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Background: Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening is a noninvasive, safe and reversible technique for targeted drug delivery to the brain. Most preclinical systems developed to perform and monitor BBB opening are comprised of a separate geometrically focused transducer and passive cavitation detector (PCD) or imaging array. This study builds upon previous work from our group developing a single imaging phased array configuration for simultaneous BBB opening and monitoring called theranostic ultrasound (ThUS), leveraging ultra-short pulse lengths (USPLs) and a novel rapid alternating steering angles (RASTA) pulse sequence design for simultaneous bilateral sonications with target-specific USPL. The RASTA sequence was further employed to evaluate the impact of USPL on BBB opening volume, power cavitation imaging (PCI) pixel intensity, BBB closing timeline, drug delivery efficiency, and safety. Methods: A P4-1 phased array transducer driven by a Verasonics Vantage ultrasound system was operated using a custom script to run the RASTA sequence which consisted of interleaved steered, focused transmits and passive imaging. Contrast-enhanced magnetic resonance imaging (MRI) confirmed initial opening volume and closure of the BBB by longitudinal imaging through 72 hours post-BBB opening. For drug delivery experiments, mice were systemically administered a 70 kDa fluorescent dextran or adeno-associated virus serotype 9 (AAV9) for fluorescence microscopy or enzyme-linked immunosorbent assay (ELISA) to evaluate ThUS-mediated molecular therapeutic delivery. Additional brain sections were also H&E-stained to evaluate histological damage, and IBA1- and GFAP-stained to elucidate the effects of ThUS-mediated BBB opening on stimulation of key cell types involved in the neuro-immune response, microglia and astrocytes. Results: The ThUS RASTA sequence induced distinct BBB openings simultaneously in the same mouse where volume, PCI pixel intensity, level of dextran delivery, and AAV reporter transgene expression were correlated with brain hemisphere-specific USPL, consistent with statistically significant differences between 1.5, 5, and 10-cycle USPL groups. BBB closure after ThUS required 2-48 hours depending on USPL. The potential for acute damage and neuro-immune activation increased with USPL, but such observable damage was nearly reversed 96 hours post-ThUS. Conclusion: ThUS is a versatile single-array technique which exhibits the potential for investigating a variety of non-invasive therapeutic delivery applications in the brain.

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Focused Ultrasound (FUS) paired with systemically-injected microbubbles (μB) is capable of transiently opening the blood–brain barrier (BBBO) for noninvasive and targeted drug delivery to the brain. FUS-BBBO is also capable of modulating the neuroimmune system, further qualifying its therapeutic potential for neurodegenerative diseases like Alzheimer’s disease (AD). Natural aging and AD impose significant strain on the brain and particularly the BBB, modifying its structure and subsequently, its functionality. The emerging focus on treating neurodegenerative diseases with FUS-BBBO necessitates an investigation into the extent that age and AD affect the BBB’s response to FUS. FUS-BBBO was performed with a 1.5-MHz, geometrically focused transducer operated at 450 kPa and paired with a bolus microbubble injection of 8 × 108 μB/mL. Here we quantify the BBBO, BBB closing (BBBC) timeline, and BBB permeability (BBBP) following FUS-BBBO in male mice with and without AD pathology, aged 10 weeks, one year, or two years. The data presented herein indicates that natural aging and AD pathology may increase initial BBBO volume by up to 34.4% and 40.7% respectively, extend BBBC timeline by up to 1.3 and 1.5 days respectively, and increase BBBP as measured by average Ktrans values up to 80% and 86.1% respectively in male mice. This characterization of the BBB response to FUS-BBBO with age and AD further clarifies the nature and extent of the functional impact of these factors and may offer new considerations for planning FUS-BBBO interventions in aged and AD populations.