Angiogenesis, a process of blood vessel formation from preexisting ones, has become a significant area of scientific enquiry due to its role in various physiological and pathological processes. Therefore methods that can stimulate fundamental steps of this process and provide a tool for testing potential therapeutic agents have been developed in the form of in vitro and in vivo assays.
Most researchers in the field of angiogenesis place greater emphasis on results from intact animals, as complex interactions between multiple cell types, the extracellular matrix, as well as hemodynamic and hemodynamic factors, can be hardly duplicated in vitro.
The earliest of in vivo assays contained diffusion chambers with Millipore filters designed to visualize and monitor neovascularization of implanted tumors. In the meantime, many in vivo assay methods have been developed that permit better quantification, and are also much easier to perform.
CAM assay
The original version of the chick chorioallantoic membrane (CAM) angiogenesis assay was introduced by experimental embryologists 50 years ago, but it still represents a centerpiece for the study of organ development. The method is performed in ovo by removing a small section of the egg shell and then placing tissue or organ grafts directly on CAM.
Nevertheless, most researchers have transferred the developing chick embryo to a plastic culture disk or other container after a 3-day incubation period. This change permits improved visualization of the CAM vasculature, allows serial measurements, and enables control and experimental molecules to be tested on the same CAM.
Angiogenic inducers (such as VEGF recombinant mouse or rat protein, TGFB1 recombinant human protein or PDGF-BB recombinant mouse or human protein) are often added to the CAM assays. They are commercially available from companies such as Thermo Fisher Scientific in different concentrations.
Corneal angiogenesis assay
The cornea is the only tissue of the body that is both transparent and avascular, which makes it ideal for observation of angiogenesis in rats, mice and rabbits. Thus corneal angiogenesis assay is still regarded as one of the best in vivo assays, and any vessels observed in the cornea after applying angiogenesis-inducing factors represent newly formed vessels.
Sponges containing proangiogenic molecules (such as FGF2 and VEGF) or slow-release polymer pellets are implanted into surgically created stromal pockets. The ingrowth of newly developed vessels from the peripheral limbal vasculature can be monitored daily, consequently allowing for precise calculation of angiogenesis rates.
Conclusive visualization of the corneal vasculature in mouse models was once established by injecting India ink, but fluorochrome-labeled dextran molecules (produced by companies such as Sigma Chemical) have become a method of choice more recently.
Matrigel plug assay
In this method, Matrigel that contains test cells (or substances) is injected under the skin where it forms a plug. This plug is then recovered after 1-3 weeks in the animal for subsequent histological examination in order to assess how many blood vessels have entered it.
Quantification is often achieved by determining the hemoglobin concentration in the Matrigel plug. There is also a rapid and unbiased RT-quantitative PCR method to investigate the angiogenic developments in the Matrigel plug in response to fibroblast growth factor-2.
BD Matrigel Matrix HC developed by BD Biosciences can be utilized to evaluate in vivo angiogenic activity of various compounds by subcutaneous injection into mice (BD Matrigel Plug Assay). As already described, the plugs are subsequently removed and explored for the formation of blood vessels.
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