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Ann Thorac Surg 2001;71:1845-1855
© 2001 The Society of Thoracic Surgeons
a Departments of Surgery, Medicine, and Pathology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
Accepted for publication March 23, 2001.
Address reprint requests to Dr Edmunds, Department of Surgery, Hospital of the University Pennsylvania, 5000 Ravdin Ct, Philadelphia, PA 19104-4283
e-mail: hank.edmunds{at}uphs.upenn.edu
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Abstract |
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Methods. The hypothesis was tested in a sheep model of anteroapical infarction that has no collateral blood flow to the area at risk. After coronary ligation or arterial occlusion for 1 or 6 hours, sheep had serial hemodynamic and quantitative echocardiographic studies before and after infarction and 2, 5, 8, and 12 weeks later. Hearts were examined by light and electron microscopy at 2 and 12 weeks; hydroxyproline and ratios of type I/III collagen were measured at 12 weeks.
Results. After coronary occlusion, left ventricular (LV) function progressively decreased and size increased to form an anteroapical aneurysm. After 1 hour of ischemia, neither resting LV size nor function changed; the infarct contained a midmyocardial scar between epicardial and endocardial muscle. After 6 hours of ischemia, LV function was significantly better than that in nonperfused sheep. Two weeks after 6 hours of ischemia, no viable myocytes were visible by light microscopy, but electron micrographs showed rare intact nucleated myocytes with scarce cytoplasmic myofibrils. At the 12th week epicardial and endocardial myocytes reappeared in the infarct. Infarct collagen type I/III ratios were 1.2 in reperfused groups and 0.7 in nonperfused sheep.
Conclusions. Delayed reperfusion causes loss and subsequent reappearance of ovine epicardial myocytes, improves collagen type I/III ratios, and attenuates LV dilatation and loss of function. One hypothesis to explain the reappearance of myocytes is that reperfusion partially reverses an incomplete apoptotic process.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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The mechanism by which delayed reperfusion of an acute infarction is beneficial is not understood; yet survival is improved [2, 6], left ventricular function may or may not improve [7, 8], infarct deformation is reduced, and ventricular dilatation is less than in patients without reperfused infarctions [8]. The question of whether the presence of surviving myocytes within the infarct is necessary for delayed reperfusion to confer a benefit has not been carefully examined in patients because variable collateral blood flow confounds the completeness of ischemia to the area at risk. Furthermore, the completeness of myocyte necrosis cannot be determined in vivo. Animal studies have not addressed this specific question and inferences from available data are confounded by short periods of ischemia [9], short-term follow-up [9–11], and use of models with preformed collateral coronary vessels to the area at risk [11, 12].
The present study was designed to compare the effect of reperfusion on postischemic ventricular remodeling in the presence and absence of surviving myocytes in the ischemic area. The hypothesis was that reperfusion would reduce infarct expansion in the absence of surviving myocytes in the area at risk.
This hypothesis was tested using an ovine infarction that uniformly remodels into an anteroapical aneurysm [13]. This model was chosen because sheep hearts do not have preformed coronary collateral vessels, infarctions have sharply defined borders, and coronary arterial anatomy varies little between sheep. Permanent occlusion of the infarct artery and 1- and 6-hour ischemic periods were studied and followed by serial measurements during 12 weeks of remodeling. The shorter ischemic period was expected to salvage some myocytes; permanent occlusion and the longer ischemic period were not expected to salvage any ovine myocytes.
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Material and methods |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Surgical protocol
All sheep were induced with sodium thiopental (7 to 20 mg/kg intravenously [IV]), intubated, anesthetized with isoflurane (1.5% to 2%), and ventilated with oxygen (Drager anesthesia monitor, North American Drager, Telford PA). All animals received glycoprolate (0.01 to 0.02 mg/kg IV), cefazolin (1 g IV), and gentamicin (200 mg IV). Postoperatively, animals received flunixin meglumine (2 mg/kg, intramuscularly [IM]) when extubated, 4 hours later and the next morning, and buprenorphine (1 to 3 µg/kg, IV or IM) 3 hours after extubation for pain relief. Sheep were treated in compliance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985).
