Measurement of Solute Transport in the Endothelial Glycocalyx Using Indicator Dilution Techniques

A new method is presented to quantify changes in permeability of the endothelial glycocalyx to small solutes and fluid flow using techniques of indicator dilution. Following infusion of a bolus of fluorescent solutes (either FITC or FITC conjugated Dextran70) into the rat mesenteric circulation, its transient dispersion through post-capillary venules was recorded and analyzed offline. To represent dispersion of solute as a function of radial position in a microvessel, a virtual transit time (VTT) was calculated from the first moment of fluorescence intensity–time curves. Computer simulations and subsequent in vivo measurements showed that the radial gradient of VTT within the glycocalyx layer (ΔVTT/Δr) may be related to the hydraulic resistance within the layer along the axial direction in a post-capillary venule and the effective diffusion coefficient within the glycocalyx. Modeling the inflammatory process by superfusion of the mesentery with 10−7 M fMLP, ΔVTT/Δr was found to decrease significantly from 0.23 ± 0.08 SD s/μm to 0.18 ± 0.09 SD s/μm. Computer simulations demonstrated that ΔVTT/Δr is principally determined by three independent variables: glycocalyx thickness (δ), hydraulic resistivity (K r) and effective diffusion coefficient of the solute (D eff) within the glycocalyx. Based upon these simulations, the measured 20% decrease in ΔVTT/Δr at the endothelial cell surface corresponds to a 20% increase in D eff over a broad range in K r, assuming a constant thickness δ. The absolute magnitude of D eff required to match ΔVTT/Δr between in vivo measurements and simulations was found to be on the order of 2.5 × 10−3 × D free, where D free is the diffusion coefficient of FITC in aqueous media. Thus the present method may provide a useful tool for elucidating structural and molecular alterations in the glycocalyx as occur with ischemia, metabolic and inflammatory events.