Animal Preparation and Protocol

This study was compliant with the guidelines of the Institutional Animal Care and Use Committee and the standards in the National Research Council’s Guide for the Care and Use of Laboratory Animals . Animals were preanesthetized with an intramuscular mixture of Telazol (4.4 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA), ketamine (2.2 mg/kg), and xylazine (2.2 mg/kg) and then intubated. Isoflurane inhaled anesthesia (induction at 4%, maintained at 1.0% to 1.8%) was then administered. The percentage oxygen mixture was kept at 24% during treatment periods in all pigs. Two femoral artery and venous catheters were placed for hemodynamic monitoring and infusions of microbubbles. An 8-Fr guide catheter was introduced into the left main artery for digital angiography and for balloon catheter insertion. Heart rate and oxygen saturation were also monitored throughout the entire experiment. Low-dose intravenous dobutamine (1–3 μg/kg/min) was used to maintain systolic arterial pressure > 80 mm Hg during the study protocol. Intravenous lidocaine boluses (40 mg followed by 20 mg × 3) and a continuous intravenous lidocaine infusion (2–4 mg/min) were used in all animals to control arrhythmias.

Acute LAD thrombotic occlusions were created in 28 pigs. Two of these pigs died of refractory ventricular fibrillation before treatment could be initiated, leaving a total sample size of 26 pigs. In 12 of the pigs, preexisting atherosclerotic lesions were created by a balloon injury in the LAD on day 1, followed by a high-fat diet (15% lard, 2% cholesterol; Harlan Laboratories, Madison, WI) lasting 52 ± 21 days.

Coronary artery thrombotic occlusions were created in the 14 normal pigs by simulating the triad of Virchow. This consists of creating endothelial injury, stasis, and a hypercoagulable state. Endothelial injury was created by advancing a balloon catheter into the LAD, after the second diagonal branch, and inflating the balloon for 30 sec three times at maximum diameters that were 110% of the measured coronary artery diameter. This balloon catheter was then withdrawn proximal to the injury site and partially inflated to reduce flow at the injury site. Then, 0.1 to 0.2 mL of clotting venous blood from the pig was injected through the balloon catheter into the site of injury to create a hypercoagulable state.

Once angiographic LAD occlusion was documented, it had to persist for ≥20 min. If spontaneous recanalization occurred, small thrombus injections were again administered through the balloon catheter until persistent occlusion was observed. Pigs then underwent baseline measurements of heart rate, oxygen saturation, arterial blood pressure, wall thickening, MBF with radiolabeled neutron-activated microspheres, perfusion defect size at plateau intensity (ultimate infarct size) with contrast low-MI imaging, and ST-segment elevation on 12-lead electrocardiography. Subsequently, 650 mg of aspirin was administered per nasogastric tube, followed by an intravenous heparin bolus (80 mg/kg) and a bolus injection of half-dose fibrinolytic agent (0.25 mg/kg tenecteplase or 1 mg/kg tissue plasminogen activator; Genentech, South San Francisco, CA). The normal pigs were then randomized to receive either no additional treatment (the control group, subsequently referred to as group I; n = 7) or a continuous intravenous infusion of MRX-801 (NuvOx Pharma Inc., Tucson, AZ) with intermittent high-MI impulses applied whenever microbubbles were visualized within the risk area (group II).

In the 12 atherosclerotic pigs, coronary artery thrombi were created at 52 ± 21 days after the day 1 balloon injury using the same protocol described for groups I and II. The second balloon injury occurred at the same location as the day 0 balloon injury. Specific angiographic markers were used (e.g., location in distance from a diagonal branch) to ensure that the thrombus initiation site on day 50 was at the site of balloon injury at day 0. After a 20-min documented angiographic occlusion, pigs received the same regimen of aspirin, heparin, and half-dose lytic agent (tenecteplase or tissue plasminogen activator). Subsequently, the pigs received either no additional treatment (group III) or the same MRX-801 infusion with intermittent high-MI impulses applied to the risk area (group IV).

Ultrasound and Microbubble Protocol

The microbubbles used for all studies were a lipid-encapsulated formulation (MRX-801). These microbubbles have a diameter of 1.0 ± 0.1 μm, with concentration of 1.5 × 10 ¹⁰ to 3.0 × 10 ¹⁰ /mL. The microbubble infusions for both therapeutic and diagnostic studies were prepared by diluting 2 mL of MRX-801 in 100 mL of 0.9% saline and infusing at a rate of 2.5 to 3.0 mL/min.

