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Ann Thorac Surg 1996;62:1691-1696
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Emory University School of Medicine, The Carlyle Fraser Heart Center, Emory/Crawford Long Hospital, Atlanta, Georgia
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentReferences |
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Methods. Leukocyte depletion 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% ± 19% of endothelin-induced constriction versus control, 46% ± 14%; p < 0.05) and acetylcholine (endothelial-dependent, receptor-dependent relaxation: leukocyte-depleted, 83% ± 11% versus control, 44% ± 15%; p < 0.05).
Conclusions. Leukocyte-mediated endothelial reperfusion injury can be attenuated by leukocyte depletion during reperfusion.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Reperfusion 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 [7–9] and because leukocyte depletion of the reperfusate attenuates reperfusion injury [8, 10, 11], the polymorphonuclear leukocyte has been implicated as the major effector 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 depletion of both the cardiopulmonary bypass (CPB) circuit and cardioplegic solution results in protection of global mechanical function or microvascular reactivity.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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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 cross-clamped 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 delivery 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 defibrillated as needed and maintained in a beating, 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 leukocyte-depleting 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. Leukocyte-depleting 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 conductances 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, Rynsburg, 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 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,© 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 µm 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 dual-chamber Plexiglas organ chamber and cannulated with dual glass micropipettes (tip measuring 40 to 80 µm 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 10W-5050, 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 preconstricted using endothelin I (1 x 10-10 mol/L to 1 x 10-8 mol/L), yielding constriction to 30% to 40% 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, receptor-dependent agent (acetylcholine). The order of administration 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 - preconstricted diameter)/(baseline diameter - preconstricted diameter)].
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.
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Results |
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).
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). 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 leukocyte-depleted versus the control group (Fig 3).
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] 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 endothelial-independent agonists.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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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-1), and the integrins (CD11/CD18 complex). These adhesion molecules follow a time-dependent course of expression after reperfusion [20]. 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 CD11/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 30% of its maximum expression by 60 minutes. Inhibition of P-selectin with monoclonal antibody (PB1.3) resulted in a 43% decrease in infarct size versus therapy with nonbinding, nonactive monoclonal antibody (p < 0.01) [21]. E-selectin, the other 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 preliminary 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 function [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 coronary blood flow [8], which is consistent with improved preservation of endothelial function. We analyzed vessels that were less than 200 µm 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 endothelium-derived 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 ischemia/reperfusion 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]. 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.
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Address reprint requests to Dr Guyton, Carlyle Fraser Heart Center, Emory/Crawford Long Hospital, 550 Peachtree St, Suite 7700, Atlanta, GA 30365-2225.
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