Three-dimensional super resolution ultrasound imaging with a multi-frequency hemispherical phased array.

BACKGROUND: High resolution imaging of the microvasculature plays an important role in both diagnostic and therapeutic applications in the brain. However, ultrasound pulse-echo sonography imaging the brain vasculatures has been limited to narrow acoustic windows and low frequencies due to the distortion of the skull bone, which sacrifices axial resolution since it is pulse length dependent. PURPOSE: To overcome the detect limit, a large aperture 256-module sparse hemispherical transmit/receive array was used to visualize the acoustic emissions of ultrasound-vaporized lipid-coated decafluorobutane nanodroplets flowing through tube phantoms and within rabbit cerebral vasculature in vivo via passive acoustic mapping and super resolution techniques. METHODS: Nanodroplets were vaporized with 55 kHz burst-mode ultrasound (burst length = 145 µs, burst repetition frequency = 9-45 Hz, peak negative acoustic pressure = 0.10-0.22 MPa), which propagates through overlying tissues well without suffering from severe distortions. The resulting emissions were received at a higher frequency (612 or 1224 kHz subarray) to improve the resulting spatial resolution during passive beamforming. Normal resolution three-dimensional images were formed using a delay, sum, and integrate beamforming algorithm, and super-resolved images were extracted via Gaussian fitting of the estimated point-spread-function to the normal resolution data. RESULTS: With super resolution techniques, the mean lateral (axial) full-width-at-half-maximum image intensity was 16 ± 3 (32 ± 6) µm, and 7 ± 1 (15 ± 2) µm corresponding to ∼1/67 of the normal resolution at 612 and 1224 kHz, respectively. The mean positional uncertainties were ∼1/350 (lateral) and ∼1/180 (axial) of the receive wavelength in water. In addition, a temporal correlation between nanodroplet vaporization and the transmit waveform shape was observed, which may provide the opportunity to enhance the signal-to-noise ratio in future studies. CONCLUSIONS: Here, we demonstrate the feasibility of vaporizing nanodroplets via low frequency ultrasound and simultaneously performing spatial mapping via passive beamforming at higher frequencies to improve the resulting spatial resolution of super resolution imaging techniques. This method may enable complete four-dimensional vascular mapping in organs where a hemispherical array could be positioned to surround the target, such as the brain, breast, or testicles.

Diagnostic and Therapeutic Updates in Leptomeningeal Disease.

PURPOSE OF REVIEW: Leptomeningeal disease (LMD) is a devastating complication of advanced metastatic cancer associated with a poor prognosis and limited treatment options. This study reviews the current understanding of the clinical presentation, pathogenesis, diagnosis, and treatment of LMD. We highlight opportunities for advances in this disease. RECENT FINDINGS: In recent years, the use of soluble CSF biomarkers has expanded, suggesting improved sensitivity over traditional cytology, identification of targetable mutations, and potential utility for monitoring disease burden. Recent studies of targeted small molecules and intrathecal based therapies have demonstrated an increase in overall and progression-free survival. In addition, there are several ongoing trials evaluating immunotherapy in LMD. Though overall prognosis of LMD remains poor, studies suggest a potential role for soluble CSF biomarkers in diagnosis and management and demonstrate promising findings in patient outcomes with targeted therapies for specific solid tumors. Despite these advances, there continues to be a gap of knowledge in this disease, emphasizing the importance of inclusion of LMD patients in clinical trials.

A PVDF Receiver for Acoustic Monitoring of Microbubble-Mediated Ultrasound Brain Therapy.