In pigs randomized to receive ultrasound, real-time low-MI biplane images were obtained using a matrix-array 1.6-MHz 3D transducer (Power Modulation at an MI of 0.2; Philips Medical Systems, Andover, MA), which permitted visualization of the perfusion defect size ( Figure 1 ). A 3D array of high-MI impulses (MI, 1.2) was delivered from the same transducer at a frame rate of 5 Hz for 5 sec (a total of 25 frames) after the visualization of microbubbles within any portion of the peripheral or central portions of the risk area (defined by the extent of the wall thickening abnormality). The guided high-MI impulses were applied for a time period that was considered sufficient to clear the myocardium (both normal and abnormal) of microbubbles, permitting the visual analysis of replenishment within the risk area and normal myocardial segments. Although the 25 frames of high-MI impulses did occasionally result in some evidence of microbubble destruction within the left ventricular cavity, there was rapid reappearance of contrast within the cavity immediately after the return to low-MI imaging. Risk area was divided into central and peripheral portions by arbitrarily dividing it into thirds, with the outer thirds being described as the peripheral portions. There were no instances in which, within 15 sec, we did not observe at least some replenishment within at least the peripheral portion of the risk area. Treatments continued for 30 min. Venous samples for activated clotting times were obtained before the initiation of treatment and at approximately 30 min into randomized treatments.

Depiction of the transthoracic coverage of the 3D transducer used for the application of guided high-MI impulses after acute LAD thrombotic occlusion. Blue arrows depict the hypoperfused zone identified with biplane low-MI imaging after LAD thrombotic occlusion.

MBF Measurements

To determine how guided high-MI impulses and microbubbles affect peri-infarct MBF even in the absence of epicardial recanalization, MBF measurements were measured in groups I and II using left atrial injections of neutron-activated microspheres (diameter, 15 μm; BioPAL, Inc., Worcester, MA) just before the initiation of treatment, at 25 min into treatment, and at 60 min after the completion of treatment. After sacrifice, the excised heart was divided into eight subendocardial and subepicardial segments ( Figure 2 ). MBF analysis from these eight segments demonstrated that there was a consistent segment with very low flow (corresponding to the central portion of the risk area) and two segments bordering on this central segment that had reduced flow, and hence we defined these as the peri-infarct regions. Peri-infarct MBF was defined as the ratio of averaged MBF within the two bordering segments surrounding the segment with lowest blood flow divided by reference MBF within two normal segments in the lateral and inferolateral walls.

Segmentation of the short axis of the left ventricle for MBF measurements with radiolabeled microspheres. Peri-infarct flow was determined by the ratio of flow in the two peri-infarct zones ( open arrows ) divided by the normal zones ( closed arrows ).

Postmortem Measurements

After the 90-min angiographic studies and final MBF measurements, the pigs were sacrificed. In those pigs treated with high-fat diets, the coronary arteries were dissected and analyzed with hematoxylin and eosin staining for the presence of intracoronary thrombus, dissection, and percentage intimal hyperplasia.

Outcome Measurements

In all pigs, epicardial recanalization was assessed by angiography using left main coronary artery injections of 5 mL of iodinated contrast at 30, 60, and 90 min after the initiation of treatment protocols. Assessments of flow in the LAD were visually estimated by a blinded experienced reviewer using the Thrombolysis In Myocardial Infarction (TIMI) criteria. Twelve-lead electrocardiography was performed at baseline before treatment and at 30, 60, and 90 min. Maximal ST-segment elevation was compared at each time point. One pig in group II was excluded from the ST-segment recovery analysis because it did not exhibit ST deviation in any lead after angiographic occlusion of the LAD. Wall thickening within the central portion of the risk area on a two-dimensional short-axis view (midpapillary muscle level) was computed offline by a blinded reviewer, who measured end-diastolic and end-systolic wall thickness before treatment and at 60 min into treatment. Percentage wall thickening was defined as the difference in thickness measurements divided by end-diastolic wall thickness times 100%. All measurements were made by an independent reviewer blinded to treatment protocol.

