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Ann Thorac Surg 2001;71:827-831
© 2001 The Society of Thoracic Surgeons
a Department of Cardiosurgery, J. Strus Hospital Pozna
, Pozna, Poland
b Department of Intensive Care and Cardiology, Karol Marcinkowski University School of Medical Sciences, Pozna, Poland
Accepted for publication May 24, 2000.
Address reprint requests to Dr Siminiak, Department of Intensive Care and Cardiology, University School of Medical Sciences, 49 Przybyszewskiego St, 60-355 Pozna, Poland
e-mail: mbalin{at}polbox.com
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Abstract |
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) and nitric oxide in patients undergoing CABG, and to verify whether crystalloid and blood cardioplegia can modify such stimulation.
Methods. Coronary sinus, peripheral arterial, and venous plasma samples were collected from 50 patients who underwent CABG and were divided into 2 equal groups which received either crystalloid or blood cardioplegia: directly before myocardial ischemia and aortic cross-clamping; at the beginning of reperfusion after aortic clamp release; and 30 minutes after reperfusion. O and nitric oxide production by PMN was evaluated by standard methods.
Results. There was a significant (p < 0.05) increase in O production by PMN incubated with plasma obtained from the coronary sinus immediately after reperfusion in patients receiving crystalloid cardioplegia compared to blood cardioplegia. No difference was observed in plasma stimulation of nitric oxide production by PMN in the 2 groups of patients at different times during the procedure.
Conclusions. Cardioplegia may affect release of neutrophil-oriented stimuli from ischemic myocardium and modify neutrophil activation during coronary artery bypass grafting.
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Introduction |
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After migration through the vascular wall, PMNs release large amounts of harmful agents, such as proteolytic enzymes and oxygen free radicals, which can damage surrounding cells and extracellular structures. PMN mediated biochemical damage of ischemic myocardium is likely to involve the release of free oxygen radicals [3, 4]. Activated PMN, with the use of membrane-related nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase produce large amounts of superoxide anion (O) [5], being an intermediate product for hydrogen peroxide, which could subsequently react with O leading to the generation of hydroxyl radical and singlet oxygen [5]. The cytotoxic effects of free oxygen radicals are related to their reaction with membrane lipids and nucleic acids [6]. Membrane dysfunction leads to alteration in intracellular ionic calcium levels and subsequent cell injury [7]. Furthermore, free oxygen radicals, especially the superoxide anion, can inhibit the action of nitric oxide (NO), which, in turn, has been shown to be involved in the modification of neutrophil activation within the ischemic myocardium [8]. It has been shown that PMNs also produce NO [9].
There is an increasing body of evidence indicating that neutrophils play a pivotal role in the development of postischemic myocardial dysfunction in patients undergoing coronary artery bypass grafting (CABG) [10]. PMN activation has been suggested to be related to the organ dysfunction in CABG patients [10]. Intraoperative procedures may also modify PMN function. Although neutrophil activation in patients undergoing CABG has been documented already in several studies, it is not clear when the cells become activated as a result of their direct contact with ischemic/reperfusion myocardium or extracorporeal circulation circuit, or whether PMN activation is an effect of specific stimuli released during CABG procedure.
The aim of the current study was to evaluate plasma-mediated neutrophil stimulation in patients undergoing coronary artery bypass grafting and to verify whether crystalloid and blood cardioplegia may modify such stimulation.
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Material and methods |
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Patients were randomly divided into 2 equal groups (A and B) to receive equal proportion of different cardioplegic solution used during the CABG procedure.
