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Reperfusion injury after temporary coronary occlusion
J
THoRAc CARDIOVASC SURG
1988;95:960-8
Reperfusion injury after temporary coronary occlusion In 24 anesthetized open-chest dogs, we examined the time course of changes in contractile function, diastolic muscle stiffness (sonomicrometry), tissue water content, and ultrastructure after 1 hour of occlusion of the left anterior descending coronary artery and after 2 hours of unmodified reperfusion. One hour of occlusion of the left anterior descending artery replaced active shortening with passive bulging (21.4% ± 2.9% versus -5.9% ± 0.9%, p < 0.05) in the involved segment. There was no increase in either subendocardial water content (78.6% ± 0.1 % versus 79.7% ± 0.7%) or operative muscle stiffness (2.80 ± 0.72 versus 2.36 ± 0.42 mm Hgjmm) after the occlusion period. There were only mild to moderate ultrastructural alterations suggestive of reversible injury. In sharp contrast, reperfusion was associated with a 2.48 % increase in subendocardial water content (p < O.O~ a 42 % increase in diastolic muscle stiffness (3.34 ± 0.42 mm Hgjmm, p < 0.05~ and greater ultrastructural damage. We conclude that myocardial injury is significantly extended with unmodified blood reperfusion after temporary coronary occlusion.
J. Vinten-Johansen, PhD, William E. Johnston, MD, Stephen A. Mills, MD, Kirk B. Faust, MD, Kim R. Geisinger, MD, Richard J. DeMasi, MD, and A. Robert Cordell, MD, Winston-Salem. n.c.
Ltal coronary occlusion reduces oxygen and substrate delivery and fails to wash out metabolites in the involved myocardium. The degree of myocardial damage is related to the severity and duration of the occlusion. 1-4 Biochemical and morphologic alterations are reversible with brief occlusions lasting 12 to 15 minutes, although contractile dysfunction persists.v? However, irreversible changes culminating in necrosis begin to occur in the subendocardium after 40 minutes of ischemia and progress in a "wavefront pattern" to involve the subepicardium after 4 to 6 hours of occlusion.' Reperfusion
From the Departments of Surgery (Section on Cardiothoracic Surgery), Anesthesia, and Pathology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, N.C. Supported in part by a grant-in-aid from the American Heart Association, North Carolina Affiliate (No. 84-85-A7) and National Institutes of Health Grant HL 36377. Presented in part at the Seventieth Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, Mo., April 15, 1986. Received for publication Sept. 19, 1986. Accepted for publication July 27, 1987. Address for reprints: J. Vinten-Johansen, PhD, Section on Cardiothoracic Surgery, Bowman Gray School of Medicine, 300 South Hawthorne Rd., Winston-Salem, NC 27103. OAf)
salvages myocardium that would otherwise be destined for necrosis if reperfusion were not established and results in smaller infarct sizes. However, reperfusion does not completely avoid infarct development after I to 2 hours of coronary occlusion. I, 4, 5 Recent studies have shown that the extent of necrosis in the area placed at risk by acute occlusion can be reduced if appropriate interventions are introduced at the onset of reperfusion." 7 When reperfusion was modified by controlling the composition of the initial reperfusate (i.e., blood cardioplegia) and the reperfusion pressure, myocardial necrosis was reduced by approximately 50% to 75%, with restoration of immediate contractile function."!' This reduction of postischemic damage with modified reperfusion is predicated on the salvageability of the myocardium at the time reperfusion is established. Although reperfusion salvages myocardium, it may also advance the extent of injury beyond that present before reperfusion. However, progression in the extent of tissue injury between ischemia and reperfusion has not been clearly shown in the setting of transient coronary occlusion. This study tests the hypothesis that myocardial injury after transient coronary occlusion is extended beyond that apparent before reperfusion. Correlative changes in contractile function, diastolic compliance (muscle stiffness), tissue edema,
Volume 95 Number 6 June 1988
Reperfusion injury
961
Table I. Hemodynamics for control, ischemia, and reperfusion periods in group III dogs undergoing 1 hour of LAD occlusion and 2 hours of reperfusion Arterial
Control Ischemia Reperfusion
CO
HR
LVSP (mmHg)
LVEDP (mmHg)
Sys (mmHg)
Dias (mmHg)
MAP (mmHg)
+dPjdt
-dPjdt
(Ljmin)
144 ± 8 141 ± 8 141 ± 7
127 ± 6 118 ± 6 110 ± 5*
7.1 ± 0.8 11.0 ± 1.0* 9.6 ± 1.2
131 ± 5 124 ± 6 112 ± 6*
III ± 5 106 ± 5 94 ± 6*
118 ± 5 III ± 5 99 ± 6*
2554 ± 235 1992 ± 166 1815 ± 117*
3245 ± 248 2608 ± 193 2213 ± 158
2.75 ± 0.22 2.18 ± 0.13* 2.08 ± 0.13*
Values are means ± standard error of the mean; LYSP.left ventricular peak systolic pressure; LYEOP, left ventricular end-diastolic pressure; Sys, systolic pressure; Dias, diastolic pressure; MAP, mean arterial pressure; CO, cardiac output. 'p < 0.05 versus control.
and ultrastructure were measured between the ischemic and reperfusion phases in a canine model of occlusion and reperfusion of the left anterior descending (LAD) coronary artery. We found that, although changes in systolic function occurred during ischemia, the predominant increase in diastolic muscle stiffness, accumulation of edema, and abnormalities in ultrastructure were manifested primarily after reperfusion.
Methods Twenty-four dogs of both sexes, weighing between 13.5 and 26.0 kg (mean 17.9 ± 1.0 kg), were anesthetized with sodium pentobarbital (30 mg/kg, given intravenously) supplemented continuously with a 1 to 2 rng/kg/hr infusion. The dogs were intubated with a cuffed endotracheal tube and ventilated with oxygen-enriched room air with a Harvard volume-cycled ventilator (Harvard Apparatus Co., Inc., S. Natick, Mass.). Ventilation was adjusted to maintain carbon dioxide tension between 35 and 45 mm Hg and pH between 7.38 and 7.42. Catheters were inserted into the right femoral artery and vein for blood sampling and administration of drugs and fluids, respectively. A 7.5F flow-directed thermodilution cardiac output catheter was introduced into the right jugular vein and positioned in the pulmonary artery. The chest was incised at the left fifth intercostal space. Umbilical tape snares were placed around the superior and inferior vena cavae for bicaval occlusion to reduce preload for assessment of segmental muscle stiffness, which is the inverse of compliance." The pericardium was widely incised and sutured to the chest wall to cradle the heart. Millar MPC-500 micromanometer-tipped catheters (Millar Instruments, Inc., Houston, Texas) were inserted into the proximal aorta via the left internal mammary artery and into the left ventricle via the apical dimple to measure instantaneous aortic and left ventricular pressures, respectively. The LAD coronary artery distal to the first diagonal branch was dissected free and isolated with a loose silicone rubber snare. Two pairs of 5 MHz ultrasonic dimension gauges, 2 mm in diameter, were implanted subendocardially in the myocardial segments perfused by the LAD (ischemic zone) and circumflex (nonischemic zone) coronary arteries to measure segmental fractional shortening (Triton model 120 sonomicrometer, Triton Equipment Corp., San Diego, Calif.). A standard limb lead II electrocardiogram was continuously monitored. Calculations. All analog signals were recorded on a twelvechannel Hewlett-Packard fiberoptic recorder (Hewlett-Pack-
ard Company, Andover, Mass.) and on FM magnetic tape for later analog-to-digital conversion and processing (Labtech Notebook software). Hemodynamic measurements included instantaneous left ventricular and aortic systolic and diastolic pressures, left ventricular peak positive and negative pressure rise (dP/dt), and thermodilution cardiac output obtained in triplicate by 3 ml iced saline injections. End-diastole and end-systole of the dimension signals were timed from the upstroke of the left ventricular pressure pulse and the dicrotic notch of the aortic pressure, respectively. Active systolic segmental shortening (SS) was calculated as follows: 100 X ([EDL - ESLJ/EDL), where EDL and ESL are enddiastolic and end-systolic segment lengths, respectively. Regional passive diastolic muscle stiffness characteristics were determined by progressivelydecreasing the left ventricular end-diastolic pressure and segment length by bicaval occlusion. The resulting pressure-segment length data points were fit to an exponential curve. Segment lengths per se cannot be used for this analysis because the prolonged systolic stretching of the bulging ischemic segment results in segment length elongation at a given end-diastolic pressure that may be independent of changes in actual regional muscle stiffness. Therefore, the curvilinear characteristics of the pressuresegment length curve were used to assess muscle stiffness of the ischemic-reperfused segment. According to the method of Gaasch and associates.P-" the tangent to the curve at 4 nun Hg was drawn. The slope of this tangent describes the operative muscle stiffness of the myocardial segment. An increase in the slope of the tangent indicates an increase in muscle stiffness (i.e., loss of compliance), whereas a decrease in the slope indicates a decrease in muscle stiffness. At the end of the experiment, transmural biopsy samples were taken from the ischemic and nonischemic zones by a high-speed drill and divided into subepicardial and subendocardial halves to determine tissue water content by drying at 85° C for 48 hours. All biopsy sites were repaired and the coronary occlusions replaced. Next, 3 ml of 2% gentian violet solution was injected into the left atrium. After the dye had been allowed to circulate for 15 seconds, the heart was arrested with bolus injection of potassium chloride and excised. The correct placement of the crystals was verified by the presence (nonischemic zone) or absence (ischemic zone) of gentian violet staining in the surrounding myocardium. The quantity of area placed at risk by coronary occlusion was determined by planimetry and calculated as the nonstained area/total left ventricular area times 100. Samples for electron microscopic study were taken from the
The Journal of Thoracic and Cardiovascular Surgery
96 2 Vinten-Johansen et al.
