A pilot study to assess markers of renal damage in the rodent kidney after exposure to 7 MHz ultrasound pulse sequences designed to cause microbubble translation and disruption

Acoustic radiation force has been proposed as a mechanism to enhance microbubble concentration for therapeutic and molecular imaging applications. It is hypothesized that once microbubbles are localized, bursting them with acoustic pressure could result in local drug delivery. It is known that low-frequency, high-amplitude acoustic energy combined with cavitation nuclei can result in bioeffects. However, little is known about the bioeffects potential of acoustic parameters involved in radiation force and microbubble destruction pulse sequences applied at higher frequencies. In this pilot study, rat kidneys are exposed to high-duty cycle, low-amplitude pulse sequences known to cause substantial bubble translation due to radiation force, as well as high-amplitude short pulse sequences known to cause microbubble destruction. Both studies are performed at 7 MHz on a clinical ultrasound system, and implemented in three-dimensions (3-D) for entire kidney exposure. Analysis of biomarkers of renal injury and renal histopathology indicate that there was no significant renal damage due to these ultrasound parameters in conjunction with microbubbles within the study group.     

Localized in Vivo Model Drug Delivery with Intravascular Ultrasound and Microbubbles

An intravascular ultrasound (IVUS) and microbubble drug delivery system was evaluated in both ex vivo and in vivo swine vessel models. Microbubbles with the fluorophore DiI embedded in the shell as a model drug were infused into ex vivo swine arteries at a physiologic flow rate (105 mL/min) while a 5-MHz IVUS transducer applied ultrasound. Ultrasound pulse sequences consisted of acoustic radiation force pulses to displace DiI-loaded microbubbles from the vessel lumen to the wall, followed by higher-intensity delivery pulses to release DiI into the vessel wall. Insonation with both the acoustic radiation force pulse and the delivery pulse increased DiI deposition 10-fold compared with deposition with the delivery pulse alone. Localized delivery of DiI was then demonstrated in an in vivo swine model. The theoretical transducer beam width predicted the measured angular extent of delivery to within 11%. These results indicate that low-frequency IVUS catheters are a viable method for achieving localized drug delivery with microbubbles.     

Ultrasound-driven microbubble oscillation and translation within small phantom vessels

The use of ultrasound radiation force to manipulate microbubbles in blood vessels has attracted recent interest as a method to increase the efficiency of ultrasonic molecular imaging and drug delivery. However, recent studies indicate that microbubble oscillation is diminished within small blood vessels, and therefore we investigate microbubble oscillation and translation within 12 μm vessels using high-speed photography. With each 0.1- to 1-MPa ultrasound pulse, microbubbles (radius of 1, 1.5 and 2 μm) within 12 μm tubes translate 5 to 10 times less than those within 200 μm tubes. Application of a pulse train with a high pulse repetition frequency displaces bubbles to the wall of 12- and 200-μm tubes within an interval (1 s) that is reasonable for clinical translation. Modeling of coupled oscillation and translation for unconstrained microbubbles, based on a modified Rayleigh-Plesset (RP) and the trajectory equations, is compared with experimental observations and demonstrates agreement for the larger displacements observed within the 200 μm tubes. This study has implications for contrast-assisted ultrasound applications, aiding the manipulation of targeted microbubbles and for further theoretical understanding of the complex bubble dynamics within constrained vessel. (E-mail: kwferrara@ucdavis.edu)     

The Mechanical Properties of Ex Vivo Bovine and Porcine Crystalline Lenses: Age-Related Changes and Location-Dependent Variations

The mechanical properties of ex vivo animal lenses from three groups were evaluated: old bovine (25–30 mo old, n = 4), young bovine (6 mo old, n = 4) and young porcine (6 mo old, n = 4) eye globes. We measured the dynamics of laser-induced microbubbles created at different locations within the crystalline lenses. An impulsive acoustic radiation force was applied to the microbubble, and the microbubble displacements were measured using a custom-built high pulse repetition frequency ultrasound system. Based on the measured dynamics of the microbubbles, Young's moduli of bovine and porcine lens tissue in the vicinity of the microbubbles were reconstructed. Age-related changes and location-dependent variations in the Young's modulus of the lenses were observed. Near the center, the old bovine lenses had a Young's modulus approximately fivefold higher than that of young bovine and porcine lenses. The gradient of Young's modulus with respect to radial distance was observed in the lenses from three groups.     

