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The VUIIS ultrasound efforts have focused on quantitative characterization of blood flow using microbubble contrast enhanced sonography and 3D Doppler imaging. Microbubbles are micron-sized (1-10 mm in diameter) bubbles of perfluropropane with a stabilizing shell. By introducing exogenous microbubbles into the vasculature, the backscattered intensity of blood is enhanced, and microbubble contrast enhanced sonography (MCES) can be used to assess tissue blood flow (1). Simple mathematical models have been developed to analyze ultrasound signal intensity patterns obtained by imaging continuously before, during, and after the injection of microbubbles (2). These models can be used to assess tissue blood flow, and vascular integrity, branching patterns, and density (3).

Assessment of Renal Vascularity using 3D Power-Doppler Sonography: 3D Power Doppler sonography provides measures of tissue volume and vascularity potentially enabling the non-invasive assessment of disease-induced changes in renal perfusion (4). A recent study by Kuwa et al. demonstrated a good correlation between power Doppler image intensity and invasive measures of renal cortical blood flow in swine (5).
(Preliminary Power Doppler Studies)
To determine the potential of this approach to measure renal perfusion we collected power Doppler ultrasound images of a normal adult mouse kidney. Figure U1 illustrates an example 3D Power Doppler image (a) and the reconstructed 3D angiography (b). This method is rapid and non-invasive, hereby sonographic quantification will be beneficial in assessing the changes of renal vascularity.

(Developmental Power Doppler Studies)
Although power Doppler has shown potential to assess renal vascular abnormalities in humans and some animals, its utility in mice needs further investigation and optimization. To validate its applicability in mice we will compare the power Doppler derived vascularity indices with measurements of global renal blood flow, as determined by PAH clearance and glomerular filtration rate as determined from FITC inulin clearance in chronic renal disease models such as the ischemia/perfusion and kidney transplant models. We will also evaluate its potential to monitor acute therapeutic response by comparing its derived parameters with simultaneous measures of renal blood flow using the Oxylite system in mice receiving injections of well-characterized vasoreactive compounds including norepinephrin, angiotension II and bradykinin.

Assessment of Renal Blood Flow and Vascular Properies using Microbubble Contrast-enhanced Sonography (MCES): Microbubbles, developed as contrast agents for ultrasound, were initially envisaged as useful for increasing the intensity of echoes and thus rescuing Doppler studies. However, subsequent studies have demonstrated that they can act as tracers and can be used to measure blood flow (2, 6). The transit of microbubbles across an organ or tissue can be used to estimate hemodynamic alterations. The fragility of microbubbles can be turned to advantage by being exploited to create a negative bolus by exposing a tissue slice to a high power beam. The rate of refilling of this slice by circulating microbubbles can then be followed with a low-intensity monitoring beam and the resulting rising exponential curve analysed to extract indices of both the reperfusion rate (the slope) and the fractional vascular volume (the asymptote). Novel non-linear modes allow vessels down to the level of the microcirculation to be imaged, allowing interrogation of a wide spectrum of tissue beds (7). The product of these is a measure of true tissue perfusion. The method can be used to assess tissue blood flow (BF) and vascular integrity, branching patterns, and density (2, 3, 7, 8). The method will allow, for the first time, real time in vivo visualization of the true renal microcirculation. It will be of great interest to assess renal perfusion in the regions of interest using MCES. Indeed, this method has successfully been applied to assessment of renal vascular properties in mouse kidney (VisualSonics,
(Preliminary MCES Studies)
Figure U2 presents an application of this technique to interrogate the blood flow status of a LLC tumor on the hind limb of a mouse. Panels A and B display signal intensity time courses taken from the highly (filled circle) and moderately (filled square) enhancing ROIs labeled in B-mode image shown in panel C. These time courses represent the replenishment of microbubbles into the field of view as measured by video intensity for the first 22s after the high mechanical index flash pulse. The solid curves were obtained by fitting the data sets with an appropriate mathematical model (3). Panel C is a parametric map of BF as estimated. Of note is the paucity of enhancement seen in the most superficial areas of the tumor. This indicates this region of the tumor is poorly vascularized and/or poorly perfused as few, if any, microbubbles were delivered to this region. MRI data of this same mouse found the same phenomenon (data not shown). According to the manufacture’s guidelines, the kidney MCES study will be performed with a longer baseline, at least 180-200 frames of baseline after hitting pre-trig then injecting the bolus, to minimize the effects of respiratory motion.

