Investigation of the Acoustic Vaporization Threshold of Lipid-Coated Perfluorobutane Nanodroplets Using Both High-Speed Optical Imaging and Acoustic Methods

A combination of ultrahigh-speed optical imaging (5 × 10⁶ frames/s), B-mode ultrasound and passive cavitation detection was used to study the vaporization process and determine both the acoustic droplet vaporization (ADV) and inertial cavitation (IC) thresholds of phospholipid-coated perfluorobutane nanodroplets (PFB NDs, diameter = 237 ± 16 nm). PFB NDs have not previously been studied with ultrahigh-speed imaging and were observed to form individual microbubbles (1–10 μm) within two to three cycles and subsequently larger bubble clusters (10–50 μm). The ADV and IC thresholds did not statistically significantly differ and decreased with increasing pulse length (20–20,000 cycles), pulse repetition frequency (1–100 Hz), concentration (10⁸–10¹⁰ NDs/mL), temperature (20°C–45°C) and decreasing frequency (1.5–0.5 MHz). Overall, the results indicate that at frequencies of 0.5, 1.0 and 1.5 MHz, PFB NDs can be vaporized at moderate peak negative pressures (<2.0 MPa), pulse lengths and pulse repetition frequencies. This finding is encouraging for the use of PFB NDs as cavitation agents, as these conditions are comparable to those required to achieve therapeutic effects with microbubbles, unlike those reported for higher-boiling-point NDs. The differences between the optically and acoustically determined ADV thresholds, however, suggest that application-specific thresholds should be defined according to the biological/therapeutic effect of interest.     

Functional ultrasound-triggered phase-shift perfluorocarbon nanodroplets for cancer therapy

In recent years, because of their unique properties, the use of perfluorocarbon nanodroplets (PFC NDs) in ultrasound-mediated tumor theranostics has attracted increasing interest. PFC is one of the most stable organic compounds with high hydrophobicity. Phase-shift PFC NDs can be transformed into highly echogenic microbubbles for ultrasound and photoacoustic imaging by ultrasound and laser light. In addition, in the process of acoustic droplet vaporization, PFC NDs with cavitation nuclei can be combined with a variety of ultrasound technologies to produce cavitation effects for tumor ablation, antivascular therapy and release of therapeutic agents loaded in nanodroplets. Moreover, they can also be used to overcome tumor hypoxia by virtue of high oxygen solubility. In this review, first the preparation and stabilization of PFC NDs are summarized and then the issues and outlook are discussed. More importantly, multifunctional platforms based on PFC NDs for cancer diagnostics, therapy and theranostics are reviewed in detail.     

Characterization of Submicron Phase-change Perfluorocarbon Droplets for Extravascular Ultrasound Imaging of Cancer

Because many tumors possess blood vessels permeable to particles with diameters of 200 nm, it is possible that submicron perfluorocarbon droplets could constitute a novel extravascular ultrasound contrast agent capable of selectively enhancing tumors. Under exposure to bursts of ultrasound of sufficient rarefactional pressure, droplets can undergo vaporization to form echogenic microbubbles. In this study, phase-change thresholds of 220-nm–diameter droplets composed of perfluoropentane were studied in polyacrylamide gel phantoms maintained at temperatures of 21–37°C, exposed to high-pressure bursts of ultrasound with frequencies ranging from 5–15 MHz and durations of 1 μs to 1 ms. The thresholds were found to depend inversely and significantly (p < 0.001) on ultrasound frequency, pulse duration, and droplet temperature, ranging from 9.4 ± 0.8 MPa at 29°C for a 1-μs burst at 5 MHz to 3.2 ± 0.5 MPa at 37°C for a 1-ms burst at 15 MHz. The diameters of microbubbles formed from droplets decreased significantly as phantom stiffness increased (p < 0.0001), and were independent of pulse duration, although substantially more droplets were converted to microbubbles for 1-ms pulse durations compared with briefer exposures. In vivo experiments in a mouse tumor model demonstrated that intravenously injected droplets can be converted into highly echogenic microbubbles 1 h after administration.     

