Leukocyte Depletion of Blood Cardioplegia Attenuates Reperfusion Injury1

L Depletion of Blood Cardioplegia Attenuates Reperfusion Injury Frank E. Schmidt, Jr, MD, Malcolm J. MacDonald, MD, Charles O. Murphy, MD, W. Morris Brown III, MD, John Parker Gott, MD, and Robert A. Guyton, MD Division of Cardiothoracic Surgery, Emory University School of Medicine, The CarlyleFraserHeart Center, Emory/Crawford Long Hospital,Atlanta, Georgia

Background. Leukocytes are associated with myocardial injury during reperfusion after ischemia. Short periods of leukocyte depletion during reperfusion result in persistent attenuation of postischemic myocardial dysfunction. Methods. Leukocyte depIetion was examined in a canine model of regional myocardial ischemia and reperfusion. The extracorporeal circuit and cardioplegia circuits underwent leukocyte depletion by mechanical filtration. Animals were instrumented for baseline global function before 90-minute occlusion of the left anterior descending coronary artery. Global function during ischemia and at 5, 30, 60, and 90 minutes after a 60-minute cardioplegic arrest using continuous blood cardioplegia was assessed in leukocyte-depleted (n = 9) and control (n = 10) groups.

Results. No significant difference between groups was seen for systemic leukocyte counts, global function, or water content. Endothelial function was significantly protected as assessed by response to both calcium ionophore (endothelial-dependent, receptor-independent relaxation: leukocyte-depleted, 72~0 A 19% of endothelininduced constriction versus control, 469!o* 140/o;p < 0.05) and acetylcholine (endothelial-dependent,receptor-dependent relaxation: leukocyte-depleted, 83Y0 * 117. versus COntrOl,440/. * 150/.; ~ < 0.().5). Conclusions. Leukocyte-mediated endothelial reperfusion injury can be attenuated by leukocyte depletion during reperfusion.

R

tion of global mechanical function or microvascular reactivity.

eperfusion of ischemic myocardium results in additional damage superimposed upon that caused by ischemia alone [1]. Prior studies have demonstrated that reperfusion injury results in endothelial dysfunction [2], expression of endothelial and leukocyte adhesion molecules [3], complement activation [4], production of free radicals [5], and the activation and accumulation of leukocytes [6]. Because reperfusion injury can be ameliorated by inhibition of the mediators of leukocyte sequestration [7-91 and because leukocyte depletion of the reperfusate attenuates reperfusion injury [8, 10, 11], the polymorphonuclear leukocyte has been implicated as the major effecter of reperfusion injury [12]. Interestingly, brief periods of leukocyte depletion during reperfusion impart lasting protective effect even if leukocytes are reintroduced [13]. Cardiac operations using continuous blood cardioplegia afford a unique clinical opportunity to control both the conditions of reperfusion and the composition of the reperfusate after a period of acute ischemia. This study was designed to determine whether leukocyte depietion of both the cardiopulmonary bypass (CPB) circuit and cardioplegic solution results in protecPresentedat the Thirty-SecondAnnualMeetingof The Society of Thoracic Surgeons, Orlando, FL, Jan 29-31, 1996.

Addressreprintrequeststo Dr Guyton,CarlyleFraser Heart Center, Emory/CrawfordLong Hospital, 550 Peachtree St, Suite 7700, Atlanta, GA 30365-2225.

01996 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

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(Ann Thorac Surg 1996;62:1691-7)

