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Ann Thorac Surg 1996;62:1580-1586
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, University of California, Los Angeles, Medical Center, Los Angeles, California
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Abstract |
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Methods. Neonatal piglet heart-lung blocks were preserved with Euro-Collins solution for 12 hours, rewarmed at room temperature for 1 hour, and reperfused for 10 minutes with either whole blood (n = 5), whole blood containing L-arginine (10 mmol/L; n = 6), or leukocyte-depleted blood (n = 6) on an isolated, blood-perfused, working heart-lung circuit. After the initial 10 minutes, all blocks received whole blood for 4 hours. Control blocks were continuously perfused on the circuit without intervening ischemia (n = 6).
Results. The partial pressure of oxygen in the whole blood group (113.8 ± 33.1 mm Hg) was significantly less than in controls (417.3 ± 6.2 mm Hg; p < 0.01). Lung compliance was significantly less in the whole blood group (0.8 ± 0.2 mL/cm H2O) than in controls (2.9 ± 0.4 mL/cm H2O; p < 0.01). The L-arginine and leukocyte-depleted blood groups showed no significant difference from controls.
Conclusions. L-Arginine infusion during reperfusion improves pulmonary function, making it a simple alternative to leukocyte depletion.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Despite recent advances in methods of lung preservation [1], the lungs remain extremely vulnerable to reperfusion injury. As endothelial damage is thought to mediate this injury, the discovery of nitric oxide (NO) in vascular endothelial cells has opened new avenues for its prevention. Nitric oxide is synthesized from the amino acid L-arginine by NO synthase [2], and is crucial to the maintenance of vascular homeostasis through the relaxation of vascular smooth muscle [3], and inhibition of leukocyte adhesion [4] and platelet aggregation [5]. It has been reported that NO levels measured at the surface of the transplanted lung decline immediately upon reperfusion [6], resulting in lung damage and a consequent decline in pulmonary function. Although previous studies in rats have demonstrated that enhancement of the NO pathway by supplementing the preservation solution with cyclic guanosine monophosphate [6], nitroglycerin [7], or L-arginine [8] can improve pulmonary function, the beneficial effects of L-arginine infusion during reperfusion remain unclear. Studies in myocardial protection have shown that infusion of L-arginine during the early phase of reperfusion improves the recovery of endothelial function in the myocardium [9, 10]. We hypothesized, therefore, that enhancement of the NO pathway by infusion of L-arginine during the initial phase of reperfusion could ameliorate lung reperfusion injury as well. Furthermore, although it is known that leukocyte depletion during reperfusion prevents lung damage, the clinical applicability of this technique remains limited, as it generally requires cardiopulmonary bypass. In this study, we investigate the effects of L-arginine infusion on lung reperfusion injury, and compare them with the effects of reperfusion with leukocyte-depleted blood in an isolated, blood-perfused piglet working heart and lung model.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Harvest
Piglets were anesthetized with intramuscular ketamine hydrochloride (150 mg/kg) and acepromazine maleate (25 mg/kg), and intubated with a 3.5F endotracheal tube through tracheotomy. Mechanical ventilation (Servo Ventilator 900C; Siemens-Elema, Sweden) at a fraction of inspired oxygen of 1.0, a tidal volume of 60 to 70 mL, and a rate of 20 breaths/min was established. The chest was opened through a median sternotomy, and the pericardium and pleura were dissected to expose the heart and lungs. After ligation of the hemiazygos vein, the following vessels were encircled: descending aorta, left subclavian artery, innominate artery, left and right brachiocephalic veins, azygos vein, and superior and inferior venae cavae. Pursestring sutures were placed on the left atrium and the main pulmonary artery for venting and cannulation. The inferior pulmonary ligaments were dissected to release the lungs. After injection of heparin (1,000 units) and cefazolin sodium (200 mg) in the main pulmonary artery, the innominate artery was cannulated and connected to a pressure monitor. In the control group, the innominate artery cannula was connected to the perfusion circuit (described below) and continuously perfused by arterial blood from an adult support pig at a mean pressure of 60 mm Hg. The main pulmonary artery was also cannulated for infusion of pulmonary preservation solution. After infusion of prostaglandin E1 (30 µg) into the main pulmonary artery, the left subclavian artery, left and right brachiocephalic veins, azygos vein, and superior and inferior venae cavae were ligated. After the inferior vena cava and the left atrial appendage were incised to decompress the heart, the descending thoracic aorta was cross-clamped and 4°C University of Wisconsin solution was infused into the innominate artery cannula at a pressure of 40 mm Hg for 2 minutes. Simultaneously, 4°C modified Euro-Collins solution (150 mL), containing 37.5 µg of prostaglandin E1 in the first 75 mL and 75 mg of methylpredonisone in the second 75 mL, was infused into the pulmonary artery by gravity at an infusion pressure of 25 mm Hg. During this period, the heart and lungs were topically cooled with 4°C normal saline solution, and the lungs were ventilated.