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Table II. Segmental length data for ischemic and nonischemic zones in group III dogs
Reperfusion
Fig. 1. Fractional systolic shortening in the ischemic (e----.) and nonischemic (0 ----0) segments during control, after 60 minutes of coronary occlusion (ischemia), and after 120 minutes of reperfusion. *p < 0.05 versus control.
central ischemic zone and the nonischemic zone after occlusion or after reperfusion in randomly selected experiments by a Travenol Tru-Cut biopsy needle (Travenol Laboratories, Inc., Deerfield, Ill.) and fixed in 2.5% glutaraldehyde. Thick sections stained with toluidine blue were reviewed to select areas for ultrastructural examination. Thin sections were double stained with uranyl acetate and lead citrate. Sections were inspected at 2500 and 7000 power. Five to six electron photomicrographs were made for each specimen at a magnification of 7000X to evaluate mitochondrial and sarcolemmal structure. In addition, several photomicrographs were taken from each specimen to analyze sarcolemmal, nuclear, and intercellular structure. The pathologist was unaware of the origin of the sample at the time of examination. Experimental protocol. The condition of each dog was allowed to stabilize for 30 minutes after completion of the operation. The dogs were then divided into three separate groups. Group I: Controls. Biopsy samples from the region perfused by the LAD were obtained for analysis of tissue water content from eight dogs that did not undergo either ischemia or reperfusion. Group II: Ischemia. Eight dogs underwent I hour of LAD occlusion without reperfusion. Lidocaine (75 mg) was administered before LAD occlusion. At the end of the ischemic period, biopsy samples were taken for assessment of tissue water content. Group III: Ischemia plus reperfusion. Eight dogs underwent I hour of LAD occlusion followed by 2 hours of reperfusion. Lidocaine (75 mg) was given before occlusion and reperfusion. Sequential hemodynamic and segmental function measurements were taken. In randomly selected dogs, tissue samples were taken after the occlusion and reperfusion periods for electron microscopic analysis. At the end of the experiment, biopsy samples were taken for tissue water content. Data were analyzed by the Statistical Analysis Systems program (SAS Institute, Cary, N.C.). Tissue water content data were analyzed by analysis of variance for group differences followed by Duncan's multiple range test to locate the source of difference." Within each group, differences between epicardial and endocardial water contents were analyzed by Student's t test. Sequentially obtained hemodynamic, functional, and operative stiffness data (group III) were analyzed
EDL
Ischemic zone 12.88 Control 14.61 Ischemia 13.66 Reperfusion Nonischemic zone Control 12.02 Ischemia 12.12 Reperfusion 12.44
ESL
%SS
± 0.87 ± 1.01 ± 0.93
10.19 ± 0.85 15.47 ± 1.08* 14.51 ± 0.97*
21.40 ± 2.86 -5.90 ± 0.91* -6.41 ± 1.05*
± 1.10 ± 1.31 ± 1.04
10.94 ± 0.94 10.52 ± 1.37 10.80 ± 0.88
9.10 ± 0.90 13.84 ± 3.73* 13.08 ± 0.83
EDL. end-diastolic length; ESL. end-systolic length; %55. percent fractional systolic shortening. *p < 0.05 versus centrol.
by the general linear model multivariate analysis for repeated measures followed by Wilk's lambda test. Significance was accepted at the 0.05 level of probability. All procedures complied with the "Guiding Principles in Care and Use of Animals," adopted by the Council of the American Physiologic Society, and with the "Guide for the Care and Use of Laboratory Animals" (Publication No. 78-23). The protocol was reviewed by the institutional research and animal care committees.
Results Hemodynamics. Hemodynamic data for the eight dogs in group III undergoing ischemia and reperfusion are summarized in Table I. Ligation of the LAD involved 35.2% ± 2.5% of the total left ventricular mass in the ischemic zone. After 1 hour of LAD occlusion,left ventricular end-diastolic pressure increased significantly and cardiac output decreased by 21% (p < 0.05). Heart rate and left ventricular and arterial blood pressures did not change significantly during the ischemic period. After 2 hours of reperfusion, however, left ventricular systolic pressure and arterial systolic and diastolic pressures decreased significantly from control values. Function. Active systolic shortening in the ischemic zone before ligation was rapidly replaced by passive lengthening, which persisted for the entire hour of ligation and averaged -33.7% ± 5.1% of the control systolic shortening (p < 0.001, Table 11). There was a moderate but insignificant increase in end-diastolic segment length during occlusion. After 2 hours of reperfusion, the ischemic segment continued to lengthen passively with systolic shortening averaging -36.0% ± 6.3% of control (Fig. 1). In the nonischemic segment, end-diastolic segment length increased slightly (p > 0.10) and segmental shortening increased significantly in compensation of the ischemic zone. This hypercontractile state in the nonischemic segment continued throughout reperfusion (Fig. 1). Tissue water content. Tissue water content in the
Volume 95 Number 6 June 1988
control group was similar in the epicardial and endocardial regions, averaging 78.18% ± 0.12% and 78.60% ± 0.14%, respectively. There was no significant increase in tissue water content in either region after I hour of LAD occlusion (Fig. 2). However, after 2 hours of reperfusion, water content had increased significantly by 2.37% in the epicardium and by 2.48% in the endocardium. The normally perfused, nonischemic segment showed only a slight decrease of approximately I % in tissue water content in the endocardium during the ischemic period. Operative muscle stiffness. A graph of left ventricular end-diastolic pressure-segment length data during gradual preload reduction in one experiment (6-4-85) is shown in Fig. 3, A. Data were curve-fit before LAD occlusion (control), after I hour of LAD occlusion (ischemia), and after 2 hours of reperfusion. Fig. 3, B showsthe tangent to the curve at 4 mm Hg taken before occlusion. The slope of the tangent (M>/ ~L) describes the passive muscle stiffness during that period." 14 With I hour of LAD occlusion, the left ventricular enddiastolic pressure-segment length curve shifted rightward, consistent with systolic elongation or myocardial creep. However, there was no change in the passive stiffnessof the muscle (Fig. 3, C). With reperfusion, on the other hand, there was a significant increase (p < 0.02) in the stiffness of the reperfused segment. For a given change in left ventricular end-diastolic segment length, there was a larger increase in end-diastolic pressure than that observed either before or after I hour of LAD occlusion. Ultrastructure. Myocardial tissue appeared less damaged after coronary occlusion than after reperfusion (Fig. 4). After 1 hour of coronary occlusion, sarcomeres appeared largely in register with contraction bands visible in only a few samples. Mild mitochondrial swelling, with some matrix clarification and disorganization of the cristae, were observed. In addition, moderate clumping of nuclear chromatin and mild intracellular and intercellular edema were noted in some samples. In contrast, more severe alterations in ultrastructure were apparent after reperfusion. Overall, myocytes that showed evidence of irreversible damage were those that had been reperfused. These changes included fragmentation of the sarcolemma, presence of contraction bands, and mitochondrial aberrations characterized by swelling, appearance of flocculent densities, matrix clarification, and disruption of the cristae. Contraction bands were more numerous than in paired samples taken after the hour of LAD occlusion before reperfusion. In addition, intracytoplasmic fluid accumulation appeared more accentuated in reperfused tissue than in its paired ischemic sample. Degenerative alterations were more
Reperfusion injury
963
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Fig. 2. Percent tissue water is shown for the ischemic (upper panel) and nonischemic (lower panel) segments. EPl. Epicardial; ENDO. endocardial. *p < 0.05 compared to control.
diffuse and more intense in endocardial, than in epicardial samples in both ischemic and ischemic-reperfused tissue biopsy specimens. Discussion Reperfusion, while essential in preventing the progression of necrosis from encompassing the entire transmural area at risk, is also associated with deleterious changes that increase the extent of myocellular injury. In our study, the ischemic segment demonstrated no increase in either total tissue water or muscle stiffness and showed less severe ultrastructural abnormalities after the ischemic period. After reperfusion, however, changes consistent with severe myocardial injury were manifested. Specifically, there was a significant increase in tissue water content and passive muscle stiffness, both suggesting a loss of muscle compliance and potentially irreversible injury. Derangements in the myocellular ultrastructure were supportive of these reperfusion alterations. These data support the concept of reperfusion injury in the setting of acute coronary occlusion and suggest that reperfusion injury is a significant component of the total myocardial damage after acute coronary occlusion and reperfusion.
