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* Department of Cardiothoracic Surgery, Patras University School of Medicine, Patras, Greece 26500;
Department of Angio-thoracic Surgery, Evangelismos General Hospital, Athens, Greece 10676;
Department of Anaesthesia and Intensive Care, Patras University School of Medicine, Patras, Greece 26500;
Patras University School of Pharmacy, Patras, Greece 26500; and
¶ Laboratory of Histology & Embryology, Medical School, University of Athens, Goudi, Athens, Greece 11527
Abstract
In experimental lung transplantation, the reduction of endogenous surfactant properties occurs after graft preservation and transplant reperfusion. The aim of this study was to evaluate the efficacy of donor lung pretreatment with exogenous surfactant on graft damage after ischemia and reperfusion. Fourteen (control group A, n = 8; study group B,n= 6) young female white pigs (mean weight 27 ± 3.5 kg) were used in a newly developed autotransplantation model within situcold ischemia. In study group B, before thoracotomy, 1.5 ml/kg surfactant apoprotein-A-free surfactant was administrated into the left main bronchus via flexible bronchoscopy. Belzer UW solution was used for lung preservation. Cold ischemia was achieved for 3 hr with interlobar lung parenchyma temperature at 8 ± 1.3°C, and central temperature maintained at 37.20 ± 0.5°C. Animals were sacrificed after 3 hr of graft reperfusion. At the end of reperfusion, pulmonary vascular resistance index (was 447.80 dyn/sec.cm5.m2(±66.8) in group A vs 249.51 in group B (P< 0.001) and serum nitric oxide was adequately preserved. The mean alveolar surface area estimated by computerized morphometry was 5280.84 (4991.1) µm2(group A) vs 3997.89 (3284.70) µm2(group B;P< 0.005). Histology revealed milder macrophage and lymphocyte infiltration in group B at the end of reperfusion. Pretreatment of donor lung with an surfactant apoprotein-A -free surfactant agent appears to be beneficial in terms of maintaining serum NO and reducing hemodynamic disturbances. Furthermore, alveolar histology and stereomorphology are better preserved.
Key Words: lung transplantation • surfactant • nitric oxide • SP-A • experimental reperfusion injury
Optimal lung preservation and storage are important prerequisites for successful lung transplantation (1, 2). Laboratory studies have shown that severe damage occurs to endothelial cells and to type II alveolar cells during ischemia (3, 4). Animal studies have shown that both synthesis and activity of surfactant are altered after lung transplantation. In particular, although total concentrations of phospholipids remain stable, the following changes may occur: i) decrease of surfactant’s heavy subtype; ii) decrease of the percentage of phosphatidylcholine in total phospholipids; and iii) reduction in the amount of surfactant-associated protein A (SP-A)(5, 6). Furthermore, analysis of the bronchoalveolar lavage revealed impairment of biophysical surfactant properties after clinical (7) and experimental lung transplantation (8, 9).
Surfactant alterations have been suggested to contribute significantly to the pathophysiology of transplantation-associated lung injuries. Therefore, procedures that stabilize the pulmonary surfactant system may prove to be crucial for optimal lung preservation. The purpose of our study was to evaluate whether pretreatment of donor lung with surfactant results in enhanced lung preservation. Furthermore, we assessed the effect of surfactant pretreatment to lung hemodynamics and serum nitric oxide (NO) levels.
Material and Methods
Experimental Animals.
Fourteen female pigs (landscape white pigs) with a body weight between 25 and 30 kg (mean weight 27 ± 3.5 kg) were used. All animals received humane care in compliance with the Principles of Laboratory Animal Care, the Guide for the Care and Use of Laboratory Animals prepared by the National Institute of Laboratory Animal Resources and published by the National Institutes of Health (publication 85–23, revised 1985), according to the guidelines of the European union (86/609). The experimental protocol was approved by The Patras University Ethical Committee.
Experimental Protocol.
A sterile, nonpyrogenic, modified natural bovine lung extract (Beractant, Survanta Abbott, Abbott Park, IL) was used as the surfactant agent. Animals were randomly assigned to two groups. After intubation, tracheostomy was performed and baseline hemodynamic measurements were recorded. In control Group A (n = 8) no pretreatment was applied. In study Group B (n = 6) the surfactant agent was administered selectively to the left main bronchus (1.5 ml/kg) using flexible bronchoscopy. Volume-controlled mechanical ventilation of the lung followed for approximately 30 min to achieve proper surfactant distribution in the left broncoalveolar tree.
