In vivo microvascular flow quantification in the mouse brain using row–column ultrasound localization microscopy and directed graph analysis

Brain perfusion relies on a complex vascular network of arteries, veins, and capillaries to meet its constant demand for oxygen and nutrients. Disruption of this microvascular system is a hallmark of many neurological disorders, including small vessel disease, stroke, and brain tumors. As such, high-resolution in vivo imaging of cerebral microvascular flow and structure remains critical to understanding these pathologies. Among them, ultrasound localization microscopy (ULM) allows noninvasive imaging of microvascular network down to small arterioles and venules at subwavelength resolution using injected microbubbles, but the approach remains mainly limited to 2D imaging with few volumetric implementations. In this study, we explore in vivo transcranial 3D ULM of the mouse brain using row-column arrays (RCA) and introduce an analysis framework to build a flow-directed vascular graph from the ULM microbubble tracking data, allowing to differentiate between subgraphs of artery-like and vein-like vascular segments. Using this framework, we are able to quantify flow and radius relationships for each subgraph in different anatomical regions. This high-sensitivity framework enables in vivo microvascular imaging and quantification in mice and provides a scalable platform for preclinical neurovascular studies in health and disease.