Presence Of Ultrasound Contrast Agents Research Articles

Ultrasound-mediated transdermal drug delivery of fluorescent nanoparticles and hyaluronic acid into porcine skin in vitro**Project partially supported by the National Natural Science Foundation of China (Grant Nos. 81127901, 81227004, 81473692, 81673995, 11374155, 11574156, 11274170, 11274176, 11474001, 11474161, 11474166, and 11674173), the Natural Science Foundation of Jiangsu Province, China (Grant No. BK2011812), the Fundamental Research Funds for the Central Universities, and

Transdermal drug delivery (TDD) can effectively bypass the first-pass effect. In this paper, ultrasound-facilitated TDD on fresh porcine skin was studied under various acoustic parameters, including frequency, amplitude, and exposure time. The delivery of yellow–green fluorescent nanoparticles and high molecular weight hyaluronic acid (HA) in the skin samples was observed by laser confocal microscopy and ultraviolet spectrometry, respectively. The results showed that, with the application of ultrasound exposures, the permeability of the skin to these markers (e.g., their penetration depth and concentration) could be raised above its passive diffusion permeability. Moreover, ultrasound-facilitated TDD was also tested with/without the presence of ultrasound contrast agents (UCAs). When the ultrasound was applied without UCAs, low ultrasound frequency will give a better drug delivery effect than high frequency, but the penetration depth was less likely to exceed 200 μm. However, with the help of the ultrasound-induced microbubble cavitation effect, both the penetration depth and concentration in the skin were significantly enhanced even more. The best ultrasound-facilitated TDD could be achieved with a drug penetration depth of over 600 μm, and the penetration concentrations of fluorescent nanoparticles and HA increased up to about 4–5 folds. In order to get better understanding of ultrasound-facilitated TDD, scanning electron microscopy was used to examine the surface morphology of skin samples, which showed that the skin structure changed greatly under the treatment of ultrasound and UCA. The present work suggests that, for TDD applications (e.g., nanoparticle drug carriers, transdermal patches and cosmetics), protocols and methods presented in this paper are potentially useful.

Sonoporation efficacy on SiHa cells in vitro at raised bath temperatures-experimental validation of a prototype sonoporation device.

BackgroundA device was devised which aimed to reduce the time and expertise required to perform sonoporation on adherent cell cultures. This prototype device was used to examine the superficial effect of bath temperature on sonoporation efficacy.MethodsThe prototype device consisted of six ultrasound transducers affixed beneath an Opticell stage. Six transducers with nominal diameters of 20 mm were constructed and the acoustic field of each was characterized using hydrophone scanning. A near field treatment plane was chosen for each transducer to minimize field heterogeneity in the near field.Cervical cancer-derived SiHa cells were exposed to nine different treatments in the presence of plasmid DNA-expressing green fluorescent protein (GFP). Ultrasound treatment with Definity ultrasound contrast agent (US+UCA) present, ultrasound treatment without contrast agent present (US), and a sham ultrasound treatment in the presence of ultrasound contrast agent (CA) were each performed at bath temperatures of 37, 39.5, and 42 °C. Each treatment was performed in biological triplicate. GFP expression and PARP expression following treatment were measured using fluorescent microscopy and digital image processing. Cell detachment was measured using phase contrast microscopy before and after treatment.ResultsMean (± s.d.) transfection rates for the US+UCA treatment were 5.4(±0.92), 5.8(±1.3), and 5.3(±1.1) % at 37, 39.5, and 42 °C, respectively. GFP expression and cell detachment were both significantly affected by the presence of ultrasound contrast agent (p < 0.001, p < 0.001). Neither GFP expression, PARP expression, or detachment differed significantly between bath temperatures.ConclusionsBath temperature did not impact the efficacy of sonoporation treatment on SiHa cells in vitro. The prototype device was found to be suitable for performing sonoporation on adherent cell cultures and will reduce the time and expertise required for conducting sonoporation experiments on adherent cell cultures in the future.

