Chondrocytes are mechanosensitive cells that require mechanical stimulation for proper growth and function in in vitro culture systems. Ultrasound (US) has emerged as a technique to deliver mechanical stress; however, the intracellular signaling components of the mechanotransduction pathways that transmit the extracellular mechanical stimulus to gene regulatory mechanisms are not fully defined. We evaluated a possible integrin/mitogen-activated protein kinase (MAPK) mechanotransduction pathway using Western blotting with antibodies targeting specific phosphorylation sites on intracellular signaling proteins. US stimulation of chondrocytes induced phosphorylation of focal adhesion kinase (FAK), Src, p130 Crk-associated substrate (p130Cas), CrkII, and extracellular-regulated kinase (Erk). Furthermore, pre-incubation with inhibitors of integrin receptors, Src, and MAPK/Erk kinase (MEK) reduced US-induced Erk phosphorylation levels, indicating integrins and Src are upstream of Erk in an US-mediated mechanotransduction pathway. These findings suggest US signals through integrin receptors to the MAPK/Erk pathway via a mechanotransduction pathway involving FAK, Src, p130Cas, and CrkII.
Chondrocyte; Ultrasound; Mechanotransduction; Integrin; focal adhesion kinase (FAK); Src; p130 Crk-associated substrate (p130Cas); CrkII; extracellular-regulated kinase (Erk)
The blood-brain barrier (BBB) inhibits the entry of the majority of chemotherapeutic agents into the brain. Previous studies have illustrated the feasibility of drug delivery across the BBB using focused ultrasound (FUS) and microbubbles. Here, we investigated the effect of FUS-enhanced delivery of doxorubicin on survival in rats with and 9L gliosarcoma cells inoculated in the brain. Each rat received either: (1) no treatment (control; N=11), (2) FUS only (N=9), (3) i.v. liposomal doxorubicin (DOX only; N=17), or (4) FUS with concurrent i.v. injections of liposomal doxorubicin (FUS+DOX; N=20). Post-treatment MRI showed that FUS+DOX reduced tumor growth compared to DOX only. Further, we observed a modest but significant increase in median survival time after a single treatment FUS+DOX treatment (p=0.0007), whereas neither DOX nor FUS had any significant impact on survival on its own. These results suggest that combined ultrasound-mediated BBB disruption may significantly increase the antineoplastic efficacy of liposomal doxorubicin in the brain.
focused ultrasound; blood-brain barrier; doxorubicin; 9L gliosarcoma; survival
The efficacy of using subharmonic emissions from Sonazoid microbubbles (GE Healthcare, Oslo, Norway) to track portal vein pressures and pressure changes was investigated in 14 canines using either slow- or high-flow models of portal hypertension (PH). A modified Logiq 9 scanner (GE Healthcare, Milwaukee, WI) operating in subharmonic mode (ftransmit:2.5MHz, freceive:1.25MHz) was used to collect RF data at 10-40% incident acoustic power levels with 2-4 transmit cycles (in triplicate), before and after inducing PH. A pressure catheter (Millar Instruments, Inc., Houston, TX) provided reference portal vein pressures. At optimum insonification, subharmonic signal amplitude changes correlated with portal vein pressure changes; r ranged from -0.82 to -0.94 and from -0.70 to -0.73 for PH models considered separately or together, respectively. The subharmonic signal amplitudes correlated with absolute portal vein pressures (r: -0.71 to -0.79). Statistically significant differences between subharmonic amplitudes, before and after inducing PH, were noted (p≤0.01). Portal vein pressures estimated using SHAPE did not reveal significant differences (p>0.05) with respect to the pressures obtained using the Millar pressure catheter. Subharmonic aided pressure estimation may be useful clinically for portal vein pressure monitoring.
