Focused Ultrasound Sonication Research Articles

Effect of Microbubble Size, Composition, and Multiple Sonication Points on Sterile Inflammatory Response in Focused Ultrasound-Mediated Blood-Brain Barrier Opening.

Blood-brain barrier opening (BBBO) using focused ultrasound (FUS) and microbubbles (MBs) has emerged as a promising technique for delivering therapeutics to the brain. However, the influence of various FUS and MB parameters on BBBO and subsequent sterile inflammatory response (SIR) remains unclear. In this study, we investigated the effects of MB size and composition, as well as the number of FUS sonication points, on BBBO and SIR in an immunocompetent mouse model. Using MRI-guided MB + FUS, we targeted the striatum and assessed extravasation of an MRI contrast agent to assess BBBO and RNaseq to assess SIR. Our results revealed distinct effects of these parameters on BBBO and SIR. Specifically, at a matched microbubble volume dose (MVD), MB size did not affect the extent of BBBO, but smaller (1 μm diameter) MBs exhibited a lower classification of SIR than larger (3 or 5 μm diameter) MBs. Lipid-shelled microbubbles exhibited greater BBBO and a more pronounced SIR compared to albumin-shelled microbubbles, likely owing to the latter's poor in vivo stability. As expected, increasing the number of sonication points resulted in greater BBBO and SIR. Furthermore, correlation analysis revealed strong associations between passive cavitation detection measurements of harmonic and inertial MB echoes, BBBO, and the expression of SIR gene sets. Our findings highlight the critical role of MB and FUS parameters in modulating BBBO and subsequent SIR in the brain. These insights inform the development of targeted drug delivery strategies and the mitigation of adverse inflammatory reactions in neurological disorders.

The impact of pulse repetition frequency on microbubble activity and drug delivery during focused ultrasound-mediated blood-brain barrier opening

Objective. Pulsed focused ultrasound (FUS) can deliver therapeutics to the brain by using intravenous microbubbles (MBs) to open the blood-brain barrier (BBB). MB emissions indicate treatment outcomes, like BBB opening (harmonics) and damage (broadband). Typically, a pulse repetition frequency (PRF) of 1 Hz is used, but the effect of PRF on MBs is not fully understood. We investigated the effect of PRF on MB activity and tracer delivery. Approach. The effect of PRF (0.125, 0.25, 0.5, 1, and 2 Hz) on MB activity was monitored through harmonic and wideband emissions during FUS sonications of the rat brain at 274.3 kHz. BBB opening was quantified through fluorescence imaging to estimate the concentration of Trypan Blue (TB) dye following a 75-pulse FUS exposure for PRFs of 1 and 0.25 Hz. Main results. At a fixed acoustic pressure, the percentage change in maximum harmonic amplitude compared to the control (PRF = 1 Hz) decreased with increasing PRF, with a median change of 73.8% at 0.125 Hz and −38.3% at 2 Hz. There was no difference in the pressure threshold for broadband emissions between PRFs of 0.25 and 1 Hz. PRF = 0.25 Hz, led to a 68.2% increase in the mean concentration of TB measured after FUS, with a 53.9% increase in the mean harmonic sum, compared with PRF = 1 Hz. Harmonic emissions-based control at PRF = 0.25 Hz yielded similar TB delivery, with less damage at histology, compared with 1 Hz. Significance. For a fixed number of FUS pulses, reducing the PRF was shown to increase the magnitude of harmonic emissions and TB delivery, but not the threshold for broadband emissions. While further research is necessary to understand the mechanisms involved, these results may be useful to improve clinical safety margins and sensitivity to detecting small harmonic signals from cavitating MBs.

Feasibility of measuring blood-brain barrier permeability using ultra-short echo time radial magnetic resonance imaging.

