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The gelatin sponge–chorioallantoic membrane assay

Nature Protocols volume 1, pages 85–91 (2006)Cite this article

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Abstract

Here we present a method for the quantification of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM) based on the implantation of a gelatin sponge on the top of the growing CAM on day 8 of development. After implantation, the sponge is treated with a stimulator of blood vessel formation in the absence or presence of an angiogenesis inhibitor. On day 12, blood vessels that are growing into the sponge are counted at macroscopic and microscopic levels. The estimated timeline for carrying out this protocol is 10 d. The presence of a vascular network in the CAM requires a careful analysis to distinguish new capillaries from pre-existing ones. This limitation does not occur in the avascular cornea assay, which may also take advantage of different genetic backgrounds when carried out in transgenic or knockout mice. Nevertheless, the gelatin sponge–CAM assay is simple, inexpensive and suitable for large-scale screening.

*Note: In the version of the article initially published online, references 6 and 7 were incorrect. The correct references are: 6. Serbedzija, G.N., Flynn, E. & Willet, C.E. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 3, 519–528 (2000). 7. Ribatti, D., Vacca, A., Roncali, L. & Dammacco, F. The chick embryo chorioallantoic membrane as a model for in vivo research on angiogenesis. Int. J. Dev. Biol. 40, 1189–1897 (1996). The error has been corrected in all versions of the article.

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Figure 1: CAM vessels (arrow) beneath the square window are photographed in ovo on day 3 of development.
Figure 2: An illustrative sponge–CAM assay in which a proangiogenic stimulus, an angiostatic compound and a vehicle were applied on top of the CAM on day 8.
Figure 3: Evaluation of a proangiogenic response by macroscopic semiquantitative scoring.
Figure 4: Evaluation of a proangiogenic response by macroscopic semiquantitative scoring of vessel branching.
Figure 5: Effect of VEGF-overexpressing V12-MCF-7 cells on CAM vascularization.
Figure 6: Effect of ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), on the angiogenic response induced by FGF-2.

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  • 10 August 2006

    In the version of the article initially published online, references 6 and 7 were incorrect. The correct references are:

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Acknowledgements

This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC, National and Regional Funds), Milan; MIUR (Interuniversity Funds for Basic Research, FIRB 2001, Center of Excellence IDET, and PRIN 2005), Rome; Istituto Superiore di Sanità (Oncotechnological Project), Rome; Fondazione Italiana per la Lotta al Neuroblastoma, Genoa, Italy.

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Authors and Affiliations

  1. Department of Human Anatomy and Histology, University of Bari Medical School, Piazza Giulio Cesare 11, Policlinico, Bari, 70124, Italy

    Domenico Ribatti & Beatrice Nico

  2. Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Piazza Giulio Cesare 11, Policlinico, Bari, 70124, Italy

    Angelo Vacca

  3. Department of Biomedical Sciences and Biotechnology, General Pathology and Immunology, University of Brescia Medical School, Viale Europa 11, Brescia, 25123, Italy

    Marco Presta

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  1. Domenico Ribatti
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Correspondence to Domenico Ribatti.

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Ribatti, D., Nico, B., Vacca, A. et al. The gelatin sponge–chorioallantoic membrane assay. Nat Protoc 1, 85–91 (2006). https://doi.org/10.1038/nprot.2006.13

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