超声破坏微泡促进骨骼肌血管新生的时间-效应关系研究

目的 探讨超声破坏微泡促进大鼠骨骼肌血管新生的时间-效应关系.方法 将48只SD大鼠随机分为4组(超声破坏微泡组、单纯超声组、单纯微泡组、对照组),超声破坏微泡组经尾静脉输入自制的脂质全氟丙烷微泡造影剂0.5 ml,同时用1.0 MHz、2.0 W/cm2的超声在其大腿骨骼肌局部作用3min;单纯超声组仅在骨骼肌局部用同等的超声能量作用;单纯微泡组仅由尾静脉输入脂质全氟丙烷微泡造影剂0.5 ml;对照组不作任何处理.实验结束后的第3、7、10、14、21、28 d,各组分别取两只大鼠处死,取局部骨骼肌行石蜡切片HE染色观察显微结构的变化,CD34免疫组化计数微血管密度(MVD),酶联免疫吸附试验(ELISA)定量评价血管内皮生长因子(VEGF)的表达.结果 超声破微泡组有较多的血管新生;单纯超声组新生血管较少;单纯微泡组和对照组几乎无血管新生.随着时间的变化,超声破坏微泡组的MVD值和VEGF表达在第10 d达到高峰;单纯超声组的高峰出现在第14 d.结论 超声破坏微泡可刺激骨骼肌中内源性VEGF较快、较多地分泌,从而更好地促进新生血管生成.     

Vibro-acoustography: quantification of flow with highly-localized low-frequency acoustic force

The intersection of two ultrasound beams with slightly different frequencies results in generation of a localized radiation force and stimulates emission of audio signals from targeted objects. Vibro-acoustography uses this phenomenon to probe elastic properties of objects. Vibro-acoustography of contrast microbubbles in degassed water produced quantitative flow measurements from analysis of their acoustic emission. We used a dual-beam transducer generating bursts of 40-kHz vibrations. The vibrations resulted from interference of 3.48-MHz and 3.52-MHz confocal beams intersecting at the center of a thin plastic conduit. We tested flows of 13,48, 85, and 120 mL/min of contrast microbubbles at concentrations from 1.2 x 10(5) to 6 x 10(9) bubbles/mL. The amplitude of the acoustic emission was linear with microbubble concentrations up to a value of 3.6 x 10(5) bubbles/mL. A replenishment method for microbubble contrast and flow rate analysis was used with radiation force bursts deployed at 0.05, 0.1, 0.2, (.5, 1, and 2-second pulsing intervals. The relation between the pulsing intervals and the peak amplitude was fitted by an exponential curve and a rate constant calculated for each tested flow rate. The rate constant values were linearly correlated with the tested flows. The vibro-acoustography method provides objective, quantitative, and highly-localized assessment of flow using contrast microbubbles.     

