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II7
Reperfusion Injury in the Ischemic Myocardium Renu
Virmani,
MD, Frank Andrew
D. Kolodgie,
MS, Mervyn
Fat-b, MD, and Russell
B. Forman,
MD, PhD,*
M. Jones
From the Department of Cardiovascular Pathology, Armed Forces Institute of Pathology, Washington, DC, and *Department of Cardiology, Vanderbilt University, Nashville, Tennessee
Myocardial reperfusion injury is defined as the conversion of reversibly injured myocytes to irreversibly injured cells following temporary coronary artery occlusion. Although not universally accepted, the concept of lethal reperfusion injury is strongly supported by studies that temporally link an interventional therapy administered in the perireperfusion period to myocardial salvage. Myocardial reperfusion may be due to the deleterious consequences of cellular edema, calcium overload, free-radical generation, neutrophil infiltration, and microvascular damage. Current studies suggest that perfluorochemicals and adenosine (agents that preserve endothelium and attenuate neutrophil chemotaxis) are the most promising compounds that reduce infarct size in experimental animal models and may warrant clinical trials in man.
Over the past decade, there has been a resurgence of interest in reperfusion injury. Although early reperfusion remains the most effective way of reducing infarct size and mortality, an increasing number of investigations suggest that reperfusion may have deleterious consequences such as calcium overload, accumulation of neutrophils and other cellular elements, edema, hemorrhage, and liberation of oxygenderived free radicals. It is thought that these factors may individually or in concert produce microvascular injury jeopardizing blood flow to potentially viable or reversibly injured myocytes, resulting in irreversible myocyte injury and infarct expansion. Therefore, it is essential to understand to what extent, and under what circumstances myocardial reperfusion may be deleterious to ascertain the appropriate adjuvant pharmacologic therapy to limit reperfusion injury.
This work was supported in part from funding provided by the American Registry of Pathology, Washington, DC. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the US Army, Navy, or Department of Defense. Manuscript received April 18, 1991; accepted October 7, 1991. Address for reprints: Renu Vimmi, MD, Department of Cardiovascular Pathology, Armed Forces Institute of Pathology, Washington, DC 20306.
01992
by
Elsevier Science Publishing Co., Inc.
Determinants
of Myocardial Infarct Size
Myocardial infarction has been shown in animal models to progress from the endocardium to the epicardium with increasing durations of coronary occlusion. This phenomenon has been termed the wavefront progression of infarction (1,2). A short duration of coronary occlusion (40 minutes) invariably results in hemorrhage into the ischemic subendocardium. In contrast, hemorrhage is rarely observed with permanent coronary artery occlusion (61-63). Ultrastructurally, Kloner and associates have shown that within any region of the ischemic myocardium, irreversible myocyte injury occurs before vascular endothelial injury and that loss of vascular integrity occurs in regions where there is already irreversible myocyte injury (64). Also, evidence of microvascular damage is not prominent until 60 to 90 minutes after coronary artery occlusion (30,64). Hemorrhage is invariably confined to subendocardial regions of the infarcted myocardium. In areas of normal or moderately depressed flows, there is no evidence of hemorrhage (63). Gross hemorrhage into the infarcted myocardium may increase left ventricular stiffness, affecting ventricular function, and it has been suggested that hemorrhage may extend and increase myocardial infarct size (51). However, hemorrhage is confined to areas of irreversibly damaged myocardium where there has been severe vascular injury and, therefore, does not produce further necrosis of surrounding myocytes (30,65). In experiments of ischemia and reperfusion in our laboratory, hemorrhage beyond the area of necro-
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sis has not been observed (20,26,66,67). Also, thrombolytic agents have not been reported to cause an expansion of infarcts in animal models (68,69).
