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(Radiology. 1999;211:698-708.)
© RSNA, 1999

Experimental Studies

Measurement of the Distribution Volume of Gadopentetate Dimeglumine at Echo-planar MR Imaging to Quantify Myocardial Infarction: Comparison with 99mTc-DTPA Autoradiography in Rats1

Håkan Arheden, MD, PhD, Maythem Saeed, DVM, PhD, Charles B. Higgins, MD, Dong-Wei Gao, MD, Jens Bremerich, MD, Rolf Wyttenbach, MD, Michael W. Dae, MD and Michael F. Wendland, PhD

1 From the Department of Radiology, Box 0628, University of California San Francisco Medical Center, 505 Parnassus Ave, Rm L308, San Francisco, CA 94143-0628. From the 1997 RSNA scientific assembly. Received Mar 26, 1998; revision requested May 5; revision received Aug 10; accepted Nov 5. Supported in part by National Institutes of Health grant no. R01 HL52569. H.A. supported by the Swedish Heart Lung Foundation (55501), the Swedish Medical Association (4.0), Hellmuth Herz Foundation, and the Swedish Royal Physiographic Society; J.B. supported by the ADUMED-foundation; and R.W. supported by the Swiss National Science Foundation as research fellows. Address reprint requests to C.B.H.


   Abstract
TOP
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
PURPOSE: To measure the fractional distribution volume of gadopentetate dimeglumine in normal and reperfused infarcted myocardium at magnetic resonance (MR) imaging by using the fractional distribution volume of technetium 99m–diethylenetriaminepentaacetic acid (DTPA) as an independent reference.

MATERIALS AND METHODS: Rats were subjected to 1 hour of coronary artery occlusion and 1 hour of reperfusion before inversion-recovery echo-planar imaging or autoradiography. Regional change in relaxation rate ({Delta}R1) ratios for myocardium over blood were compared with radioactivity ratios for myocardium over blood after the injection of 99mTc-DTPA.

RESULTS: Both {Delta}R1 and radioactivity ratios demonstrated equilibrium distribution and hence represent partition coefficients ({lambda}). The fractional distribution volumes were greater in infarcted myocardium (0.90 ± 0.05 for gadopentetate dimeglumine and 0.89 ± 0.04 for 99mTc-DTPA) than in normal myocardium (0.23 ± 0.02 for gadopentetate dimeglumine and 0.16 ± 0.01 for 99mTc-DTPA). Area at risk at autoradiography was not significantly different from that at histomorphometry. The infarction size defined by using triphenyltetrazolium chloride was 13% ± 4 smaller than that defined by using autoradiography.

CONCLUSION: The fractional distribution volumes of gadopentetate dimeglumine and 99mTc-DTPA are similar and indicate extracellular distribution in normal myocardium and intracellular as well as extracellular distribution in reperfused infarction. Because the failure of cells to exclude these agents is indicative of necrosis, contrast medium–enhanced MR imaging may be useful to quantify myocardial infarction.

Index terms: Heart, experimental studies, 511.12143, 511.12172 • Magnetic resonance (MR), contrast enhancement, 511.121412, 511.121413, 511.12143 • Magnetic resonance (MR), echo planar, 511.121416 • Myocardium, infarction, 511.771 • Myocardium, ischemia, 511.1949 • Radionuclide imaging, experimental studies, 511.12143, 511.12172


   Introduction
TOP
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Size, transmurality, and location are known measures of the extent of myocardial infarction. However, myocardial injury is not an all-or-none response. In acute myocardial ischemia, early successful reperfusion may save a substantial part of the myocardium at risk (1,2). Within the area at risk, the injury may be graded so that some cells become reversibly injured (stunned) while others perish. A noninvasive method that could be used to map the ischemically injured region, differentiate between reversible and irreversible injury, and quantitate the degree of myocardial injury in a postischemic region could be of use to guide therapy.

The concept of assessing myocardial injury by using contrast medium–enhanced magnetic resonance (MR) imaging has recently been introduced (3–7). The method is based on the distribution of low-molecular-weight MR contrast medium throughout the extracellular space but exclusion from the intracellular space in normal myocardium, while in reperfused infarcted myocardium, cells fail to exclude the contrast medium (Fig 1). Several studies have measured gadolinium content ex vivo in normal and infarcted myocardium, either directly (8,9) or indirectly (3,5,10).



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  		Figure 1. Schematic drawing of the experimental hypothesis. Extracellular tracers, like gadopentetate dimeglumine and 99mTc-DTPA (the gray areas), distribute rapidly in the extracellular space in normal myocardium, which constitutes about 20% of the myocardial tissue volume—that is, its fractional distribution volume is about 0.2. In reperfused infarcted myocardium, where myocardial cells have lost membrane integrity, the extracellular tracer passively distributes also into the intracellular space of irreversibly injured cells, and the fractional distribution volume approaches 1, or plasma levels. RBC = red blood cell.

 
Pereira et al (3) demonstrated that indirect measurements of the tissue content of gadopentetate dimeglumine in excised myocardium was inversely related to myocardial viability as early as 2 hours and up to 3 weeks after reperfusion by using thallium 201 as a marker of viability. Donahue et al (11) demonstrated that tissue bulk content of gadopentetate dimeglumine can be measured with MR imaging by using a standard inversion-recovery sequence.

