Interleukin-6 and lactate dehydrogenase expression in a novel ex vivo rocking model of equine corneal epithelial wound healing.

Abstract

To establish a physiologically relevant ex vivo model of equine corneal epithelial wound healing. Fourteen equine corneas were randomly assigned to one of two groups: wounded (n=8) or unwounded (n=6) controls. In the wounded group, the axial corneal epithelium was removed by applying a 6mm filter paper disk soaked in 1N-NaOH for 60s. Corneas were subsequently cultured using an air-liquid interface model. Evaluation of corneal healing was performed daily, and culture medium was collected. Corneas were randomly assigned to undergo processing via histopathology and RNAscope in situ hybridization for interleukin-6 (IL-6) and alpha-smooth muscle actin (αSMA) expression at T24, T48, and T72h after wounding. Media of the cultured corneas were evaluated for the presence of lactate dehydrogenase (LDH) by a colorimetric assay. The ulcerated area of the wounded corneas decreased over time and all corneas healed within 72h. Histologically, normal corneal architecture was observed including healthy epithelium (in areas other than the ulcerated ones), minimal stromal edema, intact endothelium, and Descemet's membrane. IL-6 expression was increased in wounded corneas compared with unwounded controls. LDH expression was elevated for both wounded and unwounded corneas at T24 but decreased substantially and was not detected at T48 in media from wounded and unwounded corneas, respectively. No αSMA expression was detected from either wounded or unwounded corneas. The equine air-liquid interface, ex vivo, corneal epithelial wound healing model is effective and physiologically relevant. This model can be used in future studies evaluating various corneal therapies.