The Microvascular Unit of the 6-Day Chick Chorioallantoic Membrane: A Fluorescent Confocal Microscopic and Ultrastructural Morphometric Analysis of Endothelial Permselectivity

The chorioallantoic membrane (CAM) of the chick embryo provides a unique model for investigating endothelial permselectivity during normal angiogenesis. Chick embryos were incubated using established shell-less culture techniques for intravital and ultrastructural observations at Day 6 of the normal 21-day gestation. Morphometric analyses of the precapillary, capillary, and postcapillary microvascular segments served to demonstrate a continuous endothelium of uniform cytoplasmic thickness and few plasmalemmal vesicles. Average widths and depths of the endothelial junctional clefts were also homogeneous, except the significantly longer junctional depths of the second-order postcapillaries. The frequency of endothelial junctions per unit length of segmental luminal surface, like the vesicle densities, were uniformly less than those reported previously for adult continuous endothelia. Qualitative observations of systemically microinjected FITC-dextrans (40, 70, and 150 KDa), using real-time fluorescent confocal microscopy, failed to identify substantial interstitial accumulation of any dextrans during 10-min perfusion periods. Ultrastructural examination of the same dextran probes, on the other hand, served to detect small, sporadic interstitial foci of the dextran tracers. Such foci created a wide scattering of particle counts in the extravascular space, and extravascular accumulation was not sufficient to elicit an observable fluorescent signal. These results are consistent with the interpretation that during early stages of the normal angiogenic process, macromolecules ≥40,000 MW can traverse the CAM segmental microvascular endothelium. The extent of extravasation, however, was less than that reported previously during pathologic angiogenesis associated with wound healing or tumor growth.