Investigating the endothelial glycocalyx in health and disease in dogs

Objectives: The objectives of this study were; firstly to visualise the endothelial glycocalyx using Alcian Blue perfusion for the first time in dogs and secondly to evaluate glycocalyx damage in dogs with mitral valve disease (MVD) and hypercoagulability compared to control dogs. Methods: Uterine and testicular artery samples were perfused with 0.1% Alcian Blue/2.5% glutaraldehyde/0.1M sodium cacodylate and fixed with 2.5% glutaraldehyde/0.1M sodium cacodylate prior to visualisation using transmission electron microscopy. Glycocalyx damage was evaluated in residual blood samples from dogs with MVD (diagnosed on echocardiography by a board-certified cardiologist), hypercoagulability (G>8 dynes/sec on thromboelastography) and healthy control dogs undergoing blood donation. Glycocalyx damage was quantified by measuring a glycocalyx component, hyaluronan, using a commercially available ELISA validated for dogs. Hyaluronan concentrations were compared across groups using the Kruskal-Wallis test. Post-hoc analysis between groups was performed using Mann-Whitney U tests with Bonferroni corrections. Results: Glycocalyx was successfully visualised in testicular artery from 1/5 dogs (median[range] glycocalyx depth 68.2[32.1-122.9]nm) and uterine artery from 1/1 dogs (47.6[26.1-129.4]nm). The median(range) hyaluronan concentration in dogs with MVD (n=27), hypercoagulability (n=27) and controls (n=54) was and 51.5(8.7-152.0)ng/ul respectively. There was a significant difference across groups (P<0.001), between control and MVD dogs (P<0.001), control and hypercoagulable dogs (P<0.001) but not MVD and hypercoagulable dogs (P=0.186). Statement: Definitive endothelial glycocalyx visualisation will facilitate its future study ex vivo. Significantly increased hyaluronan concentrations in dogs with MVD and hypercoagulability support the presence of glycocalyx damage in these diseases and its diagnostic and therapeutic potential.