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J AM COLL CARDIOL 1037 1983;1(4) 1037-46 Reperfusion Of Ischemic Myocardium: Ultrastructural and Histochemical Aspects JUTTA SCHAPER, MD, WOLFGANG SCHAPER, MD Bad Nauheim, West Germany The effects of reperfusion on ischemic myocardium generally depend OQ the severity of the preceding ischemic injury. Reperfusion of myocardium, irreversibly injured by ischemia, produces further progression of myocardial necrosis that is accompanied by simultaneously occurring stimulation of interstitial cell proliferation resulting in scar formation. Reperfusion of reversibly injured myocardium leads to structural improvement and reorganization. Thus, it may be stated from the ultrastructural part of this study that reperfusion of ischemic myocardium induces 1) slow structural recuperation after reversible injury, and 2) accelerated cellular destruction and symptoms of scar formation after irreversible ischemic injury. We observed that the reduced tissue content of nicotinamide adenine dinucleotide (NAD), rather than reduced dehydrogenase activity, is the basis of histochemical reactions employing tetrazolium salts. Directly measured NAD tissue content in ischemic tissue correlated well with the degree of ultrastructural injury and Emergency coronary bypass surgery and aggressive thrombolytic therapy are gaining widespread interest and are currently advocated and used in treatment of unstable angina and impending myocardial infarction (1-8). Reduction or complete prevention of myocardial infarction is the rationale for the proposed beneficial effects of reperfusion (9-12), which are still controversial. Jennings and Reimer (13) and Schaper (14), in recent reviews on reperfusion of ischemic myocardium, showed that reflow is able to limit infarct size within 3 hours and may still be successful up to 5 to 6 hours after onset of the ischemic event. Others (15-18) claim that the reperfusion itself has harmful effects on ischemic cardiac tissue. This conviction may be partially a matter of the experimental model of ischemia used, that is, the globally From the Department of Experimental Cardiology, Max-Planck-Institute, Bad Nauheim, West Germany. Address for reprints: Jutta Schaper, MD, Max-Planck-Institute, Department of Experimental Cardiology, Benekestrasse 2, 0-6350 Bad Nauheim, West Germany. © 1983 by the Amencan College of Cardiology with macroscopic differential staining. Occlusion of two small coronary arteries in the same heart followed by reperfusion of only one artery (identical occlusion times for both arteries) showed identical infarct sizes for reperfused and nonreperfused myocardium for occlusion times of 3 and 6 hours. When the effects of occlusion times of less than 3 hours are studied with tetrazoliuni salts, a difficulttechnical problem arises: during that time, tissue-NAD concentrations have not decreased enough to enable differential staining. Reperfusion leads to washout of NAD, thus producing differential staining; this may be a harmful effect of reperfusion. However, because early reperfusion leads to significant structural and functional recovery and to small infarcts, reperfusion injury is unlikely to occur. Both ultrastructural and histochemical evidence suggest that reperfusion is beneficial for reversibly injured tissue but accelerates the decay of irreversibly injured tissue. ischemic heart (18) or the model of the oxygen or calcium (Ca 2 +) paradox (19). Bulkley and Hutchins (20,21) reported the occurrence of necrotic foci in the presence of patent grafts in human myocardium, which they ascribed to the harmful effect of reflow after the intraoperative nonperfusion period. Montoya et al. (22) described hemorrhage into the myocardium of patients undergoing revascularization for unstable angina. Deleterious effects of myocardial reperfusion due to hemorrhage have been described in the canine heart by Breshnahan et al. (23), who also observed an extension of the origional infarct size. Several investigators (24-26) showed that infarct size extension due to reperfusion is only present after longer periods of coronary artery occlusion, and Corday et al. (27) suggested that the enlargement of infarct size was merely caused by hemorrhage into necrotic myocardium but not gy damage to salvageable tissue. Mclvamara et al. (26) also claimed that hemorrhage into the infarct is probably not automatically deleterious. Muller et al. (28) stated that reperfusion with blood seems to be the optimal method to salvage ischemic myocardium in human beings, but in their 0735-1097/83/0401037-10$03 .00 1038 J AM COLL CARDIOL SCHAPER AND SCHAPER 1983;1(4):1037-46 opinion, a definite confirmation of the beneficial effects of reperfusion was still needed. In numerous animal experiments, the possible beneficial or harmful effects of reperfusion on ischemic myocardium were investigated and many abnormalities were noted. The common denominator observed in ischemic reperfused cardiac tissue was that functional recovery occurred only very slowly and often was absent (29-35). Many different pathophysiologic factors have been implicated In causing delayed functional recovery. Impaired mitochondrial function (29,31,36,37), possibly associated with mitochondrial Ca2+ loading (30,31,38), but also Ca 2 + loading alone (39-42) have been discussed as possible mechanisms. Kotaka et al. (43) showed that mitochondrial dysfunction during coronary reperfusion was partially caused by acyl coenzyme A accumulation. Wood et al. (29) reported loss of glycogen and decreased phosphorylase activity, and Hess et al. (44) described decreased Ca2 + uptake by the sarcoplasmic reticulum and decreased activity of myofibrillar adenosine triphosphatase (ATPase). Delayed resynthesis of the depleted adenine nucleotide pool (30-32,45,46) has also been strongly associated with delayed recovery. Reimer et al. (46) showed that the adenine nucleotide pool was markedly dcreased after 15 minutes of reversible ischemia and that it was still slightly suppressed after 4 days of reflow. Microvascular defects causing persistence of reduced blood flow to the ischemic region (34,4750) and edema formation (51-53) combined with Ca2 + loading (53) have also been associated with slow recovery during reperfusion after an ischemic event. Only a few studies have described the effects of reperfusion on ischemic myocardium from an ultrastructural point of view. Jennings and his co-workers (54-59) reported most of the data on myocardial ultrastructure in ischemia and reperfusion and showed very clearly that tissue reversibly injured by ischemia shows structural recovery after reperfusion, whereas irreversibly injured tissue deteriorates further during reflow. Our own studies (60-62) on the ultrastructural changes in regional ischemia in the dog heart agreed with those of Jennings et al. From their work and ours, it becomes evident that it is nearly impossible to estimate the effects of coronary reperfusion on previously ischemic myocardium when the differentiation of reversible versus irreversible ischemic injury is lacking. Duration of coronary artery occlusion has been used in many studies (17 ,37,45,63) as an indicator of the degree of injury produced. However, because time is only one of several factors determining the development of myocardial ischemia and infarction (14), a more direct method needs to be used to investigate the effects of reperfusion. The present study, therefore, was undertaken to investigate the effects of ischemia of varying duration and of reperfusion in the same heart by electron microscopy. Ultrastructural evaluation of tissue obtained by small needle biopsy offers the unique opportunity to determine the degree of sichemic injury and the phase of recorvery during reperfusion with high accuracy and in a continuous manner using very small tissue samples. Methods Experimental design. In IS anesthetized open chest dogs, the anterior descending branch of the left coronary artery was occluded for 45, 90 or 180 minutes. At the end of the ischemic interval, transmural needle biopsy specimens from the center of the ischemic area were taken; thereafter, the arterial ligature was opened and the reperfusion period started. The chest was closed and the animals were sacrificed after 48 hours of reperfusion after transmural biopsy samples were again taken from the same previously ischemic area of the still beating heart. From the large number of dogs used in this experimental series, only three for each ischemic interval (each interval being defined as one group) will be described here. The experiments presented were deliberately selected from each group for their special and very typical features representative of the entire population. Preparation of tissue for electron microscopy. All tissue samples were subdivided into a subendocardial and a subepicardial part. Both samples were separately numbered and treated during the embedding, cutting and evaluation procedures. All biopsy material was immediately fixed in 3% cold glutaraldehyde, prepared with 0.1 M cacodylate buffer at pH 7.4. Immersion fixation for 2 to 6 hours was followed by frequent rinsing in cold cacodylate buffer during 3 consecutive days; thereafter the tissue was embedded in Epon following fixation in 2% osmic acid anhydride and dehydration in a series of ethanol plus substitution by propylenoxide using a Wakura automatic tissue processor. Semithin (1 to 2 ~m) sections were prepared and stained with toluidine blue from all samples. Artifact-free areas were selected from these for the preparation of thin sections (500 to 600 A) for electron microscopy. These sections were attached to uncoated copper grids, stained with uranyl acetate and lead citrate and viewed in a Philips EM 300 electron microscope. All micrographs, 30 to 40 per sample, were evaluated using our standardized system (62,64). Results and Discussion The ultrastructural results for each group are shown in Table 1 and the typical symptoms of cellular recovery or degradation are described and illustrated in the text. Grading of Ultrastructural Alterations in Ischemic and