Focused Ultrasound-Mediated Blood–Brain Barrier Opening Best Promotes Neuroimmunomodulation through Brain Macrophage Redistribution

Neuroimmunomodulation is a promising form of drug-free treatment for neurological diseases ranging from Alzheimer’s disease to depression. The evidence supporting the efficacy of focused ultrasound (FUS) neuroimmunomodulation is encouraging; however, the method has yet to be standardized, and its mechanism remains poorly understood. Methods of FUS neuroimmunomodulation can be categorized into three paradigms based on the parameters used. In the first paradigm, focused ultrasound blood–brain barrier opening (FUS-BBBO) combines FUS with microbubbles (MB) to transiently and safely induce BBB opening. In the second paradigm, focused ultrasound neuromodulation (FUS-N) harnesses the acoustic effects of FUS alone (without MB). In the third paradigm, focused ultrasound with microbubbles without BBBO (FUS + MB) combines MB with FUS below the BBBO pressure threshold—harnessing the mechanical effects of FUS without opening the barrier. Due to the recent evidence of brain macrophage modulation in response to FUS-BBBO, we provide the first direct comparison of brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s disease (AD) pathology. Flow cytometry and single-cell sequencing are employed to identify FUS-BBBO as the FUS paradigm, which maximizes brain macrophage modulation, including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between treatment cavitation dose and brain macrophage phagocytosis. Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to provide the first characterization of brain macrophage distribution in response to FUS-BBBO. Given their relevance within neurodegeneration and perturbation response, we emphasize the analysis of three brain macrophage populations—disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find and validate the redistribution of each population with an overall trend toward increased interaction with the brain–cerebrospinal fluid barrier (BCSFB) after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology. This study addresses the prior lack of FUS neuroimmunomodulation paradigm optimization and mechanism characterization, identifying that FUS-BBBO best modulates brain macrophage response via complex redistribution.