New Insights into Mechanisms of Sonothrombolysis Using Ultra-High-Speed Imaging

Thrombotic arterial occlusion is the principal etiology for acute cardiovascular syndromes such as stroke, myocardial infarction and unstable angina. Exposing the thrombus to ultrasound and microbubbles facilitates thrombus disruption, making “sonothrombolysis” a potentially powerful therapeutic strategy for thromboembolic diseases. However, optimization of such a strategy, and hence clinical translation, is constrained by an incomplete understanding of mechanisms by which ultrasound-induced microbubble vibrations disrupt blood clots. We posit that previously reported sonothrombolytic efficacy using inertial cavitation regimes was due, at least in part, to mechanical clot disruption by oscillating microbubbles. To test this hypothesis, we optically characterized lipid microbubble interactions with thrombus in the presence of ultrasound using a recently developed ultra-high-speed microscopy imaging system to visualize microbubble acoustic behaviors at megahertz frame rates. A microscope/acoustic stage designed for the system allowed an experimentally created thrombus and microbubbles to be insonified at a co-localized acoustic and optical focus during synchronized high-speed imaging. Under inertial cavitation conditions, large-amplitude microbubble oscillations caused thrombus deformation and pitting. Acoustic radiation forces (Bjerknes forces) further augmented microbubble-thrombus interaction. These observations suggest that a direct mechanical effect of oscillating lipid microbubbles on an adjacent thrombus may play a role in mediating clot disruption in the presence of specific ultrasound conditions.     

Dual Frequency Method for Simultaneous Translation and Real-Time Imaging of Ultrasound Contrast Agents Within Large Blood Vessels

A dual frequency excitation method for simultaneous translation and selective real-time imaging of microbubbles is presented. The method can distinguish signals originating from free flowing and static microbubbles. This method is implemented on a programmable scanner with a broadband linear array. The programmable interface allows for dynamic variations in the acoustic parameters and aperture attributes, enabling application of this method to large blood vessels located at varying depths. The performance of the method was evaluated in vitro (vessel diameter 2mm) by quantifying the sensitivity of the method to various acoustic, microbubble, and fluid flow parameters. It was observed that the static microbubble response maximized at the approximate resonance frequency of the microbubble population (estimated from a coulter counter measurement), thus, signifying the need for dual frequency excitation. The static microbubble signal declined from 25 to 12 dB with increasing centerline flow velocities (2.65–15.9cm/s); indicating the applicable range of flow velocities. The maximum intensity of the static microbubbles signal scaled with variations in the microbubble concentration. The rate of increment of static microbubble signal was independent of microbubble concentration. It was deduced that the rate of increment of the static microbubble signal is primarily a function of the pulse frequency, whereas the maximum static microbubble signal intensity is dependent on three parameters: (a) the pulse frequency, (b) the flow velocity and (c) the microbubble concentration. The proposed dual frequency sequence may enable the application of radiation force for optimizing the effect of targeted imaging and modulating drug delivery in large blood vessels with high flow velocities. (E-mail: avp2b@virginia.edu)     

超声辐照和SonoVue微泡在介导hAng-1基因体外转染时的作用和量效关系探讨

目的 探讨超声辐照和SonoVue微泡分别使用和联用在介导hAng-1基因体外转染过程中的作用以及辐照强度和微泡浓度对转染效率和细胞活性的影响.方法 实验分四组A组:单纯超声辐照+质粒组;B组:微泡+质粒组;C组:超声辐照+微泡+质粒组和空白对照组D组. C组内转染参数分别设置为超声照射强度0.5、1.0 、1.5和2.0 W/cm~2,微泡浓度5%、10%、20%、30%和40%.将连接有eGFP-C_3-hAng-1质粒的SonoVue微泡对293T细胞进行转染,48 h后检测各组基因转染效率和细胞存活率. 结果转染48 h后C组转染效率最高,荧光阳性细胞数最多,强度最大;A组转染效率很低,见少量荧光表达;B、D组无明显基因转染发生.随着超声照射强度和微泡浓度的增加,基因转染效率会逐步升高,具有统计学意义.微泡浓度大于20%、超声照射强度超过1.5 W/cm~2后基因转染效率不再升高甚至降低,细胞死亡率显著增高(P<0.01).结论 SonoVue微泡介导外源基因转染必须联合超声辐照才能获得较好的转染效率.对于hAng-1基因和SonoVue微泡,选择声强1.5 W/cm~2,微泡浓度20%是相对最佳转染条件.     

