Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

Focused ultrasound excites action potentials in mammalian peripheral neurons in part through the mechanically gated ion channel PIEZO2.

Neurons of the peripheral nervous system (PNS) are tasked with diverse roles, from encoding touch, pain, and itch to interoceptive control of inflammation and organ physiology. Thus, technologies that allow precise control of peripheral nerve activity have the potential to regulate a wide range of biological processes. Noninvasive modulation of neuronal activity is an important translational application of focused ultrasound (FUS). Recent studies have identified effective strategies to modulate brain circuits; however, reliable parameters to control the activity of the PNS are lacking. To develop robust noninvasive technologies for peripheral nerve modulation, we employed targeted FUS stimulation and electrophysiology in mouse ex vivo skin-saphenous nerve preparations to record the activity of individual mechanosensory neurons. Parameter space exploration showed that stimulating neuronal receptive fields with high-intensity, millisecond FUS pulses reliably and repeatedly evoked one-to-one action potentials in all peripheral neurons recorded. Interestingly, when neurons were classified based on neurophysiological properties, we identified a discrete range of FUS parameters capable of exciting all neuronal classes, including myelinated A fibers and unmyelinated C fibers. Peripheral neurons were excited by FUS stimulation targeted to either cutaneous receptive fields or peripheral nerves, a key finding that increases the therapeutic range of FUS-based peripheral neuromodulation. FUS elicited action potentials with millisecond latencies compared with electrical stimulation, suggesting ion channel­mediated mechanisms. Indeed, FUS thresholds were elevated in neurons lacking the mechanically gated channel PIEZO2. Together, these results demonstrate that transcutaneous FUS drives peripheral nerve activity by engaging intrinsic mechanotransduction mechanisms in neurons [B. U. Hoffman, PhD thesis, (2019)].

Focused ultrasound-mediated blood-brain barrier opening is safe and feasible with moderately hypofractionated radiotherapy for brainstem diffuse midline glioma.

BACKGROUND: Diffuse midline glioma (DMG) is a pediatric tumor with dismal prognosis. Systemic strategies have been unsuccessful and radiotherapy (RT) remains the standard-of-care. A central impediment to treatment is the blood-brain barrier (BBB), which precludes drug delivery to the central nervous system (CNS). Focused ultrasound (FUS) with microbubbles can transiently and non-invasively disrupt the BBB to enhance drug delivery. This study aimed to determine the feasibility of brainstem FUS in combination with clinical doses of RT. We hypothesized that FUS-mediated BBB-opening (BBBO) is safe and feasible with 39 Gy RT. METHODS: To establish a safety timeline, we administered FUS to the brainstem of non-tumor bearing mice concurrent with or adjuvant to RT; our findings were validated in a syngeneic brainstem murine model of DMG receiving repeated sonication concurrent with RT. The brainstems of male B6 (Cg)-Tyrc-2J/J albino mice were intracranially injected with mouse DMG cells (PDGFB+, H3.3K27M, p53-/-). A clinical RT dose of 39 Gy in 13 fractions (39 Gy/13fx) was delivered using the Small Animal Radiation Research Platform (SARRP) or XRAD-320 irradiator. FUS was administered via a 0.5 MHz transducer, with BBBO and tumor volume monitored by magnetic resonance imaging (MRI). RESULTS: FUS-mediated BBBO did not affect cardiorespiratory rate, motor function, or tissue integrity in non-tumor bearing mice receiving RT. Tumor-bearing mice tolerated repeated brainstem BBBO concurrent with RT. 39 Gy/13fx offered local control, though disease progression occurred 3-4 weeks post-RT. CONCLUSION: Repeated FUS-mediated BBBO is safe and feasible concurrent with RT. In our syngeneic DMG murine model, progression occurs, serving as an ideal model for future combination testing with RT and FUS-mediated drug delivery.

Combining brain perturbation and neuroimaging in non-human primates.