In a recent study from this laboratory (12), inversion-recovery echo-planar MR imaging was used to monitor the change in relaxation rate ({Delta}R1) in myocardium and left ventricular blood after the administration of gadopentetate dimeglumine. Apparent equilibrium-phase distribution between blood and myocardium was reached after 5 minutes, and thus compensation for clearance processes could be achieved by relating {Delta}R1 for a tissue to that for blood. With the assumption that the distribution volume of gadopentetate dimeglumine in blood is 1 - hematocrit, it was suggested that the necrotic cell fraction within the reperfused territory could be quantified.

However, quantifying tissue content of gadopentetate dimeglumine in vivo by using inversion-recovery echo-planar imaging is indirect because MR imaging allows measurement of the effect on water of gadopentetate dimeglumine rather than the gadopentetate dimeglumine itself. Diethylenetriaminepentaacetic acid (DTPA) labeled with radionuclides has gained wide acceptance as an extracellular tracer; however, to our knowledge, there are no published data on technetium 99m DTPA being used to quantify the extracellular space in myocardium or the degree of myocardial injury. Therefore, the purposes of the present study were to (a) measure and compare the relative tissue content of gadopentetate dimeglumine in normal and reperfused infarcted myocardium by means of inversion-recovery echo-planar imaging with the more direct measurements of 99mTc-DTPA as an independent reference and (b) calculate fractional distribution volumes of these tracers in normal and reperfused infarcted myocardium in a model of reperfused infarction known to produce substantial necrosis (1), with the ultimate purpose of quantifying myocardial infarction after reperfusion.


   MATERIALS AND METHODS
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Extracellular Tracers
DTPA bound to either gadolinium or 99mTc was used as an extracellular tracer. Gadopentetate dimeglumine (Magnevist; Schering, Berlin, Germany) was obtained as the clinical formulation 0.5 mol/L. The 99mTc-DTPA was prepared in our laboratory. The amount of free to bound 99mTc determined immediately after reconstitution was always less than 1% as measured by means of instant thin-layer chromatography with silicon gel (Gelman Sciences, Ann Arbor, Mich).

Animal Preparation
All experimental procedures were performed in accordance with the National Institutes of Health guidelines for humane handling of animals and received prior approval from the Committee of Animal Research at this institution.

Fifty-one female Sprague-Dawley rats (190–340 g; Simonsen Labs, Gilroy, Calif) were anesthetized with 50 mg of sodium pentobarbital (Nembutal Sodium Solution; Abbott Laboratories, Chicago, Ill) per kilogram of body weight and received mechanical ventilation after tracheotomy. In 36, the chest was opened at the left fourth intercostal space, and a snare ligature was placed around the anterior branch of the left main coronary artery. The artery was occluded for 1 hour followed by 1 hour of reperfusion known to produce necrosis of a substantial part of the area at risk (1). A catheter was placed in the left jugular vein to deliver gadopentetate dimeglumine, 99mTc-DTPA, or phthalocyanine blue. Another catheter was placed in a carotid artery to monitor blood pressure and heart rate and to draw blood samples for radioactivity and hematocrit measurements.

Arterial pressure (systolic, diastolic, and mean) and heart rate were monitored in all animals before administration of the tracers. Animals with a mean arterial blood pressure less than 60 mm Hg were excluded from the study because autoregulation of myocardial blood flow is lost under this level (13,14). The effect of injection of 99mTc-DTPA on blood pressure and heart rate was measured in a subset of five animals.

MR Imaging Experiments
The animals subjected to occlusion and reperfusion (n = 6) and the control animals (n = 6) were placed supine in a birdcage resonator with a 5.6-cm diameter. Copper leads were inserted into a forelimb and the lower part of the abdomen to provide electrocardiographic gating. Electrocardiographically gated images were acquired by using a 2.0-T imaging system (Omega CSI; Bruker Instruments, Fremont, Calif). An echo-planar imaging sequence preceded by a nonselective composite (90x - 180y - 90x) inversion pulse, calibrated for each animal, with subsequent gradient spoiling and a variable delay (inversion time) for relaxation evolution, was used to estimate T1. The parameters were a repetition time of at least 7,000 msec and an echo time of 10 msec, an inversion time of 20–1,000 msec, a matrix of 64 x 64 data points acquired in 32.7 msec, a section thickness of 2 mm, and a field of view of 50 x 50 mm.

To measure regional T1, a set of 10–15 images (Fig 2) was obtained within a 2-minute interval in which the inversion time interval varied typically between 20 and 1,000 msec to define the inversion-recovery null point (TInull) for each region of interest (left ventricular chamber blood and normal and reperfused injured myocardium). We injected 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight intravenously 60 minutes after reperfusion. Serial measurements of T1, calculated from the relationship T1 = TInull/ln 2, were obtained immediately before and at 4, 14, and 29 minutes after the injection of contrast medium to determine the effect of time on the regional distribution of gadopentetate dimeglumine. The {Delta}R1 after the administration of gadopentetate dimeglumine was calculated as 1/T1postcontrast - 1/T1precontrast.



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  		Figure 2. Inversion-recovery echo-planar MR images (repetition time, 7,000 msec; echo time, 10 msec) obtained after the administration of 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight of a rat heart subjected to 1 hour of occlusion of the anterior branch of the left main coronary artery followed by 1 hour of reperfusion. The images in this set are chosen to demonstrate that different regions of interest pass the null point of longitudinal magnetization recovery at different inversion time settings. At an inversion time (TI) of 0.1 second, before any substantial recovery has occurred, all tissues have negative signal and are bright. At an inversion time of 0.2 second, the lateral wall of the left ventricle (arrowheads), subjected to ischemia and reperfusion, passes through the null point first (nil signal), because it has the highest gadopentetate dimeglumine content. At an inversion time of 0.3 second, the blood, containing less gadolinium than the reperfused infarcted myocardium but more than the normal myocardium, passes through the null point. At an inversion time of 0.4 second, the normal myocardium, containing less gadolinium than both the blood and the reperfused infarcted myocardium (arrowheads), appears dark as it passes the null point.