Material and Methods Dirofilaria-negative dogs were used as study animals in one of two randomly assigned experimental groups that either did or did not employ leukocyte filters. After premeditation with 4 mg/kg morphine sulfate the animals were anesthetized using 20 mg/kg pentobarbital and endotracheally intubated. The femoral artery was cannulated for arterial blood gas analysis and pressure monitoring. The femoral vein was cannulated for central venous access. A median sternotomy was performed, the azygos vein ligated, and the pericardium opened. The heart was then instrumented with a left ventricular conductance catheter (Webster Laboratories, Baldwin Park, CA) inserted via the aortic root into the left ventricular cavity. A left ventricular micromanometer (Millar Instruments, Houston, TX) was inserted through a stab wound in the left ventricular apex. Catheter placement was checked by fluoroscope. An ultrasonic flowmeter (Transonic Instruments, Inc, Ithaca, New York) was placed on the ascending aorta. Baseline ventricular function data were obtained by occluding the inferior vena cava to alter preload. Pressure and volume data were obtained, which allowed determination of the slope of the preload recruitable strokework relationship (PRSW). The left anterior descending coronary artery (LAD) was 0003-4975/96/$15.00 PH S0003-4975(96)00736-9

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SCHMIDT ET AL LEUKOCYTE DEPLETION AND REPERFUSION

identified just distal to the first diagonal artery and occluded with a snare after systemic heparinization. Ventricular function data were also obtained after 10 and 45 minutes of ischemia. The left subclavian artery was used for arterial inflow for CPB. Bicaval cannulation was used for venous return. Dirofilaria-positive dogs were used as a source of homologous blood to prime the CPB circuit. Cardiopulmonary bypass was begun at 75 minutes of ischemia, and the animals were cooled to 28”C. The aorta was crossclamped and the heart arrested with antegrade administration of 4:1 blood-crystalloid cardioplegia containing a final concentration of 20 mEq/L potassium chloride. A coronary sinus catheter was then placed and retrograde delivery of an 8 mEq/L potassium chloride blood cardioplegic solution was begun and continued throughout the remainder of the cross-clamp period. After a total of 90 minutes of LAD occlusion, the snare was removed. Retrograde delive~ was adjusted to maintain a mean perfusion pressure of 40 to 45 mm Hg. Arrest was maintained for 60 minutes, and the animals were rewarmed 10 minutes before cross-clamp removal. The animals were defibrillate as needed and maintained in a beatin~ nonworking state for 10 additional minutes. Ventricular function data were obtained 5,30, 60, and 90 minutes after taper from CPB. In the leukocyte-depleted group, homologous blood from the donor animal was passed through leukocytedepleting filters (RC-400; Pall, East Hills, NY) as it was infused into the cardiotomy reservoir as part of the prime of the cardiopulmonary bypass circuit. Leukocytedepleting filters were also incorporated into the arterial line of the bypass circuit (LG-6; Pall) and into the cardioplegia line (BC1; Pall). Baseline blood samples were taken from both groups before institution of CPB and after 2, 15, 30, 45, and 60 minutes of bypass from the arterial inflow line for determination of leukocyte counts and differentials. Samples were similarly taken 5, 30, 60, and 90 minutes after separation from CPB. In the leukocyte-depleted group, blood samples were also taken before and after the cardioplegia line filter for determination of leukocyte counts. Left ventricular volume was assessed using the conductance catheter technique. An electrical field was generated within the left ventricular chamber using the dual field method [14]. The measured conductance is related to the volume of the ventricle and the conductivity of the medium by the following equation: Vuc(t) = (L2/s)G(t), where Vuc is the uncorrected conductance derived volume, L is the interelectrode distance, s is the specific conductivity of the medium, and G(t) is the time-varying sum of the segmental conductance plus one-third of the conductance of the first segment. Conductivity of the medium was determined by one of the modules of the signal processor-conditioner (Leycom Sigma 5/DF; Cardiodynamics, Rynsbur& the Netherlands). The catheter was connected to a signal processor-conditioner, which supplied a 20 kHz, 30 mA current to the terminal field generating electrodes. The intervening electrodes of the

