Danny M. Skyba

Quantification of Myocardial Blood Flow With Ultrasound-Induced Destruction of Microbubbles Administered as a Constant Venous Infusion

BACKGROUND Ultrasound can cause microbubble destruction. If microbubbles are administered as a continuous infusion, then their destruction within the myocardium and measurement of their myocardial reappearance rate at steady state will provide a measure of mean myocardial microbubble velocity. Conversely, measurement of their myocardial concentration at steady state will provide an assessment of microvascular cross-sectional area. Myocardial blood flow (MBF) can then be calculated from the product of the two. METHODS AND RESULTS Ex vivo and in vitro experiments were performed in which either flow was held constant and pulsing interval (interval between microbubble destruction and replenishment) was altered, or vice versa. In vivo experiments were performed in 21 dogs. In group 1 dogs (n=7), MBF was mechanically altered in a model in which coronary blood volume was constant. In group 2 dogs (n=5), MBF was altered by direct coronary infusions of vasodilators. In group 3 dogs (n=9), non-flow-limiting coronary stenoses were created, and MBF was measured before and after the venous administration of a coronary vasodilator. In all experiments, microbubbles were delivered as a constant infusion, and myocardial contrast echocardiography was performed using different pulsing intervals. The myocardial video intensity versus pulsing interval plots were fitted to an exponential function: y=A(1-e[-betat]), where A is the plateau video intensity reflecting the microvascular cross-sectional area, and beta reflects the rate of rise of video intensity and, hence, microbubble velocity. Excellent correlations were found between flow and beta, as well as flow and the product of A and beta. CONCLUSIONS MBF can be quantified with myocardial contrast echocardiography during a venous infusion of microbubbles. This novel approach has potential for measuring tissue perfusion in any organ accessible to ultrasound.

Direct In Vivo Visualization of Intravascular Destruction of Microbubbles by Ultrasound and its Local Effects on Tissue

BACKGROUND Our aim was to observe ultrasound-induced intravascular microbubble destruction in vivo and to characterize any resultant bioeffects. METHODS AND RESULTS Intravital microscopy was used to visualize the spinotrapezius muscle in 15 rats during ultrasound delivery. Microbubble destruction during ultrasound exposure caused rupture of < or = 7-microm microvessels (mostly capillaries) and the production of nonviable cells in adjacent tissue. The number of microvessels ruptured and cells damaged correlated linearly (P<0.001) with the amount of ultrasound energy delivered. CONCLUSIONS Microbubbles can be destroyed by ultrasound, resulting in a bioeffect that could be used for local drug delivery, angiogenesis, and vascular remodeling, or for tumor destruction.

Delivery of Colloidal Particles and Red Blood Cells to Tissue Through Microvessel Ruptures Created by Targeted Microbubble Destruction With Ultrasound

BACKGROUND We have previously shown that the application of ultrasound to thin-shelled microbubbles flowing through small microvessels (<7 microm in diameter) produces vessel wall ruptures in vivo. Because many intravascular drug- and gene-delivery vehicles are limited by the endothelial barrier, we hypothesized that this phenomenon could be used to deliver drug-bearing vehicles to tissue. METHODS AND RESULTS An exteriorized rat spinotrapezius muscle preparation was used. Intravascular fluorescent red blood cells and polymer microspheres (PM) (205 and 503 nm in diameter) were delivered to the interstitium of rat skeletal muscle through microvessel ruptures created by insonifying microbubbles in vivo. On intravital microscopy, mean dispersion areas per rupture for red blood cells, 503-nm PM, and 205-nm PM were 14.5x10(3) microm2, 24. 2x10(3) microm2, and 27.2x10(3) microm2, respectively. PM dispersion areas were significantly larger than the mean dispersion area for red blood cells (P<0.05). CONCLUSIONS Microvessel ruptures caused by insonification of microbubbles in vivo may provide a minimally invasive means for delivering colloidal particles and engineered red blood cells across the endothelial lining of a targeted tissue region.

Interactions between microbubbles and ultrasound: In vitro and in vivo observations

OBJECTIVES We attempted to examine the interactions between ultrasound and microbubbles. BACKGROUND The interactions between microbubbles and ultrasound are poorly understood. We hypothesized that 1) ultrasound destroys microbubbles, and 2) this destruction can be minimized by limiting the exposure of microbubbles to ultrasound. METHODS We performed in vitro and in vivo experiments in which microbubbles were insonated at different frequencies, transmission powers and pulsing intervals. Video intensity decay was measured in vitro and confirmed by measurements of microbubble size and concentrations. Peak video intensity and mean microbubble myocardial transit rates were measured in vivo. RESULTS Imaging at lower frequencies and higher transmission powers resulted in more rapid video intensity decay (p = 0.01), and decreasing exposure of microbubbles to ultrasound minimized their destruction in vitro. Although these effects were also noted in vivo with venous injections of microbubbles, they were not seen with aortic root or direct coronary artery injections. CONCLUSIONS Ultrasound results in microbubble destruction that is more evident at lower frequencies and higher acoustic powers. Reducing the exposure of microbubbles to ultrasound minimizes their destruction. This effect is most marked in vivo with venous rather than aortic or direct coronary injections of microbubbles. These findings could lead to effective strategies for myocardial perfusion imaging with venous injections of microbubbles.

