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(Circulation. 1998;97:795-804.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Progression of Myocardial Necrosis During Reperfusion of Ischemic Myocardium

Kaname Matsumura, MD; Richmond W. Jeremy, MBBS, PhD; Jutta Schaper, MD; ; Lewis C. Becker, MD

From the Division of Cardiology (R.W.J., L.C.B.) and the Division of Nuclear Medicine (K.M.), Departments of Medicine and Radiology, The Johns Hopkins Medical Institutions, Baltimore, Md, and the Department of Experimental Cardiology, Max Planck Institut, Bad Nauheim, Germany (J.S.).

Correspondence to Dr Lewis C. Becker, Halsted 500, Johns Hopkins Medical Institutions, 600 N Wolfe St, Baltimore, MD 21205.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—The occurrence of myocyte necrosis during reperfusion of ischemic myocardium is controversial. This study measured myocardial 2-deoxyglucose uptake, correlated with histology, to determine whether loss of viability occurred during reperfusion of ischemic myocardium.

Methods and Results—In 12 anesthetized dogs, the left anterior descending coronary artery was occluded for 90 minutes before 4 hours reperfusion. Myocardial blood flow was measured by microspheres and the tracers ¹⁴C-2-deoxyglucose and ¹⁸F-2-deoxyglucose were injected intravenously after 5 and 180 minutes of reperfusion, respectively. After 240 minutes, the heart was stained with thioflavin-S (size of no-reflow zone) and triphenyl-tetrazolium chloride (TTC, extent of necrosis). Samples from normal, salvaged, and necrotic myocardium were counted for ¹⁴C- and ¹⁸F-deoxyglucose and microspheres. With the use of a three-compartment model of 2-deoxyglucose uptake, the rate constant k₃ for phosphorylation of ¹⁴C- and ¹⁸F-2-deoxyglucose was calculated for each sample. Viability was defined as k₃ 0.125 min^-1 (predictive accuracy 88% versus electron microscopy and 97% versus TTC). Among 58 samples from no-reflow regions, 97% were nonviable after 5 minutes of reperfusion (k₃=0.096±0.027 min^-1). Among 164 samples from salvaged myocardium, 95% were viable after both 5 and 180 minutes of reperfusion (k₃=0.170±0.056 min^-1 P<.01 versus no-reflow). Among 179 samples from infarcted myocardium, mean k₃ after 5 minutes of reperfusion was 0.184±0.070 min^-1 and 65% of samples were viable, but after 180 minutes of reperfusion mean k₃ had decreased to 0.077±0.032 min^-1 (P<.0001) and 98% of samples were nonviable.

Conclusions—A large proportion of samples from infarcted myocardium are viable at the end of the ischemic period but lose viability during the first hours of reperfusion.

Key Words: metabolism • myocardial infarction • ischemia • reperfusion • radioisotopes

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Coronary reperfusion improves ventricular function and survival after infarction,^{1 2} but concern persists that damaged but otherwise viable myocytes may undergo necrosis during reperfusion.^{3 4} Although interventional studies with scavengers of oxygen radicals,^{5 6} inhibition or removal of neutrophil leukocytes,^{7 8} and administration of adenosine⁹ suggest that myocardial necrosis does occur during reperfusion, direct evidence has been lacking. Sequential measurements of viability, using a marker of basic cellular metabolism, are required to address this question. Radionuclide-labeled 2-deoxyglucose is used as a tracer of glucose uptake and phosphorylation in brain¹⁰ and heart.¹¹ Although rapidly phosphorylated by hexokinase,¹² 2-deoxyglucose is not a substrate for further glycolytic metabolism and is trapped in the cell.¹³ The rate constant for phosphorylation of ¹⁸F-2-deoxyglucose by hexokinase (k₃) is correlated with glucose metabolism in the reperfused myocardium.¹⁴ We used 2-deoxyglucose for sequential measurements of viability in reperfused myocardium, with injection of ¹⁴C-2-deoxyglucose immediately after reperfusion and ¹⁸F-2-deoxyglucose 3 hours later. Uptake of 2-deoxyglucose was compared with histochemical and ultrastructural evidence of reversible and irreversible myocardial injury and correlated with collateral blood flow during ischemia to differentiate lethal injury occurring during ischemia from that occurring during reperfusion.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Thirty-three mongrel dogs of either sex (weight, 20 to 27 kg) were studied after an overnight fast. The dogs were anesthetized with sodium thiamylal (12.5 mg/kg IV) followed by chloralose (14 mg/kg IM) in urethane (136 mg/kg). Polyvinyl catheters were placed in the right femoral artery and vein for reference sampling of microspheres and administration of intravenous fluids, respectively. After left thoracotomy, a catheter was placed in the left atrium for microsphere injections and a catheter-tip pressure transducer (5F, Millar Instruments) was advanced to the left ventricle from the left atrium. An inflatable occluder was placed around the proximal LAD. Hemodynamics were continuously recorded on chart paper (Gould Instruments).

