Visualization and molecular characterization of whole-brain vascular networks with capillary resolution

Structural elucidation and molecular scrutiny of cerebral vasculature is crucial for understanding the functions and diseases of the brain. Here, we introduce SeeNet, a method for near-complete three-dimensional visualization of cerebral vascular networks with high signal-to-noise ratios compatible with molecular phenotyping. SeeNet employs perfusion of a multifunctional crosslinker, vascular casting by temperature-controlled polymerization of hybrid hydrogels, and a bile salt-based tissue-clearing technique optimized for observation of vascular connectivity. SeeNet is capable of whole-brain visualization of molecularly characterized cerebral vasculatures at the single-microvessel level. Moreover, SeeNet reveals a hitherto unidentified vascular pathway bridging cerebral and hippocampal vessels, thus serving as a potential tool to evaluate the connectivity of cerebral vasculature. Structural and molecular elucidation of cerebrovascular network is promising for understanding energy supply system in the brain. Here, the authors describe labeling and tissue clearing techniques that visualize the whole-brain vasculature in a molecularly characterizable manner.