Longitudinal Awake Mouse Brain Imaging Using Functional Ultrasound and Functional Ultrasound Localization Microscopy.

Functional ultrasound (fUS), ultrasound localization microscopy (ULM), and functional ULM (fULM) are three emerging brain ultrasound imaging techniques that measure the cerebrovascular structure and function at different spatial scales. Here we establish an awake, longitudinal fUS-ULM-fULM imaging framework to study structural vascularity, microvascular flow velocity, mesoscale hemodynamic responses, and microvascular functional responses in the same animals across 5 months. No significant linear longitudinal trend was detected for any of the ultrasound measurements in control animals, with structural measures showing lower longitudinal variability than functional readouts. Mean flow velocity showed the strongest longitudinal consistency and the lowest pooled within-subject variability. Behavioral testing and GFAP/Iba1 staining further revealed no detectable memory impairment or chronic neuroinflammatory response during the 5-month serial imaging setup. Application of fUS-ULM-fULM in 5xFAD-positive mice revealed a temporally ordered, cross-scale pattern of cerebrovascular dysfunction, with cerebral blood flow velocity declining first followed by impaired stimulus-evoked responses at the microvascular and then mesoscale levels. No vascular structural changes were detected across 5 months. Overall, this study established a stable longitudinal multimodal ultrasound imaging framework for tracking the awake mouse brain and demonstrated the utility for detecting cerebrovascular structural and functional variations in a preclinical Alzheimer's disease mouse model.