Quantification of Endothelial αvß3 Expression with High-Frequency Ultrasound and Targeted Microbubbles: In Vitro and In Vivo Studies.

Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques. The precursory molecular or cellular pro-angiogenic events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. This study was designed to investigate the feasibility of ultrasound molecular imaging of endothelial αvß3 expression in vitro and in vivo using αvß3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvß3 expression was confirmed by immunofluorescence in a murine endothelial cell line and detected using the targeted UCA and ultrasound imaging at 18-MHz transmit frequency. In the in vivo study, expression of endothelial αvß3 integrin in murine carotid artery vessels and microvessels of the salivary gland was quantified using targeted UCA and high-frequency ultrasound in seven animals. Our results indicated that endothelial αvß3 expression was significantly higher in the carotid arterial wall containing atherosclerotic lesions than in arterial segments without any lesions. We also found that the salivary gland can be used as an internal positive control for successful binding of targeted UCA to αvß3 integrin. In conclusion, αvß3-targeted UCA allows non-invasive assessment of the expression levels of αvß3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.

Quantification of bound microbubbles in ultrasound molecular imaging.

Molecular markers associated with diseases can be visualized and quantified noninvasively with targeted ultrasound contrast agent (t-UCA) consisting of microbubbles (MBs) that can bind to specific molecular targets. Techniques used for quantifying t-UCA assume that all unbound MBs are taken out of the blood pool few minutes after injection and only MBs bound to the molecular markers remain. However, differences in physiology, diseases, and experimental conditions can increase the longevity of unbound MBs. In such conditions, unbound MBs will falsely be quantified as bound MBs. We have developed a novel technique to distinguish and classify bound from unbound MBs. In the post-processing steps, first, tissue motion was compensated using block-matching (BM) techniques. To preserve only stationary contrast signals, a minimum intensity projection (MinIP) or 20th-percentile intensity projection (PerIP) was applied. The after-flash MinIP or PerIP was subtracted from the before-flash MinIP or PerIP. In this way, tissue artifacts in contrast images were suppressed. In the next step, bound MB candidates were detected. Finally, detected objects were tracked to classify the candidates as unbound or bound MBs based on their displacement. This technique was validated in vitro, followed by two in vivo experiments in mice. Tumors (n = 2) and salivary glands of hypercholesterolemic mice (n = 8) were imaged using a commercially available scanner. Boluses of 100 µL of a commercially available t-UCA targeted to angiogenesis markers and untargeted control UCA were injected separately. Our results show considerable reduction in misclassification of unbound MBs as bound ones. Using our method, the ratio of bound MBs in salivary gland for images with targeted UCA versus control UCA was improved by up to two times compared with unprocessed images.

Subharmonic, non-linear fundamental and ultraharmonic imaging of microbubble contrast at high frequencies.

There is increasing use of ultrasound contrast agent in high-frequency ultrasound imaging. However, conventional contrast detection methods perform poorly at high frequencies. We performed systematic in vitro comparisons of subharmonic, non-linear fundamental and ultraharmonic imaging for different depths and ultrasound contrast agent concentrations (Vevo 2100 system with MS250 probe and MicroMarker ultrasound contrast agent, VisualSonics, Toronto, ON, Canada). We investigated 4-, 6- and 10-cycle bursts at three power levels with the following pulse sequences: B-mode, amplitude modulation, pulse inversion and combined pulse inversion/amplitude modulation. The contrast-to-tissue (CTR) and contrast-to-artifact (CAR) ratios were calculated. At a depth of 8 mm, subharmonic pulse-inversion imaging performed the best (CTR = 26 dB, CAR = 18 dB) and at 16 mm, non-linear amplitude modulation imaging was the best contrast imaging method (CTR = 10 dB). Ultraharmonic imaging did not result in acceptable CTRs and CARs. The best candidates from the in vitro study were tested in vivo in chicken embryo and mouse models, and the results were in a good agreement with the in vitro findings.