Functional Activity and Endothelial-Lining Integrity of Ex Vivo Arteries Exposed to Ultrasound-Mediated Microbubble Destruction

Ultrasound-mediated microbubble destruction (UMMD) is a promising strategy to improve local drug delivery in specific tissues. However, acoustic cavitation can lead to harmful bioeffects in endothelial cells. We investigated the side effects of UMMD treatment on vascular function (contraction and relaxation) and endothelium integrity of ex vivo Wistar rat arteries. We used an isolated organ system to evaluate vascular responses and confocal microscopy to quantify the integrity and viability of endothelial cells. The arteries were exposed for 1–3 min to ultrasound at a 100 Hz pulse-repetition frequency, 0.5 MPa acoustic pressure, 50% duty cycle and 1%–5% v/v microbubbles. The vascular contractile response was not affected. The acetylcholine-dependent maximal relaxation response was reduced from 78% (control) to 60% after 3 min of ultrasound exposure. In arteries treated simultaneously with 1 min of ultrasound exposure and 1%, 2%, 3% or 5% microbubble concentration, vascular relaxation was reduced by 19%, 58%, 80% or 93%, respectively, compared with the control arteries. Fluorescent labeling revealed that apoptotic death, detachment of endothelial cells and reduced nitric oxide synthase phosphorylation are involved in relaxation impairment. We demonstrated that UMMD can be a safe technology if the correct ultrasound and microbubble parameters are applied. Furthermore, we found that tissue-function evaluation combined with cellular analysis can be useful to study ultrasound–microbubble–tissue interactions in the optimization of targeted endothelial drug delivery.     

Ultrafast Imaging of Ultrasound Contrast Agents

The disappearance of ultrasound contrast agents after disruption can provide useful information on their environment. However, in vivo acoustical imaging of this transient phenomenon, which has a duration on the order of milliseconds, requires high frame rates that are unattainable by conventional ultrasound scanners. In this article, ultrafast imaging is applied to microbubble tracking using a 128-element linear array and an elastography scanner. Contrast agents flowing in a wall-less tissue phantom are insonified with a high-intensity disruption pulse followed by a series of plane waves emitted at a 5 kHz PRF. A collection of compounded images depicting the evolution of microbubbles is obtained after the echoes are beamformed in silico. The backscattering of the microbubbles appears to increase in the first image after disruption (4 ms) and decrease following an exponential decay in the next hundred milliseconds. This microbubble dynamic depends on the length and amplitude of the high-intensity pulse. Furthermore, confined microbubbles are found to differ significantly from their free-flowing counterparts in their dissolution curves. The high temporal resolution provided by ultrafast imaging could help distinguish targeted microbubbles during molecular imaging. (E-mail: olicou@gmail.com)     

Nanointerrogation of ultrasonic contrast agent microbubbles using atomic force microscopy

Predicting the acoustic response of an encapsulated microbubble to ultrasound requires an accurate assessment of the mechanical properties of the microbubble shell. Atomic force microscopy (AFM) provides an unprecedented spatial and force resolution of the order of Angstroms and subnanonewtons, respectively. It is introduced here as a means to interrogate microbubbles manufactured for ultrasonic imaging. The advantage of AFM over scanning electron microscopy (SEM) is that the microbubbles need not be subjected to a low temperature or low-pressure environment. The microbubbles were interrogated in a liquid environment, which could potentially be a simulated physiological environment. AFM was used in tapping mode imaging to reveal topographical detail of biSphere microbubbles. Because microbubbles are large objects compared with the overall size of usual AFM tips, a convolution between the AFM tip and the microbubble was typical of the acquired topographies. However, a part of the top half of the bubble was imaged with nanometer resolution, and roughness measurements are reported. Force-distance curves were captured using contact mode AFM. The range of stiffness or effective spring constant of biSphere was found to be between 1 and 6 N m(-1). In conclusion, the AFM is proposed here for the first time as a tool to image the surface of bubbles at the nanometer range in liquid and to perform reproducible measurements on the mechanical properties of individual microbubbles.     