Using aseptic technique, a protective rubber buttress was sewn to the epicardium over the homonymous (designated "LAD" in this report) and second diagonal (D2) coronary arteries approximately 40% from the apex through a left anterolateral thoracotomy. Polypropylene snares were then placed around the coronary arteries [14]. An ultrasonic flow probe (number 20A; Transonic Systems, Ithaca, NY) was placed around the aortic root and two epicardial pacemaker wires were sewn to the left atrium. The wound was closed and animals recovered.
Baseline data
After 10 to 14 days, sheep were induced, as described above, anesthetized with isoflurane (1.5% to 2%), intubated, and placed supine. Surface electrocardiogram was continuously monitored. A Swan-Ganz catheter (131h-7F, Baxter Healthcare Corp, Irvine, CA) was introduced into the pulmonary artery through the left internal jugular vein. High-fidelity pressure transducers (Spc-350, Millar Instruments Inc, Houston, TX) were inserted from the femoral arteries into the left ventricle (LV) and ascending aorta. Animals were disconnected from the ventilator and the heart was paced atrially at 120 beats/min for all measurements and echocardiograms.
Aortic flow, LV pressure, and thermodilution cardiac outputs were measured under steady-state conditions in triplicate and averaged. Stroke work was calculated from simultaneous measurements of stroke volume (aortic flow probe) and LV pressure as previously described [15].
Infarction and ischemia/reperfusion
After base line echocardiographic and hemodynamic measurements, coronary arterial snares were exteriorized. Heparin (20,000 units IV) was given. Snares were tightened to produce complete ischemia of the anteroapical LV comprising approximately 24% of the LV mass [13]. Snares were tied tight and reburied in group 1 animals and removed after beginning reperfusion in both reperfusion groups. Postinfarction hemodynamic and echocardiographic measurements were made 50 minutes after onset of snare occlusion in all sheep. Group 3 animals were awakened 1 hour after onset of ischemia; 6 hours later snares were removed and the wound was closed using local lidocaine anesthesia.
Sheep are extremely prone to postinfarction ventricular arrhythmias [13, 15]. Postinfarction and reperfusion arrhythmias are treated prophylactically, but expected attrition during the first 24 hours is approximately 50%. Animals receive amiodarone (150 mg IV, 30 minutes before infarction), lidocaine (2 to 4 mg/kg per minute), magnesium (1 g), calcium chloride (200 mg), and potassium supplementation to keep the potassium concentration above 4.5 mg/dL before infarction. Mean blood pressure was maintained above 80 mm Hg using small infusions of phenylephrine; cardiac output was maintained greater than 2.0 L/min with an epinephrine infusion. Hearts are defibrillated electrically when necessary, but defibrillation is only rarely successful. Animals are observed in the laboratory suite 2 to 3 days after infarction and daily thereafter.
Serial measurements
Sheep were anesthetized, intubated, and ventilated for serial hemodynamic and echocardiographic measurements at 2, 5, 8, and 12 weeks after infarction. The protocol described under base line data was followed.
Echocardiography
Subdiaphragmatic two-dimensional echocardiographic images were obtained through a sterile midline or subcostal laparotomy using a 1.8 to 4.2 MHz broadband probe (S4) and SONOS 5500 ultrasound system (Agilent Technologies, Andover, MA) and were recorded on 
-inch videotape at 30 Hz (Panasonic AG-6300 VHS recorder, Matsushita Electric, Japan) [16]. Left ventricular short axis images at three levels (at the tips of the papillary muscles, at the bases of the papillary muscles, and at the apex) and two orthogonal long-axis views were obtained. Left ventricular apical long-axis views were used to calculate LV cavity volumes by biplane Simpson’s method.
