EXPERIMENTAL STUDIES
Spontaneous redistribution after reperfusion
A unique property of AIP 201, an ultrasound contrast agent
Andre Z. Linka, MDa,
Danny M. Skyba, PhDa,
Richard J. Price, PhD*,
Kevin Wei, MD, FACCa,
Thomas C. Skalak, PhD* and
Sanjiv Kaul, MD, FACCa
a Cardiovascular Division, University of Virginia, Charlottesville, Virginia, USA
* Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA
Manuscript received March 25, 1998;
revised manuscript received July 18, 1998,
accepted July 22, 1998.
Address for correspondence: Dr. Sanjiv Kaul, Cardiovascular Division, Box 158, University of Virginia Medical Center, Charlottesville, Virginia 22908
sk{at}virginia.edu
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Abstract
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Objectives. We sought to determine the mechanism of spontaneous redistribution of AIP 201 microbubbles after reperfusion from a single left heart injection performed during coronary occlusion.
Background. AIP 201, an ultrasound contrast agent consisting of 10-µm sized microbubbles, has demonstrated spontaneous myocardial redistribution in preliminary studies.
Methods. Myocardial video intensity (VI) and radiolabeled microsphere-derived myocardial blood flow (MBF) were measured serially after reperfusion in seven dogs undergoing an AIP 201 injection during coronary occlusion. The behavior of these bubbles was also assessed in the rat spinotrapezius muscle using intravital microscopy (IM), both with and without ultrasound. The effect of ultrasound on these bubbles was also determined in vitro.
Results. A spontaneous and gradual increase in myocardial VI was noted after reperfusion, which was related to the magnitude of increase in MBF to that region (r = 0.82, p < 0.001). On IM, most of the microbubbles were seen entrapped in small arterioles. Some larger arterioles had aggregates of microbubbles that periodically became dislodged and moved downstream. This behavior was not affected in vivo by ultrasound. In vitro, however, microbubble aggregation was noted only during ultrasound exposure.
Conclusions. The magnitude of redistribution of AIP 201 microbubbles to the reperfused myocardium is related to changes in MBF and occurs from their dislodgement from microbubble aggregates entrapped in large arterioles. In vitro microbubble aggregation seen during ultrasound exposure was not reproduced in vivo. These results may have important implications for studying the effects of interventions in acute coronary syndromes and after coronary artery bypass graft surgery.
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Abbreviations and Acronyms
| | DTAF | = 5-([4,6-dichlorotriazin-2-YL] amino)-fluorescein hydrochloride | | IM | = intravital microscopy | | LAD | = left anterior descending coronary artery | | LCx | = left circumflex coronary artery | | LV | = left ventricular | | MBF | = myocardial blood flow | | MCE | = myocardial contrast echocardiography | | MI | = mechanical index | | VI | = video intensity |
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AIP 201 is an ultrasound contrast agent that consists of air-filled microbubbles with a mean concentration of 1.5 x 107·ml–1, a mean diameter of 10 µm and a 1-µm thick shell composed of denatured albumin. The large size of the microbubbles precludes their intravenous use; however, they provide excellent myocardial opacification on ultrasound examination when injected into the aortic root or left heart because they become entrapped in the myocardial microcirculation. Their thick shell prevents inward or outward diffusion of air as well as their destruction by ultrasound (1). These properties result in long-lasting myocardial opacification on ultrasound examination (1).
AIP 201 does not cause adverse hemodynamic effects when injected into the left atrium in doses required to produce optimal myocardial opacification (1). It can also be used to accurately define risk area during coronary occlusion and infarct size after reperfusion (1). In pilot studies we observed that when it was injected during coronary occlusion, as expected, a lack of myocardial opacification was noted within the occluded bed. After reperfusion, however, a spontaneous and gradual increase in myocardial opacification of the previously occluded bed was seen, which was not associated with recirculation of the agent as evidenced by the lack of contrast effect in the cardiac chambers. To our knowledge, this unique phenomenon of redistribution has not been reported before with any other ultrasound contrast agent.