Effects of Nonlinear Propagation in Ultrasound Contrast Agent Imaging

This paper investigates two types of nonlinear propagation and their effects on image intensity and contrast-to-tissue ratio (CTR) in contrast ultrasound images. Previous studies have shown that nonlinear propagation can occur when ultrasound travels through tissue and microbubble clouds, making tissue farther down the acoustic path appear brighter in pulse inversion (PI) images, thus reducing CTR. In this study, the effect of nonlinear propagation through tissue or microbubbles on PI image intensity and CTR are compared at low mechanical index. A combination of simulation and experiment with SonoVue microbubbles were performed using a microbubble dynamics model, a laboratory ultrasound system and a clinical prototype scanner. The results show that, close to the bubble resonance frequency, nonlinear propagation through a bubble cloud of a few centimeter thickness with a modest concentration (1:10000 dilution of SonoVue microbubbles) is much more significant than through tissue-mimicking material. Consequently, CTR in regions distal to the imaging probe is greatly reduced for nonlinear propagation through the bubble cloud, with as much as a 12-dB reduction compared with nonlinear propagation through tissue-mimicking material. Both types of nonlinear propagation cause only a small change in bubble PI signals at the bubble resonance frequency. When the driving frequency increases beyond bubble resonance, nonlinear propagation through bubbles is greatly reduced in absolute values. However because of a greater reduction in nonlinear scattering from bubbles at higher frequencies, the corresponding CTR is much lower than that at bubble resonance frequency. (E-mail: mengxing.tang@imperial.ac.uk)     

Ultrasound-targeted microbubble destruction enhances naked plasmid DNA transfection in rabbit Achilles tendons in vivo

The study was to investigate the probability of increasing the transfection of the gene in tendons by ultrasound-targeted microbubble destruction (UTMD), and to search for the most suitable transfection conditions. A mixture of microbubbles and enhanced green fluorescent protein (EGFP) plasmids was injected into rabbit Achilles tendons by different administration routes and the tendons were ultrasound pulse by different ultrasonic conditions in order to determine the most appropriate conditions. Then, the rabbits were divided into four groups: (1) ultrasound + microbubbles + plasmid; (2) ultrasound+ plasmid; (3) microbubble + plasmid; (4) plasmid only. EGFP expression in the tendons and other tissues, and the damage to tendon and paratenon were all observed. The results showed that EGFP expression in the tendon was higher by ultrasound pulse with 2 W cm(-2) of output intensity and a 20% duty cycle for 10 min. Local injection was determined to be the better administration route. Among the four groups, EGFP expression in Group 1 was higher than that in other groups. EGFP expression was highest on seventh day, then it gradually decrease over time, and lasted more than 56 days. EGFP expression was not found in other tissues. There was no obvious injury caused by UTMD. Under suitable conditions, it is feasible to use UTMD as a safe and effective gene transfection therapy for tendon injuries.     

Nonlinear behaviors of contrast agents relevant to diagnostic and therapeutic applications

The nonlinear properties of an encapsulated microbubble of a contrast agent were studied theoretically and experimentally. A modified nonlinear differential equation (Herring equation) was used to describe the radial oscillation of the microbubble and solved numerically. It was found that the nonlinear resonance frequency, at which the peak radial oscillation amplitude occurs, was a decreasing function of the acoustic amplitude of a driving ultrasonic pulse. Optical images of the contrast agent microbubbles under various ultrasonic exposure conditions: 1. sham exposure; 2. 2-MHz spatial peak acoustic pressure = 200 kPa, I(SATA) = 260 mW/cm(2), duty cycle = 7.5%, repetition period = 0.0266 ms; 3. 0.5-MHz spatial peak acoustic pressure = 200 kPa, I(SATA) = 130 mW/cm(2), duty cycle = 7.5%, repetition period = 0.1067 ms; have also shown that the lower-frequency ultrasound (US) excitation (0.5 MHz) is more effective in disruption of the microbubbles due to acoustic inertial cavitation than the higher frequency US (2 MHz).     