Brain perturbation studies allow detailed causal inferences of behavioral and neural processes. Because the combination of brain perturbation methods and neural measurement techniques is inherently challenging, research in humans has predominantly focused on non-invasive, indirect brain perturbations, or neurological lesion studies. Non-human primates have been indispensable as a neurobiological system that is highly similar to humans while simultaneously being more experimentally tractable, allowing visualization of the functional and structural impact of systematic brain perturbation. This review considers the state of the art in non-human primate brain perturbation with a focus on approaches that can be combined with neuroimaging. We consider both non-reversible (lesions) and reversible or temporary perturbations such as electrical, pharmacological, optical, optogenetic, chemogenetic, pathway-selective, and ultrasound based interference methods. Method-specific considerations from the research and development community are offered to facilitate research in this field and support further innovations. We conclude by identifying novel avenues for further research and innovation and by highlighting the clinical translational potential of the methods.

Modeling Pulse Wave Propagation Through a Stenotic Artery With Fluid Structure Interaction: A Validation Study Using Ultrasound Pulse Wave Imaging.

Pulse wave imaging (PWI) is an ultrasound-based method that allows spatiotemporal mapping of the arterial pulse wave propagation, from which the local pulse wave velocity (PWV) can be derived. Recent reports indicate that PWI can help the assessment of atherosclerotic plaque composition and mechanical properties. However, the effect of the atherosclerotic plaque's geometry and mechanics on the arterial wall distension and local PWV remains unclear. In this study, we investigated the accuracy of a finite element (FE) fluid-structure interaction (FSI) approach to predict the velocity of a pulse wave propagating through a stenotic artery with an asymmetrical plaque, as quantified with PWI method. Experiments were designed to compare FE-FSI modeling of the pulse wave propagation through a stenotic artery against PWI obtained with manufactured phantom arteries made of polyvinyl alcohol (PVA) material. FSI-generated spatiotemporal maps were used to estimate PWV at the plaque region and compared it to the experimental results. Velocity of the pulse wave propagation and magnitude of the wall distension were correctly predicted with the FE analysis. In addition, findings indicate that a plaque with a high degree of stenosis (>70%) attenuates the propagation of the pulse pressure wave. Results of this study support the validity of the FE-FSI methods to investigate the effect of arterial wall structural and mechanical properties on the pulse wave propagation. This modeling method can help to guide the optimization of PWI to characterize plaque properties and substantiate clinical findings.

Fast qualitative two-dimensional mapping of ultrasound fields with acoustic cavitation-enhanced ultrasound imaging.

Characterization of ultrasound fields is a routine procedure for both diagnostic and therapeutic ultrasound. Quantitative field mapping with a calibrated hydrophone and multi-axis positioning system can be difficult and time consuming. In this study, the use of acoustic cavitation field mapping as a qualitative surrogate to acoustic pressure field mapping, albeit without acoustic pressure values is demonstrated. This technique allows for fast qualitative mapping of ultrasound fields and thereby functionality of the corresponding transducers, in a matter of seconds. In addition, this technique could be used to rapidly image in vivo acoustic cavitation fields during therapeutic ultrasound applications.

Assessment of arterial stiffness in periodontitis using a novel pulse wave imaging methodology.

AIM: We investigated the cross-sectional relationship between periodontal status and arterial stiffness, assessed through a novel Pulse Wave Imaging methodology. METHODS: Eighty volunteers were enrolled (39% male, age range 24-78 years) and 33 pairs were formed of periodontitis patients/periodontally healthy controls, matched by age and gender. A full-mouth periodontal examination was performed and the degree of stiffness of the right and left carotid arteries was assessed by measuring pulse wave velocity (PWV) and the uniformity in pulse wave propagation (R2 ). Wilcoxon signed-rank tests for paired observations were used to compare periodontitis patients and healthy controls. Univariate and multivariate analyses were performed to analyze the association between PWV and R2 and potential explanatory variables. RESULTS: Patients with periodontitis had a statistically significantly lower uniformity in wave propagation (R2 ) than controls (p = .01), but PWV did not differ between the two groups. Univariate analysis showed a significant negative association between R2 and periodontitis, body mass index and smoking; periodontitis remained statistically associated with R2 in the multivariate analyses. CONCLUSIONS: Patients with periodontitis and no established cardiovascular disease presented with lower degree of uniformity in the transmission of the pulse wave through the carotid arteries, suggesting an association between periodontitis and arterial stiffness/functional alterations.