The real-time monitoring of spectral characteristics of microbubble (MB) acoustic emissions permits the prediction of increases in blood-brain barrier (BBB) permeability and of tissue damage in MB-mediated focused ultrasound (FUS) brain therapy. Single-element passive cavitation detectors provide limited spatial information regarding MB activity, greatly affecting the performance of acoustic control. However, an array of receivers can be used to spatially map cavitation events and thus improve treatment control. The spectral content of the acoustic emissions provides additional information that can be correlated with the bio-effects, and wideband receivers can thus provide the most complete spectral information. Here, we develop a miniature polyvinylidene fluoride (PVDF thickness = 110 µm, active area = 1.2 mm2) broadband receiver for the acoustic monitoring of MBs. The receiver has superior sensitivity (2.36-3.87 V/MPa) to those of a commercial fibre-optic hydrophone in the low megahertz frequency range (0.51-5.4 MHz). The receiver also has a wide -6 dB acceptance angle (54 degrees at 1.1 MHz and 13 degrees at 5.4 MHz) and the ability to detect subharmonic and higher harmonic MB emissions in phantoms. The overall acoustic performance of this low-cost receiver indicates its suitability for the eventual use within an array for MB monitoring and mapping in preclinical studies.

Establishing density-dependent longitudinal sound speed in the vertebral lamina.

Focused ultrasound treatments of the spinal cord may be facilitated using a phased array transducer and beamforming to correct spine-induced focal aberrations. Simulations can non-invasively calculate aberration corrections using x-ray computed tomography (CT) data that are correlated to density (ρ) and longitudinal sound speed (cL). We aimed to optimize vertebral lamina-specific cL(ρ) functions at a physiological temperature (37 °C) to maximize time domain simulation accuracy. Odd-numbered ex vivo human thoracic vertebrae were imaged with a clinical CT-scanner (0.511 × 0.511 × 0.5 mm), then sonicated with a transducer (514 kHz) focused on the canal via the vertebral lamina. Vertebra-induced signal time shifts were extracted from pressure waveforms recorded within the canals. Measurements were repeated 5× per vertebra, with 2.5 mm vertical vertebra shifts between measurements. Linear functions relating cL with CT-derived density were optimized. The optimized function was cL(ρ)=0.35(ρ-ρw)+ cL,w m/s, where w denotes water, giving the tested laminae a mean bulk density of 1600 ± 30 kg/m3 and a mean bulk cL of 1670 ± 60 m/s. The optimized lamina cL(ρ) function was accurate to λ/16 when implemented in a multi-layered ray acoustics model. This modelling accuracy will improve trans-spine ultrasound beamforming.

An Ultrasound-Guided Hemispherical Phased Array for Microbubble-Mediated Ultrasound Therapy.

GOAL: To develop a low-cost magnetic resonance imaging (MRI)-free transcranial focused ultrasound (FUS) system for microbubble-mediated therapy. METHODS: A 128-element 11 MHz array for skull localization was integrated within a 256-module multi-frequency (306/612/1224 kHz) dual-mode phased array. The system's transcranial transmit and receive performance was evaluated with ex-vivo human skullcaps using phase aberration corrections calculated from computed tomography (CT)-based simulations via ultrasound-based (USCT) and landmark-based (LMCT) registrations, and a gold-standard fixed source emitter (FSE)-based method. RESULTS: Displacement and rotation registration errors of 1.4 ± 0.4 mm and 2.1 ± 0.2 ° were obtained using USCT, resulting in sub-millimeter transmit targeting errors driven at 306 kHz (0.9 ± 0.2 mm) and 612 kHz (0.9 ± 0.3 mm), and source localization errors of 1.0 ± 0.3 mm and 0.6 ± 0.2 mm at receive frequencies of 306 kHz and 612 kHz, respectively (mean ± SD). Similar errors were obtained using LMCT and no significant differences between these two approaches were found on either transmit (p = 0.64/0.99) or receive (p = 0.45/0.36) at 306 kHz/612kHz. During volumetric multi-point exposures, approximately 70% and 60% of the transmit frames in which microbubble activity was detected via FSE were recovered using USCT when imaging at the second-harmonic and half-harmonic, respectively, compared to 60% and 69% using LMCT. CONCLUSION: This low-cost ultrasound-guided transcranial FUS system affords USCT skull registration with accuracy comparable to LMCT methods. SIGNIFICANCE: Such systems have great potential to advance the adoption of microbubble-mediated FUS brain therapy by improving access to the technology.

The mechanical potential of ultrasound on nervous tissue.

The Reflections series takes a look back on historical articles from The Journal of the Acoustical Society of America that have had a significant impact on the science and practice of acoustics.

