An Inverse Method Using Cross-Spectral Matrix Fitting for Passive Cavitation Imaging.

High-intensity focused ultrasound (HIFU) can produce cavitation, which requires monitoring for specific applications such as sonoporation, targeted drug delivery, or histotripsy. Passive acoustic mapping has been proposed in the literature as a method for monitoring cavitation, but it lacks spatial resolution, primarily in the axial direction, due to the absence of a time reference. This is a common issue with passive imaging compared to standard pulse-echo ultrasound. In order to improve the axial resolution, we propose an adaptation of the cross spectral matrix fitting (CMF) method for passive cavitation imaging, which is based on the resolution of an inverse problem with different regularizations that promote sparsity in the reconstructed cavitation maps: Elastic Net (CMF-ElNet) and sparse Total Variation (CMF-spTV). The results from both simulated and experimental data are presented and compared to state-of-the-art approaches, such as the frequential delay-and-sum (DAS) and the frequential robust capon beamformer (RCB). We show the interest of the method for improving the axial resolution, with an axial full width half maximum (FWHM) divided by 3 and 5 compared to RCB and DAS, respectively. Moreover, CMF-based methods improve contrast-to-noise ratio (CNR) by more than 15 dB in experimental conditions compared to RCB. We also show the advantage of the sparse Total Variation (spTV) prior over Elastic Net (ElNet) when dealing with cloud-shaped cavitation sources, that can be assumed as sparse grouped sources.

3-D Passive Cavitation Imaging Using Adaptive Beamforming and Matrix Array Transducer With Random Apodization.

With the development of promising cavitation-based treatments, the interest in cavitation monitoring with passive acoustic mapping (PAM) is significantly increasing. While most of studies regarding PAM are performed in 2-D, 3-D imaging modalities are getting more attention relying on either custom-made or commercial matrix probes. Unless specific phased-arrays are used for a specific application, limitations due to probe apertures often results in poor performances of the 3-D mapping, due to the use of a delay-and-sum (DAS) classic beamformer, which results in strong artifacts and large main lobe sizes. In this article, 3D-PAM is achieved by performing adaptive beamforming in the frequency domain (FD) in 3-D, and using a random sparse apodization of a commercial matrix array driving only 256 elements among the 1024 available. It reduces the computation time and makes use of only one 256-channel research platform. Three beamformers have been implemented in 3-D and in the FD: the DAS beamformer, which corresponds to the beamformer used in previous 3D-PAM studies, the robust capon beamformer (RCB), an adaptive algorithm widely used in 2D-PAM for its high performances, and the MidWay (MW) beamformer, an adaptive algorithm with a computation complexity equivalent to the one of DAS. These algorithms are evaluated both in simulations and experiments with a harmonic source at different positions, and are also applied to real cavitation signals. The results show that, in the case of matrix arrays of small aperture such as generic commercial matrix probes, the DAS beamformer leads to large main lobe sizes, while adaptive beamformers largely improve the performances of the mapping. The low computation time and its parameter-free character make MW beamformer a good compromise for 3D-PAM applications. It thus appears that a random sparse apodization combined with adaptive beamforming is a good solution to achieve high-performance 3D-PAM with manageable devices.

Stimulation of dental implant osseointegration by low-Intensity pulsed ultrasound: An in vivo preliminary study in a porcine model.

PURPOSE: Several studies have evaluated the interest of Low Intensity Pulsed Ultrasound (LIPUS) in the osseointegration of dental implants in murine or rabbit models. However, the thinness and narrowness bones make it difficult to study the effect of LIPUS. The purpose of this study is to assess the ability of LIPUS to stimulate bone formation in contact with a titanium dental implant in a porcine model. METHODS: Eight adults mini-pigs were used. An implant is placed on each tibial crest in the metaphysis. The right side was treated with LIPUS at 1 MHz and 300 mW/cm2 of acoustic intensity during 15 minutes per day on 5 consecutive days and during 42 days. The left side was not treated. The Bone Volume/Total Volume ratio (BV/TV), the Intersection Surface (IS) of the volume of interest by the binarized bone and the Trabecular bone Thickness (TbTh) around the implant were analyzed. RESULTS: At 42 days, BV/TV ratio is significantly higher on the treated side (42,1+/-8,76% versus 32,31+/-10,11%, p < 0,02); as well as TbTh with 0,13+/-0,01 mm versus 0,10+/-0,01 mm (p < 0,01). IS is also significantly higher on the treated side (40,7 +/- 12,68 mm2 versus 33,68+/-9,44 mm2 at 200 µm from the implant surface; p < 0,01). CONCLUSION: The present study showed that LIPUS can significantly increase bone formation and accelerate the healing process at the bone-implant interface in a porcine model. Its low toxicity, low immunogenicity and non-invasion make it a complementary treatment of choice for improving the bone formation around titanium implants.