Assessing the Stability of Aortic Aneurysms with Pulse Wave Imaging.

Purpose To assess whether the stability of murine aortic aneurysms is associated with the homogeneity of pulse wave propagation within the saccular wall. Materials and Methods All animal procedures were approved by the institutional Animal Care and Use Committee. Apolipoprotein E and tissue inhibitor of metalloproteinases-1 knockout mice (n = 26) were infused with angiotensin II by using subcutaneously implanted osmotic pumps, with an additional control mouse used for histologic examination (n = 1). Pulse wave imaging (PWI) was performed just before infusion and 15 days after infusion by using 40-MHz ultrasonography at 8000 frames per second (with electrocardiographic gating). Aneurysm appearance on B-mode images was monitored every 2-3 days for 30 days. On the basis of B-mode images obtained after 30 days, aneurysms were deemed to have been unstable if they had ruptured; otherwise, they were deemed stable. Statistical significance was assessed by using two-tailed t tests. Results In normal aortas, the pulse waves propagated at relatively constant velocities (mean ± standard deviation, 2.8 m/sec ± 0.9). Fifteen days after infusion, all mice had developed aneurysms, with significant (P < .001/12) changes in maximum anterior-posterior diameter (increase of 54.9% ± 2.5) and pulse wave velocity (PWV) (decrease of 1.3 m/sec ± 0.8). While there was no significant difference in these parameters (P = .45 for diameter and P = .55 for PWV) between stable aneurysms (n = 12) and unstable aneurysms (n = 14), the standard deviation of the high-resolution PWV was significantly higher (P < .001/12) in unstable aneurysms (5.7 m/sec ± 1.6) than in stable ones (3.2 m/sec ± 0.9). Conclusion High-resolution PWI was used to measure the local homogeneity of pulse wave propagation within the saccular wall, which is lower in unstable aneurysms than in stable ones. Hence, if proven to add additional information beyond size and appearance in human studies, PWI could potentially be used to assess the stability of aneurysms by providing information that is complementary to the anatomic data obtained with conventional B-mode imaging. © RSNA, 2016 Online supplemental material is available for this article.

Imaging the electromechanical activity of the heart in vivo.

Cardiac conduction abnormalities remain a major cause of death and disability worldwide. However, as of today, there is no standard clinical imaging modality that can noninvasively provide maps of the electrical activation. In this paper, electromechanical wave imaging (EWI), a novel ultrasound-based imaging method, is shown to be capable of mapping the electromechanics of all four cardiac chambers at high temporal and spatial resolutions and a precision previously unobtainable in a full cardiac view in both animals and humans. The transient deformations resulting from the electrical activation of the myocardium were mapped in 2D and combined in 3D biplane ventricular views. EWI maps were acquired during five distinct conduction configurations and were found to be closely correlated to the electrical activation sequences. EWI in humans was shown to be feasible and capable of depicting the normal electromechanical activation sequence of both atria and ventricles. This validation of EWI as a direct, noninvasive, and highly translational approach underlines its potential to serve as a unique imaging tool for the early detection, diagnosis, and treatment monitoring of arrhythmias through ultrasound-based mapping of the transmural electromechanical activation sequence reliably at the point of care, and in real time.

Noninvasive and localized neuronal delivery using short ultrasonic pulses and microbubbles.

