Clin. Cardiol. 13, 213-217 (1990)

Reperfusion Injury M. c . FISHBEIN. M.D. Department of Pathology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California, USA

Summary: This article reviews the early and late morphologic changes associated with reperfusion of ischemic myocardium. If instituted within minutes of coronary artery occlusion, all reversibly injured myocardium is salvaged. Once some irreversibly injured myocardium is present, the usually bland region of coagulation necrosis is transformed into an edematous, hemorrhagic zone with “contraction-band’’ necrosis and vascular obstruction (noreflow phenomenon). Whether or not these changes occur in otherwise salvageable myocardium is controversial. Data from studies with conflicting results are presented. Popular proposed mechanisms of reperfusion injury include the no-reflow phenomenon and free radical-mediated injury. No reflow has been related to direct vascular injury, compression of capillaries by edema fluid, and obstruction of vascular channels by leukocytes. Free radicals, which inactivate enzymes and destroy membranes, are primarily oxygen derived, and produced by neutrophils, endothelial cells, and myocardial cells. Whether or not reperfusion injury exists is still debated; if it does, the mechanism of injury remains to be proven. Ongoing research in this field will augment our knowledge of cell death and interventions to delay or prevent it. Key words: myocardial ischemia, myocardial infarction, myocardial salvage, coronary occlusion, reperfusion injury, free radicals, free radical scavengers

Address for reprints: Michael C. Fishbein, M.D. Department of Pathology Cedars-Sinai Medical Center 8700 Beverly Blvd. Los Angeles, CA 90048, USA Recieved: March 3 I , 1989 Accepted: April 24, 1989

Introduction Early restoration of blood flow to ischemic myocardium represents a major recent therapeutic advance in clinical medicine. I Thrombolytic agents administered early in the course of myocardial infarction re-establish blood flow, reduce infarct size, limit left ventricular dysfunction, and reduce early and late mortality.* Experimental studies in dogs indicate that reperfusion instituted within 20 min after coronary occlusion causes a delayed, but eventual complete return of normal myocardial blood flow, function, and m ~ r p h o l o g y .Why ~ . ~ then is there concern and controversy over so-called “reperfusion injury”?

Morphology of Reperfusion If 40 min or more, rather than 20, elapse before flow is restored, there may be no restoration of normal myocardial blood flow, myocardial function may not improve rapidly, and may even worsen acutely, and the morphologic signs of myocardial injury may be more pronounced than would be expected by a permanent occlusion of the same d ~ r a t i o n Reperfusion .~ of ischemically injured myocardium often results in the transformation of a bland, anemic-appearing infarct into a markedly hemorrhagic swollen necrotic zone. A number of impressive biochemical and morphologic changes occur in necrotic myocardium with reperfusion: tissue water and calcium increase m a r k e d l ~there , ~ is massive loss of myocardial enzymes and other proteins (referred to as the washout phenomen ~ n ) and , ~ marked changes in the morphologic pattern of injury occur (Fig. I). Ultrastructurally, one observes cell swelling that is more marked than with permanent ischemic injury, with large subsarcolemmal blebs present within the necrotic cells. Instead of the amorphous matrix densities that occur with permanent occlusion, more granular electron dense deposits, which have been shown to contain calcium. are present within the mitochondria. Contraction bands, a fusion of adjacent sarconieres, another hallmark of reperfusion injury, become apparent

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FIG.I Light microscopic example of contraction-band necrosis (lower half of micrograph) showing dense trmversc bands in fibers separated by spaces representing edema (heniatoxylin and eosin. X80).

F I G2 Electron microscopic example of contraction-hand nccrosis. Note fusion of sarcomeres. enlarged mitochondria with dense calcium-containing granules. and hlchs representing intracellular edema.

