3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography.

OBJECTIVE: Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the lumen and directly applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed. METHODS: Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained glycocalyx preparations. A tannic acid staining method and a novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method). RESULTS: 3D electron tomography reveals details of the architecture of the glycocalyx just above the endothelial cell layer. The LaDy GAGa method visually appears to show more complete coverage and more depth than the Tannic Acid staining method. CONCLUSION: The tomographic reconstructions show a potentially significant improvement in determining glycocalyx structure over standard transmission electron microscopy.

Glycocalyx volume: a critical review of tracer dilution methods for its measurement.

A clinical measure of endothelial glycocalyx structure would have great potential importance, because lesions of the glycocalyx may be the first changes to occur in diabetes and in a wide range of vascular diseases. A method recently described by Nieuwdorp et al. for estimating the volume of the luminal glycocalyx of the entire human vascular system would seem to be the first attempt to develop a measure of this kind. It is based on the tracer dilution principle, and this review considers the principles and conditions that underlie this method and the extent to which the conditions appear to have been fulfilled in this case. Our analysis raises two questions about 1) the estimation of the concentration of the tracer (dextran 40) at zero time and 2) the estimation of plasma volume, both of which can be answered by changes in experimental protocol. A third question, concerning the partition coefficient of the tracer between plasma and the fluid within the glycocalyx, cannot be answered at the present time, and until it has been resolved, glycocalyx volume cannot be estimated from the dilution of a macromolecular tracer.