Reperjused Cardiac Tissue Electron microscopic investigations of ischemic and reperfused myocardium allow exact definition of the stage of progression of cellular damage and degree of recovery during reperfusion when all ultrastructural changes are evaluated in a standardized way. On the basis of earlier studies in ischemia (64), normal myocardium (Fig. 1) was differ- REPERFUSION OF ISCHEMIC MYOCARDIUM J AM cou, CARDIOl 1983;1(4):1037-46 1039 1 Figure l. Normal myocardium . Magnification 30.000 x . Figure 2. Reversible slight Ischemic injury . Magnification 13.800 x , mset rnagmfication 48.000 x , Figure 3. Reversible moderate Ischemic mJury. Magnification 13.800 x ; mset magnification 30.000 x . Figure 4. Reversible severe Ischemic mjury. Magnificanon 13,800 x : inset magnification 20,400 x , Figure 5. Irreversible ischerruc mjury. Magruficauon 13,800 x ; inset magnification 48.000 x . entiated from myocardium undergoing slight (Fig. 2), moderate (Fig. 3) and severe (Fig. 4) reversible ischemic injury and irreversible injury (Fig. 5). The estimation of ultrastructural changes in mitochondria, nuclei and myofilaments provides the most reliable and reproducible identification of the degree of ischemic injury; the systematic assessment of all other subcellular cornpo- nents , including blood vessels and extravascular space of the myocardium, contributes substantial information on pathogenetic mechanisms operative in the progression of ischemia and infarction, According to our experience with ultrastructural changes in ischemic myocardium obtained in approximately 120 dogs (62), it appears that regional ischemia may provoke a cascade of events on the cellular 1040 J AM COLL CARDIOL 1983;1(4):1037-46 . ~ SCHAPER AND SCHAPER . ,.. ." Figure 7. Endothelial prohferation and presence of increased numbers of intravascular neutrophils during reperfusion. Magnificanon 9,900 x. Figure 6. Early stage of structural recovery during coronary reperfusion. Magnification 9,900 x . Inset shows incomplete recovery of mitochondria; magnification 24,000 x. level characterized by a close interaction among myocytes, all interstitial and vascular cells and cells from the circulating blood. Structural recovery during reperfusion after reversible ischemic injury. The early stage of structural recuperation is shown in Figure 6. Myocardial mitochondria were still electron lucent; they contained only a few matrix granules and a reduced number of cristae, and the cells exhibited numerous lipid droplets. Many microvessels showed endothelial proliferative activity and contained a disproportionately high number of neutrophils (Fig. 7) and platelets. Intermediate stages were characterized by slight mitochondrial clearing (Fig. 8), while complete recovery of cardiac cells was indicated by mitrochondria exhibiting a dense matrix and numerous tortuous cristae, a typical symptom of high metabolic activity (Fig. 9). At all stages of cellular recovery, these cells showed a significantly increased number of primary and secondary lysosomes and many very active parts of the Golgi apparatus (Fig. 8 and 9). Progression of lesions during reperfusion after irreversible injury. Different stages of cellular degradation after irreversible ischemic injury were observed after 48 hours of reperfusion. Destruction of myocytes depended on the duration of the preceding ischemic interval and on myocardial localization; that is, the subendocardial samples showed further developed necrosis and repair processes than did the subepicardial tissue. After short-term ischemia, destroyed myocytes were found next to intact recovered cells (Fig. 10), but after longer ischemic periods, all cells from one particular sample were irreversibly injured. Typical ultrastructural findings in necrotizing cardiac muscle cells were disappearance of nuclei, dissolution or condensation with phospholipid of mitochondria and disappearance of myofilaments, especially Z band and I band material or persisting contracture bands (Fig. 10 to 12). The following cell types were involved in cellular degradation and simultaneous repair processes during reperfusion: neutrophil granulocytes, blood platelets and monocytes, the latter transforming into tissue macrophages, proliferating fibroblasts and endothelial cells. These cells were numerous and contained a large number of primary and secondary lysosomes (Fig. 10 to 12). Importance of Cardiac Lysosomes in Ischemia and Reperfusion Lysosomal changes were especially obvious in the tissue studied and, therefore, they will be described in a more detailed manner. With increasing severity of ischemia, the number of primary lysosomes gradually decreased within myocardial cells, whereas the amount of secondary lysosomal structures, that is, phagosomes, autophagic vacuoles and phospholipid-lipofuscin complexes, remained unaltered. After irreversible injury occurred, intact lysosomes were increasingly less evident and virtually absent at late stages of ischemia. Neither intact primary lysosomes nor an increased amount of secondary lysosomal structures