(Fig. 2 ) . Reperfusion hemorrhage occurs, probably as a result of restitution of flow through severely injured microvasculature allowing leakage of intravascular fluids and cells into interstitial spaces. Reperfusion accelerates evidence of cell injury so that changes of infarction become apparent sooner and appear worse. Associated with these changes may be severe ventricular arrhythmias. If blood flow is restored to the reperfused zone, a marked diminution in flow persists; the so-called “no-reflow phenomenon”.6 This has been postulated to be due to: ( 1) myocardial cell swelling and/or contracture, which compresses capillaries, ( 2 ) capillary endothelial cell damage with swelling, preventing flow, and (3) obstruction of small vessels by leukocytes which adhere to the injured endothelial cells. The recognition of these undesirable effects of reperfusion led investigators to postulate the existence of “reperfusion injury,” a parodoxical increase in myocardial injury that would not occur in the absence of reflow. As one can imagine, this concept is difticult to study because during any single experiment it is not possible to determine the viability of all myocardial regions before reperfusion, and then subsequently determine the fate of these regions after reperfusion.

cular injury, was injected prior to deflation of the balloon to identify the area of vascular injury prior to reperfusion. Carbon black adheres only to injured blood vessels. The protocol consisted of 5 % h of ischemia, followed by a 5-min injection of carbon, 30 min for the carbon to circulate and clear from normal vessels, and 30 min of reperfusion before sacrifice of the dogs. Figure 3 shows carbon labeling in the central and subendocardial portion of the infarct. There is no vascular injury in the noninfarcted myocardium. Similar findings are shown in Figure 4. In this low-power photomicrograph there is a normal septum, irreversible injury in the anterior wall seen at this low power by edema separating myocardial fibers, and hemorrhage limited to the subendocardium within the ischemic zone. When measured by planimetry, the areas of vascular injury were always smaller than and within

Effects of Reperfusion on Infarct Size In 1980 our group attempted to determine whether reperfusion hemorrhage actually extends infarction or occurs in myocardium that was already irreversibly injured before r e p e r f u ~ i o nWe . ~ attempted to identify areas of vascular injury prior to reperfusion to determine if this injury was extended by the process of reperfusion. In an ingenious experiment designed by Dr‘ Ganz, a closed-chest balloon occlusion of the left anterior descending coronary artery was utilized. The balloon has central catheter through which carbon black, a marker ofvas-

FIG,3 Transverse slice of left ventricle from a q-,erfused dog showing labeling subendocardial portion the infarct, delineated by the v c technique. NO camon labeling of injured VCSsels occurs outside of the area of necrosis.

M . C. Fishbein: Reperfusion

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FIG.4 Low-power photomicrogrdph showing a reperfused infarct. Note that the hemorrhage is in the subendocardial region within the infarct. recognizable as the region of the anterior wall (a) with edema separating fibers. Noninfarcted septum (s) is also shown (hematoxylin and eosin, X4).

the borders of the infarcted zone (area of vascular injury = 10 5 % of LV; MI size = 20 + 5% of LV, p < .01). McNamara ef al. using a similar model with various time periods after coronary ligation prior to reperfusion,s and others using different models have demonstrated similar finding^.^.'^ Thus, from the above studies, there was no convincing evidence that reperfusion per se resulted in an increase in the amount of myocardial necrosis, in spite of being associated with striking morphologic changes within the infarcts. What about the late effects of reperfusion after necrosis is complete? Is such reperfusion still beneficial by preventing infarct extension or expansion or facilitating the healing process, or is late reperfusion detrimental with the associated hemorrhage and edema causing late extension of infarction or delay in healing? Again, there have been a number of experimental studies indicating that reperfusion does not impair and may even improve healing after myocardial In a study from our group by Geft et al. this question was studied in dogs which had reperfusion after 24 h of coronary occlusion. Reperfusion was intentionally instituted late, so that any beneficial effects observed could not be ascribed to a reduction in infarct size. We studied infarct size and transmural extent, aneurysm formation and wall thinning, amount of resorption of necrotic myocardium, degree of inflammatory cell infiltration, degree of scar tissue proliferation, and the amount of hemorrhage and calcification. Function in these dogs was also studied serially by echocardiography which eventually showed no differences in the two groups. Figure 5 shows representative low-power photomicrographs from a dog with a permanent occlusion and one with reperfhion. In this trichromestained section in which myocardium stains red and connective tissue stains blue one can see that infarct size and thickness are similar and the rim of connective tissue at the edge is the same in both dogs. When all the effects