The purposes of this study were to further characterize this phenomenon of spontaneous redistribution of AIP 201 microbubbles after reperfusion and to elucidate its mechanism. An additional aim was to determine whether this phenomenon is influenced by exposure to ultrasound.
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Methods
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The experiments were approved by the Animal Research Committee at the University of Virginia and conformed to the "Position of the American Heart Association on Research Animal Use," adopted by the Association in November 1984. They were performed in eight adult mongrel dogs and 12 Sprague-Dawley rats. The dogs were used to assess temporal changes in myocardial opacification after reflow from a single injection of AIP 201 (Andaris Ltd.) administered during coronary occlusion, with reference to changes in myocardial blood flow (MBF). The rats were used to study the behavior of AIP 201 in the microcirculation with intravital microscopy (IM) both in the absence and presence of ultrasound. Finally, in vitro experiments were performed to elucidate the physical effects of ultrasound energy on the microbubbles.
Experimental preparations.
Canine model
Dogs were anesthetized with 30 mg·kg–1 of sodium pentobarbital (Abbott Laboratories), intubated and ventilated with room air by means of a respirator pump (Model 607, Harvard Apparatus). Additional anesthesia was administered during the experiment as needed. A 7F catheter was placed in the femoral artery to obtain pressure measurements and withdrawal of arterial reference samples for radiolabeled microsphere MBF analysis. A similar catheter was also placed in each femoral vein for intravenous administration of fluids and drugs as needed. A left lateral thoracotomy was performed, and the heart was suspended in a pericardial cradle. The proximal portions of either the left anterior descending coronary artery (LAD) or left circumflex coronary artery (LCx) were dissected free from the surrounding tissues, and an occluder was placed around one of them. A 7F catheter was placed in the left atrium for pressure measurement and injections of microbubbles and radiolabeled microspheres.
Rat model
The Sprague-Dawley rats anesthetized with an intramuscular injection of a mixture of 1% alpha-chloralose and 13.3% urethane (0.6 ml·100g–1 body weight) were placed on a heating pad. The left femoral vein was cannulated for administration of additional anesthesia as needed, and the right carotid artery was similarly cannulated to enable microbubble injection. The right spinotrapezius muscle was prepared for IM as previously described (2,3). The 0.25-mm thick muscle was exteriorized, leaving the anterior edge with the main feeding artery and vein intact. It was continuously superfused with warm (37°C) Ringer’s solution (pH 7.4) saturated with 5% carbon dioxide and 95% nitrogen.
The preparation was moved to a custom-designed stage with a water bath, which had a small aperture for placement of the exteriorized muscle (Fig. 1). Its inside walls were lined with baffled sponge rubber, which served as an ultrasound absorber. The muscle was carefully drawn through the aperture into the interior of the bath and spread over a transparent coverslip placed on a hollow pedestal. A water-tight seal was created between the margins of the aperture and the muscle using nonreactive grease. The bath was then filled with warmed Ringer’s solution, and the preparation was allowed a minimum of 15 min to stabilize before observations were initiated.
Ultrasound imaging.
Ultrasound examinations were performed using a phased array system (HDI 3000cv, Advanced Technology Laboratories) capable of harmonic imaging (transmitted at 2.3 MHz and received at 4.6 MHz). Gain settings were optimized at the beginning of each experiment and were held constant throughout. A dynamic range of 60 dB and a frame rate of 30 Hz were used. Images were recorded on 1.25-cm S-VHS videotape (Panasonic AG-MD830, Matsushita Electric).
Canine experiments
A saline bath acted as an acoustic interface between the transducer and the heart. The transducer was positioned over the heart by means of a clamp affixed to the procedure table. A mechanical index (MI) of 0.7 to 0.9 was used. Short-axis images at the mid-papillary muscle and the orthogonal long-axis levels were obtained. These images were transferred from videotape to the video memory of a computer where video intensity (VI) was measured from regions of interest (>500 pixels) defined over the LAD and LCx beds in both the long- and short-axis views. Measurements from three consecutive end-diastolic frames were averaged for each view from which the mean VI was calculated.
