Search

Abstract Background Studies published in the 1970s by Mostafa S. Fahim and colleagues showed that a short treatment with ultrasound caused the depletion of germ cells and infertility. The goal of the current study was to determine if a commercially available therapeutic ultrasound generator and transducer could be used as the basis for a male contraceptive. Methods Sprague-Dawley rats were anesthetized and their testes were treated with 1 MHz or 3 MHz ultrasound while varying power, duration and temperature of treatment. Results We found that 3 MHz ultrasound delivered with 2.2 Watt per square cm power for fifteen minutes was necessary to deplete spermatocytes and spermatids from the testis and that this treatment significantly reduced epididymal sperm reserves. 3 MHz ultrasound treatment reduced total epididymal sperm count 10-fold lower than the wet-heat control and decreased motile sperm counts 1,000-fold lower than wet-heat alone. The current treatment regimen provided nominally more energy to the treatment chamber than Fahim's originally reported conditions of 1 MHz ultrasound delivered at 1 Watt per square cm for ten minutes. However, the true spatial average intensity, effective radiating area and power output of the transducers used by Fahim were not reported, making a direct comparison impossible. We found that germ cell depletion was most uniform and effective when we rotated the therapeutic transducer to mitigate non-uniformity of the beam field. The lowest sperm count was achieved when the coupling medium (3% saline) was held at 37 degrees C and two consecutive 15-minute treatments of 3 MHz ultrasound at 2.2 Watt per square cm were separated by 2 days. Conclusions The non-invasive nature of ultrasound and its efficacy in reducing sperm count make therapeutic ultrasound a promising candidate for a male contraceptive. However, further studies must be conducted to confirm its efficacy in providing a contraceptive effect, to test the result of repeated use, to verify that the contraceptive effect is reversible and to demonstrate that there are no detrimental, long-term effects from using ultrasound as a method of male contraception.

For more than a decade, the application of acoustic radiation force (ARF) has been proposed as a mechanism to increase ultrasonic molecular imaging (MI) sensitivity in vivo. Presented herein is the first noninvasive in vivo validation of ARF-enhanced MI with an unmodified clinical system. First, an in vitro optical-acoustical setup was used to optimize system parameters and ensure sufficient microbubble translation when exposed to ARF. 3-D ARF-enhanced MI was then performed on 7 rat fibrosarcoma tumors using microbubbles targeted to α(v)β₃ and nontargeted microbubbles. Low-amplitude (<25 kPa) 3-D ARF pulse sequences were tested and compared with passive targeting studies in the same animal. Our results demonstrate that a 78% increase in image intensity from targeted microbubbles can be achieved when using ARF relative to the passive targeting studies. Furthermore, ARF did not significantly increase image contrast when applied to nontargeted agents, suggesting that ARF did not increase nonspecific adhesion. [ABSTRACT FROM AUTHOR]

Alzheimer's Disease (AD) is the most common form of dementia and one of the leading causes of death. AD is known to be correlated to tortuosity in the microvasculature as well as decreases in blood flow throughout the brain. However, the mechanisms behind these changes and their causal relation to AD are poorly understood. Methods: Here, we use volumetric ultrasound localization microscopy (ULM) to non-invasively and quantitatively compare the microvascular morphology and flow dynamics of five wildtype (WT) and five APP NL-G-F Knock-in mice, a mouse model of AD, across a 1cmx1cmx1cm brain volume and in four specific brain regions: the hippocampal formation, thalamus, hypothalamus, and cerebral cortex. Results: Comparisons between groups showed a significant increase in tortuosity, as measured by the Sum of Angles Metric (SOAM), throughout the brain (p < 0.01) and the hypothalamus (p = 0.01), in mice with AD. While differences in mean velocity (p < 0.01) and blood flow (p=0.04) were detected across the whole brain, their effect size was small and no differences were detected in the four selected regions. There was a significant decrease in the linear log relationship between vessel diameter and blood flow, with AD mice experiencing a lower slope than WT mice across the whole brain volume (p = 0.02) and in the hippocampal formation (p = 0.05), a region affected by Amyloid Beta plaques in this mouse model. The AD mice had higher blood flows in smaller vessels and smaller blood flows in larger vessels than the WT mice. Conclusions: This preliminary demonstrates that the imaging technique can be used for non-invasive, longitudinal, volumetric assessment of AD, which may allow for investigation into the poorly understood microvascular degeneration associated with AD through time as well as the development of early diagnostic techniques., Competing Interests: Competing Interests: P.A.D. and G.F.P. declare inventorship on pending patents describing techniques for super-resolution imaging., (© The author(s).)

A long-standing goal of neuroimaging is the non-invasive volumetric assessment of whole brain function and structure at high spatial and temporal resolutions. Functional ultrasound (fUS) and ultrasound localization microscopy (ULM) are rapidly emerging techniques that promise to bring advanced brain imaging and therapy to the clinic with the safety and low-cost advantages associated with ultrasound. fUS has been used to study cerebral hemodynamics at high temporal resolutions while ULM has been used to study cerebral microvascular structure at high spatial resolutions. These two methods have complementary spatio-temporal characteristics, making them ideally suited for multimodal imaging, but both suffer from limitations associated with transcranial ultrasound imaging. Here, these two methods are combined on the same data acquisition, completely non-invasively, using contrast-enhancements, which solves the dual challenges of sensitivity during transcranial imaging and the ability to implement super-resolution. From this combined approach, the cerebral blood flow, activated brain region, brain connectivity, vessel diameter, and vessel velocity were all calculated from the same data acquisition. During stimulation periods, there was a statistically significant (p<0.0001) increase in cerebral blood flow, diameter, and global velocity, but a decrease in velocity in the activated region. Additionally, the global flow increased (p=0.11) and connectivity decreased (24.7%) when compared to baseline. This multimodal approach allows for the study of the relationship between cerebral hemodynamics (30 ms resolution) and the microvasculature (14.6 μm resolution) using one ultrasound scan., (© 2024. The Author(s).)

Low-boiling point perfluorocarbon nanodroplets (NDs) are valued as effective sonosensitive agents, encapsulating a liquid perfluorocarbon that would instantaneously vaporize at body temperature without the NDs shell. Those NDs have been explored for both therapeutic and diagnostic purposes. Here, phospholipid-shelled nanodroplets containing octafluoropropane (C 3 F 8 ) or decafluorobutane (C 4 F 10 ) formed by condensation of microbubbles were thoroughly characterized before blood-brain (BBB) permeabilization. Transmission electron microscopy (TEM) and cryo-TEM were employed to confirm droplet formation while providing high-resolution insights into the droplet surface and lipid arrangement assessed from electron density observation after condensation. The vaporization threshold of NDs was determined with a high-speed camera, and the frequency signal emitted by the freshly vaporized bubbles was analyzed using cavitation detection. C 3 F 8 NDs exhibited vaporization at 0.3 MPa (f 0  = 1.5 MHz, 50 cycles), and emitted signals at 2 f 0 and 1.5 f 0 from 0.45 MPa onwards (f 0  = 1.5 MHz, 50 cycles), while broadband noise was measured starting from 0.55 MPa. NDs with the higher boiling point C 4 F 10 vaporized at 1.15 MPa and emitted signals at 2 f 0 from 0.65 MPa and 1.5 f 0 from 0.9 MPa, while broadband noise was detected starting from 0.95 MPa. Both ND formulations were used to permeabilize the BBB in healthy mice using tailored ultrasound sequences, allowing for the identification of optimal applications for each NDs type. C 3 F 8 NDs proved suitable and safe for permeabilizing a large area, potentially the entire brain, at low acoustic pressure. Meanwhile, C 4 F 10 droplets facilitated very localized (400 μm isotropic) permeabilization at higher pressure. This study prompts a closer examination of the structural rearrangements occurring during the condensation of microbubbles into NDs and highlights the potential to tailor solutions for different brain pathologies by choosing the composition of the NDs and adjusting the ultrasound sequence., Competing Interests: Declaration of competing interest P.A.D. declares inventorship on patents describing low boiling point nanodroplet technologies, some of which have been licensed to Triangle Biotechnology. P.A.D. declares he is a co-founder and board member for Triangle Biotechnology. BL and AN are cofounders and stockholders of the company TheraSonic developing an ultrasound device for blood-brain barrier opening. All other authors declare that they do not have any competing interest. The figures used in this manuscript were partially generated using the Servier Medical Art image bank provided by Servier under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/) and the BioRender.com website., (Copyright © 2024. Published by Elsevier B.V.)

