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&#xa0;vivo. METHODS: The corneas of 40 canine eyes (n&#x2009;=&#x2009;20 dogs) enucleated <24&#x2009;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&#x2009;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&#x2009;&#xb1;&#x2009;standard deviation (range) outcomes were: MCD, 2544&#x2009;&#xb1;&#x2009;541 cells/mm(1750-3922 cells/mm); MCA, 431&#x2009;&#xb1;&#x2009;97&#x2009;&#x3bc;m(251-626&#x2009;&#x3bc;m); polymegathism, 17&#x2009;&#xb1;&#x2009;2% (14%-22%); pleomorphism, 84&#x2009;&#xb1;&#x2009;3% (80%-90%). No significant differences (p&#x2009;&#x2265;&#x2009;.122) were noted for any outcome between male versus female or brachycephalic versus non-brachycephalic dogs. Young dogs (<10&#x2009;years) had lower MCA (p&#x2009;=&#x2009;.044), lower pleomorphism (p&#x2009;=&#x2009;.003), and higher MCD (p&#x2009;=&#x2009;.035) when compared to older dogs (&#x2265;10&#x2009;years). Age was significantly (p&#x2009;&#x2264;&#x2009;.049) correlated with MCA (r&#x2009;=&#x2009;0.467), MCD (r&#x2009;=&#x2009;-0.476), polymegathism (r&#x2009;=&#x2009;0.444), and pleomorphism (r&#x2009;=&#x2009;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&#xa0;vivo. Our findings can be used in future studies to deepen our understanding of the corneal endothelium in health and disease.