Rat experiments
The ultrasound transducer was positioned in the water bath (Fig. 1). The image depth was set between 4 and 5.7 cm, and the muscle preparation was magnified using a built-in zoom function. The MI was varied from 0.4 to 1.8.
In vitro experiments
A custom-designed stage was prepared, and 10 ml of ultrasound gel was placed on one end of the stage. The tip of the transducer was then placed in the gel and positioned so the ultrasound beam could reach the microbubbles under the coverslip on the slide mounted on the stage, which could be observed on microscopy using a magnification of x20.
Radiolabeled microsphere MBF measurement.
Myocardial blood flow was measured using left atrial injections of  2·106 11-µm radiolabeled microspheres (Dupont Medical Products) suspended in 4 ml of 0.9% saline and 0.01% Tween-80 (4). Reference samples were withdrawn from the femoral artery over 130 s with a constant rate withdrawal pump (Model 944, Harvard Apparatus). At the end of the experiment, the short-axis left ventricular (LV) slice corresponding to the ultrasound image was cut into 16 wedge-shaped pieces, and each piece was further divided into epicardial, mid-wall and endocardial thirds. The tissue and reference blood samples were counted in a well counter with a multichannel analyzer (Model 1282, LKB Wallac). Corrections were made for activity spilling from one energy window to another.
Myocardial blood flow to each epicardial, midcardial and endocardial segment was calculated from the equation: Qm = (Cm·Qr)/Cr, where Qm is blood flow to the myocardial segment (ml·min–1); Cm is tissue counts; Qr is the rate of arterial sample withdrawal (ml·min–1); and Cr is arterial reference sample counts (4). Transmural MBF (ml·min–1·g–1) to each of the 16 wedge-shaped pieces was calculated as the quotient of the summed flows to the segments within that piece and their combined weight. Myocardial blood flow to the LAD and LCx beds was calculated by averaging MBF within the segments representing the central 75% of each bed defined on MCE during coronary occlusion.
Intravital microscopy.
IM was performed using a Zeiss customized microscope (ACM, Zeiss Inc.). Either a x20 or x63 water-immersion objective was used. Data were acquired using a video camera (Model CCD-72, Dage-MTI Inc.) mounted on the microscope and were recorded on videotape using a S-VHS recorder (Model AG-1730, Matsushita). An image intensifier (Genllsys, Dage-MTI Inc.) was used to improve the signal-to-noise ratio, and the final output was displayed on a high resolution monitor (Model PVM-137, Sony Corp.).
Protocols.
Canine experiments
Five minutes after coronary occlusion, 10 to 15 ml (0.3 to 0.5 ml·kg–1) of AIP 201 was injected into the left atrium during simultaneous ultrasound imaging (1), followed by injection of radiolabeled microspheres. Coronary occlusion was maintained for 0.5 to 3 h in individual dogs, followed by up to 3 h of reperfusion. Imaging was performed periodically during reperfusion, at which time radiolabeled microspheres were also injected. Just before termination of the experiment, a second dose of AIP 201 was injected during simultaneous ultrasound imaging, followed by the final injection of radiolabeled microspheres. At the end of the experiment, the heart was excised for radiolabeled microsphere MBF analysis.
Rat experiments
To facilitate their identification in the microcirculation, microbubbles were labeled with fluorescein-DTAF (Sigma Chemical) (5). Approximately 2 ml of AIP 201 was withdrawn from a vented vial into a syringe containing 0.5 to 1.0 mg of DTAF, which was then gently rotated to allow mixing of the microbubbles and the DTAF. The solution was then reinjected into the vented vial containing the remainder of the microbubbles, and the vial was placed on a mechanical roller for 10 min to ensure thorough mixing. In preliminary studies, it was determined that a single dose of 3·106 microbubbles (0.2 ml) could be injected into the carotid artery without compromising the microcirculation and also providing reasonable opacification of the muscle on ultrasound examination.
In the first seven rats, microbubble behavior was studied within the microcirculation without exposure to ultrasound. Before their injection, the entire spinotrapezius muscle was previewed. After carotid artery injection of AIP 201, imaging was switched from transillumination to epi-illumination at a peak wavelength of 510 nm, which is the excitation wavelength for DTAF.