Impaired mucociliary transport is a distinguishing sign of cystic fibrosis, but current methods of evaluation are invasive or expose young patients to ionizing radiation. Contrast-enhanced ultrasound imaging may provide a feasible alternative. We formulated a cationic microbubble ultrasound contrast agent, to optimize adhesion to the respiratory mucus layer when inhaled. Potential toxicity was evaluated in human bronchial epithelial cell (hBEC) cultures following a 24-hour exposure, compared to positive and negative control conditions. In vivo tolerability and pulmonary image enhancement feasibility were evaluated in mice, comparing oropharyngeal administration of contrast agent to saline control. When induced to flow across mucus plated on microscope slides, cationic microbubbles demonstrated greater affinity for target samples than standard microbubbles. Cationic microbubbles elicited no proinflammatory or cytotoxic response in hBECs, nor were any cross-links to the cilia observed. Unlike standard microbubbles, cationic microbubbles mixed into the mucus layer, without epithelial absorption, and were observed to move with the mucus layer by the action of mucociliary transport. When administered to mice, cationic microbubbles enhanced sonographic visualization of the trachea, and were well-tolerated with no adverse effects. This developmental work supports the safety and feasibility of a mucus-targeting contrast agent that may be useful for pulmonary ultrasound applications., Competing Interests: Declarations. Competing interests: Drs. Tsuruta, Weitz, Durham, Clapp, Hill, Dayton, and Caughey are co-inventors on a pending patent describing a microbubble ultrasound contrast agent optimized for muco-adhesion and pulmonary imaging. Dr. Dayton declares ownership interest in SonoVascular and Triangle Biotechnology, as well as intellectual property licenses and royalties from Revvity, Triangle Biotechnolgy, and SonoVascular. Drs. Sears, Rebuli, and Ostrowski report no competing interests., (© 2024. The Author(s).)

Two-dimensional ultrasound transducers enable the acquisition of fully volumetric data that have been demonstrated to provide greater diagnostic information in the clinical setting and are a critical tool for emerging ultrasound methods, such as super-resolution and functional imaging. This technology, however, is not without its limitations. Due to increased fabrication complexity, some matrix probes with disjoint piezoelectric panels may require initial calibration. In this manuscript, two methods for calibrating the element positions of the Vermon 1024-channel 8 MHz matrix transducer are detailed. This calibration is a necessary step for acquiring high resolution B-mode images while minimizing transducer-based image degradation. This calibration is also necessary for eliminating vessel-doubling artifacts in super-resolution images and increasing the overall signal-to-noise ratio (SNR) of the image. Here, we show that the shape of the point spread function (PSF) can be significantly improved and PSF-doubling artifacts can be reduced by up to 10 dB via this simple calibration procedure., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: P.A.D. and G.F.P. declare inventorship on pending patents relevant to super-resolution microvascular imaging. O.C. holds patents in the field of ultrasound localization microscopy. O.C. is one founder and shareholder of the ResolveStroke startup.

The blood-brain barrier (BBB) presents a formidable challenge in delivering therapeutic agents to the central nervous system. Ultrasound-mediated BBB disruption has emerged as a promising non-invasive technique to enhance drug delivery to the brain. This manuscript reviews fundamental principles of ultrasound-based techniques and their mechanisms of action in temporarily permeabilizing the BBB. Clinical trials employing ultrasound for BBB disruption are discussed, summarizing diverse applications ranging from the treatment of neurodegenerative diseases to targeted drug delivery for brain tumors. The review also addresses safety considerations, outlining the current understanding of potential risks and mitigation strategies associated with ultrasound exposure, including real-time monitoring and assessment of treatment efficacy. Among the large number of studies, significant successes are highlighted thus providing perspective on the future direction of the field., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Paul A. Dayton reports a relationship with Triangle Biotechnology that includes: equity or stocks. Paul A. Dayton has patent licensed to Revvity. Paul A. Dayton has patent pending to Triangle Biotechnology. Paul A. Dayton has patent licensed to SonoVascular. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

When microbubble contrast agents are excited at low frequencies (less than 5 MHz), they resonate and produce higher-order harmonics due to their nonlinear behavior. We propose a novel scheme with a capacitive micromachined ultrasonic transducer (CMUT) array to receive high-frequency microbubble harmonics in collapse mode and to transmit a low-frequency high-pressure pulse by releasing the CMUT plate from collapse and pull it back to collapse again in the same transmit-receive cycle. By patterning and etching the substrate to create glass spacers in the device cavity we can reliably operate the CMUT in collapse mode and receive high-frequency signals. Previously, we demonstrated a single-element CMUT with spacers operating in the described fashion. In this article, we present the design and fabrication of a dual-mode, dual-frequency 1-D CMUT array with 256 elements. We present two different insulating glass spacer designs in rectangular cells for the collapse mode. For the device with torus-shaped spacers, the 3 dB receive bandwidth is from 8 to 17 MHz, and the transmitted maximum peak-to-peak pressure from 32 elements at 4 mm focal depth was 2.12 MPa with a 1.21 MPa peak negative pressure, which corresponds to a mechanical index (MI) of 0.58 at 4.3 MHz. For the device with line-shaped spacers, the 3-dB receive bandwidth at 150 V dc bias extends from 10.9 to 19.2 MHz. By increasing the bias voltage to 180 V, the 3 dB bandwidth shifts, and extends from 11.7 to 20.4 MHz. The transmitting maximum peak-to-peak pressure with 32 elements at 4 mm was 2.06 MPa with a peak negative pressure of 1.19 MPa, which corresponds to an MI of 0.62 at 3.7 MHz.

Superharmonic contrast imaging (SpHI) suppresses tissue clutter and allows high-contrast visualization of the vasculature. An array-based dual-frequency (DF) probe has been developed for SpHI, integrating a 21-MHz, 256-element microultrasound imaging array with a 2-MHz, 32-element array to take advantage of the broadband nonlinear responses from microbubble (MB) contrast agents. In this work, ultrafast imaging with plane waves was implemented for SpHI to increase the acquisition frame rate. Ultrafast imaging was also implemented for microultrasound B-mode imaging (HFPW B-mode) to enable high-resolution visualization of the tissue structure. Coherent compounding was demonstrated in vitro and in vivo in both imaging modes. Acquisition frame rates of 4.5 kHz and 187 Hz in HFPW B-mode imaging were achieved for imaging up to 21 mm with one and 25 angles, respectively, and 3.5 kHz and 396 Hz in the SpHI mode with one and nine coherently compounded angles, respectively. SpHI images showed suppression of tissue clutter prior to and after the introduction of MBs in vitro and in vivo. The nine-angle coherently compounded 2-D SpHI images of contrast-filled flow channel showed a contrast-to-tissue ratio (CTR) of 26.0 dB, a 2.5-dB improvement relative to images reconstructed from 0° steering. Consistent with in vitro imaging, the nine-angle compounded 2-D SpHI of a Lewis lung cancer tumor showed a 2.6-dB improvement in contrast enhancement, relative to 0° steering, and additionally revealed a region of nonviable tissue. The 3-D display of the volumetric SpHI data acquired from a xenograft mouse tumor using both 0° steering and nine-angle compounding allowed the visualization of the tumor vasculature. A small vessel visible in the compounded SpHI image, measuring around [Formula: see text], is not visualized in the 0° steering SpHI image, demonstrating the superiority of the latter in detecting fine structures within the tumor.

Acoustic hologram (AH) lenses are typically produced by high-resolution 3-D printing methods, such as stereolithography (SLA) printing. However, SLA printing of thin, plate-shaped lens structures has major limitations, including vulnerability to deformation during photocuring and limited control of acoustic impedance. To overcome these limitations, we demonstrated a nanoparticle-epoxy composite (NPEC) molding technique, and we tested its feasibility for AH lens fabrication. The characterized acoustic impedance of the 22.5% NPEC was 4.64 MRayl, which is 55% higher than the clear photopolymer (2.99 MRayl) used by SLA. Simulations demonstrated that the improved pressure transmission by the higher acoustic impedance of the NPEC resulted in 21% higher pressure amplitude in the region of interest (ROI, -6-dB pressure amplitude pixels) than the photopolymer. This improvement was experimentally demonstrated after prototyping NPEC lenses through a molding process. The NPEC lens showed no significant deformation and 72% lower thickness profile errors than the photopolymer, which otherwise experienced deformed edges due to thermal bending. Beam mapping results using the NPEC lens validated the predicted improvement, demonstrating 24% increased pressure amplitude on average and 10% improved structural similarity (SSIM) with the simulated pressure pattern compared to the photopolymer lens. This method can be used for AH lens applications with improved pressure output and accurate pressure field formation.

