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Ann Thorac Surg 1996;62:1617-1621
© 1996 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

Intratracheal Surfactant Administration Preserves Airway Compliance During Lung Reperfusion

Thoracic and Cardiovascular Research Laboratory, Department of Surgery, University of Virginia Health Sciences Center, Charlottesville, Virginia

Accepted for publication June 27, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Decreased airway compliance after lung transplantation has been observed with severe ischemia-reperfusion injury. Further, it has been shown that the surfactant system is impaired after lung preservation and reperfusion. We hypothesized that surfactant replacement after allograft storage could preserve airway compliance during reperfusion.

Methods. Rabbit lungs were harvested after flush with 50 mL/kg of cold saline solution. Immediate control lungs were studied with an isolated ventilation/perfusion apparatus using venous rabbit blood recirculated at 40 mL/min, room-air ventilation at 20 breaths/min, and constant airway pressure (n = 8). Twenty-four–hour control lungs were preserved at 4°C for 24 hours and then similarly studied (n = 7). Surfactant lungs underwent similar harvest and preservation for 24 hours, but received 1.5 mL/kg of intratracheal surfactant 5 minutes before reperfusion (n = 10). Airway pressure and flow were recorded continuously during 30 minutes of reperfusion. Tidal volume and airway compliance were calculated at 30 minutes.

Results. Tidal volume was 33.67 ± 0.57, 15.75 ± 5.72, and 29.83 ± 1.07 mL in the immediate control, 24-hour control, and surfactant groups, respectively (p = 0.004, surfactant versus 24-hour control). Airway compliance was 1.94 ± 0.27, 0.70 ± 0.09, and 1.46 ± 0.10 mL/mm Hg in the immediate control, 24-hour control, and surfactant groups, respectively (p = 0.002, surfactant versus 24-hour control).

Conclusions. We conclude that surfactant administration before reperfusion after 24 hours of cold storage preserves tidal volume and airway compliance in the isolated ventilated/perfused rabbit model of lung reperfusion injury.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Surfactant, the agent responsible for reducing surface tension at the air-liquid interface in all healthy mammalian lungs, has been extensively studied and characterized in recent years. This work has led to the development of commercially available surfactant analogues, which represent a novel new class of drug for use by neonatologists, pulmonologists, and thoracic surgeons. Several types of surfactant, both natural and synthetic, have now been approved in the United States for the treatment of neonates with infantile respiratory distress syndrome, a condition known to be associated with reduced pulmonary compliance from inadequate surfactant activity [1]. After surfactant replacement therapy, significant reductions in morbidity and mortality from infantile respiratory distress syndrome have been realized [2]. These encouraging results have produced intense interest in the potential role of surfactant in the management of other lung diseases characterized by similar compliance and gas exchange derangements. Clinical trials are now under way evaluating the use of surfactant in the management of patients with adult respiratory distress syndrome. Early results have been encouraging [3]. Because the lung dysfunctions associated with both adult respiratory distress syndrome and lung reperfusion injury after transplantation have many clinical and pathologic features in common, thoracic surgeons have also begun to consider the possible role of surfactant replacement therapy in managing patients in whom manifestations of severe reperfusion injury develop after lung transplantation. To further evaluate the role of surfactant replacement in lung reperfusion injury, we devised a model of isolated rabbit lung function modified from Ueno and colleagues [4]. We hypothesized that the administration of exogenous surfactant before allograft reperfusion would prevent the characteristic decrease in lung volumes and compliance frequently noted in reperfusion-injured lungs.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 86-23, revised 1985).

