Establishment of assessment of rabbit model of total limbal stem cell deficiency

Abstract

Background The deficiency of limbus stem cells will cause various kinds of the disorder of eye surface,in serious condition,it will lead to corneal conjunctivalization,chronic inflammation,lasting deficiency of corneal epithelium and corneal opacity accompanying long-term visual loss.Objective The aim of this study was to investigate how to establish and evaluate the successful model of total limbal stem cell deficiency (LSCD).Methods Twenty Japanese white rabbits aged 3-5 months were selected.The limbus corneal epithelium was removed from 1 mm inner to 2 mm outer of the limbus 100-150 μm in deep,and the total central epithelium and shallow stroma were simultaneously removed in the left eyes using scalpel.The experimental eyes were examined by slitlamp 2,3,4,5 weeks after operation,and the inflammatory score was performed according to corneal opacity,corneal neovascularization and corneal flurescein dye using international universal rating criteria of LSCD model.Cornea tissue was obtained in various time points mentioned above to examine the structural change and goblet cells.The expression of cytokeratin 3(CK3) in the cornea was detected using indirect immuno-fluorescence staining.Fisher Exact Probability method was used to compare the successful rate of models in different time points.Results The successful rate of models rate was 12.5％,62.5％,81.3％ and 87.5％,respectively 2,3,4,5 weeks after operation,showing a significant difference between the 3-week group and 2-week group (P =0.009).However,no significant differences were found in the model successful rate between the 3-week group and the 4-week group,the 4-week group and the 5-week group (P =0.465,0.200).There was a little difference between the 3-week group and the 5-week group(P=0.049).The mean time of achieving successful model was(3.21 ±0.80) weeks.The result of hematoxylin & eosin staining showed the edema of cornea stroma,a mass of inflammatory cells infiltration and new vessels.Goblet cells could be found by Periodic acid-Schiff staining 5 weeks after operation.Expression of CK3 was absent in the corneas of successful model eyes.Conclusions The LSCD model can be successfully established by cutting off the epithelium of limbus and central cornea,and the average time of model formation is about 3.2 weeks. Key words: Limbal stem cell deficiency; Animal model; Rabbit