The risk area was approximated by defining the circumferential extent of the wall thickening abnormality. The effects of the guided high-MI impulses on ultimate infarct size were determined during continuous low-MI imaging and a continuous infusion of MRX-801 before the initiation of treatment and at 90 min after treatment. To determine ultimate infarct size, planimetered measurements of end-systolic defect size (hypoperfused area) were determined at plateau intensity during the continuous infusion of microbubbles. This has been correlated with ultimate infarct size. All measurements were obtained by an experienced reviewer (S.G.) blinded to treatment assignment. MBF ratios in the peri-infarct zones (normalized to unaffected myocardium) were compared with baseline values at the end of the randomized treatment (30 min) and sixty min after the completion of treatment.

Statistical Comparisons

Results for groups I and II were compared with each other, as were results for groups III and IV. Comparisons of angiographic patency rates between the two treatment groups were made using χ ² tests. Changes in percentage wall thickening and ST-segment resolution within groups were determined using paired t tests. Changes in peri-infarct radiolabeled MBF ratios and contrast echocardiography–derived defect size at plateau intensity (ultimate infarct size) were also compared within groups using paired t tests. Interobserver and intraobserver variability in perfusion defect size at plateau intensity was computed using Bland-Altman tests.

Hemodynamic and Histologic Findings

Table 1 demonstrates arterial pressure, oxygen saturation, heart rate, left atrial pressures, and activated clotting time measurements at baseline and 90 min into treatment. No differences were observed in any of these parameters between groups I and I or between groups III and IV. The median duration of thrombotic occlusion before treatment was 24 min (mean, 26 ± 7 min) and was not different among the four groups ( Table 1 ). In the atherosclerotic pigs, mean fasting cholesterol levels increased from 62 ± 28 to 487 ± 216 mg/dL at 52 ± 21 days after initiation of the high-cholesterol diet.

Four of the pigs in the study required 360 J direct-current cardioversion to convert ventricular fibrillation to sinus rhythm. Three pigs developed ventricular tachycardia, which terminated spontaneously. The 40-mg intravenous bolus was administered to all pigs immediately after the onset of isolated ventricular depolarizations. Twenty-milligram boluses (up to five) were given subsequent to this at 10-min to 15-min intervals, along with a lidocaine drip at 3 to 4 mg/min.

Postmortem histologic examinations of the coronary arteries in group IV pigs revealed eccentric plaques at the LAD balloon injury site in five of the six pigs placed on a high-fat diet, with stenosis severity ranging from 0% to 25% in two pigs, 26% to 50% in two pigs, and 50% to 75% in one pig. Focal areas of healed dissection in the LAD (from the day 1 balloon injury) were seen in three group III pigs. Coronary artery histology was available in five of the six control group III pigs. Two of the five exhibited either focal eccentric atherosclerotic lesions or intimal polymorphic leukocytes, and all five had thrombi at the site of the lesions. No group III pigs had evidence of healed dissection.

Angiographic, Electrocardiographic, and Wall Thickening Measurements

Table 2 displays the angiographic and electrocardiographic measurements and wall thickening recovery within the risk area after treatment in groups I and II. Baseline TIMI scores were 0 in all pigs before treatment. Angiographic recanalization rates and TIMI grade 3 flow were the same in group I and group II pigs (two of seven [29%] in each group) at 60 and 90 min into treatment. There were no differences in measured ST-segment elevation or wall thickening within the risk area before the initiation of treatment in groups I and II ( Table 2 ). However, group II pigs had significant reductions in ST-segment elevation already at 30 min after the initiation of treatment. Wall thickening within the risk area improved only in group II.

Table 2

WT, ST-segment changes, and angiographic recanalization rates before and after treatment

Variable	Group I	Group II
WT (%)
Baseline	3 ± 6	6 ± 5
60 min	5 ± 4	12 ± 6 ∗
ST-segment elevation (mm)
Baseline	5.8 ± 2.0	7.4 ± 2.3
30 min	5.1 ± 3.1	4.4 ± 2.5 ∗
60 min	4.2 ± 2.1	3.9 ± 2.5 ∗
90 min	3.7 ± 2.1 ∗	3.5 ± 2.0 ∗
Angiographic recanalization
30 min	2 (29%)	4 (57%)
60 min	2 (29%)	2 (29%)
90 min	2 (29%)	2 (29%)

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