The Journal of Thoracic and Cardiovascular Surgery
964 Vinten-Johansen et al.
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Fig. 3. A, End-diastolic pressure-segment length data in one experiment (6-4-85) obtained before coronary occlusion (control), after I hour of LAD occlusion (ischemia), and after 2 hours of reperfusion. Note the rightward shift in absolute segment length without an apparent shift in slope of the curve. After reperfusion, there is an increase in slope, indicating an increase in muscle stiffness.
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Fig. 3. B, Method of calculating operative muscle stiffness from the slope of the tangent drawn at 4 mm Hg end-diastolic pressure.
Reperfusion injury is well described in globally ischemic hearts, where it is encountered as a consequence of inadequate myocardial protection. 16. 18 Characteristic changes during reperfusion include (1) an increase in myocardial edema, (2) a loss of ventricular compliance, and (3) a depression in oxygen utilization capacity caused by reduction in mitochondrial oxidative phosphorylation 19 and significant increases in myofibrillar and mitochondrial calcium. 16 The results from our study confirm that a similar manifestation of reperfusion damage is seen in the setting of coronary occlusion and reperfusion. The reperfused segment was severely edem-
Fig. 3. C, Operative muscle stiffness, determined by the method described in B, before occlusion (control), after I hour of LAD occlusion, and after 2 hours of reperfusion. Note the significant increase (*p < 0.05) in operative muscle stiffness only after reperfusion. LVEDP. Left ventricular end-diastolic pressure.
atous and had lost compliance. The electron micrographs in the current study would suggest that segmental oxygen utilization may be depressed because of mitochondrial deterioration and dysfunction. A depressed postischemic oxygen utilization has, in fact recently been demonstrated after reperfusion of the ischemic zone after temporary coronary occlusion.20 In contrast to global left ventricular ischemic-reperfusion injury in which impaired systolic and diastolic function are expressed as compromised hemodynamics (low cardiac output, hypotension), regional ischemia may not critically affect systemic hemodynamics. In the present study, total loss of contractile function in the ischemic segment was accompanied by only modest reductions in global hemodynamics, such as left ventricular pressure, positive dP jdt, and cardiac output. In addition, a loss of compliance in the involved zone was not associated with higher left ventricular end-diastolic pressure (Table I, reperfusion). Other studies also have shown that global indices of performance do not accurately reflect the extent of regional injury and are not significantly altered in compensated regional infarction." Although global ventricular performance may be slightly depressed during coronary occlusion," systemic hemodynamics are maintained by compensatory hyperkinesia in the nonischemic segment"!" 23. 24 so long as the involved area does not exceed 40% of the total left ventricular mass. One major mechanism underlying this compensatory hyperkinesia is a local Starling response to ventricular dilatation caused by contractile dysfunction in the ischemic zone." 26 Although end-diastolic length in the nonischemic zone did not consistently increase in our study, systolic shortening did significant-
Volume 95 Number 6 June 1988
Reperfusion injury
965
Fig. 4. Electron micrographs from the epicardial region after I hour of coronary occlusion (A) and 2 hours of reperfusion (8). A shows mild to moderate clarification of mitochondria (M) with some separation of cristae and some swelling. Myofibrillar architecture is present in many areas but disrupted in others. There is evidence of very mild first-stage formation of contraction bands (C). 8 shows loss of sarcomere architecture caused by increased degree of contraction banding. Mitochondria have regained electron opacity but severe vacuolizations have developed that were not present after ischemia. (Original magnification x7000.)
ly increase consistent with a hyperkinetic response. However, a significant fraction of this hyperkinesia is expended in stretching the compliant ischemic segment during isovolumetric systole, which results in less effective ejection, reduced cardiac output, and elevated end-diastolic pressure. With the loss of compliance during reperfusion, however, a contractile effort is not expended unproductively in stretching the noncompliant segment, which potentially contributes to improved performance and lower end-diastolic pressures in our study. In addition, resumption of some degree of function with reperfusion in less ischemic "border zones" may also have contributed to apparently improved performance relative to the period of coronary occlusion. In the absence of reflow, mitochondrial damage was modest and myofibrillar derangements were largely absent. With reperfusion, however, these reversible changes progressed to largely irreversible derangements. This acceleration in ultrastructural injury with reperfusion was observed previously by Kloner and associates," but its relation to a temporally separate, progressive reperfusion injury was not appreciated. Recent evidence supporting a reperfusion-dependent progression in subcellular damage was reported by Sjostrand and col-
leagues," using a low-protein denaturation fixation process. In this study, up to 6 hours of regional ischemia caused only moderate, reversible deterioration in subcellular architecture, whereas reperfusion was marked by a dramatic transition to severe, irreversible damage. In addition to acceleration of ultrastructural damage, a similar reperfusion-dependent increase in total tissue or intracellular calcium has been observed. 19, 29-32 This calcium-loading phenomenon may be a key factor in the transition from reversible to irreversible damage.": 34 With electron microprobe analysis, there was relatively normal distribution of calcium in mitochondrial, myofibrillar, and cytosolic compartments after an ischemic period. However, with reperfusion, calcium accumulated in mitochondria and around the myofibrils." Several studies have shown that compliance is lost in severely injured myocardium. 16. 18,35.36 The results from this study extend these observations by showing that the predominant loss in segmental compliance occurs only after reperfusion. During coronary occlusion, the absolute end-diastolic segment length at a given pressure increased, possibly related to wall thinning," sarcomere elongation," or myocardial creep'<" caused by the persistent systolic paradoxical stretch. However, seg-
966
The Journal of Thoracic and Cardiovascular
Vinten-Johansen et al.
Surgery
mental compliance characteristics were not altered from the preischemic condition. The loss of compliance occurred only after reperfusion, possibly as a result of an increase in tissue water or the development of contraction bands, as observed in our study. In addition, the intracellular accumulation of calcium has also been linked to the decrease in postischemic compliance." A similar time course is seen in the loss of compliance, and the accumulation or intercompartmental shift in calcium has been reported." 30, 32, 40 Loss of compliance during reperfusion has also been attributed to augmented vascular turgor that occurs with reperfusion and edema." It may be expected that a less compliant segment would not stretch passively during systole, that is, would be dyskinetic. With a severe loss of compliance, such dyskinesia and absence of paradoxical bulging are noted. However, with less severe loss of compliance, some degree of distensibility is retained with less stretch during systole. In our study, end-systolic stretch was less after reperfusion (14.51 ± 0.97 mm) than after LAD occlusion (15.47 ± 1.08 mm) for similar end-systolic ventricular pressures. This reduction in systolic stretch in reperfused segments is masked by the apparent equality in negative systolic shortening (paradoxical bulging) because of the normalization using smaller end-diastolic segment lengths. Whether reperfusion injury is an unmasking of ischemic injury or represents an active, progressive process requires further experimental verification. The strongest support for a reperfusion injury phenomenon comes from studies showing that the degree of eventual postischemic injury can be reduced by interventions introduced only during the period of reperfusion."!': 16, 18 Recently, surgical reperfusion of acute coronary occlusion demonstrated significant reduction in infarct size and edema and partially restored fractional shortening in the involved segment after 1 or 2 hours of coronary occlusion."!' The increased salvage of myocardium may be attributed to the precise control of the conditions (cardiac work, coronary perfusion pressure) and composition (hypocalcemic, hyperosmolar, amino acidenhanced blood cardioplegia) of the initial reperfusion period. Other studies in which oxygen radical scavengers were added to the reperfusing blood demonstrated a significant reduction in infarct size,"7, 23 These studies of modified reperfusion emphasize that the total myocardial injury after coronary occlusion can be reduced by interventions initiated during the reperfusion period. The reduction in postischemic myocardial injury is predicated on the presence of salvageable tissue at the time of reperfusion. The results of the present study suggest an important T{)lp.