Investigation of vaporized submicron perfluorocarbon droplets as an ultrasound contrast agent

Acoustically activated submicron droplets of liquid perfluorocarbon are investigated as a new class of ultrasound contrast agent. In the liquid state, intravascular droplets can extravasate within tumours. Activation is then accomplished by using bursts of ultrasound to vaporize the droplets. We use acoustical and optical techniques to assess the characteristics of vaporized droplets and the resulting microbubbles in vitro, including size, conversion threshold, echogenicity and nonlinearity. Under exposure to single 5-50 cycle bursts of ultrasound at 7.5 MHz and mechanical index <1.0, droplets with mean diameter of 400 nm convert into microbubbles with mean diameter of 1.4 μm at 1 ms after vaporization, expanding to 5.6 μm by 1 s. The growth of microbubbles produced by vaporization causes a characteristic time-dependent increase in linear and nonlinear echogenicity, enabling selective detection with conventional bubble-specific imaging. These results suggest that submicron perfluorocarbon droplets, activated in situ, may be a candidate for an extravascular ultrasound contrast agent.     

The speed of sound and attenuation of an IEC agar-based tissue-mimicking material for high frequency ultrasound applications

This study characterized the acoustic properties of an International Electromechanical Commission (IEC) agar-based tissue mimicking material (TMM) at ultrasound frequencies in the range 10–47 MHz. A broadband reflection substitution technique was employed using two independent systems at 21°C ± 1°C. Using a commercially available preclinical ultrasound scanner and a scanning acoustic macroscope, the measured speeds of sound were 1547.4 ± 1.4 m?s⁻¹ and 1548.0 ± 6.1 m?s⁻¹, respectively, and were approximately constant over the frequency range. The measured attenuation (dB?cm⁻¹) was found to vary with frequency f (MHz) as 0.40f + 0.0076f². Using this polynomial equation and extrapolating to lower frequencies give values comparable to those published at lower frequencies and can estimate the attenuation of this TMM in the frequency range up to 47 MHz. This characterisation enhances understanding in the use of this TMM as a tissue equivalent material for high frequency ultrasound applications.     

Size Measurement of Tissue Debris Particles Generated from Pulsed Ultrasound Cavitational Therapy – Histotripsy

Extensive mechanical tissue fractionation can be achieved using successive high intensity ultrasound pulses (“histotripsy”). Histotripsy has many potential medical applications where noninvasive tissue removal is desired (e.g., tumor ablation). There is a concern that debris generated by histotripsy-induced tissue fractionation might be an embolization hazard. The aim of this study is to measure the size distribution of these tissue debris particles. Histotripsy pulses were produced by a 513-element 1 MHz array transducer, an 18-element 750 kHz array transducer and a 788 kHz single element transducer. Peak negative pressures of 11 to 25 MPa, pulse durations of 3 to 50 cycles, pulse repetition frequencies of 100 Hz to 2 kHz were used. Tissue debris particles created by histotripsy were collected and measured with a particle sizing system. In the resulting samples, debris <6 μm in diameter constituted >99% of the total number of tissue particles. The largest particle generated by one of the parameter sets tested was 54 μm in diameter, which is smaller than the clinic filter size (100 μm) used to prevent embolization. The largest particles generated using other parameter sets were larger than 60 μm but the value could not be specified using our current setup. Exposures with shorter pulses produced lower percentages of large tissue debris (>30 μm) in comparison to longer pulses. These results suggest that the tissue debris particle size distribution is adjustable by altering acoustic parameters if necessary. (E-mail: zhenx@umich.edu)     

The Effect of Binding on the Subharmonic Emissions from Individual Lipid-Encapsulated Microbubbles at Transmit Frequencies of 11 and 25 MHz