Focused Ultrasound-Induced Blood-Spinal Cord Barrier Opening Using Short-Burst Phase-Keying Exposures in Rats: A Parameter Study.

Transient opening of the blood-spinal cord barrier has the potential to improve drug delivery options to the spinal cord. We previously developed short-burst phase-keying exposures to reduce focal depth of field and mitigate standing waves in the spinal canal. However, optimal short-burst phase-keying parameters for drug delivery have not been identified. Here, the effects of pressure, treatment duration, pulse length, burst repetition frequency and burst length on resulting tissue effects were investigated. Increased in situ pressures (0.23-0.33 MPa) led to increased post-treatment T1-weighted contrast enhancement in magnetic resonance imaging (p = 0.015). Increased treatment duration (120 vs. 300 s) led to increased enhancement, but without statistical significance (p = 0.056). Increased burst repetition frequency (20 vs. 40 kHz) yielded a non-significant increase in enhancement (p = 0.064) but corresponded with increased damage observed on histology. No difference was observed in enhancement between pulse lengths of 2 and 10 ms (p = 0.912), corresponding with a sharp drop in the recorded second harmonic signal during the first 2 ms of the pulse. Increasing the burst length from two to five cycles (514 kHz) led to increased enhancement (p = 0.014). Results indicate that increasing the burst length may be the most effective method to enhance drug delivery. Additionally, shorter pulse lengths may allow more interleaved targets, and therefore a larger treatment volume, within one sonication.

Therapeutic Agent Delivery Across the Blood-Brain Barrier Using Focused Ultrasound.

Specialized features of vasculature in the central nervous system greatly limit therapeutic treatment options for many neuropathologies. Focused ultrasound, in combination with circulating microbubbles, can be used to transiently and noninvasively increase cerebrovascular permeability with a high level of spatial precision. For minutes to hours following sonication, drugs can be administered systemically to extravasate in the targeted brain regions and exert a therapeutic effect, after which permeability returns to baseline levels. With the wide range of therapeutic agents that can be delivered using this approach and the growing clinical need, focused ultrasound and microbubble (FUS+MB) exposure in the brain has entered human testing to assess safety. This review outlines the use of FUS+MB-mediated cerebrovascular permeability enhancement as a drug delivery technique, details several technical and biological considerations of this approach, summarizes results from the clinical trials conducted to date, and discusses the future direction of the field.

Characterization of ultrasound-mediated delivery of trastuzumab to normal and pathologic spinal cord tissue.

Extensive studies on focused ultrasound (FUS)-mediated drug delivery through the blood-brain barrier have been published, yet little work has been published on FUS-mediated drug delivery through the blood-spinal cord barrier (BSCB). This work aims to quantify the delivery of the monoclonal antibody trastuzumab to rat spinal cord tissue and characterize its distribution within a model of leptomeningeal metastases. 10 healthy Sprague-Dawley rats were treated with FUS + trastuzumab and sacrificed at 2-h or 24-h post-FUS. A human IgG ELISA (Abcam) was used to measure trastuzumab concentration and a 12 ± fivefold increase was seen in treated tissue over control tissue at 2 h versus no increase at 24 h. Three athymic nude rats were inoculated with MDA-MB-231-H2N HER2 + breast cancer cells between the meninges in the thoracic region of the spinal cord and treated with FUS + trastuzumab. Immunohistochemistry was performed to visualize trastuzumab delivery, and semi-quantitative analysis revealed similar or more intense staining in tumor tissue compared to healthy tissue suggesting a comparable or greater concentration of trastuzumab was achieved. FUS can increase the permeability of the BSCB, improving drug delivery to specifically targeted regions of healthy and pathologic tissue in the spinal cord. The achieved concentrations within the healthy tissue are comparable to those reported in the brain.

A Porcine Model of Transvertebral Ultrasound and Microbubble-Mediated Blood-Spinal Cord Barrier Opening.