Surgical Technique.
Animals were subjected to left lung in situ autotransplantation. A Swan–Ganz and an arterial catheter were as introduced in the right femoral vein and artery. A left thoracotomy was performed. The left main pulmonary artery and bronchus were isolated at the pulmonary hilum. The pericardium was opened and the origins of the left pulmonary veins were isolated at their entrance to the left atrium. After heparinization (300 IU/kg), a cannula was placed in the pulmonary artery, which was proximally cross-clamped. The left atrium was clamped on the border to the left pulmonary vein and an incision was made for fluid drainage. The left lung was flushed with cold University of Wisconsin solution (60 ml/kg). Ventilation continued during the flush perfusion. After completion of perfusion, the left main bronchus was kept clamped and the lung was semi-inflated. The lung was left in situ and kept isolated at approximately 4°C covered with cold swabs inside an isotherm and waterproof bag. The temperature in the left interlobar space was continuously measured and when it exceeded 8°C, additional cold normal saline and ice were applied to the towels over the isotherm bag. Warm normal saline (38.5°C) was infused in the pleural space so as to keep the central temperature between 37 and 38.5°C. Total ischemic time of the left lung was set at 3 hr. During this period, core and rectal temperatures were constantly monitored. Subsequently, the lung was reperfused by removing the left pulmonary artery clamp. The pulmonary vein incision was repaired after adequate venting.
Cardiopulmonary Assessment.
Assessment of cardiopulmonary function included measurements of heart rate, cardiac output by thermodilution, pulmonary artery pressure, wedge capillary pressure, central venous pressure, arterial pressure, continuous SVO2, arterial blood gases, and urine output measurements (2, 10, 11). Pulmonary vascular resistance index (PVRI) was also calculated (12, 13). Body surface area was calculated according to the following formula: body surface area (m2) = weight (g)2/3 • K • 10-4; K=9 for pigs (13).
Evaluation time points were set at the completion of instrumentation and hilar preparation, and were 60, 120, and 180 min after induced ischemia and 60, 120, and 180 min after reperfusion.
Histology–Morphometry.
After 3 hr of reperfusion, lung tissue was obtained for histological and morphometrical analysis and was fixed in Karnovsky’s solution, made up in a phosphate buffer 0.1 M, pH 7.4, for 12 hr at 4°C. The tissue was subsequently fixed in 1% aquatic osmium tetroxide for 1 hr at 4°C, dehydrated in a series of ethanols, embedded in Epon–Araldite, and left for polymerization at 60°C for 24 hr.
Tissue sections (1 µm thick) were cut using a Leica Ultracut R ultratome equipped with glass knives and stained with 1% toluidine blue in 1% borax. For sampling procedures, a Zeiss Axiolab light microscope equipped with a high-resolution color video camera (Sony CCD Iris, high resolution) was used. All observations were made at a magnification of 5–40x and sampling areas were selected at random using a multipurpose ocular test grid. For each animal 150 to 250 alveoli were analyzed. Morphometric study was carried out using the Image Pro plus 3.1 (Media Cybernetics) and a Pentium III, 800 MHz PC (14). Images for were observed under a Zeiss Axiovert S100 inverted microscope and photographs were taken using a 35-mm camera and a Kodak Tmax-100 ASA film. Film negatives were scanned in an Agfa Duoscan T2500 and images were saved as .tiff.
Nitrite Determination.
Nitrites were measured at the onset of the experiment, at the end of ischemia and at the end of reperfusion. Serum was centrifuged at 20,000g for 10 min at 4°C. for the determination of nitrites. Nitrites were subsequently measured in 500 µl of the supernatant using Griess reagent, as previously described (15). Nitrite values were expressed in µM.
Statistical Analysis.
