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 (Delta VTT/Delta r) 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, Delta VTT/Delta r was found to decrease significantly from 0.23 +/- 0.08 SD s/microm to 0.18 +/- 0.09 SD s/microm. Computer simulations demonstrated that Delta VTT/Delta r is principally determined by three independent variables: glycocalyx thickness (delta), 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 Delta VTT/Delta r at the endothelial cell surface corresponds to a 20% increase in D(eff) over a broad range in K(r), assuming a constant thickness delta. The absolute magnitude of D(eff) required to match Delta VTT/Delta r between in vivo measurements and simulations was found to be on the order of 2.5 x 10(-3) x 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.