Dissociation between two-dimensional echocardiographic left ventricular wall motion and myocardial salvage in early experimental acute myocardial infarction in dogs

Dissociation Between Two-Dimensional Echocardiographic Left Ventricular Wall Motion and Myocardial Salvage in Early Experimental Acute Myocardial Infarction in Dogs HAIM HAMMERMAN, MD, JOSEPH E. O'BOYLE, BS, CAROL COHEN, BS, ROBERT A. KLONER, MD, PhD, and ALFRED F. PARISI, MD

This study was designed to evaluate whether the effects of coronary reperfusion with or without a pharmacologic agent could be detected in the early hours after infarction by 2-dimensional (2-D) echocardiography applied in a manner analogous to its clinical use. Proximal left anterior descending coronary occlusion was performed in 24 dogs, and the dogs were then randomized into 3 groups. In group 1 (n = 8), coronary occlusion was maintained for 6 hours; in group 2 (n = 8), coronary occlusion was maintained for 2 hours and was followed by 4 hours of reperfusion; in group 3 (n = 8), 2 hours of coronary occlusion were followed by 4 hours of reperfusion but methylprednisolone (30 mg/kg intravenously) was also administered 15 minutes after coronary occlusion. At 6 hours, 2-D images were obtained through the closed chest wall and the

percentage of the left ventricular wall motion abnormalities was determined at 4 short-axis levels. The mass at risk was defined by in vivo Monastral blue injection and infarction by triphenyltetrazolium chloride staining. The mass of necrosis was 74 44% (mean -I- standard error of the mean) of the mass at risk in group 1 and was smaller in groups 2 and 3, 44 -I- 6% and 35 -I- 4%, respectively (p <0.01). Percent necrosis of the left ventricle was 22 -t- 3% in group 1, 15 -I- 3% in group 2 (difference not significant) and 10 4- 2% in group 3 (p <0.05 vs group 1). Percent wall motion abnormalities were 26 4- 1% for group 1, 26 4- 1% for group 2 and 25 4- 2 % for group 3. Thus, significant tissue salvage could not be detected during the early stages of reperfusion by 2-D echocardiographic wall motion analysis. (Am J Cardiol 1984;54:875-879)

The effect of myocardial reperfusion after coronary occlusion on left ventricular (LV) wall motion has received very little attention using 2-dimensional echocardiography (2-D echo) in a manner analogous to its clinical application, i.e., in closed-chest animals. Furthermore, to our knowledge, the possible synergistic effects of reperfusion and infarct size-reducing antiinflammatory pharmacologic agents has not yet been

studied using 2-D echo. In the experimental setting 2-D echo is ideally suited to evaluate the functional consequences of myocardial ischemia since it is both noninvasive and produces real time images. Moreover, the use of 2-D echo to detect regional LV wall motion abnormalities has been well documentedJ-6 Numerous studies, both qualitative7-17 and quantitative, 18-23have shown that 2-D echo can be used to measure the extent of LV involvement during the first few hours following acute myocardial infarction (AMI). With the recent advent of techniques that restore blood flow to jeopardized ischemic myocardium resulting in the limitation of myocardial necrosis, 24-26 it is important to determine if salvaged myocardium can be detected through improved wall motion. Since significant changes in wall motion with any type of therapy should be clearly visible with 2-D echo, this study was designed to evaluate, via an integrated analysis of wall motion at 4 short-axis cross-sectional levels, whether the effects of tissue salvage due to reperfusion with or without an adjuvant pharmacologic agent improve myocardial wall motion within the first 6 hours after myocardial injury. A ran-

