Ann Thorac Surg 2000;70:1671-1674
© 2000 The Society of Thoracic Surgeons
Original articles: general thoracic
Aprotinin improves pulmonary function during reperfusion in an isolated lung model
Menen A. Mathias, MDa,
Curtis G. Tribble, MDa,
Jeffrey F. Dietz, BSa,
Richard P. Nguyena,
Kimberly S. Shockey, MSa,
John A. Kern, MDa,
Irving L. Kron, MDa
a Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health Sciences Center, Charlottesville, Virginia, USA
Address reprint requests to Dr Kron, Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health Sciences Center, Box 3111, MR4 Bldg, Charlottesville, VA 22908;
e-mail: ikron{at}virginia.edu
Presented at the Poster Session of the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
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Abstract
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Background. We hypothesized that the use of aprotinin would ameliorate the reperfusion injury observed after lung transplantation because of a reduction in the inflammatory response.
Methods. We used an isolated, whole blood-perfused, ventilated rabbit lung model to study the effects of aprotinin during reperfusion. The control animals (group A, n = 8) underwent lung harvest after pulmonary arterial prostaglandin E1 injection and Euro-Collins preservation flush before saline storage for 18 hours at 4°C. The experimental groups received either a low dose (3,000 KIU/mL; group B, n = 8) or a high dose (10,000 KIU/mL; group C, n = 8) of aprotinin added to the pulmonary flush before storage. Each lung was reperfused at 37°C at a rate of 60 mL/min.
Results. The arterial partial pressure of oxygen values of group B (low-dose aprotinin) were significantly higher than those of group A (control) after 10 minutes of reperfusion (69.19 ± 5.69 mm Hg versus 264.30 ± 48.59 mm Hg, respectively, p = 0.001). Similar results were recorded at 20 and at 30 minutes of reperfusion. Similarly, after 10 minutes of reperfusion, the differences between groups A and C were 69.19 ± 5.69 mm Hg versus 235.91 ± 28.63 mm Hg, respectively (p = 0.001).
Conclusions. The addition of aprotinin to the Euro-Collins pulmonary flush significantly improves arterial oxygenation in the early reperfusion period. The enhanced oxygenation suggests that aprotinin may offer protection against early reperfusion injury.
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Introduction
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The optimization of lung allograft function is one of the driving forces in lung transplantation research. Since the first successful isolated lung transplant in 1963, the introduction of immunosuppression drugs and improved lung preservation solutions has diminished the incidence of severe graft dysfunction [1]. Data from several sources reveals that approximately 4,000 isolated lung transplant procedures are performed worldwide with a 1-year survival rate between 70% and 80%. The majority of patients who die early (before 3 months) after transplant succumb to graft failure or infection. This is at least 10% to 20% of transplant recipients. There has been a great deal of attention focused on ischemia–reperfusion injury in the transplanted lung [2–5]. The manifestations of this pulmonary ischemia–reperfusion injury can be characterized by increased pulmonary vascular resistance, decreased oxygenation capacity, worsened compliance, and edema formation. The initial ischemic insult to the lung correlates with the production of cytokines and increased expression of adhesion molecules by hypoxic parenchymal and endothelial cells. Accumulating evidence suggests that the specific reperfusion component of the injury cascade is mediated in large part by neutrophil–endothelial adherence and subsequent neutrophil-mediated organ injury. In fact, when the neutrophils are activated by adherence to the endothelial tissue, they secrete reactive oxygen species and proteolytic enzymes. The end result is then the profound structural and functional breakdown of delicate lung parenchyma.
Because the transplanted lung is highly susceptible to the deleterious effects of ischemia–reperfusion injury, an agent that may combat this process is much sought after in lung transplantation research. In general, organ preservation injury is associated with lysosomal enzyme release, activation of proteolytic enzymes, and endothelial cell damage. For this reason, aprotinin, a serine protease inhibitor that is in wide clinical use to minimize perioperative blood loss in cardiac operations, may be ideally suited to reduce the effects of lung reperfusion injury as it also suppresses the release of lysosomal enzymes and inhibits their activities [4].
In this study, a dose range of aprotinin that should produce hemostatic and antiinflammatory effects was added to the lung preservation flush before the cold ischemic storage time period. The hypothesis tested was that aprotinin-treated lungs had enhanced oxygenation capacity when compared to those stored in standard clinical preservation solutions.
