BASIC
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EXPERIMENTAL RESEARCH
Exogenous Surfactant Attenuates Lung Injury From Gastric-Acid Aspiration During Ex Vivo Reconditioning in Pigs Theresa Khalife´-Hocquemiller,1 Edouard Sage,1 Peter Dorfmuller,1 Sacha Mussot,1 Daniel Le Houe´rou,1 Saadia Eddahibi,1 and Elie Fadel1,2 Background. Lung injury (LI) due to gastric-acid aspiration is associated with poor posttransplantation outcomes. We investigated the effects of ex vivo lung perfusion (EVLP) reconditioning and surfactant administration on LI due to gastric-acid aspiration. Methods. Thirty piglets were allocated at random to five groups: the lungs were studied 24 hr after gastric juiceYinduced LI of the left lower lobe (LLL), LI followed by EVLP (4 hr), or LI followed by LLL surfactant lavage immediately before EVLP; sham animals were studied 24 hr after saline infusion alone or followed by EVLP. Gross anatomy, hemodynamics, and aerodynamics were evaluated; neutrophil and bacterial counts were determined in bronchoalveolar lavage (BAL) fluid and blood. LLLs were evaluated based on a semi-quantitative histologic score, apoptotic cell death (TUNEL), and inflammatory cytokine levels. Results. The sham and sham-EVLP groups were not significantly different. Compared with sham, LI animals had irreversible atelectasis, higher lung infection rates (PG0.0001) and BAL neutrophil percentages (PG0.0001), lower PaO2 (P=0.0006), higher IL-1 (P=0.022) and IL-8 (P=0.006), higher apoptotic cell percentages (P=0.007), and worse histologic severity scores (PG0.0001). EVLP alone did not improve these findings. Adding surfactant before EVLP returned PaO2, pulmonary vascular resistance, and apoptotic-cell percentage to sham-EVLP values but only partially improved the histologic severity score. Conclusion. Local surfactant infusion immediately before EVLP improved the function of donor lungs injured by gastric juice aspiration. This strategy may hold promise for decreasing the shortage of donor lungs. Keywords: EVLP, Lung injury, Lung aspiration. (Transplantation 2014;97: 413Y418)
ung transplantation is a well-established treatment for patients with end-stage respiratory failure, and the numbers of lung transplant procedures and candidates have increased steadily over the last three decades (1). Although the lung donor pool has been successfully expanded by the use of marginal donors in the past 10 years (2, 3), the demand for lungs far exceeds the supply (4). Currently, 15% to
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This study was funded by the nonprofit donation-funded organization ADETEC. The authors declare no conflicts of interest. 1 Laboratoire de Recherche Chirurgicale and INSERM U999, Hoˆpital Marie Lannelongue, Universite´ Paris Sud, Le Plessis Robinson, France. 2 Address correspondence to: Elie Fadel M.D., Department of Thoracic and Vascular Surgery and Heart-Lung Transplantation, Hoˆpital MarieLannelongue (Paris-Sud University), 133 Avenue de la Resistance, 92350 Le Plessis Robinson, France. E-mail: [email protected] T.K.-H., S.M., and E.F. participated in designing the study and writing the manuscript. E.S., P.D., D.L.H., and S.E. participated in designing and executing the study. Received 16 August 2013. Revision requested 12 September 2013. Accepted 18 November 2013. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9704-413 DOI: 10.1097/01.TP.0000441320.10787.c5
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20% of lungs from multiorgan donors are deemed suitable for lung transplantation (5). Gastric content aspiration is associated with increased risks of lung transplant infection and dysfunction. Lung injury (LI) from gastric content aspiration is related to both chemical damage and obstruction by food particles (6) and is a common reason for lungs being considered unsuitable for transplantation. Ex vivo lung perfusion (EVLP) is a promising technique for rendering injured donor lungs suitable for transplantation (7Y9). Preclinical studies of animal and human lungs have established that lung function can be safely preserved by EVLP for as long as 12 hr (8, 10). This time can be used not only to prevent lung damage but also to manage LI with the goal of making the lungs suitable for transplantation. However, we previously reported that reconditioning with EVLP alone failed to improve LI due to gastric acid aspiration in pigs (11). Surfactant is a phospholipid and protein layer produced by type II alveolar cells. Surfactant lowers surface tension, thereby preventing alveolar collapse. Surfactant inactivation is involved in ischemia/reperfusion injury, one of the main causes of primary graft dysfunction. Exogenous surfactant is used to treat hyaline membrane disease in children and www.transplantjournal.com
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FIGURE 1. PaO2/FiO2 ratio in the three groups treated with ex vivo lung perfusion (EVLP): lung injury induced by gastric-content instillation (LI-EVLP), sham group given saline instillation (Sham-EVLP), and lung injury followed by both surfactant instillation and EVLP (LI-Surf-EVLP). During EVLP, repeated measures showed a significant PaO2/FiO2 decrease (PG0.0001) in the LI-EVLP group compared with the sham-EVLP group. In the LI-Surf-EVLP group, PaO2/ FiO2 improved significantly compared with the LI-EVLP group (PG0.0001) but remained lower than sham-EVLP values (P=0.0145).