portal hypertension; subharmonic aided pressure estimation; noninvasive pressure estimation; ultrasound contrast agents; subharmonic imaging
A set of five tissue-mimicking phantoms with cylindrical inclusions were produced for assessing long-term stability of geometry and elastic properties and assessing accuracy of determination of elastic properties. The base aqueous materials were either gelatin or a mixture of agar and gelatin. Stiffness was controlled by selection of the volume percent consisting of microscopic safflower oil droplets. Cylinder diameters remained unchanged within 1% or 2% over many months. Strain ratios from elastograms of the phantoms were stable over many months, implying that elastic contrasts were also stable. Test samples, called production samples, for measurement of Young’s moduli were made at the time of manufacture of each phantom and were stored separately from one another. Each production sample was homogeneous and consisted of either inclusion material or background material. For all five phantoms, it was found that the elastic contrast computed using Young’s modulus values determined using the production samples accurately represented the true elastic contrasts in the corresponding phantom. This finding was established by the fact that the (true) elastic contrasts determined using samples excised from the phantoms themselves agreed with the elastic contrasts obtained using the homogeneous production samples.
Elastography; Elasticity; Phantom; Stability; Oil; Dispersion; Gelatin; Agar
We demonstrate two methods for vasa vasorum imaging using contrast-enhanced intravascular ultrasound, which can be performed using commercial catheters. Plaque neovascularization was recognized as an independent marker of coronary artery plaque vulnerability. IVUS-based methods to image the microvessels available to date require high bandwidth (−6 dB relative frequency bandwidth >70%), which are not routinely available commercially. We explored the potential of ultraharmonic imaging and chirp reversal imaging for vasa vasorum imaging. In vitro recordings were performed on a tissue-mimicking phantom using a commercial ultrasound contrast agent and a transducer with a center frequency of 34 MHz and a −6 dB relative bandwidth of 56%. Acoustic peak pressures <500 kPa were used. A tissue-mimicking phantom with channels down to 200 μm in diameter was successfully imaged by the two contrast detection sequences while the smallest channel stayed invisible in conventional intravascular ultrasound images. Ultraharmonic imaging provided the best contrast agent detection.
Intravascular ultrasound; Ultrasound contrast agents; Ultrasound contrast imaging; Ultraharmonic; Pulse inversion; Coded excitation; Chirp; Vasa vasorum
Tissue elasticity is related to pathology and therefore has important medical applications. Radiation force from a focused ultrasound beam has been used to produce shear waves in tissues for shear wave speed and tissue elasticity measurements. The feasibility of shear wave speed measurement using radiation force for an unfocused ultrasound beam is demonstrated in this study with a linear and a curved array transducer. Consistent measurement of shear wave speed was achieved over a relatively long axial extent (z = 10-40 mm for the linear array, and z = 15-60 mm for the curved array) in 3 calibrated phantoms with different shear moduli. In vivo measurements on the biceps of a healthy volunteer show consistent increase of shear wave speed for the biceps under 0, 1, 2, and 3 kg loading. Advantages and limitations of unfocused push are discussed.
Elasticity; Shear wave; Ultrasound radiation force; Unfocused
Non-pharmacological and non-surgical transcranial modulation of the nerve function may provide new opportunities in evaluation and treatment of cranial nerve diseases. This study investigates the possibility of using low-intensity transcranial focused ultrasound (FUS) to selectively stimulate the rat abducens nerve located above the base of the skull. FUS (frequencies of 350 kHz and 650 kHz) operating in a pulsed mode was applied to the abducens nerve of Sprague-Dawley rats under stereotactic guidance. The abductive eyeball movement ipsilateral to the side of sonication was observed at 350 kHz, using the 0.36 msec tone burst duration (TBD), 1.5 kHz pulse repetition frequency (PRF), and the overall sonication duration of 200 msec. Histological and behavioral monitoring showed no signs of disruption in the blood brain barrier (BBB) as well as no damage to the nerves and adjacent brain tissue resulting from the sonication. As a novel functional neuro-modulatory modality, the pulsed application of FUS has potential in diagnostic and therapeutic applications in diseases of the peripheral nervous system.
