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Myocardial Distribution of Indium-111- Antimyosin Fab and Technetium-99M-Sestamibi in Experimental Nontransmural Infarction

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Myocardial Distribution of Indium-111- Antimyosin Fab and Technetium-99M-Sestamibi in Experimental Nontransmural Infarction Myocardial Distribution of Indium-111- Antimyosin Fab and Technetium-99m-Sestamibi in Experimental Nontransmural Infarction Andreas J. Morguet, Dieter L., Münz,Hermann H. Klein, Sibylle Pich, Anne Conrady, Klaus Nebendahl, Heinrich Kreuzer, and Dieter Emrich Departments of Cardiology and Pulmonology, Nuclear Medicine and Experimental Animal Research, Georg August University, Göttingen,Germany Indium-111-labeled monoclonal antimyosin Fab frag Early revascularization in acute myocardial infarction results ments bind to myosin in myocytes which have lost their in normal, necrotic and partially damaged and partially sal membrane integrity and are used to indicate myocardial vaged ("intermediate") myocardium. By combining a perfusion damage (1-4). The distribution of myocardial blood flow tracer and a marker for myocardial injury, we attempted to can be assessed using the new perfusion tracer "Tc- differentiate between these three types of cardiac tissue. The sestamibi (5-8). LAD was occluded in nine pigs for 45 min and then reperfused. After 48 and 72 hr, 74 MBq 111ln-antimyosin Fab and 740 In the present study, both tracers were administered in MBq ""Tc-sestamibi, respectively, were injected intrave combination in a porcine nontransmural infarction model in order to differentiate between normal, necrotic and nously. Normally perfused myocardium was labeled with flu- partially damaged and partially salvaged ("intermediate") orescein and the heart excised. Three to four slices were cut from the apex. Tetrazolium staining revealed the zone of myocardium. necrosis. Tracer distribution on double-nuclide scintigrams of the slices also reflected the three different myocardial zones. MATERIALS AND METHODS Guided by fluorescence and macrohistochemistry, tissue samples were excised from each zone. In relation to normal Radiopharmaceuticals The antimyosin kit (Myoscint*) used in this study was obtained myocardium, mean activity in the intermediate zone was 0.82 ±0.20 for "Tc-sestamibi and 2.84 ± 1.31 for 111ln-anti- from Centocor Europe, Leiden, The Netherlands. It contains 0.5 myosin Fab. Activity in necrotic myocardium was 0.30 ±0.19 mg of monoclonal murine Rl IDlO-Fab-DTPA. The antibody and 3.95 ±2.47, respectively. These results show that 111ln- fragments were labeled with 74 MBq of high-purity '"In-chloride antimyosin Fab fragments not only accumulate in necrotic but by incubation at room temperature for 10 min. Labeling effi also in intermediate myocardium. Therefore, an overestima- ciency determined by thin-layer chromatography was always tion of infarct size may occur if 111ln-antimyosin Fab fragments higher than 95%. are used alone without a perfusion tracer. Technetium-99m-sestamibi was prepared from a kit (Cardiol ite9) supplied by DuPont de Nemours, Inc., Billerica, MA. The J NucíMed 1992; 33:223-228 vial containing 1 mg of (Cu(2-methoxy-isobutyl-isonitrile)4)BF4 was labeled with 740 MBq of [g9mTc]pertechnetate by boiling in a water bath for 10 min. Labeling efficiency checked by thin- layer chromatography was always found to be higher than 95%. n the patient presenting with acute myocardial infarc I Animal Preparation tion, early revascularization using thrombolytic therapy or Nine young farm pigs with a mean body weight of 44.0 kg percutaneous transluminal coronary angioplasty is usually (range 36-54 kg) were anesthesized using metomidate, piritram- attempted in order to minimize infarct size. Such inter ide and ventilation with nitrous oxide and oxygen. A median ventions, however, often result in nontransmural myocar thoracotomy was performed. Between its middle and distal third, dial infarction. The initiation of further therapeutic meas the left anterior descending coronary artery (LAD) was occluded ures depends on the ratio of still viable, but jeopardized to with an external clamp. After 45 min, coronary perfusion was re necrotic myocardium within the area at risk. established leaving a thread as a marker at the site of occlusion. Myocardial scintigraphy may be used to risk stratify Ventricular fibrillation was terminated in five animals by electri patients in the subacute stage of myocardial infarction. cal cardioversion. The chest was closed, and the animals were allowed to recover. Indium-111-antimyosin Fab (74 MBq) was injected in an Received Apr. 24,1991; revision accepted Sept. 9,1991. intravenous ear catheter 48 hr after the release of coronary For reprints contact: Andreas J. Morguet, MD, Dept. of Cardiology and Pulmonology, Center of Internal Medicine, Georg August University, Robert- occlusion. Koch-Str. 40, D-3400 Göttingen, Germany. On Day 3 after surgery, the chest was reopened and 740 MBq Antimyosin and Sestamibi in Nontransmural Ml •Morguet et al 223 of "Tc-sestamibi were administered intravenously 72 hr after the start of reperfusion. Sixty minutes later, the LAD was oc cluded again at the initial site and 10 ml of 