Numerical investigation of the nonlinear attenuation and dissipation of acoustic waves in a medium containing ultrasound contrast agents

The presence of microbubble ultrasound contrast agents (UCAs) in path of an ultrasound beam increases the attenuation of the medium. A detailed knowledge about the medium attenuation in the presence of UCAs is critical for optimizing ultrasound applications. In this study, a model incorporating the nonlinear attenuation of microbubbles is developed by deriving the complex and real part of the wave number from the Calfish model. Results showed that when UCAs are sonicated by their linear resonance frequency (fr), the effective attenuation of the medium can potentially decrease as the pressure increases, in agreement with experimental observations of the attenuation of monodisperse microbubbles. When sonicated with their pressure dependent resonance frequency (fs), the effective attenuation of the medium is smaller than the case of sonication with fr, but only below a pressure threshold (Ps). Above Ps, the effective attenuation increases abruptly (e.g. ~3 fold) and becomes significantly more than the attenu...

Contrast-enhanced ultrasound imaging of the vasa vasorum of carotid artery plaque.

The vasa vasorum of carotid artery plaque is a novel marker of accurately evaluating the vulnerability of carotid artery plaque, which was associated with symptomatic cerebrovascular and cardiovascular disease. The presence of ultrasound contrast agents in carotid artery plaque represents the presence of the vasa vasorum in carotid artery plaque because the ultrasound contrast agents are strict intravascular tracers. Therefore, contrast-enhanced ultrasound (CEUS) is a novel and safe imaging modality for evaluating the vasa vasorum in carotid artery plaque. However, there are some issues that needs to be assessed to embody fully the clinical utility of the vasa vasorum in carotid artery plaque with CEUS.

Contrast agent-free sonoporation: The use of an ultrasonic standing wave microfluidic system for the delivery of pharmaceutical agents.

Sonoporation is a useful biophysical mechanism for facilitating the transmembrane delivery of therapeutic agents from the extracellular to the intracellular milieu. Conventionally, sonoporation is carried out in the presence of ultrasound contrast agents, which are known to greatly enhance transient poration of biological cell membranes. However, in vivo contrast agents have been observed to induce capillary rupture and haemorrhage due to endothelial cell damage and to greatly increase the potential for cell lysis in vitro. Here, we demonstrate sonoporation of cardiac myoblasts in the absence of contrast agent (CA-free sonoporation) using a low-cost ultrasound-microfluidic device. Within this device an ultrasonic standing wave was generated, allowing control over the position of the cells and the strength of the acoustic radiation forces. Real-time single-cell analysis and retrospective post-sonication analysis of insonated cardiac myoblasts showed that CA-free sonoporation induced transmembrane transfer of fluorescent probes (CMFDA and FITC-dextran) and that different mechanisms potentially contribute to membrane poration in the presence of an ultrasonic wave. Additionally, to the best of our knowledge, we have shown for the first time that sonoporation induces increased cell cytotoxicity as a consequence of CA-free ultrasound-facilitated uptake of pharmaceutical agents (doxorubicin, luteolin, and apigenin). The US-microfluidic device designed here provides an in vitro alternative to expensive and controversial in vivo models used for early stage drug discovery, and drug delivery programs and toxicity measurements.

Cavitation-Enhanced Extravasation for Drug Delivery

A flow-through tissue-mimicking phantom composed of a biocompatible hydro-gel with embedded tumour cells was used to assess and optimize the role of ultrasound-induced cavitation on the extravasation of a macromolecular compound from a channel mimicking vessel in the gel, namely a non-replicating luciferase-expressing adenovirus (Ad-Luc). Using a 500 KHz therapeutic ultrasound transducer confocally aligned with a focussed passive cavitation detector, different exposure conditions and burst mode timings were selected by performing time and frequency domain analysis of passively recorded acoustic emissions, in the absence and in the presence of ultrasound contrast agents acting as cavitation nuclei. In the presence of Sonovue, maximum ultraharmonic emissions were detected for peak rarefactional pressures of 360 kPa, and maximum broadband emissions occurred at 1250 kPa. The energy of the recorded acoustic emissions was used to optimise the pulse repetition frequency and duty cycle in order to maximize either ultraharmonic or broadband emissions while keeping the acoustic energy delivered to the focus constant. Cell viability measurements indicated that none of the insonation conditions investigated induces cell death in the absence of a therapeutic agent (i.e. virus). Phase contrast images of the tissue-mimicking phantom showed that short range vessel disruption can occur when ultra-harmonic emissions (nf0/2) are maximised whereas formation of a micro-channel perpendicular to the flow can be obtained in the presence of broadband acoustic emissions. Following Ad-Luc delivery, luciferase expression measurements showed that a 60-fold increase in its bioavailability can be achieved when broadband noise emissions are present during insonation, even for modest contrast agent concentrations. The findings of the present study suggest that drug delivery systems based on acoustic cavitation may help enhance the extravasation of anticancer agents, thus increasing their penetration distance to hypoxic regions and poorly vascularised tumour regions.

