Therapeutic Ultrasound Improves Myocardial Blood Flow and Reduces Infarct Size in a Canine Model of Coronary Microthromboembolism

Therapeutic Ultrasound Improves Myocardial Blood Flow and Reduces Infarct Size in a Canine Model of Coronary Microthromboembolism Mrinal Yadava, MD, D. Elizabeth Le, MD, FASE, Igor V. Dykan, MD, Marjorie R. Grafe, MD, PhD, Matthew Nugent, BS, Azzdine Y. Ammi, PhD, David Giraud, MS, Yan Zhao, MD, Jessica Minnier, PhD, and Sanjiv Kaul, MD, FASE, Portland, Oregon

Background: Therapeutic ultrasound (TUS) has been used to lyse infarct-related coronary artery thrombus. There has been no study examining the effect of TUS specifically on myocardial microthromboemboli seen in acute myocardial infarction and acute coronary syndromes. The aim of this study was to test the hypothesis that TUS improves myocardial blood flow (MBF) and reduces infarct size (IS) in this situation by dissolving myocardial microthrombi. Methods: An open-chest canine model of myocardial microthromboembolism was created by disrupting a thrombus in the left anterior descending coronary artery, and 1.05- and 0.25-MHz TUS (n = 7 each) delivered epicardially for 30 min was compared with control (n = 6). MBF and IS (as a percentage of left anterior descending coronary artery perfusion bed size) were measured 60 min after treatment. In addition, immunohistochemistry was performed to assess microthrombi, and histopathology was performed to define inflammation. Results: Transmural, epicardial, and endocardial myocardial blood volume and MBF (measured using myocardial contrast echocardiography) and percentage wall thickening were significantly higher 60 min after receiving TUS compared with control. The ratio of IS to left anterior descending coronary artery perfusion bed size was significantly smaller (P = .03) in the 1.05-MHz TUS group (0.14 6 0.04) compared with the control (0.31 6 0.06, P = .04) and 0.25-MHz (0.36 6 0.08) groups. MBF versus percentage wall thickening exhibited a linear relation (r = 0.65) in the control and 1.05-MHz TUS groups but not in the 0.25-MHz TUS group (r = 0.29). The presence of myocardial microemboli in vessels >10 mm in diameter was significantly reduced in the 1.05-MHz TUS group compared with the other two groups. The distribution and intensity of inflammation was higher in the 0.25-MHz TUS group compared with the other groups. Conclusions: TUS at 1.05 MHz is effective in restoring myocardial blood volume and MBF, thus reducing IS by clearing the microcirculation of microthrombi. IS reduction is not seen at 0.25 MHz, despite improvement in MBF, which may be related to the increased inflammation noted at this frequency. Because both acute myocardial infarction and acute coronary syndromes are associated with microthromboembolism, these results suggest that TUS could have a potential adjunctive role in the treatment of both conditions. (J Am Soc Echocardiogr 2019;-:---.) Keywords: Coronary thrombosis, Microthrombi, Therapeutic ultrasound, Infarct size

From Knight Cardiovascular Institute (M.Y., D.E.L., I.V.D., M.N., A.Y.A., D.G., Y.Z., J.M., S.K.), the Department of Pathology (M.R.G.), and the Department of Biostatistics (J.M.), Oregon Health and Science University; Portland Veterans Administration Medical Center (M.Y., D.E.L., M.N.), Portland, Oregon. Conflicts of Interest: None. Drs. Yadava and Le are joint first authors. Reprint requests: Sanjiv Kaul, MD, FASE, Knight Cardiovascular Institute, Oregon Health and Science University, UHN-62, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (E-mail: [email protected]). 0894-7317/$36.00 Copyright 2019 by the American Society of Echocardiography. https://doi.org/10.1016/j.echo.2019.09.011

Management of acute myocardial infarction (AMI) revolves around early revascularization to achieve restoration of myocardial blood flow (MBF). Despite successful recanalization of the epicardial infarct-related artery (IRA), myoocardial perfusion remains impaired in a substantial proportion of patients (‘‘no-reflow’’ phenomenon).1-3 Numerous strategies have been used to improve tissue perfusion and reduce eventual infarct size (IS) during AMI. Of these, therapeutic ultrasound (TUS), both alone and in combination with thrombolytics, has been investigated with varying degrees of success.4-10 Almost all experimental and clinical TUS attempts at sonothrombolysis have targeted epicardial coronary thrombus.4-10 However, it is well known that microthromboemboli to the myocardium resulting from spontaneous or wire- and/or balloon-induced coronary 1

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thrombus disruption can contribute to the no-reflow pheAMI = Acute myocardial nomenon following AMI.11-14 infarction These microthrombi can cause microvascular plugging, IRA = Infarct-related artery resulting in lower Thrombolysis IS = Infarct size in Myocardial Infarction grade from flow stagnation in the IRA. LAD = Left anterior There have been no previous descending coronary artery attempts at using TUS LCx = Left circumflex specifically on myocardial coronary artery microthrombi and gauging its LV = Left ventricular effect on MBF and/or IS. We hypothesized that previMBF = Myocardial blood flow ous sonothrombolytic success in MBV = Myocardial blood AMI was probably due in part volume to dissolution of myocardial microthrombi, which consequently MCE = Myocardial contrast relieved microvascular plugging, echocardiography decreased flow stagnation in the PI = Pulsing interval IRA, and improved flowTUS = Therapeutic induced clearance of the epicarultrasound dial coronary thrombus. We created a canine model of WT = Wall thickening myocardial microthromboembolism and studied the effect of TUS on MBF and IS. We used two (1.05 and 0.25 MHz) of four frequencies found by us to be most effective in an in vitro thrombus model (the other two being 0.04 and 2.0 MHz).15 Other TUS parameters used were also based on the results of the study. Abbreviations