10% fluorescein were injected into the right ventricle to label the normally perfused myocardium (9). Twenty seconds later, the heart was excised, filled with a warm 2% agarose solution and immediately cooled in crushed ice. After allowing the solution to gelatinize for a few minutes, four slices of 4 mm thickness were cut from the area at risk in the apical and supraapical part of the heart parallel to the atrioventricular groove (only three slices could be obtained in Animal #8). Each of the 35 slices was exposed to ultraviolet light and its surface macroscopically mapped and photographed. Normal myocardium showed green fluorescence and the area at risk appeared dark blue. The slice was then incubated in a nitroblue tetrazolium solution for 30 min at room temperature in order to reveal macroscopically necrotic myocardium within the area at risk which appeared as bright tissue along darkly stained myocar FIGURE 1. Myocardialslicecut from the supraapicalpart of a dium (10,11). Once more, the surface of the slice was mapped porcine heart (slice 3 from Animal #9) after LAD occlusion and and photographed. The zone within the area at risk between reperfusion exposed to ultraviolet light (LV = left ventricle, RV = necrotic and normal cardiac tissue was defined as "intermediate" right ventricle). Normal myocardium shows green fluorescence myocardium. In each zone, the site designated for later excision after in vivo labeling with fluorescein. The area at risk without of a tissue sample was marked with a pin. perfusion during coronary occlusion appears dark blue. Note that the subepicardial tissue of the anterior wall is located within the Scintigraphy area at risk (arrows). Two hours after excision of the heart, the cardiac slices of one experiment were all positioned tightly under a large field of view gamma camera equipped with a medium-energy parallel-hole a sharp border with the surrounding dark area of macro- collimator (Picker International, Highland Heights, OH). Double- histochemically normal tissue (Fig. 2). nuclide scintigraphy was performed using a 20% window encom Comparison of the fluorescence and the tetrazolium passing the 247 keV photopeak of '"In and simultaneously a aspects of each slice always revealed a zone of cardiac 10% window centered on the 140-keV photopeak of "Te. A tissue between necrotic and normal myocardium within total of 80,000 to 120,000 counts in the "Te channel and of the area at risk. This intermediate myocardium tended to 50,000 to 70,000 counts in the '"In window were acquired per be located in the subepicardial layers of the anterior ven cardiac slice in a 64 x 64 pixel matrix within 60 min. tricular wall (Figs. 1 and 2). Tissue Samples Myocardial Imaging After imaging, a tissue sample with a minimum size of 4 x 4 The double-tracer scintigrams of the cardiac slices x 4 mm3 was excised from normal, necrotic and intermediate showed a markedly higher uptake of "Tc-sestamibi in myocardium of each slice. All 105 samples were weighed and both intermediate and normal myocardium compared to measured in a well counter. Analogous to scintigraphy, the energy the area of necrosis. The photon yield of "'In per pixel windows were set for both tracers simultaneously. The count rate was lower than that of "Tc. It was not possible to in the "Tc channel was corrected for spill-over from Compton- scattered '"In photons. distinguish between intermediate and necrotic tissue within the area at risk on the basis of ' "In-antimyosin Fab uptake alone. However, on all slices there was a region RESULTS with overlapping "Tc and '"In activity. This area corre sponded to the intermediate zone previously determined Fluorescence and Macrohistochemistry by fluorescence and tetrazolium staining (Fig. 3). Thus, Macroscopically, normal, necrotic and intermediate three areas could be delineated on the scans: a zone with myocardium could be clearly delineated on the surface of mostly "Tc-sestamibi uptake corresponding to normal each cardiac slice. There was always a sharp border be myocardium, an area of predominantly '"In-antimyosin tween the green fluorescent normal myocardium and the Fab accumulation corresponding to necrotic tissue and a dark blue area at risk (Fig. 1). zone of significant uptake of both tracers corresponding to After tetrazolium staining, the surface of all myocardial slices showed bright non-reducing necrotic tissue within intermediate myocardium. the area at risk which extended into the anterior wall of Sample Evaluation the left and partially into the right ventricle and the The activity of "Tc-sestamibi and '"In-antimyosin interventricular septum. Usually, this area was not com Fab per gram of myocardium measured in every tissue pletely coherent and composed of disseminated isles of sample was divided by the activity of each tracer in normal necrotic tissue of varying size. However, there was always tissue defined as unity. The activity ratios of intermediate- 224 The Journal of Nuclear Medicine •Vol. 33 •No. 2 •February 1992 accumulation but there was no linear correlation. The cloud of paired values on the graph, however, consisted of two parts, one representing intermediate, the other ne crotic myocardium.
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