Development and initial application of a fully integrated photoacoustic micro-ultrasound system.

Photoacoustic (PA) imaging for biomedical applications has been under development for many years. Based on the many advances over the past decade, a new photoacoustic imaging system has been integrated into a micro-ultrasound platform for co-registered PA-ultrasound (US) imaging. The design and implementation of the new scanner is described and its performance quantified. Beamforming techniques and signal processing are described, in conjunction with in vivo PA images of normal subcutaneous mouse tissue and selected tumor models. In particular, the use of the system to estimate the spatial distribution of oxygen saturation (sO2) in blood and co-registered with B-mode images of the surrounding anatomy are investigated. The system was validated in vivo against a complementary technique for measuring partial pressure of oxygen in blood (pO2). The pO2 estimates were converted to sO2 values based on a standard dissociation curve found in the literature. Preliminary studies of oxygenation effects were performed in a mouse model of breast cancer (MDA-MB-231) in which control mice were compared with mice treated with a targeted antiangiogenic agent over a 3 d period. Treated mice exhibited a >90% decrease in blood volume, an 85% reduction in blood wash-in rate, and a 60% decrease in relative tissue oxygenation.

Novel single-chain antibody-targeted microbubbles for molecular ultrasound imaging of thrombosis: validation of a unique noninvasive method for rapid and sensitive detection of thrombi and monitoring of success or failure of thrombolysis in mice.

BACKGROUND: Molecular imaging is a fast emerging technology allowing noninvasive detection of vascular pathologies. However, imaging modalities offering high resolution currently do not allow real-time imaging. We hypothesized that contrast-enhanced ultrasound with microbubbles (MBs) selectively targeted to activated platelets would offer high-resolution, real-time molecular imaging of evolving and dissolving arterial thrombi. METHODS AND RESULTS: Lipid-shell based gas-filled MBs were conjugated to either a single-chain antibody specific for activated glycoprotein IIb/IIIa via binding to a Ligand-Induced Binding Site (LIBS-MBs) or a nonspecific single-chain antibody (control MBs). Successful conjugation was assessed in flow cytometry and immunofluorescence double staining. LIBS-MBs but not control MBs strongly adhered to both immobilized activated platelets and microthrombi under flow. Thrombi induced in carotid arteries of C57Bl6 mice in vivo by ferric chloride injury were then assessed with ultrasound before and 20 minutes after MB injection through the use of gray-scale area intensity measurement. Gray-scale units converted to decibels demonstrated a significant increase after LIBS-MB but not after control MB injection (9.55±1.7 versus 1.46±1.3 dB; P<0.01). Furthermore, after thrombolysis with urokinase, LIBS-MB ultrasound imaging allows monitoring of the reduction of thrombus size (P<0.001). CONCLUSION: We demonstrate that glycoprotein IIb/IIIa-targeted MBs specifically bind to activated platelets in vitro and allow real-time molecular imaging of acute arterial thrombosis and monitoring of the success or failure of pharmacological thrombolysis in vivo.

Assessment and monitoring tumor vascularity with contrast-enhanced ultrasound maximum intensity persistence imaging.