From the Departments of Medicine and Pathology, Harvard Medical School, Brigham and Women's Hospital, and West Roxbury/Brockton Veterans Administration Medical Center, Boston, Massachusetts. This study was supported by the Medical Research Service of the Veterans Administration, Washington, D.C., and in part by Grants HL 23140, 28048 and SCOR 26215 from the National Heart, Lung, and Blood institute, Bethesda, Maryland. This work was done during the tenure of Dr. Kloner as an Established Investigator from the American Heart Association with funds contributed in part by the Massachusetts Heart Association, Needham, Massachusetts. This work was done during Dr. Hammerman's tenure as a fellow of the Stanley J. Sarnoff Society, Bethesda, Maryland. Manuscript received February 16, 1984; revised manuscript received May 31, 1984, accepted June 5, 1984. Address for reprints: Alfred F. Parisi, MD, West Roxbury Veterans Administration Medical Center, 1400 VFW Parkway, West Roxbury, Massachusetts 02132. 875

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TWO-DIMENSIONALECHO AND TISSUE SALVAGE

domized, p r o s p e c t i v e protocol t h a t k e p t e v a l u a t o r s of 2-D echocardiographic recordings blinded as to t h e t h e r a p e u t i c i n t e r v e n t i o n s was used. Methods

Experimental preparation: Mongrel dogs of either sex with a mean weight of 25 4- 10 kg (± standard deviation) were anesthetized with sodium pentobarbital (25 mg/kg intravenonsly). Two-dimensional echocardiographic examinations were performed through the chest wall with the dog in the right decubitus position. 27 Dogs were selected for study on the basis of an initial 2-dimensional echocardiographic examination that showed clear definition of LV epicardial and endocardial targets. The dogs were intubated, placed on a Harvard respirator and ventilated with room air. A thoracotomy was performed in the frithleftintercostalspace. The proximal poztion of the left anterior descending coronary artery was dissected free from surrounding tissue. Polyvinyl catheters were placed in the femoral artery for pressure measurements, and into the leftatrium for in vivo Monastral blue injection at the end of the experiment. Experimental protocol: After administration of lidocaine, 1.5 mg/kg intravenously, the left anterior descending coronary artery was occluded distal to the origin of the first diagonal branch by a Schwartz arterial clamp. A second similar dose of lidocaine was administered 5 minutes after coronary occlusion in all dogs. Immediately after coronary occlusion was performed each dog was randomized to I of 3 groups by opening a sealed envelope which contained the treatment assignment. In group 1 (8 dogs), coronary occlusion was maintained throughout the experiment for 6 hours. In group 2 (8 dogs), coronary occlusion was maintained for 2 hours and was followed by 4 hours of reperfusion. For group 3 (8 dogs), 2 hours of coronary occlusion were followed by 4 hours of reperfusion and methylprednisolone (MP), 30 mg/kg intravenously, was administered 15 minutes after coronary occlusion. This dosing regimen of MP is not associated with impaired healing of infarcts, in contrast to higher doses administered multiple times, which may result in impaired healing. Heart rate and arterial pressure were continuously recorded throughout the experiment. Echo-

iO0 k,.

80 _

cardiograms were performed before occlusion and immediately before sacrifice. At 6 hours, Monastral blue, I mg/kg, was injected into the left atrium with the coronary artery occluded for determination of in vivo area at risk. 28 The hearts were then arrested in diastole by potassium chloride injection and excised. The left ventricle was dissected free from the surrounding tissue, and sectioned parallel to the atrioventricular groove into 5-ram transverse sections. The heart slices were incubated in 1% triphenyltetrazolium chloride (Sigma) in phosphate buffer for 10 minutes. The in vivo area at risk (the area unstained by Monastral blue) and the area of necrosis (the area unstained by triphenyltetrazolium chloride) were determined by planimetry for each slice and multiplied by the weight of the slice to allow determination of both volume and percentage of LV mass at risk, mass of necrosis and the ratio of mass of necrosis to mass at risk. Echocardiographic analysis: After the chest was closed, echocardiograms at 4 short-axis levels--mitral valve, high papillary muscles, low papillary muscles and apex--and a long-axis view were obtained with a Varian V-3000 ultrasonograph using a 2.25-MHz transducer. Images were stored for subsequent analysis using a Panasonic NV3160 reel-to-reel video recorder and Scotch 361, 1/2-inch videotape. A simultaneous electrocardiogram was also recorded. Each 2-D echocardiographic study was assigned a 5-digit random number so that when the recorded study was analyzed subsequently, the observer would be blinded to all essential information (e.g., dog number, date or time interval of study). Analogous to previous studies, 2 observers, each blinded to the morphologic results, qualitatively analyzed each 6-hour study for wall motion/wall thickening abnormalities, independent of each other. Areas with impaired relative centripetal motion that failed to thicken or that thinned were judged abnormal. Wall motion/thickening defects at each short-axis level were expressed as a percent of the circumference (C) of a circle. The percent of LV mass with abnormal myocardial motion was calculated as: 0.36 (%Cmv) + 0.27 (°~Chp) "}" 0.23 (%Clp) + 0.14 (%Cap), where my ffi mitral valve cross-sectional level, hp ffi high papillary muscle level, lp = low papillary muscle level and ap = apical level. The coefficients in this formula are based on the relative weights of mv, hp, lp and ap anatomic sections in 10 dogs from another series. 29

