Canine corneal endothelial cell analysis using vital dyes and light microscopy

Purpose To evaluate the use of vital dyes and light microscopy for assessing canine corneal endothelial morphology ex vivo. Methods The corneas of 40 canine eyes ( n = 20 dogs) enucleated <24 h following euthanasia or death were isolated and flat‐mounted on a slide. Corneal endothelium was stained via 0.25% trypan blue followed by 0.5% alizarin red (pH 4.2), photographed, then the following morphological features were calculated using ImageJ: mean cell density (MCD), mean cell area (MCA), polymegathism (coefficient of variation of cell area), and pleomorphism (% hexagonality). Results Mean ± standard deviation (range) outcomes were: MCD, 2544 ± 541 cells/mm 2 (1750–3922 cells/mm 2 ); MCA, 431 ± 97 μm 2 (251–626 μm 2 ); polymegathism, 17 ± 2% (14%–22%); pleomorphism, 84 ± 3% (80%–90%). No significant differences ( p ≥ .122) were noted for any outcome between male versus female or brachycephalic versus non‐brachycephalic dogs. Young dogs (<10 years) had lower MCA ( p = .044), lower pleomorphism ( p = .003), and higher MCD ( p = .035) when compared to older dogs (≥10 years). Age was significantly ( p ≤ .049) correlated with MCA ( r = 0.467), MCD ( r = −0.476), polymegathism ( r = 0.444), and pleomorphism ( r = 0.609). Conclusions The combination of vital dyes and light microscopy allowed for clear visualization and evaluation of the corneal endothelium in canine eyes ex vivo. Our findings can be used in future studies to deepen our understanding of the corneal endothelium in health and disease.