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Material and methods
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Harvest procedure
The isolated rabbit lung model previously used in our laboratory was used in this study [6–10]. Fortunately, in our laboratory we were afforded an ex vivo, isolated lung model that allows reasonable approximation of clinical lung transplantation. New Zealand white rabbits of both sexes (3.0 to 3.5 kg) were randomly assigned to the individual groups. Each animal was anesthetized adequately with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) mixture. Tracheal intubation was performed through a tracheostomy and mechanical ventilation continued throughout the procedure on room air with a constant pressure ventilator (Ventilator RSP 1002; Kent Scientific Corporation, Litchfield, CT). The animals were ventilated with room air at a respiratory rate usually of 20 breaths/min and complete paralysis was maintained with vecuronium. A median sternotomy and a thymectomy were performed to provide adequate exposure of the thorax. All vena cavae were loosely encircled with ligatures to prepare for later occlusion. Both the pulmonary artery and the aorta were similarly encircled with ligatures. At this juncture, heparin (500 U/kg) was given intravenously through a marginal vein ear catheter. A 30-µg dose of prostaglandin E1 was given into the pulmonary artery followed by immediate ligation of the vena cavae. A pulmonary artery catheter was placed through a right ventriculotomy and held in place with a pursestring suture. Immediately after the cannula was secured, the cold preservation flush (50 mL/kg) was allowed to flow freely into the pulmonary artery from an adequate height above the model. In the control group, the animals received an aliquot of 150 mL of Euro-Collins without aprotinin. In the low-dose experimental group, the animals received a dose of aprotinin (10 mL, 3,000 KIU/mL) mixed with Euro-Collins (140 mL), which corresponds with the manufacturer’s guidelines for dosing by weight. In the high-dose group, the animals received (30 mL,10,000 KIU/mL) mixed with Euro-Collins (120 mL), which corresponds to the dose that is thought to exhibit antiinflammatory effects. These doses were administered with cold preservation flush through the direct delivery method, as found in previous experiments in the literature [4]. We used the liquid form of the drug as supplied by the Bayer Corporation. Before topical cooling with ice-cold saline slush, a left ventriculotomy was performed for the outflow catheter to be inserted into the left atrium. This outflow catheter was later connected to a transducer for the measurement of left atrial pressures during the reperfusion period. After completion of the flush, the inflow and outflow cannulas were clamped. The lung–heart block was then excised and stored at end-inspiration in a saline vehicle at 4°C for 18 hours. All experimental protocols were reviewed and approved by an institutional animal use committee. The animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" formulated by the National Institutes of Health (NIH publication no. 85-23, revised 1985).
Reperfusion procedure
The lung–heart block was retrieved after the cold ischemic storage period, suspended in a warm, humidified tissue chamber, and reconnected to the ventilator with a 95% oxygen/5% carbon dioxide mixture at a respiratory rate of 20 breaths/min. The pulmonary artery catheter was connected to the venous blood reperfusion circuit and the outflow tripod catheter was connected to the pressure transducer. New Zealand white rabbits (4 to 4.5 kg) served as fresh venous blood donors. The lungs were then reperfused with this venous blood from a reservoir. A second nonrecirculated fresh venous blood reservoir was used for measurement of single-pass oxygenation values during the reperfusion stage. At 10, 20, and 30 minutes, samples of arterial and venous blood were obtained for blood gas analyses. A 30-mL sample of venous blood was passed through the pulmonary vasculature at each interval to obtain accurate values of the pulmonary venous oxygen content. Also, the oxygen contact with exposed blood surfaces inside the reservoir containers was minimized by continuous passive infusion of 100% nitrogen. The perfusion circuit (Kent Scientific Corporation) allows recirculation of 150 mL of blood warmed to 37°C using a roller pump (model 7521-40; Cole-Parmer Instrument Company, Chicago, IL) at a rate of 60 mL/min. At the completion of the 30-minute reperfusion period, samples of lung tissue were taken and weighed before flash freezing in liquid nitrogen and storing at -80°C.
Statistical analysis
Statistical analyses were performed using analysis of variance and the Kruskal-Wallis nonparametric test on SPSS software (SPSS Inc, Chicago, IL). Significant differences were determined using the Bonferroni multiple comparison analysis. Differences were considered statistically significant if the p value was less than 0.05. The data expressed as the mean plus or minus the standard error of the mean.