All values were expressed as mean measurement ± SEM. Comparisons of continuous data among the groups were performed by repeated measurement analysis of variance. Where differences were found, the significance testing was performed by post-hoc tests (Bonferroni/Dunn): P < 0.05 was considered significant. To evaluate the groups in a nonparametric way, we used the Mann-Whitney U test and when the possibility was P < 0.05, we concluded that the groups were dissimilar (16). Analyses were performed using SPSS for Windows, release 9.0.0 (SPSS Inc, Chicago, IL).
Results
Hemodynamics.
Heart rate, mean arterial pressure, central venous pressure, and cardiac index were monitored continuously during the reperfusion period and the values remained in a steady state during the given period (data not shown). All animals in both groups survived the 3-hr observation period.
The PVRI base line mean value was measured at approximately 205.18 dyne • sec/cm5m2 (SD ± 66.8, normally distributed population). During the entire postreperfusion period, a highly significant increase in PVRI was observed in the control group A compared to the study group B, as shown in Figure 1
. Administration of exogenous SP-A-free surfactant appeared to result in consistent preservation of PVRI. In group A, during the entire postreperfusion period, the PVRI values increased steadily, reaching a 1.26-fold increase at 3 hr after onset of reperfusion and a 2.18-fold increase compared with baseline values (P = 0.001). In Group B, we did not find significant differences in PVRI compared with control measurements during the entire postreperfusion period (P = 0.2). The increase in PVRI values was only 1.21-fold compared to baseline values (Fig. 1).
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Histology–Morphometry.
In Group A, emphysema-like lesions were present and homogeneously distributed within the lung parenchyma (Fig. 3). In Group B, the morphology and the integrity of the alveoli appeared better preserved than in Group A. At the end of reperfusion, chronic inflammatory cell (lymphocytes and macrophages) infiltration was milder in Group B than in Group A.
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Discussion
During the past decade, our knowledge of the surfactant system has grown immensely (17). The liquid covering the alveolar epithelial surface consists of a pulmonary surfactant film overlying an aqueous subphase. In the normal lung, the role of the pulmonary surfactant film is to reduce the air-liquid interfacial tension to prevent alveolar collapse and to maintain a gas exchange surface (17).
In lung transplantation, the most popular method of donor lung preservation includes hypothermal lavage of the organ through the pneumonic artery, followed by hypothermal preservation of the transplanted graft. However, this procedure has not been optimized and sometimes may be responsible for premature lung insufficiency immediately after reperfusion, resulting in death of the transplanted patients (18, 19).
Tissue response to transplantation procedures (re-implantation response) has many similarities to the acute pneumonic impairment caused by different factors (20) that lead to abnormal surfactant activity (21). In experimental models of lung transplantation, synthesis of the surfactant agent is affected. In vitro measurements show a reduction in surfactant properties after preservation or reperfusion of the graft (3, 20). Recent studies have shown that, during lavage with the cold solution used for preservation, the ratio of lekithine/sphigomyeline and the concentration of dipalmitoyphosphatidyl-choline are reduced whereas the concentrations of total protein and albumin are increased (4). Similar findings are observed during acute lung impairment (21–23). These data suggest that abnormalities in the surfactant’s activity start at the moment of the graft lavage with the cold preservative solution and are aggravated during the preservation and transplantation phases (4). Based on the above, we decided to administer surfactant to the graft prior to the onset of cold perfusion.
Laplace’s law states that pressure (P) for spherical bubbles of air in a liquid, such as alveoli surrounded by blood, is equal 2T/R, where T is the surface tension of the liquid and R is the radius of the bubble. In bubbles of different sizes, the pressure will be higher in the smaller bubbles resulting in a gradient driving air from small to larger bubbles. If this is the case with the alveoli, the smaller alveoli would collapse into larger alveoli and, thus, the lung will have only a few large alveoli. The latter will have a small surface area in toto and, as a consequence, the gas diffusion into or out of the blood will be impaired. The role of the surfactant is to reduce the surface tension in smaller alveoli and stabilize the ratio 2T/R of Laplace’s law. This results in equalization of the pressure in all alveoli and precludes small alveoli from collapsing into larger ones.
In our experimental model, surfactant administration to donor lungs before hypothermal lavage stabilizes the alveoli by preventing emphysema-like lesions. By maintaining the alveolar morphology in the surfactant group, a smaller alveolar surface area was sustained and smaller distributions of the surface area values were obtained.