Storage
Heart and lungs were harvested by division of the aorta, left subclavian artery, left and right brachiocephalic veins, azygos vein, superior and inferior venae cavae, and trachea. Heart-lung blocks were stored in 4°C Euro-Collins solution with the lungs fully inflated with 100% oxygen.
Reperfusion and Ventilation
Reperfusion and ventilation were achieved using the isolated, blood-perfused working heart and lung circuit depicted in Figure 1
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Arterial blood gas was measured every 30 minutes (model ABL 330 Base Laboratory; Radiometer America Inc, La Verne, CA), with appropriate ventilator adjustments or correction of metabolic acidosis with sodium bicarbonate. Hematocrit was measured every 2 hours, and correction of hemodilution was achieved using whole blood obtained from a previous support animal to achieve a hematocrit of 30%. Heparin (10,000 units) was administered every hour for anticoagulation. Cefazolin sodium (1 g) was infused every 12 hours. Rectal temperature was continuously monitored and maintained at 37°C (Medi-Therm Model No. MTA-4700, Automatic/Manual-Hyper/Hypothermia; Gaymar Industries Inc, Orchard Park, NY).
In the WBL, ARG, and LDB groups, heart-lung blocks were exposed to room temperature for 1 hour before reperfusion, whereas the temperature of the storage solution was monitored every 20 minutes. Reperfusion of the heart-lung blocks was achieved by connecting the innominate artery cannula to the heated reservoir/bubble trap, with the distal aorta cross-clamped and perfusion pressure maintained at 40 mm Hg. After the right atrium was deaired, the inferior vena cava was ligated. Once the heart became stable, the perfusion pressure was gradually increased to 60 mm Hg and maintained there. Simultaneously, mechanical ventilation at an inspired oxygen fraction of 1.0, a tidal volume of 60 to 70 mL, a rate of 20 breaths/min, and a positive end-expiratory pressure of 1 cm H2O was initiated by connection of the endotracheal tube to the ventilator used at the time of harvest.
During the initial 10 minutes, three distinct reperfusion techniques were employed: Heart-lung blocks in the WBL group received unmodified whole blood. In the ARG group, L-arginine (Sigma Chemical Corp, St. Louis, MO) dissolved in normal saline solution was delivered to the heated reservoir/bubble trap in combination with whole blood to achieve a blood to L-arginine ratio of 4:1, and a final reperfusion concentration of L-arginine of 10 mmol/L. Five-milliliter samples were obtained from the pulmonary artery and aortic column upon completion of L-arginine infusion for nitrite/nitrate level determination. Heart-lung blocks in the LDB group received leukocyte-depleted blood via flow through two filters (Pall RC400 Leukocyte Removal Filters; Pall Biomedical Inc, Fajardo, PR) placed in parallel between the roller pump and the heated reservoir/bubble trap [11]. Blood samples were drawn before reperfusion and 5 minutes after the start of reperfusion to determine the degree of leukocyte depletion. Automated complete blood counts (Coulter S-PLUS STKR; Coulter Electronics, Hialeah, FL) and manual white blood cell counts were performed on all samples.
After the initial 10 minutes of reperfusion, all heart-lung blocks received unmodified whole blood. The aorta was cannulated and attached to an open column of tubing, which was adjusted to maintain an aortic pressure of 60 mm Hg. Blood from the aortic column was directed through a 40-µm blood filter (Ultipor EC3840; Pall BioSupport Corp, Glen Cove, NY) before being returned to the support animal. A fiberoptic catheter (model 110-4; Camino Laboratories, San Diego, CA) was placed in the left atrium; the preamplifier output (model 420; Camino Laboratories) was delivered to a Gould transducer amplifier (model 13-4615-50; Gould Inc, Cleveland, OH), monitored on an oscilloscope (model 300-218; Hewlett-Packard Co, Andover, MA), and recorded (Astromed MT-9000 chart recorder; Astromed Inc, West Warwick, RI). The main pulmonary artery was also cannulated and its pressure was simultaneously monitored with a Spectramed pressure transducer (model P23XL; Spectramed Inc, Oxnard, CA).
