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Toward necrotic cell fraction measurement by contrast-enhanced MRI of reperfused ischemically injured myocardium
Toward Necrotic Cell Fraction Measurement by Contrast-enhanced MRI of Reperfused Ischemically Injured Myocardium Michael F. Wendland, PhD, Maythem Saeed, DVM, PhD H6kan Arheden, MD, PhD, D-W Gao, MD, Emmanuelle Canet, DVM, PhD Jens Bremerich, MD, Michael W. Dae, MD, Charles B. Higgins, MD
Previously, low-molecular-weight extracellular MRI contrast media such as GdDTPA (1) or GdDTPA-BMA (2) were found to nearly equilibrate between blood and myocardium distribution volumes in rats subjected to 60minute coronary artery occlusion followed by 60-minute reflow (reperfused myocardial infarction). The word "nearly" is important in this statement because biological systems such this one, in which plasma-borne indicators flow into and out of the system, never achieve true equilibrium. However, near equilibration may exist if the transport between blood and tissue of the indicator material is very fast compared to terminal clearance from the blood. The evidence supporting equilibration consisted of (a) observed constant proportionality between AR1 (change in T1 relaxation rate which is proportional to the quantity of Gd) of myocardial water and that of blood during the first 30 minutes after intravenous administration (1,2) and (b) failure of increased doses of contrast agent to alter that proportionality (1). Disequilibration would cause this proportionality to increase over time and to increase with administered dose. At equilibration, the relative content of contrast agent in myocardium and blood is determined by the relative fractional distribution volumes in the respective tissues.
Acad Radiol 1998; 5(suppl 1):Sd2-$44
1From the Department of Radiology, Box 0628, University of California, 505 Parnassus Ave, Rm HSW-207, San Francisco, CA 94143. Supported by NIH grant HL52569-01. H,A. supported by the Swedish Heart Lung Foundation (55501), the Swedish Medical Association (4,0), the Hellmuth Herz Foundation, and the Swedish Royal Physiologic Society. J.B. supported by the ADUMED Foundation, E.C, supported by grants from the French Foreign Office and Philippe Foundation, Address reprint requests to M.F,W, ©AUR, 1998
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The fractional distribution volume in blood is easily determined (1.0 - hematocrit); that in the heart can be estimated from the product of the myocardium-to-blood proportionality (A R1 ratio) with the fractional blood distribution. In animals subjected to 60-minute occlusion followed by 60-minute reperfusion, the fractional distribution volume for infarcted myocardium was close to 1.0 (1,2) consistent with failure of virtually all myocardial cells in the injured region to exclude the contrast agent. The current study was conducted to (a) test the hypothesis that measured AR1 of myocardium and blood after MRI contrast agent can noninvasively quantify gradations of ischemic damage in postischemic myocardium; and (b) confirm the MRI findings using a similar but radioactive tracer, Tc-99m DTPA.
MRI Studies Female Sprague Dawley rats (250-320 g) were anesthetized (sodium pentobarbital, 50 mg/kg, intraperitoneal administration), and a tracheotomy was performed for mechanical ventilation (rodent ventilator; Harvard Instruments, South Natick, Mass). The chest was opened at the fourth intercostal space to expose the heart. Reperfused myocardial infarction of varied severity was prepared by occlusion of the anterior branch of the left main coronary artery for 20 (n = 12), 30 (n = 6), 40 (n = 8), and 60 (n = 8) minutes followed by reperfusion for 60 minutes. Rats were placed in a "birdcage" radio-frequency coil, connected to an ECG monitor (Accusync 6L; Advanced Medical Research, Milford, Conn) and placed at the center of a 2.0-T magnet for MRI (Bruker Omega CSI system; Bruker Instruments, Fremont, Calif). ECG-gated inversion-recovery echo-planar images (TPUTE -- 6,000/10 msec, FOV = 50 mm, matrix = 64 x 64, transaxial view)
were obtained at the mid-left ventricle. T1 values of myocardium and blood in the left ventricle cavity were repeatedly measured by increasing the time to inversion (TI) from 20 to 1,300 msec to define the inversion-recovery null point as described previously (1,2). Each set of images required about 2.5 minutes. Image sets were obtained before administering GdDTPA-BMA and at 5-minute intervals afterward. T1 was calculated as TInJln2. AR1 (1/ Tlpost - 1/Tlpre) was determined for LV blood and normal and injured myocardium at six time points during the initial 30 minutes after contrast injection. After imaging was complete, each animal was sacrificed by lethal injection of saturated KC1 solution, and the heart was excised, sectioned, and soaked in triphenyl tetrazolinium chloride to define the infarction zone.