Experimental and numerical investigation of pulsed flows in asevere aortic stenosed model.

Steady and pulsatile aortic stenotic flows through stenosis tubes were experimentally and numerically investigated. The objective was the understanding of the fluid dynamics in arterial geometries most relevant in the context of atherosclerosis. Axisymmetric phantoms corresponding to significant artery stenosis of 50% in diameter and severe aortic stenosis of 75% were respectively machined from silicon. A water flow circuit was established, a steady flow was provided by gravity and a pulsed flow by a pulsatile pump. At inlet Reynolds numbers in the range of 85 to 1125, flows at the stenosis region were investigated using two-component Particle Image Velocimetry (PIV). For the unsteady flow, three different heartbeats (60, 69 and 90 beats per minute) were considered. The k-ω shear-stress-transport first-order turbulence model in Computational Fluid Dynamics (CFD) commercial software was adopted for simulations. Experimental measurements of the velocity fields show good agreements with CFD for both steady and pulsed flows. Recirculation regions were found near the stenosis in both cases. Reverse flow through the stenosis was also observed in pulsatile flow during the end diastolic phase of the cycle. CFD simulations allowed us to accurately assess wall shear stress in the stenotic region where the optical measurements are very noisy. High values of wall shear stress (with high variations both in space and time), are observed, which are indicators of possible future aortic wall damage.

Stimulation of oral mucosal regeneration by low intensity pulsed ultrasound: an in vivo study in a porcine model.

PURPOSE: Many studies have shown the ability of low intensity pulsed ultrasound (LIPUS) to stimulate the bone, cartilage and tendon regeneration but only a few studied LIPUS interest in the regeneration of the oral mucosa. The purpose of this study is to assess the ability of LIPUS to stimulate the regeneration of the palatal mucosa in a porcine model. METHODS: Ten adults mini-pigs were used. Two mucosal wounds were realised on the left and right side of the palate of each pig. The right side was treated with LIPUS at 1 MHz of frequency and 300 mW/cm2 of acoustic intensity. The left side was not treated. The morphology of the wound was evaluated using a polymer silicone molding. RESULTS: The difference between two sides was significant from day 7 with a p value < 0.0001. At day 21, the wound is completely healed on all pigs with LIPUS. The control soft tissue defect exposed a healing of 80%. CONCLUSIONS: The present study showed that the use of LIPUS on the oral mucosa accelerates the healing of the masticatory mucosa.

Use of the Cross-Spectral Density Matrix for Enhanced Passive Ultrasound Imaging of Cavitation.

Passive ultrasound imaging is of great interest for cavitation monitoring. Spatiotemporal monitoring of cavitation bubbles in therapeutic applications is possible using an ultrasound imaging probe to passively receive the acoustic signals from the bubbles. Fourier-domain (FD) beamformers have been proposed to process the signals received into maps of the spatial localization of cavitation activity, with reduced computing times with respect to the time-domain approach, and to take advantage of frequency selectivity for cavitation regime characterization. The approaches proposed have been mainly nonadaptive, and these have suffered from low resolution and contrast, due to the many reconstruction artifacts. Inspired by the array-processing literature and in the context of passive ultrasound imaging of cavitation, we propose here a robust estimation of the second-order statistics of data through spatial covariance matrices in the FD or cross-spectral density matrices (CSMs). The benefits of such formalism are illustrated using advanced reconstruction algorithms, such as the robust Capon beamformer, the Pisarenko class beamformer, and the multiple signal classification approach. Through both simulations and experiments in a water tank, we demonstrate that enhanced localization of cavitation activity (i.e., improved resolution and contrast with respect to nonadaptive approaches) is compatible with the rapid and frequency-selective approaches of the FD. Robust estimation of the CSM and the derived adaptive beamformers paves the way to the development of powerful passive ultrasound imaging tools.

TRPV4 promotes acoustic wave-mediated BBB opening via Ca2+/PKC-δ pathway.