Ann Thorac Surg 1996;62:1691-7

catheter measured the conductance of five segments, the height of which could be adjusted according to the size of the ventricle. Data were recorded via an analog-to-digital conversion board (Data Translation, Inc, Marlboro, MA) and processed, stored, and analyzed using an interactive program developed in this laboratory (James M. Bradford, PhD, Computer Assisted Research Laboratory Analysis,O Emory University, 1995). The system was formatted to collect 6-second recordings of 16 channels of physiologic data. The interactive features of this program allowed visual confirmation of the hemodynamic data. Global left ventricular function was quantitated by assessing the slope of the PRSW. This relationship was obtained by computer integration of the area within the left ventricular pressure-volume loops and plotting this area (stroke work) versus the end-diastolic volume. The PRSW line was obtained as a least squares linear regression of multiple cardiac cycles during acute variation of the preload. After the last functional evaluation, the heart was rapidly excised and placed in a 4°C buffer solution. Microvascular reactivity was assessed in both the ischemic (LAD distribution) and nonischemic regions (circumflex distribution). Microarterial vessels (100 to 200 pm in diameter) were dissected from branches of the LAD and branches of the left circumflex artery using a 60x (Nikon SMZ-U, Tokyo, Japan) dissecting microscope. Vessels were then placed in an isolated dualchamber Plexiglas organ chamber and cannulated with dual glass micropipettes (tip measuring 40 to 80 ~m in diameter) and secured with 10-0 nylon monofilament suture. Krebs’/HEPES buffer solution warmed to 37°C was continuously circulated through the organ chamber and a 100 mL reservoir. The microvessels were pressurized to 20 mm Hg using a mercury manometer in a no-flow state. The organ chamber was then mounted on the stage of an inverted microscope (Nikon Diaphot Phase Contrast-2) and connected to a video camera (Burle 1OW-5O5O,Lancaster, PA). The vessel image was projected onto a television monitor, and a video dimension analyzer (Living Systems Instrumentation, Burlington, VT) was used to measure the baseline diameter of the lumen. Vessels were allowed to bathe in the organ chamber for 60 minutes before any intervention. Vessels were then preconstructed using endothelin I (1 X 10–10 mol/L to 1 X 10–* mol/L), yielding constriction to so~o to 407. of the baseline diameter. Acetylcholine, sodium nitroprusside, and calcium ionophore were applied extraluminally to elicit the maximum dilation response from the vessels. After titration to the maximum response from a vessel, a sequential three log increase in concentration was used to demonstrate that the maximum response had been achieved. The initial concentration of all drugs was 1 X 10-10 mol/L. Endothelial preservation was assessed using an endothelial-independent dilating agent (sodium nitroprusside), an endothelial-dependent, receptor-independent agent (calcium ionophore), and an endothelial-dependent, receptordependent agent (acetylcholine). The order of adminis-

Ann Thorac Surg 1996;62:1691-7

tration of acetylcholine and sodium nitroprusside was randomly assigned. Calcium ionophore was administered last due to difficulty in completely washing it from the vessel. Vessels were washed and allowed to equilibrate for 10 to 15 minutes between interventions. Vessels from the ischemic LAD and nonischemic circumflex coronary artery distributions were measured concurrently. Microvascular relaxations are expressed as the percent relaxation of the endothelin-I-induced constriction of the vessel diameter relative to the baseline diameter according to the following equation: [(vessel diameter after application of agonist – preconstructed diameter)/ (baseline diameter – preconstructeddiameter)]. Myocardial water content was determined from samples of the ischemic LAD distribution and the nonischemic circumflex distribution. Samples were weighed and placed in an oven for desiccation. After a stable weight was obtained the percent water content of the tissue was determined by the following equation: Tissue percent water content = [(wet weight – dry weight)/wet weight] x

100.

Statistical analyses were performed by a biostatistician (R. S. Clark, PhD, Emory University). Analysis of variance with repeated measures was used for all ventricular function, microvascular, and leukocyte data. Myocardial water content was compared using one-way analysis of variance. All values are expressed as mean * the standard deviation. Significant differences were said to exist at the p less than 0.05 level.

