N oninvasively applied pulses of ultrasound and microbubbles can locally deliver molecules to the brain (1,2). Ultrasound has been shown to shrink brain tumors and improve cognitive function in animal models of Alzheimer disease (3-5). However, despite promising results, there remain concerns about potentially harmful effects from permeability changes to the blood-brain barrier (6,7). Focused ultrasound delivers molecules from the bloodstream to the brain by mechanically stimulating the vessels with acoustically active microbubbles. The microbubbles, composed of a lipid shell and stabilized gas core (1-10 µm), are administered intravenously (8). Pressure oscillations from the ultrasound drive the microbubbles to expand and contract, transporting molecules across the blood-brain barrier. In animals and in clinical trials, the most common method to drive microbubbles to disrupt the blood-brain barrier is with long-pulse sequencing (9-11), characterized by long pulses emitted in a slow sequence (6,12-14). Although parameters within this regimen attain the best performance-safety balance through thorough optimization, limitations exist. First, treatment is unequal within the beam (12,15), resulting in drugs delivered in some, but not all, targeted areas. Second, unwanted biologic responses can be triggered, such as neuronal damage, red blood cell extravasation, and hemorrhage (6,16,17). A recent clinical study has shown the presence of potential microhemorrhages (shown as hypointense areas on T2 MRI scans) using pulses that last in the range of milliseconds in humans (18). Third, it takes 4-48 hours for the permeability of the blood-brain barrier to return to normal (14,19),

An emerging approach with potential in improving the treatment of neurodegenerative diseases and brain tumors is the use of focused ultrasound (FUS) to bypass the blood–brain barrier (BBB) in a non-invasive and localized manner. A large body of pre-clinical work has paved the way for the gradual clinical implementation of FUS-induced BBB opening. Even though the safety profile of FUS treatments in rodents has been extensively studied, the histological and behavioral effects of clinically relevant BBB opening in large animals are relatively understudied. Here, we examine the histological and behavioral safety profile following localized BBB opening in non-human primates (NHPs), using a neuronavigation-guided clinical system prototype. We show that FUS treatment triggers a short-lived immune response within the targeted region without exacerbating the touch accuracy or reaction time in visual-motor cognitive tasks. Our experiments were designed using a multiple-case-study approach, in order to maximize the acquired data and support translation of the FUS system into human studies. Four NHPs underwent a single session of FUS-mediated BBB opening in the prefrontal cortex. Two NHPs were treated bilaterally at different pressures, sacrificed on day 2 and 18 post-FUS, respectively, and their brains were histologically processed. In separate experiments, two NHPs that were earlier trained in a behavioral task were exposed to FUS unilaterally, and their performance was tracked for at least 3 weeks after BBB opening. An increased microglia density around blood vessels was detected on day 2, but was resolved by day 18. We also detected signs of enhanced immature neuron presence within areas that underwent BBB opening, compared to regions with an intact BBB, confirming previous rodent studies. Logistic regression analysis showed that the NHP cognitive performance did not deteriorate following BBB opening. These preliminary results demonstrate that neuronavigation-guided FUS with a single-element transducer is a non-invasive method capable of reversibly opening the BBB, without substantial histological or behavioral impact in an animal model closely resembling humans. Future work should confirm the observations of this multiple-case-study work across animals, species and tasks.

Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening is currently being investigated in clinical trials. Here, we describe a portable clinical system with a therapeutic transducer suitable for humans, which eliminates the need for in-line magnetic resonance imaging (MRI) guidance. A neuronavigation-guided 0.25-MHz single-element FUS transducer was developed for non-invasive clinical BBB opening. Numerical simulations and experiments were performed to determine the characteristics of the FUS beam within a human skull. We also validated the feasibility of BBB opening obtained with this system in two non-human primates using U.S. Food and Drug Administration (FDA)-approved treatment parameters. Ultrasound propagation through a human skull fragment caused 44.4 § 1% pressure attenuation at a normal incidence angle, while the focal size decreased by 3.3 § 1.4% and 3.9 § 1.8% along the lateral and axial dimension, respectively. Measured lateral and axial shifts were 0.5 § 0.4 mm and 2.1 § 1.1 mm, while simulated shifts were 0.1 § 0.2 mm and 6.1 § 2.4 mm, respectively. A 1.5-MHz passive cavitation detector transcranially detected cavitation signals of Definity microbubbles flowing through a vessel-mimicking phantom. T 1 -weighted MRI confirmed a 153 § 5.5 mm 3 BBB opening in two non-human primates at a mechanical index of 0.4, using Definity microbubbles at the FDA-approved dose for imaging applications, without edema or hemorrhage. In conclusion, we developed a portable system for non-invasive BBB opening in humans, which can be achieved at clinically relevant ultrasound exposures without the need for in-line MRI guidance. The proposed FUS system may accelerate the adoption of non-invasive FUS-mediated therapies due to its fast application, low cost and portability. (