To simulate ischemia-reperfusion, the distal portion of the main artery was occluded, followed by microbubble injection and IM. Five to 35 min later, the occlusion was reversed and the muscle was reexamined for up to 90 min. In two rats, adenosine (10–4 molar solution) was superfused on the muscle during reperfusion. To study the effect of muscle contraction on microbubble rheology, a pacemaker (Model 5320, Medtronic) was attached to the muscle in one rat, and the voltage was increased from 0.4 to 20.0 mA and the pacing rate was increased from 50 to 150·min–1.
In the next seven rats, microbubble behavior within the microcirculation was examined before and after the application of ultrasound. The number of microbubbles lodged within each field of view in the entire muscle was counted before and 30 to 80 min after ultrasound exposure using various MIs.
Statistical methods.
Data are expressed as the mean value ± SD. Repeated measures analysis of variance was used to compare VI measurements during coronary occlusion and reperfusion. Comparisons between MCE and radiolabeled microsphere-derived MBF measurements were made using linear regression analysis. Comparisons of paired and unpaired data from IM experiments were performed using nonparametric tests (Wilcox and Mann-Whitney). Statistical significance was defined as p < 0.05.
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Results
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Canine experiments.
Of the eight dogs, one died immediately after reperfusion from ventricular fibrillation and was excluded from analysis. Figures 2 and 3 depict short- and long-axis views from one of the remaining seven dogs that underwent LAD occlusion. During coronary occlusion, a perfusion defect was seen after a single injection of AIP 201 (Fig. 2A and 3A). By the time this image was acquired (<1 min after injection), all the bubbles had cleared from the LV cavity. Despite the rapid clearance of microbubbles from the LV cavity, it took at least 5 min to achieve maximal myocardial opacification in the nonoccluded control bed. No further change in VI was noted in either the occluded or the control bed for up to 3 h of coronary occlusion.
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Figure 2 Short-axis views from a dog undergoing LAD occlusion (A) and reperfusion (B to D). Images in panels A to C are obtained after the first injection of AIP 201, and the image in panel D was obtained after the second injection of AIP 201. The perfusion defect in panel A is indicated by an arrow. Panels B and C were obtained 10 and 120 min after reperfusion and show a progressive increase in VI in the LAD bed. See text for details.
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A spontaneous increase in VI was noted in the LAD bed 10 min after reperfusion, even without a second injection of microbubbles (Figs. 2B and 3B), and no recirculation was noted in the LV cavity. This increase in VI was even greater 120 min later (Fig. 2C and 3C). When a second injection of AIP 201 was performed, as expected, myocardial opacification was greater in the entire myocardium (Fig. 2D and 3D). However, the VI ratios between the reperfused and control bed were not different from those observed just before the second injection (Fig. 2C and 3C).
The temporal changes in MCE data are depicted in Figure 4. As would be expected, the VI in the occluded bed is less than half of that in the nonoccluded bed. After reperfusion it increases progressively over time and normalizes at a mean of 94 min, without a second injection of AIP 201. Video intensity in the control bed remains stable over this same period. When a second injection of AIP 201 is given just before termination of the experiment, VI in both the beds increases, but the ratio between the two beds does not change.
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Figure 4 Changes in mean VI in the occluded and control beds in the seven dogs analyzed. *Significant (p < 0.003) differences in VI.  No differences in VI.
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Figure 5 depicts the relation between radiolabeled microsphere-derived MBF and VI ratios from the hypoperfused and control beds during coronary occlusion and at various intervals after reperfusion after the first injection of AIP 201. Data obtained after the second injection of AIP 201, before termination of the experiment, are also depicted. There is a linear relation indicating that the VI ratios between the two beds reflect the MBF ratios between these beds.
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Figure 5 Relation between MBF ratio (x axis) and VI ratio (y axis) between the hypoperfused and normal beds from all ultrasound and radiolabeled microsphere measurements. Open and closed squares represent data from coronary occlusion and a mean of 36 min after reperfusion obtained after the first injection of AIP 201, whereas open and closed circles represent data at a mean of 94 min and 100 min after reperfusion obtained from the first and second AIP 201 injections, respectively.