 
After the conclusion of the imaging, the heart was excised in each case and stained histochemically with triphenyltetrazolium chloride (TTC) to confirm the presence, extent, and location of infarct. A direct quantitative comparison of sizes could not be performed owing to the limited spatial resolution of echo-planar imaging.

Radioisotope Experiments
After the occlusion and reperfusion protocol and immediately prior to the intravenous injection of approximately 0.5 mCi (1.85 x 107 Bq) of 99mTc-DTPA, the individual preparation was diluted (1:1 volume) with 0.9% sodium chloride solution (Baxter, Deerfield, Ill). To investigate the effect of time on the tissue distribution of 99mTc-DTPA in blood and myocardium, three groups of animals subjected to coronary occlusion and reperfusion were sacrificed at 5 (n = 8), 15 (n = 8), and 30 (n = 8) minutes after injection. In another group of animals, the effect of the occlusion and reperfusion protocol itself on tissue distribution in normal myocardium was investigated by injecting 99mTc-DTPA and sacrificing them at 5 minutes (n = 9) after injection. The distribution of 99mTc-DTPA in normal myocardium in this group was compared with the distribution in noninjured myocardium in the animals in the occlusion and reperfusion protocol.

Immediately before the animal was sacrificed, a 1-mL arterial blood sample was drawn to measure the hematocrit and radioactivity of whole blood and plasma. This was followed by reocclusion of the anterior branch of the left main coronary artery and intravenous injection of 0.25 mL of phthalocyanine blue, an intravascular dye that distributes with flowing blood, to distinguish normally perfused myocardium from myocardium at risk.

The heart was excised, the right ventricle was trimmed, and the left ventricle was transected at the midventricular level along the short axis. The apical portion was used to collect myocardial tissue samples from the area at risk (nonstained) and from normally perfused myocardium (blue stained). Skeletal muscle was obtained from the thigh to measure the distribution of 99mTc-DTPA in an independent tissue. All specimens (blood, plasma, myocardium, and skeletal muscle) were weighed and their radioactivity measured by using an automatic gamma counter system (Searle Analytic, Tampa, Fla) with the energy window set between 100 and 180 keV. The basal portion of the heart was embedded in embedding medium (Tissue Tek; Sakura Finetek USA, Torrance, Calif), immediately frozen, and subsequently sliced with a microtome (Cryocut 1899; Cambridge Instrument, Nussloch, Germany) into several 20-µm-thick slices and placed on a photostimulable storage phosphor imaging plate (Molecular Dynamics, Sunnyvale, Calif) for 1–2 hours. The phosphor imaging plate was subsequently scanned and digitized (PhosphorImager: 445 SI; Molecular Dynamics), and the data were evaluated with a commercial program (Molecular Dynamics).

The average image count density (counts per pixel) in regions of interest from injured and normal myocardium were measured on the autoradiographs (Fig 3) obtained from the scanned phosphor imaging plates. The area with increased count density always consisted of a core with high count density surrounded by a rim with moderately increased count density. The combined area of the core and the rim was objectively outlined in the digitized image as that with a count density at least 2 SD higher than the mean count density of the normal area (Fig 3).



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  		Figure 3. Profile of image count density, through a section (X) on an autoradiograph, of a 20-µm-thick midventricular slice from a rat subjected to 1 hour of coronary artery occlusion followed by 1 hour of reperfusion. Three levels of image count density typically were seen: Low count density corresponds to normal (N) myocardium, moderately increased count density (N + 2 SD) in a rim corresponds to putative ischemic injury, and high count density (N x 4) in the core corresponds to infarction.

 
To digitally outline the core, a threshold value was chosen to correspond to the visual impression of the border between the rim and the core. This threshold was four times the average count density in the normal myocardium and was typically located at the steepest part of the profile of image count density (Fig 3). Results obtained in this way are dependent on the threshold levels used; however, because the demarcation between the core and the rim was clear-cut, neither the results nor the conclusions were critically dependent on this factor.

Measurement of Area at Risk
To outline the normally perfused area (phthalocyanine blue stained) and the area at risk (nonstained), the same 20-µm-thick cross-sectional midventricular slices used for autoradiography were scanned on a flatbed scanner (Silver Scanner IV; La Cie, Hillsboro, Ore) connected to a computer (Quadra 650; Macintosh, Cupertino, Calif). In the resultant digital image, the left ventricular total cross-sectional area at the midventricular level and the unstained area (area at risk) were measured by means of computerized planimetry (NIH-Image v 1.59; National Institutes of Health, Bethesda, Md).

Infarcted Area Seen with TTC and Autoradiography
To compare the infarcted area seen with histochemical staining with the area with high count density on the autoradiograph, six rats were subjected to the occlusion and reperfusion protocol (1 hour of coronary occlusion and 1 hour of reperfusion) immediately followed by the injection of approximately 0.5 mCi (1.85 x 107 Bq) of 99mTc-DTPA. Thirty minutes later, the coronary artery was reoccluded, phthalocyanine blue was infused, and the animal was sacrificed by means of the injection of saturated potassium chloride. The heart was excised and transected at the midventricular level. The upper part was immediately frozen and sliced in 20-µm-thick slices to obtain autoradiographs as described above. The lower mirror portion was incubated for 7 minutes in 2% TTC solution, which stains viable myocardium red and leaves infarcted myocardium unstained (15).