Blood-spinal cord barrier opening, using focused ultrasound and microbubbles, has the potential to improve drug delivery for the treatment of spinal cord pathologies. Delivering and detecting ultrasound through the spine is a challenge for clinical translation. We have previously developed short burst, phase keying exposures, which can be used in a dual-aperture configuration to address clinical scale targeting challenges. Here we demonstrate the use of these pulses for blood-spinal cord barrier opening, in vivo in pigs. Methods: The spinal cords of Yorkshire pigs (n=8) were targeted through the vertebral laminae, in the lower thoracic to upper lumbar region using focused ultrasound (486 kHz) and microbubbles. Four animals were treated with a combination of pulsed sinusoidal exposures (1.0-4.0 MPa, non-derated) and pulsed short burst, phase keying exposures (1.0-2.0 MPa, non-derated). Four animals were treated using ramped short burst, phase keying exposures (1.8-2.1 MPa, non-derated). A 250 kHz narrowband receiver was used to detect acoustic emissions from microbubbles. Blood-spinal cord barrier opening was assessed by the extravasation of Evans blue dye. Histological analysis of the spinal cords was used to assess tissue damage and excised vertebral samples were used in benchtop experiments. Results: Ramped short burst, phase keying exposures successfully modified the blood-spinal cord barrier at 16/24 targeted locations, as assessed by the extravasation of Evans blue dye. At 4 of these locations, opening was confirmed with minimal adverse effects observed through histology. Transmission measurements through excised vertebrae indicated a mean transmission of (47.0 ± 7.0 %) to the target. Conclusions: This study presents the first evidence of focused ultrasound-induced blood-spinal cord barrier opening in a large animal model, through the intact spine. This represents an important step towards clinical translation.

Simultaneous Intravital Optical and Acoustic Monitoring of Ultrasound-Triggered Nanobubble Generation and Extravasation.

Ultrasound-activated nanobubbles are being widely investigated as contrast agents and therapeutic vehicles. Nanobubbles hold potential for accessing the tumor extravascular compartment, though this relies on clinically debated passive accumulation for which evidence to date is indirect. We recently reported ultrasound-triggered conversion of high payload porphyrin-encapsulated microbubbles to nanobubbles, with actively enhanced permeability for local delivery. This platform holds implications for optical/ultrasound-based imaging and therapeutics. While promising, it remains to be established how nanobubbles are generated and whether they extravasate intact. Here, insights into the conversion process are reported, complemented by novel simultaneous intravital and acoustic monitoring in tumor-affected functional circulation. The first direct acoustic evidence of extravascular intact nanobubbles are presented. These insights collectively advance this delivery platform and multimodal micro- and nanobubbles, extending their utility for imaging and therapeutics within and beyond the vasculature.

Localized anesthesia of a specific brain region using ultrasound-responsive barbiturate nanodroplets.

Background: Targeted neuromodulation is a valuable technique for the study and treatment of the brain. Using focused ultrasound to target the local delivery of anesthetics in the brain offers a safe and reproducible option for suppressing neuronal activity. Objective: To develop a potential new tool for localized neuromodulation through the triggered release of pentobarbital from ultrasound-responsive nanodroplets. Method: The commercial microbubble contrast agent, Definity, was filled with decafluorobutane gas and loaded with a lipophilic anesthetic drug, before being condensed into liquid-filled nanodroplets of 210 ± 80 nm. Focused ultrasound at 0.58 MHz was found to convert nanodroplets into microbubbles, simultaneously releasing the drug and inducing local anesthesia in the motor cortex of rats (n=8). Results: Behavioral analysis indicated a 19.1 ± 13% motor deficit on the contralateral side of treated animals, assessed through the cylinder test and gait analysis, illustrating successful local anesthesia, without compromising the blood-brain barrier. Conclusion: Pentobarbital-loaded decafluorobutane-core Definity-based nanodroplets are a potential agent for ultrasound-triggered and targeted neuromodulation.

Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery.