Basis for detection of stenosis using venous administration of microbubbles during myocardial contrast echocardiography: bolus or continuous infusion?

OBJECTIVES This study sought to determine the basis of detection of stenosis by myocardial contrast echocardiography using venous administration of microbubbles and to define the relative merits of bolus injection versus continuous infusion. BACKGROUND The degree of video intensity (VI) disparity in myocardial beds supplied by stenosed and normal coronary arteries can be used to quantify stenosis severity after venous administration of microbubbles. However, the comparative merits of administering microbubbles as a bolus injection or continuous infusion has not been studied. METHODS Coronary stenoses of varying severity were created in either the left anterior descending or the left circumflex coronary artery in 18 dogs. Imagent US (AF0150) was given as a bolus injection in 10 dogs (Group I) and as both a bolus injection and a continuous infusion in 8 dogs (Group II). For bolus injections, peak VI was derived from time-intensity plots. During continuous infusion, microbubble velocity and microvascular cross-sectional area were derived from pulsing interval versus VI plots. Myocardial blood flow (MBF) was determined using radiolabeled microspheres. RESULTS During hyperemia, VI ratios from the stenosed versus normal beds correlated with radiolabeled microsphere-derived MBF ratios from those beds for both bolus injections (r = 0.81) and continuous infusion (r = 0.79). The basis for detection of stenosis common to both techniques was the decrease in myocardial blood volume distal to the stenosis during hyperemia. The advantage of continuous infusion over bolus injection was the abolition of posterior wall attenuation and the ability to quantify MBF. CONCLUSIONS Both bolus injection and continuous infusion provide quantitative assessment of relative stenosis severity. Compared with bolus injection, continuous infusion also allows quantification of MBF and data acquisition without attenuation of any myocardial bed.

Hemodynamic characteristics, myocardial kinetics and microvascular rheology of FS-069, a second-generation echocardiographic contrast agent capable of producing myocardial opacification from a venous injection

OBJECTIVES We sought to 1) study the effects of FS-069 on cardiac and systemic hemodynamic function, myocardial blood flow, left ventricular wall thickening and pulmonary gas exchange when injected intravenously; and 2) compare the myocardial kinetics and microvascular rheology of FS-069 and Albunex when injected directly into a coronary artery. BACKGROUND FS-069 is a second-generation echocardiographic contrast agent composed of perfluoropropane-filled albumin microspheres; it is capable of consistent and reproducible myocardial opacification from a venous injection. METHODS Nine dogs were used to study the effects of FS-069 on hemodynamic function, pulmonary gas exchange, left ventricular wall thickening and myocardial blood flow and to characterize its myocardial kinetics when injected intravenously. These dogs were also used to compare the myocardial kinetics of FS-069 with those of Albunex during intracoronary injections. Nine Sprague-Dawley rats were used to compare the microvascular rheology of these two contrast agents, and in vitro modeling was performed to assess whether the microvascular findings of FS-069 can explain its echocardiographic behavior during direct coronary injections. RESULTS There were no effects of 30 rapid venous injections of FS-069 (every 20 s) on cardiac output; mean aortic, pulmonary or left atrial pressures; and peak positive and negative first derivative of left ventricular pressure (dP/dt). Similarly, there were no effects of this agent on radiolabeled microsphere-measured regional myocardial blood flow, left ventricular wall thickening or pulmonary gas exchange. When injected intravenously, the myocardial transit of this agent resembled a gamma-variate form. When diluted FS-069 was injected directly into the coronary artery; however, its transit resembled the integral of gamma-variate function, with persistent myocardial opacification lasting several minutes, which was different from that of Albunex. Intravital microscopy revealed that, unlike Albunex, when no bubbles are entrapped within the microcirculation after an arterial injection, a very small fraction of the diluted, larger FS-069 microbubbles are entrapped. In vitro modeling confirmed that this small fraction of microbubbles can result in persistent myocardial opacification. CONCLUSIONS FS-069 produces no changes in hemodynamic function, myocardial blood flow, left ventricular wall thickening or pulmonary gas exchange when injected intravenously in large amounts. When diluted FS-069 is injected into the coronary artery, a very small fraction of the larger bubbles are entrapped within the microcirculation, resulting in a persistent contrast effect. Thus, although FS-069 is a safe intravenous echocardiographic contrast agent, it cannot provide information on myocardial blood flow when injected directly into a coronary artery.

Quantification of myocardial perfusion with myocardial contrast echocardiography during left atrial injection of contrast. Implications for venous injection.