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necrosis, metabolic derangements, and contractile dysfunction in the setting of transient coronary occlusion. Its role in determining both the transition from reversible to irreversible myocellular injury and the time course of necrosis would be critical to our understanding of the salvageability of ischemic myocardium before restoration of blood flow. Previous studies that systematically characterized the transition from reversible to irreversible injury were performed in ischemic-reperfused tissue in which reperfusion injury may have already contributed to tissue injury. I, 4, 42 In addition, further studies are needed to define the time course of progression of reperfusion injury. This information is important to the development of strategies designed to reduce the pathophysiologic expression of reperfusion injury by appropriate modification of the conditions and composition of the initial phase of reperfusion. The concepts of reperfusion modification may be applied in surgical reperfusion or in the catheterization laboratory, where reperfusate modification may be achieved by means of the angioplasty catheter before its removal from the coronary artery. We wish to express our thanks to A. C. Shircliffe and the staff of the Surgery Research Unit, and to Stewart Barnett, BS, Consultation on statistical analysis was provided by George Howard, MPHS, REFERENCES I, Kloner RA, Ellis SG, Lange R, Braunwald E. Studies of experimental coronary artery reperfusion: effects on infarct size, myocardial function, biochemistry, ultrastructure and microvascular damage. Circulation 1983;68(Pt 2):18-15. 2. Braunwald E, KIoner RA. The stunned myocardium: prolonged, postischemic ventricular dysfunction. Circulation 1982;66:1146-9. 3. Reimer KA, Lowe JE, Rasmussen MM, Jennings RB. The wavefront phenomenon of ischemic cell death. I. Myocardial infarct size vs duration of coronary occlusion in dogs. Circulation 1977;56:786-94. 4. Jennings RB, Reimer KA. Factors involved in salvaging myocardium: effect of reperfusion of arterial blood. Circulation 1983;68(Pt 2):125-36. 5. Ellis SG, Henschke CI, Sandor T, Wynne J, Braunwald E, KIoner RA. Time course of functional and biochemical recovery of myocardium salvaged by reperfusion. J Am Coli Cardiol 1983;4:1047-55. 6. Jolly SR, Kane WJ, Baile MB, Abrams GD, Lucchesi BR. Canine myocardial reperfusion injury: its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 1984;54:277-85. 7. Mitsos SE, Fantone JC, Gallagher KP, et al. Canine myocardial reperfusion injury: protection by a free radical scavenger, N-2-mercaptopropionyl glycine. J Cardiovasc Pharmacol 1986;8:978-88.
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Immediate functional recovery and avoidance of reperfusion injury with surgical revascularization of short-term coronary occlusion. Circulation 1985;72:431-9. 9. Vinten-Johansen J, Edgerton TA, Hansen KJ, Carroll P, Mills SA, Cordell AR. Surgical revascularization of acute (1 hour) coronary occlusion: blood versus crystalloid cardioplegia. Ann Thorac Surg 1986;42:247-54. 10. Vinten-Johansen J, Faust KB, Mills SA, Cordell AR. Surgical revascularization of acute evolving myocardial infarction without blood cardioplegia fails to restore postischemic function in the involved segment. Ann Thorac Surg 1987;44:66-72. 11. Vinten-Johansen J, Okamoto F, Rosenkranz ER, Buckberg GD, Bugyi H, Leaf J. Studies of controlled reperfusion after ischemia. V. Superiority of surgical versus medical reperfusion after regional ischemia. J THORAC CARDIOVASC SURG 1986;92:525-34. 12. Rankin JS, Arentzen CE, Ring WS, Edwards CH III, McHale PA, Anderson RW. The diastolic mechanical properties of the intact left ventricle. Fed Proc 1980; 39:141-7. 13. Gaasch WH, Levine HJ, Quinones MA, Alexander lK. Left ventricular compliance: mechanisms and clinical implications. Am 1 Cardiol 1976;38:645-53. 14. Gaasch WH, Apstein CS, Levine Hl. Diastolic properties of the left ventricle: basic and clinical implications, Boston: Martinus Nijhoff Publishing, 1985: 143-70. IS. Woolf CM. Principles of biometry. Princeton, New lersey: D. Van Nostrand Co, Inc., 1968:101. 16. Rosenkranz ER, Buckberg GD. Myocardial protection during surgical coronary reperfusion. J Am Coli Cardiol 1983; I: 1235-46. 17. Rosenkranz ER, Okamoto F, Buckberg GD, VintenJohansen J, Robertson JM, Bugyi H. Safety of prolonged aortic clamping with blood cardioplegia. II. Glutamate enrichment in energy-depleted hearts. 1 THORAC CARDlOVASC SURG 1984;88:402-10. 18. Follette DM, Fey K, Buckberg GD, et al. Reducing postischemic damage by temporary modification of reperfusate calcium, potassium, pH, and osmolarity. 1 THORAC CARDIOVASC SURG 1981;82:221-38. 19. Ferrari R, Williams A, DiLisa F. The role of mitochondrial function in the ischaemic and reperfused myocardium. In: Caldarera CM, Harris P, eds. Advances in studies on heart metabolism. Bologna: Cooperative Libraria Universitaria Editrice, 1982:245-55. 20. Vinten-Johansen J, Sloan BD III, Campbell ML, Mills SA, Cordell AR. Defective postischemic O 2 utilization in reperfused, "stunned" myocardium [Abstract]. Fed Proc 1986;45:530. 21. Johnson RE, Dorsey LM, Moye sr, Hatcher CR lr, Guyton RA. Cardioplegic infusion: the safe limits of pressure and temperature. J THORAC CARDIOVASC SURG 1982;83:813-23. 22. Little WC, Park RC, Freeman GL. Effects of regional ischemia and ventricular pacing on LV dP/dtmax--enddiastolic volume relation. Am 1 Physiol 1987;252:H933-
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23. Mitsos SE, Askew TE, Fantone lC, Kunkel SL, Lucchesi BR. Protective effects of N-2-mercaptoproprionyl glycine (MPG) against myocardial reperfusion injury following neutrophil depletion in the dog. Circulation 1985;72(Pt 2):III 120. 24. Theroux P, Franklin D, Ross J lr, Kemper WS. Regional myocardial function during acute coronary artery occlusion and its modification by pharmacologic agents in the dog. Circ Res 1974;35:896-908. 25. Lew WYW, Chen Z, Guth B, Covell JW. Mechanisms of augmented segment shortening in nonischemic areas during acute ischemia of the canine left ventricle. Circ Res 1985;56:351-8. 26. Lew WYW. Influence of ischemic zone size on nonischemic area function in the canine left ventricle. Am J Physiol 1987;252:H990-7. 27. Kloner RA, Ganote CE, Whalen D, Jennings RB. Effect of a transient period of ischemia on myocardial cells. II. Fine structure during the first few minutes of reflow. Am 1 Pathol 1974;74:399-422. 28. Sjostrand F, Allen BS, Buckberg GD, et al. Studies of controlled reperfusion after ischemia. IV. Electron microscopic studies: importance of embedding techniques in quantitative evaluation of cardiac mitochondrial structure during ischemia and reperfusion. J THORAC CARDIOVASC SURG 1986;92:513-24. 29. Shen AC, Jennings RB. Kinetics of calcium accumulation in acute myocardial ischemic injury. Am 1 Pathol 1972; 67:441-52. 30. Walsh LG, Tormey 1M. Subcellular shifts of electrolytes during myocardial ischemia and reperfusion [Abstract]. Fed Proc 1986;45:773. 31. Nayler WG, Ferrari R, Williams A. Protective effect of pretreatment with verapamil, nifedipine and propranolol on mitochondrial function in the ischemic and reperfused myocardium. Am 1 Cardiol 1980;46:242-8. 32. Trump BF, Mergner WJ, Kahng MW, Saladino AJ. Studies on the subcellular pathophysiology of ischemia. Circulation 1976;53(Pt 2):I17-26. 33. Jennings RB, Reimer KA. Lethal myocardial ischemic injury. Am 1 Pathol 1981;102:241-55. 34. Nayler WG. The role of calcium in the ischemic myocardium. Am J Pathol 1981;102:262-70. 35. Pirzada FA, Ekong EA, Vokonas PS, Apstein CS, Hood WB Jr. Experimental myocardial infarction. XIII. Sequential changes in left ventricular pressure-length relationship in the acute phase. Circulation 1976;53: 970-4. 36. Pirzada FA, Weiner JM, Hood WB Jr. Experimental myocardial infarction. 14. Accelerated myocardial stiffening related to coronary reperfusion following ischemia. Chest 1978;74: 190-5. 37. Haendchen RV, Corday E, Torres M, Maurer G, Fishbein MC, Meerbaum S. Increased regional end-diastolic wall thickness early after reperfusion: a sign of irreversibly damaged myocardium. J Am Coli Cardiol 1984;3:144453. 38. Crozatier B, Ashraf M, Franklin D, Ross 1 Jr. Sarcomere
96 8
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sarcomere overstretch in dyskinetic ventricular regions. J Mol Cell Cardiol 1977;9:785-97. 39. Edwards CH II, Rankin JS, McHale PA, Ling D, Anderson RW. Effects of ischemia on left ventricular regional function in the conscious dog. Am J Physiol 1980;240:H413-20. 40. Nayler WG, Poole-Wilson PA, Williams A. Hypoxia and calcium. J Mol Cell Cardiol 1979;11:683-706.
Thoracic and
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41. Vogel WM, Briggs LL, Apstein CS. Separation of inherent diastolic myocardial fiber tension and coronary vascular erectile contributions to wall stiffness of rabbit hearts damaged by ischemia, hypoxia, calcium paradox and reperfusion. J Mol Cell Cardiol 1985;17:57-70. 42. Schaper J, Schaper W. Reperfusion of ischemic myocardium: ultrastructural and histochemical aspects. J Am Coli Cardiol 1983;1:1037-46.
Bound volumes available to subscribers Bound volumes of THE JOURNAL OFTHORACIC AND CARDIOVASCULAR SURGERY are available to subscribers (only) for the 1988 issues from the Publisher, at a cost of $51.00 ($68.00 international) for Vol. 95 (January-June) and Vol. 96 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 60 days after publication of the last issue of the volume. The binding is durable buckram with the JOURNAL name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact The C. V. Mosby Company, Circulation Department, 11830 Westline Industrial Drive, St. Louis, Missouri 63146, USA; phone (800) 325-4177, ext. 351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular JOURNAL subscription.