The area of high count density on the autoradiograph (core) and the area at risk on the flatbed scanned image, both obtained from a 20-µm slice, were measured as described above. The mirror surface from the lower portion of the transected left ventricle was scanned on a flatbed scanner, and the infarcted area (area not stained with TTC) and the area at risk (area not stained with phthalocyanine blue) were measured.

Calculation of Fractional Distribution Volume
If apparent equilibrium distribution of gadopentetate dimeglumine and 99mTc-DTPA between myocardium and blood exists, then the fractional distribution volumes of these tracers can be calculated, assuming equal density of whole blood and myocardial tissue, as follows: fDV = {Delta}R1 ratio(myocardium/blood) x (1 - hct) and fDV = radioactivity ratio(myocardium/blood) x (1 - hct), where fDV is the fractional distribution volume, hct is the hematocrit, radioactivity is in counts per gram per minute, {Delta}R1 ratio is the MR-derived partition coefficient ({lambda}), and radioactivity ratio is the radioisotope-derived partition coefficient ({lambda}).

The fractional distribution volume of 99mTc-DTPA in reperfused infarcted myocardium was also calculated by using the image density on the autoradiographs as fDV = radioactivity ratio(normal myocardium/blood) x (1 - hct) x image density ratio(infarct/normal).

The reason to use two methods to calculate the fractional distribution volume in reperfused infarcted myocardium (the second two equations) was that tissue specimens sampled from the area at risk were likely to contain a mixture of infarcted and noninfarcted myocardium, which could lead to an underestimation of the fractional distribution volume in the core of the injury. This was avoided by objectively outlining the infarcted myocardium (core) on the autoradiograph and multiplying the ratio of the core to normal myocardium on the autoradiograph with the fractional distribution volume in the corresponding specimen from normal myocardium.

Statistics
Data are presented as the mean ± SEM, with the number of observations within parentheses. The significance of differences between group mean values was determined by analysis of variance and deemed to be significant if P was less than .05 with subsequent multiple comparisons between groups with use of the Tukey test. Numerical analysis of agreement between measurements in the same animals was performed according to the method of Bland and Altman (16).


   RESULTS
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Heart Rate and Blood Pressure
Before the administration of 99mTc-DTPA, heart rate and systolic, diastolic, and mean blood pressures were 248 beats per minute ± 10 and 111 mm Hg ± 3, 88 mm Hg ± 4, and 98 mm Hg ± 3, respectively. In five animals, these parameters also were monitored after the administration of 99mTc-DTPA without observation of any significant changes in heart rate or systolic or diastolic blood pressure, which were three beats per minute ± 8 and -1 mm Hg ± 3 and -4 mm Hg ± 2, respectively.

MR Imaging
The relaxation rates of left ventricular blood and normal and infarcted myocardium increased significantly (P < .05) after administration of 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight. The fastest relaxation rate was found in infarcted myocardium, which indicates that this region had the highest content of gadopentetate dimeglumine, followed by left ventricular blood and then normal myocardium (Fig 2). Both relaxation rates and signal intensity decreased with time in all regions of interest as a result of plasma clearance after the injection of gadopentetate dimeglumine. However, the {Delta}R1 ratios for myocardium and blood for both normal and infarcted myocardium did not change significantly (P < .05) during the measurements, which indicates apparent equilibrium distribution of gadopentetate dimeglumine between myocardium and blood (Fig 4, filled circles).



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  		Figure 4. Plot of {Delta}R1 and radioactivity ratios of myocardium and blood at various times after the injection of gadopentetate dimeglumine and 99mTc-DTPA after 60 minutes of coronary occlusion followed by 60 minutes of reperfusion. The {Delta}R1 ratios are from repetitive measurements in the same animals, and the radioactivity ratios are the mean ± SEM from three groups of animals. The {Delta}R1 and radioactivity ratios for myocardium and blood stayed constant over time, which suggests equilibrium-phase distribution of gadopentetate dimeglumine and 99mTc-DTPA. This means that {Delta}R1 ratios and radioactivity ratios represent partition coefficients ({lambda}) and allows calculation of fractional distribution volumes. The time-averaged {Delta}R1 ratios for normal myocardium and blood were slightly higher than the corresponding radioactivity ratios for the three groups (P < .05). For reperfused infarcted myocardium and blood, the {Delta}R1 and radioactivity ratios were not significantly different.

 
There was no significant difference in {Delta}R1 between normal myocardium in animals subjected to the occlusion and reperfusion protocol and {Delta}R1 in myocardium in control animals (Table). The {Delta}R1 ratio of infarcted myocardium to normal myocardium was 4.07 ± 0.40 (n = 6). Presence of myocardial infarction was confirmed in each animal by means of postmortem TTC staining and agreed qualitatively with the extent and location at MR imaging.


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  		Partition Coefficients ({lambda}) and Fractional Distribution of Volumes of Gadopentetate Dimeglumine and 99mTc-DTPA
 
Radioisotope Experiments
On the autoradiograph (Fig 5, A) obtained from the same slice of the left ventricle used to measure the area at risk (Fig 5, B), typically three levels of image count density were observed: low, moderately increased, and high (for definition, see Materials and Methods and Fig 3). The core of the injured myocardium (high count density) was always surrounded by a rim of myocardium with moderately increased count density. The area with low count density (Fig 5, A) corresponded to the normally perfused area on the blue dye image (Fig 5, B) and thus represents normal myocardium. The combined area of the core and the rim on the autoradiograph (Fig 5, C, region of interest 1) was 49% ± 2 (n = 24) of the total left ventricular cross-sectional area. This area (core and rim) was not significantly different from the area at risk, 51% ± 2 (n = 24) defined in the same slices by using phthalocyanine blue, and thus likely represents ischemically injured myocardium.



