Jiri Sklenar

Imaging Tumor Angiogenesis With Contrast Ultrasound and Microbubbles Targeted to αvβ3

Background Angiogenesis is a critical determinant of tumor growth and metastasis. We hypothesized that contrastenhanced ultrasound (CEU) with microbubbles targeted to &agr;v‐integrins expressed on the neovascular endothelium could be used to image angiogenesis. Methods and Results Malignant gliomas were produced in 14 athymic rats by intracerebral implantation of U87MG human glioma cells. On day 14 or day 28 after implantation, CEU was performed with microbubbles targeted to &agr;v&bgr;33 by surface conjugation of echistatin. CEU perfusion imaging with nontargeted microbubbles was used to derive tumor microvascular blood volume and blood velocity. Vascular &agr;v‐integrin expression was assessed by immunohistochemistry, and microbubble adhesion was characterized by confocal microscopy. Mean tumor size increased markedly from 14 to 28 days (2±1 versus 35±14 mm2, P<0.001). Tumor blood volume increased by ≈35% from day 14 to day 28, whereas microvascular blood velocity decreased, especially at the central portions of the tumors. On confocal microscopy, &agr;v&bgr;3‐targeted but not control microbubbles were retained preferentially within the tumor microcirculation. CEU signal from &agr;v&bgr;3‐targeted microbubbles in tumors increased significantly from 14 to 28 days (1.7±0.4 versus 3.3±1.0 relative units, P<0.05). CEU signal from &agr;v&bgr;3‐targeted microbubbles was greatest at the periphery of tumors, where &agr;v‐integrin expression was most prominent, and correlated well with tumor microvascular blood volume (r=0.86). Conclusions CEU with microbubbles targeted to &agr;v&bgr;3 can noninvasively detect early tumor angiogenesis. This technique, when coupled with changes in blood volume and velocity, may provide insights into the biology of tumor angiogenesis and be used for diagnostic applications. (Circulation. 2003;108:336‐341.)

Myocardial contrast echocardiography.

“An untroubled mind, no longer seeking to consider what is right and what is wrong; A mind beyond judgements, watches and understands.” The Buddha (translated from the Dhammapada ) The purpose of this article is to describe our personal experience in translating observations made in the experimental laboratory using MCE into the clinical setting. It is not intended to be an exhaustive review of MCE, for which readers are referred elsewhere.1 2 3 The work of others in MCE and related subjects will be mentioned only when it has influenced our own work. Our bench-to-bedside experience with MCE over the past 15 years will be discussed under these six broad categories: (a) technical issues; (b) AMI, (c) detection of CAD, (d) applications in the operating room, (e) quantification of myocardial perfusion, and (f) work in progress. Historically, it has not been possible to directly assess myocardial perfusion with echocardiography. Its clinical focus has involved the evaluation of cardiac chamber size and function, valve morphology and kinetics, pericardial space and great vessels, and intracavitary blood flow velocities. Yet, echocardiography is highly suited for the evaluation of myocardial perfusion for the following reasons: (a) It has very good spatial resolution (<1 mm in the axial direction), which is far superior to that offered by SPECT and positron emission tomography, although not as good as magnetic resonance imaging and ultrafast cine computed tomography; (b) its temporal resolution is excellent (30 to 120 Hz) and exceeds that of other commonly used imaging technologies; (c) for an imaging modality, it is inexpensive and has low overhead costs; (d) it is an integral tool in the day-to-day activities of clinical cardiologists, who can obtain advanced training in its use without needing to learn an entirely new technology. The study of myocardial perfusion with echocardiography involves …

Assessment of Transmural Distribution of Myocardial Perfusion With Contrast Echocardiography

BACKGROUND We hypothesized that by using our newly defined method of destroying microbubbles and measuring their rate of tissue replenishment, we could assess the transmural distribution of myocardial perfusion. METHODS AND RESULTS We studied 12 dogs before and after creation of left anterior descending coronary artery stenoses both at rest and during hyperemia (n=62 stages). Microbubbles were administered as a constant infusion, and myocardial contrast echocardiography (MCE) was performed with the use of different pulsing intervals. The video intensity versus pulsing interval plots derived from each myocardial pixel were fitted to an exponential function: y=A(1-ebetat), where A reflects microvascular cross-sectional area (or myocardial blood volume), and beta reflects mean myocardial microbubble velocity. The product A . beta represents myocardial blood flow (MBF). Average values for these parameters were derived from the endocardial and epicardial regions of interest placed over the left anterior descending coronary artery bed. Radiolabeled microsphere-derived MBF was also measured from the same regions. There was poor correlation between radiolabeled microsphere-derived MBF and A-endocardial/epicardial ratios (EER) (r=0.46). The correlation with beta-EER was better (r=0. 69, P<0.01). The best correlation with radiolabeled microsphere-derived MBF-EER was noted with A . beta-EER (r=0.88, P<0. 01). CONCLUSIONS The transmural distribution of myocardial perfusion can be accurately assessed with MCE with the use of our newly described method of tissue replenishment of microbubbles after their ultrasound-induced destruction. In the model studied, an uncoupling of the transmural distribution of MBF and myocardial blood volume was observed during reversal of the MBF-EER.