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FIG 5 Examples of transverse slices of left ventricle from dogs with permanent occlusion (top), and reperfusion after 24 h of occlusion (bottom), sacrificed 7 days later. No morphologic differences were observed in the two groups (trichrome stain, X2).

of late reperfusion were compared with permanent occlusion there were no statistically significant differences in the two groups. Thus, in this study, late reperfusion after necrosis was complete had no beneficial or detrimental effects on subsequent infarct healing. Thus, there is ample evidence that reperfusion per se does not extend the amount of myocardial injury beyond that which would occur in the presence of permanent occlusion. Although this statement is generally accepted, it does not negate the fact that reperfusion is associated with striking morphologic and biochemical changes within the myocardium, which could be interpreted as detrimental. Thus, a modified concept of reperfusion injury has evolved. The hypothesis is that within an ischemic zone there is one population of cells which is irreversibly injured, and another with varying degrees of less severe, reversible injury. The irreversibly injured cells obviously die, whether or not reperfusion is instituted. Reperfusion, however, may salvage a certain proportion of those cells that are only reversibly injured. Thus, reperfusion injury refers to death of otherwise reversibly injured cells which, in the presence of some intervention in addition to reperfusion, might survive rather than succumb.

Mechanisms of Reperfusion Injury By what mechanisms does reperfusion injury occur? Many investigators have focused on the so-called “noreflow phenomenon” and the vascular injury which has