Induction of Working Mode
Once cardiac function stabilized, the right atrium was cannulated through the superior vena cava and deaired. The induction of working mode was achieved by perfusion of femoral venous blood from a second heated reservoir into the right atrium, and by termination of the perfusion through the innominate artery. The aortic column and the mechanical ventilation were adjusted to maintain an aortic pressure of 80 mm Hg, and an inspired oxygen fraction of 0.60, tidal volume of 60 to 70 mL, respiration rate of 20 breaths/min, and positive end-expiratory pressure of 1.0 cm H2O. The height of the heated reservoir was adjusted to maintain a mean left atrial pressure of 3 mm Hg. Heart-lung blocks were thus placed in working mode for 4 hours.
Assessment of Cardiac Function
Cardiac function was assessed every 30 minutes for the first 2 hours and every hour thereafter. Function of each heart was assessed by measurement of cardiac output (aortic column overflow) at a constant aortic pressure of 80 mm Hg and a mean left atrial pressure of 6 mm Hg. Stroke-work index (erg/g) was calculated [(mean aortic pressure - mean left atrial pressure) x cardiac output x 1,333]/(heart rate x heart weight).
Assessment of Pulmonary Function
Blood gas samples from the right atrial reservoir and the aortic outflow column were taken every 30 minutes to measure the partial pressure of venous oxygen and arterial oxygen, respectively. Lung compliance (mL/cm H2O) was continuously monitored with the Bicore neonatal pulmonary monitor (model CP-100 Pulmonary Monitor; Bicore Monitoring Systems, Irvine, CA). Pulmonary artery pressure was measured in conjunction with cardiac function, and pulmonary vascular resistance (dyne•s•cm-5) was calculated as [(mean pulmonary arterial pressure - mean left atrial pressure) x 80]/cardiac output.
Lung Water Content
At the conclusion of the experiment, multiple biopsy samples from each lung lobe (right upper, middle, and lower; left upper and lower) were taken for assessment of lung water content (wet/dry weight ratios). Wet/dry ratios were determined by weighing samples immediately after biopsies (wet weight), and again after 48 hours of desiccation at 90°C (dry weight). The wet/dry weight ratios from each of the five lobes were averaged to obtain a mean value for each piglet.
Nitrite/Nitrate Measurement
Nitrite/nitrate assay was performed as previously reported [12]. After collection, whole-blood samples were immediately placed on ice and then centrifuged at 4°C for 10 minutes at 3,200 rpm. The supernatant was obtained and kept at 4°C. Aliquots of 100 µL were then injected into the measuring apparatus and any nitrite or nitrate was rapidly reduced to nitric oxide by vanadium at 98°C. Oxygen-free purified nitrogen was delivered into a reaction flask and then the nitrogen flowed through the system and was drawn into the Dasibi Chemiluminescence NOX Analyzer (model 2108; Dasibi, Glendale, CA) at a constant rate of 200 mL/min with the aid of a vacuum pump. The detector is sensitive to nitrogen dioxide radical, the photon-emitting product generated by the reaction between ozone and nitric oxide. The analyzer was calibrated before each use with a standard mixture of 825 ppb nitric oxide in oxygen-free nitrogen (Scott-Marrin, Inc, Riverside, CA).
Statistical Analysis
Statistical significance of data measured repeatedly for each parameter was assessed with analysis of variance for repeated measures. When statistically significant differences were detected, Scheffé F tests were applied to analyze differences among the four groups. All data are expressed as mean ± standard error.
Animal Care
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985).
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Results |
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Nitric Oxide Production
Nitrite/nitrate levels in the pulmonary artery and aortic column were 21.5 ± 2.4 µmol/L and 18 ± 2.7 µmol/L, respectively. There was no significant gradient in nitrite/nitrate level between the two sites.
Cardiac Function
Although stroke work index in the ARG and LDB groups tended to be less than that of the control group, there was no significant difference in stroke work index among the four groups (p = 0.5956) (Fig 2).
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Comment |
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L-Arginine serves as a biosynthetic precursor of NO, which is central to the maintenance of vascular homeostasis. Nitric oxide regulates pulmonary vascular resistance via the relaxation of vascular smooth muscle [2]. This is supported by the finding that NW-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, results in increased pulmonary vascular resistance in isolated, perfused pig and human lungs [3]. Nitric oxide is also known to inhibit leukocyte adhesion. The inhibition of NO synthesis by NW-nitro-L-arginine methyl ester causes leukocytes to adhere to the venular endothelium and promotes leukocyte migration; this adhesion is reversed by L-arginine [4]. NW-nitro-L-arginine methyl ester also results in a rapid increase in vascular fluid filtration and vascular protein clearance, and an increase in microvascular permeability [13]. Finally, NO inhibits platelet aggregation. In vitro treatment with NG-monomethyl-L-arginine promotes platelet aggregation, whereas L-arginine has an inhibitory effect [5]. This decrease in platelet aggregation may improve the microcirculation in the pulmonary vascular bed. The maintenance of normal NO levels is, therefore, crucial to the preservation of pulmonary endothelial function.