Focused ultrasound activation of systemically administered microbubbles is a noninvasive and localized drug delivery method that can increase vascular permeability to large molecular agents. Yet the range of acoustic parameters responsible for drug delivery remains unknown, and, thus, enhancing the delivery characteristics without compromising safety has proven to be difficult. We propose a new basis for ultrasonic pulse design in drug delivery through the blood-brain barrier (BBB) that uses principles of probability of occurrence and spatial distribution of cavitation in contrast to the conventionally applied magnitude of cavitation. The efficacy of using extremely short (2.3 µs) pulses was evaluated in 27 distinct acoustic parameter sets at low peak-rarefactional pressures (0.51 MPa or lower). The left hippocampus and lateral thalamus were noninvasively sonicated after administration of Definity microbubbles. Disruption of the BBB was confirmed by delivery of fluorescently tagged 3-, 10-, or 70-kDa dextrans. Under some conditions, dextrans were distributed homogeneously throughout the targeted region and accumulated at specific hippocampal landmarks and neuronal cells and axons. No histological damage was observed at the most effective parameter set. Our results have broadened the design space of parameters toward a wider safety window that may also increase vascular permeability. The study also uncovered a set of parameters that enhances the dose and distribution of molecular delivery, overcoming standard trade-offs in avoiding associated damage. Given the short pulses used similar to diagnostic ultrasound, new critical parameters were also elucidated to clearly separate therapeutic ultrasound from disruption-free diagnostic ultrasound.

Feasibility of Phase Velocity Imaging Using Multi Frequency Oscillation-Shear Wave Elastography.

OBJECTIVE: To assess viscoelasticity, a pathologically relevant biomarker, shear wave elastography (SWE) generally uses phase velocity (PV) dispersion relationship generated via pulsed acoustic radiation force (ARF) excitation pulse. In this study, a multi-frequency oscillation (MFO)- excitation pulse with higher weight to higher frequencies is proposed to generate PV images via the generation of motion with energy concentrated at the target frequencies in contrast to the broadband frequency motion generated in pulsed SWE (PSWE). METHODS: The feasibility of MFO-SWE to generate PV images at 100 to 1000 Hz in steps of 100 Hz was investigated by imaging 6 and 70 kPa inclusions with 6.5 and 10.4 mm diameter and ex vivo bovine liver with and without the presence of an aberration layer and chicken muscle ex vivo, and 4T1 mouse breast tumor, in vivo with comparisons to PSWE. RESULTS: MFO-SWE-derived CNR was statistically higher than PSWE for 6 kPa (both with and without aberration) and 70 kPa (with aberration) inclusions and derived SNR of the liver was statistically higher than PSWE at higher frequency (600-1000 Hz). Quantitatively, at 600-1000 Hz, MFO-SWE improved CNR of inclusions (without and with) aberration on an average by (8.2 and 156)% and of the tumor by 122%, respectively, and improved SNR of the liver (without and with) aberration by (20.2 and 51.5)% and of chicken muscle by 72%, respectively compared to the PSWE. CONCLUSIONS AND SIGNIFICANCE: These results indicate the advantages of MFO-SWE to improve PV estimation at higher frequencies which could improve viscoelasticity quantification and feature delineation.

Polyvinyl Alcohol Phantoms With Heterogeneous Plaques: Estimation of Pulse Wave Velocity at the Stenotic Region Using Pulse Wave Imaging.

OBJECTIVE: Plaque characterization is essential for stroke prevention. In the study reported herein, we describe a heterogeneous phantom manufacturing technique with varying plaque compositions of different stiffness using polyvinyl alcohol (PVA) to emulate stenotic arteries and evaluated the use of pulse wave imaging (PWI) to assess plaque stiffness by comparing derived pulse wave velocities, with the goal of assessing plaque vulnerability and identifying high-risk patients for stroke. METHODS: Five stenotic phantoms (50% stenosis) were fabricated by pouring PVA solutions into 3-D-printed molds. Two of the phantoms had heterogeneous plaque compositions of soft (E0 = 13 kPa) and intermediate (E0 = 40 kPa) materials and of stiff (E0 = 54 kPa) and intermediate materials. Ultrasound sequences were acquired as the arterial phantoms were connected to a pulsating pump, and PWI was performed on the ultrasound acquisition using normalized cross-correlation to track the pulse-induced phantom wall distension propagations. Pulse wave velocities were estimated by fitting a linear regression line between the arrival time of the peak acceleration of the wall distension waveform and the corresponding location. RESULTS: Arterial phantoms with heterogeneous plaque stiffness were successfully fabricated. Pulse wave velocities of 2.06, 2.21, 2.49, 2.67 and 3.31 m/s were found in the phantom experiments using PWI for homogeneous soft plaque, the heterogeneous soft and intermediate plaque, homogeneous intermediate plaque, the heterogeneous stiff and intermediate plaque and homogeneous stiff plaque, respectively. CONCLUSION: A novel arterial phantom building technique was reported with varying heterogenous plaque compositions of different stiffness. The feasibility of using PWI to evaluate plaque stiffness in stenotic arteries was determined and found that PWI can distinguish between plaques of distinct stiffness and composition.