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Results
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Throughout the 30-minute reperfusion period, the two groups (B and C) of lungs that were preserved with Euro-Collins and aprotinin demonstrated significant improvement in arterial oxygenation. As shown in Figure 1 the arterial partial pressure of oxygen of group A lungs (control) was significantly higher than that of group B lungs (low-dose aprotinin) after 10 minutes of reperfusion (69.19 ± 5.69 mm Hg versus 264.30 ± 48.59 mm Hg, p = 0.001), after 20 minutes of reperfusion (70.0 ± 5.73 mm Hg versus 269.83 ± 50.53 mm Hg, p = 0.001), and after 30 minutes of reperfusion (72.43 ± 8.43 mm Hg versus 285.50 ± 61.08 mm Hg, p = 0.001). Similarly, for group A lungs (control) the oxygen arterial partial pressure was significantly higher than that of group C lungs (high-dose aprotinin) after 10 minutes of reperfusion (69.19 ± 5.69 mm Hg versus 235.91 ± 28.63 mm Hg, p = 0.001), as well as at 20 minutes of reperfusion (70.00 ± 5.73 mm Hg versus 325.09 ± 49.79 mm Hg, p = 0.001), and at 30 minutes of reperfusion (72.43 ± 8.43 mm Hg versus 347.45 ± 55.42 mm Hg, p = 0.001) (Fig 1). When further comparisons were made, the arterial partial pressure of oxygen of group B lungs was not significantly higher than that of group C lungs in each of the time intervals that measurements were taken.
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Fig 1. Representation of arterial oxygen tensions in millimeters of mercury at 10-, 20-, and 30-minute intervals after reperfusion of the ischemic lungs. The columns are from left to right: controls, low-dose aprotinin (Apr)-treated lungs and high-dose aprotinin-treated lungs.
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Comment
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Because reperfusion injury continues to be a challenging source of lung allograft failure, the pursuit of efficient means to curtail this phenomenon is the main focus of this study. The discovery of key cellular and molecular events mediating the phenomenon of ischemia–reperfusion injury has been an important goal of lung transplantation research in the past few years. It is now widely accepted that leukocyte activation, free radical generation, and cytokine release are events causing significant pulmonary dysfunction. These events result in a number of specific physical manifestations such as inadequate arterial oxygenation, interstitial edema, decreased pulmonary compliance, and eventual graft failure. Fortunately, the isolated, ventilated lung model used in this study is unique in that we can isolate some of the phenomena surrounding clinical lung transplantation without some of these confounding variables present in vitro. It has been suggested that pharmacologic means be implemented to suppress the inflammatory cascade and to curtail these physiologic derangements. In one recent study, ulinastatin, a human urinary protease inhibitor and serine protease inhibitor like aprotinin, was efficacious in attenuating neutrophil elastase release. In this in vivo study, this drug also appears to limit the postperfusion lung injury associated with cardiopulmonary bypass [11]. Another agent that has been examined is the neutrophil elastase inhibitor ONO-5046, which is postulated to prevent the acute lung injury caused by acid aspiration into the lungs. Several studies were conducted and the drug was shown to have slower increases in pulmonary artery pressure as well as lowering of the permeability index of the ONO-5046-treated rabbit lungs and may be useful in attenuating endotoxin-induced acute lung injury [12–14]. In this project, the drug aprotinin was chosen because of its antiinflammatory effects because of serine protease inhibition. Aprotinin is also commonly used in cardiac operations to promote hemostasis.
Aprotinin appears to combat some of the deleterious effects of extracorporeal circulation. Tumor necrosis factor-
is synthesized as a membrane-bound precursor and is released after cleavage by a serine protease inhibitor; thus, when aprotinin is used it precludes the release of this proinflammatory cytokine. Other studies have shown that tumor necrosis factor- enhances endogenous nitric oxide production, whereas aprotinin deters nitric oxide production [5].
Aprotinin appears to have hemostatic and antiinflammatory effects when the drug is at a kallikrein-inhibiting concentration. Aprotinin inhibits the initiation of both coagulation and fibrinolysis, as well as the release of the vasoactive peptide bradykinin. It appears that when kallikrein inhibition occurs, the production of the direct precursor of bradykinin, high molecular weight kininogen, is blocked. According to recent reports, because bradykinin increases during the ischemia–reperfusion period, its suppression by aprotinin should ameliorate reperfusion injury [15].
There has been some previous literature regarding aprotinin and lung reperfusion injury. One study noted that after aprotinin administration, there was improved oxygenation, reduced edema formation, and significantly increased compliance [4].