focused ultrasound; abducens nerve; cranial nerve VI; brain; neuromodulation; peripheral nervous system
Widespread implementation of quantitative muscle ultrasonography in assessing skeletal muscle pathology is limited by an inability to replicate results between different ultrasound systems. We have developed a measurement of skeletal muscle pathology, calibrated muscle backscatter (cMB), which should be reproducible between different ultrasound systems. We compared the reliability of grayscale and cMB measurements between different ultrasound systems, configurations and region-of-interest (ROI) sizes. cMB of skeletal muscle was reliably measured (intraclass correlation coefficient [ICC] ≤0.98) despite very dissimilar grayscale levels (ICC ≤0.54). cMB reliability was highest between systems using similar settings (ICC: 0.82–0.98) and was lowest when transducer type varied (ICC: 0.47–0.71). Reliability was better from ROIs spanning a narrow range of depths compared with larger ranges. cMB measurements are more reliable than grayscale between different ultrasound systems and configurations. Measuring cMB could improve widespread implementation of quantitative ultrasound in assessments of skeletal muscle pathology.
Ultrasound; Muscle; Myopathy; Backscatter; Reliability; Quantitative; Children; Adults
Xerostomia (dry mouth), resulting from radiation damage to the parotid glands, is one of the most common and distressing side effects of head-and-neck cancer radiotherapy. A noninvasive, objective imaging method to assess parotid injury is lacking, but much needed in the clinic. Therefore, we investigated echo histograms to quantitatively evaluate the morphologic and microstructural integrity of the parotid glands. Six sono-graphic features were derived from the echo-intensity histograms to assess the echogenicity, homogeneity and heterogeneity of the parotid gland: (1) peak intensity value (Ipeak), (2) −3-dB intensity width (W3-dB), (3) the low (<50% Ipeak) intensity width (Wlow), (4) the high (>50% Ipeak) intensity width (Whigh), (5) the area of low intensity (Alow) and (6) the area of high intensity (Ahigh). In this pilot study, 12 post-radiotherapy patients and seven healthy volunteers were enrolled. Significant differences (p < 0.05) were observed in four sonographic features between 24 irradiated and 14 normal parotid glands. In summary, we developed a family of sonographic features derived from echo histograms and demonstrated the feasibility of quantitative evaluation of radiation-induced parotid-gland injury.
Xerostomia; Ultrasound; Parotid gland; Radiation toxicity; Echo histogram; Head-and-neck cancer; Sonographic features
We developed new miniature ring array transducers integrated into interventional device catheters such as used to deploy atrial septal occluders. Each ring array consisted of 55 elements operating near 5 MHz with interelement spacing of 0.20 mm. It was constructed on a flat piece of copper-clad polyimide and then wrapped around an 11 French O.D. catheter. We used a braided cabling technology from Tyco Electronics Corp to connect the elements to the Volumetrics Medical Imaging (VMI) real-time 3D ultrasound scanner. Transducer performance yielded a –6 dB fractional bandwidth of 20% centered at 4.7 MHz without a matching layer versus average bandwidth of 60% centered at 4.4 MHz with a matching layer. Real time 3D rendered images of an en face view of a Gore Helex septal occluder in a water tank showed a finer texture of the device surface from the ring array with the matching layer.
2D array transducer; real-time 3D imaging; septal occluder
4D (3D spatial+time) echocardiography is gaining widespread acceptance at clinical institutions for its high temporal resolution and relatively low cost. We describe a novel method for computing dense 3D myocardial motion with high accuracy. The method is based on a classical variational optical flow technique, but exploits modern developments in optical flow research to utilize the full capabilities of 4D echocardiography. Using a variety of metrics, we present an in-depth performance evaluation of the method on synthetic, phantom, and intraoperative 4D Transesophageal Echocardiographic (TEE) data. When compared with state-of-the-art optical flow and speckle tracking techniques currently found in 4D echocardiography, the method we present shows notable improvements in error. We believe the performance improvements shown can have a positive impact when the method is used as input for various applications, such as strain computation, biomechanical modeling, or automated diagnostics.