Myocardial Distribution and Retention of 2-Deoxyglucose
Because ¹⁴C-2-deoxyglucose and ¹⁸F-2-deoxyglucose are not structurally identical, the distribution of the two tracers in reperfused myocardium was studied in three dogs. After 90 minutes of LAD occlusion, the dogs received simultaneous intravenous ¹⁴C-2-deoxyglucose (25 µCi; specific activity, 59 mCi/mmol; Sigma) and ¹⁸F-2-deoxyglucose (0.25 to 1.5 mCi; Division of Nuclear Medicine, Johns Hopkins Medical Institutions). Tracers were injected after 5 minutes of reperfusion in one dog, after 15 minutes in the second, and after 3 hours in the third. At 60 minutes after tracer injection, the LAD was reoccluded and monastral blue dye injected into the left atrium to define the ischemic region. Multiple (n=30 to 40) biopsies (30 to 85 mg wet wt) were obtained from reperfused myocardium for counting of ¹⁴C and ¹⁸F activities.

Retention of ¹⁴C-2-deoxyglucose in reperfused myocardium was studied in three other dogs. After 90 minutes of LAD occlusion and 5 minutes of reperfusion, ¹⁴C-2-deoxyglucose was given intravenously. Transmural biopsies (15 to 30 mg wet wt) were taken in triplicate from LAD and circumflex artery territories by biopsy drill at 1, 2, and 3 hours after tracer injection. The heart was arrested after 4 hours and further biopsies taken. The biopsies were weighed and counted for ¹⁴C activity. In two other dogs, myocardial retention of ¹⁸F-2-deoxyglucose was compared with myocardial blood flow during ischemia and reperfusion. The LAD was occluded for 90 minutes and blood flow measured by radionuclide-labeled 15–µm microspheres (¹⁴¹Ce, ¹¹³Sn, ⁴⁶Sc; Du Pont Co) injected into the left atrium, with reference sampling from the right femoral artery, at 10 minutes before reperfusion. After 10 minutes reperfusion, ¹⁸F-2-deoxyglucose was injected intravenously. A biopsy was excised by scalpel from reperfused myocardium 35 minutes later and divided into endocardial and epicardial halves. After 3 hours of reperfusion, blood flow was measured with microspheres before repeat biopsy of reperfused myocardium and the normal circumflex artery territory. Samples were weighed and counted for ¹⁸F activity and microspheres.

Comparison of ¹⁸F-2-Deoxyglucose Uptake With Histology
In seven dogs the LAD was occluded for 90 minutes and myocardial blood flow measured by microspheres at 10 minutes before reperfusion. After 5 minutes of reperfusion, ¹⁸F-2-deoxyglucose was injected intravenously, followed by a second microsphere injection. After 35 minutes of reperfusion, the heart was arrested by intravenous potassium chloride, excised, and sectioned into short-axis slices. Multiple transmural sections from normal and reperfused regions were divided into fifths from endocardium to epicardium (30 to 100 mg wet wt per sample). Samples were randomly selected from the control and reperfused myocardium in each dog for electron microscopy. A small section of each sample was immersed in cold (4°C) 3% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.4, and kept in fixative at 4°C for 24 hours before rinsing in 0.1 mol/L cacodylate buffer (with saccharose added, pH 7.4) and storage at 4°C before examination. The remainder of the sample was counted for ¹⁸F activity and microspheres.

Operators studied all samples by light and electron microscopy, without knowledge of sample location or regional blood flow. Samples were embedded in epon with the use of a Wakura automatic tissue processor, after fixation in 2% osmic acid anhydride, dehydration in an ethanol series, and substitution by propylenoxide. Semithin (1 to 2 µm) sections were stained with toluidine blue. Artifact-free areas were selected for preparation of thin sections (50 to 60 nm), which were attached to uncoated copper grids, stained with uranyl acetate and lead citrate, and viewed in a Phillips EM 300-electron microscope. For each myocardial sample, 30 to 40 micrographs were examined according to previously established criteria.^{15 16} Reversible injury was identified by absence of contraction bands, an intact sarcolemma, absence of mitochondrial amorphous densities, and absence of nuclear clearing and shrinkage. Irreversible injury was identified by the presence of amorphous densities, matrix clearing and/or cristae breakage in the mitochondria, clearing and shrinkage of nuclei, and/or disruption of the sarcolemma. A sample was deemed to have suffered irreversible injury if >50% of the micrographs from that sample showed evidence of irreversible injury.

Sequential Measurements of 2-Deoxyglucose Uptake in Reperfused Myocardium
Myocardial uptake of 2-deoxyglucose after 5 minutes and 3 hours of reperfusion was studied in 18 dogs in which the LAD was occluded for 90 minutes before free reperfusion was allowed. Myocardial blood flow was measured by microspheres 10 minutes before reperfusion and 10 minutes and 3 hours after reperfusion. After 5 minutes of reperfusion, ¹⁴C-2-deoxyglucose (25 µCi) was injected intravenously and after 3 hours of reperfusion, ¹⁸F-2-deoxyglucose (1 mCi; range, 0.25 to 2 mCi) was injected intravenously. The ¹⁸F-2-deoxyglucose was used as the second tracer because of the short half-life of ¹⁸F. One hour after injection of ¹⁸F-2-deoxyglucose, the fluorescent dye thioflavine-S (2% solution) was injected into the left atrium to define no-reflow zones. Two minutes later the LAD was reoccluded and monastral blue dye (20 mL) was injected into the left atrium to define the ischemic risk region. The heart was then arrested with potassium chloride and excised for tissue sampling and measurement of infarct size.