Radiotracer Studies Six groups of rats (n = 8 per group) were given 0.5 mCi of Tc-99m DTPA after being subjected to various periods of coronary occlusion and 60-minute reperfusion. Three groups had 60-minute occlusion and were sacrificed at 5, 15, and 30 minutes after receiving the radiotracer. The remaining groups had occlusion durations of 20, 30, and 40 minutes and were sacrificed at 15 minutes after receiving the tracer. Immediately before sacrifice, an aliquot of arterial blood was withdrawn for radioactivity and hematocrit measurements. The coronary was reoccluded and phthalocyanine blue dye was administered to define the risk area. Then rats were sacrificed by lethal injection of KC1 solution, and the heart was excised for radioactivity measurements. Each heart was rinsed carefully and the left ventricle cavity was cleaned with a cotton swab. The right ventricle was removed and the left ventricle was sectioned at the midline. The basal half was embedded (O.C.T. embedding compound, Tissue Tek; Sakura Finetek USA, Torrance, Calif), immediately frozen in dry ice, and sectioned at 20gm thickness using a microtome (Cryocut 1899; Cambridge Instrument GmbH, Nussloch, Germany). Eight to 10 such sections were placed on a photostimulable storage-phosphor imaging plate (Molecular Dynamics, Sunnyvale, Calif) for 1-2 hours. Plates were scanned and digitized (PhosphorImager 445 SI; Molecular Dynamics), and the data were evaluated using company-supplied software. The apical half was dissected to provide samples of deep blue stained normal myocardium and unstained ischemic myocardium. Tissue samples of myocardium and blood were weighed, and radioactivity was measured in a gamma well counter (Searle Analytic, Iowa).
MRI Results AR1 values for the regions of interest, LV blood, normal myocardium, and infarcted myocardium decreased in parallel fashion during the 30 minutes of observation as the agent was cleared from the circulation. Ratios of AR1 myocardium/blood were constant over time after contrast administration for both injured and normal myocardium, consistent with equilibration of contrast agent between blood and myocardium. The ratio values for the injured myocardium increased with occlusion duration, from 0.75 + 0.07 for 20-minute occlusion to 1.36 + 0.05 for 30 minute, 1.57 + 0.10 for 40 minute, and 1.70 _+0.12 for 60 minute. Normal myocardium was 0.35 + 0.01 for all animals without significant difference between treatment groups. The 20-minute occlusion group was particularly interesting because an abnormal region was identified on MRI but in most cases not on triphenyltetrazolium chloride (TTC) stains. On MRI the injured region typically exhibited a AR1 ratio of 0.75 + 0.07, twofold greater than that of normal myocardium while the TTC stain was usually negative for infarction. In some cases (four of 12), a small subregion was observed having a AR1 ratio of 1.20 + 0.06 and TTC stain exhibited a small region with reduced staining intensity. The implication of this observation is that a small region of irreversible injury has formed in these four cases within a larger edematous risk area composed of reversibly injured myocardium. If so, the imaging procedure has demarcated reversibly injured myocardium, a capability which to our knowledge has not previously been reported.
Radiotracer Results In hearts subjected to 60-minute coronary occlusion/ reperfusion, the injured region on autoradiographs was characterized as a large region with high intensity surrounded by a periphery of less intense radioactivity, putatively an edematous fringe. Dissected specimens of injured myocardium exhibited lower radioactivity (normalized to normal myocardium) than noted on autoradiographs because dissected specimens contained both infarcted and reversibly injured myocardium. Therefore the ratio of radioactivity in normal myocardium to blood was obtained from gamma counts of respective specimens while the analogous ratio for infarcted myocardium was obtained from autoradiographic derived radioactivity ratios of infarcted to normal myocardium.
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There was no difference in radioactivity per gram of injured and normal myocardium, normalized to radioactivity of blood, between those given 0.5 mCi Tc-99m DTPA at 5, 15, and 30 minutes prior to sacrifice. The relative content of tracer was unaffected by clearance of the tracer from the circulation. This confirmed our earlier finding that AR1 ratio, myocardium/blood, of infarcted or normal myocardium is constant over time following extracellular MR contrast agents such as GdDTPA or GdDTPA-BMA. In animals subjected to 20-, 30-, 40-, and 60-minute occlusion followed by reperfusion and given Tc-99m DTPA, the ratio of radioactivity in postischemic myocardium to that in blood increased with occlusion duration (0.59 + 0.07, 1.09 + 0.12, 1.64 + 0.19, and 1.70 + 0.11, respectively) and was similar to analogous AR1 ratio values. The radioactivity ratio for normal myocardium in these animals (0.30 + 0.01) was similar to the AR1 ratio observed on MRI. The calculated values for fractional distribution volume of GdDTPA-BMA and Tc-99m DTPA in normal and postischemic myocardium were therefore quite similar (Figure).