The operative procedures were performed using cardiopulmonary bypass at moderate hypothermia (30°C to 32°C) with hemodilution and topical hypothermia. Cardiac arrest was induced in group A with antegrade St. Thomas Hospital crystalloid cardioplegia (Plegisol [Braun, Melsungen, Germany], containing NaCl 110 mmol/l, KCl 16 mmol/l, MgCl2 16 mmol/l, CaCl2 1,2 mmol/l) or antegrade blood cardioplegia in group B according to Buckberg [11], and maintained with additional doses administered at approximately 15 to 20 minute intervals. The mean number of grafts was 3.32 in group A and 3.68 in group B. After grafting, the patients were rewarmed to 36°C and separated from cardiopulmonary bypass by the gradual reduction of venous return to the bypass circuit. The bypass circuit period ranged mean 81 ± 20 minutes (ns) in group A, and 91 ± 29 minutes (ns) in group B; the period of complete ischemia–aortal clamping was 31 ± 16 minutes (ns) in group A and 47 ± 19 minutes (ns) in group B. The reperfusion period from aortal clamp release to bypass circuit stopping was 30 to 89 minutes (mean 43 minutes). All procedures in both groups were performed by the same operating team. There were no statistical differences regarding age, sex, duration of ischemia (aortal cross-clamping) and duration of procedure between the groups. None of the patients received steroids, nonsteroidal antiinflammatory agents, lidocaine, or other medications, which might affect the inflammatory response.
Peripheral vein blood samples were drawn into tubes containing heparin (2 IU/ml) before the installation of cardiopulmonary bypass (V0). Other samples were collected from the basilic vein (V), the radial artery (A), and the coronary sinus (S) directly before myocardial ischaemia–aortal cross-clamping (A1, V1, S1), at the beginning of reperfusion–aortal clamp release (A2, V2, S2), and at 30 minutes after reperfusion (A3, V3, S3), respectively. All samples were immediately centrifuged and the plasma was deeply frozen until analysis. Neutrophils were isolated from peripheral blood samples obtained from healthy volunteers. Polymorphonuclear neutrophils were isolated by a single-step centrifugation procedure on Gradisol (Polfa, Kutno, Poland) gradient, which is a modification of the method described by Böyum [12]. After centrifugation, neutrophils were washed twice and suspended in 10 ml of saline. Red blood cells were lysed with hemolytic buffer (0.828 g NH4Cl and 0.1 g KHCO3 dissolved in 100 ml of distillated water with 20 µl of 1N NaOH). The neutrophils were resuspended in Hank’s solution (Sigma, St. Louis, MO). Superoxide anion production was estimated by cytochrome c reduction with a slightly modified method of Bellavitae and colleagues [13]. Results are expressed in nmol of superoxide anion produced by 106 neutrophils in 30 minutes. Nitric oxide production was evaluated by colorimetric method using hydroxylamin with Griess solution [14]. Results are expressed in mmol of nitric oxide produced by 106 neutrophils in 60 minutes.
Since our data were not normally distributed, as assessed by the Kolomogorov–Smirnov test, statistical evaluation was performed by the nonparametric Mann–Whitney test. Data are given as mean ± SEM.
Informed consent to participate in the study, which was accepted by the local Ethics Committee, was obtained from each subject.
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Results |
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There was a significant (p < 0.05) increase in O production by PMN incubated with plasma obtained from coronary sinus (Fig 1) immediately after reperfusion in patients receiving crystalloid cardioplegia during coronary artery bypass surgery (25.1 ± 1.9 nmol/5 x 106/30 minutes) in comparison with PMN incubated in plasma obtained from patients receiving blood cardioplegia (17.8 ± 1.8). In crystalloid and blood cardioplegia patients, plasma-mediated stimulation of PMN superoxide anion production in coronary sinus samples obtained before aorta cross-clamping were 15.5 ± 1.5 and 17.1 ± 2.1, respectively, and, at 30 minutes after reperfusion, were 18.4 ± 1.3 and 16.8 ± 1.0, respectively, the difference was not significant (Fig 1).
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Comment |
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, as well as endothelin-1 and other cytokines [4, 15]. It is believed that neutrophils play a central role in this inflammatory process. These factors, together with direct contact of the blood with synthetic surfaces of the cardiopulmonary bypass circuit, may activate neutrophils [16]. Furthermore, interventions intended to inactivate or to deplete neutrophils have been shown to reduce the tissue injury during CABG or experimental ischemia-reperfusion [17, 18]. The clinical relevance of leukocyte activation during CABG has been demonstrated recently in studies investigating the use of leukocyte-depleted cardioplegia or anti-adhesion strategies. It has been shown that leukocyte depletion as an adjunct to terminal blood cardioplegia was associated with better clinical outcome in patients with left ventricular hypertrophy [19]. Similarly, the inhibition of leukocyte-endothelial interaction by monoclonal antibodies against leukocyte adhesion molecules seems to have a beneficial effect on myocardial function after heart surgery [20]. Deist and associates [21] showed that hypothermia delays but does not prevent neutrophil activation during cardiopulmonary bypass. On the other hand, Mezzetti and colleagues [22] conclude that intermittent antegrade warm blood cardioplegia protects the myocardium from ischemia-reperfusion injury better than intermittent antegrade cold blood cardioplegia; this phenomenon may be partly due to the decreased tissue oxidant burden mediated by intermittent warm blood cardioplegia.