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been documented in myocardium after reperfusion.6. Similar negative findings were reported by Gallager et Others have focused on the loss of volume regulation in ul. ,28 and Przklenk and Kloner.27Although methodologmyocardial tissue after ischemic injury which could lead ic differences can always be invoked to explain differing results, it appears that the role of free radicals and to perivascular swelling, which upon reperfusion could increase to compress small arterioles or capillaries. l 7 The scavengers in reperfusion injury remains to be determined marked calcium influx that occurs is also thought by some with certainty. to be responsible for reperfusion injury. i 8 . 1 9 While many Whether or not reperfusion injury is proven to exist, mechanisms have been suggested, there has been a recent and whether or not therapy directed against the neutrophil, shift toward the endothelium as the target for intervenor the oxygen-derived free radical, will decrease so-called reperfusion injury remains to be determined. In any case, tions to limit reperfusion injury. Engler et ul., in several reports, have provided evidence these experimental efforts will increase our understandthat leukocyte capillary plugging plays a major role in the ing of the mechanisms of myocardial injury during its evono-reflow phenomenon.20Leukocytes accumulate in the lution, and its attempted arrest by reperfusion. microcirculation after ischemic injury and adhere to the endothelium. Release of neutrophil products and neutrophil-endothelial interactions could result in enAcknowledgment dothelial damage. A variety of interventions designed to remove neutrophils or interfere with their function have The word-processing skills of Cora Galban are gratebeen shown to reduce infarct size after reperfusion. 2 1 fully acknowledged. The most current, and popular, potential culprit in the pathogenesis of reperfusion injury is the “oxygen-derived free radical.”22 Free radicals are simple molecules with References an odd number of electrons. They cause injury to membranes by lipid peroxidation and inactivation of enzymes. I . Ganz W, Buchbinder N , Marcus H, Mondkar A, Maddahi J. Charuzi Y , O’Connor L, Shell W. Fishbein MC, Kass R, The major contributor to free radicals in the myocardium Miyamoto A, Swan HJC: lntracoronary thrombolysis in evolvafter reperfusion is oxygen which, in the presence of a ing myocardial infarction. Am Heurr J 101, 4 (1981) xanthine oxidase system, breaks down into an unstable 2. ISIS-2 (Second International Study of Infarct Survival) Coloxygen-free radical. The natural defense against superoxlaborative Group: Randomized trial of intravenous strepide injury is the conversion of these free radicals to hydrotokinase. oral aspirin. both or neither, among 17,187 patients with suspected acute myocardial infarction: ISIS-2. J Ani Coll gen peroxide by an enzyme called superoxide dismutase, Currfiol 12(suppl A), 3A (1988) and then in turn, the conversion of the hydrogen perox3. Kloner RA, Ganote CE, Whalen DA, Jennings RB: Effect of ide formed to oxygen and water by catalase and peroxia transient period of ischemia on myocadial cells. 11. Fine stmcdase. Therefore, superoxide dismutase and catalase have ture during the first few minutes of reflow. Am J Purhol 74. been used in attempts to decrease reperfusion injury. 399 (1974) Beneficial effects on infarct size have been reported from 4. Heyndrickx GR, Baig H, Nellens P, Leusen I , Fishbein MC. Lucchesi’s and more recently from ~ t h e r s . * ~ . ~ ~ Vatner SF: Depression of regional blood flow and wall thickening after brief coronary occlusions. Am J Phvsiol234, H653 Since iron is involved in the generation of injurious ac(1978) tive hydroxyl radicals by the Haber-Wise and Fenton reac5 . Vatner SF, Baig H, Manderj WT, Mamko PR: Effects ofcomtions. iron binding agents such as deferoxiniine may also nary artery reperfusion on myocardial infarct size calculated be useful in reducing free radical production.26There is froin creatine kinase. J Clin lrivesr 61, 1048 (1978) evidence that oxygen-free radicals are produced by neu6. Kloner R A , Ganote CE, Jennings RB: The “no-retlow” trophils, endothelial cells and myocardial cells, both durphenomenon after temporary coronary occlusion in the dog. J Clin Invest 54, 1496 (1974) ing ischemia and r e p e r f ~ s i o n .The ~ ~ site of action of 7. Fishbein MC, Y-Rit J , Lando V, Kanmatsuse K, Mercier JC. oxygen-free radical scavengers has not been proven. Ganz W: The relationship o f vascular injury and myocardial Nevertheless,.the encouraging results with agents which hemorrhage to necrosis after reperfusion. Circularion 62, I274 tend to interfere with this system have been pursued en( 1980) thusiastically in intervention studies following reperfusion. 8. McNamara JJ, Lacm RV, Yee M, Smith GT: Hemorrhagic inIn spite of the enthusiasm for interventions reported to farction and coronary reperfusion. J Thoruc Curdiovusc Surg reduce infarct size after reperfusion, it is only fair to state 81, 498 (1981) that there are also negative ~ t u d i e s . ~ Indeed, ~ - ~ O the con9. Higginson LAJ, White F, Heggtveit HA, Sanders TM, Rloor CM, Covell JW: Determinants of myocardial hemorrhage afcept of the reperfusion injury is not accepted by all invester coronary reperfusion in the anesthetized dog. Circuluriori tigators. Work from Dr. Jennings’ group has failed to 65, 62 (1982) demonstrate that superoxide disrnutase limits infarct size 10. Hoffman M , Hofmann M, Genth K, Schaper W: The influence after 40 min of ischemia followed by 4 days of reperfuof reperfusion on infarct size after experimental coronary arion.^^ These findings were consistent with previous tery occlusion. Bus Res Cardiol 75, 572 (1980) studies from this laboratory in which allopurinol. a xan1 I , Geft IL, Fishbein MC, Hashida J , Ninomiya K , Nishizawa S, thine oxidase inhibitor, also did not limit infarct Haendchen R, Vankatesh N , Y-Rit J , Yano J , Ganz W: Ef-