1.o
D
GdDTPA-BMA, Normal ~
GdDTPA-BMA, Ischemic
•
99mTcDTPA - Normal
99mTcDTPA, Ischemic
•
.............................................................................................................................. T....................................... ; ~
fDV 0.6
..................
0.4
...................
0,0 0°2
......(z i "".=-:i........
20
30 40 Occlusion duration (min)
,
60
C h a n g e in fractional distribution v o l u m e (fDVOof GdDTPABMA a n d Tc-99m DTPA with occlusion duration in postisc h e m i c rat hearts, fDV was c a l c u l a t e d as t h e p r o d u c t of measured m y o c a r d i u m - t o - b l o o d ratios of the indicator materials a n d measured plasma v o l u m e in b l o o d (1 - Hct; Hct was 0.45 + 0.01, n = 36). This figure demonstrates d e t e c t e d progressive severity of m y o c a r d i a l injury from mild d a m a g e (mostly reversible injury) t o c o m p l e t e infarction of t h e a r e a a t risk in the rat.
time after reperfusion (reperfusion injury), and evaluating the beneficial effects of therapeutic agents designed to preserve the myocardium.
CONCLUSION !EFERENCE,c
These results strongly suggest that gradations in severity of ischemic injury can be quantified in vivo by contrast-enhanced MRI during the postischemic. This method may be useful in distinguishing between reversible and irreversible myocardial injury, monitoring potential change in the status of myocardial damage which may evolve over
$44
1. Wendland MF, Saeed M, Lauerma K, et al. Alterations in T1 of normal and reperfused infarcted myocardium after GdBOPTA versus GdDTPA on inversion recovery EPI. Magn Reson Med 1997; 37:448456, 2. Wendland MF, Saeed M, Geschwind JF, Mann JS, Brasch RC, Higgins CB, Comparison of intravascular, extracellular and intracellular MR contrast media in hearts prepared with reperfused myocardial infarction. A e a d Radio11996; 3:$402-$404.
Previously, low-molecular-weight extracellular MRI contrast media such as GdDTPA (1) or GdDTPA-BMA (2) were found to nearly equilibrate between blood and myocardium distribution volumes in rats subjected to 60minute coronary artery occlusion followed by 60-minute reflow (reperfused myocardial infarction). The word "nearly" is important in this statement because biological systems such this one, in which plasma-borne indicators flow into and out of the system, never achieve true equilibrium. However, near equilibration may exist if the transport between blood and tissue of the indicator material is very fast compared to terminal clearance from the blood. The evidence supporting equilibration consisted of (a) observed constant proportionality between AR1 (change in T1 relaxation rate which is proportional to the quantity of Gd) of myocardial water and that of blood during the first 30 minutes after intravenous administration (1,2) and (b) failure of increased doses of contrast agent to alter that proportionality (1). Disequilibration would cause this proportionality to increase over time and to increase with administered dose. At equilibration, the relative content of contrast agent in myocardium and blood is determined by the relative fractional distribution volumes in the respective tissues.
Acad Radiol 1998; 5(suppl 1):Sd2-$44
1From the Department of Radiology, Box 0628, University of California, 505 Parnassus Ave, Rm HSW-207, San Francisco, CA 94143. Supported by NIH grant HL52569-01. H,A. supported by the Swedish Heart Lung Foundation (55501), the Swedish Medical Association (4,0), the Hellmuth Herz Foundation, and the Swedish Royal Physiologic Society. J.B. supported by the ADUMED Foundation, E.C, supported by grants from the French Foreign Office and Philippe Foundation, Address reprint requests to M.F,W, ©AUR, 1998
$42
The fractional distribution volume in blood is easily determined (1.0 - hematocrit); that in the heart can be estimated from the product of the myocardium-to-blood proportionality (A R1 ratio) with the fractional blood distribution. In animals subjected to 60-minute occlusion followed by 60-minute reperfusion, the fractional distribution volume for infarcted myocardium was close to 1.0 (1,2) consistent with failure of virtually all myocardial cells in the injured region to exclude the contrast agent. The current study was conducted to (a) test the hypothesis that measured AR1 of myocardium and blood after MRI contrast agent can noninvasively quantify gradations of ischemic damage in postischemic myocardium; and (b) confirm the MRI findings using a similar but radioactive tracer, Tc-99m DTPA.