The left ventricle was isolated and sectioned into five transverse slices (8 to 10 mm thick), which were weighed and examined under ultraviolet light to define no-reflow zones (absent thioflavin-S fluorescence). Endocardial and epicardial surfaces of each myocardial slice and borders of the ischemic region and the no-reflow zones were traced on acetate sheets. Transmural sections were excised at multiple sites in the LAD and circumflex territories of each heart and divided into fifths (70 to 100 mg wet wt per sample). A total of 60 to 70 samples were obtained from each heart, and sample sites were recorded on the acetate sheets. The myocardial slices were incubated in 2,3,5-TTC solution at 37°C for 30 minutes to differentiate infarcted myocardium (absent or negative TTC staining) from salvaged myocardium (brick-red or positive TTC staining).^{17 18 19} The borders of infarct and noninfarct regions were traced on the corresponding acetate sheets and planimetered to measure the ischemic risk region, infarct region, and no-reflow region.

Radionuclide Measurements
Myocardial samples were weighed and counted with flow reference samples and radionuclide standards in an NaTl crystal well counter (Packard 5986) set for photopeaks of ¹⁸F, ¹⁴¹Ce, ¹¹³Sn, and ⁴⁶Sc. Decay-corrected counts were corrected for crossover between radionuclides and blood flow calculated according to standard methods.²⁰ Samples were then solubilized (Protosol, Du Pont) and incubated at 50°C for 48 hours, before addition of 10 mL scintillation cocktail (EconoFluor, Du Pont) and liquid scintillation counting (Packard Tri-Carb 2660) for ¹⁴C activity. To eliminate any error in ¹⁴C measurement due to the presence of gamma emitters in the samples, calibration curves were determined for beta activity observed in the presence of ¹⁴¹Ce, ¹¹³Sn, and ⁴⁶Sc. After counting for ¹⁸F activity, tissue samples were counted twice in both gamma and beta counters at an interval of 4 weeks, with decay correction for each nuclide. Beta count activity due to the gamma emitters was then subtracted from total observed beta counts to derive the true ¹⁴C count activity.

Because reperfusion of lethally injured myocardium is associated with tissue edema and an increase in tissue wet weight of 25% during the first few hours,²¹ radionuclide count data for microspheres and ¹⁴C-2-deoxyglucose (injected during ischemia or early reperfusion) in samples from the infarct region were corrected to allow for a 25% increase in tissue wet weight during reperfusion.

The measure of viability of a myocardial sample is the phosphorylation rate of 2-deoxyglucose. When the time from tracer injection to sampling exceeds 30 minutes, the amount of nonmetabolized tracer in the sample approaches zero.²² With the use of a three-compartment model of myocardial uptake and phosphorylation of 2-deoxyglucose,¹⁴ the overall reaction rate in the sample of interest R_i can be described as:

where BG=blood glucose level; K₁ⁱ=rate of transport of 2-deoxyglucose into myocyte from plasma; k₂ⁱ=rate of reverse transport of 2-deoxglucose from myocyte to plasma; and k₃ⁱ=rate of phosphorylation of 2-deoxyglucose by hexokinase. Because the lumped constant (LC) and arterial input function are the same for all samples in each heart, the ratio of reaction rates in two samples can be calculated as the ratio of tracer activities:

where C_iT=tracer content in sample of interest; and C_rT=tracer content in reference sample.

The rate constants of 2-deoxyglucose uptake and phosphorylation in reperfused canine myocardium have been previously described,¹⁴ allowing calculation of k₃ⁱ for the infarct region. The previous data show that k₃ increases in normal myocardium after an infarct, but k₃ in salvaged postischemic myocardium remains comparable to that of the control state. Therefore, the salvaged (TTC-positive) myocardium was used as the reference region. For all samples from reperfused myocardium the rate constants used were K₁^r=0.61 mL · min^-1 · g^-1, k₂^r=0.87 min^-1. For samples from normal myocardium, the rate constants used were K₁ⁱ=0.83 mL · min^-1 · g^-1 and k₂ⁱ=1.44 min^-1.¹⁴ The same rate constants were used for calculation of k₃ for ¹⁴C- and ¹⁸F-2-deoxyglucose.

