial cells were absent in 2-week-old Cod1 mice;vacuoles were showed in the surface of the conneal epithelial layer,comeal neovascularization were detected in the comeal stroma,and some endothelial cell layer was also absent in 8-week-old Codl mice.(3)collagen fibrils were loose at the basal layer of corneal stroma in 2-week-old Codl mice;the epithelium was markedly thinner,the collagen fibrils at the bottom of the stromal layer were loose,and corneal neovascularization were also detected in the corneal stroma in 8-week-old Cod2 mice.3 Immunohistochemical staining analysis of Coa1 and Cod2 miceFirst,the cornea of 8-week-old Coa1 and Cod2 mice was analyzed by immunohistochemistry with anti-CD31 antibody,and the results showed that CD31 was expressed in the corneal stroma of Coal and Cod2 mice,which verified the existence of corneal neovascularization.We next stained for a corneal epithelium cell differentiation marker Keratin 12(K12),an epithelial cell differentiation marker Keratin 14(K14),and a specific epidermal differentiation marker,Keratin 10(K10)by immunohistochemistry in on the cornea of Cocil and Cod2 mice.The results showed that:(1)K12 expression was weak and negative locally in corneal epithelial of 2-week-old Coal mice,and was positive in 8-week-old Codl mice;K12 expression was negative locally in corneal epithelial cells of 2-week-old Cod2 mice,while K12 expression was prominent in 8-week-old Cod2 mice.(2)K14 was expressed most prominently in all corneal epithelium of Cod1 and Cod1 mice at 2 weeks and 8 weeks.(3)K10 expression was not detected in Cod1 and Cod2 mice corneal epithelium.Conclusion:In this study,we obtained two inherited corneal disease mouse models by ENU mutagenesis called Cod1 and Cod2.HE and immunohistochemical analysis were used to study the characteristics of cornea.Our research lays the foundation for the study of human corneal disease using.