An all-ultrasound cranial imaging method to establish the relationship between cranial FUS incidence angle and transcranial attenuation in non-human primates in 3D.

Focused ultrasound (FUS) is a non-invasive and non-ionizing technique which deploys ultrasound waves to induce bio-effects. When paired with acoustically active particles such as microbubbles (MBs), it can open the blood brain barrier (BBB) to facilitate drug delivery otherwise inhibited due to the presence of BBB. One of the parameters that affects the FUS beam propagation is the beam incidence angle on the skull. Prior work by our group has shown that, as incidence angles deviate from 90°, FUS focal pressures attenuate and result in a smaller BBB opening volume. The incidence angles calculated in our prior studies were in 2D and used skull information from CT. The study presented herein develops methods to calculate incidence angle in 3D in non-human primate (NHP) skull fragments using harmonic ultrasound imaging without using ionizing radiation. Our results show that ultrasound harmonic imaging is capable of accurately depicting features such as sutures and eye-sockets of the skull. Furthermore, we were able to reproduce previously reported relationships between the incidence angle and FUS beam attenuation. We also show feasibility of performing ultrasound harmonic imaging in in-vivo non-human primates. The all-ultrasound method presented herein combined with our neuronavigation system stands to increase more widespread adoption of FUS and render it accessible by eliminating the need for CT cranial mapping.

Optimization of the Tracking Beam Sequence in Harmonic Motion Imaging.

Harmonic motion imaging (HMI) is an ultrasound elastography technique that estimates the viscoelastic properties of tissues by inducing localized oscillatory motion using focused ultrasound (FUS). The resulting displacement, assumed to be inversely proportional to the tissue local stiffness, is estimated using an imaging array based on RF speckle tracking. In conventional HMI, this is accomplished with plane-wave (PW) imaging, which inherently suffers from low lateral resolution. Coherent PW compounding (PWC) leverages spatial and temporal resolution using synthetic focusing in transmit. In this study, we introduced focused imaging with parallel tracking in HMI and compared parallel tracking of various transmit F-numbers (F/2.6, 3, 4, and 5) qualitatively and quantitatively with PW and PWC imaging at various compounded angle ranges (6°, 12°, and 18°). An in silico model of a 56-kPa spherical inclusion (diameter: 3.6 mm) embedded in a 5.3-kPa background and a 5.3-kPa elastic phantom with cylindrical inclusions (Young's moduli: 22-56 kPa, diameters: 2.0-8.6 mm) were imaged to assess different tracking beam sequences. Speckle biasing in displacement estimation associated with parallel tracking was also investigated and concluded to be negligible in HMI. Parallel tracking in receive (Rx) resulted in 2%-7% and 8%-12% increase compared to PW imaging ( ) in HMI contrast and contrast-to-noise ratio in silico and phantoms. Focused imaging with parallel tracking in Rx was concluded to be most robust among PW and PWC imaging for displacement estimation, and its preclinical feasibility was demonstrated in postsurgical human cancerous breast tissue specimens and in vivo murine models of breast cancer.

Functional Cerebral Neurovascular Mapping During Focused Ultrasound Peripheral Neuromodulation of Neuropathic Pain.