Statistics
The proportion of samples in reperfused myocardium that were viable with ¹⁴C-2-deoxyglucose (early reflow) was compared with the proportion that were viable with ¹⁸F-2-deoxyglucose (late reflow) by ² analysis. Hemodynamic and regional myocardial blood flow measurements during ischemia and reperfusion were compared by ANOVA.²³ Myocardial 2-deoxyglucose contents were compared between control and postischemic regions in each dog by ANOVA. The proportions of samples that appeared to undergo necrosis during the ischemic period, or necrosis during reperfusion, or remained viable were compared with collateral blood flow by regression analysis. Results are reported as mean±SD, and a value of P<.05 is described as significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Uptake and Distribution of 2-Deoxyglucose
The distribution of ¹⁴C-2-deoxyglucose and ¹⁸F-2-deoxyglucose in reperfused myocardium, after simultaneous injection of both tracers, is shown in Fig 1. Data are shown for 108 myocardial samples from three dogs. The slope (0.93) reflects slightly lower ¹⁸F-2-deoxyglucose uptake after 5 minutes of reperfusion, but neither the group regression nor individual regressions differed from the line of identity, indicating that tissue distributions and retention of the tracers were equivalent. Retention of ¹⁴C-2-deoxyglucose in normal and reperfused myocardium is shown for another three dogs in Fig 1. One hour after injection, mean ¹⁴C-2-deoxyglucose content in reperfused myocardium was half of that in normal myocardium (P<.01). Myocardial count activities were similar to those in subsequent experiments. During 3 hours after injection, there was no significant change in mean ¹⁴C activity in normal or reperfused myocardium. Biopsies obtained 4 hours after reperfusion had ¹⁴C activity similar to that of biopsies taken after 1 hour. In the other two dogs, which had both flow and ¹⁸F-2-deoxyglucose uptake measurements, samples were grouped according to myocardial blood flow. Samples with severe ischemia (collateral flow <10% of control) and impaired reperfusion (flow <40% of control), which are likely to have the most severe necrosis, had similar ¹⁸F-2-deoxyglucose content after 35 minutes (40% of control) and after 3 hours (41% of control) reperfusion. These data show that radiolabeled 2-deoxyglucose, injected during early reperfusion, is retained over 4 hours in reperfused myocardium.

View larger version (23K): [in this window] [in a new window]   Figure 1. Top, Uptake of 14C-2-deoxyglucose and 18F-2- deoxyglucose after simultaneous injection of both tracers. Count activities in reperfused myocardium were normalized to mean activity of normal region for comparison between dogs. Lines of identity (dashed) and group regression (solid) are shown. Bottom, Retention of 14C-2-deoxyglucose in normal (open symbols) and reperfused (closed symbols) myocardium. Data are shown for each of three dogs.

Comparison of 2-Deoxyglucose Uptake With Histopathology
The uptake of ¹⁸F-2-deoxyglucose in normal and reperfused myocardium was compared with electron microscopy findings in seven dogs. Blood flow to control (1.45±0.54 mL/min per gram) and reperfused (1.54±0.34 mL/min per gram) myocardium was similar, but ¹⁸F-2-deoxyglucose content was less in reperfused myocardium (61 465±30 328 counts/min per gram) than in the control region (106 760±71 013 counts/min per gram, P<.05). Samples were randomly selected from control (n=6) and reperfused (n=28) regions. All control samples had the ultrastructural features of viable myocardium (Fig 2A). Among samples from reperfused myocardium, 8 manifested reversible ischemic injury (Fig 2B) and 20 had irreversible injury (Fig 2C). The mean k₃ for these samples were 0.320±0.152 min^-1 for control, 0.194±0.070 min^-1 for reversible injury, and 0.098±0.055 min^-1 for irreversible injury (P<.01 versus reversible injury). The individual k₃ values were compared with the electron microscopy findings to determine which value of k₃ was the best discriminator between reversible and irreversible injury (Fig 3). A value of k₃=0.125 min^-1 appeared to be the best indicator of viability (sensitivity=93%, specificity=85%, predictive accuracy=88%). If a value of k₃=0.100 min^-1 was used, specificity decreased to 60%, and if a value of k₃=0.150 min^-1 was used, sensitivity decreased to 86%. For the subsequent serial studies of 2-deoxyglucose uptake, samples with k₃ <0.125 min^-1 were considered nonviable and those with k₃ 0.125 min^-1 were considered viable.

View larger version (84K): [in this window] [in a new window]   Figure 2. A, Ultrastructure of normal myocardium with intact sarcolemma and normal nuclear chromatin. The k3 for this sample was 0.296. B, Reversibly injured myocardium with clumping of nuclear chromatin and mitochondrial swelling but intact sarcolemma. The k3 for this sample was 0.212. C, Irreversibly injured myocardium with mitochondrial swelling and loss of cristae with calcium precipitates and marked nuclear changes. The k3 for this sample was 0.085. B and C are from the same dog.

View larger version (18K): [in this window] [in a new window]   Figure 3. Sensitivity and specificity of different levels of k3 for detection of viability in reperfused myocardium, in comparison with electron microscopy findings.