Harvest Procedure
Twenty-five New Zealand White rabbits weighing 3.0 to 3.5 kg were used as lung donors. Each animal was anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (5 mg/kg). A tracheostomy was performed and mechanical ventilation instituted with a constant-pressure ventilator (RSP1002; Kent Scientific Corp, Litchfield, CT) using room air and a rate of 20 breaths/min. A median sternotomy was then performed and thymectomy carried out. The two superior and 1 inferior venae cavae were loosely encircled with ligatures and the pericardium opened. Both the pulmonary artery (PA) and aorta were dissected free and similarly encircled. A pursestring suture was then placed in the free wall of the right ventricle, and the rabbit was heparinized (500 U/kg). After injection of 30 µg of prostaglandin E₁ (Alprostadil; Upjohn Co, Kalamazoo, MI) into the pulmonary artery, the cavae were interrupted and the onset of ischemia noted. The PA was then cannulated through the right ventricular pursestring and both the right ventricular and PA ligatures were tied around the cannula. After the left ventricle was vented and the aorta was ligated, 50 mL/kg of saline solution at 4°C was infused into the PA from a height of 30 cm. Topical cooling was achieved with cold saline slush. During PA flush, the left atrium was cannulated through a left ventricular pursestring. After completion of the PA flush, the inflow and outflow cannulas were clamped. Care was taken to leave the pleurae intact until the completion of the flush to avoid parenchymal injury. The tracheostomy tube was then clamped at end-inspiration, and the heart-lung block was excised. The lungs were stored at 4°C in saline solution.

Reperfusion Procedure
Thirty heparinized and anesthetized New Zealand White rabbits served as venous blood donors. Saline solution was added to each aliquot of blood to achieve a hematocrit of 28% to 30%. During reperfusion, the lungs were suspended by a force transducer in a warmed, humidified chamber, and room air ventilation at 20 breaths/min was reestablished using a constant-pressure ventilator. The initial maximum inspiratory pressure was set to achieve a tidal volume of 10 mL/kg of donor rabbit weight and subsequently held constant. Any lung with evidence of air leak was excluded. The inflow and outflow cannulas were then connected to a venous blood-filled perfusion circuit with care taken to avoid the introduction of air bubbles. The circuit (Kent Scientific Corp) was designed to recirculate 200 mL of warmed blood using a roller pump (7521-40; Cole Palmer Instrument Co, Chicago, IL) and a blood filter (2C7600; Baxter, Deerfield, IL) at a rate of 40 mL/min. Continuous recording of PA pressure, pulmonary venous pressure, lung weight, airway flow, and airway pressure was carried out using a dynamic data acquisition program (Workbench PC; Strawberry Tree, Inc, Sunnydale, CA) run on a PC (470A; Compaq Prolinea, Houston, TX). This program allowed immediate calculation of tidal volume, pulmonary vascular resistance (pulmonary vascular resistance = 80 x (pulmonary artery pressure - pulmonary venous pressure)/flow), and dynamic airway compliance (dynamic airway compliance = tidal volume/airway pressure). The pulmonary venous pressure was maintained between 5 and 8 mm Hg by changing the height of an outflow reservoir in the circuit. Pulmonary venous blood samples were collected for blood gas analysis (Corning 178 pH/Blood Gas Analyzer, Medfield, MA) at 1, 10, 20, and 30 minutes after the start of reperfusion. Oxygen contact with exposed blood surfaces inside of the reservoir containers was minimized by the continuous passive infusion of 100% nitrogen.

Experimental Protocol
Eight double-lung blocks were immediately reperfused after harvest (immediate controls, IC). Seven lungs were stored for 24 hours at 4°C and then reperfused (24-hour controls, 24C). Ten lungs were similarly stored for 24 hours at 4°C, but received 1.5 mL/kg of surfactant (Infasurf; Ony, Inc, Amherst, NY) administered intratracheally 5 minutes before reperfusion (24-hour surfactant, 24S). All lungs were reperfused for 30 minutes. At the completion of the study, histologic specimens were taken from the right lower lobe and placed in formalin. Samples of the left lower lobe were weighed and dried for calculation of wet-to-dry weight ratios.

The surfactant preparation employed was an organic solvent extract of calf lung lavage suspended in 0.9% saline solution. This surfactant contains phospholipids, neutral lipids, fatty acids, and two hydrophobic, low-molecular-weight proteins known as SP-B and SP-C. Each milliliter of the surfactant preparation contains 35 mg of phospholipids and less than 1 mg of protein. The primary lipid is phosphatidylcholine (>75%).

