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Experimental Studies |
1 From the Departments of Diagnostic Radiology (S.I.C., S.H.C., C.H.L., H.Y.K., T.H.L.) and Diagnostic Pathology (G.Y.G.) and the Nuclear Magnetic Resonance Laboratory (S.T.K., K.H.L.), University of Ulsan College of Medicine, Asan Medical Center, 388-1 Poongnap-Dong, Songpa-Gu, Seoul 138-736, Korea; and Schering, Berlin, Germany (H.J.W.). Received January 22, 1999; revision requested March 22; final revision received October 4; accepted October 26. Supported in part by research grant 98-045 from the Asan Institute for Life Sciences. Address correspondence to T.H.L. (e-mail: thlim@www.amc.seoul.kr).
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Abstract |
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MATERIALS AND METHODS: Nine cats were subjected to 90 minutes of occlusion of the left anterior descending coronary artery followed by 90 minutes of reperfusion. Contrast material–enhanced T1-weighted spin-echo images were obtained for 12 hours in five cats and 6 hours in four cats. Pathologic examinations of the resected specimens were performed with 2'3'5-triphenyl tetrazolium chloride (TTC) histochemical staining and electron microscopy. The size of enhanced area on MR images was compared with that of irreversibly damaged myocardium with TTC staining. The time course of signal enhancement was evaluated.
RESULTS: The size of enhanced area on MR images was well correlated with that of irreversibly damaged myocardium with TTC staining. Maximum enhancement occurred 1–3 hours after administration of the contrast material, with mean enhancement of 171% that of normal myocardium. Electron microscopic examinations showed severe myocardial damage in the irreversibly damaged myocardium but only mild edematous changes in the reversibly damaged myocardium.
CONCLUSION: MR images enhanced with bis-gadolinium mesoporphyrins provide accurate sizing of irreversibly damaged myocardium with a strong and persistent signal enhancement in the reperfused myocardium.
Index terms: Animals • Bis-gadolinium mesoporphyrins • Magnetic resonance (MR), contrast enhancement • Metalloporphyrins • Myocardium, infarction, 511.771 • Myocardium, MR, 511.1214
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Introduction |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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In a previous study, MR images enhanced with the necrosis-avid contrast agent, bis-gadolinium mesoporphyrins, delineated irreversibly damaged from reversibly damaged myocardium and also showed strong, persistent signal enhancement of the irreversibly damaged myocardium in the occlusive myocardium in a rat model (12). To our knowledge, however, it has not been tested whether enhancement with this contrast material can help accurate mapping of irreversibly damaged myocardium in the reperfused myocardium. Therefore, we performed this study (a) to correlate the size of enhanced areas on T1-weighted images enhanced with bis-gadolinium mesoporphyrins to that of the infarct area at 2'3'5-triphenyl tetrazolium chloride (TTC) histopathologic examination for assessment of irreversibly damaged myocardium, (b) to evaluate the time course of signal enhancement at MR imaging enhanced with this contrast material, and (c) to assess the pathologic status of myocardium by means of electron microscopic examinations of the reperfused myocardium.
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MATERIALS AND METHODS |
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After a left lateral thoracotomy along the fifth intercostal space, pericardiotomy was performed by means of a midline incision, and a pericardial cradle was prepared by attaching the margins of the dissected pericardium at the adjacent thoracic wall. The left anterior descending coronary artery was isolated distal to the first diagonal branch, and a snare loop was made with 4-0 silk placed in a slender plastic tube. The occluder was attached to the end of the snare loop. Occlusion or reperfusion of the left anterior descending coronary artery was produced by simply fastening or releasing the snare loop. Obstruction of the left anterior descending coronary artery was confirmed by observing the change in color of the myocardium at risk during preliminary test occlusion. The cats underwent 90 minutes of occlusion of the left anterior descending coronary artery followed by 90 minutes of reperfusion.
Contrast Material
Bis-gadolinium mesoporphyrins was injected via the femoral vein at a dose of 0.1 mmol per kilogram of body weight. The synthesis methods, chemical structure, physiochemical properties, and imaging behaviors of this contrast agent have been previously described in detail (8).
MR Imaging
MR imaging was performed with a 1.5-T imager (Magnetom Vision; Siemens Medical Systems, Erlangen, Germany) with a 27-cm-diameter circularly polarized head array coil. During MR imaging, heart rates were kept between 140 and 170 beats per minute. Electrocardiography-triggered breath-hold turbo spin-echo T2-weighted MR images were obtained along the short axis of the heart before injection of the contrast material. Images in the sagittal plane were also acquired in order to obtain additional information of myocardial status. The acquisition parameters for T2-weighted MR images were as follows: repetition time of 400–600 msec (varied according to the heart rate) and echo time of 82 msec (400–600/82), echo train length of 33, acquisition time of 9–10 seconds, matrix size of 132 x 256, field of view of 210 x 280 mm, and section thickness of 5 mm.