Dobutamine echocardiography for determining the extent of myocardial salvage after reperfusion. An experimental evaluation.

BackgroundAlthough dobutamine echocardiography is being increasingly used to determine the presence of viable myocardium in patients who have undergone successful reper-fusion therapy, the physiological basis for such a use has not been clearly defined. Because postischemic myocardium has contractile reserve, we hypothesized that the absolute degree of wall thickening induced by dobutamine during reflow would be directly related to the amount of myocardium that has escaped necrosis. Methods and ResultsThree groups of 12 dogs each were studied at baseline and during 2 to 6 hours of coronary artery occlusion and 15 minutes of reperfusion. In group 1 dogs, which did not receive dobutamine during any of these stages, percent wall thickening at these stages was 32±6%, −2±6%, and 5 ±6%, respectively, and there was no relation between infarct size and percent wall thickening during reflow (r=.20, P=.51). In group 2 dogs, which received 15 μg/kg per minute of dobutamine at all stages, wall thickening at these stages was 40±8%, 0±8%, and 19±10%, respectively, and a good inverse correlation was noted between infarct size and percent wall thickening during reflow (r= −.81, P=.001). In group 3 dogs, in which wall thickening during reflow was measured both before and during infusion of 15 μg/kg per minute of dobutamine, it was 5±8% and 18±14%, respectively, at these stages. Although the correlation between infarct size and percent wall thickening was poor in the absence of dobutamine (r=.36, P=.26), an excellent inverse correlation was noted between the two in the presence of dobutamine (r= −.93, P<.001). A fair inverse correlation was also noted between infarct size and the absolute change in wall thickening induced by dobutamine (r= −.72, P<.01). Maximal wall thickening was noted at a dobutamine dose of 15 μg/kg per minute, and lower doses did not elicit thickening in the presence of larger infarcts despite the presence of viable myocardium. ConclusionsWhen myocardial necrosis coexists with post-ischemic myocardial dysfunction and no residual coronary stenosis, the absolute degree of wall thickening during dobutamine can be used to determine the extent of myocardium that has escaped necrosis. The dose of dobutamine needed to elicit maximal thickening of the postischemic myocardium is related to the amount of myocardial necrosis.

Successful and reproducible myocardial opacification during two-dimensional echocardiography from right heart injection of contrast.

Background Myocardial contrast echocardiography currently involves intra-arterial injection of contrast. For this technique to have a broader application, it is necessary that myocardial opacification be achieved from a venous injection of contrast. Methods and Results To achieve myocardial opacification after right-side injection of contrast, two groups of open-chest anesthetized dogs were studied. Group 1 included nine dogs in whom microbubbles of various sizes, concentrations, and volumes were injected into the left atrium to determine microbubble characteristics that influence myocardial opacification. Group 2 included eight dogs in whom the effect of the combination of microbubble characteristics and myocardial blood flow on myocardial opacification was evaluated after right atrial injection of contrast. Background-subtracted time-intensity plots were generated from the myocardium to measure peak videointensity. In the group 2 dogs, digital subtraction and color coding were used to further highlight the contrast effect. The number, concentration, and size of the microbubbles all independently affected (p < 0.01) peak myocardial videointensity after left atrial injection of contrast on multivariate analysis. Highly concentrated microbubbles (4.4 to 5.1 billion/ml) given during dipyridamole-induced coronary hyperemia was most frequently (88%) associated with myocardial opacification after right atrial injection of contrast and was the best predictor of this result on multivariate analysis (x2=9.01, p = 0.003). No changes were noted in left atrial, left ventricular, and pulmonary artery pressures despite injection of large numbers of microbubbles into the right atrium. Conclusions Successful and reproducible myocardial opacification can be achieved during myocardial contrast echocardiography after right atrial injection of contrast. These findings could have far-reaching implications in the use of myocardial contrast echocardiography in acute and chronic ischemic syndromes in humans.

Coronary Reserve Abnormalities in the Infarcted Myocardium Assessment of Myocardial Viability Immediately Versus Late After Reflow by Contrast Echocardiography