OBJECTIVES: Contrast-enhanced ultrasound imaging is increasingly being used in the clinic for assessment of tissue vascularity. The purpose of our study was to evaluate the effect of different contrast administration parameters on the in vivo ultrasound imaging signal in tumor-bearing mice using a maximum intensity persistence (MIP) algorithm and to evaluate the reliability of in vivo MIP imaging in assessing tumor vascularity. The potential of in vivo MIP imaging for monitoring tumor vascularity during antiangiogenic cancer treatment was further evaluated. MATERIALS AND METHODS: In intraindividual experiments, varying contrast microbubble concentrations (5 × 105, 5 × 106, 5 × 107, 5 × 108 microbubbles in 100 µL saline) and contrast injection rates (0.6, 1.2, and 2.4 mL/min) in subcutaneous tumor-bearing mice were applied and their effects on in vivo contrast-enhanced ultrasound MIP imaging plateau values were obtained using a dedicated small animal ultrasound imaging system (40 MHz). Reliability of MIP ultrasound imaging was tested following 2 injections of the same microbubble concentration (5 × 107 microbubbles at 1.2 mL/min) in the same tumors. In mice with subcutaneous human colon cancer xenografts, longitudinal contrast-enhanced ultrasound MIP imaging plateau values (baseline and at 48 hours) were compared between mice with and without antiangiogenic treatment (antivascular endothelial growth factor antibody). Ex vivo CD31 immunostaining of tumor tissue was used to correlate in vivo MIP imaging plateau values with microvessel density analysis. RESULTS: In vivo MIP imaging plateau values correlated significantly (P = 0.001) with contrast microbubble doses. At 3 different injection rates of 0.6, 1.2, and 2.4 mL/min, MIP imaging plateau values did not change significantly (P = 0.61). Following 2 injections with the same microbubble dose and injection rate, MIP imaging plateau values were obtained with high reliability with an intraclass correlation coefficient of 0.82 (95% confidence interval: 0.64, 0.94). In addition, in vivo MIP imaging plateau values significantly correlated (P = 0.01; R² = 0.77) with ex vivo microvessel density analysis. Tumor volumes in treated and nontreated mice did not change significantly (P = 0.22) within 48 hours. In contrast, the change of in vivo MIP imaging plateau values from baseline to 48 hours was significantly different (P = 0.01) in treated versus nontreated mice. CONCLUSIONS: Contrast-enhanced ultrasound MIP imaging allows reliable assessment of tumor vascularity and monitoring of antiangiogenic cancer therapy in vivo, provided that a constant microbubble dose is administered.

A new 15-50 MHz array-based micro-ultrasound scanner for preclinical imaging.

Most institutions now have a suite of imaging tools to follow mouse models of human disease. Micro-ultrasound is one of these tools and is second after whole-mouse fluorescence or bioluminescent imaging, in terms of installed systems. We report in this paper the first commercially available array transducer-based ultrasound imaging system that enables micro-ultrasound imaging at center frequencies between 15 and 50 MHz. At the heart of the new scanner is a laser-machined high-frequency 256 element, linear transducer array capable of forming dynamic diffraction limited beams. The power of the linear array approach is embodied in the uniform high resolution maintained over the full field of view. This leads to greatly expanded scope for real-time functional imaging that is demonstrated in this paper. The unprecedented images made with the new imaging system will enable many new applications not previously possible. These include real-time visualization of flow in the mouse placenta, visualization of flow development in the embryo, studies of embryonic to adult cardiac development/disease, and studies of real-time blood flow in mouse models of tumour angiogenesis.

Fabrication and performance of a 40-MHz linear array based on a 1-3 composite with geometric elevation focusing.

The fabrication and performance of a 256-element high-frequency (40-MHz) linear array is described. The array was fabricated using a high-frequency 1-3 PZT-polymer composite material developed in our laboratory. The spacing of the pillars in the composite was chosen to match the 40-microm center-to-center element spacing of the array electrodes. The element electrodes were created using photolithography, and connections to the electrodes were made using ultrasonic wire bonding. The array was focused in the elevation direction by geometrically shaping the composite material using a cylindrical die with a 6-mm radius of curvature. The resulting transducer produced pulses with a -6 dB two-way bandwidth of 50% and a peak-to-peak pressure of 503 kPa when excited with a +/-30 V monocycle pulse. The measured one-way (-6 dB) directivity for a single array element was 24 degrees and the -3 dB one-way elevation beamwidth was measured to be 130 microm. The radiation pattern for a focused 64-element subaperture was measured by mechanically translating the aperture above a needle hydrophone. A -3 dB one-way beamwidth of 97 microm was found at a depth of 6 mm. The one-way radiation pattern decreased smoothly to less than -30 dB at a lateral distance of 640 microm.

Noninvasive ultrasonic measurement of regional and local pulse-wave velocity in mice.