THoRAc CARDIOVASC SURG
1988;95:960-8
Reperfusion injury after temporary coronary occlusion In 24 anesthetized open-chest dogs, we examined the time course of changes in contractile function, diastolic muscle stiffness (sonomicrometry), tissue water content, and ultrastructure after 1 hour of occlusion of the left anterior descending coronary artery and after 2 hours of unmodified reperfusion. One hour of occlusion of the left anterior descending artery replaced active shortening with passive bulging (21.4% ± 2.9% versus -5.9% ± 0.9%, p < 0.05) in the involved segment. There was no increase in either subendocardial water content (78.6% ± 0.1 % versus 79.7% ± 0.7%) or operative muscle stiffness (2.80 ± 0.72 versus 2.36 ± 0.42 mm Hgjmm) after the occlusion period. There were only mild to moderate ultrastructural alterations suggestive of reversible injury. In sharp contrast, reperfusion was associated with a 2.48 % increase in subendocardial water content (p < O.O~ a 42 % increase in diastolic muscle stiffness (3.34 ± 0.42 mm Hgjmm, p < 0.05~ and greater ultrastructural damage. We conclude that myocardial injury is significantly extended with unmodified blood reperfusion after temporary coronary occlusion.
J. Vinten-Johansen, PhD, William E. Johnston, MD, Stephen A. Mills, MD, Kirk B. Faust, MD, Kim R. Geisinger, MD, Richard J. DeMasi, MD, and A. Robert Cordell, MD, Winston-Salem. n.c.
Ltal coronary occlusion reduces oxygen and substrate delivery and fails to wash out metabolites in the involved myocardium. The degree of myocardial damage is related to the severity and duration of the occlusion. 1-4 Biochemical and morphologic alterations are reversible with brief occlusions lasting 12 to 15 minutes, although contractile dysfunction persists.v? However, irreversible changes culminating in necrosis begin to occur in the subendocardium after 40 minutes of ischemia and progress in a "wavefront pattern" to involve the subepicardium after 4 to 6 hours of occlusion.' Reperfusion
From the Departments of Surgery (Section on Cardiothoracic Surgery), Anesthesia, and Pathology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, N.C. Supported in part by a grant-in-aid from the American Heart Association, North Carolina Affiliate (No. 84-85-A7) and National Institutes of Health Grant HL 36377. Presented in part at the Seventieth Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, Mo., April 15, 1986. Received for publication Sept. 19, 1986. Accepted for publication July 27, 1987. Address for reprints: J. Vinten-Johansen, PhD, Section on Cardiothoracic Surgery, Bowman Gray School of Medicine, 300 South Hawthorne Rd., Winston-Salem, NC 27103. OAf)
salvages myocardium that would otherwise be destined for necrosis if reperfusion were not established and results in smaller infarct sizes. However, reperfusion does not completely avoid infarct development after I to 2 hours of coronary occlusion. I, 4, 5 Recent studies have shown that the extent of necrosis in the area placed at risk by acute occlusion can be reduced if appropriate interventions are introduced at the onset of reperfusion." 7 When reperfusion was modified by controlling the composition of the initial reperfusate (i.e., blood cardioplegia) and the reperfusion pressure, myocardial necrosis was reduced by approximately 50% to 75%, with restoration of immediate contractile function."!' This reduction of postischemic damage with modified reperfusion is predicated on the salvageability of the myocardium at the time reperfusion is established. Although reperfusion salvages myocardium, it may also advance the extent of injury beyond that present before reperfusion. However, progression in the extent of tissue injury between ischemia and reperfusion has not been clearly shown in the setting of transient coronary occlusion. This study tests the hypothesis that myocardial injury after transient coronary occlusion is extended beyond that apparent before reperfusion. Correlative changes in contractile function, diastolic compliance (muscle stiffness), tissue edema,
Volume 95 Number 6 June 1988
Reperfusion injury
961
Table I. Hemodynamics for control, ischemia, and reperfusion periods in group III dogs undergoing 1 hour of LAD occlusion and 2 hours of reperfusion Arterial
Control Ischemia Reperfusion
CO
HR
LVSP (mmHg)
LVEDP (mmHg)
Sys (mmHg)
Dias (mmHg)
MAP (mmHg)
+dPjdt
-dPjdt
(Ljmin)
144 ± 8 141 ± 8 141 ± 7
127 ± 6 118 ± 6 110 ± 5*
7.1 ± 0.8 11.0 ± 1.0* 9.6 ± 1.2
131 ± 5 124 ± 6 112 ± 6*
III ± 5 106 ± 5 94 ± 6*
118 ± 5 III ± 5 99 ± 6*
2554 ± 235 1992 ± 166 1815 ± 117*
3245 ± 248 2608 ± 193 2213 ± 158
2.75 ± 0.22 2.18 ± 0.13* 2.08 ± 0.13*
Values are means ± standard error of the mean; LYSP.left ventricular peak systolic pressure; LYEOP, left ventricular end-diastolic pressure; Sys, systolic pressure; Dias, diastolic pressure; MAP, mean arterial pressure; CO, cardiac output. 'p < 0.05 versus control.
and ultrastructure were measured between the ischemic and reperfusion phases in a canine model of occlusion and reperfusion of the left anterior descending (LAD) coronary artery. We found that, although changes in systolic function occurred during ischemia, the predominant increase in diastolic muscle stiffness, accumulation of edema, and abnormalities in ultrastructure were manifested primarily after reperfusion.
Methods Twenty-four dogs of both sexes, weighing between 13.5 and 26.0 kg (mean 17.9 ± 1.0 kg), were anesthetized with sodium pentobarbital (30 mg/kg, given intravenously) supplemented continuously with a 1 to 2 rng/kg/hr infusion. The dogs were intubated with a cuffed endotracheal tube and ventilated with oxygen-enriched room air with a Harvard volume-cycled ventilator (Harvard Apparatus Co., Inc., S. Natick, Mass.). Ventilation was adjusted to maintain carbon dioxide tension between 35 and 45 mm Hg and pH between 7.38 and 7.42. Catheters were inserted into the right femoral artery and vein for blood sampling and administration of drugs and fluids, respectively. A 7.5F flow-directed thermodilution cardiac output catheter was introduced into the right jugular vein and positioned in the pulmonary artery. The chest was incised at the left fifth intercostal space. Umbilical tape snares were placed around the superior and inferior vena cavae for bicaval occlusion to reduce preload for assessment of segmental muscle stiffness, which is the inverse of compliance." The pericardium was widely incised and sutured to the chest wall to cradle the heart. Millar MPC-500 micromanometer-tipped catheters (Millar Instruments, Inc., Houston, Texas) were inserted into the proximal aorta via the left internal mammary artery and into the left ventricle via the apical dimple to measure instantaneous aortic and left ventricular pressures, respectively. The LAD coronary artery distal to the first diagonal branch was dissected free and isolated with a loose silicone rubber snare. Two pairs of 5 MHz ultrasonic dimension gauges, 2 mm in diameter, were implanted subendocardially in the myocardial segments perfused by the LAD (ischemic zone) and circumflex (nonischemic zone) coronary arteries to measure segmental fractional shortening (Triton model 120 sonomicrometer, Triton Equipment Corp., San Diego, Calif.). A standard limb lead II electrocardiogram was continuously monitored. Calculations. All analog signals were recorded on a twelvechannel Hewlett-Packard fiberoptic recorder (Hewlett-Pack-
ard Company, Andover, Mass.) and on FM magnetic tape for later analog-to-digital conversion and processing (Labtech Notebook software). Hemodynamic measurements included instantaneous left ventricular and aortic systolic and diastolic pressures, left ventricular peak positive and negative pressure rise (dP/dt), and thermodilution cardiac output obtained in triplicate by 3 ml iced saline injections. End-diastole and end-systole of the dimension signals were timed from the upstroke of the left ventricular pressure pulse and the dicrotic notch of the aortic pressure, respectively. Active systolic segmental shortening (SS) was calculated as follows: 100 X ([EDL - ESLJ/EDL), where EDL and ESL are enddiastolic and end-systolic segment lengths, respectively. Regional passive diastolic muscle stiffness characteristics were determined by progressivelydecreasing the left ventricular end-diastolic pressure and segment length by bicaval occlusion. The resulting pressure-segment length data points were fit to an exponential curve. Segment lengths per se cannot be used for this analysis because the prolonged systolic stretching of the bulging ischemic segment results in segment length elongation at a given end-diastolic pressure that may be independent of changes in actual regional muscle stiffness. Therefore, the curvilinear characteristics of the pressuresegment length curve were used to assess muscle stiffness of the ischemic-reperfused segment. According to the method of Gaasch and associates.P-" the tangent to the curve at 4 nun Hg was drawn. The slope of this tangent describes the operative muscle stiffness of the myocardial segment. An increase in the slope of the tangent indicates an increase in muscle stiffness (i.e., loss of compliance), whereas a decrease in the slope indicates a decrease in muscle stiffness. At the end of the experiment, transmural biopsy samples were taken from the ischemic and nonischemic zones by a high-speed drill and divided into subepicardial and subendocardial halves to determine tissue water content by drying at 85° C for 48 hours. All biopsy sites were repaired and the coronary occlusions replaced. Next, 3 ml of 2% gentian violet solution was injected into the left atrium. After the dye had been allowed to circulate for 15 seconds, the heart was arrested with bolus injection of potassium chloride and excised. The correct placement of the crystals was verified by the presence (nonischemic zone) or absence (ischemic zone) of gentian violet staining in the surrounding myocardium. The quantity of area placed at risk by coronary occlusion was determined by planimetry and calculated as the nonstained area/total left ventricular area times 100. Samples for electron microscopic study were taken from the
The Journal of Thoracic and Cardiovascular Surgery
96 2 Vinten-Johansen et al.
