Author + information
- Received October 7, 2002
- Revision received April 7, 2003
- Accepted April 24, 2003
- Published online August 6, 2003.
- Hideki Kunichika, MD, PhD*,
- Barry Peters, MD*,
- Bruno Cotter, MD*,
- Hisashi Masugata, MD*,
- Naomi Kunichika, MD, PhD*,
- Paul L Wolf, MD* and
- Anthony N DeMaria, MD, MACC*,* ()
- ↵*Reprint requests and correspondence:
Dr. Anthony N. DeMaria, Division of Cardiology, UCSD Medical Center, 200 West Arbor Drive, San Diego, California 92103-8411, USA.
Objectives We examined whether delayed post-injection imaging of a new ultrasound contrast agent (BR-14) could produce prolonged opacification and hyperenhancement of myocardium subjected to coronary occlusion/reperfusion.
Background We hypothesized that ultrasound exposure destroyed BR-14 and eliminated visualization of sustained myocardial opacification from retained microbubbles.
Methods We studied eight open-chest dogs with 3 h of left anterior descending coronary artery (LAD) occlusion followed by 3 h of reperfusion. Myocardial contrast echocardiography (MCE) was performed before occlusion and 120 min after the onset of both occlusion and reperfusion. Ultrasound imaging was initiated 15 min after injection. Myocardial blood flow (MBF) was assessed by microspheres.
Results Pre-occlusion images revealed uniform opacification of left ventricular myocardium greater than that of the cavity, with a mean intensity of the LAD bed of 8.66 ± 1.38 dB. During occlusion, MCE resulted in the appearance of a perfusion defect in the LAD risk area (intensity 2.08 ± 1.10 dB). After 120 min of reperfusion, the LAD risk-area myocardium manifested dense opacification of a higher intensity (“hot spot”) than baseline (13.7 vs. 8.7 dB), but with reduced MBF consistent with accumulation of a high concentration of microbubbles. Increased MCE intensity was associated with a greater myeloperoxidase score.
Conclusions These data establish that contrast opacification by BR-14 may be selectively retained within the perfusion bed of a coronary artery subjected to occlusion/reperfusion. Such opacification exhibits defects with occlusion, manifests hyperenhanced intensity (hot spot) with reperfusion, is associated with the level of myeloperoxidase activity, and conforms to the area of myocardium subjected to altered flow.
Myocardium subjected to coronary occlusion/reperfusion has characteristically appeared as an opacification defect by myocardial contrast echocardiography (MCE) (1,2). BR-14 is a recently developed contrast agent that is capable of producing sustained myocardial opacification after injection (3). Intravital microscopy has demonstrated that the prolonged persistence of BR-14 is due to retention of microbubbles within the capillary bed. However, previous studies in our laboratory, using a constant infusion of BR-14 with 15 destroy-refill sequences of the cardiac cycle, yielded a time–intensity curve similar to those curves observed with conventional, free-flowing microbubble agents (4). We therefore hypothesized that exposure to ultrasound destroyed BR-14 microbubbles and diminished the ability to visualize sustained myocardial opacification. We further speculated that delayed imaging of BR-14 would yield typical perfusion defects during coronary occlusion, but that delayed imaging during reperfusion might produce a “hot spot” of high-intensity opacification in the risk area due to increased microbubble concentration. In this study, we quantified the intensity of myocardial opacification produced by delaying ultrasound imaging for a 15-min interval after BR-14 injection. We further examined whether such images would be of value in assessing the risk area of myocardium exposed to injury in the setting of coronary occlusion/reperfusion.
The present study was approved by the Animal Research Committee of the University of California at San Diego and conformed to the “Position of the American Heart Association on Research Animal Use.” Eight mongrel dogs (26.5 ± 1.8 kg) were anesthetized and ventilated to keep arterial blood gases and pH within normal limits. The right femoral artery and vein were cannulated for arterial pressure monitoring and contrast agent injection, respectively. The heart was exposed through a left lateral thoracotomy and suspended in a pericardial cradle. The proximal portion of the left anterior descending coronary artery (LAD) was dissected free from the surrounding tissue, and a transit-time flow probe (series 2RB, Transonics System, Ithaca, New York) connected to a digital flow meter (model T201, Transonics System) was placed snugly around the vessel. An atraumatic vascular clamp produced coronary occlusion of the proximal LAD.
