Simultaneous Determination of Microvascular Endothelial Glycocalyx Structure and Albumin Flux in vivo

Endothelial Glycocalyx coats the luminal surface of all blood vessels and is a key regulator of permeability. We aimed to design a combined confocal‐fluorescent imaging method for assessing structural‐functional relationships between glycocalyx and albumin flux (P sBSA ) in systemic mesenteric microvessels of rats in vivo . Mesenteric microvessels were cannulated using double barrel theta style micropipettes and perfused with unlabeled 4% BSA and Alexa Fluor 488 labeled BSA in sequence, which was imaged using fluorescent microscopy to calculate Ps BSA . Endothelial glycocalyx depth was then quantified by changing the perfusates to trimethylamino‐diphenylhexatriene ( TMA‐DPH: membrane label ) and tetramethylrhodamine‐isothiocyanate‐conjugated Wheat Germ Agglutinin ( TRITC‐WGA lectin: glycocalyx label ), and imaged using Confocal Microscopy. To validate the technique the glycocalyx structure was modulated in real time using neuraminidase or vehicle control. Neuraminidase decreased glycocalyx depth (peak to peak) (p < 0.05, two‐way ANOVA & unpaired t‐test) and its fractional distribution (fractional coverage) (p < 0.05, unpaired t‐test) in a time dependent manner which directly correlated to a paired significant increase in P sBSA in the same microvessels in vivo (p < 0.05 by Pearson's correlation) (p < 0.001 by two‐way ANOVA). We have effectively demonstrated a technique for linking structural estimates of glycocalyx to functional measurements of albumin flux in vivo and our results indicate an activity and time dependent functional response to neuraminidase cleavage of sialoglycoproteins.