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Reperfusion
Fig. 1. Fractional systolic shortening in the ischemic (e----.) and nonischemic (0 ----0) segments during control, after 60 minutes of coronary occlusion (ischemia), and after 120 minutes of reperfusion. *p < 0.05 versus control.
central ischemic zone and the nonischemic zone after occlusion or after reperfusion in randomly selected experiments by a Travenol Tru-Cut biopsy needle (Travenol Laboratories, Inc., Deerfield, Ill.) and fixed in 2.5% glutaraldehyde. Thick sections stained with toluidine blue were reviewed to select areas for ultrastructural examination. Thin sections were double stained with uranyl acetate and lead citrate. Sections were inspected at 2500 and 7000 power. Five to six electron photomicrographs were made for each specimen at a magnification of 7000X to evaluate mitochondrial and sarcolemmal structure. In addition, several photomicrographs were taken from each specimen to analyze sarcolemmal, nuclear, and intercellular structure. The pathologist was unaware of the origin of the sample at the time of examination. Experimental protocol. The condition of each dog was allowed to stabilize for 30 minutes after completion of the operation. The dogs were then divided into three separate groups. Group I: Controls. Biopsy samples from the region perfused by the LAD were obtained for analysis of tissue water content from eight dogs that did not undergo either ischemia or reperfusion. Group II: Ischemia. Eight dogs underwent I hour of LAD occlusion without reperfusion. Lidocaine (75 mg) was administered before LAD occlusion. At the end of the ischemic period, biopsy samples were taken for assessment of tissue water content. Group III: Ischemia plus reperfusion. Eight dogs underwent I hour of LAD occlusion followed by 2 hours of reperfusion. Lidocaine (75 mg) was given before occlusion and reperfusion. Sequential hemodynamic and segmental function measurements were taken. In randomly selected dogs, tissue samples were taken after the occlusion and reperfusion periods for electron microscopic analysis. At the end of the experiment, biopsy samples were taken for tissue water content. Data were analyzed by the Statistical Analysis Systems program (SAS Institute, Cary, N.C.). Tissue water content data were analyzed by analysis of variance for group differences followed by Duncan's multiple range test to locate the source of difference." Within each group, differences between epicardial and endocardial water contents were analyzed by Student's t test. Sequentially obtained hemodynamic, functional, and operative stiffness data (group III) were analyzed
EDL
Ischemic zone 12.88 Control 14.61 Ischemia 13.66 Reperfusion Nonischemic zone Control 12.02 Ischemia 12.12 Reperfusion 12.44
ESL
%SS
± 0.87 ± 1.01 ± 0.93
10.19 ± 0.85 15.47 ± 1.08* 14.51 ± 0.97*
21.40 ± 2.86 -5.90 ± 0.91* -6.41 ± 1.05*
± 1.10 ± 1.31 ± 1.04
10.94 ± 0.94 10.52 ± 1.37 10.80 ± 0.88
9.10 ± 0.90 13.84 ± 3.73* 13.08 ± 0.83
EDL. end-diastolic length; ESL. end-systolic length; %55. percent fractional systolic shortening. *p < 0.05 versus centrol.
by the general linear model multivariate analysis for repeated measures followed by Wilk's lambda test. Significance was accepted at the 0.05 level of probability. All procedures complied with the "Guiding Principles in Care and Use of Animals," adopted by the Council of the American Physiologic Society, and with the "Guide for the Care and Use of Laboratory Animals" (Publication No. 78-23). The protocol was reviewed by the institutional research and animal care committees.
Results Hemodynamics. Hemodynamic data for the eight dogs in group III undergoing ischemia and reperfusion are summarized in Table I. Ligation of the LAD involved 35.2% ± 2.5% of the total left ventricular mass in the ischemic zone. After 1 hour of LAD occlusion,left ventricular end-diastolic pressure increased significantly and cardiac output decreased by 21% (p < 0.05). Heart rate and left ventricular and arterial blood pressures did not change significantly during the ischemic period. After 2 hours of reperfusion, however, left ventricular systolic pressure and arterial systolic and diastolic pressures decreased significantly from control values. Function. Active systolic shortening in the ischemic zone before ligation was rapidly replaced by passive lengthening, which persisted for the entire hour of ligation and averaged -33.7% ± 5.1% of the control systolic shortening (p < 0.001, Table 11). There was a moderate but insignificant increase in end-diastolic segment length during occlusion. After 2 hours of reperfusion, the ischemic segment continued to lengthen passively with systolic shortening averaging -36.0% ± 6.3% of control (Fig. 1). In the nonischemic segment, end-diastolic segment length increased slightly (p > 0.10) and segmental shortening increased significantly in compensation of the ischemic zone. This hypercontractile state in the nonischemic segment continued throughout reperfusion (Fig. 1). Tissue water content. Tissue water content in the
Volume 95 Number 6 June 1988
control group was similar in the epicardial and endocardial regions, averaging 78.18% ± 0.12% and 78.60% ± 0.14%, respectively. There was no significant increase in tissue water content in either region after I hour of LAD occlusion (Fig. 2). However, after 2 hours of reperfusion, water content had increased significantly by 2.37% in the epicardium and by 2.48% in the endocardium. The normally perfused, nonischemic segment showed only a slight decrease of approximately I % in tissue water content in the endocardium during the ischemic period. Operative muscle stiffness. A graph of left ventricular end-diastolic pressure-segment length data during gradual preload reduction in one experiment (6-4-85) is shown in Fig. 3, A. Data were curve-fit before LAD occlusion (control), after I hour of LAD occlusion (ischemia), and after 2 hours of reperfusion. Fig. 3, B showsthe tangent to the curve at 4 mm Hg taken before occlusion. The slope of the tangent (M>/ ~L) describes the passive muscle stiffness during that period." 14 With I hour of LAD occlusion, the left ventricular enddiastolic pressure-segment length curve shifted rightward, consistent with systolic elongation or myocardial creep. However, there was no change in the passive stiffnessof the muscle (Fig. 3, C). With reperfusion, on the other hand, there was a significant increase (p < 0.02) in the stiffness of the reperfused segment. For a given change in left ventricular end-diastolic segment length, there was a larger increase in end-diastolic pressure than that observed either before or after I hour of LAD occlusion. Ultrastructure. Myocardial tissue appeared less damaged after coronary occlusion than after reperfusion (Fig. 4). After 1 hour of coronary occlusion, sarcomeres appeared largely in register with contraction bands visible in only a few samples. Mild mitochondrial swelling, with some matrix clarification and disorganization of the cristae, were observed. In addition, moderate clumping of nuclear chromatin and mild intracellular and intercellular edema were noted in some samples. In contrast, more severe alterations in ultrastructure were apparent after reperfusion. Overall, myocytes that showed evidence of irreversible damage were those that had been reperfused. These changes included fragmentation of the sarcolemma, presence of contraction bands, and mitochondrial aberrations characterized by swelling, appearance of flocculent densities, matrix clarification, and disruption of the cristae. Contraction bands were more numerous than in paired samples taken after the hour of LAD occlusion before reperfusion. In addition, intracytoplasmic fluid accumulation appeared more accentuated in reperfused tissue than in its paired ischemic sample. Degenerative alterations were more
Reperfusion injury
963
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diffuse and more intense in endocardial, than in epicardial samples in both ischemic and ischemic-reperfused tissue biopsy specimens. Discussion Reperfusion, while essential in preventing the progression of necrosis from encompassing the entire transmural area at risk, is also associated with deleterious changes that increase the extent of myocellular injury. In our study, the ischemic segment demonstrated no increase in either total tissue water or muscle stiffness and showed less severe ultrastructural abnormalities after the ischemic period. After reperfusion, however, changes consistent with severe myocardial injury were manifested. Specifically, there was a significant increase in tissue water content and passive muscle stiffness, both suggesting a loss of muscle compliance and potentially irreversible injury. Derangements in the myocellular ultrastructure were supportive of these reperfusion alterations. These data support the concept of reperfusion injury in the setting of acute coronary occlusion and suggest that reperfusion injury is a significant component of the total myocardial damage after acute coronary occlusion and reperfusion.