Real-time MCE imaging
Echocardiography was performed with a commercial instrument (HDI 5000, Philips Ultrasound, Andover, Massachusetts) using a broadband 4-2 MHz transducer. Color-coded harmonic power pulse inversion images were obtained with ultrasound transmitted at 2 MHz and received at 4 MHz in the short-axis view at the papillary muscle level using low-energy (mechanical index 0.1) real-time imaging at 15 frames/s (5,6). Pulse repetition frequency was fixed at 2,500 Hz. Instrument settings were held constant for each experiment. A latex bag filled with degassed saline functioned as an acoustic interface between the heart and transducer, which was positioned to image the LAD perfusion territory.
Delayed MCE with BR-14
Using a bolus intravenous injection of 0.2 ml BR-14 (Bracco Research S.A., Basel, Switzerland), MCE was performed in eight dogs. BR-14 is a third-generation ultrasound contrast agent consisting of perfluorobutane-containing microbubbles stabilized by a phospholipid monolayer. In two dogs, continuous imaging was performed after a 15-min delay immediately following BR-14 injection both before coronary occlusion and after 2 h of reperfusion to determine the duration of opacification with constant exposure to ultrasound. Additionally, all eight dogs underwent a protocol in which ultrasound imaging was terminated at the time of BR-14 injection for a period of 15 min, whereupon 5 to 10 cardiac-cycle digital cine loops were obtained. To measure real-time MCE signal intensity, the images were digitally captured and analyzed off-line using HDI laboratory software. Myocardial signal intensity (dB) for the first three cardiac cycles was measured from a 25-mm2region of interest placed in the center of the risk area, at both lateral margins of the risk area, in an adjacent normal perfusion area, and in the center of the left ventricular (LV) cavity. The area of abnormal opacification during occlusion and reperfusion was manually traced for three end-diastolic images.
Assessment of risk area
The location and size of the risk-area myocardium was identified during coronary occlusion, both as the unstained region after blue dye injection and as the region of unopacified myocardium by MCE during continuous infusion of 0.5 ml/min BR-1 (SonoVue, Bracco Research S.A.). BR-1 is based on stabilized sulfur hexafluoride microbubbles, which behave identically to red blood cells, with a rapid, uninterrupted transit through the microcirculation. Recordings were obtained 2 min after initiating infusion to ensure that the plateau intensity had been reached. To measure the risk-area myocardium from MCE, frames preceding and following high-energy (mechanical index 0.8) “FLASH” frames were digitally captured. The area of the largest clearly demarcated contrast defect was manually traced for the final five end-diastolic images of the 15-cycle FLASH refilling sequence and was expressed as the mean value.
Myocardial blood flow (MBF) measurements
Myocardial blood flow was measured by standard techniques employing left atrial injection of fluorescent microspheres (Molecular Probes, Eugene, Oregon) while reference femoral artery samples were withdrawn. After the animal was euthanized, the heart was sliced, and the cross-sectional segment corresponding to the short-axis image was cut into 12 wedge-shaped transmural tissue pieces, each of which underwent microsphere counting.
With the guidance of long needles placed as markers, a LV short-axis slice 6 to 8 mm thick was cut out at the same level at which the echocardiograms were recorded in three dogs. To determine the infarct area, each slice was incubated in 1.5% triphenyltetrazolium chloride (TTC) at 37°C for 15 min, and the specimen was then fixed in 10% formalin for 48 h for histologic analysis. In two dogs, blinded quantitative histologic analysis of each segment was also performed by a light microscopic method with hematoxylin and eosin and myeloperoxidase stains. Scores of none (0) to severe (+3) for contraction-band necrosis, interstitial edema, intramyocardial hemorrhage, neutrophil infiltration, coagulation necrosis, and myeloperoxidase activity were obtained individually for each segment. Intracapillary erythrocyte stasis, defined as the occurrence of capillaries packed with erythrocytes, was also scored.
After a period of stabilization following instrumentation, baseline hemodynamic and MCE recordings were performed (Fig. 1). Thirty minutes after baseline recordings, the proximal LAD was occluded. Occlusion was maintained for 3 h to produce myocardial injury, after which time, the clamp was removed and 3 h of reperfusion was implemented. At 120 min of both occlusion and reperfusion, MCE was performed. Delayed imaging was additionally performed in three dogs after 30, 60, 90, 120, and 180 min of reperfusion.