BACKGROUND: Nociceptive pain is required for healthy function, yet, neuropathic pain (disease or injury) can be severely debilitating. Though a wide-array of treatment options are available, they are often systemic and/or invasive. As a promising neuromodulation treatment, Focused ultrasound (FUS) is a noninvasive and highly spatially-targeted technique shown to stimulate neural activity, yet, effects on pain signaling are currently unknown. OBJECTIVE: Develop and validate a method for studying FUS nerve stimulation modulation of pain-evoked neural responses in vivo. METHODS: We developed a high-resolution functional ultrasound (fUS) method capable of mapping cortical responses in healthy and neuropathic pain mice in response to FUS neuromodulation treatment. RESULTS: FUS-evoked hemodynamic responses are correlated with the intensity of peripheral neuromodulation. We confirm functional connectivity is altered in neuropathic mice and demonstrate that FUS can modulate neuropathic pain-evoked hemodynamics. CONCLUSIONS: The findings presented herein provides evidence for an FUS-based nerve pain method and validates the fUS technique developed for monitoring pain-evoked hemodynamics. SIGNIFICANCE: We anticipate that the findings presented herein describe a noninvasive and flexible nerve modulation technique for pain mitigation, furthering evidence for clinical translation.

Functional ultrasound (fUS) imaging of displacement-guided focused ultrasound (FUS) neuromodulation in mice.

Focused ultrasound (FUS) stimulation is a promising neuromodulation technique with the merits of non-invasiveness, high spatial resolution, and deep penetration depth. However, simultaneous imaging of FUS-induced brain tissue displacement and the subsequent effect of FUS stimulation on brain hemodynamics has proven challenging thus far. In addition, earlier studies lack in situ confirmation of targeting except for the magnetic resonance imaging-guided FUS system-based studies. The purpose of this study is 1) to introduce a fully ultrasonic approach to in situ target, modulate neuronal activity, and monitor the resultant neuromodulation effect by respectively leveraging displacement imaging, FUS, and functional ultrasound (fUS) imaging, and 2) to investigate FUS-evoked cerebral blood volume (CBV) response and the relationship between CBV and displacement. We performed displacement imaging on craniotomized mice to confirm the in targeting for neuromodulation site. We recorded hemodynamic responses evoked by FUS and fUS revealed an ipsilateral CBV increase that peaks at 4 s post-FUS. We saw a stronger hemodynamic activation in the subcortical region than cortical, showing good agreement with the brain elasticity map that can also be obtained using a similar methodology. We observed dose-dependent CBV response with peak CBV, activated area, and correlation coefficient increasing with ultrasonic dose. Furthermore, by mapping displacement and hemodynamic activation, we found that displacement colocalizes and linearly correlates with CBV increase. The findings presented herein demonstrated that FUS evokes ipsilateral hemodynamic activation in cortical and subcortical depths and the evoked hemodynamic responses colocalized and correlate with FUS-induced displacement. We anticipate that our findings will help consolidate accurate targeting as well as an understanding of how FUS displaces brain tissue and affects cerebral hemodynamics.

Modulation of cardio-respiratory activity in mice via transcranial focused ultrasound.

OBJECTIVE: The objective of this study was to investigate the effect of FUS on autonomic nervous system activity, including heart and respiratory rates, and to separate the thermal modulation from combined thermal and mechanical FUS effects. METHODS: The thalamus and hypothalamus of wild-type mice were sonicated with a continuous-wave, 2 MHz FUS transducer at pressures of 425 and 850 kPa for 60 seconds. Cardiac and respiratory rates were monitored as signs of autonomic nervous activity. FUS-induced changes in autonomic activity were compared to FUS targeted to a spatially-distant motor region and to laser-induced heating. RESULTS: FUS delivered to the primary target over the thalamus and hypothalamus at 850 kPa reversibly increased the respiratory rate by 6.5±3.2 breaths per minute and decreased the heart rate by 3.2±1.8 beats per minute. No significant changes occurred in this region at 425 kPa or when targeting the motor regions at 850 kPa. Laser heating with the same temperature rise profile produced by 850 kPa sonication resulted in cardiorespiratory modulation similar to that of FUS. CONCLUSIONS: FUS is capable of reversibly and non-invasively modulating cardiorespiratory activity in mice. Localized changes in temperature may constitute the main cause for this activity, though further investigation is warranted into the distinct and complementary mechanisms of mechanically- and thermally-induced FUS neuromodulation. Close monitoring of vital signs during FUS neuromodulation may be warranted to monitor systemic responses to stimulation.

Transcranial Functional Ultrasound Imaging Detects Focused Ultrasound Neuromodulation Induced Hemodynamic Changes in Mouse and Nonhuman Primate Brains In Vivo.