METHODS Animal Preparation The study protocol was approved by the Institutional Animal Care and Use Committee at Oregon Health and Science University and conformed to the American Heart Association guidelines for animal research. Twenty adult male mongrel dogs (weight 30-35 kg) were studied (six in a control group and seven each in the 0.25-MHz and 1.05-MHz TUS groups). We randomized the control and 1.05-MHz TUS groups and later added the 0.25-MHz group for completion. Dogs were intubated and ventilated with room air. Sodium pentobarbital was used to induce (40 mg$ kg 1) and maintain (200 mg$ h 1) anesthesia throughout the experiment. Morphine infusion (2 mg$ kg 1 min 1) was used for analgesia. Heart rate, oxygen saturation, end-tidal carbon dioxide, and temperature were continuously monitored (Advisor Vital Signs Monitor; Surgivet, Norwell, MA). A 6-Fr sheath was placed in the right femoral artery and connected to a pressure transducer for arterial pressure measurement. Catheters were placed in the right femoral and peripheral veins for administration of drugs, fluids, and ultrasound contrast agents. A median sternotomy was performed, and the heart was suspended in a pericardial cradle. The left anterior descending coronary artery (LAD) and left circumflex coronary artery (LCx) were dissected free from surrounding structures in their proximal and mid portions. Ultrasonic time-of-flight flow probes (Series SC; Transonics, Ithaca, NY) were placed on the proximal segments of both coronary arteries and were connected to a digital flow meter (model T206; Transonics). Hemodynamics and epicardial coronary blood flow were digitally recorded using a multichannel recorder

Journal of the American Society of Echocardiography - 2019

and displayed online (ES-2000; Gould Electronics, Chandler, AZ) throughout the experiment. Two adjustable occluders were placed on the LAD close to each other, proximal to the first diagonal branch and to where the flow probe was placed. They were tightened to create occlusion, which was confirmed by the absence of flow on the flow probe. The segment of the LAD between the two occluders was crushed externally using forceps to cause endothelial injury. Subsequently, 1 to 1.5 mL of thrombosed venous blood was injected into this segment, followed by 250 U of thrombin (T6634; Sigma-Aldrich, St. Louis, MO). The occluders were left in place for 100 min to facilitate thrombus formation. Coronary Angiography Coronary angiography was performed via the left femoral artery using a Judkins left 3.5-Fr catheter. Omnipaque (GE Healthcare, Princeton, NJ), 300 mg mL 1, was used for coronary opacification. Images were obtained in standard orthogonal projections to visualize all sections of the epicardial coronary arteries for confirming presence or absence of coronary thrombus. Myocardial Contrast Echocardiography Intermittent harmonic imaging was performed using a phased-array system (Sonos 5500; Philips Medical Systems, Andover, MA) with ultrasound transmitted at 1.3 MHz and received at 3.6 MHz. The transducer was clamped at the midpapillary level, and a bath filled with degassed 0.9% sodium chloride served as the acoustic interface between transducer and heart. Dynamic range, overall gain, depth, and focus optimized at the beginning of each experiment for high– mechanical index, intermittent imaging (mechanical index 1.0) were held constant throughout the experiment. Ultrasound contrast was prepared by sonicating an aqueous lipid dispersion of polyoxyethylene-40-stearate and distearoyl phosphatidylcholine saturated with decafluorobutane gas. Microbubble concentration was measured by electrical sensing zone using a Coulter counter (Multisizer III; Beckman Coulter, Brea, CA). Microbubbles were diluted in 0.9% sodium chloride to a concentration of 1  107 microbubbles $ mL 1. They were infused intravenously at a rate of 1 mL $ min 1. End-systolic images were acquired at pulsing intervals (PI) ranging from one to 20 cardiac cycles by gating to the electrocardiogram. Five images at a PI of one cardiac cycle without ultrasound contrast served as background images. Subsequently, five images each at PIs of one, two, three, five, eight, 10, and 20 cardiac cycles were obtained with contrast. Images acquired at each PI were aligned using computer cross-correlation. The spatial extent of the LAD perfusion bed was defined by injection of ultrasound contrast into the proximal LAD at the end of the experiment. This bed was the region of interest because microthrombi would embolize only into this territory. LAD perfusion bed size was calculated as the percentage of the left ventricular (LV) area in the short-axis view. For MBF measurement, background-subtracted images at different PIs were used from the central 50% of the LAD and LCx perfusion beds (for the latter, the region not opacified when ultrasound contrast was injected into the LAD). Plots of acoustic density versus PI were fitted to the exponential function y = A (1 e bt), where y is the acoustic density at PI t, A is the plateau acoustic density representing myocardial blood volume (MBV), and b is the rate of rise of acoustic density denoting the mean myocardial flow velocity.16 MBV fraction was calculated by dividing