increases pulmonary oxygenation in acute respiratory distress syndrome due to direct LI (12). In a pig model, EVLP with surfactant lavage improved lung function after acid aspiration (13). However, the surfactant was given immediately after acid instillation, before LI development. In practice, aspirationinduced LI in multiorgan donors is diagnosed at the stage of established radiologic and histologic LI. Here, our objective was to determine whether surfactant lavage immediately before starting EVLP improved established LI induced 24 hr earlier by gastric acid instillation. To this end, we conducted a study in a preclinical pig model.
RESULTS Compared with the sham animals, LI animals studied 24 hr after gastric content instillation into the left lower lobe
FIGURE 2. Pulmonary vascular resistance in the three groups treated with ex vivo lung perfusion (EVLP): lung injury induced by gastric-content instillation (LI-EVLP), sham group given saline instillation (Sham-EVLP), and lung injury followed by both surfactant instillation and EVLP (LISurf-EVLP). During EVLP, repeated measures showed a significant PVR increase (P=0.0002) in the LI-EVLP group compared with the Sham-EVLP group. In the LI-Surf-EVLP group, PVR returned to sham values.
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(LLL) as previously described by Meer and colleagues (14) had similar hemodynamic variables and PaO2/FiO2 ratio values but significantly higher blood neutrophil counts (34 750T10 474/mm3 vs. 19 880T3641/mm3; P=0.0199) and bronchoalveolar lavage (BAL) neutrophil percentages (65% vs. 11%; PG0.0001). BAL cultures were negative in the sham animals and positive (9105 bacteria/mL) in 66% of the LI animals (PG0.0001). Repeated-measures analysis of variance was performed to compare four EVLP time points: before starting EVLP (24 hr after LI induction) then 1, 3, and 4 hr after starting EVLP. At the 4-hr time point, compared with the sham-EVLP group (saline instillation followed by EVLP), the LI-EVLP group (gastric acid instillation followed by EVLP) had significantly higher pulmonary vascular resistance (PVR) (P=0.0002) and significantly lower PaO2/FiO2 (PG0.0001). At the same 4-hr time point, the LI-Surf-EVLP group (gastric acid instillation followed by surfactant administration and EVLP) showed a return of PVR to sham-EVLP values and a significant improvement in PaO2/FiO2 compared with the LI-EVLP group (PG0.0001). However, PaO2/FiO2 values in the LI-SurfEVLP group remained lower than in the sham-EVLP group (P=0.0145) (Figs. 1 and 2). The percentage of neutrophils in BAL fluid after 4 hr of EVLP remained elevated in the LI-EVLP group compared with the sham-EVLP group (69% vs. 5%; P=0.0065). After surfactant lavage, the BAL neutrophil percentage remained elevated, similar to that in the LI-EVLP group (57% vs. 69%; P=NS) and higher than in the sham-EVLP group (P=0.0047). BAL-fluid bacteriologic cultures were negative in both sham groups and positive (9105) in 57% of LI animals and 60% of LI-EVLP animals (PG0.0001). Surfactant lavage failed to decrease the BAL infection rate (51%; P=NS). Bacteria found in BAL were Escherichia coli, Pseudomonas, and Aeromonas. On gross anatomy, LLLs were similar in the two sham groups. All right lower lobes (RLLs) were normal. The LLLs in the LI group showed a collapsed appearance that was not reversible by manual ventilation. After 4 hr of EVLP, the collapse was exacerbated, and both hemorrhage and edema were increased (Fig. 3).
FIGURE 3. Gross appearance of the left lower lobe in the LI-EVLP group.