Serial Studies of 2-Deoxyglucose Uptake in Reperfused Myocardium
Among 18 dogs included in the group, two had ventricular fibrillation shortly after reperfusion and were not resuscitated. Four dogs with collateral blood flows >30% control flows and no evidence of infarction on TTC staining were excluded from analysis. Data are reported for 12 dogs that completed 90 minutes of ischemia and 4 hours of reperfusion with TTC evidence of infarction. Heart rate did not change from before ischemia (136±19 bpm) to 3 hours of reperfusion (135±28 bpm), but mean arterial pressure was lower during ischemia (92±22 mm Hg) than before ischemia (102±24 mmHg) or after 3 hours of reperfusion (102±20 mm Hg, P<.05 versus ischemia). Blood flow in the circumflex territory was 1.07±0.57 mL/min per gram after 5 minutes of reperfusion and 1.03±0.55 mL/min per gram after 3 hours. Collateral flow during LAD occlusion was 0.07±0.04 mL/min per gram, increasing to 1.19±0.34 mL/min per gram during early reperfusion. After 3 hours, flow in the LAD myocardium was 0.68±0.23 mL/min per gram (P<.05 versus early reperfusion). Collateral flow to the TTC-positive region was greater than flow to the TTC-negative region (P<.01) (Table 1). Blood flow was reduced in the no-reflow zones, but there was no other difference in flow between TTC-negative and TTC-positive regions during reperfusion.

View this table: [in this window] [in a new window]   Table 1. Regional Blood Flow in Reperfused Myocardium

A total of 850 myocardial samples were examined (340 from the control circumflex territory and 510 from the reperfused LAD territory). Among samples from reperfused myocardium, 237 were from TTC-negative regions, including 58 from the no-reflow zone, and 164 were from TTC-positive regions. There were 109 samples from borders of TTC-negative and TTC-positive myocardium, which were not included in the data analysis. Among the 401 samples from reperfused myocardium, the ¹⁸F-2-deoxyglucose k₃ threshold of 0.125 min^-1 identified 235 of the 237 TTC-negative samples as nonviable and 155 of the 164 TTC-positive samples as viable (sensitivity for identifying viable myocardium, 93%; specificity, 99%; predictive accuracy, 97%).

Contrasting examples of 2-deoxyglucose uptake during early and late reperfusion are shown in Figs 4 and 5. Data from one heart are shown in Fig 4. Samples are grouped by origin from TTC-positive, TTC-negative, or no-reflow regions. This heart had reduced ¹⁴C-2-deoxyglucose uptake in TTC-negative myocardium after 5 minutes of reperfusion (Fig 4, A and B). In TTC-negative samples the k₃ for ¹⁴C-2-deoxyglucose was 0.082±0.019 min^-1, and all but one of these samples had k₃<0.125 min^-1, indicating necrosis by 5 minutes after reperfusion. The same TTC-negative samples all had k₃<0.125 min^-1 for ¹⁸F-2-deoxyglucose (Fig 4, C and D), with k₃=0.067±0.015 min^-1 (NS versus ¹⁴C-2-deoxyglucose). Eight samples from no-reflow regions all had k₃<0.125 min^-1 at both early and late reperfusion. The data in this heart are consistent with irreversible injury occurring during ischemia.

View larger version (22K): [in this window] [in a new window]   Figure 4. Uptake of 14C-2-deoxyglucose and 18F-2-deoxyglucose in reperfused myocardium of one heart. The TTC-negative region had low uptake of both 14C-2-deoxyglucose (A) and 18F-2-deoxyglucose (C). The TTC-negative samples were below the viability threshold (k3=0.125 minutes-1 shown as dashed line) for both 14C-2-deoxyglucose (B) and for 18F-2-deoxyglucose (D), suggesting that necrosis was complete by the end of the ischemic period.

View larger version (20K): [in this window] [in a new window]   Figure 5. Uptake of 14C-2-deoxyglucose and 18F-2-deoxyglucose in reperfused myocardium of another heart. The TTC-negative samples had uptake of 14C-2-deoxyglucose similar to TTC-positive samples with k3 values above the viability threshold (k3=0.125 min-1 shown as dashed line) (A and B). Later, TTC-negative samples had low uptake of 18F-deoxyglucose (C) with k3 values below the viability threshold (D), consistent with the occurrence of irreversible injury during reperfusion.

Data for a different heart are shown in Fig 5. There was avid uptake of ¹⁴C-2-deoxyglucose during early reperfusion in both TTC-positive and TTC-negative regions (Fig 5A). The calculated k₃ for ¹⁴C-2-deoxyglucose in the TTC-negative region was 0.228±0.077 min^-1. All samples in the TTC-negative region had k₃>0.125 min^-1, indicating viability at 5 minutes of reperfusion (Fig 5B). After 3 hours of reperfusion the k₃ for ¹⁸F-2-deoxyglucose in the same TTC-negative samples was 0.057±0.016 min^-1 (P<.001 versus ¹⁴C-2-deoxyglucose), and all TTC-negative samples had k₃<0.125 min^-1, indicating irreversible injury by the time of ¹⁸F-2-deoxyglucose injection (Fig 5, C and D). The data in this heart are consistent with the occurrence of necrosis during the reperfusion period.

