Development and Assessment of a Novel Canine Ex Vivo Corneal Model

• Published in: Current eye research 2017-06, Vol.42 (6), p.813-821
• Main Authors: Proietto, Laura R., Whitley, R. David, Brooks, Dennis E., Schultz, Gregory E., Gibson, Daniel J., Berkowski, William M., Salute, Marc E., Plummer, Caryn E.
• Format: Article
• Language: English
• Subjects: Animals; Cell Movement; Cell Survival; Cells, Cultured; Cornea; Cornea - ultrastructure; Corneal Diseases - diagnosis; Disease Models, Animal; Dogs; Epithelium, Corneal - ultrastructure; ex vivo; fibrosis; Fibrosis - pathology; Immunohistochemistry; Microscopy, Electron, Scanning; Microscopy, Electron, Scanning Transmission; Organ Culture Techniques; Reproducibility of Results; Wound Healing
• ISSN: 0271-3683; 1460-2202
• DOI: 10.1080/02713683.2016.1262428

Purpose: To develop a novel ex vivo extended culture model of canine corneal epithelial cell wound healing. Materials and Methods: Canine corneoscleral rims (CSR) were obtained and, after preparation for culture, were placed on a nutating scaffold and incubated in physiological conditions. In experiment 1, eight CSR in a serum-containing antimicrobial-fortified medium were monitored for epithelial integrity and bacterial infection up to 28 days in culture. CSR were assessed histologically at the end of the culture period end points 0, 7, 14, and 28 days with accompanying scanning electron microscopic (SEM) and transmission electron microscopic (TEM) evaluation. Samples for microbial culture were obtained at days 0, 3, 7, 14, and 28. In experiment 2, uniform 8-mm-diameter superficial corneal epithelial wounds were created and monitored for re-epithelialization in the same culture conditions or in a serum-free protein equivalent medium, with four CSR per group. Standardized digital images were obtained with cobalt filter at the time of fluorescein staining and media change every six hours. Image J imaging software was used to measure the area of fluorescein retention. Re-epithelialization rates were calculated and CSR then fixed for immunohistochemistry (IHC). Results: All corneas survived to end points as described in experiment 1 with no evidence of contamination or compromised epithelial integrity. Histologically, a multilayered epithelium was maintained and corneal edema was not appreciated until day 14. SEM examination revealed epithelial cell layer confluence and migrating epithelial cells of normal cellular morphology with normal cell-cell interactions on TEM. In experiment 2, all eight corneas healed with a healing rate of 0.702 ± 0.130 mm 2 /h (1.25 mm/day epithelial cell migration rate) and were positive in IHC evaluation for markers of corneal fibrosis. Conclusion: This ex vivo canine corneal wound healing model is an appropriate and clinically relevant tool for assessment and modulation of epithelial wound healing.