Completeness of ischemia was verified by contrast echocardiography in all animals except in the first 2 sheep of group 3 (coronary angiography was used in these animals). Contrast (0.1 mL mixed in 10 mL blood, Optison, Mallinckrodt, St. Louis, MO) was injected into the left main coronary ostium 20 to 30 minutes after the onset of ischemia. High fidelity transaxial long and short axis images at the level of the infarct were obtained using second harmonic echocardiographic imaging (SONOS 5500, Agilent) and stored on magneto-optical disk and videotape.
Validation of contrast echocardiography and completeness of ischemia
Fifteen million colored 15.5-µm diameter microspheres (NuFlow, North Hollywood, CA) were injected into 6 nonprotocol, anesthetized, intubated sheep to validate the absence of collateral circulation and completeness of ischemia during snare occlusion. Injections were made 10 minutes before snares were drawn tight, 30 minutes before reperfusion and 30 minutes after release of the snares 1 (n = 2) or 6 hours (n = 4) after onset of ischemia. Animals were euthanized 1 hour (n = 1), or 2.5 weeks (n = 5) later. Myocardial sections were obtained (see below), weighed, and dissolved in potassium hydroxide overnight. Different colored microspheres were recovered and counted in a Coulter Counter (Z1, Coulter Co, Hialea, FL).
Autopsy and pathologic studies
After the last serial study, the heart was arrested with 500 mL of cold crystalloid cardioplegic solution. Simultaneously the inferior vena cava was transected. The arrested heart was rapidly excised, placed into a basin of saline and ice, and trimmed of extraneous tissue. The trimmed heart was patted dry, weighed, and manually cut base to apex to expose the entire LV. Caliper measurements of wall thickness were made at the center of the apical infarct. Full thickness myocardial sections were obtained for collagen studies, hydroxyproline content, and light and electron microscopy from the center of the infarct, the border zone, and posterior LV wall. Additional samples were frozen in liquid nitrogen and stored at -80°C in a monitored freezer.
One group 1 sheep was euthanized after the 2-week study for new onset hindlimb paralysis. One group 2 animal was euthanized 1 hour after reperfusion and another at 17 days. One group 3 sheep died 4 hours after reperfusion; 4 were euthanized 2.5 weeks later and another died of unknown causes at 15 days. These 9 hearts had histologic studies as did 19 protocol hearts.
Light microscopy
Tissue was fixed in 5% buffered formalin, paraffin embedded, sectioned at 3 to 4 micrometers, and stained with either hematoxylin and eosin or Masson’s trichrome.
Electron microscopy
Small tissue pieces of fresh heart were fixed in half Karnovsky’s fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, and embedded in epoxy resin. Forty- and 90-nm thick sections were cut with an ultramicrotome, double stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope (Hitachi H-600, Nissei Sanyo, Gaithersburg, MD).
Measurement of collagen, types I and III
Collagen type and hydroxyproline were measured in the infarct in 5 sheep from each group euthanized at 12 weeks. Five-micrometer paraffin sections were placed on glass slides. After deparaffinization and dehydration, antigen retrieval was done in a microwave oven. Rabbit anti-collagen I and III antibodies (Research Diagnostics Inc, Flanders, NJ) were added to separate slides after blocking for endogenous peroxidase and rinsing. Following rinsing, slides were exposed to secondary biotinylated anti-rabbit antibody and streptavidin-biotin-peroxidase complex.
