RESUMEN
Background Corneal collagen cross-linking (CXL) shows good clinical effects for keratoconus,and de-epithelized CXL appears to be benefit to the distribution and absorption of riboflavin in cornea stroma.However,de-epithelization of CXL will increase the infective risk and corneal healing time.It is very important to understand and control the affecting factors of corneal repair after de-epithelization of CXL.Objective This study was to evaluate the characteristics of corneal epithelial repair and analyze the relevant factors affecting corneal healing time after de-epithelized CXL.Methods A series-cases observational study was performed.De-epithelized CXL was performed on 77 eyes of 68 keratoconus patients in Shandong Eye Hospital from September 2013 to September 2015 under the approval of Ethic Committee of this hospital and informed consent of each patient.The age,corneal curvature,corneal thickness,breakup time of tear film (BUT),corneal front astigmatism (Astig) and epithelial healing time of the patients were recorded after surgery.The correlations between corneal epithelium healing time and above-mentioned factors were analyzed.Results De-epithelized CXL was smoothly finished in all the eyes.The corneal epithelium healing time was 2-12 days after surgery,with the average healing time 5 (4,6) days.The mean age,thickness at corneal thinnest point,minimal cornea curvature (Kf),maximal corneal curvature (Ks),corneal average curvature (Km) and Astig was 22.00 (18.00,25.00) years,436 (412,470) μm,47.40 (44.70,50.45) D,52.10 (49.00,54.55) D,50.00 (47.15,53.15) D and-3.30 (-5.45,1.70) D,respectively.Spearman rank correlation analysis showed significant negative correlations between corneal epithelium healing time and BUT or the thickness at corneal thinnest point (BUT:rs =-0.334,P =0.003;corneal thickness:rs =-0.417,P =0.000),and thesignificant positive correlations were found between corneal epithelium healing time and Km,Kf and Ks (Km:rs =0.449,P =0.000;Kf:rs =0.300,P =0.008;Ks:rs =0.432,P =0.000).There were no considerable correlarions between corneal epithelium healing time and age or Astig (age:rs =0.023,P =0.845;Astig:rs =-0.190,P =0.098).Multiple linear regression analysis were carried out to study the dependent variable and independent factors.Because of the multiple co-linearity between variables,this paper corrects the model by using ridge regression.There is significant negative correlation between BUT,corneal thickness and corneal healing time,respectively (both at P＜0.05),corneal curvature Km and Kf is positively correlated with corneal healing time (both at P ＜ 0.05).Conclusions The corneal thickness,Kf,Km,as well as BUT are influencing factors of epithelial healing after CXL.
RESUMEN
Background Limbal stem cells (LSCs) play an important role on the stability of corneal epithelium and corneal transparency.Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor can promote cell proliferation and reduce apoptosis,such as human embryonic stem cells and karatin epithelial cells.Objective This study was to investigate the improving effect of Y-27632,a ROCK inhibitor,on the activity of rabbit LSCs in corneal preservation medium.Methods Corneal preservation solution was prepared by adding 12.5％ chondroitin sulfate,10.0％ low molecular dextran,20.0 mg/L dexamethasone,100 mg/L tobramycin sulfate,9.5 g/L Hepes and 0.375 mg/L L-glutamine in MEM.The corneas of New Zealand white rabbits were collected and preserved in the corneal preservation solution with or without Y-27632 for 4,7,14 days,and the density and morphology of corneal endothelial cells were examined by using 0.25％ trypan blue staining and 0.2％ alizarin red staining.Isolated corneal epithelial cells were seeded on 3T3 feeder layer and cultured for 7-10 days until colonies formation.Colony shape of LSCs was observed under the light microscope,and colony-formation efficiency was analyzed after Giemsa staining by Image J software.Results The morphology and density of corneal endothelial cells were normal in the corneal preservation solution with and without Y-27632 for 4 days.In the seventh day after preservation,the cells remained the regular hexagon in shape in the preservation solution with Y-27632,however,the cellular membrane was slightly shrinking with the positive staining for alizarin red in the preservation solution without Y-27632.The density of corneal endothelial cells in the corneal preservation solution without Y-27632 was (2 262-± 75) cells /mm2,while in the preservation solution with Y-27632 was (2 425 ±95) cells/mm2(P＜0.001).The cloning spheres of LSCs were similar in preservation solution both with and without Y-27632 in the freshly isolated cornea or preserved corneas and exhibited more cells inside.But in 7 days and 14 days after preservation,the cloning spheres were much smaller in the preservation solution without Y-27632 group than those in the preservation with Y-27632 group.No significant differences were found in the cloning-formation rate and survival rate of corneal epithelial cells in corneas freshly isolated or preserved for 4 days in both groups (all at P＞0.05).In 7 days and 14 days after preservation,the active rates of corneal epitheli.al cells were (73.00±2.12)％ and (56.00±0.71)％ in the preservation solution with Y27632,which were significantly higher than (66.00 ± 4.00) ％ and (49.00 ± 0.71) ％ in the preservation solution without Y-27632,showing statistically significant differences between them (t =3.098,P =0.018;t =9.798,P =0.000).In addition,the cloning-formation rates of LSCs were (11.05±0.21)％ and (3.10±1.97)％ in the preservation solution with Y-27632 in 7 days and 14 days after preservation,revealing significantly elevation in comparison with (2.05 ± 1.20) ％ and (0.40 ±0.14) ％ in the preservation solution without Y-27632 (t =18.107,P =0.000;t=3.184,P=0.017).Conclusions Y-27632 promotes the vitality and cloning-formation ability of LSCs in corneal preservation medium,suggesting its potential use during storage of cornea.