Results The animals were easily tapered from CPB. The anterior left ventricular wall was dyskinetic in all animals before reperfusion as expected from LAD coronary artery occlusion. Global left ventricular function was not depressed either during the ischemic period or at 90 minutes of reperfusion. The slope of the PRSW increased during the ischemic period an average of 20.3Y0 at 10 minutes of LAD occlusion and 26.57. at 45 minutes. After weaning from CPB, global function as assessed by the slope of the PRSW relationship was comparable with baseline and recovered to levels above baseline in both groups (Table 1). Systemic leukocyte counts were obtained at 2, 15, 30, 45, and 60 minutes of bypass. In both groups there was a large decrement in circulating leukocytes after the initiation of Cl’B. The leukocyte counts continued to decrease during the first 30 minutes of CPB but plateaued and remained below baseline levels during the remaining 30 minutes on CPB. In both groups there was a gradual increase in circulating leukocyte levels after CPB, although the levels did not return to baseline. There was no significant difference between the leukocyte-depleted and nondepleted animals in relation to systemic leukocyte counts either before, durina or after CPB (Fig 1). Leukocyte counts were measured in the cardioplegia circuit both before and after filtration. There was a significant decrease in leukocyte counts in the cardiople-

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Table 1. Comparisonof GlobalFunction” Slope PRSW Time

LD

Control

100%

Baseline 10 min ischemia 45 min ischemia 5 min after wean 30 min after wean 60 min after wean 90 min after wean

100% 153% 142% 106% 118!4 137% 146%

97%t 19% 1150/0* 90% * 130% t 116% t 134% ?

36% 33’% 47% 44% 35%

? L ~ * t *

62% 54% 37% 39% 47% 720/a

‘ Global function assessed by the preload recruitable strokework relationship (PRSW) is shown for the labeled times for both the leukocytedepleted (LD) and control groups. Measurements are shown as percent of baseline function. There are no significant differences between the two groups at any time point.

gia solution after filtration (before fikration, 757 ~ 80 cells/mL; after filtration, 41 ~ 8 cells/mL; p < 0.05). The efficiency of leukocyte filtration was consistent throughout the cardioplegic period (Fig 2). Differential leukocyte counts obtained from the extracorporeal circuit did not reveal preferential filtration of either cell type as there was no significant difference in the ratio of polymorphonuclear leukocytes to lymphocytes in the leukocytedepleted versus the control group (Fig 3). Microvascular reactivity was significantly preserved in the leukocyte-depleted group compared with the control group in the ischemic LAD region (p < 0.05) as assessed by microvascular responses to both endotheliumdependent vasoactive agents (calcium ionophore [Fig 4] and acetylcholine [Fig 5]). Non-endothelial-dependent relaxation was not different between groups (Fig 6). There was no significant difference in microvascular reactivity between groups in the nonischemic circumflex region for either endothelial-dependent or endothelialindependent agonists.

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TIMES COLLECTED Fig 1. Total leukocytecountsdrawnfrom the arterialline of the cardiopulmonarybypasscircuitexpressedas percent of baseline. The ticmarkon the x-axis illustratesthe end of the 60-minute cardiopulmonary bypass (CPB) period and the beginning of the 90-minute postarrestperiod. There was no signi$cant difference (W in SYStemic leukocytecountswhen leukocytedepletion (LD) was employed.

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SCHMIDT ETAL LEUKOCYTE DEPLETION AND REPERFUSION

Ann Thorac Surg 1996;62:1691-7

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Comment Control of the conditions of reperfusion and the composition of the reperfusate has been shown to have a dramatic effect on preservation of myocardial viability and function after a period of ischemia [15]. The neutrophil is involved in the amplification of the destructive inflammatory activation by its release of cytokines [16]. It has a role in the “no-reflow” phenomenon [17] and releases enzymes that produce oxygen-derived free radicals as well as cytotoxic enzymes responsible for direct myocyte necrosis [18] after ischemia and reperfusion. The endothelium plays a crucial interactive role in this process with impairment of both autocrine and paracrine function resulting in rapid neutrophil adherence and accumulation [19]. Adherence of leukocytes is mediated via surface ligands on both the neutrophils and endothelial cells. These molecules belong to three families: the selectins (E, P, and L), the immunoglobulin superfamily (intercellular adhesion molecule-l), and the integrins (CD1l/CD18 complex). These adhesion molecules follow a timedependent course of expression after reperfusion [20].