Low power images of stained slides for each collagen type were obtained from the infarct or ischemia/reperfusion region with a stereomicroscope (Leica DMLB, Germany) at (10x) and digitized (150 pixels/mm), using a digital camera (DMC LE, Polaroid Corp, Cambridge, MA). Sample fields were 1,900,000 pixels. Blue component was selected after red-green-blue image decomposition. The gray-scale mean optical density of remote myocardium was measured in each slide (Fig 1) and a histogram was generated (Image J software, National Institutes of Health, Bethesda, MD). Positive collagen staining was defined as integrated optical density values in excess of the mean background value +2 SD [16]. Collagen-stained areas were segmented on each image by interactive thresholding above the 2-SD positive limit. A collagen index for collagen type I and collagen type III [14] was calculated by measuring the number of collagen-staining pixels for each field and dividing by the total number of pixels. An average of four fields of scar tissue was sampled for each slide (Fig 1). The collagen type I/III ratio was calculated for each field.
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Statistics
Measurements are reported as means and standard errors. Differences between groups are compared by three-way multivariate analysis of variance (ANOVA; group, time) with Bonferroni adjustment for repeated measures (NCSS 97, NCSS Statistical Software, Kaysville, UT). When the group effect was significant, one-way ANOVA was used for within-group comparisons. If the time effect was significant by one-way ANOVA, differences between before infarction measurements and measurements at subsequent times were compared by the paired t statistic. When the group effect was significant, differences between groups at the same times were compared by the unpaired t statistic. Significance was considered to be p < 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Echocardiograms
Base line quantitative and contrast echocardiograms demonstrated normal wall motion of the anteroapical region of the heart in every sheep (Fig 2A) and normal contrast enhancement in 5 of 5 sheep. All animals had contrast echocardiography after tightening snares and demonstrated no perfusion and loss of apical contractility in the area at risk (Fig 2B). Group 2 animals demonstrated reflow into the ischemic region 20 minutes after release of snares (Fig 2C). In group 3 animals, hemodynamic and contrast echocardiograms were not obtained early after reperfusion because of vulnerability to ventricular arrhythmias, but were obtained at 2 weeks to verify reperfusion. All demonstrated full contrast in the infarct region.
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In group 1 animals, LV systolic and diastolic volumes progressively increased 134% and 102%, respectively, over 12 weeks (Table 2). Both volumes in reperfused groups differed from group 1 animals by 2 weeks. Left ventricular volumes did not differ between the two reperfused groups until the 12th week (Fig 3A).
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Hemodynamic measurements
In group 1 animals LV end-diastolic pressure progressively increased from 3.5 ± 0.9 to 10.3 ± 0.7 mm Hg (Table 2). A significant rise occurred at 5 weeks. In both reperfused groups, LV end-diastolic pressure did not change significantly over 12 weeks and there were no differences between groups 2 and 3.
Resting cardiac output and stroke work progressively decreased over 12 weeks in group 1 sheep, but neither measurement changed significantly in either reperfused group. Significant differences developed between group 1 and both reperfused groups between 2 and 5 weeks (Table 2, Fig 3B).
Pathology
Reperfusion caused interstitial edema and hemorrhage within the area at risk in the group 2 sheep euthanized 1 hour after snare release and in the group 3 sheep that developed ventricular fibrillation 5 hours after reperfusion.
Figure 4 demonstrates representative gross pathologic specimens obtained at autopsy and light microscopic sections at the infarct edges. The extent of infarcted myocardium was approximately the same in all 19 sheep. Caliper wall thickness measurements at the apex of the infarct were 1.5 ± 0.2, 5.1 ± 0.3, and 3.7 ± 0.2 mm for groups 1, 2, and 3, respectively (p < 0.01 for all comparisons).
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Figure 5 demonstrates representative trichrome sections of the infarct epicardium of a representative group 3 sheep at 19 days (Fig 5A) and at the 12th week (Fig 5B). Light microscopy revealed no viable epicardial myocytes in any section of group 3 animals studied 2.5 weeks after ischemia/reperfusion (n = 5). At the 12th week, unequivocal muscle cells, not showing mitosis, were present in the epicardial layer in all sheep.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Ischemia produces an immediate outward bulging of the anteroapical region [15]. Within minutes myocytes no longer shorten [20]. After an ischemic period sufficient to disrupt capillary integrity, reperfusion engorges myocardium, stiffens the infarct, and reduces outward strain and regional wall stress by increasing regional wall thickness and decreasing the radius of curvature [21]. This early change in the material properties of ischemic myocardium attenuates infarct deformation and LV dilatation, helps to maintain a more normal LV shape, and more evenly redistributes the increase in end-systolic wall stress over the entire ventricle.
