A novel ex vivo model of equine corneal wound healing

Corneal disease is a significant cause of decreased vision or blindness in the horse worldwide. Numerous topical and systemic drugs over a prolonged period of time may be used to treat an equine corneal ulcer. Despite the high prevalence of corneal disease and ulceration in the horse, and the complexity of corneal wound healing, research is limited and more heavily devoted to in vivo studies whose limitations include small sample sizes and ethical considerations. Bench top laboratory in vitro studies are limited in number and overlook the complexity of corneal wound healing by assessing each corneal layer separately from its neighbor. The objective of this study was to develop a novel ex vivo model of equine corneal wound healing that maintained structural integrity, and is physiologically relevant, mimicking in vivo events. In this randomized and controlled study, fourteen equine corneas were harvested within two hours of humane euthanasia for reasons unrelated to this study. All donors were screened and corneas were included if they were free of ocular and systemic disease. Corneas were immediately processed and corneoscleral rims excised 2 mm posterior to the limbus. Corneas were randomly assigned to one of the following groups: wounded (n=8) or unwounded (n=6) controls. Each pair of eyes was divided such that corneas from the same horse were assigned to a different group. In the event that only one of the two eyes was included, the eye was assigned to the wounded group. In the ‘wounded’ group, the axial cornea was wounded for 60 seconds with a 6 mm disk of filter paper soaked in 1N sodium hydroxide (NaOH). Epithelial ulceration was confirmed employing fluorescein stain. Subsequently, all corneas were cultured using an air-liquid interface model in complete media. The corneal cups were placed on a rocker set at a 6 degree incline and incubated at 37 ÃÂðC with 5% CO2 humidity. The media bathed the cornea 8 times per minute to simulate normal horse blinking. Evaluation of corneal healing was performed daily and included fluorescein staining and fluorescein retention (i.e. ulcer measurements). Corneas from both groups were randomly assigned to undergo further processing via histopathology at designated time points of 24 hours (T24; n=5), T48 (n=5), and T72 (n=4) post wounding, and stained with hematoxylin and eosin. Structural integrity and pathologic changes to the corneal epithelium, stroma, Descemet’s membrane and endothelium were assessed. Interleukin-6 (IL-6) expression was then evaluated by means of RNAscope in situ hybridization. All wounded corneas healed within 72 hours. Histologically, normal corneal architecture was observed including intact epithelium, minimal stromal edema, and presence of endothelium. Increased IL-6 expression was noted in wounded corneas compared to unwounded controls and was predominantly in the metabolically active basal epithelial cell layer. As the corneal wound healing progressed, IL-6 expression decreased. The equine air-liquid interface, ex vivo, corneal wound healing model is an effective and physiologically relevant model. This model can be utilized to reduce or eliminate the use of live horses for corneal wound healing studies, while providing an effective platform for corneal wound healing research in this species

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

Purpose To establish a physiologically relevant ex vivo model of equine corneal epithelial wound healing. Methods 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 6 mm filter paper disk soaked in 1N-NaOH for 60 s. 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 T72 h after wounding. Media of the cultured corneas were evaluated for the presence of lactate dehydrogenase (LDH) by a colorimetric assay. Results The ulcerated area of the wounded corneas decreased over time and all corneas healed within 72 h. 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. Conclusions 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.This is the published version of the following article: Wehrman, Rita Fay, Ulrike Genschel, Adhithiya Charli, Anumantha G. Kanthasamy, Rachel Anne Allbaugh, and Gil Ben‐Shlomo. "Interleukin‐6 and lactate dehydrogenase expression in a novel ex vivo rocking model of equine corneal epithelial wound healing." Veterinary Ophthalmology 24, no. 5 (2021): 509-519. DOI: 10.1111/vop.12935. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivsLicense, which permits use and distribution in anymedium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.© 2021 The Authors. Posted with permission