This document describes the chick chorioallantoic membrane (CAM) assay, which is used to study angiogenesis. The CAM assay involves applying test agents/drugs to the CAM of a developing chick embryo and observing their effects on blood vessel growth over time. Specifically, it counts the number of branch points in the vascular network with and without the test agent to determine pro- or anti-angiogenic effects. The document outlines two methodologies - an in ovo (in egg) method and an ex ovo (outside egg) method. It provides details on preparation, application of test agents to the CAM, incubation, evaluation of vascular response, and expression of results.
This document describes the chick chorioallantoic membrane (CAM) assay, which is used to study angiogenesis. The CAM assay involves applying test agents/drugs to the CAM of a developing chick embryo and observing their effects on blood vessel growth over time. Specifically, it counts the number of branch points in the vascular network with and without the test agent to determine pro- or anti-angiogenic effects. The document outlines two methodologies - an in ovo (in egg) method and an ex ovo (outside egg) method. It provides details on preparation, application of test agents to the CAM, incubation, evaluation of vascular response, and expression of results.
This document describes the chick chorioallantoic membrane (CAM) assay, which is used to study angiogenesis. The CAM assay involves applying test agents/drugs to the CAM of a developing chick embryo and observing their effects on blood vessel growth over time. Specifically, it counts the number of branch points in the vascular network with and without the test agent to determine pro- or anti-angiogenic effects. The document outlines two methodologies - an in ovo (in egg) method and an ex ovo (outside egg) method. It provides details on preparation, application of test agents to the CAM, incubation, evaluation of vascular response, and expression of results.
This document describes the chick chorioallantoic membrane (CAM) assay, which is used to study angiogenesis. The CAM assay involves applying test agents/drugs to the CAM of a developing chick embryo and observing their effects on blood vessel growth over time. Specifically, it counts the number of branch points in the vascular network with and without the test agent to determine pro- or anti-angiogenic effects. The document outlines two methodologies - an in ovo (in egg) method and an ex ovo (outside egg) method. It provides details on preparation, application of test agents to the CAM, incubation, evaluation of vascular response, and expression of results.
PREPARED BY: YSABEL ANN C. DE OCAMPO, RPH CAM Assay • Chick Chorioallantoic Assay • Screening assay for potential pro- and anti-angiogenic agents
ANGIOGENESIS • Formation of new blood vessels out of pre-existing capillaries • Assessment is an indirect assessment of the carcinogenicity of a drug sample CAM Assay • Used in the studies of: • Angiogenic effect of drugs (by examining the response in the rapidly growing CAM) • Angiogenesis inhibitors (Fibroblast growth factor-2) CAM Assay • Principle:
• Capillaries appear in the yolk sac
of the fertilized egg at 28 hours and grow rapidly over the next 6-8 days which is the best time to introduce the test chemical agent and observe its effect on the 12th day Chorioallantoic Membrane • Develops from 4th to 5th ID in the domestic fowl (Gallus domesticus) • Fusion of mesodermal layers of chorion and allantois • Resembles the primary respiratory organ of the developing avian embryo • Serves as an organ for gaseous exchange between the pores of the eggshell and dense network of capillaries subjacent in a “placenta-like” fashion. • It finally covers almost the complete interior surface of the egg shell by ID 12. METHODOLOGY: Version 1 (In Ovo) • PREPARATION OF FILTER PAPER DISCS
1. Punch filter paper using a puncher (approx. 5 mm. in diameter).
2. Sterilize by autoclaving or exposing to UV light for at least 15 minutes. METHODOLOGY: Version 1 (In Ovo) • ASSAY PROPER
1. Incubate the fertilized egg for 8-10 days at 37oC and 70% humidity 2. Cut 1x1 cm window in the egg shell to expose the CAM 3. Place the treated filter disc directly onto the CAM 4. Seal the treated eggs with plastic tape and incubate for two days METHODOLOGY: Version 1 (In Ovo) • ASSAY PROPER
5. After 48 hours of incubation, harvest the CAM carefully removing the hard shell leaving intact the soft membranes covering the embryo 6. Transfer the shell-less embryo to a glass petri dish 7. Examine CAM for the number of blood vessel branch points METHODOLOGY: Version 1 (In Ovo) • VISUAL ASSESSMENT AND PHOTOGRAPHY
1. Examine the CAM at the site of application for angiogenesis
2. Draw 4 quadrants about the area of the CAM 3. Manually count the blood vessel branch points at each area of the quadrant counting clockwise 4. CAM is photographed for documentation using 40 to 80 x total magnification and the ocular lens METHODOLOGY: Version 1 (In Ovo) • RESULTS • CAM vascularity can be expressed as % of control
No. of branch points (treated) – No. of branch points (control)
x 100 No. of branch points (control) Baptista, P., Kanaras, A., Heuer-Jungemann, A., Roma-Rodrigues, C., & Fernandes, A. (2016). Peptide-coated gold nanoparticles for modulation of angiogenesis in vivo. International Journal of Nanomedicine, 2633. doi:10.2147/ijn.s108661 METHODOLOGY: Version 2 (Ex Ovo) 1. Fertilized White Leghorn chicken eggs staged according to Hamburger and Hamilton are placed in an incubator as soon as embryogenesis starts and are kept under constant humidity at 37oC. 2. On day 3, a square window is opened in the shell after removal of 2-3 mL of albumen to detach the CAM from the shell itself and the underlying CAM vessels are disclosed. METHODOLOGY: Version 2 (Ex Ovo) 3. The window is sealed with a class and incubation goes on until the day of the experiment. This technique may preserve amore physiological environment; however, it limits the are for use and observation. 4. The embryo and its extraembryonic membranes are transferred to a petri dish on day 3 or 4 incubation and CAM develops at the top as a flat membrane and reaches the edge of the dish to provide two dimensional monolayer onto which multiple grafts can be placed. METHODOLOGY: Version 2 (Ex Ovo) • Survival rate of eggs cultured ex ovo is the major success limiting step in this technique • The ex ovo method may be preferred to the in vivo method because ◦ It allows the quantification of the response over a wider area of the CAM ◦ Large number of samples can be tested at any one time ◦ The time required for a response to occur is shorter (2-3 days)