The purpose of this study is to evaluate the feasibility of using 3-dimensional (3D) ultra-short echo time (UTE) radial imaging method for measurement of the permeability of the blood-brain barrier (BBB) to gadolinium-based contrast agent. In this study, we propose to use the golden-angle radial sparse parallel (GRASP) method with 3D center-out trajectories for UTE, hence named as 3D UTE-GRASP. We first examined the feasibility of using 3D UTE-GRASP dynamic contrast-enhanced (DCE)-magnetic resonance imaging (MRI) for differentiating subtle BBB disruptions induced by focused ultrasound (FUS). Then, we examined the BBB permeability changes in Alzheimer's disease (AD) pathology using Alzheimer's disease transgenic mice (5xFAD) at different ages. For FUS experiments, we used four Sprague Dawley rats at similar ages where we compared BBB permeability of each rat receiving the FUS sonication with different acoustic power (0.4-1.0MPa). For AD transgenic mice experiments, we included three 5xFAD mice (6, 12, and 16 months old) and three wild-type mice (4, 8, and 12 months old). The result from FUS experiments showed a progressive increase in BBB permeability with increase of acoustic power (p <.05), demonstrating the sensitivity of DCE-MRI method for detecting subtle changes in BBB disruption. Our AD transgenic mice experiments suggest an early BBB disruption in 5xFAD mice, which is further impaired with aging. The results in this study substantiate the feasibility of using the proposed 3D UTE-GRASP method for detecting subtle BBB permeability changes expected in neurodegenerative diseases, such as AD.

An MR-compatible fiber-optic probe for measuring focused ultrasound-induced temperature rises without viscous heating artifacts

Focused ultrasound (FUS) beam interactions with thermocouples and fiber optic (FO) probes cause a “viscous heating artifact” (VHA), which prevent accurate temperature measurements during acoustic sonication. This work demonstrates a novel fiber-optic temperature probe which is insensitive to VHA, validated in an MR-guided FUS treatment setting. The FO probe (OSENSA Innovations; PRB-140, 0.14 mm diameter) was inserted with an 18G catheter into a tissue-mimicking gelatin phantom. The FO probe tip was located with high-resolution MR images (0.25 × 0.25 × 0.5 mm3) for targeting with FUS. Continuous-wave FUS sonications (50 W, 20 s) were delivered at a distance of ∼1.5 mm from the FO probe tip using a 256 phased-array transducer (Imasonic, France; 1 MHz, 2.1 × 2.3 × 9.8 FWHM spot size) and FUS-induced heating was measured with 3D MR temperature imaging (MRTI; 0.5 × 0.5 × 1 mm3 resolution, 3.9 s acquisition). The VHA effect was not observed in the PRB-140 FO probe measurement data. The FO probe heating and cooling curves closely matched those measured by MRTI, with a root mean-squared error (RMSE) of 0.61 °C. In contrast, the RMSE of VHA-sensitive FO probe was 6.00 °C for a similar acoustic power output. This ultrasound artifact-immune FO temperature probe is highly advantageous for MR temperature sequence development and precise temperature monitoring in FUS treatment applications.

Magnetic Resonance Imaging Monitoring of Thermal Lesions Produced by Focused Ultrasound

Abstract Background: The main goal of the study was to find the magnetic resonance imaging (MRI) parameters that optimize contrast between tissue and thermal lesions produced by focused ultrasound (FUS) using T1-weighted (T1-W) and T2-weighted (T2-W) fast spin echo (FSE) sequences. Methods: FUS sonications were performed in ex vivo porcine tissue using a single-element FUS transducer of 2.6 MHz in 1.5 and 3 T MRI scanners. The difference in relaxation times as well as the impact of critical MRI parameters on the resultant contrast-to-noise ratio (CNR) between coagulated and normal tissues were assessed. Discrete and overlapping lesions were inflicted in tissue with simultaneous acquisition of T2-W FSE images. Results: FUS lesions are characterized by lower relaxation times than intact porcine tissue. CNR values above 80 were sufficient for proper lesion visualization. For T1-W imaging, repetition time values close to 1500 ms were considered optimum for obtaining sufficiently high CNR at the minimum time cost. Echo time values close to 50 ms offered the maximum lesion contrast in T2-W FSE imaging. Monitoring of acute FUS lesions during grid sonications was performed successfully. Lesions appeared as hypointense spots with excellent contrast from surrounding tissue. Conclusion: MRI monitoring of signal intensity changes during FUS sonication in grid patterns using optimized sequence parameters can provide useful information about lesion progression and the success of ablation. This preliminary study demonstrated the feasibility of the proposed monitoring method in ex vivo porcine tissue and should be supported by in vivo studies to assess its clinical potential.

Evaluation of acoustic-thermal simulations of invivo magnetic resonance guided focused ultrasound ablative therapy.

