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.