Data are expressed as the mean value ± SD. Analyses of the data were performed in a blinded fashion, without knowledge of any confirmatory data. Comparisons of MCE data among all stages were performed using repeated-measures analysis of variance. Differences were considered significant at p < 0.05.
All experiments were completed successfully without hemodynamic changes or adverse events. Each experiment yielded technically adequate data for analysis.
Signal intensity from MCE
The MCE data at each stage are summarized in Table 1. Similar to our previous report, during continuous imaging, dense opacification filled the LV cavity immediately after BR-14 injection and was followed by the subsequent appearance of less intense signals of relatively uniform distribution within the myocardium (4). For the delayed imaging, LV cavity opacification was markedly decreased, whereas that of the myocardium persisted. Therefore, myocardial intensity exceeded that of the cavity at 15 min (7.8 ± 1.2 vs. 3.0 ± 1.4 dB, p < 0.05). During LAD occlusion, the risk area manifested the anticipated perfusion defect in delayed images, with a marked reduction in risk area versus normal intensity (2.08 ± 1.10 vs. 7.18 ± 1.54 dB, p < 0.05). During reperfusion, however, the risk-area myocardium manifested a marked increase in signal intensity relative to the normal region (13.7 ± 1.2 vs. 8.0 ± 0.6 dB, p < 0.05) (Fig. 2). Also, the signal intensity of the center of the infarction area was significantly higher than that of noninfarcted border myocardium within the risk area (13.7 ± 1.2 vs. 10.6 ± 1.2 dB, p < 0.05). Following reperfusion, accumulation of microbubbles developed gradually in the reperfused myocardium. The images after 120 and 180 min of reperfusion revealed that the LAD risk-area myocardium manifested dense opacification of a higher intensity than that at baseline (Table 2). Thus, during reperfusion, the risk-area myocardium was identified as a localized region of dense, bright opacification, or “hot spot.” Continuous exposure of BR-14 to ultrasound following the delayed imaging protocol resulted in elimination of myocardial opacification after 1 to 2 min of exposure in the both normal and reperfused myocardium.
Assessment of size of region at-risk area
The location and size of myocardial segments that manifested decreased opacification during occlusion and increased intensity during reperfusion, as visualized by MCE with BR-14, were compared with the risk area identified by BR-1 (Table 1). Both the defect and enhanced segments occupied the same location as the risk area. During coronary occlusion, the defect size observed with BR-14 was smaller than the risk area, but no significant difference in size was observed between the risk area and region with high-intensity signals during reperfusion (490.6 ± 49.3 vs. 490.4 ± 72.6 mm2). A comparison of the size of the myocardial region showing increased intensity with BR-14 during reperfusion and the risk-area myocardium identified by blue dye injection were closely correlated (y = 0.91x + 9.91, r = 0.90) (Fig. 3).
Coronary flow and MBF
Data on MBF by microspheres and LAD flow by Doppler are summarized in Table 3for each stage. During LAD occlusion, MBF was severely reduced within the risk area, but some blood flow from collateral channels was detectable. During reperfusion, epicardial LAD flow returned to baseline, whereas MBF within the risk area was reduced from baseline.
The percentage of infarcted myocardium measured by TTC of the total risk area, identified by blue dye, was 43 ± 3%. By light microscopy, there were no significant differences in contraction-band necrosis, interstitial edema, or coagulation necrosis between the center and margins of the infarct area. However, the degree of intramyocardial hemorrhage, neutrophil infiltration, myeloperoxidase score, and intracapillary erythrocyte stasis were higher in the center than in the margin of the infarct area. A comparison of the myeloperoxidase stain score and the signal intensity of the hot spot exhibited a good correlation (Fig. 4).
Until now, all microbubble contrast agents have behaved as free-flowing red blood cells and depicted flow impairment as perfusion defects. In the present study, we evaluated the potential of BR-14 to opacify the myocardium after a 15-min ultrasound-free interval following contrast injection. We observed that the signal intensity ratio of the normal myocardium to LV cavity exceeded 2.1 at 15 min after contrast injection, indicating that BR-14 has the characteristics of prolonged myocardial residence. Moreover, during coronary reperfusion, we observed that delaying exposure of BR-14 to ultrasound energy produced images containing a high accumulation of signal intensity in the risk-area myocardium. Thus, although previous MCE depicted occluded/reperfused myocardium as an opacification defect, BR-14 visualized such myocardium as a hot-spot region of hyperenhanced, high-intensity signals in the delayed imaging protocol.