BACKGROUND The aim of this study was to determine whether myocardial contrast echocardiography (MCE) during exogenous vasodilation can accurately delineate infarct size, and hence the extent of myocardial viability, both immediately (15 minutes) and late (3 hours) after reperfusion when postreflow coronary hyperema is still present. METHODS AND RESULTS Twenty-one open-chest anesthetized dogs underwent 3 to 6 hours of coronary occlusion followed by reperfusion. MCE was performed 15 minutes after reflow before and during infusion of 0.2 mg.kg-1.min-1 adenosine i.v.. In 12 dogs, infarct size was measured at this time. In the remaining 9 dogs, reperfusion was continued for 3 hours, when MCE was repeated before and after an infusion of 0.56 mg.kg-1.min-1 dipyridamole i.v. and infarct size was measured. In the absence of adenosine, MCE perfusion defect at 15 minutes underestimated infarct sizes at both 15 minutes and 3 hours, whereas in the presence of adenosine, the estimate of infarct size was more accurate. Similarly, in the absence of dipyridamole, although MCE perfusion defect underestimated infarct size (both measured 3 hours after reflow), in the presence of dipyridamole, the estimate of infarct size was more accurate. CONCLUSIONS By unmasking abnormalities in flow reserve within the infarct bed, MCE in conjunction with coronary vasodilators can accurately predict infarct size both 15 minutes and 3 hours after reperfusion. Thus, MCE can be used for assessing the extent of myocardial viability both immediately and late after reperfusion when postreflow coronary hyperemia is still present.

Method for the Quantitation of Myocardial Perfusion During Myocardial Contrast Two-Dimensional Echocardiography

This article describes the hardware and software components of two systems designed for quantitative analysis of data obtained during myocardial contrast two-dimensional echocardiography. One system is meant for off-line analysis of data, whereas the other is designed for on-line analysis, especially in the operating room. The algorithms used for data transfer, selection of appropriate frames, data alignment, derivation of time-intensity plots, and curve-fitting and parameter generation are described in some detail. It is hoped that this information will be of use to others who work in the field of myocardial perfusion imaging.

Potential Advantage of Flash Echocardiography for Digital Subtraction of B-Mode Images Acquired During Myocardial Contrast Echocardiography

Optimal assessment of myocardial perfusion with contrast echocardiography by using B-mode imaging often requires image alignment and background subtraction, which are time consuming and need extensive expertise. Flash echocardiography is a new technique in which primary images are gated to the electrocardiogram and secondary images are obtained by transmitting ultrasound pulses in rapid succession after each primary image. Myocardial opacification is seen in the primary image and not in the secondary images because of ultrasound-induced bubble destruction. Because the interval between the primary and first few secondary images is very short, cardiac motion between these images should be minimal. Therefore we hypothesized that 1 or more secondary images could be subtracted from the primary image without the need for image alignment. The ability of ultrasound to destroy microbubbles was assessed by varying the sampling rate, line density, and mechanical index in 6 open-chest dogs. The degree of translation between images was quantified in the x and y directions with the use of computer cross-correlation. At sampling rates of 158 Hz or less and a mechanical index of more than 0.6, videointensity rapidly declined to baseline levels by 25 ms. Significant translation between images was noted only at intervals of more than 112 ms. It is concluded that flash echocardiography can be used for digital subtraction of baseline from contrast-enhanced B-mode images without image alignment. Background subtraction is therefore feasible on-line, potentially eliminating the need for off-line image processing in the future.

Detection of Coronary Artery Stenosis With Power Doppler Imaging

Background—Power Doppler is a new imaging method for detecting microbubbles during myocardial contrast echocardiography (MCE) based on the registration of variance resulting from ultrasound-induced nonlinear bubble behavior. We tested the hypothesis that power Doppler imaging can be used to quantify coronary stenoses. Methods and Results—Three left anterior descending (LAD) coronary stenoses of varying severity were created in each of 9 open-chest dogs. MCE was performed by continuous intravenous infusion of a nitrogen-filled bilayer shell microbubble, PB127, during triggered power Doppler imaging at incremental pulsing intervals. MCE and radiolabeled microsphere measurements were made at baseline and during each stenosis, with and without adenosine stress. Videointensities in the LAD and left circumflex (LCx) beds were plotted against pulsing interval and fit to a previously described exponential function modeling microbubble destruction and replenishment, which was used to derive parameters of bubble velocity (&bgr;) and peak plateau videointensity (A). Contrast defects matching the location of radiolabeled microsphere hypoperfusion were clearly seen, without need for image processing. The product of &bgr; and A was linearly related to LAD/LCx flow (r =0.90, P <0.0001) and inversely related to stenosis gradient (r =−0.70, P <0.0001). Endocardial/epicardial flow ratios were visualized and quantifiable. Conclusions—As with B-mode harmonics, a model of microbubble destruction/replenishment can be applied to power Doppler data as a means to detect a broad range of stenoses. Image clarity and the lack of attenuation or requirement for background subtraction are additional advantages of this imaging approach. Power Doppler MCE imaging holds promise for the detection of coronary artery disease.

Quantification of Images Obtained During Myocardial Contrast Echocardiography

This article describes currently used quantitative methods for analysis of data obtained during myocardial contrast echocardiography. The specific issues addressed are: obtaining time‐intensity curves from the myocardium in order to derive transit rates of microbubbles through the myocardium; defining spatial distribution of flow within a myocardial segment; and color‐coding algorithms used to define the extent and magnitude of hypoperfusion within a cross‐section of the heart. These methods are being adopted by several companies dealing with acquisition and analysis of echocardiographic data and should become available soon for clinical use.