Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain

ABSTRACT The formation of new blood vessels and the establishment of vascular networks are crucial during brain development, in the adult healthy brain, as well as in various diseases of the central nervous system (CNS). Here, we describe a method that enables hierarchical imaging and computational analysis of vascular networks in postnatal- and adult mouse brains. Resin-based vascular corrosion casting, scanning electron microscopy, synchrotron radiation and desktop µCT imaging, and computational network analysis are used. Combining these methods enables detailed visualization and quantification of the three-dimensional (3D) brain vasculature. Network features such as vascular volume fraction, branch point density, vessel diameter, - length, -tortuosity, and -directionality as well as extravascular distance can be obtained at any developmental stage from the early postnatal to the adult brain. Our method allows characterizing brain vascular networks separately for capillaries and non-capillaries. The entire protocol, from mouse perfusion to vessel network analysis, takes approximately 10 days. Online summary This protocol uses vascular corrosion casting, hierarchical synchrotron radiation µCT imaging, and computational image analysis to assess the three-dimensional vascular network architecture.