The role of free radicals in lethal reperfusion injury. Reactive oxygen metabolites are compounds that have unpaired electrons in their outer orbitals. Oxygenderived free radicals include the superoxide anion (.O,-) and this species, via hydrogen peroxide, can form the even more highly reactive hydroxyl ion (-OH) (44). Oxygen-derived free radicals can be cytotoxic to cellular components causing lipid peroxidation of membranes, protein degradation (chain scission, amino acid oxidation, protein-protein crosslinking), and degradation of nucleic acids (44,70). Oxgyenderived free radicals, in particular the hydroxyl ion (*OH) have been implicated in the pathogenesis of ischemia and reperfusion injury (51,71,72). Most recent support of myocardial injury stems from indirect evidence through reduction of infarct size or improved ventricular function by freeradical scavenging enzymes, antioxidants, “quenching” agents, and iron chelators (72-87) or by demonstration of a loss of the protective intracellular antioxidant systems with acute myocardial infarction (43). Recently, free radical production has been measured in the setting of ischemia and reperfusion by electron spin resonance spectroscopy (43,8792). These studies demonstrated a burst of oxygen freeradical production within the first few minutes of reperfusion (43,88-92). Another study, using electron paramagnetic resonance spectroscopy and spin-trapping agents, detected a burst of free radical formation during the first 15 minutes of ischemia and reperfusion (92). The dominant pathway of free radical production during ischemia and reperfusion is unknown. As mentioned previously, xanthine oxidase is one source of free radicals. The liberation of superoxide at reperfusion is believed to result from the oxidation of hypoxanthine, which accumulates during ischemia as ATP is degraded. Under aerobic conditions, the metabolism of hypoxanthine is mediated by xanthine dehydrogenase. During myocardial ischemia, however, xanthine dehydrogenase is converted to xanthine oxidase. The latter enzyme utilizes oxygen as its electron acceptor (as opposed to nicotinamide adenine dinucleotide [NAD], as for xanthine dehydrogenase), resulting in the formation of superoxide at the time of reperfusion (44,70,72,93-95). The superoxide radical may combine with hydrogen peroxide in the presence of catalysts such as iron and copper to form hydroxyl radicals (72). However, this pathway in endothelial cells may be species dependent, as discussed previously. Endothelial cells may not be the only source of the free radicals, and in certain pathologic conditions such as the adult respiratory distress syndrome, xanthine oxidase may be present within the blood stream (96,97). Also, activated neutrophil NADPH oxidase and myeloperoxidase both may be responsible for the production of superoxide anion and toxic hypochlorous acid (98,99). Other potential sources of free radicals include the oxidation of catecholamines re-
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leased locally during ischemia, the cyclooxygenase pathway of arachidonic acid metabolism, and the electron transport chain of mitochondria (100,101). Evidence of direct toxic effect of oxygen-derived free radicals on myocyte and endothelial cells has been observed in vitro. Burton et al. (102,103) reported endothelial vacuolization, blebs, and accumulation of membrane debris in vascular and extravascular spaces following exposure to oxygen-derived radicals. Myocardial cells show dilated T tubules and vacuolization by exposure to superoxide radicals. More severe morphologic changes consisting of myocyte swelling, disruption of mitochondria, blebing of the sarcolemma, and breaks in sarcolemmal membrane were observed following exposure to hydroxyl radicals (101,102). However, an in viva preparation of coronary ischemia and reperfusion performed at our laboratory was unable to demonstrate any ultrastructural differences in animals undergoing ischemia and reperfusion with and without the free radical scavengers, oxypurinol, and N-acetylcysteine (81,82). Overall, the ability of the free-radical scavengers to reduce infarct size is uncertain when these agents have been given either late in the ischemic period or at the time of reperfusion. However, there seems to be more convincing evidence that free-radical scavengers may play an important role in reducing myocardial stunning than in infarct size reduction. Further studies are required using standardized models to conclusively address the question of whether free radicals are important mediators of reperfusion injury. Neutrophils in reperfusion injury. Polymorphonuclear leukocytes play an important role in the demolition and repair