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Rat experiments.
In the six rats undergoing ischemia-reperfusion without ultrasound exposure, no microbubbles were seen entering the muscle distal or proximal to the occluder at any time after their injection. After reperfusion, no bubbles were seen to enter the muscle during the 90-min observation period, even after arteriolar vasodilation with adenosine.
In the six rats with data during ultrasound exposure, microbubbles were seen to be entrapped within the microcirculation after their injection through the carotid artery. Most of them were lodged as single bubbles in small arterioles ( 10 µm in diameter). Red blood cell flow and plasma flow were usually absent in these vessels (Fig. 6A). Sometimes trains of 2 to 20 bubbles could be seen within arterioles. Aggregation or clusters of bubbles (4 or 5 to almost 100) could also be found in arterioles ranging in size from 20 to 80 µm in diameter (Fig. 6B). A smaller microbubble was occasionally seen passing through a capillary.
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Figure 6 Entrapment of single microbubbles in small arterioles (A) and microbubble aggregates in larger arterioles (B). See text for details.
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The microbubble aggregates seen within the larger arterioles were associated with different flow patterns. Sometimes no flow was seen. At other times, temporary cessation of flow was seen with periodic resumption of reduced flow around the aggregates. At still other times, after a temporary reduction, normal flow resumed around the aggregates. Each instance of flow reduction was preceded by total or subtotal obstruction of the arterioles. Similarly, flow resumption invariably occurred after dilation of the arteriole at the site of obstruction, and the dilation reversed when the aggregates moved downstream.
The aggregates themselves demonstrated changes in their size and location, which were principally related to changes in flow. When flow improved entire aggregates could become dislodged from larger arterioles and move further downstream to the smaller arterioles. At other times a few microbubbles would separate from the aggregates and move downstream before they became lodged in smaller arterioles (Fig. 7). These aggregates could later become dislodged and reach even smaller arterioles.
Muscle opacification was seen on ultrasound at the time of appearance of the microbubbles within the microcirculation. Figure 8 illustrates ultrasound images of the spinotrapezius muscle from one rat before and after injection of microbubbles. No bubble destruction was seen in vivo, even during insonification at a maximal MI of 1.8. Before ultrasound exposure, 2.2 ± 1.5 bubbles per viewing field were noted and 2.1 ± 1.5 were found after ultrasound exposure (p = NS). The aggregation or disaggregation of microbubbles was also not affected by ultrasound. Pacing induced muscle contraction but did not affect microbubble lodging or behavior.
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Figure 8 Ultrasound images of the spinotrapezius muscle before (A) and after (B) microbubble injection. The central portion of the image represents the muscle. The spokelike structures are sutures used to spread out the muscle.
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In vitro experiments.
When present in low concentrations, microbubbles were slowly pushed away from the focal point of the transducer on exposure to ultrasound. When present in higher concentrations, with smaller distances between them, microbubbles moved toward each other on exposure to ultrasound, and the movement accelerated as they approached one another. This phenomenon led to the formation of aggregates of varying sizes within seconds of ultrasound exposure (Fig. 9A). After ultrasound was discontinued, disintegration of these aggregates was noted with individual microbubbles separating from each other (Fig. 9B). Finally, similar to in vivo observations, no microbubble destruction was noted in vitro, even at the maximal MI of 1.8.
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Figure 9 In vitro aggregation of microbubbles suspended in saline after ultrasound exposure (A) and disaggregation after discontinuation of ultrasound (B).
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Discussion
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The main findings of this study are that 1) after a single injection during coronary occlusion, AIP 201 microbubbles spontaneously redistribute to the myocardium after reperfusion; 2) the magnitude of redistribution is related to changes in MBF to the reperfused myocardium; 3) the mechanism of redistribution is probably related to dislodgement after reflow of microbubbles from aggregates lodged in collateral arterioles 10 to 80 µm in size; and 4) the in vitro behavior of these microbubbles is not replicated in the microcirculation. These findings may have important implications for the assessment of myocardial perfusion, particularly after interventions for acute ischemic syndromes in the cardiac catheterization laboratory, after revascularization following coronary artery bypass graft surgery, and for the assessment of collateral vessel function.
