The chick chorioallantoic membrane model (CAM) as a tool to monitor angiogenesis

The chorioallantoic membrane (CAM) is a highly vascular membrane formed by the fusion of the allantois and the chorion. The CAM is composed of three layers, the chorionic epithelium, the mesenchyme and the allantoic epithelium. During development numerous blood capillaries found in chorionic layer associate closely with air found in pores of the shell membrane of the egg allowing gas exchange by the developing embryos. Due to its ease of accessibility and vascularization, the CAM provides researchers with an excellent in vivo model for the study of angiogenesis. This study focuses on the establishment of both in ovo and ex ovo CAM preparations and reports on the ability of the developing CAM vasculature to sprout in response to pro-angiogenic stimuli grafted onto the membrane in a 3D collagen droplet. Fertilized White Leghorn eggs (Ovagen, Ballina, Co. Mayo) were activated for 4 days in a humidified rotating egg incubator at 37°C. On day 4 of development, the CAM was exposed either by scoring a window on the upper-side of the egg and gently removing a portion of the eggshell (in ovo model) or by gently cracking the shell and emptying the contents of the egg into a sterile container (ex ovo model). Eggs were inspected for a visible embryonic heartbeat, colour and quality of egg yolk, and quality of blood vessel formation prior to maturation. The eggs were then transferred to a stationary incubator at 37°C and 60% humidity. On day 11 of development angiogenic factors VEGF (75ng onplant) or FGF (62.5ng onplant) or human oral squamous carcinoma cells (1x106 onplant) were grafted onto the CAM in a semi-solidified collagen onplant prepared by adding factors to 30 #956;l neutralized collagen drops. CAMs were further cultured for 72 hours during which time angiogenesis was monitored. Following treatments, the CAM were fixed with 4% paraformaldehyde and either paraffin embedded for H amp;E staining or resin embedded for transmission electron microscopy (TEM). Both ex ovo and in ovo models have been established with average survival rates of 70% from D4 to D14 of development recorded irrespective of the model used. Following H amp;E staining, the three layers of the CAM were clearly visible and TEM reveals the highly vascular nature of the chorionic layer. Preliminary studies show increased angiogenesis following treatment with EGF or FGF and also demonstrated clear oral cancer tumour-induced angiogesis.