Targeted microbubble imaging at ultrasound frequencies above 5 MHz has applications in both a preclinical context for a range of disease processes and clinically for the assessment of atherosclerosis and superficial tumors. Although the feasibility of ultrasound molecular imaging has been well demonstrated for a range of target molecules, little work has examined the effects of binding on microbubble oscillations, which is of potential relevance to improving the sensitivity, specificity, and quantification of bound-bubble detection. In this study we investigated the influence of binding on the subharmonic response of bubbles at transmit frequencies of 11 and 25 MHz. Individual bubbles were situated adjacent to a boundary in either a bound or an unbound state, optically sized and acoustically interrogated with pressures ranging from 0.02 to 1.2 MPa. At 11 MHz, unbound bubbles (n = 53) were found to have strong subharmonic activity for sizes between 2.4 and 2.6 μm, whereas bound bubbles (n = 50) were most active from 2.6 to 3.0 μm. Destruction thresholds were found to be lower for bound bubbles. At 25 MHz, bound-bubble (n = 57) activity was found to peak at 1.9 μm as compared to 2.1 μm in the unbound cases (n = 53), with a 20% increase in amplitude. Comparison with simulations indicates that both unbound and bound bubbles undergo compression-only behavior at 11 MHz, and expansion-dominated behavior at 25 MHz. Subharmonic emissions elicited from 0 radian transmit pulses were found to be π/2 radians out of phase with those elicited from a π radian transmit pulse, suggesting inefficient subharmonic preservation from pulse inversion schemes. With the appropriate postprocessed phase correction, an increase in the subharmonic amplitude of up to 60% was shown, depending on the bubble size and transmit frequency.     

Hemorrhage in murine fetuses exposed to pulsed ultrasound

In the late-gestation fetal mouse, exposure to piezoelectric lithotripter fields at amplitudes < 1 MPa produced hemorrhages in tissues near developing bone, such as the head and limbs. This study was undertaken to determine if exposure to pulsed ultrasound at diagnostic frequencies produces similar hemorrhages in the late-gestation fetal mouse. On the 18th day of gestation, fetal mice were exposed in utero to pulsed ultrasound with a 10-micros pulse duration and 100-Hz pulse repetition frequency for a total exposure duration of 3 min. Hemorrhages occurred most often to the developing fetal head. At 1.2 MHz, a threshold for hemorrhage to the fetal head was determined at positive exposure pressures of approximately 4 MPa and corresponding negative pressures of approximately 2.5 MPa. The threshold increased with at least the first power of frequency.     

Microbubble Type and Distribution Dependence of Focused Ultrasound-Induced Blood–Brain Barrier Opening

Focused ultrasound, in the presence of microbubbles, has been used non-invasively to induce reversible blood–brain barrier (BBB) opening in both rodents and non-human primates. This study was aimed at identifying the dependence of BBB opening properties on polydisperse microbubble (all clinically approved microbubbles are polydisperse) type and distribution by using a clinically approved ultrasound contrast agent (Definity microbubbles) and in-house prepared polydisperse (IHP) microbubbles in mice. A total of 18 C57 BL/6 mice (n = 3) were used in this study, and each mouse was injected with either Definity or IHP microbubbles via the tail vein. The concentration and size distribution of activated Definity and IHP microbubbles were measured, and the microbubbles were diluted to 6 × 10⁸/mL before injection. Immediately after microbubble administration, mice were subjected to focused ultrasound with the following parameters: frequency = 1.5 MHz, pulse repetition frequency = 10 Hz, 1000 cycles, in situ peak rarefactional acoustic pressures = 0.3, 0.45 and 0.6 MPa for a sonication duration of 60 s. Contrast-enhanced magnetic resonance imaging was used to confirm BBB opening and allowed for image-based analysis. Permeability of the treated region and volume of BBB opening did not significantly differ between the two types of microbubbles (p > 0.05) at peak rarefractional acoustic pressures of 0.45 and 0.6 MPa, whereas IHP microbubbles had significantly higher permeability and opening volume (p < 0.05) at the relatively lower pressure of 0.3 MPa. The results from this study indicate that microbubble type and distribution could have significant effects on focused ultrasound-induced BBB opening at lower pressures, but less important effects at higher pressures, possibly because of the stable cavitation that governs the former. This difference may have become less significant at higher pressures, where inertial cavitation typically occurs.     