Transluminal Approach with Bubble-Seeded Histotripsy for Cancer Treatment with Ultrasonic Mechanical Effects

Bubble-seeded histotripsy (BSH) is a newly developed ultrasound-based mechanical fractionation technique using locally injected phase change nanodroplets (PCNDs) as sensitizers. The PCNDs are a kind of microbubble precursor compressed into submicron-size in droplets form, which were designed for local administration and will expand into microbubbles under ultrasound exposure. Previously, we reported that a combination of PCNDs injection and pulsed high-intensity focused ultrasound (pHIFU) with an acoustic intensity as low as about 3 kW/cm² at 1.1?MHz, which is similar to the acoustic intensity of currently available HIFU coagulation therapy, was enough to induce tissue fractionation after significant antitumor effects in an in vivo study. Toward therapeutic application of BSH to deep-seated tissues such as the pancreas, the transluminal approach, using endoscopic ultrasound was thought to be ideal. Therefore, for a preliminary examination, we developed a new transducer with a small aperture (20-?×?20-mm square) and long focal length (35?mm), operating at 2.1?MHz that could be attached to an EUS-mimicking probe. With the newly developed transducer and locally injected PCNDs, predictable tissue mechanical fractionation was observed in both ex vivo and in vivo studies at acoustic intensities that were too low to induce any significant bioeffects (around 4 kW/cm²) without using PCNDs. For in situ monitoring of the treatment site during a procedure, the degree of attenuation of microbubble motions after exposing the microbubbles to pHIFU was monitored, using ultrafast echographic imaging. Microbubble movements were observed to be largest at 25–30?s after pHIFU exposure. On the contrary, after 40?s, the movement of microbubbles decreased to the same level as at the start of the procedure, suggesting that an overdose of pHIFU exposure causes coagulation attributable to the thermal effect caused by absorption of the energy. Those results were promising for expanding the application of BSH for a transluminal approach, using a small transducer under real-time monitoring.     

Investigation of microbubble response to long pulses used in ultrasound-enhanced drug delivery

In current drug delivery approaches, microbubbles and drugs can be co-administered while ultrasound is applied. The mechanism of microbubble interaction with ultrasound, the drug and the cells is not fully understood. The aim of this study was to investigate microbubble response to long ultrasonic pulses used in drug delivery approaches. Two different in vitro set-ups were considered: with the microbubbles diluted in an enclosure and with the microbubbles flowing in a capillary tube. Acoustic streaming, which influences the observed bubble response, was observed in “typical” drug delivery conditions in the first set-up. With the capillary set-up, streaming effects were avoided and accurate bubble responses were recorded. The diffraction pattern of the source greatly influences the bubble response and in different locations of the field different bubble responses are observed. At low nondestructive pressures, microbubbles can oscillate for thousands of cycles repeatedly. At high acoustic pressures (at 1 MHz), most bubble activity disappeared within about 100 μs despite the length of the pulse, mainly due to violent bubble destruction and subsequent accelerated diffusion.     

Ultrasound Molecular Imaging of Cardiovascular Disease

Molecular imaging with ultrasound contrast agents relies on the detection of microbubbles within diseased tissue. Microbubbles produce an acoustic signal owing to their resonant properties in an ultrasound field. Microbubble targeting is accomplished by either manipulating the microbubble shell for attachment of microbubbles to activated leukocytes, or by conjugation of disease-specific ligands to the microbubble surface. Inflammation, angiogenesis, and thrombus formation are central pathophysiologic processes in many cardiovascular diseases and produce phenotypic changes in the vascular compartment that can be imaged with targeted ultrasound contrast agents. In the future, targeted contrast ultrasound could aid in the diagnosis of atherosclerosis, myocardial ischemia, transplant rejection, and thrombosis syndromes and could be used for assessing angiogenesis.     

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.