The 2-deoxyglucose k₃ values in TTC-positive and TTC-negative myocardium are summarized for the group in Table 2. In the no-reflow region, k₃ after 5 minutes of reperfusion was 0.096±0.027 min^-1 and after 3 hours 0.060±0.023 min^-1 (NS). All but 2 of these samples were classified as nonviable after 5 minutes and all were nonviable after 3 hours of reperfusion. In the TTC-negative region, k₃ decreased from 0.184±0.070 min^-1 after 5 minutes of reperfusion to 0.077±0.032 min^-1 (P<.0001) after 3 hours. After 5 minutes, 117 of these 179 samples were viable according to the k₃ threshold, but after 3 hours only 4 were viable (P<.0001). In the TTC-positive region, mean k₃ after 5 minutes (0.170±0.087 min^-1) and after 3 hours (0.170±0.056 min^-1) were similar, and 155 of these 164 samples were viable. Among 237 samples from no-reflow and TTC-negative infarct regions, 119 (50.2%) were viable at the time of ¹⁴C-2-deoxyglucose injection, but only 4 (1.7%) were viable at the time of ¹⁸F-2-deoxyglucose injection, consistent with the development of irreversible injury during reperfusion. The proportion of samples classified as viable in the no-reflow, TTC-negative, and TTC-positive regions, according to different values of k₃ are shown in Fig 6. For k₃ between 0.100 and 0.200 min^-1, almost no samples in the no-reflow zones were classified as viable by ¹⁴C-2-deoxyglucose or ¹⁸F-2-deoxyglucose. In TTC-positive myocardium, the proportions of samples classified as viable by ¹⁴C-2-deoxyglucose and ¹⁸F-2-deoxyglucose were similar, whichever value of k₃ was used. In TTC-negative myocardium, there was a marked difference between the proportions of samples classified as viable by ¹⁴C-2-deoxyglucose and those classified as viable by ¹⁸F-2-deoxyglucose.

View this table: [in this window] [in a new window]   Table 2. Regional 2-Deoxyglucose Uptake in Control and Reperfused Myocardium

View larger version (17K): [in this window] [in a new window]   Figure 6. Comparisons of the number of viable samples in infarcted and salvaged regions of the LAD territory at time of injection of 14C-2-deoxyglucose and of 18F-2-deoxyglucose. Data are shown for the proportion of viable samples in each region, according to different levels of k3. A, Samples from within the no-reflow zone. All but two samples appeared nonviable by 5 minutes of reperfusion and all were nonviable after 3 hours of reperfusion. B, Samples from the TTC-negative region. Almost all samples were necrotic after 3 hours of reperfusion, but only 35% of these samples were nonviable after 5 minutes of reperfusion. At each k3 value, many more samples were viable at the time of 14C-2-deoxyglucose injection than at the time of 18F-2-deoxyglucose injection. *P<.05, **P<.01 vs 18F-2- deoxyglucose at same k3. C, Samples from within the salvaged TTC-positive zone. There is no difference in viability determined by 14C-2-deoxyglucose and 18F-2-deoxyglucose. For the threshold of k3=0.125 min-1, nearly all samples are deemed viable at both 5 minutes and 3 hours after reperfusion.

Ischemic Necrosis, Reperfusion Necrosis, and Infarct Size
The ischemic risk region occupied 30.0±4.0% of the left ventricle. The mean infarct size was 37.6±21.0% of the risk region (range, 5.7% to 68.3%), and the mean size of the no-reflow region was 8.3±7.7% of the risk region. Total infarct size was inversely related to collateral blood flow during ischemia (r= -.87), and the extent of the no-reflow zone was also inversely related to collateral blood flow (r= -.64).

The proportion of anatomic infarct size due to necrosis during ischemia or reperfusion in each dog was calculated from the number of infarct samples that were nonviable by both ¹⁴C-2-deoxyglucose and ¹⁸F-2-deoxyglucose (ischemic necrosis) or viable by ¹⁴C-2-deoxyglucose but nonviable by ¹⁸F-2-deoxyglucose (reperfusion necrosis) for that particular dog. The proportions of infarct size due to ischemic or reperfusion necrosis varied according to the level of collateral blood flow (Fig 7). The four dogs with the lowest collateral blood flow (2.8±1.1% of control flow) had the largest infarcts (54±7% of risk region) and in these dogs 86±12% of the infarct samples were irreversibly injured by 5 minutes of reperfusion. The four dogs with intermediate collateral flows (4.8±0.9% of control) had an infarct size of 46±15% of risk region, but only 30±23% of infarct samples were irreversibly injured by 5 minutes of reperfusion. In contrast, the four dogs with the highest collateral flows (12.7±3.1% of control) had small infarcts (13±10% of risk region) and only 7±7% of infarct samples were nonviable by 5 minutes of reperfusion. As collateral blood flow increased, the proportion of infarction due to irreversible injury during reperfusion increased.

