The glymphatic system regulates cerebrospinal fluid (CSF) transport and brain waste clearance. Focused ultrasound combined with microbubbles (FUSMB) has shown feasibility for manipulating glymphatic transport, yet its mechanisms remain poorly understood. In this work, we used in vivo two-photon microscopy to reveal how FUSMB manipulates the CSF tracer transport in the mouse brain. A FUS transducer was confocally aligned with the objective of a two-photon microscope. Fluorescently labeled albumin was infused into the CSF via cisterna magna. FUS sonication was applied following an intravenous injection of microbubbles. Dynamic imaging was performed through a cranial window to record local changes in vessel and tracer dynamics. The fluorescence intensity of the CSF tracer within the treated region decreased rapidly upon FUSMB treatment. Concurrently, vessel deformation was observed, and the fastest diameter changes were observed during FUSMB treatment. A linear correlation was identified between the rate of vessel diameter change and the rate of tracer intensity change. Moreover, given the same rate of vessel diameter change, the tracer intensity changed faster around larger vessels than smaller vessels. These findings offer insight into the potential biophysical mechanism of FUSMB-mediated glymphatic transport.
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