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HIGHLIGHTS  Therapeutic ultrasound at 1.05 MHz reduces infarct size in a myocardial microemboli model  This effect is not seen at 0.25 MHz, which was also associated with increased inflammation  Therapeutic ultrasound could be used as adjunctive therapy in acute myocardial infarction myocardial A by the background-subtracted LV cavity signal at a PI of 20 sec. It was used as the value of A for the purposes of this analysis. The product A ∙ b represents MBF. Wall Thickening Analysis The method for measuring myocardial wall thickening (WT) has been previously described17 and used extensively by us in prior work.18-20 Briefly, the epicardial junction of the right ventricular posterior wall and the LV free wall was identified, from which WT measurement was initiated in each frame, allowing registration of the same endocardial and epicardial points between frames despite cardiac rotation around its long axis. A dozen targets were then outlined on the endocardium and epicardium in each frame from end-diastole to end-systole, which were then automatically connected using cubic spline interpolation. After outlining cardiac contours, 100 equidistant points were automatically defined on the epicardial outline to which a tangent was drawn, and a line perpendicular to the tangent intersected the epicardial and endocardial contours as the shortest distance between them. The resulting chord length was measured in all frames from end-diastole to end-systole, with the first chord being the reference point. Using overlays derived from myocardial contrast echocardiography (MCE), WT was averaged in the central 50% of the LAD and LCx perfusion beds. It was expressed as the percentage change in WT as a function of the original wall thickness in a regional coronary territory. Study Protocol The study protocol is illustrated in Figure 1. After animal preparation (stage A), coronary angiography was performed to define coronary anatomy. MCE was performed at the mid papillary muscle short-axis level at baseline, after which the LAD was occluded for

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120 min. MCE was performed 100 min into the occlusion (stage B), and angiography was then performed to demonstrate presence of coronary thrombus. Twenty minutes later, IRA revascularization was attempted either by mechanical force using a coronary wire or percutaneous transluminal coronary angioplasty using a balloon (stage C). Coronary angiography was repeated to confirm recanalization, and MCE was performed. Animals were then randomized to receive either TUS at 1.05 MHz or no ultrasound (control) for 30 min, after which MCE was repeated (stage D). Table 1 lists the comparison of the ultrasound transducer parameters for the two TUS treatments. Of note, 0.25-MHz TUS used a combination of three single-element transducers. Sixty minutes later (stage E), MCE was again repeated, and the LAD was cannulated and ultrasound contrast agent injected to delineate LAD perfusion territory. At the end of the experiment, animals were euthanized according to the June 2007 American Veterinary Medical Association guidelines on euthanasia; the procedure was approved by the Institutional Animal Care and Use Committee at our institution. Subsequently, this protocol was repeated in additional dogs who were treated with 0.25-MHz TUS. Postmortem Histopathology and Immunohistochemistry The midpapillary plane through which imaging was performed was marked with a suture. The heart was sectioned in short axis through this plane. The section was stained with triphenyl tetrazolium chloride to delineate infarcted myocardium.21 IS was measured on both sides of the short-axis slice, and the average was expressed as a percentage of the LV area. The slices were then formalin fixed, and those adjacent to the triphenyl tetrazolium chloride slice were evaluated for the presence of microthrombi and inflammation. Formalin-fixed blocks were embedded in paraffin. Sections (6 mm thick) were stained for microthrombi using immune staining with CD61 antibody (LSBio, Seattle, WA). Deparaffinized tissue sections were incubated with 5% normal goat serum in phosphate buffered saline with 1% bovine serum albumin and 0.3% Triton (blocking serum) for 20 min at room temperature, followed by an avidin/biotin blocking step and 15 min in 3% hydrogen peroxide dissolved in methanol. All rinses were performed with Tris-buffered saline (pH 7.6) with 0.1% Triton X-100. Sections were incubated overnight at 4 C with the primary antibody diluted 1:3,000 in the blocking serum. The secondary antibody was applied to the tissue for 30 min at room temperature (biotinylated goat antirabbit 1:200; Vector Laboratories BA1000, Burlingame, CA). Sections were incubated with avidinbiotin-peroxidase complex (Vectastain Elite kit, Vector Laboratories)

Figure 1 Study protocol. Stage A, baseline; stage B, coronary occlusion with a thrombus; stage C, dissolution of coronary thrombus; stage D, end of 30-min treatment; stage E, 60-min period following treatment.See text for details. PTCA, Percutaneous transluminal coronary angioplasty.

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Table 1 Ultrasound parameters used in the study Parameter

Peak negative pressure, MPa Mode

1.05 MHz

0.25 MHz

1 Pulsed

2 Pulsed

Number of cycles

50

25

Pulse repetition frequency

50

25

Duty cycle, %

0.25

0.25

Number of transducers

1

3

Number of active elements per transducer

3

1

Outer element power, %

30

—

Middle element power, %

50

—

100

—

Inner element power, % Transducer diameter, mm Transducer height, mm Focused/unfocussed

81.8 19 Unfocussed

included to account for within-subject correlation. The factors group and stage and the interaction of group and stage were included in the models. Overall group effect across stages and the differences between TUS groups and control individually were tested with F tests using the linear mixed model framework. For histologic data, the difference between the LAD and LCx was evaluated using the Mann-Whitney U test (nonparametric). Comparison between treatment groups was analyzed using the Kruskal-Wallis test (nonparametric) and, when applicable, Dunn’s posttest (nonparametric). For all tests, P values < .05 were considered to indicate statistical significance (two tailed).