Unique characteristics of AIP 201.
Most preformed microbubbles in commercially produced ultrasound contrast agents are small enough to pass through the pulmonary microcirculation and result in LV and myocardial opacification (6). A small fraction that are larger than the pulmonary capillary diameter are filtered by the lungs. Because they have already passed through the lungs, the microbubbles reaching the left heart can easily pass through the coronary circulation in a manner similar to red blood cells (3). Assessing an intervention aimed at altering MBF, however, requires repeated administration of such agents.
AIP 201 is currently the only ultrasound contrast agent containing preformed microbubbles that are similar to microspheres, in that they become lodged in the myocardial microcirculation after a left heart injection. This finding was confirmed in this study in the experiments using IM. The mean diameter of the bubbles is 10 ± 0.4 µm, with 70% being >7 µm and 0.5% being  20 µm. These bubbles contain room air and are encapsulated in a 1-µm shell composed of denatured albumin. Unlike other air-filled albumin microbubbles that become smaller in size after contact with blood because of outward diffusion of air (7), the thick shell of AIP 201 microbubbles prevents diffusion of gases and maintains their size in vivo. These properties result in long-lasting myocardial opacification on ultrasound examination after a left heart injection. The large microbubble size, however, precludes the use of this agent for venous administration.
Mechanism of redistribution.
Redistribution is a phenomenon described with thallium-201 (8). It occurs from uptake of the tracer from blood during repeated recirculation through the coronary system. The redistribution seen with AIP 201 cannot be explained by this phenomenon because recirculation is absent. Most microbubbles are entrapped in the coronary circulation during their first pass, and the ones that do not get entrapped are cleared from blood probably by entrapment in other organs.
There are other possible mechanisms to explain this phenomenon of redistribution. Because we noted this effect in a beating canine heart, we wanted to exclude muscle contraction as the cause of this phenomenon. Pacing of the rat spinotrapezius muscle did not result in any movement of the entrapped microbubbles. Because redistribution in the canine experiments could only be seen by performing an ultrasound examination, we also wanted to exclude the possible effect of ultrasound on microbubble behavior. Microbubble behavior was influenced by ultrasound in the in vitro experiments as a result of radiation forces (9,10). These forces result from positive (primary radiation effect) and negative (secondary radiation effect) pressures around the microbubbles, which cause them to move either outward or inward, respectively. Microbubbles have to be physically unhindered for this phenomenon to occur. It is probably for this reason that a similar behavior was not observed in the microcirculation on exposure to ultrasound. Entrapment in smaller arterioles and limited mobility in larger arterioles prevent ultrasound-induced motion.
The only other putative mechanism for redistribution involves lodging of microbubbles in larger arterioles that are not present within the myocardium but are present on its epicardial surface. After reperfusion, these microbubbles would then migrate to the formerly occluded bed. Our results support this mechanism. First we noted that opacification in the normal myocardium increased gradually over at least 5 min after left atrial injection, which was several minutes after the bubbles had cleared from the LV cavity. These results imply that all bubbles are not immediately deposited to even normally perfused myocardium, but are gradullary transported there from the coronary circulation proximal to the myocardium. This notion was further supported by IM, where microbubbles were seen as aggregates in larger arterioles from where they periodically dislodged and moved more distally to smaller arterioles.
Because the coronary artery was occluded, the only way in which microbubbles could have reached arterioles distal to it is through collateral vessels. The pressure difference between the occluded and nonoccluded beds is maximal during coronary occlusion, which would facilitate this phenomenon (11,12). It is probable that the microbubbles were entrapped in the larger epicardial collateral arterioles supplying the occluded bed and became dislodged after resumption of flow to that bed. This disaggregation of microbubbles takes several minutes to hours depending on MBF to the heart muscle. Although the rat spinotrapezius muscle is supplied by collateral vessels, any such vessels are separated from the muscle during its isolation. The absence of collateral vessels can explain the lack of redistribution to the spinotrapezius muscle after reflow, even during adenosine exposure.