After a baseline image was acquired, the contrast media was administered. T1-weighted MR contrast material–enhanced images were obtained for 12 hours in five cats and for 6 hours in four cats. A series of MR images were obtained at 10-minute intervals for 60 minutes, at 30-minute intervals for 1–6 hours, and at 60-minute intervals for 6–12 hours. Electrocardiography-triggered multisection T1-weighted spin-echo imaging was performed with the following imaging parameters: 300/25, section thickness of 5 mm, field of view of 210 x 280 mm, and one signal acquired. All images were obtained along the short axis of the heart, and images in the sagittal plane were also acquired occasionally to provide an additional confirmation of the signal enhancement of the irreversibly damaged myocardium.
Postmortem Histochemical Staining
After MR imaging studies were completed, each cat was sacrificed by means of intravenous injection of potassium chloride solution. The heart was excised and cut into five or six 5-mm-thick consecutive slices in the same planes in which the MR images were obtained. Then, specimens were immersed in a 1.5% TTC solution at 36°C and stained for 15 minutes. After staining, the specimens were stored in 10% formalin solution for 12 hours. Irreversibly damaged myocardium was defined as an area not stained with TTC. The specimens were scanned into a computer (Macintosh; Apple Computers, Cupertino, Calif) to measure the size of the infarct area and of the total left ventricle with use of public domain image processing software (IMAGE 135; National Institutes of Health, Bethesda, Md).
Image Analysis
All MR images were analyzed independently by two experienced radiologists (S.I.C., T.H.L.), and the discrepancies were resolved in consensus. The size of the area of abnormal signal intensity was measured on the computer screen, on which observers traced the entire myocardium of the left ventricle, the high-signal-intensity area on T2-weighted images, and the enhanced area on T1-weighted contrast-enhanced images. The size of the area of abnormal signal intensity on MR images and that of the irreversibly damaged myocardium with TTC staining was expressed as a percentage of the size of the total left ventricle on T1- and T2-weighted images. The size of the enhanced area on T1-weighted contrast-enhanced images was compared with that of the irreversibly damaged myocardium with TTC staining and of the high-signal-intensity area on T2-weighted images.
A paired Student t test was used to evaluate the statistical significance of differences (defined as P < .05). Also, correlation between the size of the enhanced area on T1-weighted contrast-enhanced images and that of the irreversibly damaged myocardium with TTC staining was made by means of linear regression analysis. On T1-weighted contrast-enhanced images, signal intensities were measured in regions of interest located in the enhanced and nonenhanced areas. The contrast ratio was calculated as the signal intensity in the enhanced area divided by that in the nonenhanced area. Mean contrast ratio values were obtained for each heart.
Ultrastructural Examinations with Electron Microscopy
Electron microscopic examinations were performed in three cases. Tissue from irreversibly damaged myocardium was sampled from the centers of the TTC-unstained areas that corresponded to the center of the enhanced area on the T1-weighted contrast-enhanced images. For reversibly damaged myocardium, tissue was sampled from the TTC-stained peripheral region adjacent (1–2 mm) to the TCC-unstained area. For normal myocardium, tissue was sampled from the center of the TTC-stained area of the posterior wall.
Tissue was cut into 1-mm cubes and fixed in a 2.5% buffered glutaraldehyde solution for 12–16 hours followed by additional fixation in a solution of osmotic acid at 5°C for 2 hours. The cubes were then dehydrated in graded alcohol at room temperature, passed through propylene oxide, and placed in a 1:1 mixture of propylene oxide and Epon 812 (Polyscience; Niles, Ill) for 12–16 hours before being embedded in the latter. Slices approximately 0.5 µm thick were cut by using a diamond knife (LKB Ultramicrotome; Pharmacia, Uppsala, Sweden). Thin slices were mounted on a copper grid and stained with 4% aqueous uranyl acetate and lead citrate for examination with a transmission electron microscope (JEM-1200 EX II; Jeol, Tokyo, Japan).
The electron microscopic criteria used for distinguishing between irreversibly and reversibly damaged myocardium were the same as those described previously (13). We regarded the ultrastructural findings of both electron-dense deposits in the mitochondrial matrix and disruption of the sarcolemma as indicative of irreversibly damaged myocardium. Ultrastructural findings of reversibly damaged myocardium included mild edematous myocytes, increased sarcoplasmic space, prominent I band, and mild peripheral aggregation of nuclear chromatin without any ultrastructural features of irreversibly damaged myocardium.
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RESULTS |
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DISCUSSION |
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In this study, MR imaging enhanced with bis-gadolinium mesoporphyrins helped distinguish irreversibly damaged from reversibly damaged myocardium in the reperfused myocardium in a cat model. The question addressed by the present study was whether contrast-enhanced MR imaging can be used to accurately map irreversibly damaged myocardium in the reperfused myocardum. The principal findings in this study were (a) accurate correlation between the size of the enhanced area on the contrast-enhanced T1-weighted images and that of the irreversibly damaged myocardium with TTC staining and (b) strong, persistent signal enhancement of the irreversibly damaged myocardium.