Statistical Analysis
Statistical analysis was performed for the three groups using analysis of variance on the software STATISTICA by StatSoft, Inc, Tulsa, OK. Significant differences were determined using Tukey's honestly significant difference test. Contrast analyses were performed to determine the significance of predefined questions. The data are reported as mean plus or minus the standard error of the mean.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
All of the 24-hour–preserved lungs, in both the 24C and 24S groups, were severely injured during reperfusion. This injury was manifested grossly by parenchymal hemorrhage, profound swelling, and airway edema. Histologically, all 24-hour–preserved specimens revealed marked interstitial edema, erythrocyte and leukocyte sequestration, and eosinophilic alveolar fluid accumulation. Wet-to-dry weight ratios were also significantly increased in the 24C and 24S groups compared with the IC group (Fig 1). The hemodynamic data recorded during reperfusion revealed severe pulmonary hypertension and increased pulmonary vascular resistance in the 24C and 24S groups compared with the IC group (Figs 2, 3). Pulmonary venous oxygenation data demonstrated impaired oxygenation capacity after 24 hours of cold storage compared with the IC group (Fig 4). These data reflect the severity of injury that occurred during reperfusion after 24 hours of ischemia in this model. There were no statistically significant differences between the 24C group and the 24S group with regard to gross or histologic appearance, tissue water accumulation, PA pressure, pulmonary vascular resistance, or oxygenation capacity.

View larger version (10K): [in this window] [in a new window]   Fig 1. . Wet-to-dry weight ratio: p = 0.003 for IC versus 24C and IC versus 24S, and p = not significant for 24C versus 24S. (IC = immediate control group; 24C = 24-hour control group; 24S = 24-hour surfactant group.)

View larger version (17K): [in this window] [in a new window]   Fig 2. . Pulmonary arterial pressure: p = 0.02 for IC versus 24C and IC versus 24S, and p = not significant for 24C versus 24S. (IC = immediate control group; 24C = 24-hour control group; 24S = 24-hour surfactant group.)

View larger version (17K): [in this window] [in a new window]   Fig 3. . Pulmonary vascular resistance: p = 0.008 for IC versus 24C and IC versus 24S, and p = not significant for 24C versus 14S. (IC = immediate control group; 24C = 24-hour control group; 24S = 24-hour surfactant group.)

View larger version (16K): [in this window] [in a new window]   Fig 4. . Pulmonary venous partial pressure of oxygen: p = 0.0002 for IC versus 24C and IC versus 24S, and p = not significant for 24C versus 24S. (IC = immediate control group; 24C = 24-hour control group; 24S = 24-hour surfactant group.)

View larger version (15K): [in this window] [in a new window]   Fig 5. . Tidal volume: p = 0.004 for IC versus 24C and 24S versus 24C, and p = not significant for IC versus 24S. (IC = immediate control group; 24C = 24-hour control group; 24S = 24-hour surfactant group.)

View larger version (17K): [in this window] [in a new window]   Fig 6. . Dynamic airway compliance: p = 0.002 for IC versus 24C and 24S versus 24C, and p = not significant for IC versus 24S. (IC = immediate control group; 24C = 24-hour control group; 24S = 24-hour surfactant group.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

A number of investigators have published data supporting the idea that at least a portion of the physiologic derangements characteristic of lung reperfusion injury are the result of an abnormal surfactant system after lung transplantation. Surfactant (surface active agent) is a heterogeneous substance comprising a number of unsaturated and saturated phospholipids as well as surfactant-associated proteins, which exhibits the property of reducing surface tension in the alveolus [6]. As lung volume decreases, the more liquid unsaturated phospholipids within surfactant are squeezed out of the surface matrix, thereby increasing the proportion of saturated phospholipid (predominantly dipalmitoyl-phosphatidylcholine) at the alveolar lining, which forms a solid monolayer and resists compression [2, 7]. Without appropriately functioning surfactant in adequate amounts, the pressure difference across the alveolus during expiration results in alveolar collapse and air-space fluid accumulation. A marked drop in pulmonary compliance, an increase in barotrauma, and an impairment of oxygenation capacity result.