To evaluate numerical simulations of focused ultrasound (FUS) with a rabbit model, comparing simulated heating characteristics with magnetic resonance temperature imaging (MRTI) data collected during invivo treatment. A rabbit model was treated with FUS sonications in the biceps femoris with 3D MRTI collected. Acoustic and thermal properties of the rabbit muscle were determined experimentally. Numerical models of the rabbits were created, and tissue-type-specific properties were assigned. FUS simulations were performed using both the hybrid angular spectrum (HAS) method and k-Wave. Simulated power deposition patterns were converted to temperature maps using a Pennes' bioheat equation-based thermal solver. Agreement of pressure between the simulation techniques and temperature between the simulation and experimental heating was evaluated. Contributions of scattering and absorption attenuation were considered. Simulated peak pressures derived using the HAS method exceeded the simulated peak pressures from k-Wave by 1.6 ± 2.7%. The location and FWHM of the peak pressure calculated from HAS and k-Wave showed good agreement. When muscle acoustic absorption value in the simulations was adjusted to approximately 54% of the measured attenuation, the average root-mean-squared error between simulated and experimental spatial-average temperature profiles was 0.046 ± 0.019 °C/W. Mean distance between simulated and experimental COTMs was 3.25 ± 1.37 mm. Transverse FWHMs of simulated sonications were smaller than in invivo sonications. Longitudinal FWHMs were similar. Presented results demonstrate agreement between HAS and k-Wave simulations and that FUS simulations can accurately predict focal position and heating for invivo applications in soft tissue.

BIOM-05. FIRST-IN-HUMAN PROSPECTIVE TRIAL OF SONOBIOPSY IN GLIOBLASTOMA PATIENTS USING NEURONAVIGATION-GUIDED FOCUSED ULTRASOUND

Abstract INTRODUCTION Blood-based liquid biopsy for brain cancer offers a noninvasive alternative to invasive surgical biopsies. However, the presence of the blood-brain barrier (BBB) limits the release of brain tumor-derived molecular biomarkers into the bloodstream for sensitive diagnosis. Focused ultrasound (FUS)-enabled blood-based liquid biopsy (sonobiopsy) locally disrupts the BBB and releases tumor-derived biomarkers into blood circulation. Our previous studies have demonstrated that sonobiopsy improved the detection sensitivity of glioblastoma (GBM)-specific cirulating tumor DNA (ctDNA) in animal models of GBM. This study aimed to evaluate the feasibility and safety of sonobiopsy in enriching circulating tumor biomarkers in a first-in-human, prospective clinical trial. METHODS Five GBM patients (G01-05) were enrolled in the initial study. The sonobiopy procedures were performed prior to tumor resection, using a nimble FUS device integrated with a clinical neuronavigation system. The plasma level of glial fibrillary acidic protein (GFAP) was quantified using ultrasensitive single-molecule array (Simoa), while the cell-free DNA (cfDNA) levels were quantified using Bioanalyzer. Patient-specific tumor variants were comprehensively analyzed using a personalized tumor-informed ctDNA assay (Invitae Personalized Cancer Monitoring assay). Additionally, the two most common GBM-specific tumor variants were analyzed using customized ddPCR assays. RESULTS Analysis of plasmas collected before and after FUS sonication showed sonobiopsy enhanced plasma circulating tumor biomarker levels, including a maximum increase of 1.3-fold for GFAP, 1.6-fold for the concentration of mono-nucleosomal cfDNA fragment (120–280 bp), 1.9-fold increase in patient-specific tumor variant levels, and 5.6-fold increase in the TERT mutation level. Histological analysis of surgically resected tumors confirmed the safety of the procedure. Transcriptome analysis of sonicated and nonsonicated tumor tissues found that FUS sonication mainly modulated cell physical structure-related genes. CONCLUSIONS These feasibility and safety data support the continued investigation of sonobiopsy for noninvasive molecular diagnosis of brain diseases.