INTRODUCTION: Numerous studies have shown the ability of low-energy acoustic waves such as focused ultrasound or shockwave to transiently open blood-brain barrier (BBB) and facilitate drug delivery to the brain. Preclinical and clinical evidences have well demonstrated the efficacy and safety in treating various brain disorders. However, the molecular mechanisms of acoustic waves on the BBB are still not fully understood. OBJECTIVES: The present study aimed at exploring the possible molecular mechanisms of acoustic wave stimulation on brains. METHODS BRIEFLY DESCRIBE THE EXPERIMENTAL DESIGN: The left hemisphere of the rat's brain was treated with pulsed ultrasound from a commercial focused shockwave or a planar ultrasound device, and the right hemisphere served as a control. One hour after the mechanical wave stimulation or overnight, the rats were sacrificed and the brains were harvested for protein or histological analysis. Agonists and antagonists related to the signal transduction pathways of tight junction proteins were used to investigate the possible intracellular mechanisms. RESULTS: Intracellular signal transduction analysis shows calcium influx through transient receptor potential vanilloid 4 (TRPV4) channels, and the activation of PKC-δ pathway to mediate dissociation of ZO-1 and occludin after acoustic wave stimulation. The activation of TRPV4 or PKC-δ signaling further increased the expression level of TRPV4, suggesting a feedback loop to regulate BBB permeability. Moreover, the tight junction proteins dissociation can be reversed by administration of PKC-δ inhibitor and TRPV4 antagonist. CONCLUSION: The present study shows the crucial role of TRPV4 in acoustic wave-mediated BBB permeability, specifically its effect on compromising tight junction proteins, ZO-1 and occludin. Our findings provide a new molecular perspective to explain acoustic wave-mediated BBB opening. Moreover, activation of TRPV4 by agonists may reduce the threshold intensity level of acoustic waves for BBB opening, which may prevent undesirable mechanical damages while maintaining efficient BBB opening.

Nonspherical modes nondegeneracy of a tethered bubble.

When excited at sufficiently high acoustic pressures, a wall-attached bubble may exhibit asymmetric nonspherical modes. These vibration modes can be decomposed over the set of spherical harmonics Y_{nm}(θ,ϕ) for a degree n and order m. We experimentally capture the time-resolved dynamics of asymmetric bubble oscillations in a top-view configuration. A spatiotemporal modal analysis is performed and allowed recovering the set of zonal (m=0), tesseral (0

Weighting the Passive Acoustic Mapping Technique With the Phase Coherence Factor for Passive Ultrasound Imaging of Ultrasound-Induced Cavitation.

Ultrasound (US) cavitation is currently being explored for low-invasive therapy techniques applied to a wide panel of pathologies. Because of the random behavior of cavitation, a real-time spatial monitoring system may be required. For this purpose, the US passive imaging techniques have been recently investigated. In particular, the passive acoustic mapping (PAM) beamforming method enables the reconstruction of cavitation activity maps by beamforming acoustic signals passively recorded by an array transducer. In this paper, an optimized version of PAM, PAM weighted with a phase coherence factor (PAM-PCF), is considered. A general validation process is developed including simulations on a point source and experiments on a wire. Furthermore, using a focused regulated US-induced cavitation generator, reproducible cavitation experiments are conducted in water and in agar gel. The spatial behavior of a bubble cavitation cloud is determined using the PAM-PCF beamforming method to localize the focal cavitation point in two perpendicular imaging planes.

Parametric Shape Optimization of Lens-Focused Piezoelectric Ultrasound Transducers.

Focused transducers composed of flat piezoelectric ceramic coupled with an acoustic lens present an economical alternative to curved piezoelectric ceramics and are already in use in a variety of fields. Using a displacement/pressure (u/p) mixed finite element formulation combined with parametric level-set functions to implicitly define the boundaries between the materials and the fluid-structure interface, a method to optimize the shape of acoustic lens made of either one or multiple materials is presented. From that method, two 400 kHz focused transducers using acoustic lens were designed and built with different rapid prototyping methods, one of them made with a combination of two materials, and experimental measurements of the pressure field around the focal point are in good agreement with the presented model.

Ultrafast monitoring and control of subharmonic emissions of an unseeded bubble cloud during pulsed sonication.