Early work in the late 1960s and early 1970s drew a link between allograft rejection and surfactant dysfunction [8, 9]. Since that time, however, the techniques available for studying the surfactant system after transplantation have vastly improved. More recently, Klepetko and associates [10] studied the composition of surfactant in bronchoalveolar lavage samples from dogs during lung preservation and also after transplantation. They found that the levels of dipalmitoyl-phosphatidylcholine, the most important phospholipid present in surfactant, decreased throughout the period of cold ischemia. After implantation, the dipalmitoyl-phosphatidylcholine content continued to decrease. This decrease in dipalmitoyl-phosphatidylcholine levels noted in transplanted lungs during reperfusion could be partially ameliorated by supplementing the recipient with intravenous L-carnitine, a cofactor involved in transmitochondrial fatty acid transport during surfactant synthesis. Importantly, Klepetko and associates noted improved allograft function in the group receiving supplemental L-carnitine.

Additional evidence that the constitution of surfactant becomes abnormal after transplantation was published by Veldhuizen and colleagues [11]. In a canine model of dog transplantation, they found impaired gas exchange in transplanted lungs after 12 hours of cold storage that was associated with surfactant composition abnormalities. These included increased sphingomyelin content, decreased phosphatidylglycerol content, and decreased surfactant-associated protein A levels. Ischemic injury was also associated with increased amounts of surfactant-inhibiting serum protein in lung lavage. They noted that the surfactant abnormalities identified in their model after lung transplantation resembled those found in experimental models of adult respiratory distress syndrome. They concluded that surfactant replacement therapy after lung transplantation may have an important role in treating reperfusion injury.

The first experimental evaluation of surfactant replacement after lung transplantation was carried out by Novick and colleagues [12]. They administered bovine surfactant to lungs just before reperfusion after a prolonged 38-hour period of cold storage and compared allograft function with function of untreated controls. All control animals and 5 of 8 surfactant-treated animals demonstrated evidence of severe reperfusion injury. However, 3 of the treated animals had normal lung function with maintenance of an oxygen tension/inspired oxygen fraction ratio of more than 400 mm Hg and a normal carbon dioxide tension. They concluded that surfactant replacement can result in excellent lung preservation, but the inconsistency of its effectiveness would require further study.

The potential role of exogenous surfactant replacement therapy after lung transplantation is based on a growing understanding of the mechanisms of abnormal surfactant function in experimental models of lung ischemia-reperfusion. Endogenous surfactant is produced, stored, and secreted by the alveolar type II pneumocyte. Ischemic damage to the type II pneumocyte during lung storage has been postulated to cause inadequate production of surfactant during reperfusion [3, 10]. Lung injury has also been shown to result in an abnormal distribution of surfactant subtypes after secretion [11, 13]. Complex changes in the metabolic pathways by which surfactant is recycled and cleared are thought to cause these alterations after injury.

Changes in the alveolar milieu of the reperfused lung may also have a detrimental effect on surfactant function, further justifying surfactant replacement in this setting. Both adult respiratory distress syndrome and reperfusion injury are characterized by a disruption of the capillary-endothelial barrier. Leakage of plasma protein into the alveolus is, therefore, a hallmark of both conditions. These proteins, including albumin, fibrin, fibrinogen, and hemoglobin, have been shown to have a profound inhibitory effect on surfactant function [2, 6, 14]. This effect is exacerbated when the quantity of surfactant is low or its composition is abnormal [3]. Interestingly, resistance of surfactant to this plasma protein inhibition is dependent on the presence of the surfactant-associated proteins [2]. These findings have important implications for the optimal composition of exogenous surfactant to be used in patients with lung injury after transplantation.