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* 2014 Lippincott Williams & Wilkins
FIGURE 4. Semi-quantitative histologic damage severity score determined in the left lower lobe by an observer blinded to group assignment in the two sham groups and three lung-injured groups. *Sham versus LI, PG0.0001; U, LISurf-EVLP versus LI-EVLP, PG0.0001; $, LI-Surf-EVLP versus sham, PG0.0001. LI, lung injury; EVLP, ex vivo lung perfusion; Surf, surfactant.
The wet-to-dry ratio showed no significant differences across groups. The histologic injury severity scores were not significantly different across RLLs or between LLLs in the two sham groups (1.000T1.225 vs. 2.600T1.140, P=0.346). Compared with the sham group, the LI group had a significantly higher score (15.429T4.791 vs. 1.000T1.225; PG0.0001), and the score value did not improve with EVLP (12.400T2.408, P=0.061). Surfactant instillation immediately before EVLP was
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associated with significantly lower scores compared with EVLP alone (8.300T1.337 vs. 15.429T4.791; PG0.0001) (Fig. 4). In RLLs, the number of apoptotic cells per field as assessed by TUNEL was similar in all groups. In LLLs, there was a nonsignificant trend toward higher apoptotic cell counts in the LI group compared with the sham group (4.4 vs. 2.2, P=0.067). EVLP had no effect in sham animals (2.2 in the sham-EVLP group vs. 2.2 in the sham group; P=NS) but significantly increased the apoptotic cell count in injured lungs (LI-EVLP group, 8.2 vs. 4.4 in the LI group; P=0.004). Surfactant instillation (LI-Surf-EVLP group) decreased the apoptotic cell counts (3.3 vs. 8.2 in the LI-EVLP group; PG0.0001). Similarly, cytokine levels were comparable in all RLLs. In LLLs (Fig. 5), IL-10, INFF and TNF> levels were similar in all groups; whereas, compared with the sham group, IL-1A, IL-6, and IL-8 levels were increased in the LI group (P=0.0007, P=0.011, and P=0.0052, respectively), LI-EVLP group (PG0.0001, P=0.0006, and P=0.0106, respectively), and LI-SurfEVLP group (P=0.0005, P=0.0214, and P=0.0085, respectively). Only IL-6 values were significantly lower in the LI Surf-EVLP group compared with the LI-EVLP group (P=0.0414).
DISCUSSION In this study, exogenous surfactant therapy by direct instillation via a flexible bronchoscope followed by 4 hr of EVLP attenuated the lung damage present 24 hr after gastric-content instillation. Surfactant therapy returned PVR to normal values and significantly improved both the PaO2/FiO2 ratio and the histologic injuries. Without surfactant therapy, EVLP alone did not improve graft quality.
FIGURE 5. IL-1A (A), IL-6 (B), and IL-8 (C) levels in the left lower lobes from the two sham groups and three lung-injured groups. Compared with sham animals, IL-1A, IL-6, and IL-8 levels were increased in the LI group (*P=0.0007, *P=0.011, and *P=0.0052, respectively), LI-EVLP group (*PG0.0001, *P=0.0006, and *P=0.0106, respectively), and LI-Surf-EVLP group (*P=0.0005, *P=0.0214, and *P=0.0085, respectively). IL-6 values were significantly lower in the LI-Surf-EVLP group compared with the LI-EVLP group ($P=0.0414). LI, lung injury; EVLP, ex vivo lung perfusion; Surf, surfactant.