First-in-human prospective trial of sonobiopsy in high-grade glioma patients using neuronavigation-guided focused ultrasound

Sonobiopsy is an emerging technology that combines focused ultrasound (FUS) with microbubbles to enrich circulating brain disease-specific biomarkers for noninvasive molecular diagnosis of brain diseases. Here, we report the first-in-human prospective trial of sonobiopsy in high-grade glioma patients to evaluate its feasibility and safety in enriching plasma circulating tumor biomarkers. A nimble FUS device integrated with a clinical neuronavigation system was used to perform sonobiopsy following an established clinical workflow for neuronavigation. Analysis of blood samples collected before and after FUS sonication showed that sonobiopsy enriched plasma circulating tumor DNA (ctDNA), including a maximum increase of 1.6-fold for the mononucleosome cell-free DNA (cfDNA) fragments (120–280 bp), 1.9-fold for the patient-specific tumor variant ctDNA level, and 5.6-fold for the TERT mutation ctDNA level. Histological analysis of surgically resected tumors confirmed the safety of the procedure. Transcriptome analysis of sonicated and nonsonicated tumor tissues found that FUS sonication modulated cell physical structure-related genes. Only 2 out of 17,982 total detected genes related to the immune pathways were upregulated. These feasibility and safety data support the continued investigation of sonobiopsy for noninvasive molecular diagnosis of brain diseases.

Focused ultrasound-mediated blood-brain barrier opening in Alzheimer's disease: long-term safety, imaging, and cognitive outcomes.

MRI-guided low-intensity focused ultrasound (FUS) has been shown to reversibly open the blood-brain barrier (BBB), with the potential to deliver therapeutic agents noninvasively to target brain regions in patients with Alzheimer's disease (AD) and other neurodegenerative conditions. Previously, the authors reported the short-term safety and feasibility of FUS BBB opening of the hippocampus and entorhinal cortex (EC) in patients with AD. Given the need to treat larger brain regions beyond the hippocampus and EC, brain volumes and locations treated with FUS have now expanded. To evaluate any potential adverse consequences of BBB opening on disease progression, the authors report safety, imaging, and clinical outcomes among participants with mild AD at 6-12 months after FUS treatment targeted to the hippocampus, frontal lobe, and parietal lobe. In this open-label trial, participants with mild AD underwent MRI-guided FUS sonication to open the BBB in β-amyloid positive regions of the hippocampus, EC, frontal lobe, and parietal lobe. Participants underwent 3 separate FUS treatment sessions performed 2 weeks apart. Outcome assessments included safety, imaging, neurological, cognitive, and florbetaben β-amyloid PET. Ten participants (range 55-76 years old) completed 30 separate FUS treatments at 2 participating institutions, with 6-12 months of follow-up. All participants had immediate BBB opening after FUS and BBB closure within 24-48 hours. All FUS treatments were well tolerated, with no serious adverse events related to the procedure. All 10 participants had a minimum of 6 months of follow-up, and 7 participants had a follow-up out to 1 year. Changes in the Alzheimer's Disease Assessment Scale-cognitive and Mini-Mental State Examination scores were comparable to those in controls from the Alzheimer's Disease Neuroimaging Initiative. PET scans demonstrated an average β-amyloid plaque of 14% in the Centiloid scale in the FUS-treated regions. This study is the largest cohort of participants with mild AD who received FUS treatment, and has the longest follow-up to date. Safety was demonstrated in conjunction with reversible and repeated BBB opening in multiple cortical and deep brain locations, with a concomitant reduction of β-amyloid. There was no apparent cognitive worsening beyond expectations up to 1 year after FUS treatment, suggesting that the BBB opening treatment in multiple brain regions did not adversely influence AD progression. Further studies are needed to determine the clinical significance of these findings. FUS offers a unique opportunity to decrease amyloid plaque burden as well as the potential to deliver targeted therapeutics to multiple brain regions in patients with neurodegenerative disorders.

Mechanically manipulating glymphatic transport by ultrasound combined with microbubbles

The glymphatic system is a perivascular fluid transport system for waste clearance. Glymphatic transport is believed to be driven by the perivascular pumping effect created by the pulsation of the arterial wall caused by the cardiac cycle. Ultrasound sonication of circulating microbubbles (MBs) in the cerebral vasculature induces volumetric expansion and contraction of MBs that push and pull on the vessel wall to generate a MB pumping effect. The objective of this study was to evaluate whether glymphatic transport can be mechanically manipulated by focused ultrasound (FUS) sonication of MBs. The glymphatic pathway in intact mouse brains was studied using intranasal administration of fluorescently labeled albumin as fluid tracers, followed by FUS sonication at a deep brain target (thalamus) in the presence of intravenously injected MBs. Intracisternal magna injection, the conventional technique used in studying glymphatic transport, was employed to provide a comparative reference. Three-dimensional confocal microscopy imaging of optically cleared brain tissue revealed that FUS sonication enhanced the transport of fluorescently labeled albumin tracer in the perivascular space (PVS) along microvessels, primarily the arterioles. We also obtained evidence of FUS-enhanced penetration of the albumin tracer from the PVS into the interstitial space. This study revealed that ultrasound combined with circulating MBs could mechanically enhance glymphatic transport in the brain.