Glioblastoma is an aggressive brain cancer with a very poor prognosis in which less than 6% of patients survive more than five-year post-diagnosis. The outcome of this disease for many patients may be improved by early detection. This could provide clinicians with the information needed to take early action for treatment. In this work, we present the utilization of a non-invasive, fully volumetric ultrasonic imaging method to assess microvascular change during the evolution of glioblastoma in mice. Volumetric ultrasound localization microscopy (ULM) was used to observe statistically significant ( ) reduction in the appearance of functional vasculature over the course of three weeks. We also demonstrate evidence suggesting the reduction of vascular flow for vessels peripheral to the tumor. With an 82.5% consistency rate in acquiring high-quality vascular images, we demonstrate the possibility of volumetric ULM as a longitudinal method for microvascular characterization of neurological disease.

We investigated the accuracy of CEUS for characterizing cystic and solid kidney lesions in patients with chronic kidney disease (CKD). Cystic lesions are assessed using Bosniak criteria for computed tomography (CT) and magnetic resonance imaging (MRI); however, in patients with moderate to severe kidney disease, CT and MRI contrast agents may be contraindicated. Contrast-enhanced ultrasound (CEUS) is a safe alternative for characterizing these lesions, but data on its performance among CKD patients are limited. We performed flash replenishment CEUS in 60 CKD patients (73 lesions). Final analysis included 53 patients (63 lesions). Four readers, blinded to true diagnosis, interpreted each lesion. Reader evaluations were compared to true lesion classifications. Performance metrics were calculated to assess malignant and benign diagnoses. Reader agreement was evaluated using Bowker's symmetry test. Combined reader sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for diagnosing malignant lesions were 71%, 75%, 45%, and 90%, respectively. Sensitivity (81%) and specificity (83%) were highest in CKD IV/V patients when grouped by CKD stage. Combined reader sensitivity, specificity, PPV, and NPV for diagnosing benign lesions were 70%, 86%, 91%, and 61%, respectively. Again, in CKD IV/V patients, sensitivity (81%), specificity (95%), and PPV (98%) were highest. Inter-reader diagnostic agreement varied from 72% to 90%. In CKD patients, CEUS is a potential low-risk option for screening kidney lesions. CEUS may be particularly beneficial for CKD IV/V patients, where kidney preservation techniques are highly relevant.

Objective: For the treatment of tumor hypoxia, microbubbles comprising oxygen as a majority component of the gas core with a stabilizing shell may be used to deliver and release oxygen locally at the tumor site through ultrasound destruction. Previous work has revealed differences in circulation half-life in vivo for perfluorocarbon-filled microbubbles, typically used as ultrasound imaging contrast agents, as a function of anesthetic carrier gas. These differences in circulation time in vivo were likely due to gas diffusion as a function of anesthetic carrier gas, among other variables. This work has motivated studies to evaluate the effect of anesthetic carrier gas on oxygen microbubble circulation dynamics., Methods: Circulation time for oxygen microbubbles was derived from ultrasound image intensity obtained during longitudinal kidney imaging. Studies were constructed for rats anesthetized on inhaled isoflurane with either pure oxygen or medical air as the anesthetic carrier gas., Results: Results indicated that oxygen microbubbles were highly visible via contrast-specific imaging. Marked signal enhancement and duration differences were observed between animals breathing air and oxygen. Perhaps counterintuitively, oxygen microbubbles disappeared from circulation significantly faster when the animals were breathing pure oxygen compared with medical air. This may be explained by nitrogen counterdiffusion from blood into the bubble, effectively changing the gas composition of the core, as has been observed in perfluorocarbon core microbubbles., Conclusion: Our findings suggest that the apparent longevity and persistence of oxygen microbubbles in circulation may not be reflective of oxygen delivery when the animal is anesthetized breathing air., Competing Interests: Conflict of interest P.G.D., M.B., P.A.D. and V.P. are all inventors or co-inventors on patents related to oxygen microbubble formulation and/or use. M.B. is also a founder and the chief scientific officer of Respirogen Inc. (Boulder, CO, USA). The other authors declare no competing interests., (Copyright © 2023 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Objective: Efficient, sustained and long-term delivery of therapeutics to the brain remains an important challenge to treatment of diseases such as brain cancer, stroke and neurodegenerative disease. Focused ultrasound can assist movement of drugs into the brain, but frequent and long-term use has remained impractical. Single-use intracranial drug-eluting depots show promise but are limited for the treatment of chronic diseases as they cannot be refilled non-invasively. Refillable drug-eluting depots could serve as a long-term solution, but refilling is hindered by the blood-brain barrier (BBB), which prevents drug refills from accessing the brain. In this article, we describe how focused ultrasound enables non-invasive loading of intracranial drug depots in mice., Methods: Female CD-1 mice (n = 6) were intracranially injected with click-reactive and fluorescent molecules that are capable of anchoring in the brain. After healing, animals were treated with high-intensity focused ultrasound and microbubbles to temporarily increase the permeability of the blood-brain barrier and deliver dibenzocyclooctyne (DBCO)-Cy7. The mice were perfused, and the brains were imaged via ex vivo fluorescence imaging., Results: Fluorescence imaging indicated small molecule refills are captured by intracranial depots as long as 4 wk after administration and are retained for up to 4 wk based on fluorescence imaging. Efficient loading was dependent on both focused ultrasound and the presence of refillable depots in the brain as absence of either prevented intracranial loading., Conclusion: The ability to target and retain small molecules at predetermined intracranial sites with pinpoint accuracy provides opportunities to continuously deliver drugs to the brain over weeks and months without excessive BBB opening and with minimal off-target side effects., Competing Interests: Conflict of interest P.A.D. declares that he was a co-founder of SonoVol, Inc., and a co-inventor on patents licensed to SonoVol. C.T.M. and Y.B. declare that they are co-inventors on patent applications related to refillable depots., (Copyright © 2023 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Chronic wounds frequently become infected with bacterial biofilms which respond poorly to antibiotic therapy. Aminoglycoside antibiotics are ineffective at treating deep-seated wound infections due to poor drug penetration, poor drug uptake into persister cells, and widespread antibiotic resistance. In this study, we combat the two major barriers to successful aminoglycoside treatment against a biofilm-infected wound: limited antibiotic uptake and limited biofilm penetration. To combat the limited antibiotic uptake, we employ palmitoleic acid, a host-produced monounsaturated fatty acid that perturbs the membrane of gram-positive pathogens and induces gentamicin uptake. This novel drug combination overcomes gentamicin tolerance and resistance in multiple gram-positive wound pathogens. To combat biofilm penetration, we examined the ability of sonobactericide, a non-invasive ultrasound-mediated-drug delivery technology to improve antibiotic efficacy using an in vivo biofilm model. This dual approach dramatically improved antibiotic efficacy against a methicillin-resistant Staphylococcus aureus (MRSA) wound infection in diabetic mice., Competing Interests: Declaration of interests P.A.D declares that he is a co-inventor on patents describing the formulation of low boiling-point perfluorocarbon agents, as well as a co-founder of Triangle Biotechnology, a company that has licensed this patent and in which he has equity. V.P., P.G.D., P.A.D., and S.E.R. are all co-inventors on a patent describing the use of low boiling-point phase change contrast agents for enhancing the delivery of therapeutics agents to biofilms. S.E.R is a co-inventor on a provisional patent describing the use of membrane acting agents for potentiating antibiotic efficacy. A.E.S, K.L, A.Z.V, D.T.B, and M.A.S. declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objective: Doppler ultrasound (DU) is used to detect venous gas emboli (VGE) post dive as a marker of decompression stress for diving physiology research as well as new decompression procedure validation to minimize decompression sickness risk. In this article, we propose the first deep learning model for VGE grading in DU audio recordings., Methods: A database of real-world data was assembled and labeled for the purpose of developing the algorithm, totaling 274 recordings comprising both subclavian and precordial measurements. Synthetic data was also generated by acquiring baseline DU signals from human volunteers and superimposing laboratory-acquired DU signals of bubbles flowing in a tissue mimicking material. A novel squeeze-and-excitation deep learning model was designed to effectively classify recordings on the 5-class Spencer scoring system used by trained human raters., Results: On the real-data test set, we show that synthetic data pretraining achieves average ordinal accuracy of 84.9% for precordial and 90.4% for subclavian DU which is a 24.6% and 26.2% increase over training with real-data and time-series augmentation only. The weighted kappa coefficients of agreement between the model and human ground truth were 0.74 and 0.69 for precordial and subclavian respectively, indicating substantial agreement similar to human inter-rater agreement for this type of data., Conclusion: The present work demonstrates the first application of deep-learning for DU VGE grading using a combination of synthetic and real-world data., Significance: The proposed method can contribute to accelerating DU analysis for decompression research.

