AAT Bioquest

What are the in vivo angiogenesis assays?

Posted October 10, 2023


The in vivo angiogenesis assays include: chick chorioallantoic membrane (CAM), sponge implantation, zebrafish, dorsal air sac, corneal, chamber and tumor angiogenesis models. 

  1. The chorioallantoic membrane of the chick allows an accessible system in which to study angiogenesis in vivo. It is a simple model, and provides a physiological technique for in vivo analysis of cells, reagents, and pathogens than in vitro systems do. The CAM is readily accessible outside the embryo. The chick is fairly immunotolerant, which enables the study of cross-specifix xenografts crafts including tumors, mammalian tissue explants, and cultured human cancer cells. 
  2. Zebrafish yield hundreds of embryos per mating event. These large quantities and optical clarity of developing embryos assist in the study of development and angiogenesis. Zebrafish share many genes and mechanisms of angiogenesis regulation with mammals, making them highly useful for analyzing development and function of the vasculature. More specifically, the formation of the intersegmental vessels and subintestinal veins in embryos is well characterized, making them ideal for the study of angiogenesis inhibitors. The embryos are also inexpensive to produce and easy to maintain long-term. Hundreds of offspring produced each week, making it possible to carry out large-scale screening experiments. 
  3. Implanted sponges and polymers have also been used to induce and study angiogenesis. However, the implants do not become encapsulated and elicit the generation of fused macrophages, which secret angiogenic cytokines and interfere with the test substrate. The rate and intensity of the tissue response induced by the sponge depend on factors including the type of sponge material and size. The sponge implant technique has been utilized to analyze the creation of fibrovascular tissue measuring neovascularization and wound healing, fibronectin deposition, collagen metabolism, development of granulation tissue, and kinetics of cellular proliferation. 
  4. The cornea is the only avascular transparent tissue in the body, and thus any vessels penetrating from the limbus into the corneal stroma are newly formed, visible, and can be quantified. This assay is however time consuming and technically difficult. Almost all types of corneal injury induce neovascularization. The processes of corneal neovascularization involve multiple interactions within the cornea and thus it is possible that different pathways of angiogenesis are involved in different experimental methods. 
  5. The doral air sac model showed that implantation of a chamber ring loaded with tumor cells causes angiogenic vessel formation of the murine skin attached to the ring. After treatment with a compound of interest, the chamber is detached from the skin of mice, exposing the newly formed blood vessels. Angiogenesis is then analyzed using a dissecting microscope to count vessels or photographs of the skin for quantification of vessel density. Dyes such as Evans blue can also be injected into mice, which leaks out of the angiogenic vessels and accumulates in interstitial spaces, but is retained within pre-existing vessels. 
  6. Chamber assays enable determination of whether a newly formed blood vessel is perfused and contributing to tissue oxygenation through in vivo imaging of angiogenesis. It is possible to visualize the vasculature using light transillumination if tissue is less than 300 micrometers thick, epi-illumination microscopy, or intravital microscopy. Specifically, a transparent chamber is used instead of acute preparations as angiogenesis is a process which lasts over days or weeks. Using the dorsal skinfold as a chamber implantation site has been adapted for rats, hamsters, and mice to quantify structural and functional changes in the neovasculature of tumors. 
  7. Tumor models are one of the simplest methods for analyzing tumor growth and angiogenesis in vivo. Tumor growth is assessed by measuring tumor dimensions regularly and weighing excised tumors at the end of the experiment. Although angiogenesis cannot be visualized daily, if the groups are sufficiently powered, subgroups of animals may be killed sequentially for imaging angiogenesis. The major advantage of using these models is that all tumors are established within a few weeks following cell implantation, while human cancer develops over a period of months to years. 
Additional resources

In Vitro Angiogenesis Assays