4D transesophageal echocardiography; motion estimation; 3D optical flow
Acoustic Radiation Force (ARF)-based methods have been demonstrated to be a viable tool for noninvasively estimating tissue elastic properties, and shear wave velocimetry has been used to quantitatively measure the stiffening and relaxation of myocardial tissue in open-chest experiments. Dynamic stiffness metrics may prove to be indicators for certain cardiac diseases, but a clinically-viable means of remotely generating and tracking transverse wave propagation in myocardium is needed. Intracardiac echocardiography (ICE) catheter-tip transducers are demonstrated here as a viable tool for making this measurement. ICE probes achieve favorable proximity to the myocardium, enabling the use of shear wave velocimetry from within the right ventricle throughout the cardiac cycle. This work describes the techniques used to overcome the challenges of using a small probe to perform ARF-driven shear wave velocimetry, and presents in vivo porcine data showing the effectiveness of this method in the interventricular septum. Acoustic Radiation Force (ARF)-based methods have been demonstrated to be a viable tool for noninvasively estimating tissue elastic properties, and shear wave velocimetry has been used to quantitatively measure the stiffening and relaxation of myocardial tissue in open-chest experiments. Dynamic stiffness metrics may prove to be indicators for certain cardiac diseases, but a clinically-viable means of remotely generating and tracking transverse wave propagation in myocardium is needed. Intracardiac echocardiography (ICE) catheter-tip transducers are demonstrated here as a viable tool for making this measurement. ICE probes achieve favorable proximity to the myocardium, enabling the use of shear wave velocimetry from within the right ventricle throughout the cardiac cycle. This work describes the techniques used to overcome the challenges of using a small probe to perform ARF-driven shear wave velocimetry, and presents in vivo porcine data showing the effectiveness of this method in the interventricular septum.
Ultrasonic Imaging; Acoustic Radiation Force; Intracardiac Echocardiography; Myocardial Stiffness; Shear Wave Velocimetry
Agents targeting vascular endothelial growth factor (VEGF) have been validated as cancer therapeutics, yet efficacy can differ widely between tumor types and individual patients. In addition, such agents are costly and can have significant toxicities. Rapid noninvasive determination of response could provide significant benefits. We tested if response to the anti-VEGF antibody bevacizumab (BV) could be detected using contrast-enhanced ultrasound imaging (CEUS). We used two xenograft model systems with previously well-characterized responses to VEGF inhibition, a responder (SK-NEP-1) and a non-responder (NGP), and examined perfusion-related parameters. CEUS demonstrated that BV treatment arrested the increase in blood volume in the SK-NEP-1 tumor group only. Molecular imaging of αVβ3 with targeted microbubbles was a more sensitive prognostic indicator of BV efficacy. CEUS using RGD-labeled microbubbles showed a robust decrease in αVβ3 vasculature following BV treatment in SK-NEP-1 tumors. Paralleling these findings, lectin perfusion assays detected a disproportionate pruning of smaller, branch vessels. Therefore, we conclude that the response to BV can be identified soon after initiation of treatment, often within 3 days, by use of CEUS molecular imaging techniques. The use of a noninvasive ultrasound approach may allow for earlier and more effective determination of efficacy of anti-angiogenic therapy.
bevacizumab; VEGF blockade; Wilms tumor; neuroblastoma; molecular imaging; blood volume; microbubble
The transverse carpal ligament (TCL) forms the palmar boundary of the carpal tunnel and plays an important role in carpal tunnel mechanics. TCL hypertrophy has been observed for individuals with carpal tunnel syndrome (CTS) and postulated as a potential etiological factor. Ultrasound is particularly advantageous for TCL imaging because of its capability of detecting the interfaces between the TCL and other tissues. The purposes of this study were to develop an ultrasound based method to measure the TCL thickness and to test the validity and reliability of this method. Three operators conducted two sessions of ultrasound examination on 8 cadaveric specimens and 8 healthy volunteers. A custom script was used to calculate TCL thickness along the TCL length from the ultrasound images. The ultrasound based TCL thickness of the cadaveric specimens was compared to the dissection based TCL thickness for validation. The results showed Pearson’s correlation coefficients of 0.867–0.928, intraclass correlation coefficient (ICC) values of 0.726–0.865, a standard error of measurement of 0.02–0.07 mm, and a minimal detectable difference of 0.05–0.15 mm. The high correlation coefficients and small errors indicate that the ultrasound based method is valid for measuring TCL thickness. Furthermore, ultrasound measurements showed excellent intra-operator and inter-operator reliability with ICC values as 0.826–0.933 and 0.840–0.882, respectively. The ultrasound based TCL thickness was in the range of 0.93–2.34 (1.54 ± 0.33) mm and agreed well with previous studies. The ultrasound method developed in this study is a valuable tool to examine morphological properties of healthy and pathological TCLs.