Surfactant replacement therapy after lung transplantation may also be justified by examining the increasing number of roles surfactant in now known to play that are unrelated to its surface-tension lowering properties. Both the lipid and protein components of surfactant have been shown to be important in the immune function of the lung. A number of studies have revealed that surfactant enhances macrophage phagocytosis of bacteria and viruses, and modulates the function of lymphocytes [3, 14]. Surfactant is important in maintaining a normally functioning mucociliary transport system. In studies of injured tracheal epithelium, the addition of surfactant enhances ciliary recovery, and thus potentially speeds the clearance of inhaled particulate matter. Perhaps most importantly, surfactant is known to have direct antioxidant capabilities, allowing it to scavenge reduced oxygen species and prevent subsequent free radical damage at the level of the alveolus [15]. Although none of these actions has been proved to play a role in surfactant's ability to prevent or ameliorate the effects of reperfusion injury, the possibilities warrant further study.

We have been able to show in an isolated model of rabbit lung ischemia that exogenous calf lung surfactant administered just before reperfusion is capable of ameliorating some of the effects of severe reperfusion injury. Despite pulmonary hypertension and marked edema, normal tidal volume was preserved in the surfactant-treated lungs when compared with untreated controls. Additionally, no significant decrement in pulmonary compliance was observed during reperfusion in the surfactant group; whereas a marked decrease in compliance occurred in the control group. The administration of surfactant was not capable of preventing pulmonary hypertension or edema formation, or of enhancing oxygenation capacity. However, we believe that severe reperfusion injury after lung ischemia is a multifactorial syndrome, which will require several combined therapeutic approaches, of which exogenous surfactant replacement may be a part. The combination of an agent such as surfactant, which protects and enhances airway function, with an agent designed to preserve vascular endothelium may produce the best results.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This work was funded, in part, by the National Institutes of Health under RO-1 grant HL 48242 and National Research Service Award fellowship 5 F32 HL 08940. Additional support from CNPq-Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil, is acknowledged.

We express our appreciation to Mr Anthony Herring for his invaluable technical assistance.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Kron, Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Box 310, University of Virginia Health Sciences Center, Charlottesville, VA 22908.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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2. Van Golde LMG, Batenburg JJ, Robertson B. The pulmonary surfactant system. News Physiol Sci 1994;9:13–20.[Abstract/Free Full Text]
3. Brown DL, Pattishall EN. Other uses of surfactant. Clin Perinatol 1993;20:761–89.[Medline]
4. Ueno T, Yokomise H, Oka T, et al. The effect of PGE1 and temperature on lung function following preservation. Transplantation 1991;52:626–30.[Medline]
5. Lefer AM, Lefer DJ. Pharmacology of the endothelium in ischemia-reperfusion and circulatory shock. Annu Rev Pharmacol Toxicol 1993;33:71–90.
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9. Drews JA, Tierney DF, Benfield JR. Effect of immunosuppression and lung transplantation upon surfactant. Surgery 1974;76:80–7.[Medline]
10. Klepetko W, Lohninger A, Wisser W, et al. Pulmonary surfactant in bronchoalveolar lavage after canine lung transplantation: effect of L-carnitine application. J Thorac Cardiovasc Surg 1990;99:1048–58.[Abstract]
11. Veldhuizen RAW, Lee J, Sandler D, et al. Alterations in pulmonary surfactant composition and activity after experimental lung transplantation. Am Rev Respir Dis 1993;148:208–15.[Medline]
12. Novick RJ, Veldhuizen RAW, Possmayer F, Lee J, Sandler D, Lewis JF. Exogenous surfactant therapy in thirty-eight hour lung graft preservation for transplantation. J Thorac Cardiovasc Surg 1994;108:259–68.[Abstract/Free Full Text]
13. Ikegami M, Jobe AH. Surfactant metabolism. Semin Perinatol 1993;17:233–40.[Medline]
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