Nitric oxide is known to mediate acetylcholine-associated vasodilation in humans and in several animal models. Pulmonary vasodilation results from NO activity in the nonadrenergic, noncholinergic neural system (24, 25). Inhibition of NO synthesis results in an increased vasoconstrictor response in a variety of species and in pulmonary vessels of all sizes (25). Whether continuous NO release maintains basal vascular tone remains unclear. Nitric oxide may be immediately released only when a vasoconstrictor stimulus is detected in an effort to inhibit increased pulmonary vascular tone and NO activity appears to be essential in acute and chronic hypoxic pulmonary vasoconstriction. NO has a well-documented relaxant effect on constricted pulmonary vessels but has minimal effect in nonconstricted vessels (25, 26). The reduction of NO production results in an early loss of NO-dependent vasodilation (27). However, it has become noticeable that NO is engaged in a wide range of biological processes other than vasosilation, such as neurotransmission, blood clotting, and host defense mechanisms (28). It appears to play a major part in the orchestration of the immune response (29), affects cellular responses in inflammation, and shows both anti-inflammatory and proinflammatory effects (30). Thus, NO appears to act as an inhibitor of the neutrophil–endothelial cell interaction that precedes neutrophil extravasation (31) and the consequent organ damage by oxygen free radicals, cytokines and proteases released from neutrophils (32). Furthermore, inflammatory stimuli activate nitric oxide synthase (NOS) in neutrophils and macrophagal NO suppresses lymphocyte proliferation (33).
In our experimental model, pretreatment of donor lung with surfactant resulted in a reliable and stable decrease of PVRI without a significant decrease in systemic arterial pressure. Similarly, NO serum concentrations were significantly preserved. Recent data suggest that ischemia–reperfusion injury reduces endogenous production of NO and prostacyclin by endothelial cells (27). Taking into account the biological half- life of NO (3–30 sec)(34), it is assumed that there is no reduction of NO production during the first 3 postperfusion hours.
The role of surfactant proteins in exogenous preparations is controversial. Surfactants obtained by lipid extraction, including BERACTANT (SURVANTA, ABBOTT,), do not contain the hydrophilic, large molecular weight surfactant-associated protein known as SP-A (35). The surfactant associated proteins SP-A and SP-D bind a variety of pathogens that enhance macrophage uptake and can influence the production and secretion of inflammatory cytokines and superoxide radicals from inflammatory cells, for example, alveolar macrophages (17, 36). However, in animal models, SP-A appears to have unusual functions, including recruiting neutrophils in the lung (37) and playing an important role in the innate host defense against pathogenic microorganisms (38).
The concentration of SP-A levels in bronchoalveolar lavage is significantly decreased after bronchoscopic application of an SP-A-free surfactant material, like Alveofact (39), perhaps the result of dilution of SP-A by the amount of administered surfactant. Moreover, there is a reduction in the amount of SP-A, an inhibiting effect of plasma proteins (5), as well as modification of the secretion and re-uptake of the surfactant from the type II pneumonic cells (6). In our experimental model, pretreatment of donor lung with surfactant may reduce SP-A concentrations, leading to up-regulation in NO synthesis.
Our data demonstrate that pretreatment of donor lung with an SP-A-free surfactant agent is beneficial by means of maintaining PVRI and stabilizing the alveoli, thus reducing respiratory and hemodynamic disturbances in the recipient. Further studies are required before assessing the potential benefits of this method in clinical practice.
Acknowledgments
The authors wish to thank "Digital Image Systems" for expert technical assistance with the ImagePro morphometry software and E. Panagopoulou for excellent preparation of tissue sections for histology and morphometry.
Footnotes
1 To whom requests for reprints should be addressed at 31, Chlois Str. Voula, 166 73 Athens, Greece. E-mail: ekoletsis{at}hotmail.com 
References
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  E. Koletsis, A. Chatzimichalis, E. Apostolakis, K. Kokkinis, V. Fotopoulos, M. Melachrinou, M. Chorti, J. Crockett, E. Marinos, I. Bellenis, et al. In Situ Cooling in a Lung Hilar Clamping Model of Ischemia-Reperfusion Injury Experimental Biology and Medicine, September 1, 2006; 231(8): 1410 - 1420. [Abstract] [Full Text] [PDF]
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