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Aspiration is one of the main factors that can make lungs unsuitable for transplantation. Gastric-content aspiration immediately before brain death is common either during vomiting when death is related to a neurologic event or during external cardiac massage for cardiopulmonary resuscitation. Organs are evaluated for transplantation after a few hours to several days of intensive care, at a time when blood gas values are usually acceptable. Aspiration may be suspected based on clinical history, chest imaging, or flexible bronchoscopy. Gastric fluid contains hydrochloric acid, food, pepsin, and other proteases that directly damage the alveolar-capillary membrane, causing severe LI. The resulting increased membrane permeability to serum proteins causes alveolar-space flooding by protein-rich fluid (15), alveolar hemorrhage, inhibition and degradation of surfactant, and ultimately, lung atelectasis (16). Currently, donor lungs with clinical evidence of aspiration on bronchoscopy are not used for transplantation. They represent 14% of lungs declined for transplantation, but this proportion is probably severely underestimated because not all declined lungs are evaluated (17). Ex vivo lung perfusion was first developed to assess lung function in nonYheart-beating donors before transplantation (18) and was then successfully extended to the reconditioning of donor lungs that were declined for transplantation, chiefly because of lung edema with PaO2/FiO2 impairment (4, 19). These preliminary promising results prompted experimental studies to identify means of reconditioning the lungs before transplantation (20). Ex vivo lung perfusion is now the most promising technique for overcoming the shortage of donor lungs (4), as lungs that would have been unsuitable can become suitable after EVLP reconditioning. Ex vivo lung perfusion has been used to treat LI induced by gastric-content instillation in animal models (13, 14). Inci and colleagues induced LI using a betaine-HCl/pepsin mixture (13). However, the use of gastric content is more reproducible, more stable, and more closely similar to the clinical situation in humans (14). Furthermore, in both previous studies, the animals were studied immediately after lung instillation, which did not replicate clinical practice (13, 14). We conducted our assessments 24 hr after instillation. Instillation was associated with lung infection and greater histologic damage severity. In another study, preemptive steroid treatment followed by EVLP improved lung function after gastric-content instillation (21). However, the treatment of established LI is more challenging. In pigs, BAL with diluted surfactant during EVLP improved the function of transplanted injured lungs (13). In our study, exogenous surfactant was administered undiluted, in both lungs, by instillation via a flexible bronchoscope. This method allowed us to remove food debris and to deliver a large surfactant dose (100 mL/Kg) equally to both lungs, thus protecting them from circulating inflammatory cytokines potentially released by the injured parenchyma. In contrast, Inci and colleagues (13) administered the surfactant by dilute lavage in a dose of 10 mL/Kg to achieve uniform delivery to both lungs (13). Furthermore, in contrast to Inci et al., we avoided cold storage of the lungs before EVLP to focus on aspiration-induced LI by minimizing ischemia-induced LI. These differences may explain the better histologic recovery in our study despite the greater LI severity. Interestingly, unlike previous reports demonstrating that EVLP for 2 hr started 2 hr after lung instillation of gastric
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juice did not aggravate LI (14), we found that EVLP without surfactant worsened the LI. The use of homologous blood cells in the perfusate highlighted this aggravation in the shape of parenchymal hemorrhage (Fig. 3). This difference is probably ascribable to greater alveolar-capillary membrane deterioration 24 hr compared with 2 hr after aspiration. In keeping with this possibility, the diagnosis of aspiration in clinical practice is difficult early on but becomes much easier after 24 hr (22). Furthermore, our results highlight the key role for surfactant in the repair of aspiration-induced LI and the deleterious effect of EVLP. Whether surfactant instillation alone attenuates LI should be investigated. Our findings are consistent with a recent study showing improved function of acid-injured lungs treated with surfactant instillation immediately before transplantation (23). The proinflammatory cytokines IL-1>, IL-6, and IL8 were increased in injured lung tissue, consistent with the elevated levels of these cytokines in BAL fluid in an earlier study (13). In neither study did the cytokine levels decrease significantly with surfactant therapy, despite the clinical and histologic improvements (13). The mechanisms of cytokine elevation in aspiration-induced LI are unclear. In mouse lungs with ventilator-induced injury, inflammatory mediators were identified in the lung perfusate, which induced lung dysfunction when injected into normal lungs using an isolated perfused lung technique (24). This finding may explain the lung function impairments seen after reperfusion, despite the focal nature of the initial aspiration-induced LI, as well as the deterioration in injured lung function in our study after 4 hr of EVLP. The sustained elevations in inflammatory cytokines and neutrophils in BAL fluids despite surfactant therapy in the study by Inci and colleagues (13) and in the LLL tissue in the present study suggest a palliative effect of surfactant as opposed to a reparative effect on the alveolo-capillary membrane. However, surfactant therapy may prevent further deterioration of the membrane during reperfusion. In injured lung tissue, surfactant therapy also failed to decrease the levels of inflammatory mediators, despite improvements in lung function. These results argue against an active role for these mediators and are consistent with previous results showing no correlation between mediator levels and dysfunction severity (20, 25). Ex vivo lung perfusion is an important advance in lung transplantation. Although EVLP alone failed to improve LI because of gastric-acid aspiration, it may be useful for evaluating doubtful lungs (e.g., features of Mendelson’s syndrome without purulent secretions), identifying lungs unsuitable for transplantation, and testing treatments. Surfactant instillation combined with EVLP improved PaO2 and histology. Thus, surfactant therapy of marginal donor lungs may hold promise for increasing the number of lungs suitable for transplantation.
