Longitudinal awake imaging of mouse deep brain microvasculature with super-resolution ultrasound localization microscopy

Ultrasound localization microscopy (ULM) is an emerging imaging modality that resolves microvasculature in deep tissues with high spatial resolution. However, existing preclinical ULM applications are largely constrained to anesthetized animals, introducing confounding vascular effects such as vasodilation and altered hemodynamics. As such, ULM quantifications (e.g., vessel diameter, density, and flow velocity) may be confounded by the use of anesthesia, undermining the usefulness of ULM in practice. Here, we introduce a method to address this limitation and achieve ULM imaging in awake mouse brain. Pupillary monitoring was used to support the presence of the awake state during ULM imaging. Vasodilation induced by isoflurane was observed by ULM. Upon recovery to the awake state, reductions in vessel density and flow velocity were observed across different brain regions. In the cortex, the effects induced by isoflurane are more pronounced on venous flow than on arterial flow. In addition, serial in vivo imaging of the same animal brain at weekly intervals demonstrated the highly robust longitudinal imaging capability of the proposed technique. The consistency was further verified through quantitative analysis on individual vessels, cortical regions of arteries and veins, and subcortical regions. This study demonstrates longitudinal ULM imaging in the awake mouse brain, which is crucial for many ULM brain applications that require awake and behaving animals.