was observed in myocytes during the development of necrosis. However, this situation was entirely different in moderate to severe reversible ischemic injury followed by cellular recovery during myocardial reperfusion. After 48 hours of reperfusion, many new intact primary lysosomes were present at the nuclear poles and in the periphery of the myocardial cells, and the number of secondary Iysosomes had REPERFUSION OF ISCHEMIC MYOCARDIUM J AM COLLCARDIOL 1041 1983,1(4).1037-46 Figure 9. Complete structural recovery after reversible ischemia. Note numerous lysosomal figures and active Golgi apparatus. Magnification 30,000 x. Inset shows part of a mitochondrion with prominent tortuous cristae; magnification 90,000 x. Figure 8. Intermediate state of cellular recovery after reversible ischemic injury, Note the increased number of various lysosomal structures. Magnification 24,600 x. Inset also shows different stages of lysosomes close to parts of a Golgi apparatus; magnification 54,000 x. significantly increased compared with that in the nonischemic tissue of the same heart. This finding implicates the very important role of lysosomal enzyme activity in removing, most probably by autophagocytosis, those intracellular structures that were injured by the earlier ischemic event. Leakage of proteolytic enzymes from lysosomes causing degradation at the myofibrillar level is structurally indicated by the disarrangement of sarcomeres in reperfused tissue, but apparently this process must have ended at a certain state during reperfusion, perhaps when normal cellular pH was reached again. This view is reinforced by our own ultrastructural observations on human myocardium obtained as intraoperative needle biopsy specimens from patients undergoing aortocoronary bypass surgery (65). The myocardial cells of patients with chronic coronary heart disease exhibited a very typical morphologic pattern: most of the myocytes obtained from the poststenotic area showed a decrease in size, distinct disarray of myofibrils, normal-appearing mitochondria and nuclei, but an increased number of intact primary lysosomes and a large amount of secondary lysosomal structures. Apparently, these myocytes, when underperfused during an attack of angina pectoris, were stimulated to release and, thereafter, to reproduce lysosomal enzymes to permit removal of cellular components injured by ischemia. Lysosomes in reversible versus irreversible ischemia. In summary, in irreversible cellular damage leading to myocardial infarction, lysosomes and their enzymes apparently gradually disappear from myocardial cells after contributing to the destruction of the tissue. In contrast, in reversible ischemic injury followed by long-term reperfusion, lysosomal enzyme activity may act as protective mechanism by scavenging intracellular debris, thereby contributing to the structural reorganization and recovery of ischemically injured myocytes. The cellular recovery from ischemic injury (during reperfusion) is characterized by a massive induction of lysosomes that clear the cells of debris accumulated during ischemia. The result of this endocytotic "clear up" is a live myocardial cell. Irreversibly damaged myocytes are the target of scavenger cells; their own lysosomal system apparently does not contribute to autophagocytosis. Reperfusion After Regional Ischemia: Beneficial or Harmful? From the data presented in this study (Table I), it may be concluded that the degree of injury may vary from slight to severe reversible damage in all groups. After 45 minutes, 1042 J AM COLL CARDIOL SCHAPER AND SCHAPER 1983;1(4): 1037-46 one sample showed many irreversibly injured cells, whereas after 90 and 180 minutes, irreversible injury, when present, was ubiquitous. The subendocardial samples showed more severe damage than did those from the subepicardium. The great range of severity of .ischemi~ in al~ g~oups supports the finding (14) that the time of Ischemia IS only One of several factors determining whether myocardial infarction occurs . Types of cellular reaction to reperfusion. The effects of reperfusion on ischemic cardiac tissue appeared to be very different, again independent of the time of ischemia. Three different types of cellular reaction to reperfusion were observed. I) Structural recovery after reversible ischemic injury was observed in dogs from all three groups. Improvement of cellular structures was usually present after moderate and severe reversible ischemic injury. 2) The situation did not change from that observed after the ischemic interval that usually occurred after slight to moderate reversible injury. This tissue reaction may be similar to that observed frequently in well perfused tissue during or after an ischemic insult (own observations). 