Ultrasound; transverse carpal ligament; validity; reliability
Recent advances in combining ultrasonic particle manipulation with attenuated total reflection infrared spectroscopy of yeast suspensions are presented. Infrared spectroscopy provides highly specific molecular information about the sample. It has not been applicable to in-line monitoring of cells during fermentation, however, because positioning cells in the micron-thin measurement region of the attenuated total reflection probe was not possible. Ultrasonic radiation forces exerted on suspended particles by an ultrasonic standing wave can result in the buildup of agglomerates in the nodal planes, hence enabling the manipulation of suspended cells on the microscopic scale. When a chamber setup and a prototype in-line applicable probe were used, successful control over the position of the yeast cells relative to the attenuated total reflection sensor surface could be proven. Both rate of increase and maximum mid-infrared absorption of yeast-specific bands during application of a pushing frequency (chamber setup: 1.863 MHz, in-line probe: 1.990 MHz) were found to correlate with yeast cell concentration.
Ultrasonic particle manipulation; Fourier transform infrared spectroscopy; Acoustic radiation force; Ultrasonic standing wave; Attenuated total reflection; Saccharomyces cerevisiae
Poly lactic acid (PLA) ultrasound contrast agents (CA) have been previously developed in our laboratory for ultrasound (US) imaging, as well as surface coated with doxorubicin to create a potential targeted platform of chemotherapeutic delivery using focused US. However, we have previously found it impossible to sterilize these agents while at the same time maintaining their acoustic properties, a task that would probably require fabrication within a clean facility. The purpose of this paper is to investigate the feasibility of using plasma to sterilize these CA while maintaining maximum echogenicity, a step that would greatly facilitate in vivo investigations. Effects of plasma exposure time (1, 3 and 6 minutes) and intensity (low- 10 mA, 6.8 W; medium- 15 mA, 10.5 W; and high- 25 mA, 18W) on the CA’s acoustic properties, surface morphology, zeta potential, capacity to carry chemotherapeutics, and overall sterility are described. Both increases in plasma intensity and exposure time increased CA zeta potential and also significantly increased drug payload. High intensity plasma exposure for three minutes was found to be an optimal sterilization protocol for maximal (100%) preservation of CA echogenicity. Plasma exposure resulted in sterile samples and maintained original CA enhancement of 20 dB and acoustic half-life over 75 minutes, while increasing CA zeta potential by 11 mV and doxorubicin loading efficiency by 10%. This study not only shows how a highly temperature and pressure sensitive agent can be sterilized using plasma, but also that surface modification can be used to increase surface binding of drug.
Ultrasound contrast agents; poly lactic acid; plasma sterilization; surface modification; drug delivery
Most applications of therapeutic ultrasound (US) for intracellular delivery of drugs, proteins, DNA/ RNA and other compounds would benefit from efficient uptake of these molecules into large numbers of cells without killing cells in the process. In this study we tested the hypothesis that efficient intracellular uptake of molecules can be achieved with high cell viability after US exposure in vitro. A search of the literature for studies with quantitative data on uptake and viability yielded 26 published papers containing 898 experimental data points. Analysis of these studies showed that just 7.7% of the data points corresponded to relatively efficient uptake (>50% of cells exhibiting uptake). Closer examination of the data showed that use of Definity US contrast agent (as opposed to Optison) and elevated sonication temperature at 37°C (as opposed to room temperature) were associated with high uptake, which we further validated through independent experiments carried out in this study. Although these factors contributed to high uptake, almost all data with efficient uptake were from studies that had not accounted for lysed cells when determining cell viability. Based on retrospective analysis of the data, we showed that not accounting for lysed cells can dramatically increase the calculated uptake efficiency. We further argue that if all the data considered in this study were re-analyzed to account for lysed cells, there would be essentially no data with efficient uptake. We therefore conclude that the literature does not support the hypothesis that efficient intracellular uptake of molecules can be achieved with high cell viability after US exposure in vitro, which poses a challenge to future applications of US that require efficient intracellular delivery.
