Visualising the endothelial glycocalyx in dogs

Abstract

The endothelial glycocalyx (eGlx) lines the luminal surface of endothelial cells. It is critical in maintaining vascular health and when damaged contributes to many diseases. Its fragility makes studying the eGlx technically challenging. The current reference standard for eGlx visualisation, by electron microscopy using glutaraldehyde/Alcian blue perfusion fixation, has not been previously reported in dogs. Established techniques were applied to achieve visualisation of the eGlx in the microvasculature of reproductive tissue in five healthy dogs undergoing elective neutering. Uterine and testicular artery samples underwent perfusion fixation, in the presence of Alcian blue, prior to transmission electron microscopy imaging. Image processing software was used to determine eGlx depth. EGlx was visualised in the arteries of two dogs, one testicular and one uterine, with median (range) eGlx depths of 68.2 nm (32.1-122.9 nm) and 47.6 nm (26.1-129.4 nm) respectively. Study of the eGlx is technically challenging, particularly its direct visualisation in clinical samples. Further research is needed to develop more clinically applicable techniques to measure eGlx health.

Keywords: Alcian blue; Canine; Microvasculature; Transmission electron microscopy.

Fig. 1

These images depict the process of perfusing a uterine artery sample prior to processing for TEM. A) Uterine horn with uterine artery labelled. B) Following perfusion with glucose-free mammalian Ringer-Locke’s solution to remove plasma proteins and red blood cells. C) After perfusion with Alcian blue fixative solution. OV, ovary; UA, uterine artery; UH, uterine horn.

Fig. 2

Front and side views of the equipment used to achieve Alcian blue perfusion and fixation for endothelial glycocalyx visualisation. Two 50 ml Falcon tube reservoirs were used as Alcian blue storage chambers (one flush, one fixative). These were attached via taps to a Y connector and a catheter. Controlled perfusion pressure was applied via an air pressure reservoir, the positive pressure chamber comprising a 500 ml plastic bottle maintained at 100 mmHg by a sphygmomanometer bulb. The reservoir taps allowed selection of either flush or fix reservoir via the Alcian blue perfusion pipes to the catheter.

Fig. 3

Transmission electron microscopy images of dog endothelial glycocalyx. A) Lower and B) Higher power images of dog uterine artery (NB: different sections are shown). C) Higher power image of dog testicular artery. EC, endothelial cell; eGlx, endothelial glycocalyx; RBC, red blood cell; VL, vessel lumen.