|

60

4o

V

2o-

40 .

20

I

&

Reperfusion

MP + Reperfusion

.,,j i

I

I

Permanent Occlusion

Reperfusion

MP + Reperfusion

FIGURE 1. Percent mass of necrosis as a function of mass at risk in 3 study groups. Each closed circle is the individualvalue for each dog; open circles and bars are the mean 4- standard error of the mean for each group. Mean values are significantly smaller (p <0.01) in both reperfusion groups as compared to permanent occlusion. MP = methylprednisolone.

o

Permanent Occlusion

FIGURE 2. Percent left ventricular (LV) mass of necrosis in 3 study groups. Each closed circle is the individual value for each dog; open circles and bars are the mean 4- standard error of the mean for each group. Only the mean value for the group treated with methylprednisolone (MP) and reperfuslon is significantly smaller (p <0.05) compared with the permanent occlusion group.

October 1, 1984 THE AMERICAN JOURNAL OF CARDIOLOGY Volume54

TABLE I

Summary of Mass at Risk

Permanent occlusion Reperfusion MP + reperfuslon

stamLng did not differ between 3 groups. It was 28 4- 3%

Mass Necrosis Mass at Risk

Necrosis In LV

Wall Motion Abnormalities

74 4- 4 44 4- 6" 35 4- 4"

22 4- 3 15 4- 3 10 4- 2 t

26 4- 1 26 4- 1 25 4- 2

(%)

877

(%)

(%)

* p <0.01 vs group with permanent occlusion; 1"p <0.05 vs group with permanent occlusion. Values are mean 4- standard error of the mean. LV = left ventricle; MP = methylprednlsolone,

The percentage of wall motion abnormalities was finally expressed as the mean of the 2 independent observations. Echocardiographic analyses represent the mean of independent determinations of 2 trained observers. The interobserver variability for these 2 observers was obtained for the entire series as well as for each randomized subgroup. Overall mean difference for percent ventricular involvement between the 2 observers was 3.9 + 3.1%. The mean differences were 2.6% for the controls, 4.7% for the reperfused group, and 4.5% for the reperfused dogs treated with MP. Statistical analysis: One-way analysis of variance using the general linear models procedure (corrected for multiplicity where necessary, as in the hemodynamic measurements) was used to calculate the significance of difference of infarct size measurements, echocardiographic and hemodynamic data among the groups. The calculations were performed on an IBM 4341 computer using the Statistical Analysis System (SAS). Results

Assessment of myocardial salvage: Percent LV mass at risk as assessed in vivo by Monastral blue PERMANENT LIGATION