A numerical investigation of passive acoustic mapping for monitoring bubble-mediated focused ultrasound treatment of the spinal cord.

Focused ultrasound (FUS) combined with intravenous microbubbles (MBs) has been shown to increase drug delivery to the spinal cord in animal models. Eventual clinical translation of such a technique in the sensitive spinal cord requires robust treatment monitoring to ensure efficacy, localization, safety, and provide key intraprocedural feedback. Here, the use of passive acoustic mapping (PAM) of MB emissions with a spine-specific detector array in the context of transvertebral FUS sonications is investigated in silico. Using computed tomography-derived human vertebral geometry, transvertebral detection of MBs is evaluated over varying source locations with and without phase and amplitude corrections (PACs). The impact of prefocal cavitation is studied by simulating concurrent cavitation events in the canal and pre-laminar region. Spatially sensitive application of phase and amplitude is used to balance signal strengths emanating from different axial depths in combination with multiple dynamic ranges to elicit multisource viewing. Collectively, the results of this study encourage the use of PAM in transvertebral FUS applications with PACs to not only localize sources originating in the spinal canal but also multiple sources of innate amplitude mismatches when corrective methods are applied.

Treatment of mammary cancer with focused ultrasound: A pilot study in canine and feline patients

In recent years, veterinary medicine has expanded its practices beyond conventional methods, gradually integrating the Focused Ultrasound (FUS) technology in the care of companion animals like dogs and cats. The current study aimed to examine the feasibility and provide insights into the application of thermal FUS in canine and feline mammary cancer therapy. FUS was delivered by a 2-MHz single-element spherically focused ultrasonic transducer as integrated with an existing robotic positioning device. The functionality of the FUS system and sonication protocol in efficiently and safely ablating live tissue was initially validated in a rabbit thigh model in a laboratory environment. Nine (9) dogs and cats with superficial mammary cancer were recruited through a dedicated campaign according to specific safety criteria. The veterinary patients underwent FUS ablation followed by immediate surgical resection of the entire malignancy. Histopathology examination demonstrated well-defined regions of coagulative necrosis in all treated tumors with no off-target damage. Further study with a larger patient population is needed to confirm the current findings and demonstrate the safety and feasibility of complete FUS ablation of deep-seated tumors.

Feasibility of MRI-guided focused ultrasound-mediated intranasal delivery in a large animal model

Intranasal delivery provides non-invasive delivery of drugs from the nose to the brain bypassing the blood-brain barrier (BBB). We previously reported the focused ultrasound (FUS)-mediated intranasal delivery (FUSIN) of various agents in mice using FUS. However, no study has investigated the feasibility of FUSIN in large animals. The objective of this study was to investigate the feasibility of MR-guided FUS-mediated intranasal delivery in a large animal model. Pigs were used as the large animal model due to their clinical relevance to humans. Fluorescence-labeled albumin was mixed with an MRI contrast agent and administrated intranasally to the pig using a catheter. Followed by intravenous injection of microbubbles, FUS sonication was performed at two brain regions, the cortex and brainstem. T1-weighted MR images showed enhancement of MRI contrast agent at the olfactory region of the pig nose. Fluorescence imaging displayed enhanced fluorescence intensity at the FUS-targeted brain regions. These findings suggest that MR-guided FUS can achieve noninvasive and localized delivery of intranasally administered agents to the brain in large animals.