Acoustic cavitation assisted vascular endothelial growth factor transfection in vitro.

Angiogenesis is a complex process that is mediated by growth factors such as vascular endothelial growth factor (VEGF). Researchers have reported that therapeutic ultrasound (US) is able to promote VEGF production in vitro, with the acoustic cavitation‐induced sonopration. Here, with the presence of ultrasound contrast agents, the vascular endothelia cells mixed with VEGF165 were exposed to 1‐MHz US pulses with varied acoustic parameters. The inertial cavitation (IC) activities accumulated during US exposure were quantified as IC dose (ICD) based on passive cavitation detection. The US sonoporation pores were examined with electron scan microscopy. The VEGF expression and the proliferation of the treated cells were evaluated based on ELISA and MTT assessments, respectively. The results show that (1) the ICD could be affected by US parameters (e.g., pressure, total treatment time, and PRF) and (2) the sonopration pore size, VEGF expression, and the cell proliferation were initially increased with the increasing ICD, then tended to saturate instead of achieving a maximum while the ICD kept going up. The results indicated that IC should play an important role in stimulating VEGF expression through sonopration, and ICD could be used as an effective tool to monitor and control the US‐mediated drug delivery outcomes. [This work was funded by the National Basic Research Program of China (2011CB707900) and NSF of China (11074123 and 10974093).]

Reversible blood–brain barrier disruption by repeated transcranial focused ultrasound allows enhanced extravasation

The permeability of blood–brain barrier (BBB) for albumin can be enhanced by focused ultrasound (FUS) in a targeted region when this is applied in the presence of ultrasound contrast agent (UCA). In this study, we demonstrate that, using this noninvasive treatment, Evans Blue (EB) extravasation can be enhanced by repeated sonication. Sonications were applied at an ultrasound frequency of 1 MHz with a 5% duty cycle, and a repetition frequency of 1 Hz. The brains of male Sprague–Dawley rats were subjected to FUS exposure at the same targeted site. At the same acoustic power, the extravasation caused by leakage of EB into the brain was found to be dependent on the applied sonication time. In vivo, both single and repeated sonications increased the extravasation of the albumin binding EB, especially for the repeated sonication group. In the retreatment experiment, there was a nearly twofold increase in EB extravasation in groups with a second sonication compared with the single sonication group. BBB disruption can be prolonged by repeated FUS sonication and the duration is dependent on the time point of the resonication after the first sonication. Compared to a single sonication, the MR imaging analysis and histological examination of the affected brains indicated that the pattern of contrast enhancement was changed and that vacuolation occurred after repeated sonication. This noninvasive technology offers the possibility of controlling the extent of drug delivery by means of repeated treatment and adjusting the duration and interval between sonications.

Real-time ultrasound image-monitored focal changes of the vascular function in rats by pulsed HIFU

It has been shown that B-mode ultrasound can be useful for the real-time visualization of high-intensity focused ultrasound (HIFU) treatment. The aim of this study is to demonstrate the real-time ultrasound observation of functional changes when a vessel is exposed to pulsed-HIFU in the presence of preformed microbubbles. Using in vivo experiments, 12 male Sprague-Dawley rats were sonicated by 1-MHz pulsed-HIFU in the presence of ultrasound contrast agent (UCA) at four doses (0, 150, 300, and 450 microL/kg). The microbubbles passing through the aorta can be discerned with B-mode imaging. The mean peak systolic velocity (PSV) of the blood flow, as measured by Doppler ultrasound imaging, increased in arteries when the low-dose UCA groups (0 and 150 microL/kg) were examined after pulsed-HIFU at 45 W, but decreased when the high-dose UCA groups (300 and 450 microL/ kg) were examined. Additionally, the normalized pulsatility index (PI) changes increased with the injected dose of UCA. The interactions between ultrasound and the microbubbles can be seen to change the tissue permeability of the drug. Thus, monitoring of PSV or PI might be useful as an online method to ensure the correct sonicated position and to indicate when drug delivery has occurred.