of infarcted myocardium and are important determinants of the ultimate extent of tissue necrosis (104,105). Also, infiltration of leukocytes is essential for the ultimate replacement of necrotic myocardium by fibroblasts and collagen deposition. In permanent coronary artery occlusion in humans, infiltration of leukocytes has been observed as early as 6 hours after infarction (105,106). Sommers and Jennings (107), using the dog model, have reported leukocyte margination as early as 4 hours after coronary occlusion. This margination is accelerated with myocardial reperfusion. Neutrophils are designed for the defense of the host organism; however, under certain pathologic conditions they may induce tissue injury. It has been proposed that neutrophils contribute to reperfusion injury of the ischemic myocardium during reperfusion. Neutrophil activation occurs early during myocardial ischemia and precedes the appearance of histologic evidence of myocardial injury (108). Reperfusion markedly accelerates the appearance and extent of infiltration of neutrophils into the ischemic region (10911 1). Go and coworkers, utilizing indium-labeled neutrophils, observed a threefold to fivefold increase in neutrophil infiltration with reperfusion when the duration of ischemia was longer than 40 minutes. Leukocyte infiltration was most
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prominent in the subendocardium and was observed within 10 minutes of reperfusion (111). In contrast, neutrophil infiltration is observed only after 4 to 6 hours in the permanent occlusion model and occurs predominantly at the border of the ischemic zone (107). Intercellular adherence appears necessary for leukocyte accumulation at sites of inflammation, and this process is facilitated by the CD1 l/CD18 adherence glycoprotein on the neutrophil surface ( 112). This is followed by activation, diapedesis, and extravascular migration into the interstitium. Whether enhanced neutrophil adherence during ischemia is produced by changes in neutrophils, endothelial cells, or both is controversial (112). Although a continuous physiologic interaction normally occurs between the neutrophil and endothelium, hypoxic injury has been shown to augment neutrophil adherence in vitro (112). Ischemic endothelial cells express membrane receptors for immunoglobulin and complement fragments that promote leukocyte adherence, and it has been shown that the complement fraction C,, localizes to the ischemic myocardium in vivo (112-l 14). Production of additional chemoattractants by activated neutrophils amplifies the initial inflammatory response. Activated neutrophils contain an enzyme that cleaves Cs into chemotactically active fragments (115). Neutrophils exposed to chemoattractants become more spherical, resulting in the expression of a glycoprotein on the cell surface that promotes adhesion, aggregation, and chemotaxis (116,117). Ongoing neutrophil activation is dependent on intracellular calcium concentrations (118). Chemotactic factors cause an increase in cytosolic calcium, leading to activation of phospholipases and generation of arachidonate products from cyclooxygenase and lipoxygenase pathways (118). Activated neutrophils release leukotriene B,, a potent chemoattractant that enhances vascular smooth muscle tone, potentiates platelet aggregation, promotes endothelial permeability, and also may modulate proteolytic enzyme release. The importance of leukotrienes in the inflammatory responses is suggested by the observation that lipoxygenase inhibitors attenuate neutrophil infiltration into reperfused myocardium and reduce infarct size (119). Although the processes are complex, calcium appears to play a role as a second messenger in the secretion of cytotoxic substances from activated neutrophils (120). These substances include numerous potent proteolytic enzymes and reactive oxygen species. Exposure of neutrophils to chemotactic factors such as leukotriene B4 and C5 results in release of enzymes from azurophilic and specific granules via reverse endocytosis (120). Neutrophil activation is associated with a markedly enhanced oxygen uptake by the cell (respiratory burst), resulting in the production of large quantities of reactive oxygen species (121). Both lysosomal enzymes, such as elastase, and reactive oxygen species have been shown to disrupt endothelial cell basement membranes in vitro (122,123). Neutrophil-derived oxidants may also inactivate antiproteases, such as a,-antitrypsin, present in
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the plasma (124). Neutrophil degranulation and free-radical release may therefore permit unchecked activity of proteolytic enzymes on endothelial and myocyte membranes.