Nonlinear Emission from Individual Bound Microbubbles at High Frequencies

Targeted microbubbles detected with high-frequency ultrasound can establish the molecular expression of blood vessels with submillimeter resolution. To improve microbubble-specific imaging at high frequencies, the subharmonic and second harmonic signal from individual microbubbles were measured as a function of size and pressure. Single phospholipid-shell microbubbles (1.1 to 5.0 μm in diameter) bound to gelatin, co-aligned with an optical microscope and transducer, were insonated with 30 MHz Gaussian-enveloped pulses at pressures from 20 kPa to 1 MPa with –6 dB one-way bandwidths of 11%, 20% and 45%. A subharmonic signal (15 MHz) was detected above a pressure threshold of 110 kPa—independent of bandwidth. The signal peaked for microbubbles 1.60 μm in diameter subject to 20% and 11% bandwidth pulses, and 1.80 μm for 45% bandwidth pulses, for pressures up to 400 kPa, agreeing with the notion that microbubbles insonated at twice their resonant frequency preferentially emit a subharmonic component. For pressures between 400 kPa and 1 MPa, a broader range of microbubbles emitted a subharmonic signal, and microbubbles below 1.70 μm in diameter were disrupted. The second harmonic signal measured, within the limited experimental conditions, was consistent with nonlinear propagation. Further, the results shed light on the effect of the shell on the phase of the subharmonic signal with respect to the fundamental signal. (E-mail: michael.sprague@sri.utoronto.ca)     

Acoustic Sizing of an Ultrasound Contrast Agent

Because the properties of ultrasound contrast agent populations after administration to patients are largely unknown, methods able to study them noninvasively are required. In this study, we acoustically performed a size distribution measurement of the ultrasound contrast agent Definity^®. Single lipid-shelled microbubbles were insonified at 25 MHz, which is considerably higher than their resonance frequency, so that their acoustic responses depended on their geometrical cross sections only. We calculated the size of each microbubble from their measured backscattered pressures. The acoustic size measurements were compared with optical reference size measurements to test their accuracy. Our acoustic sizing method was applied to 88 individual Definity^® bubbles to derive a size distribution of this agent. The size distribution obtained acoustically showed a mean diameter (2.5 μm) and a standard deviation (0.9 μm) in agreement within 8% with the optical reference measurement. At 25 MHz, this method can be applied to bubble sizes larger than 1.2 μm in diameter. It was observed that similar sized bubbles can give different responses (up to a factor 1.5), probably because of shell differences. These limitations should be taken into account when implementing the method in vivo. This acoustic sizing method has potential for estimating the size distribution of an ultrasound contrast agent noninvasively. (E-mail: d.maresca@erasmusmc.nl)     

HIFU-induced cavitation and heating in ex vivo porcine subcutaneous fat

The present study is motivated by the fact that there are no published studies quantifying cavitation activity and heating induced by ultrasound in adipose tissue and that there are currently no reliable techniques for monitoring successful deposition of ultrasound energy in fat in real time. High-intensity focused ultrasound (HIFU) exposures were performed in excised porcine fat at four different frequencies (0.5, 1.1, 1.6 and 3.4 MHz) over a range of pressure amplitudes and exposure durations. The transmission losses arising from reflection at the skin interface and attenuation through skin and fat were quantified at all frequencies using an embedded needle hydrophone. A 15 MHz passive cavitation detector (PCD) coaxial to the HIFU transducer was used to capture acoustic emissions emanating from the focus during HIFU exposures, while the focal temperature rise was measured using minimally invasive needle thermocouples. Repeatable temperature rises in excess of 10°C could be readily instigated across all four frequencies for acoustic intensities (Ispta) in excess of 50 W/cm² within the first 2 s of exposure. Even though cavitation could not be initiated at 1.1, 1.6 and 3.4 MHz over the in situ peak rarefactional (p_-) pressure range 0-3 MPa explored in the present study, inertial cavitation activity was always initiated at 0.5 MHz for pressures greater than 1.6 MPa (p_-) and was found to enhance focal heat deposition. A good correlation was identified between the energy of broadband emissions detected by the PCD and the focal temperature rise at 0.5 MHz, particularly for short 2 s exposures, which could be exploited as a tool for noninvasive monitoring of successful treatment delivery. (E-mail: zoe.kyriakou@eng.ox.ac.uk)     