View larger version (22K): [in this window] [in a new window]   Figure 7. A, Relationship between collateral flow (% of normal resting flow) and infarct size due to ischemic necrosis according to 2-deoxyglucose data. B, The proportion of total infarct size due to ischemic necrosis was inversely related to collateral flow. Low collateral flows were associated with large infarcts, almost all due to ischemic necrosis. C, Relationship between collateral flow and infarct size due to reperfusion necrosis, according to 2-deoxyglucose data. Regions most likely to manifest necrosis during reperfusion were those with only mild or moderate ischemia during LAD occlusion. D, The proportion of total infarct size due to reperfusion necrosis. As collateral flows increased, a greater proportion of necrosis appeared to occur during reperfusion.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study examined changes in viability of reperfused myocardium. In salvaged myocardium, uptakes of ¹⁴C-2-deoxyglucose during early reperfusion and ¹⁸F-2-deoxyglucose 3 hours later were similar and above a threshold of viability. In samples from severely injured no-reflow regions, both ¹⁴C-2-deoxyglucose and ¹⁸F-2-deoxyglucose uptakes were below the viability threshold. In most samples from the infarct region, ¹⁴C-2-deoxyglucose uptake during early reperfusion was above the viability threshold, but 3 hours later uptake of ¹⁸F-2-deoxyglucose in the same samples was reduced to levels associated with irreversible injury, consistent with the occurrence of myocyte necrosis during reperfusion. Regions with low collateral flows had nearly complete loss of viability by the end of ischemia, but regions with higher collateral flows appeared to undergo necrosis during reperfusion.

Myocardial Viability and 2-Deoxyglucose
The tracer ¹⁸F-2-deoxyglucose is widely used as a viability marker in clinical studies with PET.²⁴ The phosphorylated tracer accumulates within myocytes, reaching steady state by 60 minutes,²⁵ because dephosphorylation is slow and alternate metabolic pathways are limited.²⁶ Myocardial uptake of 2-deoxyglucose depends on tracer delivery and kinetics of 2-deoxyglucose transport and phosphorylation. We calculated k₃, the rate constant for 2-deoxyglucose phosphorylation, in individual myocardial samples, using parameters derived from PET studies of reperfused infarcts in canine hearts.¹⁴ The values of k₃ calculated for normal, salvaged, and infarcted myocardium in this study are similar to the previous PET data. To define an appropriate value of k₃ as a marker of viability, we compared tissue k₃ with ultrastructural appearance and histochemical staining of reperfused myocardium. Myocardial samples with an ultrastructural pattern of irreversible injury, after 1 hour of reperfusion, had k₃<0.125 min^-1, but samples showing reversible injury had k₃ 0.125 min^-1. Similarly, samples that were necrotic by TTC stain after 4 hours of reperfusion had k₃<0.125 min^-1, but samples that were viable by TTC stain had k₃ 0.125 min^-1. The lowest k₃ values were found in the no-reflow regions with the most severe ischemic injury. Small differences in predictive accuracy of k₃=0.125 min^-1 for detection of viability compared with electron microscopy or TTC stain reflect different numbers of samples in each comparison. It should be noted that this threshold may not be universally applicable, particularly for clinical PET studies when patients are given a glucose load.

The k₃ in the reference region and the value of the viability threshold might change during reperfusion, but our pathology comparisons indicate the same viability threshold at 35 minutes and at 4 hours after reperfusion. There was no systematic change in k₃ in the normal myocardium in this study and the previous PET study¹⁴ found no change in k₃ in salvaged myocardium. The proportion of samples deemed viable by ¹⁴C-2-deoxyglucose and ¹⁸F-2-deoxyglucose in TTC-positive myocardium were the same across a wide range of k₃ values. To avoid any bias related to selection of the threshold value of k₃, we determined the number of samples that were viable in the infarct region according to a wide range of threshold values of k₃. Irrespective of the k₃ threshold used, many samples from the TTC-negative infarct region, which were viable at the time of ¹⁴C-2-deoxyglucose injection after 5 minutes of reperfusion, were nonviable by the time of ¹⁸F-2-deoxyglucose injection 3 hours later.

Interpretation of Findings
Approximately half of myocardial samples from the TTC-negative infarct region were apparently viable during early reperfusion, but during the next 3 hours exhibited a decrease in 2-deoxyglucose phosphorylation to the levels found in necrotic myocardium from the no-reflow zone. This observation is consistent with the occurrence of myocardial necrosis during reperfusion, but several other possible interpretations should be examined. Loss of ¹⁸F-2-deoxyglucose during late reperfusion is unlikely to account for the observed differences in myocardial 2-deoxyglucose content between 5 minutes and 3 hours of reperfusion. First, our initial experiments showed that myocardial content of 2-deoxyglucose was largely unchanged during 4 hours of reperfusion. Second, if ¹⁸F-2-deoxyglucose were lost from necrotic myocytes, then ¹⁴C-2-deoxyglucose would also be lost. Third, increased metabolism of ¹⁸F-2-deoxyglucose-phosphate is unlikely, as the rate constant for dephosphorylation remains an order of magnitude below k₃ during reperfusion.¹⁴