MRI Studies Female Sprague Dawley rats (250-320 g) were anesthetized (sodium pentobarbital, 50 mg/kg, intraperitoneal administration), and a tracheotomy was performed for mechanical ventilation (rodent ventilator; Harvard Instruments, South Natick, Mass). The chest was opened at the fourth intercostal space to expose the heart. Reperfused myocardial infarction of varied severity was prepared by occlusion of the anterior branch of the left main coronary artery for 20 (n = 12), 30 (n = 6), 40 (n = 8), and 60 (n = 8) minutes followed by reperfusion for 60 minutes. Rats were placed in a "birdcage" radio-frequency coil, connected to an ECG monitor (Accusync 6L; Advanced Medical Research, Milford, Conn) and placed at the center of a 2.0-T magnet for MRI (Bruker Omega CSI system; Bruker Instruments, Fremont, Calif). ECG-gated inversion-recovery echo-planar images (TPUTE -- 6,000/10 msec, FOV = 50 mm, matrix = 64 x 64, transaxial view)
were obtained at the mid-left ventricle. T1 values of myocardium and blood in the left ventricle cavity were repeatedly measured by increasing the time to inversion (TI) from 20 to 1,300 msec to define the inversion-recovery null point as described previously (1,2). Each set of images required about 2.5 minutes. Image sets were obtained before administering GdDTPA-BMA and at 5-minute intervals afterward. T1 was calculated as TInJln2. AR1 (1/ Tlpost - 1/Tlpre) was determined for LV blood and normal and injured myocardium at six time points during the initial 30 minutes after contrast injection. After imaging was complete, each animal was sacrificed by lethal injection of saturated KC1 solution, and the heart was excised, sectioned, and soaked in triphenyl tetrazolinium chloride to define the infarction zone.
Radiotracer Studies Six groups of rats (n = 8 per group) were given 0.5 mCi of Tc-99m DTPA after being subjected to various periods of coronary occlusion and 60-minute reperfusion. Three groups had 60-minute occlusion and were sacrificed at 5, 15, and 30 minutes after receiving the radiotracer. The remaining groups had occlusion durations of 20, 30, and 40 minutes and were sacrificed at 15 minutes after receiving the tracer. Immediately before sacrifice, an aliquot of arterial blood was withdrawn for radioactivity and hematocrit measurements. The coronary was reoccluded and phthalocyanine blue dye was administered to define the risk area. Then rats were sacrificed by lethal injection of KC1 solution, and the heart was excised for radioactivity measurements. Each heart was rinsed carefully and the left ventricle cavity was cleaned with a cotton swab. The right ventricle was removed and the left ventricle was sectioned at the midline. The basal half was embedded (O.C.T. embedding compound, Tissue Tek; Sakura Finetek USA, Torrance, Calif), immediately frozen in dry ice, and sectioned at 20gm thickness using a microtome (Cryocut 1899; Cambridge Instrument GmbH, Nussloch, Germany). Eight to 10 such sections were placed on a photostimulable storage-phosphor imaging plate (Molecular Dynamics, Sunnyvale, Calif) for 1-2 hours. Plates were scanned and digitized (PhosphorImager 445 SI; Molecular Dynamics), and the data were evaluated using company-supplied software. The apical half was dissected to provide samples of deep blue stained normal myocardium and unstained ischemic myocardium. Tissue samples of myocardium and blood were weighed, and radioactivity was measured in a gamma well counter (Searle Analytic, Iowa).