Thrombosis in cardiovascular disease is an urgent global issue, but treatment progress is limited by the risks of current antithrombotic approaches. The cavitation effect in ultrasound-mediated thrombolysis offers a promising mechanical alternative for clot lysis. Further addition of microbubble contrast agents introduces artificial cavitation nuclei that can enhance the mechanical disruption induced by ultrasound. Recent studies have proposed sub-micron particles as novel sonothrombolysis agents with increased spatial specificity, safety and stability for thrombus disruption. In this article, the applications of different sub-micron particles for sonothrombolysis are discussed. Also reviewed are in vitro and in vivo studies that apply these particles as cavitation agents and as adjuvants to thrombolytic drugs. Finally, perspectives on future developments in sub-micron agents for cavitation-enhanced sonothrombolysis are shared., Competing Interests: Conflict of interest J.K., Z.X., X.J. and P.D. are co-inventors on patents describing techniques, cavitation agents and devices for sonothrombolysis. J.K., X.J. and P.D. consult and/or advise for SonoVascular, a company commercializing sonothrombolysis technologies that has licensed patents from some of the co-authors. Z.X. has conflict of interest with Histosonics, Inc., a company commercializing therapeutic ultrasound, and P.D. is a founder of Triangle Biotechnology, Inc., a company commercializing cavitation enhancing agents., (Copyright © 2023. Published by Elsevier Inc.)

Doppler ultrasound (DU) measurements are used to detect and evaluate venous gas emboli (VGE) formed after decompression. Automated methodologies for assessing VGE presence using signal processing have been developed on varying real-world datasets of limited size and without ground truth values preventing objective evaluation. We develop and report a method to generate synthetic post-dive data using DU signals collected in both precordium and subclavian vein with varying degrees of bubbling matching field-standard grading metrics. This method is adaptable, modifiable, and reproducible, allowing for researchers to tune the produced dataset for their desired purpose. We provide the baseline Doppler recordings and code required to generate synthetic data for researchers to reproduce our work and improve upon it. We also provide a set of pre-made synthetic post-dive DU data spanning six scenarios representing the Spencer and Kisman-Masurel (KM) grading scales as well as precordial and subclavian DU recordings. By providing a method for synthetic post-dive DU data generation, we aim to improve and accelerate the development of signal processing techniques for VGE analysis in Doppler ultrasound., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Le et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rationale: Structure and function of the microvasculature provides critical information about disease state, can be used to identify local regions of pathology, and has been shown to be an indicator of response to therapy. Improved methods of assessing the microvasculature with non-invasive imaging modalities such as ultrasound will have an impact in biomedical theranostics. Ultrasound localization microscopy (ULM) is a new technology which allows processing of ultrasound data for visualization of microvasculature at a resolution better than allowed by acoustic diffraction with traditional ultrasound systems. Previous application of this modality in brain imaging has required the use of invasive procedures, such as a craniotomy, skull-thinning, or scalp removal, all of which are not feasible for the purpose of longitudinal studies. Methods: The impact of ultrasound localization microscopy is expanded using a 1024 channel matrix array ultrasonic transducer, four synchronized programmable ultrasound systems with customized high-performance hardware and software, and high-performance GPUs for processing. The potential of the imaging hardware and processing approaches are demonstrated in-vivo. Results : Our unique implementation allows asynchronous acquisition and data transfer for uninterrupted data collection at an ultra-high fixed frame rate. Using these methods, the vasculature was imaged using 100,000 volumes continuously at a volume acquisition rate of 500 volumes per second. With ULM, we achieved a resolution of 31 µ m, which is a resolution improvement on conventional ultrasound imaging by nearly a factor of ten, in 3-D. This was accomplished while imaging through the intact skull with no scalp removal, which demonstrates the utility of this method for longitudinal studies. Conclusions: The results demonstrate new capabilities to rapidly image and analyze complex vascular networks in 3-D volume space for structural and functional imaging in disease assessment, targeted therapeutic delivery, monitoring response to therapy, and other theranostic applications., Competing Interests: Competing Interests: P.A.D. and G.F.P. declare inventorship on patents pending related to super-resolution imaging. P.A.D. declares he is a founder of Triangle Biotechnology, Inc., and an advisory role for SonoVascular, Inc., and is a co-inventor on intellectual property licensed to both companies. P.A.D. declares royalties from intellectual property licenses to Perkin Elmer., (© The author(s).)

This research aims to demonstrate a novel vortex ultrasound enabled endovascular thrombolysis method designed for treating cerebral venous sinus thrombosis (CVST). This is a topic of substantial importance since current treatment modalities for CVST still fail in as many as 20% to 40% of the cases, and the incidence of CVST has increased since the outbreak of the coronavirus disease 2019 pandemic. Compared with conventional anticoagulant or thrombolytic drugs, sonothrombolysis has the potential to remarkably shorten the required treatment time owing to the direct clot targeting with acoustic waves. However, previously reported strategies for sonothrombolysis have not demonstrated clinically meaningful outcomes (e.g., recanalization within 30 min) in treating large, completely occluded veins or arteries. Here, we demonstrated a new vortex ultrasound technique for endovascular sonothrombolysis utilizing wave-matter interaction-induced shear stress to enhance the lytic rate substantially. Our in vitro experiment showed that the lytic rate was increased by at least 64.3% compared with the nonvortex endovascular ultrasound treatment. A 3.1-g, 7.5-cm-long, completely occluded in vitro 3-dimensional model of acute CVST was fully recanalized within 8 min with a record-high lytic rate of 237.5 mg/min for acute bovine clot in vitro. Furthermore, we confirmed that the vortex ultrasound causes no vessel wall damage over ex vivo canine veins. This vortex ultrasound thrombolysis technique potentially presents a new life-saving tool for severe CVST cases that cannot be efficaciously treated using existing therapies., (Copyright © 2023 Bohua Zhang et al.)

Background: Doppler ultrasound is used currently in decompression research for the evaluation of venous gas emboli (VGE). Estimation of heart rate from post-dive Doppler ultrasound recordings can provide a tool for the evaluation of physiological changes from decompression stress, as well as aid in the development of automated VGE detection algorithms that relate VGE presence to cardiac activity., Method: An algorithm based on short-term autocorrelation was developed in MATLAB to estimate the heart rate in post-dive precordial Doppler ultrasound. The algorithm was evaluated on 21 previously acquired and labeled precordial recordings spanning Kisman-Masurel (KM) codes of 111-444 (KM I-IV) with manually derived instantaneous heart rates., Results: A window size of at least two seconds was necessary for robust and accurate instantaneous heart rate estimation with a mean error of 1.56 ± 7.10 bpm. Larger window sizes improved the algorithm performance, at the cost of beat-to-beat heart rate estimates. We also found that our algorithm provides good results for low KM grade Doppler recordings with and without flexion, and high KM grades without flexion. High KM grades observed after movement produced the greatest mean absolute error of 6.12 ± 8.40 bpm., Conclusion: We have developed a fully automated algorithm for the estimation of heart rate in post-dive precordial Doppler ultrasound recordings., Competing Interests: The authors of this paper declare no conflicts of interest exist with this submission., (Copyright© Undersea and Hyperbaric Medical Society.)

We present a rapid prototyping method for sub-megahertz single-element piezoelectric transducers by using 3D-printed components. In most of the early research phases of applying new sonication ideas, the prototyping quickness is prioritized over the final packaging quality, since the quickness of preliminary demonstration is crucial for promptly determining specific aims and feasible research approaches. We aim to develop a rapid prototyping method for functional ultrasonic transducers to overcome the current long lead time (>a few weeks). Here, we used 3D-printed external housing parts considering a single matching layer and either air backing or epoxy-composite backing (acoustic impedance > 5 MRayl). By molding a single matching layer on the top surface of a piezoceramic in a 3D-printed housing, an entire packaging time was significantly reduced (<26 h) compared to the conventional methods with grinding, stacking, and bonding. We demonstrated this prototyping method for 590-kHz single-element, rectangular-aperture transducers for moderate pressure amplitudes (mechanical index > 1) at focus with temporal pulse controllability (maximum amplitude by <5-cycle burst). We adopted an air-backing design (Type A) for efficient pressure outputs, and bandwidth improvement was tested by a tungsten-composite-backing (Type B) design. The acoustic characterization results showed that the type A prototype provided 3.3 kPa/Vpp far-field transmitting sensitivity with 25.3% fractional bandwidth whereas the type B transducer showed 2.1 kPa/Vpp transmitting sensitivity with 43.3% fractional bandwidth. As this method provided discernable quickness and cost efficiency, this detailed rapid prototyping guideline can be useful for early-phase sonication projects, such as multi-element therapeutic ultrasound array and micro/nanomedicine testing benchtop device prototyping.