Pulsed Focused Ultrasound Reduces Hippocampal Volume Loss and Improves Behavioral Performance in the Kainic Acid Rat Model of Epilepsy

Focused ultrasound (FUS) has the potential to modulate regional brain excitability and possibly aid seizure control; however, effects on behavior of FUS used as a seizure therapy are unknown. This study explores behavioral effects and hippocampal restoration induced by pulsed FUS in a kainic acid (KA) animal model of temporal lobe epilepsy. Twenty-nine male Sprague–Dawley rats were observed for 20 weeks with anatomical magnetic resonance imaging (MRI) and behavioral performance evaluations, comprising measures of anxiety, limb usage, sociability, and memory. FUS targeted to the right hippocampus was given 9 and 14 weeks after KA was delivered to the right amygdala. Ultrasound pulsations were delivered with the acoustic settings of 0.25 of mechanical index, 0.5 W/cm2 of intensity spatial peak temporal average (ISPTA), 100 Hz of pulse repetition frequency, and 30% of duty cycle, during three consecutive pulse trains of 10 min separated by 5-min rests. Controls included normal animals with sham injections and KA-exposed animals without FUS exposure. Longitudinal MRI observations showed that FUS substantially protected hippocampal and striatal structures from KA-induced atrophy. KA alone increased anxiety, impaired contralateral limb usage, and reduced sociability and learning. Two courses of FUS sonications partially ameliorated these impairments by enhancing exploring and learning, balancing limb usage, and increasing social interaction. The histology results indicated that two sonications enhanced neuroprotection effect and decreased the inflammation markers induced by KA. This study supports existence of both neuroprotective and beneficial behavioral effects from low-intensity pulsed ultrasound in the KA animal model of epilepsy.

TRPV1-mediated sonogenetic neuromodulation of motor cortex in freely moving mice

Background. Noninvasive and cell-type-specific neuromodulation tools are critically needed for probing intact brain function. Sonogenetics for noninvasive activation of neurons engineered to express thermosensitive transient receptor potential vanilloid 1 (TRPV1) by transcranial focused ultrasound (FUS) was recently developed to address this need. However, using TRPV1-mediated sonogenetics to evoke behavior by targeting the cortex is challenged by its proximity to the skull due to high skull absorption of ultrasound and increased risks of thermal-induced tissue damage. Objective. This study evaluated the feasibility and safety of TRPV1-mediated sonogenetics in targeting the motor cortex to modulate the locomotor behavior of freely moving mice. Approach. Adeno-associated viral vectors was delivered to the mouse motor cortex via intracranial injection to express TRPV1 in excitatory neurons. A wearable FUS device was installed on the mouse head after a month to control neuronal activity by activating virally expressed TRPV1 through FUS sonication at different acoustic pressures. Immunohistochemistry staining of ex vivo brain slices was performed to verify neuron activation and evaluate safety. Results. TRPV1-mediated sonogenetic stimulation at 0.7 MPa successfully evoked rotational behavior in the direction contralateral to the stimulation site, activated cortical neurons as indicated by the upregulation of c-Fos, and did not induce significant changes in inflammatory or apoptotic markers (GFAP, Iba1, and Caspase-3). Sonogenetic stimulation of TRPV1 mice at a higher acoustic pressure, 1.1 MPa, induced significant changes in motor behavior and upregulation of c-Fos compared with FUS sonication of naïve mice at 1.1 MPa. However, signs of damage at the meninges were observed at 1.1 MPa. Significance. TRPV1-mediated sonogenetics can achieve effective and safe neuromodulation at the cortex with carefully selected FUS parameters. These findings expand the application of this technique to include superficial brain targets.

Utilization of focused ultrasound for opening of the blood-nerve barrier

Objective. Focused ultrasound (FUS) use with and without microbubbles (MB) for investigation of the blood-nerve barrier (BNB) within the peripheral nervous system (PNS) has been performed in this study. We evaluate the feasibility of BNB opening in a rodent sciatic nerve model by direct vision FUS treatment and provide preliminary results of magnetic resonance guided FUS (MRgFUS). Approach. Twenty rodent bilateral sciatic nerves were investigated. Rodents were treated using a benchtop FUS system to directly visualize nerve FUS studies. Definity MB, Evans blue dye (EB) and latex micro beads were injected during studies. Selected animals underwent further compound muscle action potential (CMAP) studies. Sonication peak pressure (MPa), width, duty-cycle and duration as well as MB concentration were varied to investigate effective pressure threshold. Further preliminary MRgFUS studies were performed on selected animals. Immunohistochemistry and histological analysis under florescent microscopy were performed at termination of experiments to verify treatment outcomes. Main results. Three ultrasound pressures and three microbubble concentrations at a single sonication frequency (476.5 kHz) were performed under direct open targeting. Histological analysis demonstrated nerve internal architecture disruption at 1.2 MPa with 166.7 μl kg−1 while 0.3 MPa, with 40 μl kg−1 MB concentration was the lower threshold for consistently observed disruption of the BNB without anatomical microarchitecture disruption. EB leakage was confirmed at the target region in histological evaluation of nerve following MB injection and FUS sonication. Supra-harmonic emissions were detected during FUS exposures following MB injection but not at baseline reference, indicating effective MB response and stable cavitation. CMAP amplitudes showed delayed onset latency and lower amplitudes in sonicated nerves compared to control nerves without evidence of complete conduction block, suggesting a transient BNB disruption, while at lower limit pressure subtle conduction changes were observed. In MRgFUS, targeted nerves demonstrated further contrast agent leak as well as supra-harmonic frequency detection. Significance. Opening of the BNB in the PNS was achieved using FUS and MB in a rodent model. Ongoing work aims to refine FUS parameters for drug delivery into the nerve after experimental transient BNB disruption.