(Developmental MCES Studies)
Correlations in BF measurement between MCES and MRI or SPECT/PET (and Oxyflow) will be evaluated. Correlation between MCES-measured vasculariztion and renal vascular density (quantified by a histologic technique including CD31 immunohistochemistry) will be assessed through collaboration with Histology and Morphometry Core.

Targeted Contrast-Enhanced High-Frequency Ultrasonography for Assessment of Renal Vascular Responses: With recent technological advances, ultrasound is also a potential modality for molecular imaging. Ultrasonography offers several key advantages over other molecular imaging modalities: high spatial resolution, real-time imaging, the ability to obtain both anatomic and molecular information in a single imaging session, and freedom from the use of ionizing radiation. Also, the short imaging time (10 min) enables us to assess multiple molecules in a single imaging session. In addition, its non-invasive nature and unbiased tissue sampling allows clinical translation of this method. Traditional immunohistochemical analysis of target protein expression is suboptimal because it requires a high number of animals and tissue sections. Thus, molecular ultrasonography can be an effective tool for assessing renal vasculature because it couples information on molecular and cellular profiles of endothelial cells with information on perfusion and microvascular volume. One of potential applications of the molecular ultrasonography is the assessment of vasculature using the microbubbles which target endothelial antigens such as VEGFR2 (angiogenesis) and P-selectin (inflammation).
(Preliminary Studies)
Recently, we have successfully characterized tumor angiogenesis in mice in vivo using the targeting microbubbles, which are conjugated with VEGFR2 antibody (9). The VEGFR2 antibody-conjugated microbubbles were prepared using streptoavidin-coated target-ready microbubbles (VisualSonics) and biotin-VEGFR2 antibody (eBioscience) according to manufacture’s guidelines. Each mouse received boluses of both the targeted microbubbles and the control microbubbles in random order with a 30 minute interval to minimize sequence bias. The images were acquired with a Vevo770 high-frequency ultrasound system (VisualSonics) with a scanner equipped with a 40 MHz center frequency transducer. The image was acquired and analyzed by differentiating the backscattered acoustic signal due to microbubbles retained in the tumor tissue from the background (postdestruction) signals of the tissue itself and microbubbles still freely circulating in the blood stream as previously described (9). Figure U3 presents targeted MCES for imaging VEGFR2 expression on vascular endothelium in two xenografted breast cancer tumors: a highly invasive metastatic (4T1) and a non-metastatic (67NR) model. Retention of targeted contrast agent was significantly higher in 67NR tumors than in 4T1 tumors and this correlated with VEGFR2 expression as determined by immunohistochemistry.

(Developmental Molecular Ultrasound Imaging)
In addition to the application and validation of anti-VEGFR2 MCES to renal disease models, we will prepare microbubbles conjugated with anti-P selection and Lycopersion esculentum lectin which labels vascular endothelium in vivo and could potentially enable the assessment of renal inflammation and vascularization. Recently, anti-P selectin MCES has successfully applied to assessment of inflammatory responses in ischemic mouse kidneys (VisualSonics, We will further continue the efforts developing novel targeted microbubbles to assess renal vascular properties (e.g. anti-VEGFR3, anti-CD31, anti-VEGFR1 microbubbles).


A Visualsonics Vevo 770 sonographic imaging system, equipped with a 40MHz linear array transducer isavailable for these experiments. Coupling gel is applied to cover the ROI and the transducer head. After anesthetization, the animal is restrained on the imaging surface while the transducer is fixed directly over the site of ROI. A 512x512 acquisition matrix over a 10 mm field of view and a 0.100 mm image slice thickness is the imaging resolution. Scout images are obtained over the whole ROI to select the kidney for MCES characterization. MCES will employ harmonic and pulse inversion imaging techniques. After the administration of 100 ƒÝL of Definity microbubble contrast via a jugular vein catheter, the contrast agent is allowed to reach a steady-state distribution in the blood stream. For the localized destruction of microbubbles within the imaging plane, multiple flashing pulses of high mechanical index (1.0) are administered. Imaging continues before, during, and for 30-60 seconds after the flash pulse so that the replenishment of microbubbles into the field of view could be observed and subsequently modeled to return estimates of blood volume and perfusion.