1x

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C’;ossclamp+;me (minuttl) + Pre-Filter + Post-Filter Fig 2. Leukocytecountsbefore the cardioplegiafilter and thoseajler it. A significantreductionin leukocytenumbers was demonstrated throughoutthe cardioplegicperiod.

1X1O”

+ LD + Control

Fig 4. Microvasccdarresponseto the ercdothelial-dependerct, receptorindependentdilatorcala”umionophorein the ischernic(lefianterior descending artey) reg’on. Percentageof dilationfiom a preconstructedstateis shown on the y-axis and increasingconcentrationof agent is depictedon the x-axis.A significantdifferencein response to the agent was demonstratedbetweenthe leukocyte-depleted(LD) and control(C) groups.

P-selectin, which is found on platelets and endothelial cells, is constitutively stored in cytoplasmic granules and is therefore rapidly expressed after ischemia and reperfusion. It binds to cell surface oligosacharrides on the leukocytes and initiates slow rolling of the leukocytes along the endothelium, which allows interaction of the CD1l/CD18 integrin on the leukocyte with intercellular adhesion molecule-1 on the endothelium [21]. Weyrich and associates [20] demonstrated this selectin peaks in expression at 20 minutes after onset of reperfusion yet is rapidly downregulated to 30Y0 of its maximum expression by 60 minutes. Inhibition of P-selectin with monoclinal antibody (PB1.3) resulted in a A3~0 decrease in infarct size versus therapy with nonbinding nonactive monoclinal antibody (p < 0.01) [21]. E-selectin, the other

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End Study

■ LD-PMN ❑ C-PMN E LD-Lymph ❑ C-Lymph Fig 3. Percentageof polymoiphonuclearleukocytes@MN and lymphocytes(Lymph) at baseline,at the end of the cardiopulmonary bypass (CPB) period, and at the end of the study. There was no significantdiference (NW in the leukocytecompositionbefween the leukocyte-depleted(I-D)and control(C) STOMPS.

1x

10-f

1Xto’

1 x 10-’

1x

10*

1X1O*

Concentration + LD +Control Fig 5. Microvascularresponseto the endothelial-dependent, receptordependentdilatoracetylcholine.Percentageof dilation~m a preconstrietedstateis shownon the y-m”sand increasingconcentrationof agentis depictedon thex-axis.A significantdiff’ence was demonstratedin the ischemicregionbetweenthe leukocyte-depleted LD) and controlgroups.

Ann Thorac Surg 1996;62:1691-7

SCHMIDTETAL

LEUKOCYTE DEPLETION AND REPEIWUSION

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Fig 6. Microvascularresponse to the endothelial-independentdilator sodium nitroprusside.Percentageof dilationfrom a preconsti”cted stateis shown on the y-axis and increasingconcentrationof agent is depictedon the x-axis. No significantdifference(NE.)was seen between the the responseachieved in the leukocyte-depleted(LD) group and the controlgroup.

endothelially based selectin, is not upregulated by reperfusion and does not show substantial expression even at 270 minutes of reperfusion. L-selectin is constitutively stored in leukocyte cytoplasmic granules and is rapidly expressed and shed as the leukocytes become activated [21]. Intercellular adhesion molecule-1 requires gene expression for its upregulation and therefore plays little role before 3 to 4 hours of reperfusion. Removal of leukocytes from the reperfusate has been shown to limit neutrophil accumulation [12] and translate into superior preservation of myocardial function and reduction of infarct size [10, 22]. Even brief periods of leukocyte depletion (20 minutes) after ischemia/ reperfusion protect myocardial viability and preserve myocardial function despite the reintroduction of circulating leukocytes [13]. This implicates P-selectin as a primary determinant of the severity of reperfusion damage after a period of ischemia. Rapid downregulation may explain why temporary leukocyte depletion confers long-lasting protection. Prior studies of the time course of expression of the surface molecules were performed using unmodified whole blood reperfusion. The time course of selectin expression with a regimen of modified cardioplegic reperfusion is unknown and warrants further investigation. Some prelimina~ clinical correlation is provided by Sawa and associates [23], who demonstrated a decreased need for inotropic support and decreased serum creatine kinase-MB levels in patients requiring emergency revascularization using leukocyte-depleted cardioplegic reperfusion. Superior preservation of endothelial function was seen in the leukocyte-depleted group; however, there was no difference in global myocardial function between the two groups. The lack of a significant difference in indices of global function may be due to compensatory hypercontraction of the remote nonischemic myocardium that masks regional dysfunction in measures of global func-