The Journal of Thoracic and Cardiovascular Surgery
964 Vinten-Johansen et al.
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Fig. 3. A, End-diastolic pressure-segment length data in one experiment (6-4-85) obtained before coronary occlusion (control), after I hour of LAD occlusion (ischemia), and after 2 hours of reperfusion. Note the rightward shift in absolute segment length without an apparent shift in slope of the curve. After reperfusion, there is an increase in slope, indicating an increase in muscle stiffness.
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Reperfusion injury is well described in globally ischemic hearts, where it is encountered as a consequence of inadequate myocardial protection. 16. 18 Characteristic changes during reperfusion include (1) an increase in myocardial edema, (2) a loss of ventricular compliance, and (3) a depression in oxygen utilization capacity caused by reduction in mitochondrial oxidative phosphorylation 19 and significant increases in myofibrillar and mitochondrial calcium. 16 The results from our study confirm that a similar manifestation of reperfusion damage is seen in the setting of coronary occlusion and reperfusion. The reperfused segment was severely edem-
Fig. 3. C, Operative muscle stiffness, determined by the method described in B, before occlusion (control), after I hour of LAD occlusion, and after 2 hours of reperfusion. Note the significant increase (*p < 0.05) in operative muscle stiffness only after reperfusion. LVEDP. Left ventricular end-diastolic pressure.
atous and had lost compliance. The electron micrographs in the current study would suggest that segmental oxygen utilization may be depressed because of mitochondrial deterioration and dysfunction. A depressed postischemic oxygen utilization has, in fact recently been demonstrated after reperfusion of the ischemic zone after temporary coronary occlusion.20 In contrast to global left ventricular ischemic-reperfusion injury in which impaired systolic and diastolic function are expressed as compromised hemodynamics (low cardiac output, hypotension), regional ischemia may not critically affect systemic hemodynamics. In the present study, total loss of contractile function in the ischemic segment was accompanied by only modest reductions in global hemodynamics, such as left ventricular pressure, positive dP jdt, and cardiac output. In addition, a loss of compliance in the involved zone was not associated with higher left ventricular end-diastolic pressure (Table I, reperfusion). Other studies also have shown that global indices of performance do not accurately reflect the extent of regional injury and are not significantly altered in compensated regional infarction." Although global ventricular performance may be slightly depressed during coronary occlusion," systemic hemodynamics are maintained by compensatory hyperkinesia in the nonischemic segment"!" 23. 24 so long as the involved area does not exceed 40% of the total left ventricular mass. One major mechanism underlying this compensatory hyperkinesia is a local Starling response to ventricular dilatation caused by contractile dysfunction in the ischemic zone." 26 Although end-diastolic length in the nonischemic zone did not consistently increase in our study, systolic shortening did significant-
Volume 95 Number 6 June 1988
Reperfusion injury
965
Fig. 4. Electron micrographs from the epicardial region after I hour of coronary occlusion (A) and 2 hours of reperfusion (8). A shows mild to moderate clarification of mitochondria (M) with some separation of cristae and some swelling. Myofibrillar architecture is present in many areas but disrupted in others. There is evidence of very mild first-stage formation of contraction bands (C). 8 shows loss of sarcomere architecture caused by increased degree of contraction banding. Mitochondria have regained electron opacity but severe vacuolizations have developed that were not present after ischemia. (Original magnification x7000.)
ly increase consistent with a hyperkinetic response. However, a significant fraction of this hyperkinesia is expended in stretching the compliant ischemic segment during isovolumetric systole, which results in less effective ejection, reduced cardiac output, and elevated end-diastolic pressure. With the loss of compliance during reperfusion, however, a contractile effort is not expended unproductively in stretching the noncompliant segment, which potentially contributes to improved performance and lower end-diastolic pressures in our study. In addition, resumption of some degree of function with reperfusion in less ischemic "border zones" may also have contributed to apparently improved performance relative to the period of coronary occlusion. In the absence of reflow, mitochondrial damage was modest and myofibrillar derangements were largely absent. With reperfusion, however, these reversible changes progressed to largely irreversible derangements. This acceleration in ultrastructural injury with reperfusion was observed previously by Kloner and associates," but its relation to a temporally separate, progressive reperfusion injury was not appreciated. Recent evidence supporting a reperfusion-dependent progression in subcellular damage was reported by Sjostrand and col-
leagues," using a low-protein denaturation fixation process. In this study, up to 6 hours of regional ischemia caused only moderate, reversible deterioration in subcellular architecture, whereas reperfusion was marked by a dramatic transition to severe, irreversible damage. In addition to acceleration of ultrastructural damage, a similar reperfusion-dependent increase in total tissue or intracellular calcium has been observed. 19, 29-32 This calcium-loading phenomenon may be a key factor in the transition from reversible to irreversible damage.": 34 With electron microprobe analysis, there was relatively normal distribution of calcium in mitochondrial, myofibrillar, and cytosolic compartments after an ischemic period. However, with reperfusion, calcium accumulated in mitochondria and around the myofibrils." Several studies have shown that compliance is lost in severely injured myocardium. 16. 18,35.36 The results from this study extend these observations by showing that the predominant loss in segmental compliance occurs only after reperfusion. During coronary occlusion, the absolute end-diastolic segment length at a given pressure increased, possibly related to wall thinning," sarcomere elongation," or myocardial creep'<" caused by the persistent systolic paradoxical stretch. However, seg-
966
The Journal of Thoracic and Cardiovascular
Vinten-Johansen et al.
Surgery
mental compliance characteristics were not altered from the preischemic condition. The loss of compliance occurred only after reperfusion, possibly as a result of an increase in tissue water or the development of contraction bands, as observed in our study. In addition, the intracellular accumulation of calcium has also been linked to the decrease in postischemic compliance." A similar time course is seen in the loss of compliance, and the accumulation or intercompartmental shift in calcium has been reported." 30, 32, 40 Loss of compliance during reperfusion has also been attributed to augmented vascular turgor that occurs with reperfusion and edema." It may be expected that a less compliant segment would not stretch passively during systole, that is, would be dyskinetic. With a severe loss of compliance, such dyskinesia and absence of paradoxical bulging are noted. However, with less severe loss of compliance, some degree of distensibility is retained with less stretch during systole. In our study, end-systolic stretch was less after reperfusion (14.51 ± 0.97 mm) than after LAD occlusion (15.47 ± 1.08 mm) for similar end-systolic ventricular pressures. This reduction in systolic stretch in reperfused segments is masked by the apparent equality in negative systolic shortening (paradoxical bulging) because of the normalization using smaller end-diastolic segment lengths. Whether reperfusion injury is an unmasking of ischemic injury or represents an active, progressive process requires further experimental verification. The strongest support for a reperfusion injury phenomenon comes from studies showing that the degree of eventual postischemic injury can be reduced by interventions introduced only during the period of reperfusion."!': 16, 18 Recently, surgical reperfusion of acute coronary occlusion demonstrated significant reduction in infarct size and edema and partially restored fractional shortening in the involved segment after 1 or 2 hours of coronary occlusion."!' The increased salvage of myocardium may be attributed to the precise control of the conditions (cardiac work, coronary perfusion pressure) and composition (hypocalcemic, hyperosmolar, amino acidenhanced blood cardioplegia) of the initial reperfusion period. Other studies in which oxygen radical scavengers were added to the reperfusing blood demonstrated a significant reduction in infarct size,"7, 23 These studies of modified reperfusion emphasize that the total myocardial injury after coronary occlusion can be reduced by interventions initiated during the reperfusion period. The reduction in postischemic myocardial injury is predicated on the presence of salvageable tissue at the time of reperfusion. The results of the present study suggest an important T{)lp.
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necrosis, metabolic derangements, and contractile dysfunction in the setting of transient coronary occlusion. Its role in determining both the transition from reversible to irreversible myocellular injury and the time course of necrosis would be critical to our understanding of the salvageability of ischemic myocardium before restoration of blood flow. Previous studies that systematically characterized the transition from reversible to irreversible injury were performed in ischemic-reperfused tissue in which reperfusion injury may have already contributed to tissue injury. I, 4, 42 In addition, further studies are needed to define the time course of progression of reperfusion injury. This information is important to the development of strategies designed to reduce the pathophysiologic expression of reperfusion injury by appropriate modification of the conditions and composition of the initial phase of reperfusion. The concepts of reperfusion modification may be applied in surgical reperfusion or in the catheterization laboratory, where reperfusate modification may be achieved by means of the angioplasty catheter before its removal from the coronary artery. We wish to express our thanks to A. C. Shircliffe and the staff of the Surgery Research Unit, and to Stewart Barnett, BS, Consultation on statistical analysis was provided by George Howard, MPHS, REFERENCES I, Kloner RA, Ellis SG, Lange R, Braunwald E. Studies of experimental coronary artery reperfusion: effects on infarct size, myocardial function, biochemistry, ultrastructure and microvascular damage. Circulation 1983;68(Pt 2):18-15. 2. Braunwald E, KIoner RA. The stunned myocardium: prolonged, postischemic ventricular dysfunction. Circulation 1982;66:1146-9. 3. Reimer KA, Lowe JE, Rasmussen MM, Jennings RB. The wavefront phenomenon of ischemic cell death. I. Myocardial infarct size vs duration of coronary occlusion in dogs. Circulation 1977;56:786-94. 4. Jennings RB, Reimer KA. Factors involved in salvaging myocardium: effect of reperfusion of arterial blood. Circulation 1983;68(Pt 2):125-36. 5. Ellis SG, Henschke CI, Sandor T, Wynne J, Braunwald E, KIoner RA. Time course of functional and biochemical recovery of myocardium salvaged by reperfusion. J Am Coli Cardiol 1983;4:1047-55. 6. Jolly SR, Kane WJ, Baile MB, Abrams GD, Lucchesi BR. Canine myocardial reperfusion injury: its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 1984;54:277-85. 7. Mitsos SE, Fantone JC, Gallagher KP, et al. Canine myocardial reperfusion injury: protection by a free radical scavenger, N-2-mercaptopropionyl glycine. J Cardiovasc Pharmacol 1986;8:978-88.
