Corneal wound healing is modulated by topical application of amniotic fluid in an ex vivo organ culture model

Abstract

The purpose of this study was to evaluate the effects of topical human amniotic fluid (HAF) and equine amniotic fluid (EAF) on corneal reepithelialization and stromal wound healing. New Zealand white rabbit corneas (n=52) were placed in an ex vivo air-interface organ culture. An 8.5mm-diameter mark in the center of the cornea was produced with a hand trephine to select the area for epithelial scraping. A number 15 surgical blade was used to remove the epithelial layer within the demarcated area in a standardized fashion. The corneas were assigned to one of four treatment groups (n=8): fetal bovine serum (FBS), HAF, EAF, and a control group that was exposed to phosphate buffer solution (PBS). Corneal epithelial defects were imaged every 8 h for 72 h after the application of a 30 microl drop of 0.015% fluorescein. Five corneas of each treatment group were used for histology, proliferation, and apoptosis assay at 72 h after the epithelial defect was created. There was no significant difference in the mean rate of closure of the corneal epithelial defect between FBS treated corneas and controls (P>0.06). The mean epithelial defect area (MEDA) was significantly smaller in the EAF group as compared to control corneas at 24 h (P=0.016), 40 h (P=0.032), 64 h (P=0.008) and 72 h (P=0.007) following epithelial scrape. The MEDA in the HAF group was significantly smaller at 16 h (P=0.008), 64 h (P=0.0072), and 72 h (P=0.016) compared to the control group. The MEDA in the HAF and EAF groups was smaller at all time points as compared to the FBS group, but the difference was not significant. At histology, the mean keratocyte density was significantly higher in the anterior stroma in the HAF (P<0.001) and EAF groups (P=0.001) as compared to control group. The number of BrdU positive keratocytes was significantly higher in the superficial and deep stromal sub-areas in the HAF group as compared to control (P<0.001 and P=0.002, respectively). EAF and FBS treated corneas also showed a higher number of BrdU positive cells compared to control, but this difference was not significant. Finally, we did not observe any difference in the amount of TUNEL positive keratocytes among the different groups. Our data indicates that the topical application of HAF and EAF is associated with accelerated reepithelialization in this cornea organ culture model. Similarly, corneal keratocyte density appears to be less affected after epithelial injury using this treatment.