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tion [24]. This appears to be an intrinsic limitation of global indices of function in a model of regional ischemia. To further investigate this possibility, we placed regional sonomicrometer crystals in the ischemic LAD region and in the nonischemic circumflex region in the last 5 animals (3 leukocyte-depleted, 2 control). All animals showed a significant decrement in regional indices of myocardial performance versus the nonischemic regions. Simultaneous measurement of global myocardial function as measured by the slope of the PRSW relationship demonstrated performance that was improved compared with baseline. The leukocyte-depleted animals showed hypocontractility in the area of ischemia. The ischemic area in the control animals was noncontractile and dyskinetic. Five animals had evaluable regional data, and statistical analysis was not done. Endothelial microvascular reactivity was superior to control in the leukocyte-depleted group. This supports the earlier observation that leukocyte depletion results in better preservation of postreperfusion corona~ blood flow [8], which is consistent with improved preservation of endothelial function. We analyzed vessels that were less than 200 pm in diameter, which represent the resistance vessels in a canine model and thus have an enormous effect on postreperfusion flow [25]. There is also evidence that microvessels show detectable dysfunction earlier than do larger epicardial vessels after this injury [26]. Endothelial function is mediated via endotheliumderived relaxing factor, which is synonymous with nitric oxide. Continued nitric oxide synthesis by the endothelium is important as it inhibits platelet adhesion, neutrophil adhesion, homotypic aggregation, and superoxide formation [19]. Lack of this potent vasodilator may be more important in the no-reflow phenomenon than is extrinsic capillary compression [27] or mechanical plugging [17] as was previously hypothesized. The no-reflow phenomenon may contribute to further injury to damaged myocardium resulting in irreversible necrosis. Preservation of endothelium-derived relaxing factor has also been shown to directly modulate myocardial performance after ischemia and reperfusion [28]. Thus superior preservation of endothelial function has important implications for the effectiveness of reperfusion modification at preventing myocardial necrosis and preserving myocardial performance. The relative importance of each site of leukocyte depletion was not an end point of this study. The statistically significant decrease in leukocyte counts across the cardioplegia filter may be the result of all three sites of filtration and should not be taken as evidence for the lack of efficacy of systemic filtration or filtration of homologous blood. A recent study by Lazar and colleagues [29] was designed to determine the relative efficacy of site of filtration. Using a model of ischemiah-eperfusion injury, they compared wall motion and myocardial necrosis between groups in which systemic leukocyte filtration alone, cardioplegia filtration alone, filtration of both the cardioplegic and systemic circuits, or no filtration was used. They found superior wall motion scores and improved viability in the systemic filtration group [29].