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Immediate functional recovery and avoidance of reperfusion injury with surgical revascularization of short-term coronary occlusion. Circulation 1985;72:431-9. 9. Vinten-Johansen J, Edgerton TA, Hansen KJ, Carroll P, Mills SA, Cordell AR. Surgical revascularization of acute (1 hour) coronary occlusion: blood versus crystalloid cardioplegia. Ann Thorac Surg 1986;42:247-54. 10. Vinten-Johansen J, Faust KB, Mills SA, Cordell AR. Surgical revascularization of acute evolving myocardial infarction without blood cardioplegia fails to restore postischemic function in the involved segment. Ann Thorac Surg 1987;44:66-72. 11. Vinten-Johansen J, Okamoto F, Rosenkranz ER, Buckberg GD, Bugyi H, Leaf J. Studies of controlled reperfusion after ischemia. V. Superiority of surgical versus medical reperfusion after regional ischemia. J THORAC CARDIOVASC SURG 1986;92:525-34. 12. Rankin JS, Arentzen CE, Ring WS, Edwards CH III, McHale PA, Anderson RW. The diastolic mechanical properties of the intact left ventricle. Fed Proc 1980; 39:141-7. 13. Gaasch WH, Levine HJ, Quinones MA, Alexander lK. Left ventricular compliance: mechanisms and clinical implications. Am 1 Cardiol 1976;38:645-53. 14. Gaasch WH, Apstein CS, Levine Hl. Diastolic properties of the left ventricle: basic and clinical implications, Boston: Martinus Nijhoff Publishing, 1985: 143-70. IS. Woolf CM. Principles of biometry. Princeton, New lersey: D. Van Nostrand Co, Inc., 1968:101. 16. Rosenkranz ER, Buckberg GD. Myocardial protection during surgical coronary reperfusion. J Am Coli Cardiol 1983; I: 1235-46. 17. Rosenkranz ER, Okamoto F, Buckberg GD, VintenJohansen J, Robertson JM, Bugyi H. Safety of prolonged aortic clamping with blood cardioplegia. II. Glutamate enrichment in energy-depleted hearts. 1 THORAC CARDlOVASC SURG 1984;88:402-10. 18. Follette DM, Fey K, Buckberg GD, et al. Reducing postischemic damage by temporary modification of reperfusate calcium, potassium, pH, and osmolarity. 1 THORAC CARDIOVASC SURG 1981;82:221-38. 19. Ferrari R, Williams A, DiLisa F. The role of mitochondrial function in the ischaemic and reperfused myocardium. In: Caldarera CM, Harris P, eds. Advances in studies on heart metabolism. Bologna: Cooperative Libraria Universitaria Editrice, 1982:245-55. 20. Vinten-Johansen J, Sloan BD III, Campbell ML, Mills SA, Cordell AR. Defective postischemic O 2 utilization in reperfused, "stunned" myocardium [Abstract]. Fed Proc 1986;45:530. 21. Johnson RE, Dorsey LM, Moye sr, Hatcher CR lr, Guyton RA. Cardioplegic infusion: the safe limits of pressure and temperature. J THORAC CARDIOVASC SURG 1982;83:813-23. 22. Little WC, Park RC, Freeman GL. Effects of regional ischemia and ventricular pacing on LV dP/dtmax--enddiastolic volume relation. Am 1 Physiol 1987;252:H933-
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23. Mitsos SE, Askew TE, Fantone lC, Kunkel SL, Lucchesi BR. Protective effects of N-2-mercaptoproprionyl glycine (MPG) against myocardial reperfusion injury following neutrophil depletion in the dog. Circulation 1985;72(Pt 2):III 120. 24. Theroux P, Franklin D, Ross J lr, Kemper WS. Regional myocardial function during acute coronary artery occlusion and its modification by pharmacologic agents in the dog. Circ Res 1974;35:896-908. 25. Lew WYW, Chen Z, Guth B, Covell JW. Mechanisms of augmented segment shortening in nonischemic areas during acute ischemia of the canine left ventricle. Circ Res 1985;56:351-8. 26. Lew WYW. Influence of ischemic zone size on nonischemic area function in the canine left ventricle. Am J Physiol 1987;252:H990-7. 27. Kloner RA, Ganote CE, Whalen D, Jennings RB. Effect of a transient period of ischemia on myocardial cells. II. Fine structure during the first few minutes of reflow. Am 1 Pathol 1974;74:399-422. 28. Sjostrand F, Allen BS, Buckberg GD, et al. Studies of controlled reperfusion after ischemia. IV. Electron microscopic studies: importance of embedding techniques in quantitative evaluation of cardiac mitochondrial structure during ischemia and reperfusion. J THORAC CARDIOVASC SURG 1986;92:513-24. 29. Shen AC, Jennings RB. Kinetics of calcium accumulation in acute myocardial ischemic injury. Am 1 Pathol 1972; 67:441-52. 30. Walsh LG, Tormey 1M. Subcellular shifts of electrolytes during myocardial ischemia and reperfusion [Abstract]. Fed Proc 1986;45:773. 31. Nayler WG, Ferrari R, Williams A. Protective effect of pretreatment with verapamil, nifedipine and propranolol on mitochondrial function in the ischemic and reperfused myocardium. Am 1 Cardiol 1980;46:242-8. 32. Trump BF, Mergner WJ, Kahng MW, Saladino AJ. Studies on the subcellular pathophysiology of ischemia. Circulation 1976;53(Pt 2):I17-26. 33. Jennings RB, Reimer KA. Lethal myocardial ischemic injury. Am 1 Pathol 1981;102:241-55. 34. Nayler WG. The role of calcium in the ischemic myocardium. Am J Pathol 1981;102:262-70. 35. Pirzada FA, Ekong EA, Vokonas PS, Apstein CS, Hood WB Jr. Experimental myocardial infarction. XIII. Sequential changes in left ventricular pressure-length relationship in the acute phase. Circulation 1976;53: 970-4. 36. Pirzada FA, Weiner JM, Hood WB Jr. Experimental myocardial infarction. 14. Accelerated myocardial stiffening related to coronary reperfusion following ischemia. Chest 1978;74: 190-5. 37. Haendchen RV, Corday E, Torres M, Maurer G, Fishbein MC, Meerbaum S. Increased regional end-diastolic wall thickness early after reperfusion: a sign of irreversibly damaged myocardium. J Am Coli Cardiol 1984;3:144453. 38. Crozatier B, Ashraf M, Franklin D, Ross 1 Jr. Sarcomere
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sarcomere overstretch in dyskinetic ventricular regions. J Mol Cell Cardiol 1977;9:785-97. 39. Edwards CH II, Rankin JS, McHale PA, Ling D, Anderson RW. Effects of ischemia on left ventricular regional function in the conscious dog. Am J Physiol 1980;240:H413-20. 40. Nayler WG, Poole-Wilson PA, Williams A. Hypoxia and calcium. J Mol Cell Cardiol 1979;11:683-706.
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41. Vogel WM, Briggs LL, Apstein CS. Separation of inherent diastolic myocardial fiber tension and coronary vascular erectile contributions to wall stiffness of rabbit hearts damaged by ischemia, hypoxia, calcium paradox and reperfusion. J Mol Cell Cardiol 1985;17:57-70. 42. Schaper J, Schaper W. Reperfusion of ischemic myocardium: ultrastructural and histochemical aspects. J Am Coli Cardiol 1983;1:1037-46.
Bound volumes available to subscribers Bound volumes of THE JOURNAL OFTHORACIC AND CARDIOVASCULAR SURGERY are available to subscribers (only) for the 1988 issues from the Publisher, at a cost of $51.00 ($68.00 international) for Vol. 95 (January-June) and Vol. 96 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 60 days after publication of the last issue of the volume. The binding is durable buckram with the JOURNAL name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact The C. V. Mosby Company, Circulation Department, 11830 Westline Industrial Drive, St. Louis, Missouri 63146, USA; phone (800) 325-4177, ext. 351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular JOURNAL subscription.