RESULTS 15.5 25.4

Unfocused

Beam width at natural focus, mm

60

Insonification area/transducer, cm2

28.3

35 9.6

Total insonification area, cm2

28.3

28.8

for 30 min, reacted with diaminobenzidine, lightly counterstained with Mayer’s hematoxylin, then dehydrated, cleared, and coverslipped with Permount. Sections were also stained with hematoxylin and eosin for assessment of inflammation. All sections were coded with random numbers, then reviewed by an experienced pathologist. CD61-stained sections were evaluated for the presence of thrombi, defined as aggregates of CD61-positive platelets within a blood vessel. Isolated single platelets in blood vessels were not considered thrombi. Larger myocardial microvessels with multilayered walls and a lumen >10 mm in diameter (arterioles) were scored as thrombi present (1) or not identified (0). The distribution of thrombi in single layer walls, <10 mm in diameter (mostly capillaries and small venules) was scored on a scale similar to that for acute inflammation distribution described below. The distribution of acute inflammation was scored as 0 = no neutrophils, 1 = neutrophils only in epicardium, 2 = focal myocardial neutrophils (in one or two 10lens fields; one 10field approximately 2.1 mm in diameter), 3 = multifocal myocardial neutrophil infiltration (more than two fields to 50% of 10fields), or 4 = widespread myocardial neutrophil infiltration (>50% of 10fields). The intensity of acute inflammation was scored as 0 = none; 1 = few, single infiltrating neutrophils; 2 = moderate, clusters of infiltrating neutrophils; or 3 = many infiltrating neutrophils. A combined score of both distribution and intensity of inflammation was calculated. Statistical Analysis Differences in in vivo results were evaluated using t tests to determine whether groups (TUS treatments vs control) were different at baseline (stage A) and whether the effects of coronary occlusion (stage B) were different between groups. Because only heart rate was different between groups at all stages, we did not adjust for baseline values (stage A) in the main analyses. Average measurements between groups (TUS treatments vs control) were compared at stages C, D, and E via linear mixed models adjusting for initial value recorded at stage A. A random intercept was

Results were derived from all stages in all dogs, except for myocardial contrast echocardiographic measurements, for which the stored digital image files were corrupted at stage E in one dog in the control group and two dogs in the 0.25-MHz TUS group. Histopathology and immunochemistry could be performed in all animals except one control. Hemodynamic Data Table 2 illustrates hemodynamic data. There were baseline differences between groups in terms of heart rate, but it remained unchanged between stages. In contradistinction, both systolic and mean blood pressure decreased progressively between stages, becoming significantly lower than baseline at stage E in all groups and at stage D in the control group. As expected, LAD coronary blood flow was zero at stage B (coronary occlusion) but returned to normal after reperfusion in the control and 0.25-MHz TUS groups but tended to remain low in the 1.05-MHz TUS group. In comparison with other groups, significant hyperemia was noted immediately after reperfusion (stage C) in the 0.25-MHz TUS group. LCx flow declined slowly over time in all groups as mean blood pressure declined, becoming significantly lower than baseline at either stage D or E. Myocardial Contrast Echocardiography Data Table 3 depicts the MCE results. Transmural MBV (A), reflecting perfused capillary density, declined after coronary occlusion (stage B) in all groups. However, compared with the two TUS treatment groups, it remained low at all stages in the control group. Results were similar for the epicardial and endocardial MBV values. Transmural MBF velocity (b) also decreased dramatically in all groups during coronary occlusion (stage B), with recovery seen at subsequent stages, except at stage E in the control group, when it declined significantly. Similar results were seen for the epicardial and endocardial MBF velocity values. Transmural MBF (A $ b) declined during coronary occlusion (stage B) and partly recovered at subsequent stages in the TUS treatment groups but not in the control group, in which it was significantly lower than in the two TUS treatment groups. Similar results were seen for epicardial and endocardial MBF, except for stage D (immediately after TUS), when the values were lower for the 0.25-MHz treatment group. The endocardial/epicardial MBF ratio, although mildly reversed, declined in stage C only in the control group, in which it recovered in subsequent stages. Panels A and B in Figures 2-4 illustrate examples of fitted transmural, epicardial, and endocardial MCE plots from the central

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Table 2 Hemodynamic and wall thickening results Variables