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SCHMIDT ETAL LEUKOCYTE DEPLETION AND REPERFUSION

Filtration of the cardioplegia solution alone provided no benefit over control (no filtration) and no added benefit over systemic filtration alone. This implies that the most important site of filtration is the systemic circuit. The interaction of leukocytes with the endothelium and with myocytes after extravascular migration is a complex physiologic reaction that needs to be more clearly understood before its damaging effects can be prevented. Removal of leukocytes during CPB to alter the composition of the cardioplegic reperfusate improves protection of endothelial microvascular reactivity in a canine model of ischemia and reperfusion. Leukocyte depletion may be an important component of an overall plan to improve both the conditions and the composition of reperfusion to achieve maximal myocardial viability and function in the setting of surgical revascularization for acute myocardial ischemia. References 1. Braunwald E, Kloner RA. Myocardial reperfusion: a doubleedge sword? J Clin Invest 1985;76:1713–9. 2. Ku DD. Coronaryvascularreactivityafter acute myocardial ischemia. Science 1982;218:576-8. 3. Smith CW. Molecular determinants of neutrophil adhesion [Review]. Am J Respir Cell Mol Biol 1990;2:487-9. 4. Stahl GL, Reenstra WR, Frendl G. Complement-mediated loss of endothelium-dependent relaxation of porcine coronary arteries. Role of the terminal membrane attack complex. Circ Res 1995;76:575-83. 5. McCord JM. Oxygen-derived free radicals in postischemic tissue injury [Review]. N Engl J Med 1985;312:159–63. 6. Ma XL, Tsao F’S, Lefer AM. Antibody to CD-18 exerts endothelial and cardiac protective effects in myocardial ischemia and reperfusion. J Clin Invest 1991;88:1237–43. 7. Lefer DJ, Flynn DM, Phillips ML, Ratcliffe M, Buda AJ. A novel sialyl LewisX analog attenuates neutrophil accumulation and myocardial necrosis after ischemia and reperfusion. Circulation 1994;90:2390-401. 8. Byrne JG, Appleyard RF, Lee CC, et al. Controlled reperfusion of the regionally ischemic myocardium with leukocytedepleted blood reduces stunnin~ the no-reflow phenomenon, and infarct size. J Thorac Cardiovasc Surg 1992;103: 66-72. 9. Ma XI+ Lefer DJ, Lefer AM, Rothlein R. Coronary endothelial and cardiac protective effects of a monoclinal antibody to intercellular adhesion molecule-1 in myocardial ischemia and reperfusion. Circulation 1992;86:937–46. 10. Breda MA, Drinkwater DC, Laks H, et al. Prevention of reperfusion injury in the neonatal heart with leukocytedepleted blood. J Thorac Cardiovasc Surg 1989;97:654-65. 11. Pearl JM, Drinkwater DC Jr, Laks H, Stein DG, Capouya ER, Bhuta S. Leukocyte-depleted reperfusion of transplanted human hearts prevents ultrastructural evidence of reperfusion injury. J Surg Res 1992;52:298–308. 12. Litt MR. Ieremv RW, Weisman HF, Winkelstein IA, Becker LC. Ne&ophfi depletion limited’ to reperfusion “reduces

myocardial infarct she after 91Jminutes of ischemia. Evidence for neutrophil-mediated reperfusion injury. Circulation 1989;80:1816-27. 13. Wilson IC, Gardner TJ, DiNatale JM, et al. Temporary leukocyte depletion reduces ventricular dysfunction during prolonged postischemic reperfusion. J Thorac Cardiovasc Surg 1993;106:805-10. 14. Steendijk P, van der Velde ET, Baan J. Single and dual excitation of the conductance-volume catheter anaiysed in a spheroidal mathematical model of the canine left ventricle. Eur Heart J 1992; 13(Suppl E):28-34.

