Mapping of the microcirculation in the chick chorioallantoic membrane during normal angiogenesis.

The microcirculation within the chorioallantoic membrane (CAM) of the chick is particularly well suited for in vivo observation and has been used extensively as an assay to detect angiogenic activity. Although progressive chronological expansion of the CAM capillary network occurs normally during embryogenesis, descriptions of the branching patterns of CAM pre- and postcapillary microvessels during embryonic development have not been recorded. In the present study chick embryos were incubated, using an established shell-less culture technique, and observed in vivo at Days 6, 10, and 14 of embryonic development. Morphometric analyses of photomicrographs of CAM microvessels were based upon the centripetal ordering method of microvascular mapping of the first three orders of pre- and postcapillary microvessels with the capillaries serving as the initial point of reference. For both pre- and postcapillary vessels, the number of first-order vessels exceeded the number of second-order vessels which, in turn, outnumbered third-order vessels during each observation period. First- and second-order vessels progressively increased in number from Day 6 to Day 14; however, the number of third-order vessels remained essentially constant during this period. Further, the number of precapillary vessels was greater than postcapillary vessels in their respective orders at Days 6 and 10; however, by Day 14 the numbers were comparable. Average diameters and lengths of the third-order vessels were greater than the second-order vessels which, in turn, were greater than the first-order vessels in both the pre- and postcapillary compartments. Further, mean lengths of each of the three vessel orders in both compartments decreased progressively and by Day 14 were significantly less than at Day 6. Average diameters of each vessel order, on the other hand, remained unchanged from Day 6 to Day 14. Finally, intercapillary distances, based on measurements from fluorescent micrographs obtained after microinjections of fluorescein isothiocyanate (FITC)-dextran, were substantially less at Day 10 and 14 than at Day 6. Based on these morphometric data, the endothelial precursor responsible for continuous neoformation of first- and second-order microvessels during embryogenesis remains uncertain. Whether existing first-, second-, or third-order vessel endothelia serve as this precursor or histodifferentiation of existing capillaries enables continuous expansion of the first- and second-order microvessels remains to be tested.