Neurons but not glial cells overexpress ubiquitin in the rat brain following focused ultrasound-induced opening of the blood–brain barrier

Focused ultrasound-induced opening of the blood–brain barrier (BBB) in the presence of ultrasound contrast agents is a promising strategy for a targeted drug delivery to the brain. The aim of our study was to identify whether brain molecular stress pathways are targeted by ultrasound treatment. Using an upper level of acoustic pressures in combination with microbubbles, which have been previously reported as reliable for BBB opening without causing tissue damage, we found that ultrasound leads to an increased ubiquitinylation of proteins in neuronal (11±3 ubiquitin-overexpressing cells per optical field) but not glial cells 6 h post-insonation, increasing to 16 (±4) labelled cells after 24 h. No changes in the expression of Hsp70 and Hsc70 were detected over 24 h. Ultrasound treatment was followed by limited apoptosis after 24 h (32±6 cleaved-caspase 3-positive cells per optical field) in the insonated areas. Only neurons were identified in the apoptotic population. Although these observations may not be applicable for all acoustic parameters useful for BBB opening, they demonstrate that insonation of the rat brain with the parameters used in our experiments is a useful tool for BBB opening and induces specific cellular stress response restricted to neuronal cells.

Effect of ultrasound contrast agent dose on the duration of focused-ultrasound-induced blood-brain barrier disruption

It has been shown that focused ultrasound (FUS) is capable of noninvasive and reversible disruption of the blood-brain barrier (BBB) at target regions when applied in the presence of ultrasound contrast agent (UCA). The purpose of this study was to investigate the effects of UCA dose on the reversibility of BBB disruption induced by the same acoustical power of FUS. Sonications were applied at an ultrasound frequency of 1 MHz with a 5% duty cycle and a repetition frequency of 1 Hz. The brains of 66 male Sprague-Dawley rats were subjected to sonications with three doses of UCA. BBB integrity was evaluated via femoral vein injection of Evans Blue (EB) while the rats were anesthetized. The relationship between UCA dose and the region of EB extravasation was evaluated at an acoustic power of 1.43 W. The BBB disruption, as indexed by the amount of EB extravasation, was the largest immediately after the sonications. The quantity of Evans blue extravasation decreased as a function of time at various UCA doses. This study demonstrates that the appropriate dose of UCA not only enhance the BBB opening but also effectively aid in controlling the duration of BBB disruption.

Low-Intensity Ultrasound-Exposed Microbubbles Provoke Local Hyperpolarization of the Cell Membrane Via Activation of BK Ca Channels

Ultrasound (US) contrast agents have gained wide interest in gene therapy as many researchers reported increased membrane permeability and transfection efficiency by sonoporation in the presence of US contrast agents. We recently demonstrated an increase in cell membrane permeability for Ca 2+ in rat cardiomyoblast (H9c2) cells insonified in the presence of microbubbles. In the present study, we specifically investigated whether US-exposed microbubbles have an effect on the cell membrane potential and whether Ca 2+-dependent potassium (BK Ca) channels are involved. We particularly focused on local events where the microbubble was in contact with the cell membrane. H9c2 cells were cultured on US transparent membranes. US exposure consisted of bursts with a frequency of 1 MHz with a peak-to-peak pressure of 0.1 or 0.5 MPa. Pulse repetition frequency was set to 20 Hz, with a duty cycle of 0.2%. Cells were insonified during 30 s in the presence of Sonovue ™ microbubbles. The membrane potential was monitored during US exposure using the fluorescent dye di-4-aminonaphtylethenylpyridinium (di-4-ANEPPS). The experiments were repeated in the presence of iberiotoxin (100 nM), a specific inhibitor of BK Ca channels. Surprisingly, despite the previously reported Ca 2+ influx, we found patches of hyperpolarization of the cell membrane, as reflected by local increases in di-4-ANEPPS mean intensity of fluorescence (MIF) to 118.6 ± 2.5% ( p < 0.001, n = 267) at 0.1 MPa and 125.7 ± 5.9% ( p < 0.001, n = 161) at 0.5 MPa at t = 74 s, respectively, compared with “no US” (100.3 ± 3.4%, n = 52). This hyperpolarization was caused by the activation of BK Ca channels, as iberiotoxin completely prevented hyperpolarization. (MIF t74 = 100.6 ± 1.4%; p < 0.001, n = 267) and 0.5 MPa (MIF t74 = 88.8 ± 2.0%; p< 0.001, n = 193), compared with 0.1 and 0.5 MPa microbubbles without iberiotoxin. In conclusion, US-exposed microbubbles elicit a Ca 2+ influx, which leads to activation of BK Ca channels and a subsequent, local hyperpolarization of the cell membrane. This local hyperpolarization of the cell membrane may facilitate uptake of macromolecules through endocytosis and macropinocytosis. (E-mail: ljm.juffermans@vumc.nl)