Stage A

Stage B

Stage C

Control

120 6 5*

122 6 6

121 6 8

118 6 9

119 6 11

1.05 MHz

139 6 10

125 6 2

123 6 7

106 6 21†

120 6 4

0.25 MHz

148 6 6

138 6 3*

143 6 5*

134 6 3*

130 6 4

Heart rate, beats $ min

Stage D

Stage E

1

Systolic blood pressure, mm Hg 90 6 14†

88 6 16†

Control

116 6 10

116 6 4

100 6 6

1.05 MHz

119 6 3

117 6 5

108 6 10

105 6 11

95 6 4†

0.25 MHz

128 6 6

137 6 10

119 6 6

111 6 6

95 6 4†

Mean blood pressure, mm Hg Control

101 6 9

99 6 5

83 6 5

73 6 12†

73 6 13†

1.05 MHz

101 6 4

98 6 4

90 6 9

84 6 9

74 6 6†

106 6 5

108 6 6

98 6 4

92 6 5

79 6 3† 30 6 9

0.25 MHz LAD CBF, mL ∙ min

1

Control

37 6 5

0‡

35 6 8

27 6 5

1.05 MHz

25 6 3

0‡

31 6 11

15 6 4

13 6 3

0.25 MHz

32 6 4

0‡

50 6 8 *

34 6 5

27 6 3

Control

42 6 7

47 6 11

34 6 7

31 6 4†

34 6 4

1.05 MHz

43 6 6

39 6 5

35 6 6

36 6 6

37 6 6

0.25 MHz

41 6 6

47 6 7

38 6 6

33 6 3†

31 6 4†

LCx CBF, mL ∙ min

1

LAD %WT Control

39 6 0.2‡

362

1.05 MHz

38 6 0.5‡

363

11 6 3

5 6 2*

15 6 3

4 6 2*

16 6 3

7 6 3*

0.25 MHz

39 6 0.4‡

563

14 6 5

10 6 3

17 6 3

Control

39 6 0.4

39 6 0.4

39 6 0.3

39 6 0.5

39 6 0.3

1.05 MHz

38 6 0.5

37 6 0.4

37 6 0.4

37 6 0.4

38 6 0.5

0.25 MHz

39 6 0.3

39 6 0.4

39 6 0.3

39 6 0.3

40 6 0.8

LCx %WT

Data are expressed as mean 6 SEM. Numbers of animals are six in control and seven each in the 0.25-MHz and 1.05-MHz TUS groups. CBF, Coronary blood flow. *P < .05 compared with other treatments. † P < .05 compared with stage A (baseline). ‡ P < .05 compared with other stages.

50% of the LAD perfusion bed at baseline (stage A) and during reperfusion (stage E) from a dog each from the control (Figure 2), 1.05-MHz TUS (Figure 3), and 0.25-MHz TUS (Figure 4) group. Whereas in the control dog, the MBF values are about one fifth of that at baseline, in the 1.05-MHz TUS dog, they are one half of baseline, and in the 0.25-MHz dog, they are approximately one third of baseline. WT Data Table 2 depicts percentage WT data from the central 50% of the LAD and LCx beds. Percentage WT was similar in all three groups at baseline (stage A) and was substantially reduced during coronary occlusion (stage B). In control dogs, it never recovered at any stage, whereas in both TUS treatment groups it recovered at reperfusion (stages D and E) to approximately one third of the baseline value, which was significantly higher than in the control group. Percentage WT remained normal in the LCx bed in all three groups. Figure 5 illustrates the flow-function relation in all three groups at stage E (60 min after completion of treatment), in which transmural

MBF (x axis) is plotted against transmural percentage WT (y axis). This relation was linear and close in control dogs and those treated with 1.05-MHz TUS, whereas it was poor in the 0.25-MHz TUS– treated dogs.

LAD Perfusion Bed Area and IS Figures 2-4 illustrate examples of LAD perfusion bed size (panel C) and IS (panel D) in a dog each from the control group (Figure 2), 1.05-MHz treatment group (Figure 3), and 0.25-MHz treatment group (Figure 4). It is obvious that for the same approximate LAD perfusion bed size, IS was smaller in the TUS treatment groups compared with the control group, with IS being smallest in the 1.05-MHz TUS group. The ratio of IS to LAD perfusion bed size in these three examples was 0.54, 0.21, and 0.44, respectively. The overall results (Figure 6) also show that the ratio was lower (P = .03) in the 1.05-MHz group (0.14 6 0.04) compared with both the control (0.31 6 0.06) and the 0.25-MHz (0.36 6 0.08) groups.

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Table 3 Results of MCE from the LAD perfusion bed Variables

Stage A

Stage B

Stage C

Stage D

Stage E

Transmural MBV (A) Control

61 6 6

13 6 5*

24 6 5†,‡

37 6 7†,‡

32 6 5†,‡

1.05 MHz

58 6 4

33 6 5*

63 6 9

52 6 6

55 6 8

0.25 MHz

61 6 4

17 6 9*

43 6 7

31 6 6

59 6 5

Transmural MBF velocity (b) Control

0.72 6 0.14

0.25 6 0.11*

0.72 6 0.25

0.67 6 0.10

0.39 6 0.12†,‡

1.05 MHz

0.99 6 0.15

0.20 6 0.02*

0.82 6 0.13

0.65 6 0.12

0.59 6 0.12

0.25 MHz

0.98 6 0.07

0.20 6 0.14*

0.80 6 0.14

0.84 6 0.09

0.59 6 0.12

Control

45 6 11

5 6 3*

20 6 9†

27 6 7

12 6 4†,‡

1.05 MHz

56 6 4

6 6 1*

55 6 11

36 6 8

27 6 4

0.25 MHz

60 6 7

3 6 1*

40 6 9

21 6 3

36 6 9

Control

65 6 6

13 6 6*

22 6 6*

38 6 8

38 6 6

1.05 MHz

61 6 4

38 6 9*

63 6 9

51 6 6

53 6 5

16 6 8*

47 6 8

32 6 5

59 6 5

Transmural MBF (A ∙ b)

Endocardial MBV (A)

0.25 MHz

66.4

Endocardial MBF velocity (b) Control

0.76 6 0.15

0.45 6 0.24†

0.65 6 0.21

0.74 6 0.08

0.27 6 0.08†,‡

1.05 MHz

0.97 6 0.14

0.19 6 0.02*

0.71 6 0.16

0.63 6 0.13

0.53 6 0.06

0.25 MHz

0.97 6 0.11

0.18 6 0.09*

0.78 6 0.14

0.68 6 0.07

0.55 6 0.10

Control

51 6 11

10 6 6*

17 6 8†

28 6 7

11 6 5†

1.05 MHz

57 6 6

7 6 2*

56 6 11

36 6 9

28 6 3

0.25 MHz

64 6 9

3 6 2*

42 6 9

20 6 4†,‡

33 6 8

Control

61 6 7

13 6 5*

27 6 3†

42 6 9

32 6 5†

1.05 MHz

55 6 5

31 6 5*

64 6 9

50 6 9

0.25 MHz

60 6 5

21 6 9*

41 6 7

25 6 5

Endocardial MBF (A ∙ b)