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  		Figure 5. A, C, Autoradiographs and B, D, flatbed scanned images, from one midventricular slice (20-µm thickness), 15 minutes after the injection of 0.5 mCi (1.85 x 107 Bq) of 99mTc-DTPA in an animal that had been subjected to 1 hour of occlusion of the anterior branch of the left main coronary artery followed by 1 hour of reperfusion. Before the injection of saturated potassium chloride to arrest the heart in diastole, the coronary artery was reoccluded, and phthalocyanine blue was infused to delineate the nonperfused area ("area at risk"). A, The count density is greater in the postischemic region owing to higher 99mTc-DTPA content. Ant = anterior, Inf = inferior, Lat = lateral, Sept = septum. B, Flatbed scanned image shows the perfused area as dark and the area at risk as bright. C, The injured region (region of interest 1) is defined as that with a count density more than 2 SDs greater than the mean value in the normal region (N) (Fig 3). The core of the infarction (region of interest 2) is defined as that having a count density greater than that of the normal myocardium. D, For comparison, region of interest 1 from C has been superimposed on the flatbed scanned image to demonstrate that there is agreement between the injured area (core and rim) and the true area at risk.

 
The area of the core on the autoradiograph (Fig 5, C, region of interest 2) was 61% ± 3 of the area at risk (n = 24). The count density ratios of the rim and the core to normal myocardium were 2.70 ± 0.04 and 5.48 ± 0.15 (n = 24), respectively. Because the demarcation between the rim and the core was distinct (Figs 3; 5, A), the ratio between the core and normal myocardium was not critically dependent on the threshold value chosen (more than four times the count density of normal myocardium). Threshold values of 3 or 5, for example, produced ratios of core of injury to normal myocardium of 5.09 ± 0.16 and 5.78 ± 0.17, respectively, on autoradiographs.

The radioactivity ratio of the area at risk to normal myocardium obtained by gamma counting of tissue specimens was considerably lower: 3.76 ± 0.22 (n = 24) (Fig 6). The discrepancy between radioactivity ratios obtained at autoradiography on the one hand and gamma counting of tissue specimens on the other hand was because of the likelihood that tissue samples taken from the area at risk also contained noninfarcted myocardium. Therefore, comparisons between radioactivity ratios and {Delta}R1 ratios were calculated by using data obtained from the autoradiographs.



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  		Figure 6. Bar graph shows radioactivity ratio (area at risk over normal myocardium) of 99mTc-DTPA from tissue specimens. This ratio is significantly (P < .05) lower than that obtained from autoradiographs (paired data, n = 24). It is likely that specimens, taken from the area at risk, contained both infarcted and noninfarcted myocardium, whereas on the autoradiograph noninfarcted myocardium was excluded.

 
The combined data for all individual tissue specimens and all time points after the injection of 99mTc-DTPA (5, 15, and 30 minutes) displayed a range spanning more than one order of magnitude and were well described by straight lines (Fig 7). The data show that the distribution of radioisotope in reperfused infarcted myocardium is similar to that in plasma, which suggests that the tracer is not excluded from the intracellular space of infarcted cells. The difference between directly measured plasma radioactivity and calculated plasma radioactivity (from the product of whole blood counts and hematocrit) was 0.00 ± 0.07 (SD; n = 17). Thus, the accuracy of calculated plasma radioactivity was 100%, and the precision was 93%. This indicates that the radioactivity of whole blood is contained in the plasma and that no 99mTc-DTPA enters the red blood cell fraction.



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  		Figure 7. Graph shows counts per gram per minute for individual specimens of plasma, reperfused infarcted and normal myocardia, and myocardium from control animals versus plasma values of counts per gram per minute calculated from whole blood (hematocrit is 0.45 ± 0.01, n = 24). The values for reperfused infarcted myocardium are obtained by multiplying the ratio of infarcted over normal myocardium in the autoradiograph with counts per gram per minute for normal myocardium. Measured plasma values (n = 17) fall close to the line of identity (not shown), which demonstrates that calculated plasma values are accurate and precise. The values for normal myocardium in the occlusion and reperfusion group and the values for myocardium in the control animals are essentially the same. Counts per gram per minute for reperfused infarcted myocardium are close to plasma values, which suggests that infarcted cells were unable to exclude 99mTc-DTPA. All relations were linear. Plasma: y = 0.94x + 0.03 x 106, r2 = 0.98; reperfused infarct: y = 0.82x + 0.03 x 106, r2 = 0.79; normal: y = 0.13x + 0.01 x 106, r2 = 0.83; control: y = 0.15x + 0.01 x 106, r2 = 0.96. cpm/g = counts per minute per gram, hct = hematocrit.

 
The radioactivity ratios of myocardium over blood for both normal and reperfused infarcted myocardium did not change with time after injection (Fig 4, open circles), which indicates apparent equilibrium distribution of 99mTc-DTPA between myocardium and blood. There was no significant difference in the radioactivity ratios of normal myocardium compared with the myocardium of control animals (Table). The radioactivity of reperfused infarcted myocardium was greater than that of normal myocardium and close to the radioactivity of plasma (Table).

