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R David Whitley, Laura Proietto, Dennis E Brooks, Gregory S Schultz, Daniel J Gibson, William M. Berkowski, Caryn Plummer; Development and assessment of a novel ex vivo canine model of corneal epithelial wound healing. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3458.
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© ARVO (1962-2015); The Authors (2016-present)
To develop an ex vivo model of canine corneal epithelial wound healing that maintains corneal clarity, integrity and limbal stem cell function in extended culture.
Canine globes with normal ophthalmic examination were harvested from dogs euthanized for non-ophthalmic reasons within 4 hours of euthanasia. The corneal-scleral (CS) rims were placed on a scaffold in a modified 6-well culture plate containing tear media on a nutating base in an incubator set at physiologic conditions. Medium similar to that of physiologic tears was changed every 6 hours, after an initial therapy with fortified antimicrobial medium for 12 hours. In Part A of the experiment, 8 corneas were stained with full strength fluorescein and assessed every 72 hours for epithelial integrity with cobalt blue filter biomicroscopy. Two CS rims were maintained for 7 days, 4 CS rims were maintained for 14 days and 2 CS rims were maintained for 28 days in culture. A single randomly chosen cornea was submitted for microbial culture on days 3, 7, 14 and 28 days. Each CS rim was then formalin fixed for histopathology at endpoints 7, 14 and 28 days. In Part B of this experiment, 3 corneas were wounded with an 8mm trephine, and the epithelium and basement membrane was removed to exposing corneal stroma, which was confirmed by fluorescein stain, and CS rims were monitored for healing in the same culture conditions. Digital photographs were taken every 6 hours at the time of fluorescein application and medium change with standardized scale and cobalt filter. Imaging software was used to determine the defect area. Healing rates of epithelial wounds were calculated by (area of defect/area of original defect in mms)/hr. Healing occurred by day 3. At day 7, histology was performed on 1 cornea and SEM on 2 corneas.
All non-wounded corneas (n=8) survived to endpoints with no contamination or fluorescein stain indicative of compromise to the epithelium. Histologically, a multilayered epithelium was maintained. Corneal edema was not present until day 14 in the 2 corneas maintained to 28 days. All corneas with epithelial wounds (n=3) healed at a rate of 0.128 mm2/hr +/- 0.003 mm2/hr. SEM examination shows epithelial layer confluence and migrating epithelial cells with normal cellular morphology.
This is a novel model for assessment of topical therapeutics modulating epithelial wound healing in the canine.
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