Subharmonic contrast microbubble signals for noninvasive pressure estimation under static and dynamic flow conditions

Our group has proposed the concept of subharmonic aided pressure estimation (SHAPE) utilizing microbubble-based ultrasound contrast agent signals for the noninvasive estimation of hydrostatic blood pressures. An experimental system for in vitro SHAPE was constructed based on two single-element transducers assembled confocally at a 60 degree angle to each other. Changes in the first, second and subharmonic amplitudes of five different ultrasound contrast agents were measured in vitro at static hydrostatic pressures from 0-186 mmHg, acoustic pressures from 0.35-0.60 MPa peak-to-peak and frequencies of 2.5-6.6 MHz. The most sensitive agent and optimal parameters for SHAPE were determined using linear regression analysis and implemented on a Logiq 9 scanner (GE Healthcare, Milwaukee, WI). This implementation of SHAPE was then tested under dynamic-flow conditions and compared to pressure-catheter measurements. Over the pressure range studied, the first and second harmonic amplitudes reduced approximately 2 dB for all contrast agents. Over the same pressure range, the subharmonic amplitudes decreased by 9-14 dB and excellent linear regressions were achieved with the hydrostatic pressure variations (r = 0.98, p < 0.001). Optimal sensitivity was achieved at a transmit frequency of 2.5 MHz and acoustic pressure of 0.35 MPa using Sonazoid (GE Healthcare, Oslo, Norway). A Logiq 9 scanner was modified to implement SHAPE on a convex transducer with a frequency range from 1.5-4.5 MHz and acoustic pressures from 0-3.34 MPa. Results matched the pressure catheter (r2 = 0.87). In conclusion, subharmonic contrast signals are a good indicator of hydrostatic pressure. Out of the five ultrasound contrast agents tested, Sonazoid was the most sensitive for subharmonic pressure estimation. Real-time SHAPE has been implemented on a commercial scanner and offers the possibility of allowing pressures in the heart and elsewhere to be obtained noninvasively.     

Investigating the subharmonic response of individual phospholipid encapsulated microbubbles at high frequencies: a comparative study of five agents

There are a range of contrast ultrasound applications above 10 MHz, a frequency regime in which nonlinear microbubble behavior is poorly understood. Lipid-encapsulated microbubbles have considerable potential for use at higher frequencies because they have been shown to exhibit pronounced nonlinear activity at frequencies up to 40 MHz. The objective of this work was to investigate the influence of agent formulation on the subharmonic response of lipid-encapsulated microbubbles at high frequencies with a view to providing information relevant to improving contrast agent design and imaging performance. An optical-acoustical setup was used to measure the subharmonic emissions from small (d < 3 μm) individual lipid-encapsulated microbubbles as a function of transmit pressure, size and composition. In this study, five agent formulations (Definity™, MicroMarker™ and three in-house agents manipulated to exhibit different levels of shell microstructure heterogeneity) were insonified at 25 MHz over a peak negative pressure (P_n) range of 0.02–1.2 MPa. All agents exhibited distinctly different subharmonic behavior, both in terms of amplitude and active sizes. MicroMarker™ exhibited the strongest, broadest and most consistent subharmonic response, 22% greater in power than that of Definity™ and as much as 50% greater than the in-house formulations. No clear relation between in-house agents’ shell microstructure and nonlinear response was found, other than the variability in the nonlinear response itself. An analysis of the response of MicroMarker™ bubbles suggests that these bubbles exhibit “expansion-dominated” oscillations, in contrast to “compression-only” oscillations observed for similar bubbles at lower frequencies (f < 11 MHz).     