MATERIALS AND METHODS The study was approved by the Animal Care and Use Committee of the Paris Sud University.
Study Groups Thirty large white piglets weighing 23T2 kg were allocated at random to five groups of six animals each. Lung injury was induced by selectively infusing 1 mL/kg of autologous gastric content into the LLL under sterile bronchoscopic control. The lungs were harvested 24 hr later and studied (LI group) or subjected to either EVLP alone for 4 hr (LI-EVLP group) or surfactant lavage
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Khalife´-Hocquemiller et al.
* 2014 Lippincott Williams & Wilkins
of the LLL followed immediately by EVLP for 4 hr (LI-Surf-EVLPgroup). These three LI groups were compared with two sham groups, in which saline was infused into the LLL instead of gastric juice; 24 hr later the lungs were studied in one group (sham group), whereas in the other, EVLP was given for 4 hr (sham-EVLP group). The EVLP circuit was prepared before harvesting to avoid lung ischemia. As soon as the lungs were connected to the circuit, surfactant was administered before starting EVLP.
Lung Aspiration Injury The animals were fed normally on the day before the study to ensure replication of clinical conditions. Under general anesthesia, after endotracheal intubation, a gastric tube was inserted and connected to a 50-mL syringe for gastric-content aspiration. The pH of the gastric juice was assessed and titrated to less than 4.0. The gastric tube was removed, and a sterile flexible bronchoscope was inserted for bronchial tree evaluation. The bronchoscope was then introduced selectively into the LLL and used for BAL followed by selective infusion of 1 mL/kg of autologous gastric content into the lower subsegmental branches of the LLL bronchus. In the sham groups, normal saline was used. Then, the animals were allowed to recover.
Preparation for EVLP Heart-lung block harvesting was performed as described elsewhere (13). Autologous blood was collected (about 700 mL) to be added to the priming solution. A cannula with a lateral hole allowing pressure monitoring was sewn onto the PA trunk, and the lungs were flushed in situ with 1 L cold (+4-C) extracellular Celsior solution (Genzyme, Limoges, France) in the antegrade direction through the pulmonary arterial trunk. Retrograde flush was performed through the pulmonary veins. The heart-lung block was removed with the lungs inflated, following a standardized procedure. A cuffed endotracheal tube measuring 6.5 to 7.0 mm in inner diameter (Portex tracheal tube, Smiths, Hythe, Kent, UK) was inserted into the trachea and secured firmly using umbilical tape. A similar cannula was inserted through the left ventricle into the left atrium and secured by a purse suture around the inlet port. As soon as the heart-lung block was harvested and prepared, it was connected to the EVLP machine to avoid warm ischemia.
EVLP Circuit (Sorin Group, Clamart, France) All procedures were done under sterile conditions as previously described (13). Ex vivo lung perfusion duration was 4 hr. Circuit priming was initiated with 1 L of acellular normothermic perfusate (Steen Solution, Vitrolife AB, Kungsbacka, Sweden). Then, donor blood was added to obtain 10% to 20% hematocrit after cell-saver washing (Dideco, Electa concept, Sorins, Italy). We added 10 IU of insulin and 10 000 IU of sodium heparin. After deairing of the circuit, EVLP was started at low flow (100 mL/min) in normothermia. Ex vivo lung perfusion output was adjusted to maintain pulmonary artery pressure (PAP) between 15 and 20 mm Hg. Left atrial pressure was kept between 0 and 2 mm Hg by adjusting the height of the reservoir. Then, the deoxygenation system was connected and adapted to PaCO2.
centrifuged (1500/min, +4-C, 3 min), and the supernatant was stained with hematoxylin-eosin. At the end of the experiments, biopsy specimens weighing 300 to 500 mg were taken from the left and right lower lobes, from the same predefined areas, regardless of gross appearance. Fixed lung sections were processed using standard histologic techniques and embedded in paraffin. Acute LI was scored by a pathologist (P.D.), who was blinded to group assignment and used a semiquantitative damage scale based on hemorrhage, edema, atelectasis, and neutrophil and macrophage infiltration in air-spaces or vessel walls, as described by Mikawa and colleagues (26); as well as on necrosis and lymphocyte infiltration. Each of these features was scored from 0 to 4, and the item scores were then summed to obtain the damage severity score. Apoptotic cells were detected using the DeadEnd colorimetric TUNEL system kit (Promega, Madison, WI, USA) on the LLL as specified by the manufacturer. Stained cells were counted by evaluating 20 fields per sample, without knowledge of group assignment, and the mean of the 20 values was computed. The mean number of apoptotic cells per field was computed. The wet-to-dry ratio was determined for LLL and RLL samples harvested at the same level. Lung tissue samples were taken for assays of proinflammatory cytokines (IL-1A, TNF>, IL-6, IL-8, and IFNF). The samples were homogenized, sonicated, and centrifuged. Supernatants were evaluated in duplicate using specific enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. Protein content was determined using Bradford’s method. The final cytokine levels were expressed as pg/mL/Kg total protein.