Stability of alteplase in presence of cavitation

Several experimental studies have demonstrated that ultrasound (US) can accelerate enzymatic fibrinolysis and this effect is further enhanced in the presence of ultrasound contrast agents (UCA). Although UCA have been shown to be safe when administered to ischemic stroke patients, safety information of these agents in the thrombolysis setting is limited. Therefore, in this study we investigated potential adverse effects of acoustic cavitation generated by UCA on alteplase (t-PA), the drug used for treatment of ischemic stroke patients. A volume of 0.9mL of alteplase was dispensed into a custom-made polyester sample tube. For treatments in the presence or absence of cavitation either 0.1mL Optison or phosphate buffer saline was combined with alteplase. Three independent samples of each treatment group were exposed to ultrasound of 2MHz frequency at three different peak negative acoustic pressures of 0.5, 1.7, and 3.5MPa for a duration of 60min. All treatments were carried out in a cavitation detection system which was used to insonify the samples and record acoustic emissions generated within the sample. After ultrasound exposure, the treated samples and three untreated drug samples were tested for their enzymatic activity using a chromogenic substrate. The insonified samples containing Optison demonstrated cavitational activity proportional to acoustic pressure. No significant cavitation activity was observed in the absence of Optison. Enzymatic activity of alteplase in both insonified groups was comparable to that in the control group. These tests demonstrated that exposure of alteplase to 60min of 2MHz ultrasound at acoustic pressures ranging from 0.5MPa to 3.5MPa, in the presence or absence of Optison had no adverse effects on the stability of this therapeutic compound.

Quantitative evaluation of the use of microbubbles with transcranial focused ultrasound on blood–brain-barrier disruption

It has been shown that focused ultrasound (FUS) can disrupt the blood–brain barrier (BBB) noninvasively and reversibly at target locations when applied in the presence of ultrasound contrast agent (UCA). In this study, the dose-dependent effects of UCA on BBB disruption were investigated in the brains of 16 male Wistar rats sonicated by 1.0-MHz transcranial FUS, with the UCA present at four doses. The BBB disruption was evaluated quantitatively based on the extravasation of Evans blue (EB). The amount of EB extravasation in the brain increased with the quantity of UCA injected into the femoral vein prior to sonication. Moreover, the use of a suitable dose of UCA resulted in the BBB disruption being concentrated in the focal region instead of the entire brain. Our results indicate that injecting an appropriate quantity of UCA effectively increases and localizes the BBB disruption induced by transcranial FUS sonications.

Quantitative Evaluation of Focused Ultrasound with a Contrast Agent on Blood-Brain Barrier Disruption

The use of an ultrasound contrast agent (UCA) with focused ultrasound sonication has the potential to disrupt the blood-brain barrier (BBB) noninvasively and reversibly at target locations. This study investigated the effects of UCA dose and ultrasound pressure on BBB disruption. Sonications were applied at 1 MHz with a burst length of 10 ms, a 1% duty cycle and a repetition frequency of 1 Hz. The duration of the sonication was 30 s. In in vivo experiments, 16 male Wistar rats were sonicated in the presence of UCA at four doses (0, 30, 60 and 90 μL/kg). BBB integrity was evaluated by injecting Evans blue (EB) into the femoral vein of anesthetized rats. The relationship between UCA dose and the region of EB extravasation was evaluated at ultrasound pressures of 0.9 and 1.2 MPa. The BBB disruption, as quantified by the amount of EB extravasation, was significantly greater in rats injected with UCA at a dose of 60 or 90 μL/kg than at a dose of 0 or 30 μL/kg. The amount of EB extravasation increased monotonically with the quantity of UCA injected into the femoral vein before sonication. Furthermore, the BBB disruption could be enhanced in the focal region relative to the surrounding region with a higher dose of UCA (60 or 90 μL/kg). This study demonstrates that BBB disruption can be both increased and localized to the focal region by injecting an appropriate quantity of UCA before performing focused ultrasound sonications. (E-mail: winli@ntu.edu.tw)