(mean 4- standard error of the mean) in the permanent occlusion group, 31 + 5% in the reperfusion group and 26 • 4%0in the M P plus reperfusion group. The mean mass of necrosis was 74 ± 4% of the mass at risk in the permanent occlusion group, and significantly smaller in both reperfusion groups. For the reperfusion alone group it was 44 + 6% and for MP plus reperfusion group 35 + 4% (p <0.01 permanent vs reperfusion or MP plus reperfusion). There was no statistically significant difference between reperfusion group and reperfusion plus M P group. LV mass of necrosis was 22 ~- 3% in permanent occlusion group, 15 + 3% in reperfusion group and was statistically significantly smaller in the M P plus reperfusion as compared to the permanent group (10 ~= 2%, p <0.05) (Fig. 1 and 2, Table I). Thus, infarct size expressed as function of the mass at risk was significantly smaller in both reperfusion groups, while LV necrotic mass was smaller only in the M P plus reperfusion group compared with the permanent occlusion group. This reduction in infarct size was due primarily to a decrease in the transmural extent of the infarction. Figure 3 indicates where the reduction primarily occurred. In the permanent group, 7 of the 8 median slices had at least a partial transmural extent of infarction, compared with only 2 of the 8 from the M P plus reperfusion group. There was also circumferential reduction in infarct size, but this was not as striking as the transmural decrease in infarct size. Echocardiographic and motion analysis: Echocardiographic data adequate for analysis were available for all animals in all groups. The mean percent LV wall motion abnormalities was 26 ± 1% for the permanent occlusion group, 26 ~= 1% in the reperfusion group and 25 ± 2% in M P plus reperfusion group (difference not significant). Thus, no difference was found between groups by analysis of 2-D echocardiographic wall motion abnormalities (Fig. 4). H e m o d y n a m i c measurements: Heart rate, systolic and diastolic blood pressures did not differ between the

40 MP + REPERFUSION

i

5O 20

.,.,I

o FIGURE 3. Tracings of the median slices of the left ventricle in the permanent occlusion and methylprednlsolone (MP) -I- reperfusion groups. The area at risk is shown by stippling and the area of infarct is in black. This demonstrates the extent of infarct size reduction in the MP -t- reperfuslon group and that most of thls reduction was in limiting the transmural extent of infarction.

I

I

I

Permanent Reperfusion MP + Occlusion Reperfusion

FIGURE 4. Percent wall motion abnormalities as analyzed by 2-dimensional echocardlography. There is no significant difference between values among the groups. MP = methylprednisolone.

878

TWO-DIMENSIONALECHO AND TISSUE SALVAGE

T A B L E II

HemodynamlcSummary

Heart rate (beats/min)

Systolic pressure (mm Hg)

Diastolic pressure (mm Hg)

Preocclusion

2 Hrs Postocclusion

6 Hrs Postocclusion

141 4- 6

142 4- 6

144 4- 9

136 4- 7

139 -I- 9

153 4- 11

144 4- 5

124 4- 5

131 4- 7

125 4- 6

128 4- 6

132 4- 8

140 4- 10

131 4- 11

146 4- 8

144 4- 13

144 4- 11

139 4- 10

91 :t: 5

94 4- 6

96 4- 5

101 4- 8

94 4- 9

101 4- 7

96 4- 7

101 -1- 6

107 4- 6

Permanent occlusion (n = 8) Reperfusion (n = 8) MP -t- reperfusion (n = 8) Permanent occlusion (n = 8) Reperfusion (n = 7) MP -t- reperfusion (n = 7) Permanent occlusion (n = 8) Reperfusion (n = 7) MP + reperfusion (n = 7)

Values are mean 4- standard error of the mean. MP = methylprednisolone.

groups in the preocclusion phase and later during the experiments (Table II). Arterial pressure data were not available for 2 dogs because of calibration error (1 in the reperfusion group, 1 in the MP plus reperfusion group). Discussion