Cell viability; Contrast agent; Drug delivery; Intracellular uptake efficiency; Therapeutic ultrasound
Cavitation memory effects occur when remnants of cavitation bubbles (nuclei) persist in the host medium and act as seeds for subsequent events. In pulsed cavitational ultrasound therapy, or histotripsy, this effect may cause cavitation to repeatedly occur at these seeded locations within a target volume, producing inhomogeneous tissue fractionation or requiring an excess number of pulses to completely homogenize the target volume. We hypothesized that by removing the cavitation memory, i.e., the persistent nuclei, the cavitation bubbles could be induced at random locations in response to each pulse; therefore, complete disruption of a tissue volume may be achieved with fewer pulses. To test the hypothesis, the cavitation memory was passively removed by increasing the intervals between successive pulses, Δt, from 2, 10, 20, 50 and 100, to 200 ms. Histotripsy treatments were performed in red blood cell tissue phantoms and ex vivo livers using 1-MHz ultrasound pulses of 10 cycles at P−/P+ pressure of 21/59 MPa. The phantom study allowed for direct visualization of the cavitation patterns and the lesion development process in real time using high-speed photography; the ex vivo tissue study provided validation of the memory effect in real tissues. Results of the phantom study showed an exponential decrease in the correlation coefficient between cavitation patterns in successive pulses from 0.5 ± 0.1 to 0.1 ± 0.1 as Δt increased from 2–200 ms; correspondingly, the lesion was completely fractionated with significantly fewer pulses for longer Δts. In the tissue study, given the same number of therapy pulses, complete and homogeneous tissue fractionation with well-defined lesion boundaries was achieved only for Δt ≥ 100 ms. These results indicated that the removal of the cavitation memory resulted in more efficient treatments and homogeneous lesions.
Histotripsy; Cavitation memory; Efficient treatments; Homogeneous lesions
Effective real-time monitoring of high-intensity focused ultrasound (HIFU) ablation is important for application of HIFU technology in interventional electrophysiology. This study investigated rapid, high-frequency M-mode ultrasound imaging for monitoring spatiotemporal changes during HIFU application. HIFU (4.33 MHz, 1 kHz PRF, 50% duty cycle, 1 s, 2600 – 6100 W/cm2) was applied to ex-vivo porcine cardiac tissue specimens with a confocally and perpendicularly aligned high-frequency imaging system (Visualsonics Vevo 770, 55 MHz center frequency). Radiofrequency (RF) data from M-mode imaging (1 kHz PRF, 2 s × 7 mm) was acquired before, during, and after HIFU treatment (n = 12). Among several strategies, the temporal maximum integrated backscatter with a threshold of +12 dB change showed the best results for identifying final lesion width (receiver-operating characteristic curve area 0.91 ± 0.04, accuracy 85 ± 8%, as compared to macroscopic images of lesions). A criterion based on a line-to-line decorrelation coefficient is proposed for identification of transient gas bodies.