The ability to visualize prolonged myocardial opacification by BR-14 is dependent on withholding ultrasound exposure. Continuous ultrasound imaging with BR-14 resulted in a prompt decrease in myocardial intensity, likely due to cumulative destruction of circulating microbubbles, disruption of microbubbles residing within the myocardium, or a combination of effects. The strategy of deferring exposure to ultrasound for 15 min and then acquiring images from the initial cardiac cycles enabled us to amplify the deposit properties of BR-14, particularly with regard to reperfused risk-area myocardium. The results obtained by other ultrasound contrast agents subjected to a similar imaging protocol are an important area for future research.
The precise mechanism of prolonged myocardial opacification and post-occlusion/reperfusion hyperenhancement are not known with certainty. During reperfusion, the risk-area myocardium manifested dense, high-intensity opacification consistent with accumulation of a high concentration of microbubbles. Because epicardial LAD flow returned to baseline after 120 min of reperfusion, while MBF within the risk area was reduced from baseline at that time, persistent hyperemia did not exist within the risk-area myocardium to deliver more bubbles to the occlusion bed. Therefore, the increased opacification with BR-14 after occlusion/reperfusion is most likely primarily due to its character as a deposit agent rather than to post-occlusion hyperemia. During nondestructive continuous imaging, BR-14 can act as a normal, free intravascular tracer, as reported in our earlier study (4), because microbubbles are being continuously destroyed. However, in the delayed MCE imaging approach, BR-14 was observed to produce prolonged myocardial opacification in the absence of inflammation, despite a small size of the microbubbles. In the setting of normal flow, this deposit property may be related to retention of bubbles within capillaries due to a net negative charge and/or complement-mediated attachment to the endothelium (7). In regions of tissue inflammation produced by ischemia/reperfusion, lipid layer microbubbles can adhere to activated leukocytes in post-capillary venules via cell-surface integrins or opsonization with complement, resulting in prolonged myocardial opacification throughout the risk area (8–12). By avoiding bubble destruction, the delayed MCE imaging protocol may be able to expose the deposit characteristics of this type of microbubble. In our study, both the degree of myeloperoxidase stain score and the signal intensity were higher in the center of the infarct area than in the margins. These results are consistent with the concept that increased post-occlusion/reperfusion opacification is related to the degree of regional myocardial inflammation (Fig. 4). Thus, high-signal intensity may indicate inflammation, a condition associated with reperfusion injury and the “no-reflow” phenomenon. It should be noted that we assessed MCE using a model of 3-h occlusion and 3-h reperfusion. The ultimate hot-spot signal intensity may differ with variation of the duration of ischemia.
The perfusion defect area visualized with BR-14 during coronary occlusion was smaller than the risk area defined by BR-1. However, the hot-spot size with BR-14 was equal to the risk area. The risk area includes both infarcted (irreversible, presumably nonperfused) and ischemic (reversible, presumably poorly perfused by collateral vessels) tissue. During occlusion, it is likely that slow, low-volume collateral flow was sufficient to be visualized in the border zone, thereby yielding a smaller perfusion defect. The spatial extent of myocardial enhancement during reperfusion with BR-14 was similar to the risk area identified by blue dye injection. The acoustic hyperenhancement area with BR-14 during reperfusion was a marker of all abnormal myocardium, both ischemic and infarcted. These data suggest that inflammation is prevalent in the border zone of the at-risk myocardium.
Although we cannot be certain, we believe that our data may have several possible clinical implications. If this experimental work can be reproduced in patients, the ability to produce sustained myocardial opacification may be of value in imaging and quantifying perfusion defects, while simultaneously avoiding problems associated with signal attenuation. Enhanced signal accumulation during reperfusion might diminish the necessity to achieve dense opacification in adjacent normal myocardium in order to detect reduced perfusion or perfusion defects. We speculate that hot-spot imaging may be an important area for the research of the no-reflow phenomenon, as well as inflammation. Finally, the imaging protocol employed in this study may enable MCE imaging to be performed at substantial intervals following the injection of ultrasonic contrast.
- left anterior descending coronary artery
- left ventricular
- myocardial blood flow
- myocardial contrast echocardiography
- triphenyltetrazolium chloride
- Received October 7, 2002.
- Revision received April 7, 2003.
- Accepted April 24, 2003.
- American College of Cardiology Foundation
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