The mechanism of microbubble aggregation in vivo is not clear. This phenomenon has also been described with microspheres (4). Suspension in a Tween solution (polyoxyethylene sorbitan mono-oleate), which acts as a surfactant, reduces the aggregation of microspheres (4). The mechanism of microbubble aggregation could be similar to that of microspheres. Low doses of Tween are used while preparing these microbubbles to reduce aggregation. Although higher doses may be required to completely inhibit aggregation, they may result in hemodynamic disturbances (13).
Clinical implications.
Microbubbles are most likely to be used in the echocardiography laboratory and the emergency room for the detection of chronic coronary artery disease and acute ischemic syndromes (7). In these situations, it is necessary to use microbubbles that are capable of pulmonary transit rather than those requiring a direct left heart injection. There are situations, however, where access to the left heart has already been obtained for other reasons, such as during cardiac catheterization and open heart surgery. In these settings, a direct injection of microbubbles into the left heart, aorta or coronary arteries has previously provided useful information (14–18). The advantage of AIP 201 over other contrast agents is that it requires only a single injection before an intervention such as primary angioplasty for acute myocardial infarction or placement of coronary artery grafts. The microbubbles are likely to become lodged in extramyocardial arterioles supplying the occluded or poorly perfused bed, and then they embolize to smaller myocardial arterioles when flow is restored. Thus, the success of a revascularization procedure can be judged without repeated injections of microbubbles. In addition, because of the long-lasting myocardial opacification, the effect of the intervention can also be monitored after the procedure—for example, in the recovery room.
Because redistribution of these bubbles is not possible in the absence of collateral vessels, myocardial opacification after restoration of adequate flow also indicates collateral vessel function (11,12). The effects of various pharmacologic agents on collateral vessel function could be studied in humans, as could the effect of growth factors that promote angiogenesis (19,20). By acting as deposit tracers, these microbubbles also reflect relative MBF and could be used to assess serial changes in this measurement. These applications, however, need to be verified in clinical studies.
Obstruction and plugging of microvessels have disadvantages in that they can cause reductions in MBF. It has been demonstrated that up to 22 million 7- to 10-µm sized radiolabeled microspheres can be safely administered through the left atrium at rest without any adverse hemodynamic effects (21). Because only 5% of total cardiac output is distributed to the coronary circulation during rest, these results imply that 1 million microbubbles can become lodged in the myocardium without significant hemodynamic effects. These experiments were performed in normal hearts (21). The number of microbubbles that would be tolerated in situations where MBF is compromised will be less depending on the microvascular state of the myocardium. In a canine model of ischemia-reperfusion, we have previously shown that at similar doses to those used in this study, AIP 201 does not cause any hemodynamic changes, despite the instability of the preparation (1). Similar results need to be demonstrated in humans before AIP 201 can be recommended for clinical use.
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Acknowledgments
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We thank N. Craig Goodman, BS, Soroosh Firoozan, MD, and Gursel Ates, MD, for assisting in the canine experiments.
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Footnotes
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This study was supported in part by Grants R01-HL48890, R01-HL52309 and R01-HL49146 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, a grant from Andaris Ltd., Nottingham, England and an equipment grant from Advanced Technology Laboratories, Bothell, Washington. The radiolabeled microspheres were provided by Dupont-Merck, North Billerica, Massachusetts. Dr. Linka was supported by Ciba-Geigy Jubiläums-Stiftung and Theodor und Ida Herzog-Egli Stiftung, Switzerland. Dr. Skyba was supported by Postdoctoral Fellowship Grant F32-HL095410 from the National Heart, Lung, and Blood Institute, National Institutes of Health. Dr. Price was the recipient of a Postdoctoral Fellowship Training Grant from the Virginia Affiliate of the American Heart Association, Glen Allen, Virginia. Dr. Wei was the recipient of a Junior Personnel Research Fellowship from the Heart and Stroke Foundation of Canada, Ottawa, Canada.
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