Focused ultrasound (FUS) is an emerging noinvasive technique for neuromodulation in the central nervous system (CNS). To evaluate the effects of FUS-induced neuromodulation, many studies used behavioral changes, functional magnetic resonance imaging (fMRI) or electroencephalography (EEG). However, behavioral readouts are often not easily mapped to specific brain activity, EEG has low spatial resolution limited to the surface of the brain and fMRI requires a large importable scanner that limits additional readouts and manipulations. In this context, functional ultrasound imaging (fUSI) holds promise to directly monitor the effects of FUS neuromodulation with high spatiotemporal resolution in a large field of view, with a comparatively simple and flexible setup. fUSI uses ultrafast Power Doppler Imaging (PDI) to measure changes in cerebral blood volume, which correlates well with neuronal activity and local field potentials. We designed a setup that aligns a FUS transducer with a linear array to allow immediate subsequent monitoring of the hemodynamic response with fUSI during and after FUS neuromodulation. We established a positive correlation between FUS pressure and the size of the activated area, as well as changes in cerebral blood volume (CBV) and found that unilateral sonications produce bilateral hemodynamic changes with ipsilateral accentuation in mice. We further demonstrated the ability to perform fully noninvasive, transcranial FUS-fUSI in nonhuman primates for the first time by using a lower-frequency transducer configuration.

Transcranial Blood-Brain Barrier Opening in Alzheimer's Disease Patients Using a Portable Focused Ultrasound System with Real-Time 2-D Cavitation Mapping.

Background: Focused ultrasound (FUS) in combination with microbubbles has recently shown great promise in facilitating blood-brain barrier (BBB) opening for drug delivery and immunotherapy in Alzheimer's disease (AD). However, it is currently limited to systems integrated within the MRI suites or requiring post-surgical implants, thus restricting its widespread clinical adoption. In this pilot study, we investigate the clinical safety and feasibility of a portable, non-invasive neuronavigation-guided FUS (NgFUS) system with integrated real-time 2-D microbubble cavitation mapping. Methods: A phase 1 clinical study with mild to moderate AD patients (N=6) underwent a single session of microbubble-mediated NgFUS to induce transient BBB opening (BBBO). Microbubble activity under FUS was monitored with real-time 2-D cavitation maps and dosing to ensure the efficacy and safety of the NgFUS treatment. Post-operative MRI was used for BBB opening and closure confirmation as well as safety assessment. Changes in AD biomarker levels in both blood serum and extracellular vesicles (EVs) were evaluated, while changes in amyloid-beta (Aß) load in the brain were assessed through 18F-Florbetapir PET. Results: BBBO was achieved in 5 out of 6 subjects with an average volume of 983±626 mm3 following FUS at the right frontal lobe both in white and gray matter regions. The outpatient treatment was completed within 34.8±10.7 min. Cavitation dose significantly correlated with the BBBO volume (R2>0.9, N=4), demonstrating the portable NgFUS system's capability of predicting opening volumes. The cavitation maps co-localized closely with the BBBO location, representing the first report of real-time transcranial 2-D cavitation mapping in the human brain. Larger opening volumes correlated with increased levels of AD biomarkers, including Aß42 (R2=0.74), Tau (R2=0.95), and P-Tau181 (R2=0.86), assayed in serum-derived EVs sampled 3 days after FUS (N=5). From PET scans, subjects showed a lower Aß load increase in the treated frontal lobe region compared to the contralateral region. Reduction in asymmetry standardized uptake value ratios (SUVR) correlated with the cavitation dose (R2>0.9, N=3). Clinical changes in the mini-mental state examination over 6 months were within the expected range of cognitive decline with no additional changes observed as a result of FUS. Conclusion: We showed the safety and feasibility of this cost-effective and time-efficient portable NgFUS treatment for BBBO in AD patients with the first demonstration of real-time 2-D cavitation mapping. The cavitation dose correlated with BBBO volume, a slowed increase in pathology, and serum detection of AD proteins. Our study highlights the potential for accessible FUS treatment in AD, with or without drug delivery.