Observations of translation and jetting of ultrasound-activated microbubbles in mesenteric microvessels

High-speed photomicrography was used to study the translational dynamics of single microbubbles in microvessels of ex vivo rat mesenteries. The microbubbles were insonated by a single 2 μs ultrasound pulse with a center frequency of 1 MHz and peak negative pressures spanning the range of 0.8-4 MPa. The microvessel diameters ranged from 10-80 μm. The high-speed image sequences show evidence of ultrasound-activated microbubble translation away from the nearest vessel wall; no microbubble showed a net translation toward the nearest vessel wall. Microbubble maximum translation displacements exceeded 20 μm. Microjets with the direction of the jets identifiable were also observed; all microjets appear to have been directed away from the nearest vessel wall. These observations appear to be characteristic of a strong coupling between ultrasound-driven microbubbles and compliant microvessels. Although limited to mesenteric tissues, these observations provide an important step in understanding the physical interactions between microbubbles and microvessels.     

Multi-Modality Safety Assessment of Blood-Brain Barrier Opening Using Focused Ultrasound and Definity Microbubbles: A Short-Term Study

As a potentially viable method of brain drug delivery, the safety profile of blood-brain barrier (BBB) opening using focused ultrasound (FUS) and ultrasound contrast agents (UCA) needs to be established. In this study, we provide a short-term (30-min or 5-h survival) histological assessment of murine brains undergoing FUS-induced BBB opening. Forty-nine mice were intravenously injected with Definity microbubbles (0.05 μL/kg) and sonicated under the following parameters: frequency of 1.525 MHz, pulse length of 20 ms, pulse repetition frequency of 10 Hz, peak rarefactional acoustic pressures of 0.15–0.98 MPa and two 30-s sonication intervals with an intermittent 30-s delay. The BBB opening threshold was found to be 0.15–0.3 MPa based on fluorescence and magnetic resonance imaging of systemically injected tracers. Analysis of three histological measures in hematoxylin and eosin–stained sections revealed the safest acoustic pressure to be within the range of 0.3–0.46 MPa in all examined time periods post sonication. Across different pressure amplitudes, only the samples 30 min post opening showed significant difference (p < 0.05) in the average number of distinct damaged sites, microvacuolated sites, dark neurons and sites with extravasated erythrocytes. Enhanced fluorescence around severed microvessels was also noted and found to be associated with the largest tissue effects, whereas mildly diffuse BBB opening with uniform fluorescence in the parenchyma was associated with no or mild tissue injury. Region-specific areas of the sonicated brain (thalamus, hippocampal fissure, dentate gyrus and CA3 area of hippocampus) exhibited variation in fluorescence intensity based on the position, orientation and size of affected vessels. The results of this short-term histological analysis demonstrated the feasibility of a safe FUS-UCA–induced BBB opening under a specific set of sonication parameters and provided new insights on the mechanism of BBB opening. (E-mail: ek2191@columbia.edu)     

Vaporization of perfluorocarbon droplets using optical irradiation

Micron-sized liquid perfluorocarbon (PFC) droplets are currently being investigated as activatable agents for medical imaging and cancer therapy. After injection into the bloodstream, superheated PFC droplets can be vaporized to a gas phase for ultrasound imaging, or for cancer therapy via targeted drug delivery and vessel occlusion. Droplet vaporization has been previously demonstrated using acoustic methods. We propose using laser irradiation as a means to induce PFC droplet vaporization using a method we term optical droplet vaporization (ODV). In order to facilitate ODV of PFC droplets which have negligible absorption in the infrared spectrum, optical absorbing nanoparticles were incorporated into the droplet. In this study, micron-sized PFC droplets loaded with silica-coated lead sulfide (PbS) nanoparticles were evaluated using a 1064 nm laser and ultra-high frequency photoacoustic ultrasound (at 200 and 375 MHz). The photoacoustic response was proportional to nanoparticle loading and successful optical droplet vaporization of individual PFC droplets was confirmed using photoacoustic, acoustic, and optical measurements. A minimum laser fluence of 1.4 J/cm(2) was required to vaporize the droplets. The vaporization of PFC droplets via laser irradiation can lead to the activation of PFC agents in tissues previously not accessible using standard ultrasound-based techniques.     