3) Marked structural deterioration of myocardium that was already irreversibly injured by ischemia occurred, indicative of further progression of necrosis and the beginning of scar formation . In conclusion, the ultrastructural data presented in this study provide evidence that reperfusion of ischemic myocardium induces : I) structural recuperation after reversible injury , Figure 10. Heterogeneity of myocardial ultrastructure after a short period of ischemia plus reperfusion. Irreversible injury at left an~ top of the micrograph. but recovering almost intact myo~ardial cell at bottom and right. A very active interstitial cell is present m the center of the micrograph. Magnification 3,000 x. and 2) accelerated cellular destruction and symptoms of scar formation after irreversible ischemic injury. Reperfusion, therefore, is beneficial for reversibly injured tissue. Histochemistry Dehydrogenase-stain (tetrazolium salts). One of the most precise measurements of infarct size utilizes tetrazolium salts . The compounds act as electron acceptors in reactions catalyzed by dehydrogenases . Colorless soluble tetrazolium salts become intensely colored (production of formazan dye), and because these dyes are insoluble in water, they precipitate precisely at places where the reaction occurred. These methods were developed to diagnose human myocardial infarcts postmortem (66-68). The method is usually called the "dehydrogenase-stain" because of the mechanism involved. Dehydrogenases exhibit reduced activity in human myocardial infarction, that is, only normal muscle stains; infarcted muscle does not stain . The reaction can be carried out in ultrathin sections for electron microscopy, on thin sections for light microscopy and on myocardial rings for inspection and macroscopic size determination (planimetry and point counting). Addition of succinate or NAD. In recent years , the method has gained great popularity because true anatomic infarct size can be obtained more easily (compared with conventional light microscopy) and at much greater speed (69). However. most worker s did not recognize that the proposed mechanism of action (dehydrogenase activity and the lack thereof) is not operative in short-term (up to 6 hours) occlusions, because dehydrogenase activity does not appreciably decrease within 24 hours after coronary occlusion, at least not to a degree that affects the staining reaction (70). Yet, if an experimental occlusion lasts for 3 hours or longer, Figure II. Cellular degradation after irreversible ischemic injury. Myofilaments and mitochondna (*) are dissolving (top) . Adherence of a neutrophilic (right) and mfiltration by a phagocyt ic (bottom left) cell. Magnification 13,800 x REPERFUSION OF ISCHEMIC MYOCARDIUM J AM COLL CARDIOL 1043 1983:1(4).1037-46 differential staining with tetrazolium is obtained. Furthermore, we observed that in the presence of differential staining (normal tissue stained, infarcted tissue not stained), the addition of either succinate or nicotinamide adenine dinucleotide (NAD) caused a uniform dye precipitate, that is, previously nonstained infarcted tissue became stained. It was found that differential staining after relatively shortterm occlusions is caused not by the lack of dehydrogenases, but rather by the lack of their coenzymes, that is, NAD (70,71). In a series of experiments, we showed that the sum of NAD plus NADH remained constant in regionally ischemic tissue as long as the degree of damage was reversible by ultrastructural criteria. Total NAD decreased when the point of no return was reached. When the tissue concentration of total NAD fell below 200 pmol/g wwt (normal = 700), the tissue lost its ability to produce the formazan dye. This ability is quickly restored in vitro by the addition of NAD to the incubation medium. Succinate dehydrogenase, which becomes active when its substrate is added to the incubation medium, produces the insoluble formazan dye in infarcted muscle because this enzyme does not need NAD as a coenzyme, The reaction that takes place on the cut surface of myocardial cells is interpreted as an enzymatic cycling (70). Quantifying myocardial damage after short occlusion times. The question whether reperfusion is good or bad for previously ischemic heart muscle assumes practical significance when trying to quantitatively define the amount of damage inflicted by relatively short occlusion times (between 20 minutes and 3 hours). Within that time, it is not possible to measure infarct size with tetrazolium salts. It is, of course, possible to diagnose irreversible ischemic damage at the end of a short occlusion time, but without reperfusion, a size determination is difficult if not impossible. This, in essence, means that a subendocardial infarction that was caused by an occlusion of 2 hours' duration cannot be diagnosed quantitatively with tetrazolium salts unless the tissue is reperfused. Reperfusion aids identification of tissue that has undergone irreversible ischemic damage within these 2 hours of occlusion. Reperfusion removed (washed out) NAD from severely ischemic tissue and thus permitted differential staining. At this point, it may be necessary again to question whether reperfusion might have caused (in part) the effect that only indicates like a litmus test. It is also necessary to discuss the modes of disappearance of NAD from ischemic