Therapeutic ultrasound strategies that harness the mechanical activity of cavitation nuclei for beneficial tissue bio-effects are actively under development. The mechanical oscillations of circulating microbubbles, the most widely investigated cavitation nuclei, which may also encapsulate or shield a therapeutic agent in the bloodstream, trigger and promote localized uptake. Oscillating microbubbles can create stresses either on nearby tissue or in surrounding fluid to enhance drug penetration and efficacy in the brain, spinal cord, vasculature, immune system, biofilm or tumors. This review summarizes recent investigations that have elucidated interactions of ultrasound and cavitation nuclei with cells, the treatment of tumors, immunotherapy, the blood-brain and blood-spinal cord barriers, sonothrombolysis, cardiovascular drug delivery and sonobactericide. In particular, an overview of salient ultrasound features, drug delivery vehicles, therapeutic transport routes and pre-clinical and clinical studies is provided. Successful implementation of ultrasound and cavitation nuclei-mediated drug delivery has the potential to change the way drugs are administered systemically, resulting in more effective therapeutics and less-invasive treatments.

Aging exacerbates neutrophil pathogenicity in ischemic stroke.

Ischemic stroke is major cause of disability and mortality worldwide, and aging is strong risk factor for poor post-stroke outcome. Neutrophils traffic rapidly to the brain following ischemic stroke, and recent evidence has suggested that aging may alter neutrophil function after tissue injury. In this study, we hypothesize that aging enhances the pro-inflammatory function of neutrophils, directly contributing to the poorer outcomes seen in aging patients. We utilized demographic data and biological specimens from ischemic stroke patients and an experimental mouse model to determine the correlation between age, neutrophil function and stroke outcomes. In ischemic stroke patients, age was associated with increased mortality and morbidity and higher levels of neutrophil-activating cytokines. In mice, aged animals had higher stroke mortality and morbidity, higher levels of neutrophil-activating cytokines and enhanced generation of neutrophil reactive oxygen species compared to young mice. Finally, depletion of neutrophils via a specific monoclonal antibody after ischemic stroke led to long-term benefits in functional outcome in aged male and female animals, with no benefit observed in young. These results demonstrate that aging is associated with augmented neutrophil pathogenicity in ischemic stroke, and that neutrophil-targeted therapies may confer greater benefit in aged subjects.

A Spine-Specific Phased Array for Transvertebral Ultrasound Therapy: Design and Simulation.

OBJECTIVE: To design and simulate the performance of two spine-specific phased arrays in sonicating targets spanning the thoracic spine, with the objective of efficiently producing controlled foci in the spinal canal. METHODS: Two arrays (256 elements each, 500 kHz) were designed using multi-layered ray acoustics simulation: a four-component array with dedicated components for sonicating via the paravertebral and transvertebral paths, and a two-component array with spine-specific adaptive focusing. Mean array efficiency (canal focus pressure/water focus pressure) was evaluated using forward simulation in neutral and flexed spines to investigate methods that reduce spine-induced insertion loss. Target-specific four-component array reconfiguration and lower frequency sonication (250 kHz) were tested to determine their effects on array efficiency and focal dimensions. RESULTS: When neutral, two- and four-component efficiencies were [Formula: see text]% and [Formula: see text]%, respectively, spine flexion significantly increased four-component efficiency ([Formula: see text]%), but not two-component efficiency ([Formula: see text]%). Target-specific four-component re-configuration significantly improved efficiency ([Formula: see text]%). Both arrays produced controlled foci centered within the canal with similar 50% pressure contour dimensions: 10.8-11.9 mm (axial), 4.2-5.6 mm (lateral), and 5.9-6.2 mm (vertical). Simulation at 250 kHz also improved two- and four-component efficiency ([Formula: see text]% and [Formula: see text]%, respectively), but doubled the lateral focal dimensions. CONCLUSION: Simulation shows that the spine-specific arrays are capable of producing controlled foci in the thoracic spinal canal. SIGNIFICANCE: The complex geometry of the human spine presents geometrical and acoustical challenges for transspine ultrasound focusing, and the design of these spine-specific ultrasound arrays is crucial to the clinical translation of focused ultrasound for the treatment of spinal cord disease.

Enhanced Detection of Bubble Emissions Through the Intact Spine for Monitoring Ultrasound-Mediated Blood-Spinal Cord Barrier Opening.