Indirect Evidence of the Occurrence of Reperfusion Injury Various agents have been used to reduce reperfusion injury and infarct size. Anti-inflammatory agents such as indomethacin, aspirin, ibuprofen, iloprost, and prostaglandin E, have been studied in experimental myocardial reperfusion (125-13 1). Of these agents only ibuprofen, iloprost, and prostaglandin E, have been shown to reduce neutrophil infiltration and reduce infarct size (126-131). Also, neutropenia induced by neutrophil antiserum or antimetabolities (hydroxyurea) reduces infarct size (132,119). In these studies, neutropenia was induced before the induction of ischemia; therefore, the beneficial effects could occur during ischemia or reperfusion. A recent study showed that removal of neutrophils at the time of reperfusion resulted in significant infarct size reduction (133). Histologic studies have demonstrated that mechanical plugging of capillaries with neutrophils can be prevented with severe neutropenia using neutrophil filters (134). Activated complement is a potent neutrophil chemotactic factor, and agents like cobra venom, a compound that depletes complement, significantly reduce infarct size when given prior to ischemia (135). As mentioned previously, the neutrophil surface exhibits a family of heterodimeric glycoproteins (CD 1 l/CD 18 complex) that are involved in the process of cellular adhesion. CD18 is the p subunit of three heterodimers on the neutrophil surface: LFA-1 (CD1 la/CD18), Mac-l (CD1 lb-CD18), andp150,95 (CD1 lcKD18) (112). With regard to adhesion to endothelial cells, Mac- 1 and LFA-1 are the most important (112). Anti-CD 18 monoclonal antibodies have been shown to reduce infarct size (136). The mechanism of infarct size reduction with anti-CD18 antibodies may involve (i) Mac-ldependent adhesion induced by local chemotactic factors acting on circulating neutrophils, (ii) LFA- 1-dependent adhesion resulting from the action of local cytokines on endothelial cells, or (iii) LFA-1 and Mac-l-dependent diapedesis induced by chemotactic factors and cytokines acting together on neutrophils and endothelial cells (112).
Perfluorochemicals Perfluorochemical (Fluosol-DA 20%, Alpha Therapeutic Corp.), an acellular perfusate, has a small particle size (1-2 pm), low viscosity, and high oxygen-carrying capacity with potent antineutrophilic effects (137,138). Perfluorochemicals have been shown to reduce infarct size and improve contractile function in various models of occlusion and reperfusion (6,23,27,66,139-144). Fluosol-DA has been shown to reduce infarct size when delivered as an intracoronary infusion and also when administered via the intra-
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venous route in both the dog and rabbit (6,23,27,66,140, 144). Histologic examination has revealed a pronounced reduction in neutrophil infiltration within the ischemic myocardium with relative preservation of endothelial ultrastructure and reduced neutrophil plugging in capillaries (27). Endothelium-dependent relaxation of both large and small vessels has been shown to be preserved in animals treated with Fluosol-DA when compared with blood reperfusion (27). To further separate the role of neutrophil suppression from endothelial preservation, we studied the effects of blood-free reperfusion with oxygenated and unoxygenated peffluorochemical (PFC) administered over 20 minutes after 90 minutes of reperfusion (33). Endothelial blood flow in the ischemic zone decreased progressively with reperfusion in control (blood reperfusion) and unoxygenated PFC animals but was maintained with oxygenated perfluorochemicals. These findings were associated with relative preservation of endothelial cell structure with oxygenated perfluorochemicals compared with both unoxygenated PFC and control groups. The number of capillaries containing leukocytes was significantly less in animals treated with perfluorochemicals than in controls. These findings support the hypothesis that blood-free reperfusion with oxygenated perfluorochemicals improves oxygen delivery to the microvasculature and protects the endothelium from further injury. Fewer leukocytes within capillaries may also have contributed to a decrease in the no-reflow phenomenon in both peffluorochemical groups. However, endothelial preservation may be the predominant mechanism in limiting no-reflow injury with oxygenated perfluorochemical.