Detection of bare gas microbubbles by magnetic resonance (MR) at low concentrations typically used in clinical contrast-ultrasound studies was recently demonstrated using hyperCEST. Despite the enhanced sensitivity achieved with hyperCEST, in vivo translation is challenging as on-resonance saturation of the gas-phase core of microbubbles consequently results in saturation of the gas-phase hyperpolarized 129 Xe within the lungs. Alternatively, microbubbles can be condensed into the liquid phase to form perfluorocarbon nanodroplets, where 129 Xe resonates at a chemical shift that is separated from the gas-phase signal in the lungs. For ultrasound applications, nanodroplets can be acoustically reverted back into their microbubble form to act as a phase-change contrast agent. Here, we show that low-boiling point perfluorocarbons, both in their liquid and gas form, generate phase-dependent hyperCEST contrast. Magnetic resonance detection of ultrasound-mediated phase transition demonstrates that these perfluorocarbons could be used as a dual-phase dual-modality MR/US contrast agent., (© 2022 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

Pancreatic adenocarcinoma is an aggressive malignancy with limited therapeutic treatments available and a 5-y survival less than 10%. Pancreatic cancers have been found to be immunogenically "cold" with a largely immunosuppressive tumor microenvironment. There is emerging evidence that focused ultrasound can induce changes in the tumor microenvironment and have a constructive impact on the effect of immunotherapy. However, the immune cells and timing involved in these effects remain unclear, which is essential to determining how to combine immunotherapy with ultrasound for treatment of pancreatic adenocarcinoma. We used low-intensity focused ultrasound and microbubbles (LoFU + MBs), which can mechanically disrupt cellular membranes and vascular endothelia, to treat subcutaneous pancreatic tumors in C57BL/6 mice. To evaluate the immune cell landscape and expression and/or localization of damage-associated molecular patterns (DAMPs) as a response to ultrasound, we performed flow cytometry and histology on tumors and draining lymph nodes 2 and 15 d post-treatment. We repeated this study on larger tumors and with multiple treatments to determine whether similar or greater effects could be achieved. Two days after treatment, draining lymph nodes exhibited a significant increase in activated antigen presenting cells, such as macrophages, as well as expansion of CD8 + T cells and CD4 + T cells. LoFU + MB treatment caused localized damage and facilitated the translocation of DAMP signals, as reflected by an increase in the cytoplasmic index for high-mobility-group box 1 (HMGB1) at 2 d. Tumors treated with LoFU + MBs exhibited a significant decrease in growth 15 d after treatment, indicating a tumor response that has the potential for additive effects. Our studies indicate that focused ultrasound treatments can cause tumoral damage and changes in macrophages and T cells 2 d post-treatment. The majority of these effects subsided after 15 d with only a single treatment, illustrating the need for additional treatment types and/or combination with immunotherapy. However, when larger tumors were treated, the effects seen at 2 d were diminished, even with an additional treatment. These results provide a working platform for further rational design of focused ultrasound and immunotherapy combinations in poorly immunogenic cancers., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Hyperbaric pressure experiments have provided researchers with valuable insights into the effects of pressure changes, using various species as subjects. Notably, extensive work has been done to observe rodents subjected to hyperbaric pressure, with differing imaging modalities used as an analytical tool. Decompression puts subjects at a greater risk for injury, which often justifies conducting such experiments using animal models. Therefore, it is important to provide a broad view of previously utilized methods for decompression research to describe imaging tools available for researchers to conduct rodent decompression experiments, to prevent duplicate experimentation, and to identify significant gaps in the literature for future researchers. Through a scoping review of published literature, we will provide an overview of decompression bubble information collected from rodent experiments using various non-invasive methods of ultrasound for decompression bubble assessment. This review will adhere to methods outlined by the Joanna Briggs Institute Manual for Evidence Synthesis and be reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Reviews (PRISMA-ScR). Literature will be obtained from the PubMed, Embase, and Scopus databases. Extracted sources will first be sorted to a list for inclusion based on title and abstract. Two independent researchers will then conduct full-text screening to further refine included papers to those relevant to the scope. The final review manuscript will cover methods, data, and findings for each included publication relevant to non-invasive in vivo bubble imaging., Competing Interests: The authors have declared that no competing interests exist.

Shear wave elastography (SWE) is an ultrasound-based stiffness quantification technology that is used for noninvasive liver fibrosis assessment. However, despite widescale clinical adoption, SWE is largely unused by preclinical researchers and drug developers for studies of liver disease progression in small animal models due to significant experimental, technical, and reproducibility challenges. Therefore, the aim of this work was to develop a tool designed specifically for assessing liver stiffness and echogenicity in small animals to better enable longitudinal preclinical studies. A high-frequency linear array transducer (12-24 MHz) was integrated into a robotic small animal ultrasound system (Vega; SonoVol, Inc., Durham, NC) to perform liver stiffness and echogenicity measurements in three dimensions. The instrument was validated with tissue-mimicking phantoms and a mouse model of nonalcoholic steatohepatitis. Female C57BL/6J mice (n = 40) were placed on choline-deficient, L-amino acid-defined, high-fat diet and imaged longitudinally for 15 weeks. A subset was sacrificed after each imaging timepoint (n = 5) for histological validation, and analyses of receiver operating characteristic (ROC) curves were performed. Results demonstrated that robotic measurements of echogenicity and stiffness were most strongly correlated with macrovesicular steatosis (R 2  = 0.891) and fibrosis (R 2  = 0.839), respectively. For diagnostic classification of fibrosis (Ishak score), areas under ROC (AUROCs) curves were 0.969 for ≥Ishak1, 0.984 for ≥Ishak2, 0.980 for ≥Ishak3, and 0.969 for ≥Ishak4. For classification of macrovesicular steatosis (S-score), AUROCs were 1.00 for ≥S2 and 0.997 for ≥S3. Average scanning and analysis time was <5 minutes/liver. Conclusion: Robotic SWE in small animals is feasible and sensitive to small changes in liver disease state, facilitating in vivo staging of rodent liver disease with minimal sonographic expertise., (© 2022 SonoVol, Inc and University of North Carolina. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

This article presents an imaging probe with a 256-element ultrawideband (UWB) 1-D capacitive micromachined ultrasonic transducer (CMUT) array designed for acoustic angiography (AA). This array was fabricated on a borosilicate glass wafer with a reduced bottom electrode and an additional central plate mass to achieve the broad bandwidth. A custom 256-channel handheld probe was designed and implemented with integrated low-noise amplifiers and supporting power circuitry. This probe was used to characterize the UWB CMUT, which has a functional 3-dB frequency band from 3.5 to 23.5 MHz. A mechanical index (MI) of 0.33 was achieved at 3.5 MHz at a depth of 11 mm. These promising measurements are then combined to demonstrate AA. The use of alternate amplitude modulation (aAM) combined with a frequency analysis of the measured transmit signal demonstrates the suitability of the UWB CMUT for AA. This is achieved by measuring only a low level of unwanted high-frequency harmonics in both the transmit signal and the reconstructed image in the areas other than the contrast bubbles.

Ultrasound monitoring, both in the form of Doppler and 2D echocardiography, has been used post-dive to detect decompression bubbles circulating in the bloodstream. With large variability in both bubble time course and loads, it has been hypothesised that shorter periods between imaging, or even continuous imaging, could provide more accurate post-dive assessments. However, while considering applications of ultrasound imaging post-decompression, it may also be prudent to consider the possibility of ultrasound-induced bioeffects. Clinical ultrasound studies using microbubble contrast agents have shown bioeffect generation with acoustic powers much lower than those used in post-dive monitoring. However, to date no studies have specifically investigated potential bioeffect generation from continuous post-dive echocardiography. This review discusses what can be drawn from the current ultrasound and diving literature on the safety of bubble sonication and highlights areas where more studies are needed. An overview of the ultrasound-bubble mechanisms that lead to bioeffects and analyses of ultrasound contrast agent studies on bioeffect generation in the pulmonary and cardiovascular systems are provided to illustrate how bubbles under ultrasound can cause damage within the body. Along with clinical ultrasound studies, studies investigating the effects of decompression bubbles under ultrasound are analysed and open questions regarding continuous post-dive monitoring safety are discussed., (Copyright: This article is the copyright of the authors who grant Diving and Hyperbaric Medicine a non-exclusive licence to publish the article in electronic and other forms.)