Nitric oxide levels measured at the surface of the transplanted lung, however, decrease immediately upon reperfusion, leading to increased pulmonary vascular resistance and leukostasis. Accelerated destruction of NO by oxygen free radicals may be the major mechanism accounting for this decline [6]. Enhancement of the NO pathway by supplementing the preservation solution with cyclic guanosine monophosphate [6], nitroglycerin [7], or L-arginine [8] has proved effective in improving pulmonary function. Irreversible injury, however, occurs not only during preservation, but at the time of reperfusion. Isolated heart studies have demonstrated that endothelial function is impaired as early as 2.5 minutes after reperfusion [14]. Furthermore, infusion of L-arginine during the early phase of reperfusion improves the recovery of endothelial function after myocardial ischemia [9, 10]. We hypothesized, therefore, that augmentation of the NO pathway during early reperfusion might be most effective in ameliorating lung endothelial damage.
In this study, infusion of L-arginine (10 mmol/L) during the initial 10 minutes of reperfusion improves gas exchange and lung compliance, and attenuates the increase in wet/dry weight ratios. It also tends to reduce the increase in pulmonary vascular resistance. Proposed mechanisms for the protective effects of L-arginine on reperfusion injury remain speculative, but appear to involve an increase in NO production in pulmonary vascular endothelial cells [2]. The ability of NO to inhibit leukocyte adhesion may explain the higher arterial oxygenation and lower wet/dry weight ratio in heart-lung blocks infused with L-arginine compared with those reperfused with whole blood. Although the attenuation in the increase in pulmonary vascular resistance does not reach statistical significance, the trend suggests that infusion of L-arginine may increase the production of NO and limit the increase in pulmonary vascular resistance during reperfusion. Further studies with a larger sample size may be required to demonstrate statistical significance.
No significant gradient in measured nitrite/nitrate levels upon completion of L-arginine infusion was detected between the pulmonary artery and the aortic column. Should L-arginine infusion enhances NO production in the pulmonary vascular endothelial cells, nitrite/nitrate level in the aortic column would be greater than that in the pulmonary artery. There is one possible explanation for this. In this study, L-arginine was infused at first through the coronary circulation and then through the pulmonary circulation. Therefore, nitrite/nitrate level in the pulmonary artery may reflect NO production in the coronary vascular endothelial cells and mask the gradient in nitrite/nitrate levels between two sites.
Nitric oxide, which is a vasodilator and thus potentially protective, is also cytotoxic. This cytotoxic effect is due to hydroxyl radicals produced by the homolytic fission of peroxynitrite, the product of a reaction between NO and the superoxide anion [15, 16]. The basic source of the superoxide anion is xanthine oxidase. During ischemia, xanthine dehydrogenase is converted to xanthine oxidase. Substrates for xanthine oxidase are generated by ischemia-induced breakdown of adenine nucleotides to hypoxanthine. On reperfusion, oxygen is supplied to xanthine oxidase, which converts hypoxanthine to uric acid and generates the superoxide anion [17]. Therefore, xanthine oxidase activity affects the production of the superoxide anion during reperfusion. However, the greater xanthine oxidase activity in rats compared with that in humans [18] makes results from studies using rats [6–8] of questionable applicability to humans. Xanthine oxidase activity in pigs, however, is very low and identical to that in humans [18], making the isolated, blood-perfused, piglet working heart and lung circuit a more relevant animal model. The present data are therefore more applicable to humans that those obtained in rat studies. Although the present study does not show any adverse effects of infusion of L-arginine, further studies are needed to investigate the optimal duration and dosage of this infusion.
In summary, infusion of L-arginine during the initial 10 minutes of reperfusion improves pulmonary function. This improvement was the same as that achieved by reperfusion with leukocyte depleted blood. Although leukocyte depletion is effective in ameliorating lung reperfusion injury [19, 20], it is cumbersome to apply in the clinical setting. The infusion of L-arginine appears to be a simple and effective alternative to reperfusion with leukocyte-depleted blood.
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Acknowledgments |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Laks, Division of Cardiothoracic Surgery, UCLA Medical Center, Center for the Health Sciences, Rm 62-182A, 10833 Le Conte Ave, Los Angeles, CA 90095.
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= p < 0.05 and 
= p < 0.01 versus values at 0.5 hour.)