It is an established fact that impaired oxygenation capacity remains a sensitive barometer of inadequate lung function. Therefore, the marked improvement in arterial partial pressure of oxygen values is indicative of good lung allograft function in the isolated lung model. In another study, investigators have shown that aprotinin has improved oxygenation capacity as well as compliance [16].
It appears that aprotinin may offer protection of lung transplants against early reperfusion injury. Even at reduced dosages, the drug can exhibit this effect. Aprotinin can be easily incorporated into lung transplant protocols and may have the dual role of hemostasis and lung protection.
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Acknowledgments
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This study was supported by the National Institutes of Health grant RO1 HL56093, the National Institutes of Health training grant T32 HL 07849 to 01A2, type 1, and an educational gift from the Bayer Corporation, Pharmaceutical Division. We appreciate the invaluable technical assistance of Anthony J. Herring, Sheila D. Hammond, and Perry Stevens in the completion of this project.
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References
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Kaiser L., Kron I., Spray T. Mastery of cardiothoracic surgery. Technique of lung transplantation. New York: Lippincott-Raven, 1998.
-
Welbourn C.R., Goldman G., Paterson I.S., et al. Pathophysiology of ischaemia reperfusion injury. Br J Surg 1991;78:651-655.[Medline]
-
Jaquiss R.D., Huddleston C.B., Spray T.L. Use of aprotinin in pediatric lung transplantation. J Heart Lung Transplant 1995;14:302-307.[Medline]
-
Roberts R.F., Nishanian G.P., Carey J.N., et al. Addition of aprotinin to organ preservation solutions decreases lung reperfusion injury [see comments]. Ann Thorac Surg 1998;66:225-230.[Abstract/Free Full Text]
-
Hill G.E., Springall D.R., Robbins R.A. Aprotinin is associated with a decrease in nitric oxide production during cardiopulmonary bypass. Surgery 1997;121:449-455.[Medline]
-
Ross S.D., Tribble C.G., Gaughen J.R., Jr, et al. Reduced neutrophil infiltration protects against lung reperfusion injury after transplantation. Ann Thorac Surg 1999;67:1428-1434.[Abstract/Free Full Text]
-
Ross S.D., Tribble C.G., Linden J., et al. Selective adenosine-A2A activation reduces lung reperfusion injury following transplantation. J Heart Lung Transplant 1999;18:994-1002.[Medline]
-
King R.C., Binns O.A., Kanithanon R.C., et al. Low-dose sodium nitroprusside reduces pulmonary reperfusion injury. Ann Thorac Surg 1997;63:1398-1404.[Abstract/Free Full Text]
-
Buchanan S.A., Mauney M.C., Parekh V.I., et al. Intratracheal surfactant administration preserves airway compliance during lung reperfusion. Ann Thorac Surg 1996;62:1617-1621.[Abstract/Free Full Text]
-
Binns O.A., DeLima N.F., Buchanan S.A., et al. Both blood and crystalloid-based extracellular solutions are superior to intracellular solutions for lung preservation. J Thorac Cardiovasc Surg 1996;112:1515-1521.[Abstract/Free Full Text]
-
Hiyama A., Takeda J., Kotake Y., et al. A human urinary protease inhibitor (Ulinistatin) inhibits neutrophil extracellular release of elastase during cardiopulmonary bypass. J Cardiothorac Vasc Anesth 1997;11:580-584.[Medline]
-
Kaneko K., Kudoh I., Hattori S., et al. Neutrophil elastase inhibitor, ONO-5046, modulates acid-induced lung and systemic injury in rabbits. Anesthesiology 1997;87:635-641.[Medline]
-
Nishina K., Mikawa K., Takao Y., et al. ONO-5046, an elastase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg 1997;84:1097-1103.[Abstract]
-
Miyazaki Y., Inque T., Kyi M., et al. Effects of a neutrophil elastase inhibitor (ONO-5046) on acute pulmonary injury induced by tumor necrosis factor alpha(TNF) and activated neutrophils in isolated perfused rabbit lungs. Am J Respir Crit Care Med 1998;157:89-94.[Abstract/Free Full Text]
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Nagahiro I., White T., Yano M., et al. Recombinant Kunitz protease inhibitor ameliorates reperfusion injury in rat lung transplantation. Ann Thorac Surg 1998;66:351-355.[Abstract/Free Full Text]
-
Hill G.E., Diego R.P., Stammers A.H., et al. Aprotinin enhances the endogenous release of interleukin-10 after cardiac operations. Ann Thorac Surg 1998;65:66-69.[Abstract/Free Full Text]
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Top
Abstract
Introduction
