MENG-TING LINCHUEH-HUNG WULiao, Wei-HaoWei-HaoLiaoChan, Tsai-YunTsai-YunChanChen, Shao-YuShao-YuChenYang, Shu-MeiShu-MeiYangMING-YEN HSIAOYoung, Tai-HorngTai-HorngYoungWEN-SHIANG CHEN2026-04-012026-04-012026-09https://scholars.lib.ntu.edu.tw/handle/123456789/736999Introduction The glymphatic system clears brain waste but remains difficult to image, particularly perivascular space (PVS) dynamics. This study aimed to employ contrast-enhanced superb microvascular imaging (SMI) to achieve real-time visualization and quantitative evaluation of glymphatic flow dynamics in rat brains, while evaluating how contrast agent size and focused ultrasound (FUS) stimulation affect imaging performance and flow dynamics. Methods Adult rats received nanobubbles (<450 nm) or microbubbles (>1 µm) injections via the cisterna magna (CM) or intravenous (IV) routes (as positive control). The SMI-derived vascularity index (VI) was quantified at baseline and multiple post-injection time points to measure PVS-associated flow. To evaluate mechanical modulation of glymphatic transport, FUS was applied following CM nanobubbles injection. The resulting VI was quantified at baseline and post-stimulation. Results CM injection of nanobubbles resulted in a significant increase in VI compared to baseline ( p = 0.0011), whereas CM-injected microbubbles showed no signal enhancement ( p = 0.6241). The nanobubbles-derived signal was transient, peaking at 5 min post-injection ( p < 0.0001) and declining by 20 min ( p < 0.0001). Furthermore, subsequent FUS stimulation robustly amplified the nanobubbles-enhanced VI, showing a significant increase after the FUS ( p < 0.0001). Conclusions This research establishes nanobubble-enhanced SMI as a viable platform for quantifying PVS dynamics, demonstrating that nanobubbles, unlike microbubbles, overcome size-exclusion limitations. This ultrasound-based strategy bridges a critical gap between superficial optical imaging and macroscopic MRI, offering a powerful, accessible tool to probe glymphatic dysfunction in deep parenchymal structures and assess FUS-mediated modulation of glymphatic dynamics.journal-articlehttps://doi.org/10.1016/j.ultras.2026.108074