Phase-change contrast agents (PCCAs) consisting of lipid-encapsulated low-boiling-point perfluorocarbons can be used in conjunction with ultrasound for diagnostic and therapeutic applications. One benefit of PCCAs is site-specific activation, whereby the liquid core is acoustically vaporized into a bubble detectable via ultrasound imaging. For further evaluation of PCCAs in a variety of applications, it is useful to disperse these nanodroplets into an acoustically compatible stationary matrix. However, many traditional phantom preparations require heating, which causes premature thermal activation of low-boiling-point PCCAs. Polyvinyl alcohol (PVA) cryogels do not require heat to set. Here we propose a simple method for the incorporation of the low-boiling-point PCCAs using octafluoropropane (OFP) and decafluorobutane (DFB) into PVA cryogels for a variety of acoustic characterization applications. We determined the utility of the phantoms by activating droplets with a focused transducer, visualizing the lesions with ultrasound imaging. At 1 MHz, droplet activation was consistently observed at 2.0 and 4.0 MPa for OFP and DFB, respectively., Competing Interests: Conflict of interest disclosure P.A.D. reports that he is a co-inventor of low-boiling-point nanodroplet technology and a co-founder of Triangle Biotechnology, a company that has licensed this technology from University of North Carolina at Chapel Hill. P. A. D. and J. K. also declares that they have advisory relationships with SonoVascular, Inc. and are co-inventors on patents licensed to SonoVascular., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Due to the versatility of the in situ forming implant (ISFI) drug delivery system, it is crucial to understand the effects of formulation parameters for clinical translation. We utilized ultrasound imaging and pharmacokinetics (PK) in mice to understand the impact of administration route, injection volume, and drug loading on ISFI formation, degradation, and drug release in mice. Placebo ISFIs injected subcutaneously (SQ) with smaller volumes (40 μL) exhibited complete degradation within 30-45 days, compared to larger volumes (80 μL), which completely degraded within 45-60 days. However, all dolutegravir (DTG)-loaded ISFIs along the range of injection volumes tested (20-80 μL) were present at 90 days post-injection, suggesting that DTG can prolong ISFI degradation. Ultrasound imaging showed that intramuscular (IM) ISFIs flattened rapidly post administration compared to SQ, which coincides with the earlier T max for drug-loaded IM ISFIs. All mice exhibited DTG plasma concentrations above four times the protein-adjusted 90% inhibitory concentration (PA-IC90) throughout the entire 90 days of the study. ISFI release kinetics best fit to zero order or diffusion-controlled models. When total administered dose was held constant, there was no statistical difference in drug exposure regardless of the route of administration or number of injections.

The thrombolysis potential of low-boiling-point (-2 °C) perfluorocarbon phase-change nanodroplets (NDs) has previously been demonstrated on aged clots, and we hypothesized that this efficacy would extend to retracted clots. We tested this hypothesis by comparing sonothrombolysis of both unretracted and retracted clots using ND-mediated ultrasound (US+ND) and microbubble-mediated ultrasound (US+MB), respectively. Assessment data included clot mass reduction, cavitation detection, and cavitation cloud imaging in vitro. Acoustic parameters included a 7.9-MPa peak negative pressure and 180-cycle bursts with 5-Hz repetition (the corresponding duty cycle and time-averaged intensity of 0.09% and 1.87 W/cm 2 , respectively) based on prior studies. With these parameters, we observed a significantly reduced efficacy of US+MB in the retracted versus unretracted model (the averaged mass reduction rate from 1.83%/min to 0.54%/min). Unlike US+MB, US+ND exhibited less reduction of efficacy in the retracted model (from 2.15%/min to 1.04%/min on average). The cavitation detection results correlate with the sonothrombolysis efficacy results showing that both stable and inertial cavitation generated in a retracted clot by US+ND is higher than that by US+MB. We observed that ND-mediated cavitation shows a tendency to occur inside a clot, whereas MB-mediated cavitation occurs near the surface of a retracted clot, and this difference is more significant with retracted clots compared to unretracted clots. We conclude that ND-mediated sonothrombolysis outperforms MB-mediated therapy regardless of clot retraction, and this advantage of ND-mediated cavitation is emphasized for retracted clots. The primary mechanisms are hypothesized to be sustained cavitation level and cavitation clouds in the proximity of a retracted clot by US+ND.

Background: Individuals with chronic kidney disease (CKD) have decreased kidney cortical microvascular perfusion, which may lead to worsening kidney function over time, but methods to quantify kidney cortical microvascular perfusion are not feasible to incorporate into clinical practice. Contrast-enhanced ultrasound (CEUS) may quantify kidney cortical microvascular perfusion, which requires further investigation in individuals across the spectrum of kidney function., Methods: We performed CEUS on a native kidney of 83 individuals across the spectrum of kidney function and calculated quantitative CEUS-derived kidney cortical microvascular perfusion biomarkers. Participants had a continuous infusion of the microbubble contrast agent (Definity) with a flash-replenishment sequence during their CEUS scan. Lower values of the microbubble velocity ( β ) and perfusion index ( β ×A) may represent lower kidney cortical microvascular perfusion. Multivariable linear regression models tested the associations of the microbubble velocity ( β ) and perfusion index ( β ×A) with estimated glomerular filtration rate (eGFR)., Results: Thirty-eight individuals with CKD (mean age±SD 65.2±12.6 years, median [IQR] eGFR 31.5 [18.9-41.5] ml/min per 1.73 m 2 ), 37 individuals with end stage kidney disease (ESKD; age 54.8±12.3 years), and eight healthy volunteers (age 44.1±15.0 years, eGFR 117 [106-120] ml/min per 1.73 m 2 ) underwent CEUS without side effects. Individuals with ESKD had the lowest microbubble velocity ( β ) and perfusion index ( β ×A) compared with individuals with CKD and healthy volunteers. The microbubble velocity ( β ) and perfusion index ( β ×A) had moderate positive correlations with eGFR ( β : r s =0.44, P <0.001; β ×A: r s =0.50, P <0.001). After multivariable adjustment, microbubble velocity ( β ) and perfusion index ( β ×A) remained significantly associated with eGFR (change in natural log transformed eGFR per 1 unit increase in natural log transformed biomarker: β , 0.38 [95%, CI 0.17 to 0.59]; β ×A, 0.79 [95% CI, 0.45 to 1.13])., Conclusions: CEUS-derived kidney cortical microvascular perfusion biomarkers are associated with eGFR. Future studies are needed to determine if CEUS-derived kidney cortical microvascular perfusion biomarkers have prognostic value., Competing Interests: E.H. Chang reports research funding from Lantheus Medical Imaging and is on the medical advisory board for the North Carolina chapter of the National Kidney Foundation. P. Dayton reports consultancy for SonoVascular, Inc., SonoVol, Inc., and Triangle Biotechnology, Inc.; ownership interest in SonoVascular, Inc., SonoVol, Inc., and Triangle Biotechnology, Inc.; research funding from Kitware, LLC, SonoVol, Inc., and Triangle Biotechnology, Inc.; patents or royalties from SonoVascular, Inc., SonoVol, Inc., and Triangle Biotechnology, Inc.; an advisory or leadership role for SonoVascular, Inc., SonoVol, LLC, and Triangle Biotechnology, Inc.; and other interests or relationships with Pfizer, Inc. R. Walmer reports consultancy agreements with SonoVol, Inc. (entered into on November 2, 2021). A. Srivastava reports consultancy agreements with CVS Caremark and Tate & Latham (medicolegal consulting); honoraria from AstraZeneca, Bayer, and Horizon Therapeutics plc.; and participation in a speakers’ bureau for AstraZeneca. All remaining authors have nothing to disclose., (Copyright © 2022 by the American Society of Nephrology.)