MRI Results AR1 values for the regions of interest, LV blood, normal myocardium, and infarcted myocardium decreased in parallel fashion during the 30 minutes of observation as the agent was cleared from the circulation. Ratios of AR1 myocardium/blood were constant over time after contrast administration for both injured and normal myocardium, consistent with equilibration of contrast agent between blood and myocardium. The ratio values for the injured myocardium increased with occlusion duration, from 0.75 + 0.07 for 20-minute occlusion to 1.36 + 0.05 for 30 minute, 1.57 + 0.10 for 40 minute, and 1.70 _+0.12 for 60 minute. Normal myocardium was 0.35 + 0.01 for all animals without significant difference between treatment groups. The 20-minute occlusion group was particularly interesting because an abnormal region was identified on MRI but in most cases not on triphenyltetrazolium chloride (TTC) stains. On MRI the injured region typically exhibited a AR1 ratio of 0.75 + 0.07, twofold greater than that of normal myocardium while the TTC stain was usually negative for infarction. In some cases (four of 12), a small subregion was observed having a AR1 ratio of 1.20 + 0.06 and TTC stain exhibited a small region with reduced staining intensity. The implication of this observation is that a small region of irreversible injury has formed in these four cases within a larger edematous risk area composed of reversibly injured myocardium. If so, the imaging procedure has demarcated reversibly injured myocardium, a capability which to our knowledge has not previously been reported.
Radiotracer Results In hearts subjected to 60-minute coronary occlusion/ reperfusion, the injured region on autoradiographs was characterized as a large region with high intensity surrounded by a periphery of less intense radioactivity, putatively an edematous fringe. Dissected specimens of injured myocardium exhibited lower radioactivity (normalized to normal myocardium) than noted on autoradiographs because dissected specimens contained both infarcted and reversibly injured myocardium. Therefore the ratio of radioactivity in normal myocardium to blood was obtained from gamma counts of respective specimens while the analogous ratio for infarcted myocardium was obtained from autoradiographic derived radioactivity ratios of infarcted to normal myocardium.
$43
There was no difference in radioactivity per gram of injured and normal myocardium, normalized to radioactivity of blood, between those given 0.5 mCi Tc-99m DTPA at 5, 15, and 30 minutes prior to sacrifice. The relative content of tracer was unaffected by clearance of the tracer from the circulation. This confirmed our earlier finding that AR1 ratio, myocardium/blood, of infarcted or normal myocardium is constant over time following extracellular MR contrast agents such as GdDTPA or GdDTPA-BMA. In animals subjected to 20-, 30-, 40-, and 60-minute occlusion followed by reperfusion and given Tc-99m DTPA, the ratio of radioactivity in postischemic myocardium to that in blood increased with occlusion duration (0.59 + 0.07, 1.09 + 0.12, 1.64 + 0.19, and 1.70 + 0.11, respectively) and was similar to analogous AR1 ratio values. The radioactivity ratio for normal myocardium in these animals (0.30 + 0.01) was similar to the AR1 ratio observed on MRI. The calculated values for fractional distribution volume of GdDTPA-BMA and Tc-99m DTPA in normal and postischemic myocardium were therefore quite similar (Figure).
1.o
D
GdDTPA-BMA, Normal ~
GdDTPA-BMA, Ischemic
•
99mTcDTPA - Normal
99mTcDTPA, Ischemic
•
.............................................................................................................................. T....................................... ; ~
fDV 0.6
..................
0.4
...................
0,0 0°2
......(z i "".=-:i........
20
30 40 Occlusion duration (min)
,
60
C h a n g e in fractional distribution v o l u m e (fDVOof GdDTPABMA a n d Tc-99m DTPA with occlusion duration in postisc h e m i c rat hearts, fDV was c a l c u l a t e d as t h e p r o d u c t of measured m y o c a r d i u m - t o - b l o o d ratios of the indicator materials a n d measured plasma v o l u m e in b l o o d (1 - Hct; Hct was 0.45 + 0.01, n = 36). This figure demonstrates d e t e c t e d progressive severity of m y o c a r d i a l injury from mild d a m a g e (mostly reversible injury) t o c o m p l e t e infarction of t h e a r e a a t risk in the rat.
time after reperfusion (reperfusion injury), and evaluating the beneficial effects of therapeutic agents designed to preserve the myocardium.
CONCLUSION !EFERENCE,c
These results strongly suggest that gradations in severity of ischemic injury can be quantified in vivo by contrast-enhanced MRI during the postischemic. This method may be useful in distinguishing between reversible and irreversible myocardial injury, monitoring potential change in the status of myocardial damage which may evolve over
$44
1. Wendland MF, Saeed M, Lauerma K, et al. Alterations in T1 of normal and reperfused infarcted myocardium after GdBOPTA versus GdDTPA on inversion recovery EPI. Magn Reson Med 1997; 37:448456, 2. Wendland MF, Saeed M, Geschwind JF, Mann JS, Brasch RC, Higgins CB, Comparison of intravascular, extracellular and intracellular MR contrast media in hearts prepared with reperfused myocardial infarction. A e a d Radio11996; 3:$402-$404.