Mouse models of human disease are increasingly used to study the nature of cardiovascular diseases such as atherosclerosis. The pulse wave velocity (PWV) provides an indirect measure of arterial stiffness and can be useful for characterizing disease progression. In this study, the PWV was measured noninvasively in the left common carotid artery of seven young mice using two image-guided approaches: a regional transit-time (TT) method and a local flow-area (QA) method. The QA approach measures the cross-sectional area and volume flow through the vessel using high frame-rate retrospective colour flow imaging. The QA method was found to correlate well with the TT method (r2=0.80, p<0.001). The mean difference between methods was 0.05+/-0.21 m/s. This study demonstrates the feasibility of measuring both regional and local PWV in mice using image-based high-frequency ultrasound methodologies.

Detecting vascular changes in tumour xenografts using micro-ultrasound and micro-ct following treatment with VEGFR-2 blocking antibodies.

Blockade of vascular endothelial growth factor (VEGF) binding to its receptors on endothelial cells has been shown preclinically to induce tumour growth inhibition. Using ultrasound biomicroscopy (UBM) or micro-ultrasound imaging and micro-computed tomography (micro-CT) analysis, we have examined the effects of DC101, a highly specific vascular endothelial growth factor receptor-2 (VEGFR-2)-targeting antibody, in inducing growth inhibition and functional vascular changes in established melanoma (MeWo) xenografts in mice. Postprocessing of UBM imaging loops for speckle variance was introduced to estimate the level of functional blood flow in tumours. Perfused tumour area visualized by speckle variance revealed decreased blood flow within 48 h after DC101 injection (control versus DC101: 1.90 +/- 0.25% versus 1.01 +/- 0.11%, p < 0.01) and following a 3-wk DC101 therapy (control versus DC101: 0.76 +/- 0.14% versus 0.45 +/- 0.05%, p = 0.04), suggesting that VEGFR-2 blockade mediates both early and long-term effects on tumour blood flow. The growth of xenografts was significantly inhibited after treating with DC101 for 3 wk compared with controls. In addition to UBM, we examined the tumour vasculature in three-dimension (3D) using contrast-enhanced Micro-CT imaging, which displayed a reduction in the number of tumour vessels following extended VEGFR-2 blockade (vascular density of control versus DC101: 48.4 +/- 5.4% versus 20.6 +/- 1.8%). Lastly, decreased microvessel density (MVD) was noted in DC101-treated xenografts (3 wk) by performing immunohistochemical staining of endothelial marker CD34. Our study investigates tumour response to DC101 using complementing micro-ultrasound and micro-CT imaging tools.

Interframe clutter filtering for high frequency flow imaging.

This study investigates 'interframe' clutter filtering with a high frequency (HF) flow imaging system with the objective of improving the performance of HF microvascular imaging at high frame rates. An interframe filter exploits the correlation of tissue signals on the time scale of the frame rate and is, therefore, insensitive to tissue spectral broadening induced by sweeping a single element transducer over a region of tissue. In vitro experiments were conducted in a tissue-mimicking flow phantom over a range of mean flow velocities (0.5 to 70.0 mm/s). Power Doppler (PD) imaging and color flow (CF) imaging were performed for both slow (0.25 fps) and fast (20 fps) scanning acquisitions. Flow data acquired at 20 fps and interframe filtered had similar velocity and mean Doppler power values as the 0.25 fps single-frame filtered data sets. In vivo validation experiments were conducted using a 500 microm blood vessel in a human finger and detected blood flow of 2 to 3 mm/s. Further in vivo experiments examining experimental murine tumors demonstrated the feasibility of performing HF PD and CF imaging at high frame rates using interframe filtering.

Transgenic expression of Angiopoietin 1 in the liver leads to changes in lymphatic and blood vessel architecture.

