Analysis of Angiogenesis in the Developing Mouse Central Nervous System

In order to study basic mechanisms of sprouting angiogenesis, researchers worldwide rely on the use of model tissues such as rodent retina, which becomes vascularized postnatally, to study the growth of blood vessels. By definition, models have to be simple, recapitulating angiogenic processes in a stereotyped and relatively easy accessible manner, allowing the application of standardized analyses. These criteria also apply in an ideal manner to the embryonic mouse hindbrain, which becomes vascularized by sprouting angiogenesis from a preformed perineural vascular plexus, leading to the stereotypical formation of a capillary subventricular plexus. Similar to the retina model, between embryonic days 10.5 and 13.5, the hindbrain can be flat-mounted in an “open-book” preparation, allowing the analysis of the vascular bed in two-dimensional extension, of parameters like vessel density, morphology, and remodeling including branching and sprouting. In addition to sprouting angiogenesis, the hindbrain is a suitable model for investigating inductive mechanisms towards the blood–brain barrier phenotype of microvessels in the central nervous system. In this chapter, we describe how to fix, dissect, stain, and analyze the developing hindbrain vasculature.