Morphometric measurements of chorioallantoic membrane vascularity: effects of hypoxia and hyperoxia

We studied the effects of hypoxia and hyperoxia on the angiogenesis process in the chick embryo chorioallantoic membrane (CAM) using four different morphometric measurements of vascularity. Chick eggs were incubated in various oxygen atmospheres (12, 16, 21, 45, or 70% oxygen) beginning on the 7th day of development, and vascularity was measured on the 14th day. Measurements of vascularity included vessel endpoint density (VED), length density, fractional image area, and a vascular density index. All measurements were made on blood vessels in randomly selected areas of CAM using a computerized image analysis system. An opaque colloidal carbon-albumin perfusate was used as a vascular marker. All four measurements showed that vascularity of CAM was inversely related to the oxygen tension to which the embryos were subjected. The VED, an estimate of total number of pre- and postcapillary vessels, exhibited the greatest degree of change, but overall changes in vascularity were modest. Prolonged exposure to a 12% oxygen atmosphere increased VED by approximately 16%, whereas 70% oxygen decreased VED by approximately 19% compared with room air control groups. We also studied the normal growth of CAM vasculature from days 8 to 18 of development. In these studies, the values of VED increased progressively throughout the entire period of development, whereas the other measurements of vascularity reached maximum values by the 14th day. We conclude that hypoxia stimulates angiogenesis in the CAM in a dose-related manner, hyperoxia inhibits CAM angiogenesis in a dose-related manner, and VED provides a sensitive estimate of vascularity in chick CAM throughout its development.