Preclinical mouse solid tumor models are widely used to evaluate efficacy of novel cancer therapeutics. Recent reports have highlighted the need for utilizing orthotopic implantation to represent clinical disease more accurately, however the deep tissue location of these tumors makes longitudinal assessment challenging without the use of imaging techniques. The purpose of this study was to evaluate the performance of a new multi-modality high-throughput in vivo imaging system that combines bioluminescence imaging (BLI) with robotic, hands-free ultrasound (US) for evaluating orthotopic mouse models. Long utilized in cancer research as independent modalities, we hypothesized that the combination of BLI and US would offer complementary advantages of detection sensitivity and quantification accuracy, while mitigating individual technological weaknesses. Bioluminescent pancreatic tumor cells were injected into the pancreas tail of C57BL/6 mice and imaged weekly with the combination system and magnetic resonance imaging (MRI) to serve as a gold standard. BLI photon flux was quantified to assess tumor activity and distribution, and US and MRI datasets were manually segmented for gross tumor volume. Robotic US and MRI demonstrated a strong agreement (R 2  = 0.94) for tumor volume measurement. BLI showed a weak overall agreement with MRI (R 2  = 0.21), however, it offered the greatest sensitivity to detecting the presence of tumors. We conclude that combining BLI with robotic US offers an efficient screening tool for orthotopic tumor models., (© 2022. The Author(s).)

Liver metastasis is a leading cause of cancer morbidity and mortality. Thus, there has been strong interest in the development of therapeutics that can effectively prevent liver metastasis. One potential strategy is to utilize molecules that have broad effects on the liver microenvironment, such as miR-122, a liver-specific miRNA that is a key regulator of diverse hepatic functions. Here we report the development of a nanoformulation miR-122 as a therapeutic agent for preventing liver metastasis. We engineered a galactose-targeted lipid calcium phosphate (Gal-LCP) nanoformulation of miR-122. This nanotherapeutic elicited no significant toxicity and delivered miR-122 into hepatocytes with specificity and high efficiency. Across multiple colorectal cancer liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented colorectal cancer liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes involved in metastatic and cancer inflammation pathways, including several proinflammatory factors, matrix metalloproteinases, and other extracellular matrix degradation enzymes. Moreover, Gal-LCP miR-122 treatment was associated with an increased CD8 + /CD4 + T-cell ratio and decreased immunosuppressive cell infiltration, which makes the liver more conducive to antitumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA. SIGNIFICANCE: Highly specific and efficient delivery of miRNA to hepatocytes using nanomedicine has therapeutic potential for the prevention and treatment of colorectal cancer liver metastasis., (©2021 American Association for Cancer Research.)

Ultrasound localization microscopy (ULM) has been able to overcome the diffraction limit of ultrasound imaging. The resolution limit of ULM has been previously modeled using the Cramér-Rao lower bound (CRLB). While this model has been validated in a homogeneous medium, it estimates a resolution limit, which has not yet been achieved in vivo. In this work, we investigated the effects of three sources of image degradation on the resolution limit of ULM. The Fullwave simulation tool was used to simulate acquisitions of transabdominal contrast-enhanced data at depth. The effects of reverberation clutter, trailing clutter, and phase aberration were studied. The resolution limit, in the presence of reverberation clutter alone, was empirically measured to be up to 39 times worse in the axial dimension and up to 2.1 times worse in the lateral dimension than the limit predicted by the CRLB. While reverberation clutter had an isotropic impact on the resolution, trailing clutter had a constant impact on both dimensions across all signal-to-trailing-clutter ratios (STCR). Phase aberration had a significant impact on the resolution limit over the studied analysis ranges. Phase aberration alone degraded the resolution limit up to 70 and 160 [Formula: see text] in the lateral and axial dimensions, respectively. These results illustrate the importance of phase aberration correction and clutter filtering in ULM postprocessing. The analysis results were demonstrated through the simulation of the ULM process applied to a cross-tube model that was degraded by each of the three aforementioned sources of degradation.

Ultrasound molecular imaging (USMI) is a technique used to noninvasively estimate the distribution of molecular markers in vivo by imaging microbubble contrast agents (MCAs) that have been modified to target receptors of interest on the vascular endothelium. USMI is especially relevant for preclinical and clinical cancer research and has been used to predict tumor malignancy and response to treatment. In the last decade, methods that improve the resolution of contrast-enhanced ultrasound by an order of magnitude and allow researchers to noninvasively image individual capillaries have emerged. However, these approaches do not translate directly to molecular imaging. In this work, we demonstrate super-resolution visualization of biomarker expression in vivo using superharmonic ultrasound imaging (SpHI) with dual-frequency transducers, targeted contrast agents, and localization microscopy processing. We validate and optimize the proposed method in vitro using concurrent optical and ultrasound microscopy and a microvessel phantom. With the same technique, we perform a proof-of-concept experiment in vivo in a rat fibrosarcoma model and create maps of biomarker expression co-registered with images of microvasculature. From these images, we measure a resolution of 23 μm, a nearly fivefold improvement in resolution compared to previous diffraction-limited molecular imaging studies., Competing Interests: CONFLICTS OF INTEREST F. Stuart Foster and Paul A. Dayton are inventors on a patent describing dual-frequency imaging, which is now licensed to Perkin Elmer. Thomas M. Kierski, Isabel G. Newsome, Gianmarco F. Pinton, and Paul A. Dayton are inventors on a patent describing dual-frequency ultrasound localization microscopy.

Even simple behaviors arise from the simultaneous activation of multiple regions in the brain. Thus, the ability to simultaneously stimulate multiple regions within a brain circuit should allow for better modulation of function. However, performing simultaneous multifocus ultrasound neuromodulation introduces challenges to transducer design. Using 3-D Fullwave simulations, we have designed an ultrasound neuromodulation array for nonhuman primates that: 1) can simultaneously focus on multiple targets and 2) include an imaging aperture for additional functional imaging. This design is based on a spherical array, with 128 15-mm elements distributed in a spherical helix pattern. It is shown that clustering the elements tightly around the 65-mm imaging aperture located at the top of the array improves targeting at shallow depths, near the skull surface. Spherical arrays have good focusing capabilities through the skull at the center of the array, but focusing on off-center locations is more challenging due to the natural geometric configuration and the angle of incidence with the skull. In order to mitigate this, the 64 elements closest to the aperture were rotated toward and focusing on a shallow target, and the 64 elements farthest from the aperture were rotated toward and focusing on a deeper target. Data illustrated that this array produced focusing on the somatosensory cortex with a gain of 4.38 and to the thalamus with a gain of 3.82. To improve upon this, the array placement was optimized based on phase aberration simulations, allowing for the elements with the largest impact on the gain at each focal point to be found. This optimization resulted in an array design that can focus on the somatosensory cortex with a gain of 5.19 and the thalamus with a gain of 4.45. Simulations were also performed to evaluate the ability of the array to focus on 28 additional brain regions, showing that off-center target regions can be stimulated, but those closer to the skull will require corrective steps to deliver the same amount of energy to those locations. This simulation and design process can be adapted to an individual monkey or human skull morphologies and specific target locations within individuals by using orientable 3-D printing of the transducer case and by electronic phase aberration correction.

Acoustic angiography is a contrast-enhanced ultrasound technique that relies on superharmonic imaging to form high-resolution, three-dimensional maps of the microvasculature. In order to obtain signal separation between tissue and contrast, acoustic angiography has been performed with dual-frequency transducers with nonoverlapping bandwidths. This enables a high contrast-to-tissue ratio, and the choice of a high frequency receiving element provides high resolution. In this chapter, we describe the technology behind acoustic angiography as well as the step-by-step implementation of this contrast enhanced microvascular imaging technique., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Thrombo-occlusive disease is one of the leading causes of death worldwide. There has been active research on safe and effective thrombolysis in preclinical and clinical studies. Recently, the dual-frequency transcutaneous sonothrombolysis with contrast agents [microbubbles (MBs)] has been reported to be more efficient in trigging the acoustic cavitation, which leads to a higher lysis rate. Therefore, there is increasing interest in applying dual-frequency technique for more significant efficacy improvement in intravascular sonothrombolysis since a miniaturized intravascular ultrasound transducer typically has a limited power output to fully harness cavitation effects. In this work, we demonstrated this efficacy enhancement by developing a new broadband intravascular transducer and testing dual-frequency sonothromblysis in vitro. A broadband intravascular transducer with a center frequency of 750 kHz and a footprint size of 1.4 mm was designed, fabricated, and characterized. The measured -6-dB fractional bandwidth is 68.1%, and the peak negative pressure is 1.5 MPa under the driving voltage of 80 V pp . By keeping one frequency component at 750 kHz, the second frequency component was selected from 450 to 650 kHz with an interval of 50 kHz. The in vitro sonothrombolysis tests were conducted with a flow model and the results indicated that the MB-mediated, dual-frequency (750+500 kHz) sonothrombolysis yields an 85% higher lysis rate compared with the single-frequency treatment, and the lysis rate of dual-frequency sonothrombolysis increases with the difference between the two frequency components. These findings suggest a dual-frequency excitation technique for more efficient intravascular sonothrombolysis than conventional single-frequency excitation.

Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples., (© 2021 Marcel et al.; Published by Cold Spring Harbor Laboratory Press.)

As blood clots age, many thrombolytic techniques become less effective. To fully evaluate these techniques for potential clinical use, a large animal aged-clot model is needed. Previous minimally invasive attempts to allow clots to age in an in vivo large animal model were unsuccessful because of the clot clearance associated with relatively high level of cardiac health of readily available research pigs. Prior models have thus subsequently used invasive surgical techniques with the associated morbidity, animal stress and cost. We propose a method for forming sub-acute venous blood clots in an in-vivo porcine model. The age of the clots can be controlled and varied. By using an intravenous scaffold to anchor the clot to the vessel wall during the aging process, we can show that sub-acute clots can consistently be formed with a minimally invasive, percutaneous approach. The clot formed in this study remained intact for at least 1 wk in all subjects. Therefore, we established a new minimally invasive, large animal aged-clot model for evaluation of thrombolytic techniques., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Recent in vitro work has revealed that a forward-viewing intravascular (FVI) transducer has sonothrombolysis applications. However, the safety of this device has yet to be evaluated. In this study, we evaluated the safety of this device in terms of tissue heating, vessel damage and particle debris size during sonothrombolysis using microbubbles or nanodroplets with tissue plasminogen activator, in both retracted and unretracted blood clots. The in vitro and ex vivo sonothrombolysis tests using FVI transducers revealed a temperature rise of less than 1°C, no vessel damage as assessed by histology and no downstream clot particles >500 µm. These in vitro and ex vivo results indicate that the FVI transducer poses minimal risk for sonothrombolysis applications and should be further evaluated in animal models., Competing Interests: Conflict of interest disclosure X.J. has financial interest in Sonovascular, Inc. (Chapel Hill, NC, USA) which licensed an intravascular sonothrombolysis technology from North Carolina State. P.D. is an inventor on several patents describing the low-boiling-point phase change nanodroplets described here, and is a co-founder of Triangle Biotechnology, Inc. (Chapel Hill, NC, USA), which has licensed these patents. Z.X. has financial interest in HistoSonics., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Microbubble contrast agents are commonly used for therapeutic and diagnostic imaging applications. Under certain conditions, these contrast agents can coalesce on ultrasound application and form larger bubbles than the initial population. The formation of large microbubbles potentially influences therapeutic outcomes and imaging quality. We studied clinically relevant ultrasound parameters related to low-pressure therapy and contrast-enhanced ultrasound imaging to determine their effect on microbubble coalescence and subsequent changes in microbubble size distributions in vitro. Results indicate that therapeutic ultrasound at low frequencies, moderate pressures and high duty cycles are capable of forming bubbles greater than two times larger than the initial bubble distribution. Furthermore, acoustic parameters related to contrast-enhanced ultrasound imaging that are at higher frequency, low-pressure and low-duty cycle exhibit no statistically significant changes in bubble diameter, suggesting that standard contrast ultrasound imaging does not cause coalescence. Overall, this work suggests that the microbubble coalescence phenomenon can readily occur at acoustic parameters used in therapeutic ultrasound, generating bubbles much larger than those found in commercial contrast agents, although coalescence is unlikely to be significant in diagnostic contrast-enhanced ultrasound imaging. This observation warrants further expansion of parameter ranges and investigation of resulting effects., Competing Interests: Conflict of interest disclosure P.A.D. declares co-founder interest in SonoVol, Inc., and Triangle Biotechnology, Inc. Other authors declare no conflict of interest., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

We aim to develop a nanodroplet (ND)-mediated intravascular ultrasound (US) transducer for deep vein thrombosis treatments. The US device, having an efficient forward directivity of the acoustic beam, is capable of expediting the clot dissolution rate by activating cavitation of NDs injected onto a thrombus. We designed and prototyped a multi-pillar piezoelectric stack (MPPS) transducer composed of four piezoelectric stacks. Each stack was made of five layers of PZT-4 plates, having a dimension of 0.85 × 0.85 × 0.2 mm 3 . The transducer was characterized by measuring the electrical impedance and acoustic pressure, compared to simulation results. Next, in-vitro tests were conducted in a blood flow mimicking system using the transducer equipped with an ND injecting tube. The miniaturized transducer, having an aperture size of 2.8 mm, provided a high mechanical index of 1.52 and a relatively wide focal zone of 3.4 mm at 80 V pp , 0.96 MHz electric input. The mass-reduction rate of the proposed method (NDs + US) was assessed to be 4.1 and 4.6 mg/min with and without the flow model, respectively. The rate was higher than that (1.3-2.7 mg/min) of other intravascular ultrasound modalities using micron-sized bubble agents. The ND-mediated intravascular sonothrombolysis using MPPS transducers was demonstrated with an unprecedented lysis rate, which may offer a new clinical option for DVT treatments. The MPPS transducer generated a high acoustic pressure (~3.1 MPa) at a distance of approximately 2.2 wavelengths from the small aperture, providing synergistic efficacy with nanodroplets for thrombolysis without thrombolytic agents., (Copyright © 2021. Published by Elsevier B.V.)

This paper reports a novel technique using the rotational magnetic field oscillation and low-intensity sub-megahertz ultrasound stimulation of magnetic microbubbles (MMBs) to promote the nanodroplets (NDs) phase transition and improve the permeation of NDs into the blood clot fibrin network to enhance the sonothrombolysis efficiency. In this study, the influence of different treatment methods with a combination of MMBs and NDs on the thrombolysis rate of both unretracted and retracted clots were investigated, including the stable and inertial cavitation, tPA effects, MMBs/NDs concentration ratio, sonication factors (input voltage, duty cycle) and rotational magnetic field factors (flux density, frequency). We demonstrated that tPA-mediated magneto-sonothrombolysis in combining NDs with MMBs could significantly enhance in vitro lysis of both unretracted clots (85 ± 8.3%) and retracted clots (57 ± 6.5%) in a flow model with 30 min treatment. The results showed that the combination of MMBs and NDs substantially improves in vitro lysis of blood clots with an unprecedented lysis rate., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acoustic angiography is a superharmonic contrast-enhanced ultrasound imaging method that produces high-resolution, 3-D maps of the microvasculature. Previous acoustic angiography studies have used twoelement, annular,mechanicallyactuated transducers(called "wobblers") to image microvasculature in preclinical tumor models with high contrast-to-tissue ratio and resolution, but these earlywobbler transducerscould not achieve the depth and sensitivity required for clinical acoustic angiography. In this work, we present a system for performing acoustic angiography with a novel dual-frequency(DF) transducer-a coaxially stacked DF array (DFA). We evaluate the DFA system bothin vitro andin vivo and demonstrate improvements in sensitivity and imaging depth up to 13.1 dB and 10 mm, respectively, compared with previous wobbler probes.

Superharmonic imaging with dual-frequency imaging systems uses conventional low-frequency ultrasound transducers on transmit, and high-frequency transducers on receive to detect higher order harmonic signals from microbubble contrast agents, enabling high-contrast imaging while suppressing clutter from background tissues. Current dual-frequency imaging systems for superharmonic imaging have been used for visualizing tumor microvasculature, with single-element transducers for each of the low- and high-frequency components. However, the useful field of view is limited by the fixed focus of single-element transducers, while image frame rates are limited by the mechanical translation of the transducers. In this article, we introduce an array-based dual-frequency transducer, with low-frequency and high-frequency arrays integrated within the probe head, to overcome the limitations of single-channel dual-frequency probes. The purpose of this study is to evaluate the line-by-line high-frequency imaging and superharmonic imaging capabilities of the array-based dual-frequency probe for acoustic angiography applications in vitro and in vivo. We report center frequencies of 1.86 MHz and 20.3 MHz with -6 dB bandwidths of 1.2 MHz (1.2-2.4 MHz) and 14.5 MHz (13.3-27.8 MHz) for the low- and high-frequency arrays, respectively. With the proposed beamforming schemes, excitation pressure was found to range from 336 to 458 kPa at its azimuthal foci. This was sufficient to induce nonlinear scattering from microbubble contrast agents. Specifically, in vitro contrast channel phantom imaging and in vivo xenograft mouse tumor imaging by this probe with superharmonic imaging showed contrast-to-tissue ratio improvements of 17.7 and 16.2 dB, respectively, compared to line-by-line micro-ultrasound B-mode imaging.