Statistical Analysis Statistical analyses were performed using Statview Software version 5 (Abacus Concept, Berkeley, CA). Data were described as meanTSD. Values obtained at four time points during 4 hr of ex vivo evaluation were compared across groups using repeated-measures analysis of variance followed by Fisher’s test. The paired t test was used to compare prereperfusion and postreperfusion samples. For histologic scores, the nonparametric MannWhitney test was used. PG0.05 was considered significant.
REFERENCES 1.
2. 3. 4. 5.
6.
Surfactant Administration Immediately before lung perfusion initiation by the EVLP pump, undiluted exogenous surfactant (Curosurf, Chiesi Farmaceutici, Parma, Italy) in a dose of 100 mg/kg was instilled into both lungs in equal amounts via a flexible bronchoscope introduced down to each main bronchus.
7.
Measurements
9.
Hemodynamic variables and blood gases were measured before gastriccontent instillation then 1 and 24 hr later. During EVLP, PAP, pulmonary artery flow, and left atrial pressure (LAP) were monitored continuously, and blood gases were measured hourly. Pulmonary vascular resistance was calculated as PAP-LAP/flow. Leukocyte counts were determined before gastric-content instillation then 1 and 24 hr later. Bronchoalveolar lavage was performed by injecting 20 mL of normal saline solution via a flexible bronchoscope introduced into the LLL before and 24 hr after LI induction. For each BAL, the aspirated residue was
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Christie JD, Edwards LB, Kucheryavaya AY, et al. International Society of Heart and Lung Transplantation. The Registry of the International Society for Heart and Lung Transplantation: 29th adult lung and heartlung transplant report-2012. J Heart Lung Transplant 2012; 31: 1073. Aigner C, Winkler G, Jaksch P, et al. Extended donor criteria for lung transplantationVa clinical reality. Eur J Cardiothorac Surg 2005; 27: 757. Van Raemdonck D, Neyrinck A, Verleden GM, et al. Lung donor selection and management. Proc Am Thorac Soc 2009; 6: 28. Cypel M, Yeung JC, Liu M, et al. Normothermic ex vivo lung perfusion in clinical lung transplantation. N Engl J Med 2011; 364: 1431. Roman MA, Nair S, Tsui S, Dunning J, Parmar JS. Ex vivo lung perfusion: a comprehensive review of the development and exploration of future trends. Transplantation 2013; 96: 509. Downing TE, Sporn TA, Bollinger RR, et al. Pulmonary histopathology in an experimental model of chronic aspiration is independent of acidity. Exp Biol Med (Maywood) 2008; 233: 1202. Ingemansson R, Eyjolfsson A, Mared L, et al. Clinical transplantation of initially rejected donor lungs after reconditioning ex vivo. Ann Thorac Surg 2009; 87: 255. Cypel M, Yeung JC, Hirayama S, et al. Technique for prolonged normothermic ex vivo lung perfusion. J Heart Lung Transplant 2008; 27: 1319. Wierup P, Haraldsson A, Nilsson F, et al. Ex vivo evaluation of nonacceptable donor lungs. Ann Thorac Surg 2006; 81: 460. Cypel M, Rubacha M, Yeung J, et al. Normothermic ex vivo perfusion prevents lung injury compared to extended cold preservation for transplantation. Am J Transplant 2009; 9: 2262. Khalife´-Hocquemiller T, Sage E, Dorfmuller P, et al. Ex vivo perfusion worsened lung injuries induced by gastric acid aspiration in pigs. J Heart lung Transplant 2011; 30: S135. Taut FJ, Rippin G, Schenk P, et al. A Search for subgroups of patients with ARDS who may benefit from surfactant replacement therapy: a pooled analysis of five studies with recombinant surfactant protein-C surfactant (Venticute). Chest 2008; 134: 724.