Publications for Micro-Ultrasound (9)

Lyshchik A, Fleischer AC, Huamani J, Hallahan DE, Brissova M, Gore JC. Molecular imaging of vascular endothelial growth factor receptor 2 expression using targeted contrast-enhanced high-frequency ultrasonography. J Ultrasound Med (2007) 26:1575-86
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OBJECTIVE: The aim of our study was to investigate the use of targeted contrast-enhanced high-frequency ultrasonography for molecular imaging of vascular endothelial growth factor receptor 2 (VEGFR2) expression on tumor vascular endothelium in murine models of breast cancer. METHODS: Highly invasive metastatic (4T1) and nonmetatstatic (67NR) breast cancer cells were implanted in athymic nude mice. Tumors were examined in vivo with targeted contrast-enhanced high-frequency ultrasonography using a scanner with a 40-MHz probe. Randomized boluses of ultrasound contrast agents (UCAs) conjugated with an anti-VEGFR2 monoclonal antibody or an isotype control antibody (immunoglobulin G) were injected into the animals. Sonograms were analyzed by calculation of the normalized video intensity amplitudes caused by backscatter of the bound UCA. After ultrasonography, the tumor samples were harvested for analysis of VEGFR2 expression by immunoblotting and immunocytochemistry. RESULTS: The mean video intensity amplitude caused by backscatter of the retained VEGFR2-targeted UCA was significantly higher than that of the control UCA (mean +/SD: 4T1 tumors, 15 +/3.5 versus 7 +/1.6 dB; P < .01; 67NR tumors, 50 +/12.3 versus 12 +/2.6 dB; P < .01). There was a significant difference in VEGFR2-targeted UCA retention between 4T1 and 67NR tumors (normalized video intensity amplitudes, 15 +/3.5 and 50 +/12.3 dB, respectively; P < .001), and this correlated well with relative VEGFR2 expression in the two tumor types. CONCLUSIONS: Targeted contrast-enhanced high-frequency ultrasonography may enable in vivo molecular imaging of VEGFR2 expression on the tumor vascular endothelium and may be used for noninvasive longitudinal evaluation of tumor angiogenesis in preclinical studies.

Xuan JW, Bygrave M, Jiang H, Valiyeva F, Dunmore-Buyze J, Holdsworth DW, Izawa JI, Bauman G, Moussa M, Winter SF, Greenberg NM, Chin JL, Drangova M, Fenster A, Lacefield JC. Functional neoangiogenesis imaging of genetically engineered mouse prostate cancer using three-dimensional power Doppler ultrasound. Cancer Res (2007) 67:2830-9
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We report the first application of high-frequency three-dimensional power Doppler ultrasound imaging in a genetically engineered mouse (GEM) prostate cancer model. We show that the technology sensitively and specifically depicts functional neoangiogenic blood flow because little or no flow is measurable in normal prostate tissue or tumors smaller than 2-3 mm diameter, the neoangiogenesis "switch-on" size. Vascular structures depicted by power Doppler were verified using Microfil-enhanced micro-computed tomography (micro-CT) and by correlation with microvessel distributions measured by immunohistochemistry and enhanced vascularity visualized by confocal microscopy in two GEM models [transgenic adenocarcinoma of the mouse prostate (TRAMP) and PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP)]. Four distinct phases of neoangiogenesis in cancer development were observed, specifically, (a) an early latent phase; (b) establishment of a peripheral capsular vascular structure as a neoangiogenesis initiation site; (c) a peak in tumor vascularity that occurs before aggressive tumor growth; and (d) rapid tumor growth accompanied by decreasing vascularity. Microsurgical interventions mimicking local delivery of antiangiogenesis drugs were done by ligating arteries upstream from feeder vessels branching to the prostate. Microsurgery produced an immediate reduction of tumor blood flow, and flow remained low from 1 h to 2 weeks or longer after treatment. Power Doppler, in conjunction with micro-CT, showed that the tumors recruit secondary blood supplies from nearby vessels, which likely accounts for the continued growth of the tumors after surgery. The microsurgical model represents an advanced angiogenic prostate cancer stage in GEM mice corresponding to clinically defined hormone-refractory prostate cancer. Three-dimensional power Doppler imaging is completely noninvasive and will facilitate basic and preclinical research on neoangiogenesis in live animal models.