Infarction Size Defined by Using TTC Staining and Autoradiography
The infarcted area defined by using TTC and the high count density area (core) on the autoradiograph had the same appearance (Fig 8). The infarcted area defined by using TTC and the high count density area at autoradiography (core) were 61% ± 3 and 71% ± 5, respectively, of the area at risk (n = 6). The infarcted area defined by TTC was 13% ± 4 smaller (P < .05) than the core area on the autoradiograph.



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  		Figure 8. Infarcted area of high count density (core) on the autoradiograph (left panel), the true infarcted area (bright) defined by using TTC (middle panel), and the true area at risk on the scanned image (right panel) appear to have the same shape, size, and location, which suggests that the core on the autoradiograph represents infarcted myocardium.

 
Relation between MR Imaging and Radioisotope Experiments
The shape, extent, and location of the injured area were essentially the same on T1-weighted spin-echo images, echo-planar images, and autoradiographs, as demonstrated in Figure 9. The behavior of {Delta}R1 and radioactivity ratios over time were similar (Fig 4). In normal myocardium, the radioactivity ratios were smaller than the corresponding {Delta}R1 ratios (P < .05) (Table), but there was no significant difference between the two methods for reperfused infarcted myocardium (Fig 4, Table).



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  		Figure 9. Three images, at the midventricular level of the left ventricle, in an animal subjected to 1 hour of occlusion of the anterior branch of the left main coronary artery followed by 1 hour of reperfusion. The reperfused injured zone can be seen as a bright area on all images. A, T1-weighted spin-echo MR image obtained 20 minutes after the administration of 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight with a repetition time of 250 msec and an echo time of 12 msec. B, Inversion-recovery echo-planar MR image obtained 14 minutes after the intravenous injection of 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight (repetition time, 7,000 msec; echo time, 10 msec). The inversion time is set to 220 msec, which produces negative signal in normal myocardium, positive signal in reperfused infarcted myocardium (arrowheads), and nil signal in left ventricular chamber blood. C, Autoradiograph obtained 15 minutes after the intravenous injection of 0.5 mCi (1.85 x 107 Bq) of 99mTc-DTPA.

 
Fractional Distribution Volumes of Gadopentetate Dimeglumine and 99mTc-DTPA
Constant ratios of {Delta}R1 and radioactivity over time mean that these ratios represent partition coefficients ({lambda}) and thus enable calculation of fractional distribution volumes. By using MR-derived partition coefficients, the fractional distribution volume of gadopentetate dimeglumine in normal myocardium was 0.23 ± 0.02, and that in reperfused infarcted myocardium was 0.90 ± 0.05 (n = 6). The former value corresponds to the fractional volume of the extracellular space of myocardium, while the latter value approaches the fractional volume of the combined intracellular and extracellular spaces.

In normal myocardium, the fractional distribution volume for 99mTc-DTPA was slightly smaller (P < .05) than that for gadopentetate dimeglumine, while in the core of the injured myocardium the fractional distribution volumes were not significantly different and approached plasma levels (Fig 10, Table). For measurements performed with either gadopentetate dimeglumine or 99mTc-DTPA, there were no significant differences in fractional distribution volumes between normal myocardium in the animals subjected to the occlusion and reperfusion protocol and normal myocardium in the control animals (Table). The fractional distribution volume of 99mTc-DTPA in the skeletal muscle of the thigh was 0.07 ± 0.01 (n = 5).



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  		Figure 10. Bar graph shows fractional distribution volumes of 99mTc-DTPA and gadopentetate dimeglumine in normal and reperfused infarcted myocardia. The fractional distribution volumes are the same in the core of the infarction, whereas they differ slightly in normal myocardium (P < .05). The values in normal myocardium are in agreement with earlier published data (17–24) on myocardial extracellular volume. The values in the core of the reperfused injury are what can be expected from inert passively distributing agents under equilibrium conditions if most myocardial cells have lost cellular membrane integrity.

 

   DISCUSSION
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The main findings of the present study were that (a) apparent equilibrium-phase distribution between myocardium and blood exists for both gadopentetate dimeglumine and 99mTc-DTPA and allows the calculation of partition coefficients ({lambda}); (b) the quantification of the fractional tissue content of gadopentetate dimeglumine and the quantification of the fractional tissue content of 99mTc-DTPA by using independent imaging modalities agree; (c) these methods can be used to calculate fractional distribution volumes in normal and reperfused infarcted myocardium; (d) the fractional distribution volumes in reperfused infarcted myocardium were close to plasma levels—that is, the same value as the combined intra- and extracellular spaces—which suggests that essentially all myocardial cells in reperfused infarcted myocardium, as opposed to cells in normal myocardium, were unable to exclude these tracers.

Size and Location of Injury
On autoradiographs (Fig 5), it is likely that the core of the reperfused area, with high count density, represents necrotic myocardium, because the appearance of this area was similar to the TTC–negative area (Fig 8) and because the failure of cells to exclude extracellular tracers is an "excellent marker of loss of cell membrane integrity with ultrastructural changes indistinguishable from those observed in lethal injury" (25). Moreover, the core area in the present study constituted 61% ± 3 of the area at risk, which is similar to the results of Hale and Kloner (1), who found that "area of necrosis" as a percentage of area at risk was 54.3% ± 7.0 as a result of 60 minutes of coronary artery occlusion followed by 24 hours of reperfusion. The total area with increased 99mTc-DTPA content (core and rim) was not significantly different from the area at risk defined by the blue dye experiments, or 49% ± 2 versus 51% ± 2 (n = 24). The rim area may represent reversible injury with increased extracellular space following degradation of the extracellular matrix (26), reactive hyperemia, or diffuse interspersed irreversible injury. However, although the exact nature of the moderate radioisotope increase in the rim area between normal and infarcted myocardium remains unclear, it represents neither normal nor completely infarcted myocardium, and it corresponds to the area at risk.