In the aim of limiting the destructive effects of collapsing bubbles, the regime of stable cavitation activity is currently targeted for sensitive therapeutic applications such as blood-brain barrier opening by ultrasound. This activity is quantified through the emergence of the subharmonic component of the fundamental frequency. Due to the intrinsically stochastic behavior of the cavitation phenomenon, a better control of the different (stable or inertial) cavitation regimes is a key requirement in the understanding of the mechanisms involving each bubble-induced mechanical effect. Current strategies applied to stable cavitation control rely on the use of either seeded microbubbles or a long-lasting pulse to reinitiate subharmonic emission. The present work aims at developing an ultrafast (inferior to 250 µs) monitoring and control of subharmonic emissions during long-pulsed (50 ms) sonication. The use of a FPGA-based feedback loop provides reproducible level of subharmonic emissions combined with temporal stability during the sonication duration. In addition, stable cavitation events are differentiated from the broadband noise characterizing inertial cavitation activity, with perspectives in the discrimination of the involved mechanisms underlying bubble-mediated therapeutic applications.

PIV for the characterization of focused field induced acoustic streaming: seeding particle choice evaluation.

This research evaluates the use of Particle Image Velocimetry (PIV) technique for characterizing acoustic streaming flow generated by High Intensity Focused Ultrasound (HIFU). PIV qualification tests, focusing on the seeding particle size (diameter of 5, 20 and 50µm) were carried out in degassed water subjected to a focused field of 550kHz-frequency with an acoustic pressure amplitude of 5.2, 10.5 and 15.7bar at the focus. This study shows that the ultrasonic field, especially the radiation force, can strongly affect seeding particle behavior. Large particles (50µm-diameter) are repelled from the focal zone and gathered at radiation pressure convergence lines on either side of the focus. The calculation of the acoustic radiation pressure applied on these particles explains the observed phenomenon. PIV measurements do not, therefore, properly characterize the streaming flow in this case. On the contrary, small particles (5µm-diameter) velocity measurements were in good agreement with the Computational Fluid Dynamics (CFD) simulations of the water velocity field. A simple criterion approximating the diameter threshold below which seeding particles are qualified for PIV in presence of focused ultrasound is then proposed.

Hysteresis of inertial cavitation activity induced by fluctuating bubble size distribution.

Amongst the variety of complex phenomena encountered in nonlinear physics, a hysteretic effect can be expected on ultrasound cavitation due to the intrinsic nonlinearity of bubble dynamics. When applying successive ultrasound shots for increasing and decreasing acoustic intensities, a hysteretic behaviour is experimentally observed on inertial cavitation activity, with a loop area sensitive to the inertial cavitation threshold. To get a better insight of the phenomena underlying this hysteretic effect, the evolution of the bubble size distribution is studied numerically by implementing rectified diffusion, fragmentation process, rising and dissolution of bubbles from an initial bubble size distribution. When applying increasing and decreasing acoustic intensities, the numerical distribution exhibits asymmetry in bubble number and distribution. The resulting inertial cavitation activity is assessed through the numerical broadband noise of the emitted acoustic radiation of the bubble cloud dynamics. This approach allows obtaining qualitatively the observed hysteretic effect and its interest in terms of control is discussed.

Jumping acoustic bubbles on lipid bilayers.

In the context of sonoporation, we use supported lipid bilayers as a model for biological membranes and investigate the interactions between the bilayer and microbubbles induced by ultrasound. Among the various types of damage caused by bubbles on the surface, our experiments exhibit a singular dynamic interaction process where bubbles are jumping on the bilayer, forming a necklace pattern of alteration on the membrane. This phenomenon was explored with different time and space resolutions and, based on our observations, we propose a model for a microbubble subjected to the combined action of van der Waals, acoustic and hydrodynamic forces. Describing the repeated jumps of the bubble, this model explains the lipid exchanges between the bubble and bilayer.

Sonoporation of adherent cells under regulated ultrasound cavitation conditions.

A sonoporation device dedicated to the adherent cell monolayer has been implemented with a regulation process allowing the real-time monitoring and control of inertial cavitation activity. Use of the cavitation-regulated device revealed first that adherent cell sonoporation efficiency is related to inertial cavitation activity, without inducing additional cell mortality. Reproducibility is enhanced for the highest sonoporation rates (up to 17%); sonoporation efficiency can reach 26% when advantage is taken of the standing wave acoustic configuration by applying a frequency sweep with ultrasound frequency tuned to the modal acoustic modes of the cavity. This device allows sonoporation of adherent and suspended cells, and the use of regulation allows some environmental parameters such as the temperature of the medium to be overcome, resulting in the possibility of cell sonoporation even at ambient temperature.