During coronary artery bypass grafting performed with the use of extracorporeal circuit, the heart is subjected to a global ischemia. Thus, experimental models of both regional and global ischemia have their clinical counterparts. In the current study, we verified the possibility of modification of the release of stimuli for neutrophil superoxide anion and nitric oxide production by the cardioplegic solution used during CABG. We have found an increase in O production by PMN incubated with plasma obtained from coronary sinus immediately after reperfusion during coronary artery bypass surgery in patients receiving crystalloid cardioplegia in comparison to PMN incubated with the samples taken from patients receiving blood cardioplegia. However, no change in the superoxide anion production in the peripheral arterial and venous plasma has been observed. This finding suggests higher levels of PMN-oriented stimuli, released within ischemic myocardium in patients receiving crystalloid cardioplegia, as compared to patients receiving blood cardioplegia.
It is known that activated neutrophils generate and release oxygen-derived free radicals, proteolytic enzymes, and arachidonic acid metabolites, all of which are capable of affecting the function and structure of tissue organs [3]. There is substantial evidence that myocardial ischemia/reperfusion results in the activation of neutrophils and their interaction with endothelium and platelets [1, 3]. These interactions are mediated by a variety of mediators including cytokines and membrane-bound receptors.
Neutrophils may have a functional role in modulating interactions between endothelium and platelets in ischemic myocardium [4]. Both activated platelets and endothelium are a rich source of potent mediators, which modify PMN activity.
It has been documented that activated endothelial cells produce platelet activating factor (PAF), prostacyclin (PGI2), nitric oxide (NO), interleukin-8 (IL-8), and endothelins capable of modifying leukocyte activation [4].
Nitric oxide can be inhibited by free oxygen radicals, especially superoxide anions, resulting in capillary constriction and enlargement of ischemic area. Superoxide radicals are produced in high concentrations by PMNs and are potent inactivators of NO and inhibitors of its production [23]. In addition, their cytotoxic effects may cause direct cellular damage to endothelium. PMNs have been implicated in the etiology of functional and structural damage to endothelial cells [24]. There is evidence that activated PMNs not only cause vasoconstriction but also impair the response to endothelium-dependent vasodilators, while inhibition of PMN reduces the endothelial dysfunction [25].
Cell-to-cell interactions occurring in the milieu of advanced coronary atherosclerosis result in activated leukocytes that may yield substances such as superoxide anions, extend endothelial injury, and increase vascular permeability. Activated platelets in concert with endothelial injury result in severe vasoconstriction, reduction in blood flow, and local thrombosis or vasospasm. In this environment, by means of release of neutrophil-derived relaxing factor, leukocytes may induce vasorelaxation that will counter the smooth muscle contraction caused by the interaction of platelets with the deendothelialized coronary artery. Overall, interactions among leukocytes, platelets, and the blood vessel wall may determine the state of vascular tone and the tendency toward the formation of thrombus at the site of endothelial disruption [24]. Currently, we demonstrate the role of plasma-mediated stimulation of neutrophil free oxygen radical and nitric oxide production in patients receiving crystalloid and blood cardioplegia during coronary artery bypass surgery. The difference in O anion production in both study groups indicates that cardioplegia itself may affect the release of neutrophil-oriented stimuli from ischemic myocardium and thus modify neutrophil activation during coronary artery bypass grafting. This finding may eventually lead to pharmacological interventions oriented on limitation of PMN-related myocardial damage and requires further investigation.
Blood cardioplegia is now considered the standard of care in many centers, and the use of crystalloid cardioplegia has been virtually abandoned. This seems to be confirmed by the results of our study indicating better cardioprotection by blood cardioplegia.
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Acknowledgments |
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