Intravascular inertial cavitation activity detection and quantification in vivo with Optison

Inertial cavitation (IC) is an important mechanism by which ultrasound (US)-induced bioeffects can be produced. It has been reported that US-induced in vitro mechanical bioeffects with the presence of ultrasound contrast agents (UCAs) are highly correlated with quantified IC “dose” (ICD: cumulated root-mean-squared broadband noise amplitude in the frequency domain). The ICD has also been used to quantify IC activity in ex vivo perfused rabbit ear vessels. The in vivo experiments reported here using a rabbit ear vessel model were designed to: (1) detect and quantify IC activity in vivo within the constrained environment of rabbit auricular veins with the presence of Optison and (2) measure the temporal evolution of microbubble IC activity and the ICD generated during insonation treatment, as a function of acoustic parameters. Preselected regions-of-interest (ROI) in the rabbit ear vein were exposed to pulsed focused US (1.17 MHz, 1 Hz PRF). Experimental acoustic variables included peak rarefaction pressure amplitude ([PRPA]: 1.1, 3.0, 6.5 or 9.0 MPa) and pulse length (20, 100, 500 or 1000 cycles). ICD was quantified based on passive cavitation detection (PCD) measurements. The results show that: (1) after Optison injection, the time to onset of measurable microbubble IC activity was relatively consistent, approximately 20 s; (2) after reaching its peak value, the IC activity decayed exponentially and the half-life decay coefficient (t 1/2) increased with increasing PRPA and pulse length; and (3) the normalized ICD generated by pulsed US exposure increased significantly with increasing PRPA and pulse length. (E-mail: juantu@u.washington.edu)

Acoustic response of compliable microvessels containing ultrasound contrast agents

The existing models of the dynamics of ultrasound contrast agents (UCAs) have largely been focused on an UCA surrounded by an infinite liquid. Preliminary investigations of a microbubble's oscillation in a rigid tube have been performed using linear perturbation, under the assumption that the tube diameter is significantly larger than the UCA diameter. In the potential application of drug and gene delivery, it may be desirable to fragment the agent shell within small blood vessels and in some cases to rupture the vessel wall, releasing drugs and genes at the site. The effect of a compliant small blood vessel on the UCA's oscillation and the microvessel's acoustic response are unknown. The aim of this work is to propose a lumped-parameter model to study the interaction of a microbubble oscillation and compliable microvessels. Numerical results demonstrate that in the presence of UCAs, the transmural pressure through the blood vessel substantially increases and thus the vascular permeability is predicted to be enhanced. For a microbubble within an 8 to 40 µm vessel with a peak negative pressure of 0.1 MPa and a centre frequency of 1 MHz, small changes in the microbubble oscillation frequency and maximum diameter are observed. When the ultrasound pressure increases, strong nonlinear oscillation occurs, with an increased circumferential stress on the vessel. For a compliable vessel with a diameter equal to or greater than 8 µm, 0.2 MPa PNP at 1 MHz is predicted to be sufficient for microbubble fragmentation regardless of the vessel diameter; however, for a rigid vessel 0.5 MPa PNP at 1 MHz may not be sufficient to fragment the bubbles. For a centre frequency of 1 MHz, a peak negative pressure of 0.5 MPa is predicted to be sufficient to exceed the stress threshold for vascular rupture in a small (diameter less than 15 µm) compliant vessel. As the vessel or surrounding tissue becomes more rigid, the UCA oscillation and vessel dilation decrease; however the circumferential stress is predicted to increase. Decreasing the vessel size or the centre frequency increases the circumferential stress. For the two frequencies considered in this work, the circumferential stress does not scale as the inverse of the square root of the acoustic frequency va as in the mechanical index, but rather has a stronger frequency dependence, 1/va.For more information on this article, see medicalphysicsweb.org