Myocardial infarction could not be visually distinguished from normal myocardium with T1-weighted imaging. Even optimal T2-weighted imaging depicts both reversibly and irreversibly damaged myocardium with increased signal intensity (14,15). Gadopentetate dimeglumine aids the diagnosis of myocardial injury but overestimates the extent of irreversibly damaged myocardium by approximately 12% (16) and fails to differentiate irreversibly and reversibly damaged myocardium (17,18).
Previous study of MR imaging enhanced with bis-gadolinium mesoporphyrins demonstrated that (a) the size of the enhanced area on MR images matched well with that of the TTC-unstained area at postmortem examination, and (b) contrast was significantly increased between irreversibly and reversibly damaged myocardium in the occlusive myocardium in a rat model (12). Our study demonstrated that MR imaging enhanced with bis-gadolinium mesoporphyrins precisely delineated irreversibly damaged myocardium with a strong, persistent signal enhancement and that maximum enhancement of the irreversibly damaged myocardium occurred from 1 to 3 hours after administration of the contrast material, with mean enhancement of 171% of normal myocardium in the reperfused myocardium.
From 40 minutes to 12 hours after administration of bis-gadolinium mesoporphyrins, the size of the enhanced area on T1-weighted images correlated precisely with that of the irreversibly damaged myocardium with TTC staining. However, from the immediate period to 30 minutes, the enhanced area on the T1-weighted images was larger than the irreversibly damaged myocardium with TTC staining. Overestimation of infarct size from the immediate period to 30 minutes after administration of the contrast material seems to reflect distribution of the contrast material in the interstitial spaces of the reversibly and irreversibly damaged myocardium. As the washout of contrast agent from the reversibly damaged myocardium occurred, the size of the enhanced area on the T1-weighted images gradually decreased and finally correlated with that of the irreversibly damaged myocardium with TTC staining.
Maximum enhancement was seen from 1 to 3 hours after administration of bis-gadolinium mesoporphyrins, with mean enhancement of 171% of normal myocardium. The mechanism of signal enhancement by the contrast material in irreversibly damaged myocardium is still not well understood, but it can be assumed to result from some kind of binding of the compound to the sites of denatured tissue components by means of reperfused coronary flow and progressive extravascular diffusion. The porphyrin derivative obviously accumulates in necrotic tissue and tumor necrosis (11). We speculate that the entrapment may be a complex interplay of various factors such as protein binding and well-balanced excretion. Specific binding to cell debris is possible but still hypothetical. Hofmann et al (19) demonstrated that no specific binding to DNA or lipid moieties occurs. Further studies to elucidate the mechanism of accumulation are needed.
One drawback of MR imaging enhanced with bis-gadolinium mesoporphyrins is that a long waiting time is required to precisely delineate irreversibly damaged myocardium and to acquire the maximum enhancement of signal (at least 40 minutes after administration of the contrast material).
The results of electron microscopic examination indicate that irreversibly damaged myocardium was distinguished from reversibly damaged myocardium. Our results showed that the main findings of ultrastructural change in reversibly damaged myocardium were only mild edematous changes, in contrast with severe myocardial damage seen in irreversibly damaged myocardium. The ultrastructures of reversibly damaged myocardium were similar to ultrastructural changes observed previously (13). The ultrastructures of reversibly damaged myocardium show mild edematous myocytes, increased sarcoplasmic space, prominent I band, and mild peripheral aggregation of nuclear chromatin.
The ultrastructures of irreversibly damaged myocardium exhibit two characteristic features in addition to all the changes seen in reversibly damaged myocardium. First, all of the mitochondria are swollen with disorganized cristae and contain small, eosinophilic amorphous densities. The second feature is disruption of the plasmalemma of the sarcolemma. We sampled tissue of the reversibly damaged myocardium from the TTC-stained peripheral region adjacent (1–2 mm) to the TTC-unstained area. However, there was no ultrastructural finding of irreversibly damaged myocardium in the TTC-stained peripheral region. Therefore from our findings, it can be assumed that the ultrastructural changes in the TTC-stained peripheral region reflected reversible myocardial damage.
In conclusion, MR imaging enhanced with bis-gadolinium mesoporphyrins precisely delineates irreversibly damaged myocardium with a strong and persistent signal enhancement in the reperfused myocardium in a cat model.Practical application: MR imaging enhanced with bis-gadolinium mesoporphyrins will play an important role in determining myocardial viability by clearly documenting irreversibly damaged myocardium in patients undergoing reperfusion therapy.
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Acknowledgments |
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Footnotes |
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Author contributions: Guarantor of integrity of entire study, T.H.L.; study concepts, T.H.L.; study design, T.H.L., S.I.C.; definition of intellectual content, T.H.L.; literature research, S.I.C., S.H.C.; experimental studies, S.I.C., S.H.C., S.T.K., C.H.L., K.H.L.; data acquisition, S.I.C., S.H.C.; data analysis, S.I.C., G.Y.G., T.H.L.; statistical analysis, S.I.C., S.H.C.; manuscript preparation, S.I.C.; manuscript editing, H.Y.K., G.Y.G., H.J.W.; manuscript review, T.H.L.
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