Yankeelov TE, Niermann KJ, Huamani J, Kim DW, Quarles CC, Fleischer AC, Hallahan DE, Price RR, Gore JC. Correlation between estimates of tumor perfusion from microbubble contrast-enhanced sonography and dynamic contrast-enhanced magnetic resonance imaging. J Ultrasound Med (2006) 25:487-97
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OBJECTIVE: We compared measurements of tumor perfusion from microbubble contrast-enhanced sonography (MCES) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in an animal tumor model. METHODS: Seven mice were implanted with Lewis lung carcinoma cells on their hind limbs and imaged 14 days later with a Philips 5to 7-MHz sonography system (Philips Medical Systems, Andover, MA) and a Varian 7.0-T MRI system (Varian, Inc, Palo Alto, CA). For sonographic imaging 100 microL of a perfluoropropane microbubble contrast agent (Definity; Bristol-Myers Squibb Medical Imaging, Billerica, MA) was injected and allowed to reach a pseudo steady state, after which a high-mechanical index pulse was delivered to destroy the microbubbles within the field of view, and the replenishment of the microbubbles was imaged for 30 to 60 seconds. The MRI included acquisition of a T(10) map and 35 serial T(1)-weighted images (repetition time, 100 milliseconds; echo time, 3.1 milliseconds; alpha, 30 degrees ) after the injection of 100 microL of 0.2-mmol/kg gadopentetate dimeglumine (Magnevist; Berlex, Wayne, NJ). Region-of-interest and voxel-by-voxel analyses of both data sets were performed; microbubble contrast-enhanced sonography returned estimates of microvessel cross-sectional area, microbubble velocity, and mean blood flow, whereas DCE-MRI returned estimates of a perfusion-permeability index and the extravascular extracellular volume fraction. RESULTS: Comparing similar regions of tumor tissue seen on sonography and MRI, region-of-interest analyses revealed a strong (r(2) = 0.57) and significant relationship (P < .002) between the estimates of perfusion obtained by the two modalities. CONCLUSIONS: Microbubble contrast-enhanced sonography can effectively depict intratumoral heterogeneity in preclinical xenograft models when voxel-by-voxel analysis is performed, and this analysis correlates with similar DCE-MRI measurements.

Cosgrove D. Angiogenesis imaging--ultrasound. Br J Radiol (2003) 76 Spec No 1:S43-9
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Kuwa T, Cancio LC, Sondeen JL, Matylevich N, Jordan BS, McManus AT, Goodwin CW. Evaluation of renal cortical perfusion by noninvasive power Doppler ultrasound during vascular occlusion and reperfusion. J Trauma (2004) 56:618-24
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BACKGROUND: Urine output, a frequently used resuscitation end point, is presumed to represent renal cortical perfusion. However, no noninvasive method for direct measurement of renal perfusion exists. Power Doppler ultrasound (PDUS) is a method that reportedly is sensitive to low-velocity and microvascular blood flow and can depict it. This study aimed to develop a quantitative technique for PDUS image analysis, and to evaluate the ability of PDUS to quantify cortical perfusion during renal ischemia induced by vascular occlusion. METHODS: A method was developed to determine the mean gray-scale intensity of PDUS images from within the renal cortex (PDUS image intensity). This index was hypothesized to represent renal cortical microvascular blood flow. Renal cortical blood flow was determined using fluorescent microspheres in five swine. Renal artery flow was measured with an ultrasonic flow probe. Power Doppler ultrasound was performed at baseline; at 75%, 50%, and 25% of baseline renal artery flow; and during reperfusion. RESULTS: Subjectively, PDUS images showed decreases in image intensity corresponding to renal artery occlusion and increases after reperfusion. Cortical blood flow correlated well with renal artery flow (n = 25; r2 = 0.868) and with PDUS image intensity (n = 25; r2 = 0.844). CONCLUSION: Noninvasive power Doppler ultrasound image intensity correlated well with invasively measured renal cortical blood flow, and may be useful during resuscitation of injured and critically ill patients.

Krix M, Kiessling F, Vosseler S, Farhan N, Mueller MM, Bohlen P, Fusenig NE, Delorme S. Sensitive noninvasive monitoring of tumor perfusion during antiangiogenic therapy by intermittent bolus-contrast power Doppler sonography. Cancer Res (2003) 63:8264-70
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Intermittent bolus-contrast power Doppler ultrasound was used for noninvasive, quantitative monitoring of tumor perfusion during antiangiogenic therapy. Subcutaneous heterotransplants of human squamous cell carcinoma cells in nude mice were treated with a blocking antibody to vascular endothelial growth factor receptor 2 (DC101) and repeatedly examined at weekly intervals. Using replenishment kinetics of microbubbles (Levovist) tumor vascularization, including capillary blood flow, was clearly visualized by this dynamic ultrasound method allowing the determination of a comprehensive functional status of tumor vascularization (blood volume, blood flow, perfusion, and mean blood velocity) in all examined tumors. DC101 treatment decreased tumor blood flow (-64%) and volume (-73%) compared with untreated controls (+409% and +185%, respectively). Regression of functional vessel parameters was observed early well before reduction of tumor size. The treatment-related amount of reduction in tumor volume was directly correlated for the initial tumor blood flow before start of therapy and the perfusion calculated at the preceding examination. The vessel density (immunofluorescence staining with CD31 antibody at different time points) showed an excellent correlation with the calculated relative blood volume (k = 0.84, P < 0.01), thereby validating intermittent sonography as a useful monitoring method. We conclude that intermittent sonography is a promising tool for comprehensive monitoring of antiangiogenic or proangiogenic therapies, especially during early stages of treatment, thus yielding information regarding a prospective evaluation of therapy effects beyond the follow up of tumor size.