Epicardial MBV (A) 53 6 9 †,‡

56 6 8

Epicardial MBF velocity (b) Control

0.62 6 0.10†

0.51 6 0.32†

0.96 6 0.33

0.76 6 0.14

0.38 6 0.13†,‡

1.05 MHz

0.95 6 0.13

0.23 6 0.02*

,‡

0.90 6 0.19

0.60 6 0.09

0.54 6 0.09

1.05 MHz

1.00 6 0.07

0.29 6 0.10*,‡

0.77 6 0.16

0.85 6 0.13

0.69 6 0.12 11 6 4†,‡

Epicardial MBF (A ∙ b) Control

39 6 9

11 6 6*

27 6 10

32 6 9

1.05 MHz

51 6 8

7 6 1*

57 6 11

33 6 7

0.25 MHz

60 6 6

5 6 1*

36 6 8

19 6 3

25 6 4 †,‡

39 6 9

EER MBF Control

1.3 6 0.1

0.95 6 0.1

0.91 6 0.1

1.05 MHz

1.2 6 0.1

1.0 6 0.19

0.95 6 0.1

1.03 6 0.1

1.19 6 0.2

0.25 MHz

1.1 6 0.1

0.84 6 0.34

1.1 6 0.1

1.1 6 0.1

0.86 6 0.1

0.94 6 0.2

57 6 0.1†,‡

Data are expressed as mean 6 SEM. N = 6 in control and 1.05-MHz treatment group, and n = 5 in 0.25-MHz treatment group. EER, Endocardial/epicardial ratio. *P < .05 compared with other stages. † P < .05 compared with other treatments. ‡ P < .05 compared with stage A (baseline).

Immunohistochemistry and Histopathology Tables 4 and 5 list immunohistochemistry findings related to the myocardial microthrombi in vessels >10 mm (arterioles) and <10 mm (mostly capillaries). More microthrombi were seen in the

LAD compared with the LCx bed in all groups, but this difference was more evident in the 0.25-MHz TUS group (Table 4). The number of microthrombi in >10-mm vessels in the LAD bed was significantly less in the 1.05-MHz TUS group (Table 5). This finding is also

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Figure 2 Data from a control group dog not undergoing TUS treatment. (A, B) Transmural, endocardial, and epicardial plots of time vs acoustic intensity at baseline (stage A) and at the end of treatment (stage E). (C) LAD perfusion bed size defined by injection of ultrasound contrast directly into the LAD. (D) Infarction at the same short-axis slice as in (C). See text for details.

Figure 3 Data from a dog undergoing treatment with a 1.05-MHz transducer. (A,B) Transmural, endocardial, and epicardial plots of time vs acoustic intensity at baseline (stage A) and at the end of treatment (stage E). (C) LAD perfusion bed size defined by injection of ultrasound contrast directly into the LAD. (D) Infarction at the same short-axis slice as in (C). See text for details.

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Figure 4 Data from a dog undergoing treatment with a 0.25-MHz transducer. (A,B) Transmural, endocardial, and epicardial plots of time vs acoustic intensity at baseline (stage A) and at the end of treatment (stage E). (C) LAD perfusion bed size defined by injection of ultrasound contrast directly into the LAD. (D) Infarction at the same short-axis slice as in (C). See text for details. DISCUSSION

Figure 5 Scatterplots with linear fits between transmural MBF (x axis) and percentage WT (y axis) 60 min after treatment in control dogs (n = 6), dogs receiving 1.05-MHz TUS treatment (n = 6), and dogs receiving 0.25-MHz treatment (n = 5). See text for details.

illustrated in Figure 7, which shows examples of microthrombi in the LAD and LCx beds in all groups. Tables 6 and 7 list histopathologic findings related to the distribution and intensity of inflammation as well as the product of the two. It is evident that inflammation was greater in the LAD compared with the LCx bed in both treatment groups but not in the control group (Table 6). Inflammation was significantly greater in the LAD bed in the 0.25-MHz TUS group compared with the 1.05-MHz TUS and control groups (Table 7). This finding is also evident in the examples shown in Figure 8 depicting LAD and LCx beds from each group.

The novel finding of this study is that TUS aimed specifically at lysing myocardial microthrombi in the setting of AMI normalizes MBV and improves MBF, thus reducing IS, by clearing the microcirculation of microthromboemboli. These results were obtained with a 1.05MHz transducer at specified settings and could not be reproduced with a 0.25-MHz transducer despite encouraging in vitro results.15 Other permutations of frequency, pulse repetition frequency, and duty cycle may need to be investigated for further optimizing TUS for this purpose. Management of AMI revolves around early revascularization to achieve restoration of MBF. Despite successful recanalization of the IRA, however, myocardial perfusion remains impaired in a substantial proportion of patients (no-reflow phenomenon).1-3 Numerous adjunctive treatments have been used to improve tissue perfusion and reduce eventual IS during AMI. Of these, TUS, both alone and in combination with thrombolytics, has been investigated with varying degrees of success.4-10 In addition to AMI, coronary thrombosis also causes other acute coronary syndromes, and resulting myocardial microthromboembolism may play a role in tissue necrosis in these conditions.22 Furthermore, IIb/IIIa expression and microvascular fibrin formation increase after reperfusion, leading to leukocyte and platelet entrapment and microvascular obstruction.23 The beneficial effects of platelet glycoprotein IIb/IIIa receptor blockade seen in animal models24 and patients25,26 may be due in part to reduction of in situ thrombosis and consequently IS. TUS aimed at myocardial microthrombi may also dissolve in situ microthrombosis caused by ischemia.