Microbubble-enhanced hemorrhage control using high intensity focused ultrasound

Our objective was to investigate whether hemorrhage control can be achieved faster when high-intensity focused ultrasound (HIFU) is applied in the presence of ultrasound contrast agents (UCA) as compared to HIFU only application. Incisions (3cm long and 0.5cm deep) were produced in the livers of anesthetized rabbits. UCA Optison (0.18ml/kg) was injected into the mesenteric vein. A HIFU applicator (5.5MHz, 6800W/cm2 in situ) was scanned at a rate of 1–2mm/s in one direction over the incision (with multiple passes if needed), until hemostasis was achieved. Hemostasis times were 59±23s (n=21) in the presence of Optison and 70±23s (n=29) without Optison. The presence of Optison produced on average 37% reduction in hemostasis times normalized to initial bleeding rates (p<0.05), as well as 60% faster formation of the coagulum seal over the incision (p<0.05). Gross and histological observations showed similar appearance of HIFU lesions produced in the presence of Optison and HIFU lesions produced without Optison. Our results suggest potential utility of UCA for increasing efficiency of HIFU-induced hemostasis of solid organ injuries.

Effects of Ultrasound Contrast Agents on Doppler Tissue Velocity Estimation

The combination of Doppler tissue imaging and myocardial contrast echocardiography has the potential to provide information about motion and perfusion of the myocardium in a single examination. The purpose of this study was to establish how the presence of ultrasound contrast agent (UCA) affects measurements of Doppler tissue velocities in vivo and in vitro. We performed echocardiography in 12 patients with ischemic heart disease before and immediately after a slow intravenous infusion of the UCA Optison, using color Doppler tissue imaging to examine the effect of contrast agents in vivo. The myocardial peak systolic velocities and their integrals were analyzed in digitally stored cineloops before and after contrast administration. To distinguish between methodologic and physiologic factors affecting the measurement of tissue velocity in vitro, experiments with a rotating disk and a flow cone phantom were also carried out for the 3 contrast agents: Optison, Sonovue, and Sonazoid. In vivo results show that the values for peak systolic velocity increased by about 10% during contrast infusion, from mean 5.2 +/- 1.8 to 5.7 +/- 2.3 cm/s (P = .02, 95% confidence interval 2%-16%). The increase in myocardial peak systolic velocities was verified in experimental models in which the UCA increased the estimated mean velocity in the order of 5% to 20% for the motion interval of 5 to 7 cm/s, corresponding to the myocardial velocities studied in vivo. The response was similar for all 3 contrast agents and was not affected by moderate variations in concentration of the agent. We have shown that the presence UCA will affect Doppler tissue measurements in vivo and in vitro. The observed bias is presumed to be an effect of harmonic signal contribution from rupturing contrast agent microbubbles and does not indicate biologic or physiologic effects.

Ultrasound contrast agents for bleeding detection and acoustic hemostasis

Objective: To investigate the application of ultrasound contrast agents (UCA) in improving both therapeutic and diagnostic aspects of ultrasound-guided High Intensity Focused Ultrasound (HIFU) therapy. Methods: Incisions (3 cm long, 0.5 cm deep) were made in rabbit livers (in anterior surface for HIFU treatment, or posterior surface for bleeding detection). UCA Optison (∼0.1 ml/kg) was injected into mesenteric vein or ear vein. A HIFU applicator (5.5 MHz, 6400 W/cm2) was scanned manually over the incision until hemostasis was achieved. Occult bleeding was monitored with Doppler ultrasound. Results: The presence of Optison produced 37% reduction in hemostasis times normalized to initial bleeding rates. Gross and histological observations showed similar appearance of HIFU lesions produced in the presence of Optison and control HIFU lesions. The temperature reached 100°C in both HIFU only and HIFU+UCA treatments. Tension strength of hemostatic liver incisions was 0.9±0.5 N. Almost no bleeding could be detected before Optison injection. First appearance of contrast enhancement localized at the bleeding site was 15 s after Optison injection, and lasted for ∼50 s. Conclusion: The presence of UCA during HIFU treatment of liver incisions resulted in shortening of HIFU application times and better visualization of bleeding sites.