It seems likely that this boundary region contains a population of necrotic cells into which the radioisotope is distributed. This results in a lesser increase in the activity of the radioisotope in this region compared with the densely infarcted region. However, an alternate consideration is that reversibly injured myocardial cells permit the influx of a smaller amount of the tracer, which causes a boundary region of lesser isotope accumulation. Because gadopentetate dimeglumine and 99mTc-DTPA have identical distribution characteristics, this isotope at autoradiography serves as a surrogate for the distribution of the MR imaging contrast medium.

The finding that the infarcted area defined by TTC was 16% smaller than the high count density area on the autoradiograph (the core) visible by using the extracellular tracer 99mTc-DTPA is consistent with the findings of earlier studies (6,27) showing that the infarcted area defined by TTC is 20% smaller than the hyperenhanced area on MR images visible by using the extracellular tracer gadopentetate dimeglumine–bis(methylamide). This finding may be a result of the relatively short reperfusion time used in this study (90 minutes), because the size of the infarcted area defined by TTC increases with reperfusion time (15). Moreover, the infarction size defined by TTC may be an underestimation (28).

Measurement of the Fractional Distribution Volumes of Gadopentetate Dimeglumine and 99mTc-DTPA
Using signal intensity on the MR image to quantify the tissue content of low- molecular-weight MR contrast media like gadopentetate dimeglumine is complicated by the fact that the signal intensity is not proportional to the tissue gadolinium content (29,30). However, because {Delta}R1 for a tissue is proportional to the gadopentetate dimeglumine content, the quantification of the bulk tissue content of gadopentetate dimeglumine can be achieved by using T1 measurements (11).

Even if T1 is measured, there are other uncertainties associated with measuring gadopentetate dimeglumine content in vivo by means of MR imaging because of, for example, the rate of water exchange across capillary cell membranes (11,29,31) and because MR imaging measures the effect on water of gadopentetate dimeglumine rather than the gadopentetate dimeglumine itself. Therefore, the purpose of the present study was to compare MR imaging measurements of myocardial gadopentetate dimeglumine content with the more direct radioisotope measurements of myocardial 99mTc-DTPA content over time in identically prepared animals.

According to Kruhoffer (32), the extracellular volume can be determined by using a tracer that is strictly extracellular and distributes homogeneously in the entire extracellular volume, is not eliminated or excreted by the tissues, and is essentially iso-osmotic and nontoxic, and the ratio between the concentrations in the interstitial fluid and in the plasma must be a known fixed value. Chelating agents, such as DTPA, do not accumulate in cells and are rapidly and completely excreted in the urine (33), which indicates strict extracellular distribution. Both the gadopentetate dimeglumine and 99mTc-DTPA used in the current study have extracellular distributions (34,35) and similar pharmacokinetics (36,37). Neither 99mTc-DTPA nor gadopentetate dimeglumine enters the red blood cellular fraction (35,38), and this was confirmed for 99mTc-DTPA in the present study. This enables the calculation of the fractional distribution volume of these tracers in blood as 1 - hematocrit. Because there is a considerable clearance of these tracers from blood (plasma half-life of approximately 20 minutes), the condition of constant ratio between myocardium over blood had to be investigated.

After the administration of the tracers and after 60 minutes of reperfusion, constant {Delta}R1 and radioactivity ratios over time were found for both infarcted and normal myocardium, which indicates apparent equilibrium distribution. This suggests that the tracers are cleared from normal and infarcted myocardium at the same rate as from plasma and that no tracer is accumulated in the infarcted myocardium, as opposed to the classic approach in which infarct-avid radioisotope tracers (39,40) or infarct-avid MR contrast media (41,42) are used. When the condition of constant ratios of the tracers in myocardium to those in blood is met, these ratios represent partition coefficients ({lambda}), and the myocardial fractional distribution volume is simply the ratio of tracer content in tissue over tracer content in plasma.

In normal myocardium in the animals subjected to the occlusion and reperfusion protocol, the distribution volumes of gadopentetate dimeglumine and 99mTc-DTPA were 0.23 ± 0.02 and 0.16 ± 0.01, respectively. These values are in the range of values reported in the literature (17–24) for myocardial extracellular volume in rats, rabbits, dogs, and humans: 0.15–0.32, with a mean value of 0.22. Because the extracellular space in the nonischemic zone of infarcted rat heart is expanded (43), we studied the extracellular volume in control animals without finding any significant changes compared with normal myocardium in rats subjected to the occlusion and reperfusion protocol (Table). In the present study, the fractional distribution volume of gadopentetate dimeglumine in normal myocardium was 0.23 at inversion-recovery echo-planar imaging, which is similar to the values reported in humans, or 0.21–0.28 at inversion-recovery TurboFLASH imaging (44).

The extracellular volume measured with 99mTc-DTPA was somewhat lower compared with that measured with gadopentetate dimeglumine. This could have several explanations. Impurities are more likely to occur in 99mTc-DTPA preparations than in gadopentetate dimeglumine preparations. These impurities bind to plasma proteins (45) and may lead to an overestimation of the fractional distribution volume in plasma and hence an underestimation in normal myocardium. However, this is unlikely, because the amount of free to bound 99mTc in vitro was less than 1%, which leads to an in vivo impurity of 0.01% (46). The fractional tissue compartment volumes of gadopentetate dimeglumine were determined in vivo, whereas those of 99mTc-DTPA were determined ex vivo. Excision of myocardial tissue specimens leads to drainage of blood out of the microvessels and causes underestimation of the extracellular volume.