In vitro demonstration of focused ultrasound thrombolysis using bifrequency excitation.

Focused ultrasound involving inertial cavitation has been shown to be an efficient method to induce thrombolysis without any pharmacological agent. However, further investigation of the mechanisms involved and further optimization of the process are still required. The present work aims at studying the relevance of a bifrequency excitation compared to a classical monofrequency excitation to achieve thrombolysis without any pharmacological agent. In vitro human blood clots were placed at the focus of a piezoelectric transducer. Efficiency of the thrombolysis was assessed by weighing each clot before and after sonication. The efficiencies of mono- (550 kHz) and bifrequency (535 and 565 kHz) excitations were compared for peak power ranging from 70 W to 220 W. The thrombolysis efficiency appears to be correlated to the inertial cavitation activity quantified by passive acoustic listening. In the conditions of the experiment, the power needed to achieve 80% of thrombolysis with a monofrequency excitation is reduced by the half with a bifrequency excitation. The thermal effects of bifrequency and monofrequency excitations, studied using MR thermometry measurements in turkey muscle samples where no cavitation occurred, did not show any difference between both types of excitations when using the same power level.

Ultrasound enhanced PEI-mediated gene delivery through increasing the intracellular calcium level and PKC-δ protein expression.

PURPOSE: Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. It has also been shown that combining ultrasound (US) with PEI could enhance and prolong in vitro and in vivo transgene expression. However, the role US in the enhancement of PEI uptake is poorly understood. This study investigates the impact of US on PEI-mediated gene transfection. METHODS: Specific endocytosis pathway siRNA, including clathrin HC siRNA, caveolin-1 siRNA and protein kinase C-delta (PKC-δ) siRNA, are used to block the corresponding endocytosis pathways prior to the transfection of luciferase DNA/PEI polyplexes to cultured cells by 1-MHz pulsed US with ultrasound contrast agent SonoVue®. RESULTS: Transgene expression was found not to be enhanced by US treatment in the presence of the PKC-δ siRNA. We further demonstrated that PKC-δ protein could be enhanced at 6 h after US exposure. Moreover, intracellular calcium levels were found to be significantly increased at 3 h after US exposure, while transgene expressions were significantly reduced in the presence of calcium channel blockers both in vitro and in vivo. CONCLUSIONS: Our results suggest that US enhanced PEI-mediated gene transfection specifically by increasing PKC-δ related fluid phase endocytosis, which was induced by increasing the intracellular calcium levels.

Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation.

It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 µg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection.

Orbital trajectory of an acoustic bubble in a cylindrical resonator.

Acoustic cavitation-induced microbubbles in a cylindrical resonator filled with water tend to concentrate into ring patterns due to the cylindrical geometry of the system. The shape of these ring patterns is directly linked to the Bjerknes force distribution in the resonator. Experimental observations showed that cavitation bubbles located in the vicinity of this ring may exhibit a spiraling behavior around the pressure nodal line. This spiraling phenomenon is numerically studied, the conditions for which a single cavitation bubble follows an orbital trajectory are established, and the influences of the acoustic pressure amplitude and the initial bubble radius are investigated.

Control of inertial acoustic cavitation in pulsed sonication using a real-time feedback loop system.

Owing to the complex behavior of ultrasound-induced bubble clouds (nucleation, linear and nonlinear oscillations, collapse), acoustic cavitation remains a hardly controllable phenomenon, leading to poorly reproducible ultrasound-based therapies. A better control of the various aspects of cavitation phenomena for in vivo applications is a key requirement to improve emerging ultrasound therapies. Previous publications have reported on systems performing regulation of acoustic cavitation in continuous sonication when applied in vitro, but the main challenge today is to achieve real-time control of cavitation activity in pulsed sonication when used in vivo. The present work aims at developing a system to control acoustic cavitation in a pulsed wave condition using a real-time feedback loop. The experimental setup consists of a water bath in which is submerged a focused transducer (pulsed waves, frequency 550 kHz) used for sonication and a hydrophone used to listen to inertial cavitation. The designed regulation process allows the cavitation activity to be controlled through a 300 µs feedback loop. Without regulation, cavitation exhibits numerous bursts of intense activity and large variations of inertial cavitation level over time. In a regulated regime, the control of inertial cavitation activity within a pulse leads to consistent cavitation levels over time with an enhancement of the reproducibility.