Canine corneal endothelial cell analysis using vital dyes and light microscopy

Abstract

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/mm² (1750-3922 cells/mm²); MCA, 431 ± 97 μm² (251-626 μm²); 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.

Keywords: alizarin red; corneal edema; corneal endothelial degeneration; corneal endothelial dystrophy; endothelial cell density; trypan blue.

FIGURE 1

Representative photomicrograph of the canine corneal endothelium observed with light microscopy (20× magnification) following staining with 0.25% trypan blue and 0.5% alizarin red dyes.

FIGURE 2

Results of the Pearson correlation testing between the dogs age and the following corneal endothelium parameters: endothelial cell density (A), endothelial cell area (B), polymegathism (C), and pleomorphism (D).