BackgroundThe purpose of this study was to determine whether myocardial perfusion can be quantified with myocardial contrast echocardiography using left atrial (LA) injection of contrast. Methods and ResultsBased on a series of in vitro and in vivo experiments, the optimal dose of sonicated albumin microbubbles injected into the LA for establishing a linear relation between video intensity and blood volume in the anterior myocardium was determined. In 10 open-chest dogs, myocardial blood flow (MBF) was augmented by increasing myocardial blood volume (MBV) with an intravenous infusion of phenylephrine HCl. In the presence of this drug, left anterior descending artery stenosis was produced, followed by release of stenosis, to change MBF within the anterior myocardium. MBV was calculated by dividing radiolabeled microsphere-derived MBF by microbubble transit rate. There was close coupling between MBF and MBV in the anterior myocardium during LA injection of contrast (y=1.0x−0.03, SEE=1.07, r=.92, P<.001). An excellent correlation was also noted between background-subtracted peak video intensity and MBV (y=0.24x+0.73, SEE=0.36, r=.88, P<.001). On multivariate analysis, background-subtracted peak video intensity correlated best with MBV. ConclusionsMyocardial perfusion can be quantified from time-intensity curves derived from the anterior myocardium after LA injection of contrast. Background-subtracted peak video intensity in this situation correlates closely with MBV. When MBV and MBF are closely coupled, such as during inotropic stimulation of the heart, background-subtracted peak video intensity also correlates closely with MBF. Since there are similarities in the models of LA and venous injections, these data indicate that it may be feasible to quantify myocardial perfusion with myocardial contrast echocardiography after venous injection of contrast.

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.

Albumin Microbubble Persistence During Myocardial Contrast Echocardiography Is Associated With Microvascular Endothelial Glycocalyx Damage

BACKGROUND We hypothesized that the persistence of albumin microbubbles within the myocardium during crystalloid cardioplegia (CP) infusion and ischemia-reperfusion (I-R) occurs because of endothelial injury. METHODS AND RESULTS The myocardial transit rate of albumin microbubbles was measured in 18 dogs perfused with different CP solutions and in 12 dogs undergoing I-R. Electron microscopy with cationized ferritin labeling of the glycocalyx was performed in 9 additional dogs after CP perfusion and in 3 additional dogs undergoing I-R. Microbubble transit was markedly prolonged during crystalloid CP perfusion. The addition of whole blood to the CP solution accelerated the transit rate in a dose-dependent fashion (P<0.05), which was greater with venous than with arterial blood (P<0.05). The addition of plasma or red blood cells to CP solutions was less effective in improving transit rate than addition of whole blood (P<0.05). Microbubble transit rate was independent of the temperature, K+ content, pH, PO2, osmolality, viscosity, and flow rate of the perfusate. Similarly, a proportion of microbubbles persisted in the myocardium after I-R, which was related to the duration of ischemia (P<0.01) but not of reflow. Crystalloid CP perfusion and I-R resulted in extensive loss of the endothelial glycocalyx without other ultrastructural changes. This effect was partially reversed in the case of crystalloid CP when it was followed by blood CP. CONCLUSIONS Sonicated albumin microbubbles persist within the myocardium in situations in which the endothelial glycocalyx is damaged. The measurement of the myocardial transit rate of albumin microbubbles may provide an in vivo assessment of endothelial glycocalyx damage.

Myocardial perfusion characteristics and hemodynamic profile of MRX-115, a venous echocardiographic contrast agent, during acute myocardial infarction.

We sought to determine whether MRX-115, a new venous echocardiographic contrast agent, could accurately assess risk area during coronary occlusion and infarct size after reperfusion by using novel imaging modalities meant to selectively enhance contrast signals. In 12 open-chest dogs, venous injections of 0.5 ml of MRX-115 were performed during baseline and coronary occlusion and after reperfusion in the presence of exogenous hyperemia. Ultrasound was transmitted at 2 MHz and received at both 2 MHz (fundamental) and 4 MHz (harmonic) frequencies during continuous and intermittent (end-systolic only) imaging. The risk area during coronary occlusion was compared with technetium autoradiography, and the infarct size after reperfusion was compared with postmortem tissue staining. MRX-115 produced no alterations in hemodynamic or pulmonary gas exchange at any stage. During continuous (both fundamental and harmonic) and intermittent fundamental imaging, measurements of perfusion defects were precluded in many dogs by either poor signal enhancement or posterior wall attenuation. By comparison, these measurements were possible during intermittent harmonic imaging in all dogs except one, which had a very small infarction during reflow. Correlation analysis between perfusion defect size on intermittent harmonic imaging and either autoradiographic risk area or postmortem infarct size gave r values of 0.83 and 0.92, respectively. We conclude that MRX-115 is hemodynamically well tolerated and, when imaging is performed after venous injection, can accurately assess regions of hypoperfusion when combined with intermittent harmonic imaging. These results are promising for the use of this approach in patients with acute myocardial infarction.