Cardiac Ablation; High-Intensity Focused Ultrasound; Spectrum Analysis; Echo-Decorrelation; Tissue Characterization; High-Frequency Ultrasound
For over a decade, the application of acoustic radiation force (ARF) has been proposed as a mechanism to increase ultrasonic molecular imaging (MI) sensitivity in vivo. Presented herein is the first noninvasive in vivo validation of ARF-enhanced MI with an unmodified clinical system. First, an in vitro optical-acoustical setup was used to optimize system parameters and ensure sufficient microbubble translation when exposed to ARF. 3D ARF-enhanced MI was then performed on 7 rat fibrosarcoma tumors using microbubbles targeted to αvβ3 and non-targeted microbubbles. Low-amplitude (< 25 kPa) 3D ARF pulse sequences were tested and compared to passive targeting studies in the same animal. Our results demonstrate that a 78% increase in image intensity from targeted microbubbles can be achieved when using ARF relative to the passive targeting studies. Furthermore, ARF did not significantly increase image contrast when applied to non-targeted agents, suggesting that ARF did not increase non-specific adhesion.
microbubble; molecular imaging; targeted imaging; radiation force
This study investigated the characteristics of the secondary bubble cluster produced by an electrohydraulic lithotripter using high-speed imaging and passive cavitation detection techniques. The results showed that (i) the discrepancy of the collapse time between near a flat rigid boundary and in a free field of the secondary bubble cluster was not as significant as that by the primary one; (ii) the secondary bubble clusters were small but in a high bubble density and nonuniform in distribution, and they did not expand and aggregate significantly near a rigid boundary; and (iii) the corresponding bubble collapse was weaker with few microjet formation and bubble rebound. By applying a strong suction flow near the electrode tip, the production of the secondary shock wave (SW) and induced bubble cluster could be disturbed significantly, but without influence on the primary ones. Consequently, stone fragmentation efficiency was reduced from 41.2 ± 7.1% to 32.2 ± 3.5% after 250 shocks (p <0.05). Altogether, these observations suggest that the secondary bubble cluster produced by an electrohydraulic lithotripter may contribute to its ability for effective stone fragmentation.
Electrohydraulic lithotripter; Bubble cluster cavitation; Secondary shock wave; Stone fragmentation
This study is designed to investigate the feasibility for molecular imaging of endothelial CD81 expression in vitro and in vivo using the CD81-targeted ultrasound contrast agents (UCA). In the in vitro study, murine bEnd.3 cells were stimulated with phenazine methosulfate (PMS), an oxidative stress inducer. Changes in CD81 expression after stimulation were confirmed by Western blotting, tracked by using the targeted UCA and further imaged under ultrasound imaging system with 5 MHz transmit frequency. In the in vivo study, expression of endothelial CD81 proteins in murine carotid artery vessels was studied using high-frequency ultrasound system with 40 MHz transmit frequency. Our results showed that endothelial CD81 expression was gradually up-regulated with the increase of PMS concentration. Correspondingly, the accumulation of targeted UCA was gradually improved and could be inhibited significantly upon addition of free anti-CD81 antibodies. The mean video intensity (grey-level) of stimulated cells and vessels from backscatter of the CD81-targeted UCA was 17.2 (interquartile range [IQR] 15.4–19.8) and 27.2 (IQR 22.4–29.8), significantly greater than that of non-stimulated cells with 9.0 (IQR 8.6–10.8) (p < 0.01) and non-stimulated vessels with 11.3 (IQR 10.4–13.2) (p < 0.01), respectively. In conclusion, CD81-targeted UCA allows noninvasive assessment of the expression levels of CD81 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.
Ultrasound contrast agents; Molecular imaging; CD81; Protein expression; Atherosclerosis
Many recent studies on ultrasonic particle image velocimetry (Echo PIV) showed that the accuracy of two-dimensional (2-D) flow velocity measured depends largely on the concentration of ultrasound contrast agents (UCAs) during imaging. This article presents a texture-based method for identifying the optimum microbubble concentration for Echo PIV measurements in real-time. The texture features, standard deviation of gray level, and contrast, energy and homogeneity of gray level co-occurrence matrix were extracted from ultrasound contrast images of rotational and pulsatile flow (10 MHz) in vitro and in vivo mouse common carotid arterial flow (40 MHz) with UCAs at various concentrations. The results showed that, at concentration of 0.8~2 × 103 bubbles/mL in vitro and 1~5 × 105 bubbles/mL in vivo, image texture features had a peak value or trough value, and velocity vectors with high accuracy can be obtained. Otherwise, poor quality velocity vectors were obtained. When the texture features were used as a feature set, the accuracy of K-nearest neighbor classifier can reach 86.4% in vitro and 87.5% in vivo, respectively. The texture-based method is shown to be able to quickly identify the optimum microbubble concentration and improve the accuracy for Echo PIV imaging.
