Quantitative Analysis of Angiogenesis in the Allantois Explant Model

Abstract

The murine allantois represents a powerful system for the analysis of developmental angiogenesis ex vivo, producing a complex vascular network in less than a day’s time. This can be visualised by appropriate antibody staining. Angiogenesis in the explants occurs in the absence of external, confounding factors. Since the allantois is taken at an early developmental stage, it is a useful system for studying embryonic lethal mutations with no need for breeding conditional knock-outs. In addition, allantois explants are useful for testing inhibitors without any need for animal procedures. Manual quantification of angiogenesis in the complex vascular networks is time and labour intensive. We developed AngioTool, a piece of software which allows the quick, hands-off and reproducible quantification of microscopic images of vascular networks. AngioTool is available free of charge and was developed for analysis of angiogenesis in the allantois explant, although it is also suitable for other systems. Parameters measured include the overall size of the vascular network, average and total vessel length, percentage of area covered by vessels and number of endpoints. In addition, AngioTool calculates the so-called “branching index” (branch points/unit area of the explant), measuring the sprouting activity of an explant whilst correcting for its overall size. The entire experimental protocol including analysis takes ≈5 days to complete.