Focused ultrasound and microbubbles have been extensively used to generate therapeutic bioeffects. Despite encouraging in vivo results, there remains poor control of the magnitude and spatial distribution of these bioeffects due to the limited ability of conventional pulse shapes and sequences to control cavitation dynamics. Thus current cycles) emitted at a low burst repetition frequency (<10 Hz), we decomposed this burst into short pulses by adding intervals to facilitate inter-pulse microbubble movement. To evaluate how this sequence influenced cavitation distribution, we emitted short pulses

Despite the promise of microbubble-mediated focused ultrasound therapies, in vivo findings have revealed over-treated and under-treated regions distributed throughout the focal volume. This poor distribution cannot be improved by conventional pulse shapes and sequences, due to their limited ability to control acoustic cavitation dynamics within the ultrasonic focus. This paper describes the design of a rapid short-pulse (RaSP) sequence which is comprised of short pulses separated by μs off-time intervals. Improved acoustic cavitation distribution was based on the hypothesis that microbubbles can freely move during the pulse off-times. Flowing SonoVue® microbubbles (flow velocity: 10 mm/s) were sonicated with a 0.5 MHz focused ultrasound transducer using RaSP sequences (peak-rarefactional pressures: 146–900 kPa, pulse repetition frequency: 1.25 kHz, and pulse lengths: 5–50 cycles). The distribution of cavitation activity was evaluated using passive acoustic mapping. RaSP sequences generated uniform distributions within the focus in contrast to long pulses (50 000 cycles) that produced non-uniform distributions. Fast microbubble destruction occurred for long pulses, whereas microbubble activity was sustained for longer durations for shorter pulses. High-speed microscopy revealed increased mobility in the direction of flow during RaSP sonication. In conclusion, RaSP sequences produced spatiotemporally uniform cavitation distributions and could result in efficient therapies by spreading cavitation throughout the treatment area

Drug delivery in diffuse intrinsic pontine glioma is significantly limited by the blood-brain barrier (BBB). Focused ultrasound (FUS), when combined with the administration of microbubbles can effectively open the BBB permitting the entry of drugs across the cerebrovasculature into the brainstem. Given that the utility of FUS in brainstem malignancies remains unknown, the purpose of our study was to determine the safety and feasibility of this technique in a murine pontine glioma model. A syngeneic orthotopic model was developed by stereotactic injection of PDGF-B+PTEN−/−p53−/− murine glioma cells into the pons of B6 mice. A single-element, spherical-segment 1.5 MHz ultrasound transducer driven by a function generator through a power amplifier was used with concurrent intravenous microbubble injection for tumor sonication. Mice were randomly assigned to control, FUS and double-FUS groups. Pulse and respiratory rates were continuously monitored during treatment. BBB opening was confirmed with gadolinium-enhanced MRI and Evans blue. Kondziela inverted screen testing and sequential weight lifting measured motor function before and after sonication. A subset of animals were treated with etoposide following ultrasound. Mice were either sacrificed for tissue analysis or serially monitored for survival with daily weights. FUS successfully caused BBB opening while preserving normal cardiorespiratory and motor function. Furthermore, the degree of intra-tumoral hemorrhage and inflammation on H&E in control and treated mice was similar. There was also no difference in weight loss and survival between the groups (p > 0.05). Lastly, FUS increased intra-tumoral etoposide concentration by more than fivefold. FUS is a safe and feasible technique for repeated BBB opening and etoposide delivery in a preclinical pontine glioma model.

Ultrasound-driven microbubbles have been used in therapeutic applications to deliver drugs across capillaries and into cells or to dissolve blood clots. Yet the performance and safety of these applications have been difficult to control. Microbubbles exposed to ultrasound not only volumetrically oscillate, but also move due to acoustic radiation, or Bjerknes, forces. The purpose of this work was to understand the extent to which microbubbles moved and clustered due to secondary Bjerknes forces. A microbubble population was exposed to a 1-MHz ultrasound pulse with a peak-rarefactional pressure of 50-100 kPa and a pulse length of 20 ms. Microbubbles exposed to low-pressure therapeutic ultrasound were observed to cluster at clustering rates of 0.01-0.02 microbubbles per duration (in ms) per initial average inter-bubble distance (in μm), resulting in 1 to 3 clustered microbubbles per initial average inter-bubble distance (in μm). Higher pressures caused faster clustering rates and a larger number of clustered microbubbles. Experimental data revealed clustering time scales, cluster localizations, and cluster sizes that were in reasonable agreement with simulations using a time-averaged model at low pressures. This study demonstrates that clustering of microbubbles occurs within a few milliseconds and is likely to influence the distribution of stimuli produced in therapeutic applications.

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