Adenosine Adenosine is a potent endogenous coronary arteriolar vasodilator that is found in substantial quantities in endothelial cells (20,145). Adenosine appears to be potentially useful in ameliorating reperfusion injury because of its physiologic properties. These include abolition of microvasculature constriction, inhibition of various neutrophil functions (such as adherence to endothelial cells and superoxide anion production), reduction of platelet aggregation, and replenishment of high-energy stores in endothelial and myocardial cells (146,147). Selective intracoronary administration of adenosine for 1 hour, commencing 5 to 10 minutes after reperfusion, significantly reduced infarct size in animals subjected to 90 and 120 minutes of ischemia followed by 24 hours of reperfusion (45). Intravenous administration of the agent just before reperfusion in canine and rabbit models of ischemia also demonstrated a significant reduction in infarct size (4,148,150). Other studies have demonstrated that the agent significantly attenuates functional and structural abnormalities in the microvasculature after 2 hours of regional ischemia in the dog (4,149). This was associated with improved regional myocardial blood flow during the first 3 hours of reperfusion. In vitro studies have demonstrated that
VIRMANI ET AL. REPERFUSION INJURY IN THE ISCHEMlC MYOCARDIUM
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both adenosine and 2-chloradenosine reduce neutrophil adherence and cytotoxicity to cultured endothelial cells (148, 150). Although the exact mechanism whereby adenosine reduces reperfusion injury is unknown, these studies provide further evidence that the introduction of activated neutrophils to immunologically primed vasculature at reperfusion may enhance the inflammatory response, resulting in accelerated vessel injury, neutrophil plugging, and a continual decrease in microcirculatory flow. Further studies are needed to clarify the cell types involved and the precise mechanisms of action of adenosine in ameliorating reperfusion injury.
Ischemic Preconditioning It has been well recognized that the myocardium does not undergo irreversible injury if reperfusion is accomplished within 20 min of total coronary occlusion. Also, repeated periods of ischemia (~15 minutes) interspersed with reperfusion does not result in cumulative injury to the myocardium. Conversely, repeated ischemia “preconditioning” followed by prolonged ischemia of 40 minute duration renders the myocardium more resistant to infarction (151). This phenomenon has been termed ischemic preconditioning (15 1). Preconditioning has been shown to be protective in the canine, swine, and rabbit models (151-154). The underlying cause for this protection is still highly speculative. It has been suggested that reduced ATP loss and lactate accumulation may contribute to preconditioning protection (155). Murry et al. (156) have shown that preconditioning reduces myocardial energy demand during ischemia, which leads to a reduced rate of high-energy phosphate utilization and a reduced rate of anaerobic glycolysis (reduced accumulation of glucose-l-phosphate, glucose-6-phosphate, and lactate). Ultrastructural injury in preconditioned hearts develops more slowly. In the canine, no evidence of irreversible injury has been observed at 20 minutes of sustained ischemia in a preconditioned heart when compared with nonpreconditioned animals (156). At 40 minutes of sustained ischemia, preconditioned hearts showed focal irreversible myocyte injury, in contrast to nonpreconditioned controls, in which there was homogenous irreversible injury (156). Reduced liberation of oxygen-derived free radicals has also been proposed as a mechanism of preconditioning (157). Murry et al. showed that the protective effects of ischemic preconditioning was blocked by the administration of superoxide dismutase and catalase (157).
Conclusion Timely reperfusion still remains the most logical approach in the treatment of an evolving myocardial infarction. However, the beneficial effects of reperfusion following prolonged coronary occlusion may be limited by an
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incomplete return of blood flow to some areas of the ischemic myocardium, which may result in the conversion of reversibly injured myocytes to irreversible injury. Myocardial reperfusion may involve deleterious consequences including explosive cellular edema, calcium overload, freeradical generation, neutrophil infiltration, and microvascular damage. Current experimental studies suggest that therapeutic agents directed to reduce or prevent these mechanisms of injury may be beneficial in further reducing infarct size. The most promising agents appear to be peffluorochemicals and adenosine, which warrant clinical trials to further investigate the role of reperfusion injury in limiting myocardial salvage in patients undergoing emergency revascularization.
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