Echo PIV; Flow imaging; Ultrasound contrast agents; Microbubbles; Texture analysis
Strain developed under quasi-static deformation has been mostly used in ultrasound elasticity imaging (UEI) to determine the stiffness change of tissues. However, the strain measure in UEI is often less sensitive to a subtle change of stiffness. This is particularly true for Crohn’s disease where we have applied strain imaging to the differentiation of acutely inflamed bowel from chronically fibrotic bowel. In this study, a new nonlinear elastic parameter of the soft tissues is proposed to overcome this limit. The purpose of this study is to evaluate the newly proposed method and demonstrate its feasibility in the UEI. A non-linear characteristic of soft tissues over a relatively large dynamic range of strain was investigated. A simplified tissue model based on a finite element (FE) analysis was integrated with a laboratory developed ultrasound radio frequency (RF) signal synthesis program. Two dimensional speckle tracking was applied to this model to simulate the nonlinear behavior of the strain developed in a target inclusion over the applied average strain to the surrounding tissues. A nonlinear empirical equation was formulated and optimized to best match the developed strain-to-applied strain relation obtained from the FE simulation. The proposed nonlinear equation was applied to in vivo measurements and nonlinear parameters were further empirically optimized. For an animal model, acute and chronic inflammatory bowel disease was induced in Lewis rats with trinitrobenzene sulfonic acid (TNBS)-ethanol treatments. After UEI, histopathology and direct mechanical measurements were performed on the excised tissues. The extracted non-linear parameter from the developed strain-to-applied strain relation differentiated the three different tissue types with 1.96 ± 0.12 for normal, 1.50 ± 0.09 for the acutely inflamed, and 1.03 ± 0.08 for the chronically fibrotic tissue. T-tests determined that the non-linear parameters between normal, acutely inflamed and fibrotic tissue types were statistically significantly different (normal/fibrotic (P = 0.0000185), normal/acutely inflamed (P = 0.0013), and fibrotic/acutely inflamed (P = 0.0029)). This technique may provide a sensitive and robust tool to assess subtle stiffness changes in tissues such as in acutely inflamed bowel wall. (E-mail: firstname.lastname@example.org)
Ultrasound elasticity imaging; Nonlinear elasticity; Tissue mechanics; Ultrasound speckle tracking; Strain; Crohn’s disease
Ultrasound molecular imaging is a powerful diagnostic modality employing microbubbles coated with targeting ligands specific for endothelial biomarkers. The circulation persistence of ligand-bearing contrast agents is a key determinant in their contrast enhancement and targeting capability. Prior studies have shown that targeted microbubbles with ligands attached to the shell using the conventional exposed-ligand architecture (ELA) could trigger undesired ligand-induced complement activation and decreased circulation time. Microbubbles with the buried-ligand architecture (BLA), however, were found to inhibit complement activation and prolong circulation time. In the present study, we extended the stealth BLA microbubble design to size-selected (4–5 µm diameter) microbubbles targeted with cyclic RGD peptide using the post-labeling technique. Microbubble circulation persistence was measured in the healthy mouse kidney using a Visualsonics Vevo 770 scanner operating at 40 MHz in fundamental mode. The circulation persistence for targeted BLA microbubbles was significantly longer compared to their ELA counterparts and similar to no-ligand controls. Use of the BLA instead of the ELA increased the circulation half-life by approximately 2-fold. Analysis of the time-intensity and time-fluctuation curves with a two-compartment pharmacokinetic model showed a minimal degree of nonspecific vascular adhesion for any group. These results demonstrate the importance of surface architecture in the design of targeted microbubbles for ultrasound molecular imaging.
molecular imaging; targeting; RGD peptide; complement activation; mononuclear phagocyte system; clearance; mouse kidney