Impaired delivery of ¹⁸F-2-deoxyglucose to the infarct region after 3 hours of reperfusion is also unlikely to account for our findings. Blood flow to the infarct zone was mildly reduced after 3 hours, but mean flows in the TTC-negative and TTC-positive regions were similar, and myocyte uptake of ¹⁸F-2-deoxyglucose at steady state is independent of blood flow. Reperfusion of infarcted myocardium is associated with myocyte swelling and interstitial edema.²¹ The true uptake of ¹⁴C-2-deoxyglucose during early reperfusion might be underestimated when the sample is weighed after 4 hours. This error would underestimate the calculated k₃ for early reperfusion but could not explain the differences in k₃ found in this study. Correction for tissue edema in the infarcted myocardium was used in this study, but even in the absence of any such correction, 60% of samples from TTC-negative myocardium were viable after 5 minutes of reperfusion, with k₃>0.125 min^-1 for ¹⁴C-2-deoxyglucose.

Reperfusion of irreversibly injured myocytes is associated with contraction bands, cell swelling, and sarcolemmal disruption.^{27 28} Such cells would be unlikely to accumulate ¹⁴C-2-deoxyglucose, consistent with our finding in samples from the no-reflow zones. An increase in sarcolemmal permeability²⁹ leads to loss of enzymes such as creatine kinase. The reduced ¹⁸F-2-deoxyglucose content seen during later reperfusion might reflect washout of hexokinase from necrotic myocytes, but ¹⁴C-2-deoxyglucose would also be lost from the same myocytes. Our observations cannot be explained as an artifact of tissue edema or tracer washout. The most likely explanation is that myocytes, which were viable during early reperfusion, subsequently lost viability during the next 3 hours of reperfusion.

Irreversible Myocardial Injury During Reperfusion
Many samples from infarcted myocardium appear to have undergone necrosis after restoration of coronary blood flow. Myocytes can undergo necrosis in the presence of apparently adequate coronary perfusion, as with catecholamine stress,^{30 31} loss of calcium homeostasis,^{32 33} or reoxygenation after anoxia.³⁴ It is also now known that myocytes may be programmed to die through the process of apoptosis.³⁵ Although normally a mechanism for removal of senescent cells, it is possible that apoptosis may be responsible for large scale cell loss under pathological conditions.

The small residual uptake of 2-deoxyglucose observed in infarcted myocardium may represent a few surviving myocytes, or uptake in endothelial cells or fibroblasts, but the volume of these elements is small compared with myocyte volume. Neutrophil leukocytes accumulating during reperfusion^{36 37} are a potential site of 2-deoxyglucose uptake, but leukocyte uptake of ¹⁸F-2-deoxyglucose in reperfused myocardium is small compared with overall myocyte uptake.³⁸ Furthermore, any such error would result in an increase in tissue ¹⁸F-2-deoxyglucose uptake, which is opposite to our observations.

Two other studies have reported data consistent with the occurrence of irreversible myocardial injury during reperfusion. One study in rabbits, using sequential tissue staining with horseradish peroxidase and TTC, found an apparent increase in infarct size during 3 hours of reperfusion.³⁹ A canine study, using radionuclide-labeled antimyosin antibodies, found a progressive increase in antibody binding in reperfused myocardium,⁴⁰ which suggests lethal injury, although these observations could also be explained by increasing sarcolemmal permeability in infarcted myocytes.

This study does not define the mechanism of lethal myocardial injury occurring during reperfusion, although the concordance between the time course of reperfusion injury and neutrophil infiltration^{36 37} is compelling. Intervention studies have implicated neutrophil leukocytes in the pathogenesis of irreversible injury during reperfusion.^{7 8} Although it is possible that myocytes are "programmed" for inevitable necrosis during reperfusion, as a result of an irreversible ischemic insult, our data show that a significant proportion of samples from the infarct region are viable at the time of reperfusion and many interventional studies argue that reperfusion necrosis is not inevitable.

	Selected Abbreviations and Acronyms

 

LAD = left anterior descending coronary artery TTC = triphenyl-tetrazolium chloride PET = positron emission tomography

	Acknowledgments

 
This study was supported by USPHS grant 17655 (Specialized Center of Research in Ischemic Heart Disease) from the National Heart, Lung, and Blood Institute, Bethesda, Md. Dr Jeremy was supported by an Overseas Research Fellowship of the National Heart Foundation of Australia and a Telectronics Research Fellowship of the Royal Australasian College of Physicians. The authors wish to thank Dr Robert Dannals of the Division of Nuclear Medicine for the generous supply of ¹⁸F-deoxyglucose, Anthony Di Paula for laboratory assistance, Dr Hugh McCarron for statistical analyses, and Christine Holzmueller for secretarial assistance.

Received June 5, 1997; revision received September 29, 1997; accepted October 7, 1997.

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