15. Beyersdorf F, Buckberg GD. Myocardial protection in patients with acute myocardial infarction and cardiogenic shock. Semin Thorac Cardiovasc Surg 1993;5:151–61. 16. Herskowitz A, Choi S, Ansari AA, Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol 1995;146:41>28. 17. Engler RL, Schmid-Schonbein GW, Pavelec RS. Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol 1983;111:98–111. 18. Inauen W, Granger DN, Meininger CJ, et al. Anoxiareoxygenation-induced, neutrophil-mediated endothelial cell injury: role of elastase. Am J Physiol 1990;259:H925–31. 19. Vinten-Johansen J, Zhao ZQ Sato H. Reduction in surgical ischemic-reperfusion injury with adenosine and nitric oxide therapy [Review]. Ann Thorac Surg 1995;60:852–7. 20. Weyrich AS, Buerke M, Albertine KH, Lefer AM. Time course of coronary vascular endothelial adhesion molecule expression during reperfusion of the ischemic feline myocardium. J Leukoc Biol 1995;57:45–55. 21. Lefer DJ: Myocardial protective actions of nitric oxide donors after myocardial ischemia and reperfusion [Review]. New Horiz 1995;3:105-12. 22. Westlin W, Mullane KM. Alleviation of myocardial stunning by leukocyte and platelet depletion. Circulation 1989;80: 1828-36. 23. Sawa Y, Matsuda H, Shimazaki Y, et al. Evaluation of leukocyte-depleted terminal blood cardioplegic solution in patients undergoing elective and emergency coronary artery bypass grafting. J Thorac Cardiovasc Surg 1994;108:1125-31. 24. Beyersdorf F. Protection of evolving myocardial infarction and failed PTCA [Review]. Ann Thorac Surg 1995;60:833–8. 25. Nellis SH, Liedtke AJ, Whitesell L. Small coronary vessel pressure and diameter in an intact beating rabbit heart using fixed-position and free-motion techniques. Circ Res 1981;49: 342-53. 26. Quillen JE, Sellke FW, Brooks LA, Harrison DG. Ischemiareperfusion impairs endothelium-dependent relaxation of coronary microvessels but does not affect large arteries. Circulation 1990;82:586-94. 27. Wiggers CJ. The interplay of coronary vascular resistance and myocardial compression in regulating coronary flow. Circ Res 1954;2:271-9. 28. Mohan P, Brutsaert DL, Sys SU. Myocardial performance is modulated by interaction of cardiac endothelium derived nitric oxide and prostaglandins. Cardiovasc Res 1995;29: 637-40. 29. Lazar HL, Zhang X, Hamasaki T, et al. Role of leukocyte depletion during cardiopulmonary bypass and cardioplegic arrest. Ann Thorac Surg 1995;60:1745–8.

DISCUSSION DR GEORGEM. PALATIANOS(Athens,Greece): We have been using leukocyte-filtered blood cardioplegia for the last 2V2 years. We have seen that the patients in whom we use this type of cardioplegia have less arrhythmogenic activity postoperatively, less reperfusion ventricular fibrillation, and a smaller

need for antiarrhythmics. Also we have noticed less use of inotropic agents postoperatively. These are our clinical observations. I would like to ask you to comment on this protective effect of the leukocyte-depleted cardioplegic solution against the posioperative arrhythmogenic activiv.

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My second question is referring to the results you showed us. How do you explain the lack of improved myocardial contractility of the hearts perfused with leukocyte-filtered cardioplegia in your experiments? DR SCHMIDT: As far as improvement in any arrhythmogenic injury, I cannot explain that in particular other than saying that it is probably decreased injury overall. But I have no data to direct specifically at the issue of arrhythmias. As far as the difference between global and regional function, we used the preload recruitable stroke work relationship to assess our global function. This was based on the conductance catheter, which allowed us to assess baseline function and then function after our interventions. We had hoped that with the comparison with baseline this technique would be sensitive enough to show a difference in global function relative to baseline; however, apparently the left ventricular reserve was adequate enough in both groups to attain. We did, however, add segment length crystals in the last 5 animals in the study.

Becauseof the lownumbers,we didnotpresentthatinformation today. In the controlgroupthere was persistenceof systoliclength-

SCHMIDT ET AL 1697 LEUKOCYTE DEPLETION ANDREPERFUSION

ening (dyskinesis) in the ischemic region. In the leukocytedepleted group there was decreased systolic segment shortening (hypokinesis), but it did contract. Global function greater than baseline was seen at the same time that systolic lengthening (dyskinesis) was seen in the control group, which is further evidence that our measurement of global function was not sensitive enough to detect regional injury. I think the injury is there, but our methods of detecting it were not sensitive enough. DR GERALD D. BUCKBERG (Los Angeies, CA): In light of the previous paper on the use of L-arginine, evidently L-arginine by itself will produce nitric oxide and prevent the leukocyte adherence. Have you done any studies comparing your leukocyte filtration with L-arginine-type presemation to see if they were comparable or different?

DR SCHMIDT:We have not done that study. My suspicion would be that they may be complementary. We showed a difference in the leukocyte-depleted group from control, but there was still injury in the depleted group, and the addition of varginine may decrease that injury.