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Figure 6 Box-and-whisker plots of ratio with individual data points and mean (red dots) and median values (thick horizontal line) depicting the ratio between IS and LAD perfusion bed size in control dogs (n = 6), dogs receiving 1.05-MHz TUS treatment (n = 6), and dogs receiving 0.25-MHz treatment (n = 5). See text for details. Table 4 Comparison of myocardial microthrombi between LAD and LCx beds Variable

LAD

LCx

Table 5 Comparison of myocardial microthrombi on the basis of treatment

P

Control

1.05 MHz

0.25 MHz

(n = 5)

(n = 7)

(n = 6)

Vessel lumen (>10 mm)

0.8 6 0.2

0.2 6 0.2*

1.0 6 0.0†

Vessel lumen (<10 mm)

2.8 6 0.2

2.3 6 0.5

3.2 6 0.3

Vessel lumen (>10 mm)

0.2 6 0.2

0.3 6 0.2

0.0 6 0.0

Vessel lumen (<10 mm)

0.6 6 0.6

0.7 6 0.5

0.3 6 0.3

Control (n = 5) Vessel lumen (>10 mm)

0.8 6 0.2

0.2 6 0.2

.07

Vessel lumen (<10 mm)

2.8 6 0.2

0.6 6 0.6

<.04

Vessel lumen (>10 mm)

0.2 6 0.2

0.3 6 0.2

.53

Vessel lumen (<10 mm)

2.3 6 0.5

0.7 6 1.3

.06

1.05 MHz (n = 7)

0.25 MHz (n = 6) Vessel lumen (>10 mm)

1.0 6 0.0

0.0 6 0.0

<.01

Vessel lumen (<10 mm)

3.2 6 0.3

0.3 6 0.3

<.01

Data are expressed as mean 6 SEM.

An experimental study using IIb/IIIa targeted microbubbles reported better epicardial artery recanalization and microvascular perfusion compared with controls.6 The microvascular perfusion success rate was higher than that of the epicardial artery, which the authors speculated was from additional effects on possible microthrombi in the myocardium. IS was not measured in this study. The same group of investigators then reported similar effects using nontargeted bubbles in conjunction with TUS in a small number of patients with AMI.10 In that study, neither was MBF nor IS measured. Our study had important differences from these previous studies. First, we specifically targeted myocardial microthromboemboli. We disrupted the coronary thrombus in the IRA and confirmed its absence before TUS delivery. Second, we did not use ultrasound contrast for therapy, only for assessing myocardial perfusion. Our aim was to study the effects of TUS alone on myocardial microthromboemboli, something not attempted previously. It is likely that microbubbles, when given with TUS, will amplify this effect. Third, we measured MBV (capillary density) and MBF using MCE and showed that both were mostly restored by 30 min application of TUS. Fourth, we measured IS using a gold-standard method to demonstrate the beneficial effects of TUS. Reduction of IS remains the main goal of

LAD bed

LCx region

Data are expressed as mean 6 SEM. *P < .05 compared with control. † P < .05 compared with 1.05 MHz.

AMI and acute coronary syndrome treatment. A previous study reported similar results in a model of hind-limb microthrombi using a 1-MHz transducer with different ultrasound parameters and in conjunction with microbubbles.27 Finally, immunohistochemistry and histopathology confirmed that microthrombi were significantly less numerous in myocardial arterioles in the 1.05-MHz TUS group compared with the 0.25-MHz TUS and control groups. In the two previous studies mentioned above,6,10 microvascular perfusion was noted in some instances in which the IRA was said to remain occluded. This finding could be attributable to some lysis of the IRA thrombus causing flow channels within the thrombus, allowing microbubbles to reach the microcirculation. Myocardial perfusion could also result from collateral flow, but these investigators observed perfusion in the myocardial area that excludes regions supplied by collateral flow.28-30 Another reason for increased microvascular perfusion could result from the direct effect of TUS on tissue perfusion. Although mostly described using low-intensity ultrasound at low frequencies (0.027-0.040 MHz),31-34 high-intensity focused ultrasound delivered at higher frequency can also result in increased MBF.35 We showed increased MBF in a canine model of coronary occlusion using a 1.05-MHz transducer.36 More recently, we reported

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Figure 7 Representative immunohistochemical stain for CD61 of myocardium (20magnification) in the ischemic LAD (top) and nonischemic LCx (bottom) beds in an animal from each group. Thrombi are noted within vessels >10 mm in diameter (mostly arterioles) and those <10 mm in diameter (mostly capillaries). Thrombi are identified as clusters of platelets (brown) within blood vessels; single platelets are not considered thrombi. The degree of microthrombi in the 1.05-MHz treatment group was significantly less than in the control and 0.25-MHz groups. See text for scoring of microthrombi.