To investigate the possible role of the Angiopoietins in vessel remodelling, we overexpressed one of the angiopoietins, Angiopoietin-1 (Ang1), in the hepatocytes of mice by means of the conditional binary transgenic system. Animals were examined by Doppler ultrasound, and dissected livers were analyzed by immunohistochemical staining. Double transgenic mice presented with enlarged spleens and kidneys, enlarged, disorganized blood vessels located near the surface of the liver, sprouting, dilation, and disorganization of liver lymphatics, and turbulent flow in about 1/4 of the blood vessels sampled. Most of these characteristics completely resolved within 12 weeks of turning off the expression of the Ang1 transgene, illustrating a dependence on the continual presence of Ang1 for maintenance of the vascular phenotype. Conditional Angiopoietin-1 overexpression in the liver of mice leads to a phenotype highly reminiscent of portal hypertension illustrating that Ang1 can drive both vascular and lymphatic vessel remodelling and may play a role in portal hypertension.

Ultrahigh frame rate retrospective ultrasound microimaging and blood flow visualization in mice in vivo.

To overcome frame rate limitations in high-frequency ultrasound microimaging, new data acquisition techniques have been implemented for 2-D (B-scan) and color flow visualization. These techniques, referred to as retrospective B-scan imaging (RBI) and retrospective color flow imaging (RCFI) are based on the use of the electrocardiogram (ECG) to trigger signal acquisitions. B-scan and color flow images are reconstructed by retrospectively assembling the processed data on a line-by-line basis. Retrospective techniques are used to produce the first in vivo B-scan and color flow images of mouse carotid arteries at frame rates up to 10,000 fps. Retrospective B-scan images of mouse heart were also produced at frame rates of 1000 fps using a version of RBI implemented on a commercial imaging system (Vevo660, VisualSonics, Toronto, ON, Canada). This technology enables detailed in vivo biomechanical studies of dynamic tissues such as the myocardium of the mouse heart with high temporal resolution.

High-frequency, nonlinear flow imaging of microbubble contrast agents.

It has been shown that nonlinear scattering can be stimulated from microbubble contrast agents at high-transmit frequencies (14-32 MHz). This work was extended to demonstrate the feasibility of nonlinear contrast imaging through modifications of existing ultrasound biomicroscopy linear B-scan imaging instrumentation. In this study, we describe the development and evaluation of prototype coherent flow imaging instrumentation for nonlinear microbubble imaging using transmit frequencies from 10 to 50 MHz. Phantom validation experiments were conducted to demonstrate color and power flow imaging using nonlinear 10 MHz (subharmonic) scattering induced by a 20 MHz transmit frequency. In vivo flow imaging of a rabbit ear microvessel was successfully performed. This work indicates the feasibility of performing flow imaging at high frequencies using nonlinear scattering from microbubbles.

High frequency nonlinear B-scan imaging of microbubble contrast agents.

It previously was shown that it is possible to produce nonlinear scattering from microbubble contrast agents using transmit frequencies in the 14-32 MHz range, suggesting the possibility of performing high-frequency, nonlinear microbubble imaging. In this study, we describe the development of nonlinear microbubble B-scan imaging instrumentation capable of operating at transmit center frequencies between 10 and 50 MHz. The system underwent validation experiments using transmit frequencies of 20 and 30 MHz. Agent characterization experiments demonstrate the presence of nonlinear scattering for the conditions used in this study. Using wall-less vessel phantoms, nonlinear B-scan imaging is performed using energy in one of the subharmonic, ultraharmonic, and second harmonic frequency regions for transmit frequencies of 20 and 30 MHz. Both subharmonic and ultraharmonic imaging modes achieved suppression of tissue signals to below the noise floor while achieving contrast to noise ratios of up to 26 and 17 dB, respectively. The performance of second harmonic imaging was compromised by nonlinear propagation and offered no significant contrast improvement over fundamental mode imaging. In vivo experiments using the subharmonic of a 20 MHz transmit pulse show the successful detection of microvessels in the rabbit ear and in the mouse heart. The results of this study demonstrate the feasibility of nonlinear microbubble imaging at high frequencies.