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Inci I, Ampollini L, Arni S, et al. Ex vivo reconditioning of marginal donor lungs injured by acid aspiration. J Heart Lung Transplant 2008; 27: 1229. Meers CM, Tsagkaropoulos S, Wauters S, et al. A model of ex vivo perfusion of porcine donor lungs injured by gastric aspiration: a step towards pretransplant reconditioning. J Surg Res 2011; 170: 159. Hermon MM, Wassermann E, Pfeiler C, et al. Early mechanical ventilation is deleterious after aspiration-induced lung injury in rabbits. Shock 2005; 23: 59. Brackenbury AM, Puligandla PS, McCaig LA, et al. Evaluation of exogenous surfactant in HCL-induced lung injury. Am J Respir Crit Care Med 2001; 163: 1135. Meers C, Van Raemdonck D, Verleden GM, et al. The number of lung transplants can be safely doubled using extended criteria donors; A single-center review. Transpl Int 2010; 23: 628. Steen S, Liao Q, Wierup PN, et al. Transplantation of lungs from nonheart-beating donors after functional assessment ex vivo. Ann Thorac Surg 2003; 76: 244. Steen S, Ingemansson R, Eriksson L, et al. First human transplantation of a nonacceptable donor lung after reconditioning ex vivo. Ann Thorac surg 2007; 83: 2191.
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Sanchez PG, Bittle GJ, Burdorf L, et al. State of art: clinical ex vivo lung perfusion: rationale, current status, and future directions. J Heart Lung Transplant 2012; 31: 339. Meers CM, Wauters S, Verbeken E, et al. Preemptive therapy with steroids but not macrolides improves gas exchange in caustic-injured donor lungs. J Surg Res 2011; 170: 141. Dargaville PA, Mills JF, Headley BM, et al. Therapeutic lung lavage in the piglet model of meconium aspiration syndrome. Am J Respir Crit Care Med 2003; 168: 456. Inci I, Hillinger S, Arni S, et al. Surfactant improves graft function after gastric acid-induced lung damage in lung transplantation. Ann Thorac Surg 2013; 95: 1013. Jaecklin T, Engelberts D, Otulakowski G, et al. Lung-derived soluble mediators are pathogenic in ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2011; 300: 648. Sadaria MR, Smith PD, Fullerton DA, et al. Cytokine expression profile in human lungs undergoing normothermic ex-vivo lung perfusion. Ann Thorac Surg 2011; 92: 478. Mikawa K, Nishina K, Maekawa N, et al. Effect of lidocaine pretreatment on endotoxin-induced lung injury in rabbits. Anesthesiology 1994; 81: 689.
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AND
EXPERIMENTAL RESEARCH
Exogenous Surfactant Attenuates Lung Injury From Gastric-Acid Aspiration During Ex Vivo Reconditioning in Pigs Theresa Khalife´-Hocquemiller,1 Edouard Sage,1 Peter Dorfmuller,1 Sacha Mussot,1 Daniel Le Houe´rou,1 Saadia Eddahibi,1 and Elie Fadel1,2 Background. Lung injury (LI) due to gastric-acid aspiration is associated with poor posttransplantation outcomes. We investigated the effects of ex vivo lung perfusion (EVLP) reconditioning and surfactant administration on LI due to gastric-acid aspiration. Methods. Thirty piglets were allocated at random to five groups: the lungs were studied 24 hr after gastric juiceYinduced LI of the left lower lobe (LLL), LI followed by EVLP (4 hr), or LI followed by LLL surfactant lavage immediately before EVLP; sham animals were studied 24 hr after saline infusion alone or followed by EVLP. Gross anatomy, hemodynamics, and aerodynamics were evaluated; neutrophil and bacterial counts were determined in bronchoalveolar lavage (BAL) fluid and blood. LLLs were evaluated based on a semi-quantitative histologic score, apoptotic cell death (TUNEL), and inflammatory cytokine levels. Results. The sham and sham-EVLP groups were not significantly different. Compared with sham, LI animals had irreversible atelectasis, higher lung infection rates (PG0.0001) and BAL neutrophil percentages (PG0.0001), lower PaO2 (P=0.0006), higher IL-1 (P=0.022) and IL-8 (P=0.006), higher apoptotic cell percentages (P=0.007), and worse histologic severity scores (PG0.0001). EVLP alone did not improve these findings. Adding surfactant before EVLP returned PaO2, pulmonary vascular resistance, and apoptotic-cell percentage to sham-EVLP values but only partially improved the histologic severity score. Conclusion. Local surfactant infusion immediately before EVLP improved the function of donor lungs injured by gastric juice aspiration. This strategy may hold promise for decreasing the shortage of donor lungs. Keywords: EVLP, Lung injury, Lung aspiration. (Transplantation 2014;97: 413Y418)