Blood–brain barrier opening in a large animal model using closed-loop microbubble cavitation-based feedback control of focused ultrasound sonication

Focused ultrasound (FUS) in combination with microbubbles has been established as a promising technique for noninvasive and localized Blood–brain barrier (BBB) opening. Real-time passive cavitation detection (PCD)-based feedback control of the FUS sonication is critical to ensure effective BBB opening without causing hemorrhage. This study evaluated the performance of a closed-loop feedback controller in a porcine model. Calibration of the baseline cavitation level was performed for each targeted brain location by a FUS sonication in the presence of intravenously injected microbubbles at a low acoustic pressure without inducing BBB opening. The target cavitation level (TCL) was defined for each target based on the baseline cavitation level. FUS treatment was then performed under real-time PCD-based feedback controller to maintain the cavitation level at the TCL. After FUS treatment, contrast-enhanced MRI and ex vivo histological staining were performed to evaluate the BBB permeability and safety. Safe and effective BBB opening was achieved with the BBB opening volume increased from 3.8 ± 0.7 to 53.6 ± 23.3 mm3 as the TCL was increased from 0.25 to 1 dB. This study validated that effective and safe FUS-induced BBB opening in a large animal model can be achieved with closed-loop feedback control of the FUS sonication.

Sonobiopsy for minimally invasive, spatiotemporally-controlled, and sensitive detection of glioblastoma-derived circulating tumor DNA.

Though surgical biopsies provide direct access to tissue for genomic characterization of brain cancer, they are invasive and pose significant clinical risks. Brain cancer management via blood-based liquid biopsies is a minimally invasive alternative; however, the blood-brain barrier (BBB) restricts the release of brain tumor-derived molecular biomarkers necessary for sensitive diagnosis.Methods: A mouse glioblastoma multiforme (GBM) model was used to demonstrate the capability of focused ultrasound (FUS)-enabled liquid biopsy (sonobiopsy) to improve the diagnostic sensitivity of brain tumor-specific genetic mutations compared with conventional blood-based liquid biopsy. Furthermore, a pig GBM model was developed to characterize the translational implications of sonobiopsy in humans. Magnetic resonance imaging (MRI)-guided FUS sonication was performed in mice and pigs to locally enhance the BBB permeability of the GBM tumor. Contrast-enhanced T1-weighted MR images were acquired to evaluate the BBB permeability change. Blood was collected immediately after FUS sonication. Droplet digital PCR was used to quantify the levels of brain tumor-specific genetic mutations in the circulating tumor DNA (ctDNA). Histological staining was performed to evaluate the potential for off-target tissue damage by sonobiopsy.Results: Sonobiopsy improved the detection sensitivity of EGFRvIII from 7.14% to 64.71% and TERT C228T from 14.29% to 45.83% in the mouse GBM model. It also improved the diagnostic sensitivity of EGFRvIII from 28.57% to 100% and TERT C228T from 42.86% to 71.43% in the porcine GBM model.Conclusion: Sonobiopsy disrupts the BBB at the spatially-targeted brain location, releases tumor-derived DNA into the blood circulation, and enables timely collection of ctDNA. Converging evidence from both mouse and pig GBM models strongly supports the clinical translation of sonobiopsy for the minimally invasive, spatiotemporally-controlled, and sensitive molecular characterization of brain cancer.

