Ultrasound-Assisted Blood–Brain Barrier Opening Monitoring by Photoacoustic and Fluorescence Imaging Using Indocyanine Green

Objective: The blood–brain barrier (BBB) is a selectively permeable membrane that restricts drug delivery to the central nervous system. Focused ultrasound (FUS) combined with microbubbles (MBs) is a promising technique to transiently open the BBB, enabling therapeutic delivery. However, real-time monitoring of BBB permeability changes remains challenging. This study investigated the use of indocyanine green (ICG) as a bi-modal contrast agent for photoacoustic and fluorescence imaging to assess BBB opening and closure dynamics. Methods: BALB/c mice underwent FUS-mediated BBB opening with different doses of MBs and ICG administration. Photoacoustic and fluorescence imaging were performed at various time points post-FUS to evaluate ICG extravasation dynamics. Magnetic resonance imaging (MRI) with gadolinium contrast was used as the gold standard for BBB permeability assessment. The effect of MB dose and injection timing on BBB closure kinetics was analyzed. Results: Photoacoustic imaging provided reliable BBB monitoring within the first hour post-FUS, whereas fluorescence imaging was more effective at detecting ICG extravasation at 24 h. A strong correlation was observed between fluorescence intensity and MRI-based contrast enhancement, confirming BBB opening dynamics. BBB closure followed an exponential decay model, with a half-closure time of approximately 81 min. The degree of BBB opening was proportional to the MB dose administered. Conclusion: ICG-based photoacoustic and fluorescence imaging provide a non-invasive and cost-effective alternative to MRI for monitoring FUS-induced BBB opening. These techniques offer complementary temporal windows for assessment, improving the precision of BBB permeability evaluation in preclinical and potentially clinical applications.