Yeh CK, Yang MJ, Li PC. Contrast-specific ultrasonic flow measurements based on both input and output time intensities. Ultrasound Med Biol (2003) 29:671-8
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Ultrasonic contrast agents are used to assess perfusion conditions based on evaluation of the time-intensity curve. Such a curve reflects the concentration of microbubbles in the perfused area and the indicator-dilution theory is used to derive the volumetric flow rate from the measured concentration. Previous results have shown that the technique is not reliable in some conditions due to the shadowing effect. To overcome this problem, a contrast-specific technique using both the input and output time-intensity relationships is proposed; this contrasts with conventional techniques that utilize only the relationship directly from the perfused area. The proposed technique is referred to as the input-output time-intensity curve (IOTIC) method. In this work, the shadowing effect was studied experimentally and the efficacy of the IOTIC technique was assessed and compared with conventional techniques. The results indicate that the IOTIC technique eliminates the shadowing effect and provides a good correlation between the actual flow rate and measured flow-related parameters; thus, making quantitative estimation of perfusion feasible. Note that the IOTIC is applicable, based on the assumption that both the input and the output can be positioned within the same image plane; its clinical applications include situations where the perfused area cannot be effectively imaged by ultrasound (US). One example is the assessment of brain perfusion, and it will be used as a target clinical application of the IOTIC technique.

Cosgrove D, Eckersley R, Blomley M, Harvey C. Quantification of blood flow. Eur Radiol (2001) 11:1338-44
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Traditionally, Doppler ultrasound has been used to estimate blood flow as the mean velocity multiplied by the vessel area, but this is subject to significant errors and may be difficult to perform accurately. Microbubbles, developed as contrast agents for ultrasound, were initially envisaged as useful for increasing the intensity of echoes and thus rescuing Doppler studies that were technical failures because of attenuated signals or very slow flow. However, they can act as tracers and, by analogy with isotope techniques, can be used to measure blood flow with transit-time methods which exploit both arterial and venous time-intensity data. An acceptable compromise is to acquire both a tissue intensity curve and one from the feeding artery. The transit of microbubbles across an organ or tissue can be used to estimate haemodynamic alterations, e.g. the arterialisation of the supply to the liver in malignancies and cirrhosis and the delayed arterio-venous transit in the transplant kidney during rejection. The fragility of microbubbles can be turned to advantage by being exploited to create a negative bolus by exposing a tissue slice to a high power beam. The rate of refilling of this slice by circulating microbubbles can then be followed with a low-intensity monitoring beam and the resulting rising exponential curve analysed to extract indices of both the reperfusion rate (the slope) and the fractional vascular volume (the asymptote). The product of these is a measure of true tissue perfusion.

Harvey CJ, Blomley MJ, Eckersley RJ, Cosgrove DO. Developments in ultrasound contrast media. Eur Radiol (2001) 11:675-89
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Ultrasound microbubble contrast agents are effective and safe echo enhancers. An ingenious array of methods are employed to achieve stability and provide a clinically useful enhancement period. Microbubbles enhance ultrasound signals by up to 25 dB (greater than 300-fold increase) due to resonant behaviour. This is used to rescue failed Doppler studies and may be extended to image the microcirculation of tumours and the myocardium using non-linear modes. Functional studies open up a whole range of applications by using a variety of active and passive quantitation techniques to derive indices from the transit of contrast through a tissue of interest. This has been especially successful in the detection of liver metastases and cirrhosis and shows great promise as a clinical tool. It also has great potential in measuring microcirculatory flow velocity. The demonstration that some microbubbles are not just pure blood pool agents but have a hepatosplenic specific phase has extended the versatility of ultrasound. Imaging of this stationary phase with non-linear modes such as phase inversion and stimulated acoustic emission, has improved the sensitivity and specificity of ultrasound in the detection and characterisation of focal liver lesions to rival that of CT and MR.

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Last updated on 2008-01-25 Moderated by Takamune Takahashi