Another possibility is that the injection of potassium chloride to arrest the heart in diastole may result in partial displacement of 99mTc-DTPA by potassium chloride solution in the myocardial capillaries. Furthermore, there may be an unknown inherent systematic bias in the estimations of the fractional distribution of 99mTc-DTPA. However, this is not likely, because the fractional distribution volume of 99mTc-DTPA in skeletal muscle in the present study (0.07 ± 0.01, n = 5) was in close agreement with previously reported values: approximately 0.08 for gadopentetate dimeglumine in skeletal muscle of the rat (47) and 0.077–0.080 for chromium 51–ethylenediaminetetraacetic acid and 0.070–0.085 for inulin in skeletal muscle of the rabbit (19). Moreover, there was no bias between the theoretical (1 - hematocrit) and measured fractional distribution volumes of 99mTc-DTPA in plasma.

In acutely reperfused infarcted myocardium, the fractional distribution volumes for gadopentetate dimeglumine and 99mTc-DTPA were not significantly different if the fractional distribution volume of 99mTc-DTPA was calculated by using counts per gram per minute from normal myocardium multiplied by the ratio of infarcted to normal myocardium from the autoradiograph (Fig 10). However, if tissue data from the area at risk (in counts per gram per minute) were used directly, the calculated fractional distribution volume for reperfused infarcted myocardium was considerably lower. The reason for this is most likely because obtaining tissue specimens to represent reperfused infarcted tissue was guided by the area at risk. Because the area at risk also contains noninfarcted myocardium (Fig 5), tissue specimens are likely to represent a mixture of infarcted and noninfarcted myocardium. Therefore, in the calculation of the fractional distribution volumes of 99mTc-DTPA in reperfused infarcted myocardium, tissue data of counts per gram per minute from normal myocardium multiplied by the image count ratio of infarcted over normal myocardium from the autoradiographs were used. The finding that the fractional distribution volumes of gadopentetate dimeglumine and 99mTc-DTPA in infarcted myocardium were close to the plasma levels suggests that these tracers are inert and diffuse passively into myocardial cells that have lost membrane integrity.

The major limitation of this study was that the fractional tissue contents of gadopentetate dimeglumine were determined in vivo, whereas the fractional tissue contents of 99mTc-DTPA were determined ex vivo. This is the most likely explanation for why the fractional distribution volume in normal myocardium was lower for 99mTc-DTPA ex vivo than the corresponding values for gadopentetate dimeglumine in vivo. Another limitation is that the MR imaging and the radionuclide imaging were not performed in the same animals. This was not possible because the radionuclide studies required three different groups to be sacrificed at three different time points, and this was not compatible with MR imaging for 30 minutes in the same animals after the injection of contrast medium. Because the only way to provide direct evidence that {Delta}R1 ratios represent partition coefficients is to show that they are constant over time in the same animal, it was necessary to use separate groups for MR imaging and radionuclide imaging.

In conclusion, {Delta}R1 and radioactivity ratios for myocardium over blood were constant during the measurements and thus represent partition coefficients ({lambda}). This means that the clearance of gadopentetate dimeglumine and 99mTc-DTPA from myocardium paralleled their clearance from blood and that neither gadopentetate dimeglumine nor 99mTc-DTPA were sequestered in the acutely infarcted myocardium. These conditions allow the calculation of fractional distribution volumes with use of these tracers. The fractional distribution volumes of gadopentetate dimeglumine in vivo at inversion-recovery echo-planar MR imaging and of 99mTc-DTPA ex vivo with radioisotope methods agreed well in both normal and reperfused infarcted myocardium. The distribution volumes of the tracers show extracellular distribution in normal myocardium and extracellular as well as intracellular distribution in the core of the reperfused injury. The distribution in the rim, surrounding the core of the injury, indicates injury but not complete infarct.Practical applications: Because the failure of cells to exclude passive extracellular tracers, such as gadopentetate dimeglumine, is indicative of irreversible cellular injury, direct and noninvasive measurements of the fractional distribution volumes of such agents at inversion-recovery echo-planar imaging may be useful to confirm or exclude complete myocardial infarction in a reperfused ischemically injured region and to estimate the infarcted cell fraction.


   Acknowledgments
 
We gratefully acknowledge John Huberty, BSc, for preparing the radiopharmaceutical.


   Footnotes
 
Abbreviations: {Delta}R1 = change in relaxation rate DTPA = diethylenetriaminepenta- acetic acid TTC = triphenyltetrazolium chloride

Author contributions: Guarantors of integrity of entire study, M.S., C.B.H., M.F.W.; study concepts, M.S., C.B.H., M.F.W.; study design, M.S., C.B.H., M.W.D., M.F.W.; definition of intellectual content, M.S., C.B.H., M.F.W.; literature research, H.A., M.S., C.B.H., M.F.W.; experimental studies, H.A., M.S., D.W.G., J.B., R.W., M.F.W.; data acquisition, H.A., M.S., D.W.G., J.B., R.W., M.F.W.; data analysis, H.A., M.S., D.W.G., J.B., M.W.D. M.F.W.; statistical analysis, H.A., M.S., M.F.W.; manuscript preparation, H.A., M.S.; manuscript editing, H.A., M.S., C.B.H., M.F.W.; manuscript review, all authors.


   References
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 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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