OBJECTIVE: We previously developed short burst, phase keying (SBPK) focused ultrasound (FUS) to mitigate standing waves in the human vertebral canal. Here, we show microbubble emissions from these pulses can be detected through the human vertebral arch and that these pulses are effective for blood-spinal cord barrier (BSCB) opening. METHODS: At f0 = 514 kHz, circulating microbubbles were sonicated through ex vivo human vertebrae (60 kPa-1 MPa) using a dual-aperture approach and SBPK exposures engineered to incorporate pulse inversion (PI). Signals from a 250 kHz receiver were analyzed using PI, short-time Fourier analysis and the maximum projection over the pulse train. In rats (n = 14), SBPK FUS+microbubbles was applied to 3 locations/spinal cord at fixed pressures (∼0.20-0.47 MPa). MRI and histology were used to assess opening and tissue damage. RESULTS: In human vertebrae between 0.2-0.4 MPa, PI amplified the microbubble/baseline ratio at f0/2 and 2f0 by 202 ± 40% (132-291%). This was maximal at 0.4 MPa, coinciding with the onset of broadband emissions. In vivo, opening was achieved at 40/42 locations, with mean MRI enhancement of 46 ± 32%(16%-178%). Using PI, f0/2 was detected at 14/40 opening locations. At the highest pressures (f0/2 present) histology showed widespread bleeding throughout the focal region. At the lowest pressures, opening was achieved without bleeding. CONCLUSION: This study confirmed that PI can increase sensitivity to transvertebral detection of microbubble signals. Preliminary in vivo investigations show that SBPK FUS can increase BSCB permeability without tissue damage. SIGNIFICANCE: SBPK is a clinically relevant pulse scheme and, in combination with PI, provides a means of mediating and monitoring BSCB opening noninvasively.

Transforming growth factor-ß promotes basement membrane fibrosis, alters perivascular cerebrospinal fluid distribution, and worsens neurological recovery in the aged brain after stroke.

Aging and stroke alter the composition of the basement membrane and reduce the perivascular distribution of cerebrospinal fluid and solutes, which may contribute to poor functional recovery in elderly patients. Following stroke, TGF-ß induces astrocyte activation and subsequent glial scar development. This is dysregulated with aging and could lead to chronic, detrimental changes within the basement membrane. We hypothesized that TGF-ß induces basement membrane fibrosis after stroke, leading to impaired perivascular CSF distribution and poor functional recovery in aged animals. We found that CSF entered the aged brain along perivascular tracts; this process was reduced by experimental stroke and was rescued by TGF-ß receptor inhibition. Brain fibronectin levels increased with experimental stroke, which was reversed with inhibitor treatment. Exogenous TGF-ß stimulation increased fibronectin expression, both in vivo and in primary cultured astrocytes. Oxygen-glucose deprivation of cultured astrocytes induced multiple changes in genes related to astrocyte activation and extracellular matrix production. Finally, in stroke patients, we found that serum TGF-ß levels correlated with poorer functional outcomes, suggesting that serum levels may act as a biomarker for functional recovery. These results support a potential new treatment strategy to enhance recovery in elderly stroke patients.

Analysis of Multifrequency and Phase Keying Strategies for Focusing Ultrasound to the Human Vertebral Canal.

Focused ultrasound has been shown to increase the permeability of the blood-brain barrier and its feasibility for opening the blood-spinal cord barrier has also been demonstrated in small animal models, with great potential to impact the treatment of spinal cord (SC) disorders. For clinical translation, challenges to transvertebral focusing of ultrasound energy on the human spinal canal, such as a focal depth of field and standing-wave formation, must be addressed. A dual-aperture approach using multifrequency and phase-shift keying (PSK) strategies for achieving a controlled focus in human thoracic vertebrae was investigated through numerical simulations and benchtop experiments in ex vivo human vertebrae. An ~85% reduction in the focal depth of field was achieved compared to a single-aperture approach at 564 kHz. Short-burst (two-cycle) excitations in combination with PSK were found to suppress the formation of standing waves in ex vivo human thoracic vertebrae when focusing through the vertebral laminae. The results make an important contribution toward the development of a clinical-scale approach for targeting ultrasound therapy to the SC.