Focus Ultrasound‐Induced Blood‐Brain Barrier opening enhances anti‐pGlu3 Aβ mAb delivery and amyloid‐beta plaque clearance

AbstractBackgroundAlzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and hyperphosphorylated tau in the brain. Currently, therapeutic agents targeting amyloid appear promising for AD, however, delivery to the CNS is limited due to the blood‐brain‐barrier (BBB). Focused ultrasound (FUS) is a method to induce a temporary opening of the BBB to enhance delivery of therapeutic agents to CNS. Our lab has shown that an mAb (07/2a), targeting N‐terminally truncated and modified toxic Aβ species, reduced pGlu3 Aβ and general Aβ cerebral plaques in aged APP/PS1dE9 mice. Here, we asked whether FUS‐induced BBB opening enhances the delivery of 07/2a mAb and whether it has any additive effect on cognition or plaque clearance.MethodsFirst, 24 mo‐old APP/PS1dE9 mice were i.v. infused with a single dose of 300 µg 07/2a with or without hippocampal FUS sonication (n=5/group) with i.v. infused Optison microbubbles and euthanized 4 or 72 h later; brain levels of antibodies were measured by ELISA. Next, 16 mo‐old APP/PS1dE9 mice were treated weekly for 3 weeks with PBS (n=9), 500 µg 07/2a alone (n=9), FUS alone (n=7) or 07/2a + FUS combination (n=6). Behavioral testing in the Water T Maze (WTM) was performed 1‐2 weeks later followed by euthanasia.ResultsFUS treatment increased 07/2a mAb levels in brain by 5.9‐fold 4 hr (41.7 pg/mg for mAb alone, vs. 244 pg/mg mAb+FUS; t=3.48, p&lt;0.005) and 5.5‐fold (mean=31.5 pg/mg for mAb alone, vs. 173 pg/mg for mAb+FUS; t=2.9, p&lt;0.05) 72 hr post‐treatment. Immunohistochemistry confirmed a significant increase in IgG2a mAb staining in the mAb+FUS treated mice at 4 hr (p=0.007) and a strong trend in the mAb+FUS treated mice at 72 hr after treatment compared to mAb alone. Three weekly treatments of 07/2a improved cognition however, when combined with FUS, this improvement occurred faster and in a higher percentage of mice and led to reduced hippocampal plaque load compared to PBS control mice.ConclusionsTransient opening of the BBB by FUS increased 07/2a delivery to the brain, improved cognition, and enhanced Ab clearance in aged APP/PS1dE9 mice, suggesting that FUS increased efficacy of the 07/2a mAb.

EXTH-49. FOCUSED ULTRASOUND AND 5-ALA MEDIATED ELIMINATION OF DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma (DMG) is a devastating and incurable childhood brain cancer. With a median survival of only 9 to 11 months, over 90% of children affected by DMG die within two years of diagnosis. Despite decades of research and a growing understanding of the biology of these tumors, there have been no advancements in therapies for DMGs. Tumor heterogeneity and diffuse infiltration in inoperable brain regions make these tumors uniquely difficult to manage both surgically and pharmacologically. Therefore, there is an urgent need for the exploration of novel treatment regimens. Focused Ultrasound (FUS) is an emerging technology with significant clinical potentials. Sonodynamic therapy (SDT) is an up-and-coming treatment strategy aiming to non-invasively eliminate tumor cells by acting through compounds known as sonosensitizers, which render tumor cells sensitive to ultrasound energy. Recently, 5-Aminolevulinic acid (5-ALA), an FDA-approved molecule, has been proposed as a sono-sensitizing agent. 5-ALA mediated SDT prolonged survival in C6 rat glioma models by selective elimination of tumor cells upon sonication. Mechanistically, it is thought that 5-ALA uptake and metabolic conversion into Protoporphyrin IX (PpIX) occurs preferentially in tumor cells due to differential activity of enzymes involved in heme metabolism. Here, we investigated SDT in DMG cells treated with 5-ALA. PpIX fluorescence increased linearly up to 24 h upon 5-ALA treatment and accumulated significantly more (1.6-fold, p &amp;lt; 0.01) when compared to C6 cells. Consequently, FUS sonication of 5-ALA treated DMG cells at 250 kHz significantly (p &amp;lt; 0.05) decreased DMG cell viability compared to treatment with 5-ALA or FUS alone. Here, we show the first 5-ALA mediated sonodynamic effect in DMG cells, leading to enhanced cell death. Our findings provide a rationale for considering clinical investigation of 5-ALA mediated sonodynamic therapy in DMG.