myocardium. NAD can disappear from ischemic myocardium by one or more of the following routes: 1) by washout through leaky membranes; 2) by enzymatic degradation through activation of glycohydrolase (72); and 3) by depressed de novo synthesis in the presence of accelerated degradation (73). Stages of sarcolemmal damage during ischemia. Results from our previous experiments suggest that, with regard to NAD deprivation of ischemic tissue, ischemia causes a two-stage damage to sarcolemma and prob- Table 1. Ultrastructural Results of Coronary Occlusion and Reperfusion in Nine Dogs Ischemia Dog Min EPI Endo h Epi Endo I 2 3 4 5 45 45 45 90 90 90 180 180 180 Slight Mod Mod Mod Slight Mod Mod Mod Irre Sev Sev Mod Mod Irre Irre Irre Mod Irre 48 48 48 48 48 48 48 48 48 NI Slight Slight Nl Mod Slight Irre Nl Irre Slight Mod Mod Mod Irre Irre Irre Slight Irre 6 Figure p. Myocardial cell destruction after irreversible ischemic injury, dissolutionof mitochondria (*) but persistmg contracture of myofilaments and presence of an active macrophage with numerous lysosomes. Magnification 39,000 x , Reperfusion 7 8 9 Endo ~ endocardial layer: Epi = epicardial layer: h = hours: Irre = irreverstble: MIn = minutes: Mod = moderate. NI = normal. Sev = severe 1044 J AM COLL CARDIOL SCHAPER AND SCHAPER 1983,1(4): 1037-46 ably other membranes. At a first stage (very probably early irreversible injury), the membranes become leaky and on reperfusion, NAD is washed out. At a later stage (advanced necrosis), the membrane-bound enzyme glycohydrolase is activated and cleaves NAD (70-72). The presence and activity of glycohydrolase in heart, brain and kidney were demonstrated by us (70,71) with the following experiment. Freshly obtained tissue was rapidly homogenized in the presence of oxygen, and aliquots of the homogenates were taken at 1 minute intervals and assayed for NAD and NADH. The pyridine nucleotides decreased within minutes through the action of activated glycohydrolase (homogenization), and the rate of decline was very fast in brain, followed by that in heart and kidney (70,71). This is also the order of susceptibility to ischemic damage. The decrease of the pyridine nucleotides in the tissue homogenates could be completely prevented when nicotinamide (the end product) was added to the incubation medium. It is unresolved which mechanism contributes most to the observed loss of total NAD in ischemic tissue. The observation of differential staining without reflow in the presence of infarcts at time intervals longer than 3 hours after coronary occlusion would suggest activation of glycohydrolase, but tissues exposed longer to ischemia were also perfused longer by collateral flow; that is, a washout effect, even in nonreperfused myocardium, cannot be ruled out. Does reperfusion irreversibly damage ischemic tissue? Because pyridine nucleotides playa key role in energy metabolism, their loss through reperfusion at a time when the diagnosis of irreversible ischemic injury by means other than NAD histochemistry is difficult, leaves doubt whether reperfusion is so beneficial. However, the observation (made using tetrazolium salts) that up to 80% of the myocardium at risk can be salvaged if reperfusion is established within 45 minutes after occlusion (69,74) suggests that a deleterious effect of reperfusion must be very small especially in comparison with its beneficial effects. The question whether already existing infarcts can become larger by reperfusion was addressed with the following experiment (74): in the same heart (canine, open chest), two medium size coronary arteries were prepared and occluded for a period of 3 hours; thereafter one artery was reperfused but not the other. This was possible by occluding one artery 1 hour before the other to allow for a 1 hour reperfusion period. Rings of left ventricular myocardium were then incubated in p-nitro blue tetrazolium and infarct sizes were compared in each heart. In another experiment, the duration of occlusion was extended to 6 hours. The purpose of the experiment was to test the hypothesis that reperfusion may irreversibly damage ischemic tissue that may have been, at the moment of reperfusion, only reversibly injured. If this were true, the infarct at the reperfused side should have been larger than at the nonreperfused side of the same heart. This was not the case at 3 or 6 hours of occlusion. The incidence of hemorrhagic infarcts was one of eight reperfused 3 hour occlusions and eight of eight in occlusions lasting 6 hours. It should be noted that Althaus et al. (75) found that reperfused hemorrhagic infarcts in the pig produced tougher scars than did nonreperfused, nonhemorrhagic infarcts. References 1. Berg R, Selinger SL, Leonard 11, Grumwald RP, O'Grady WP. Immediate coronary bypass surgery for acute evolving infarction. J Thorac Cardiovasc Surg 1981;81:493-7. 2. Pifarre R, Spinazzola A, Nermckas R, Scanlon PJ, Tobin JR. Coronary bypass for acute evolving myocardial infarction. Arch Surg 1971;103:525-8. 3. 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