Corneal wound healing is modulated by topical application of amniotic fluid in an ex vivo organ culture model Juan Castro-Combs a , Guillermo Noguera a , Marisol Cano a , Margaret Yew a , Peter L. Gehlbach a , Jonathan Palmer b , Ashley Behrens a, * a The Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine, Baltimore, MD, USA b The School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, USA article info Article history: Received 17 July 2007 Accepted in revised form 22 April 2008 Available online 30 April 2008 Keywords: cornea wound healing reepithelialization amniotic fluid 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 ¼ 52) were placed in an ex vivo air-interface organ culture. An 8.5 mm-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 ¼ 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 ml 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 ¼ 0.016), 40 h (P ¼ 0.032), 64 h (P ¼ 0.008) and 72 h (P ¼ 0.007) following epithelial scrape. The MEDA in the HAF group was significantly smaller at 16 h (P ¼ 0.008), 64 h (P ¼ 0.0072), and 72 h (P ¼ 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 ¼ 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 ¼ 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. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Human amniotic membrane (HAM) was first used in ophthal- mology by de Rotth (1940) to repair conjunctival defects. Since then, HAM has been used to treat a wide variety of corneal disor- ders such as persistent epithelial defects (Shukla, 1962; Batmanov et al., 1990), neurotrophic corneas (Kruse et al., 1999; Lee and Tseng, 1997), chemical injuries (Sorsby and Symons, 1946; Sorsby et al., 1947), recurrent erosion syndrome and persistent epithelial defects associated with cicatricial conditions (Lee and Tseng, 1997). Positive effects on corneal reepithelialization, inflammatory response, and scar formation have been reported with the use of HAM (Kim and Tseng, 1995; Meller and Tseng, 1998). In a randomized prospective study of rabbit corneas undergoing photorefractive keratectomy excimer laser Choi et al. (1998) demonstrated a reduction of haze in the postoperative period after amniotic membrane was applied to the wound area; they hypothesized that this may be due to a reduction in inflammatory cell infiltration in the early postoperative period. Moreover, Woo et al. (2001) created large * Corresponding author. The Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine, 600 N Wolfe Street, Woods Building room 255, Baltimore, MD 21287, USA. Tel.: þ1 410 502 0461; fax: þ1 410 502 2461. E-mail address: abehrens@jhmi.edu (A. Behrens). Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer 0014-4835/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2008.04.010 Experimental Eye Research 87 (2008) 56–63