Table 6 Comparison of inflammation between LAD and LCx beds Variable

LAD

LCx

P

Distribution of inflammation

2.0 6 0.6

1.8 6 0.4

.85

Intensity of inflammation

1.4 6 0.5

1.6 6 0.5

$.999

Distribution+intensity

3.4 6 1.1

3.4 6 0.7

$.999

(n = 7)

(n = 7)

Distribution of inflammation

2.4 6 0.3

1.0 6 0.2

.03

Intensity of inflammation

1.7 6 0.3

0.7 6 0.2

.05

Distribution+intensity

4.1 6 0.6

1.7 6 0.5

.01

(n = 6)

(n = 6)

Distribution of inflammation

3.8 6 0.2

2.0 6 0.4

.03

Intensity of inflammation

2.5 6 0.3

1.2 6 0.3

.09

Distribution+intensity

6.3 6 0.5

3.2 6 0.7

.001

Table 7 Comparison of inflammation on the basis of treatment Control

1.05 MHz

0.25 MHz

(n = 5)

(n = 7)

(n = 6)

Distribution of inflammation

2.0 6 0.6

2.4 6 0.3

3.8 6 0.2*,†

Intensity of inflammation

1.4 6 0.5

1.7 6 0.3

2.5 6 0.3

Distribution+intensity

3.4 6 1.1

4.1 6 0.6

6.3 6 0.5

Distribution of inflammation

1.8 6 0.4

1.0 6 0.2

2.0 6 0.4

Intensity of inflammation

1.6 6 0.5

0.7 6 0.2

1.2 6 0.3

Distribution+intensity

3.4 6 0.7

1.7 6 0.5

3.2 6 0.7

Control (n = 5)

1.05 MHz (n = 7)

0.25 MHz (n = 6)

LAD bed

LCx bed

Data are expressed as mean 6 SEM. *P < .05 compared with control. † P < .05 compared with 1.05 MHz.

Data are expressed as mean 6 SEM. 37

similar results with a 0.25-MHz transducer. The increase in MBF in the 0.25-MHz TUS group despite a lack of microthrombi resolution may be related to the direct tissue effects of ultrasound. An unexpected finding in this group was the greater degree of inflammation in the LAD bed that could conceivably be an adverse bioeffect of ultrasound at this frequency. Pathophysiology of Microthromboembolism Descriptions of MBF during ischemia have ensued from models of either coronary occlusion alone or occlusion followed by reperfusion.

To our knowledge, this is the first study in which MBF was measured in a myocardial microthromboembolism model. In a previous animal study, coronary blood flow was reduced to 10% of baseline for 90 min, and then 42-mm microspheres were injected to create microembolization.38 The 50% increase in IS by microembolization was attributed to increased ischemia duration compared with animals that did not receive additional microspheres. Because we also created ischemia for a similar period in all groups during coronary occlusion before coronary recanalization, some of the infarction in our study could also be attributable to the duration of ischemia. Interestingly, unlike coronary occlusion, in which the endocardial/ epicardial MBF ratio is reversed,29,30,34 this ratio was close to 1 at

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Figure 8 Representative Hematoxylin and Eosin stain of myocardium (20magnification) in the ischemic LAD (top) and nonischemic LCx (bottom) regions in the same three animals depicted in Figure 7. The intensity of inflammation represented by the presence of neutrophils. The 1.05-MHz treatment group had more inflammation than the control group, and the 0.25-MHz treatment group had more inflammation compared with the control and 1.05-MHz treatment groups. See text for scoring of intensity and distribution of inflammation.

all stages in our model, even in the control group (except at stage C), probably because of microvascular obstruction throughout the entire myocardial thickness as observed on histopathology. When coronary occlusion is followed by reperfusion, myocardial stunning is noted, such that there is no relation between MBF and percentage WT.39-41 In our model of microthromboembolism, however, a linear relation between MBF and percentage WT was noted 60 min after treatment, even in the control dogs. This finding implies that unlike coronary occlusion followed by reperfusion, in which myocellular edema results in myocardial stunning,42,43 significant edema may not occur in the setting of microthromboembolization alone. However, we did not measure myocardial edema in this study. Interestingly, the flow-function relation was poorest in the dogs undergoing 0.25-MHz TUS, resembling what one would expect after reperfusion in a model of coronary occlusion (stunning). This finding may be related to greater inflammation seen in this group. Limitations The first limitation of our study was the small number of animals used. Whereas our results are significant in terms of the end point of IS, type I error is possible. Second, although we randomized the control and 1.05-MHz TUS groups, we added the 0.25-MHz group later. This may explain the baseline differences in heart rate and hyperemic response at stage C in this group of animals. Third, the variance in MBF, especially after reperfusion, is high and can be influenced by fluctuations in systemic pressure because of autoregulation exhaustion. This variance has been described previously, and it takes hours to days for MBF variance to subside.44,45 Fourth, we cannot readily explain why IS was greater in the 0.25-MHz treatment group despite achieving similar MBF as in the 1.05-MHz TUS group. One reason

could be the persistently higher heart rate in this group, resulting in higher myocardial oxygen consumption and a worse oxygen demand/supply balance compared with the other two groups. Another could be the higher peak negative pressure used (2 MPa), which may be injurious to tissue. Finally, we used a nonimaging ultrasound probe for treatment. A combined imaging and treatment 1.05MHz probe could easily be designed for this purpose, as imaging would be feasible at harmonic frequencies with such a probe.

CONCLUSION The pathophysiology of AMI in the setting of myocardial microthromboembolism is different from that in total coronary occlusion. TUS at 1.05 MHz is effective in clearing the microcirculation of microthrombi, thus restoring MBF and reducing IS. Microthrombi clearance and IS reduction are not seen at 0.25 MHz, at which additional inflammation is seen. Our results indicate that TUS should be further investigated using different ultrasound parameters to optimize its effects on myocardial microthromboemboli dissolution as well as its direct tissue effect on MBF. Because both AMI and acute coronary syndromes are associated with microthromboembolism, TUS could have a potential role as an adjunct in the treatment of both conditions.

ACKNOWLEDGMENT We thank Lijuan Liu, DVM, of the Department of Anesthesiology and Perioperative Medicine for preparation of histopathology.

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