M. C. Fishbein: Reperfusion fects of late coronary artery reperfusion after myocardial necrosis is complete. Am Heart J 107. 623 (1984) 12. Schaper J , Schaper W: Reperfusion of ischemic myocardium: Ultrastmcture and histochemical aspects. J Am Coll Curdiol I , 1037 ( 1983) 13. Roberts CS, Schoen FJ, Kloner RA: Effect of coronary reperfusion on myocardial hemorrhage and infarct healing. A m J Curdiol 52, 610 (1983) 14. Althaus V , Gurtner HP, Baur H. Hamburger S. Roos B: Consequences of myocardial reperfusion following temporary coronary occlusion in pigs. Effects on morphologic, biochemical and haemodynamic findings. Dur J Cliri Inwsr 7. 437 ( 1977) 15. Constanti C, Corday E, Lang TW, Meerbaum S. Brasch J, Kaplan L, Rubin S, Gold H, Osher J: Revascularization after 3 hours of coronary arterial occlusion. Effects on regional cardiac metabolic function and infarct size. Am J Curdiol 36, 368 (1975) 16. Bulkley BH, Hutchins GM: Myocardial consequences of coronary artery bypass graft surgery. The paradox of necrosis in areas of revascularization. Circulurioti 56, 906 ( 1977) 17. DiBona DR, Powell WJ Jr: Quantitative correlation between cell swelling and necrosis in myocardial ischemia in dogs. Circ Res 47, 653 (1980) 18. Reimer KA, Jennings RB: Verapamil in two reperfusion models of myocardial infarction. Temporary protection of severely ischemic myocardium without limitation of ultimate infarct size. Luh Inwsr 5 I , 655 (1984) 19. Campbell CA, Kloner RA. Alker K J , Braunwald E: Effect of verapamil on infarct size in dogs subjected to coronary artery occlusion with transient reperfusion. J A N ? Coll Curdid 8, I169 ( 1986) 20. Engler RL, Schmid-Schonbein GW, Pavelec RS: Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. An, J furhol I I I. 98 (1983) 21. Romson JL, Hook BG. Kunkel SL, Abrams GD, Shork A , Lucchesi BR: Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Cirtulririou 67, 1016 ( I 983) 22. McCord JM: Oxygen-derived free radicals in postischemic tissue injury. N G7,ql J Mid 312. 159 (1985)

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23. Jolly SR, Kane WJ. Bailie MB, Abrams GD. Lucchesi BR: Canine myocardial reperfusion injury: Its reduction by the combined administration of sueroxide dismutase and catalase. Circ Rrs 54, 277 (1984) 24. Chambers DE, Parks DA. Patterson G, Roy R, McCord JM, Yoshida S, Parmley LF, Downey JM: Xanthine oxidase as a source of free radical damage in myocardial ischemia. J Mol Cidl Curdiol 17. 145 (1985) 25. Ambrosio G, Becker LC. Hutchins GM, Weisman HF. Weisfeldt ML: Reduction in experimental infarct size by recombinant human superoxide dismutase: Insights into the pathophysiology of reperfusion injury. Circulation 74, 1424 (1986) 26. Ambrosio G. Zueier JL. Jacobus WE, Weisfeldt ML, Flaherty JT: Improvement of postischemic myocardial infarction and metabolism by administration of deferoxamine at the time of reflow: The role of iron in the pathogenesis of reperfusion injury. Circularion 76, 906 (1987) 27. Przklenk K , Kloner RA: “Reperfusion injury” by oxygenderived free radicals’?Effcct of superoxide dismutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function. coronary microvasculature, and regional myocardial blood flow. Circ Rrs 64,86 (1989) 28. Gallager KP, Buda AJ, Pace D, Gerren RA, Schlafer M: Failure of superoxide dismutase and catalase to alter size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion. Circuluriori 73, 1065 (1986) 29. Nejima J , Knight DR. Fallon JT, Uemura N , Manders WT, Canfield DR, Cohen MV, Vatner SF: Superoxide dismutase reduces reperfusion arrhythymias but fails to salvage regional function or myocardium at risk in conscious dogs. Circukirioti 79. 143 (1989) 30. Uraizee A, Reimer KA, Muny CE. Jennings RB: Failure of superoxidase dismutase to limit size of myocardial infarction after40 minutes of ischemia and 4 days of reperfusion in dogs. Circulution 75. 1237 (1987) 31. Reimer KA, Jennings RB: Failure of the xanthine oxidase inhibitor allopurinol to limit infarct size after ischemia and reperfusion in dogs. Circdufiori 71, 1069 (1985)

Reperfusion injury.

This article reviews the early and late morphologic changes associated with reperfusion of ischemic myocardium. If instituted within minutes of corona...
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