The problem of detecting and quantifying the extent of AMI using 2-D echo has received considerable attention in recent years. Many clinical studies involving primarily qualitative analysis have shown that 2-D echo can detect and localize infarction and the extent of wall motion abnormalities correlate with other measures of infarct size and have prognostic importance.7-17 Experimental studies have also shown promise for use of 2-D echo in AMI, but there is a tendency to overpredict infarct size using wall motion abnormalities as an index of necrosis early after injury. 2°,21Nevertheless, several studies have either had encouraging correlations with infarct size or have shown important fluctuations that may offer promise for following the course of infarction. Meltzer et a122reported a good correlation (r = 0.82) in a canine model between the extent of abnormal 2-D echo wall motion and infarct size before death (51/2hours after coronary occlusion). Furthermore, wall motion abnormalities which were initially similar in all dogs, showed changes during interventions: They were significantly improved after treatment with nitroglycerin and worsened following treatment with phenylephrine. Wyatt et al 2z reported that 2-D echocardiographic dysynergic areas correlated reasonably (r = 0.87) with nitroblue tetrazolium infarct areas in dogs. Clinical studies have shown that 2-D echo correlates well with infarct size, but overestimates it. 23 Nieminen et al 2° demonstrated that in a canine model from 2 to 48 hours after coronary occlusion, 2-D echocardiographic wall motion abnormalities improved and correlated better with infarct size at 48 hours than at 2 hours.

Few studies have assessed the impact of reperfusion on wall motion abnormalities as imaged by 2-D echo early after infarction, and there are no 2-D echo studies of the combination of an antiinflammatory pharmacologic agent such as MP in combination with reperfusion. Since early reperfusion is the goal of both lytic therapy and coronary bypass during AMI, it is important to determine whether any beneficial effects of reperfusion are readily detectable by 2-D echo as it would be used in a clinical context. Hence, this study was specifically designed to evaluate whether infarct size reduction by early coronary reperfusion or coronary reperfusion plus a pharmacologic agent could be reliably detected in the first few hours after occlusion by systematic blinded qualitative evaluations of serial 2-D echocardiographic images in a canine model. Although qualitative evaluation is not as rigorous as a strictly quantitative approach, there is ample precedent for its use. 2,6,8,9,13,15 This method may miss minor changes in wall motion and is not ideal for serial studies, but it is unlikely to miss major wall motion differences in intergroup comparisons. Our data indicate that on average, a greater than 50% reduction in infarct size occurred in the 8 dogs treated with reperfusion plus MP, with a lesser but still significant reduction in the mass necrosis/mass at risk ratio in dogs that received reperfusion alone. Despite this substantial myocardial preservation, there was no detectable functional recovery in either of the treated subgroups at 4 hours after reperfusion (Fig. 4) in the presence or absence of MP. Two other studies have examined the impact of reperfusion on regional and/or global LV function using 2-D echo. In a study evaluating the functional and biochemical effects of reperfusion, Ellis et al28 failed to detect active thickening in the central ischemic zone until 72 hours after reperfusion. Haendchen et al 3° compared reperfusion to reperfusion plus retroperfusion

October 1, 1984 THE AMERICAN JOURNAL OF CARDIOLOGY Volume 54

in animals subjected to 3 hours of left anterior descending coronary occlusion. Although their reperfused animals began to show improvement in ejection fraction between 1 and 24 hours, they failed to show thickening in their central ischemic zone up to 7 days later. The findings in our study provide independent confLrmation of these prior observations. In addition, our study reveals that early recovery of wall motion does not occur when MP therapy is combined with reperfusion despite significant tissue salvage. Furthermore, the observational procedures we have used have direct relevance to clinical situations in which 2-D echo might be used to screen for beneficial effects of tissue reperfusion early after some degree of myocardial injury. In this study the method of 2-D echocardiographic analysis was carried out in a manner analogous to clinical use--closed-chest and assessment of 4 short-axis levels, compared with previous studies in which open-chest animals were used or in which function was assessed by ultrasonic crystals. Thus, we conclude that significant tissue salvage can occur early after AMI by reperfusion and reperfusion plus low-dose MP, which is not likely to be detected by gross improvement in wall motion abnormalities in the first few hours after successful intervention. Acknowledgment: The authors gratefully acknowledge the technicalassistanceCurtis Wrenn, Howard Afford, Henry Foster and Tatiana Scantlebury and the secretarialassistance of Nancy Watterson-Diorio. Animals used in thisstudy were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animals Resources, National Research Council [ D H E W Pub. No. (NIH) 78-23, revised 1978].

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