Sonophoresis using ultrasound contrast agents for transdermal drug delivery: an in vivo experimental study

Sonophoresis temporally increases skin permeability such that various medications can be delivered noninvasively. Previous sonophoresis studies have suggested that cavitation plays an important role in enhancing transdermal drug delivery (TDD). In this study, the feasibility of controlled cavitation using ultrasound contrast agents (UCAs) at high frequency was explored through in vivo experiments in a rat model. Two commercially available UCAs, SonoVue® and Definity®, were used at 2.47 MHz and 1.12 MHz, respectively. Fluorescein isothiocyanate (FITC)-dextran with 0.1% UCA was used as the drug to be delivered through the skin. Ultrasound with a 10 ms pulse and a 1% duty cycle at 1 MPa acoustic pressure for 30 min was applied in all sonication sessions. The efficacy of sonophoresis with UCAs was quantitatively analyzed using an optical imaging system that was used to count photons emitted from fluorescein. The results showed that the proposed sonophoresis method significantly improved drug penetration compared with the traditional sonophoresis method with 4 kD, 20 kD and 150 kD FITC-dextrans at 1.12 MHz, and with 4 kD and 20 kD FITC-dextrans at 2.47 MHz. Sonophoresis for TDD was performed more effectively with the aid of UCAs. Sonophoresis with UCAs has excellent potential for broad applications in drug delivery for diseases requiring the chronic administration of medications such as diabetes.     

"Stable" inertial cavitation.

This note compares theoretical predictions of pressure waves scattered by free gas bubbles with recent acoustical determinations of cavitation thresholds for individual microbubbles of the surfactant-stabilized contrast agent Sonazoid(R). The results indicate that surfactant-coated microbubbles undergo "stable" (i.e., repetitive) inertial cavitation above a threshold of 0.3 to 0.4 MPa at 2.5 MHz, and that irreversible postcollapse bubble fragmentation usually requires much higher pressures (approximately 1.5 MPa). Adverse bioeffects can be expected in vivo far below these fragmentation pressures when contrast agents are present. With diagnostically relevant exposures, the threshold for the generation of petechiae in skeletal muscle is approximately 0.6 MPa at 2.5 MHz.     

Time- and Dose-Dependent Effects of Pulsed Ultrasound on Dermal Repair in Diabetic Mice

Chronic wounds, including diabetic, leg and pressure ulcers, impose a significant health care burden worldwide. Some evidence indicates that ultrasound can enhance soft tissue repair. However, therapeutic responses vary among individuals, thereby limiting clinical translation. Here, effects of pulsed ultrasound on dermal wound healing were assessed using a murine model of chronic, diabetic wounds. An ultrasound exposure system was developed to provide daily ultrasound exposures to full-thickness, excisional wounds in genetically diabetic mice. Wounds were exposed to 1 MHz ultrasound (2 ms pulse, 100 Hz pulse repetition frequency, 0–0.4 MPa) for 2 or 3 wk. Granulation tissue thickness and wound re-epithelialization increased as a function of increasing ultrasound pressure amplitude. At 2 wk after injury, significant increases in granulation tissue thickness and epithelial ingrowth were observed in response to 1 MHz pulsed ultrasound at 0.4 MPa. Wounds exposed to 0.4 MPa ultrasound for 3 wk were characterized by collagen-dense, revascularized granulation tissue with a fully restored, mature epithelium. Of note, only half of wounds exposed to 0.4 MPa ultrasound showed significant granulation tissue deposition after 2 wk of treatment. Thus, the db⁺/db⁺ mouse model may help to identify biological variables that influence individual responses to pulsed ultrasound and accelerate clinical translation.