Results in group 2 animals confirmed the value of preserving myocytes in the area at risk. After 1 hour of ischemia, surviving intact myocytes and presumably some of the collagen scaffold combined to strengthen the infarcted ventricular wall. The extent of ischemic damage to the collagen scaffold was not known, but some damage must occur because even short, reversible periods of ischemia cause injury [22]. Reperfusion reduces hydroxyproline content, but increases the collagen type I/III ratio to produce a thicker, less distensible infarct at 12 weeks. The sum of these incompletely investigated processes is that infarct bulging is minimal and akinetic rather than dyskinetic, and by the 12th week none of the resting hemodynamic measurements or ventricular volumes was significantly different from base line values. Ventricular shape was largely preserved and the partially infarcted ventricle remained compensated. These data confirm and support previous laboratory and clinical observations [2, 8, 9, 23].
Reappearance of myocytes after 6 hours of complete ischemia was an unexpected discovery with possible important implications for concepts of irreversible cell death [19, 20]. Three possible sources of the new myocytes are migration from surrounding epicardial myocytes, proliferation (hyperplasia) of surrounding myocytes, or revitalization of partially damaged cells.
The observation of myocyte ghosts containing a nucleus, mitochondria, and cell membrane with few or no cytoplasmic elements (Fig 6B) supports the possibility of survival of intact cellular remnants after 6 hours of complete ischemia/reperfusion and reconstitution over the next 12 weeks. Increasing evidence indicates that apoptosis is an integral process of ischemic cell death and is independent of necrosis [24]. In rabbits, reperfusion further increases apoptosis in the center of the infarct [25]; in rats, caspase inhibitors decrease infarct size [26]. Accumulating evidence is also consistent with the notion that apoptosis in myocytes occurs over a longer period than previously thought [19, 27], may not always be complete with DNA fragmentation (ie, apoptosis interruptus [28]), and may be associated predominantly with cytoplasmic protein damage [29, 30]. Ischemic myofibrillarlytic cells are an extreme example of cytoplasmic loss with an intact nucleus [31]. Because the nuclear blueprint remains in these cells, it is conceivable that myocytes surviving an ischemic and reperfusion injury may regenerate cytoplasmic structures and myofibrils once a consistent source of energy production is restored [28, 30]. It is not known whether the abundant epicardial myocytes seen at 12 weeks are only regenerated cells or whether regenerated cells also proliferate.
In sheep, 6 hours of ischemia essentially destroys all myocytes in the area at risk, but reperfusion significantly alters subsequent remodeling. These findings support our hypothesis. Infarct bulging is attenuated and the infarct area is akinetic and not dyskinetic. Resting function is partially preserved and LV dilatation is less than nonperfused sheep, but is not completely arrested. Although resting hemodynamics did not change over 12 weeks, ventricular volumes slowly, but progressively, increased and ejection fractions decreased. Hydroxyproline content was reduced, but the infarct wall was thicker and the ratio of type I/III collagen was higher, indicating a stiffer, less deformable infarction. Nevertheless, regional wall stresses remained higher than reparative biochemical processes in the extracellular matrix and reappearance of epicardial myocytes can be overcome. Although infarct expansion progresses more slowly, unarrested expansion eventually leads to dilated cardiomyopathy and heart failure and provides the rationale for restraining expansion by surgical intervention [14].
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Acknowledgments |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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= Nonperfused sheep; • = 1-hour ischemia; 
= 6-hour ischemia. See 