ung transplantation is a well-established treatment for patients with end-stage respiratory failure, and the numbers of lung transplant procedures and candidates have increased steadily over the last three decades (1). Although the lung donor pool has been successfully expanded by the use of marginal donors in the past 10 years (2, 3), the demand for lungs far exceeds the supply (4). Currently, 15% to
L
This study was funded by the nonprofit donation-funded organization ADETEC. The authors declare no conflicts of interest. 1 Laboratoire de Recherche Chirurgicale and INSERM U999, Hoˆpital Marie Lannelongue, Universite´ Paris Sud, Le Plessis Robinson, France. 2 Address correspondence to: Elie Fadel M.D., Department of Thoracic and Vascular Surgery and Heart-Lung Transplantation, Hoˆpital MarieLannelongue (Paris-Sud University), 133 Avenue de la Resistance, 92350 Le Plessis Robinson, France. E-mail: [email protected] T.K.-H., S.M., and E.F. participated in designing the study and writing the manuscript. E.S., P.D., D.L.H., and S.E. participated in designing and executing the study. Received 16 August 2013. Revision requested 12 September 2013. Accepted 18 November 2013. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9704-413 DOI: 10.1097/01.TP.0000441320.10787.c5
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20% of lungs from multiorgan donors are deemed suitable for lung transplantation (5). Gastric content aspiration is associated with increased risks of lung transplant infection and dysfunction. Lung injury (LI) from gastric content aspiration is related to both chemical damage and obstruction by food particles (6) and is a common reason for lungs being considered unsuitable for transplantation. Ex vivo lung perfusion (EVLP) is a promising technique for rendering injured donor lungs suitable for transplantation (7Y9). Preclinical studies of animal and human lungs have established that lung function can be safely preserved by EVLP for as long as 12 hr (8, 10). This time can be used not only to prevent lung damage but also to manage LI with the goal of making the lungs suitable for transplantation. However, we previously reported that reconditioning with EVLP alone failed to improve LI due to gastric acid aspiration in pigs (11). Surfactant is a phospholipid and protein layer produced by type II alveolar cells. Surfactant lowers surface tension, thereby preventing alveolar collapse. Surfactant inactivation is involved in ischemia/reperfusion injury, one of the main causes of primary graft dysfunction. Exogenous surfactant is used to treat hyaline membrane disease in children and www.transplantjournal.com
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FIGURE 1. PaO2/FiO2 ratio in the three groups treated with ex vivo lung perfusion (EVLP): lung injury induced by gastric-content instillation (LI-EVLP), sham group given saline instillation (Sham-EVLP), and lung injury followed by both surfactant instillation and EVLP (LI-Surf-EVLP). During EVLP, repeated measures showed a significant PaO2/FiO2 decrease (PG0.0001) in the LI-EVLP group compared with the sham-EVLP group. In the LI-Surf-EVLP group, PaO2/ FiO2 improved significantly compared with the LI-EVLP group (PG0.0001) but remained lower than sham-EVLP values (P=0.0145).
increases pulmonary oxygenation in acute respiratory distress syndrome due to direct LI (12). In a pig model, EVLP with surfactant lavage improved lung function after acid aspiration (13). However, the surfactant was given immediately after acid instillation, before LI development. In practice, aspirationinduced LI in multiorgan donors is diagnosed at the stage of established radiologic and histologic LI. Here, our objective was to determine whether surfactant lavage immediately before starting EVLP improved established LI induced 24 hr earlier by gastric acid instillation. To this end, we conducted a study in a preclinical pig model.
RESULTS Compared with the sham animals, LI animals studied 24 hr after gastric content instillation into the left lower lobe
FIGURE 2. Pulmonary vascular resistance in the three groups treated with ex vivo lung perfusion (EVLP): lung injury induced by gastric-content instillation (LI-EVLP), sham group given saline instillation (Sham-EVLP), and lung injury followed by both surfactant instillation and EVLP (LISurf-EVLP). During EVLP, repeated measures showed a significant PVR increase (P=0.0002) in the LI-EVLP group compared with the Sham-EVLP group. In the LI-Surf-EVLP group, PVR returned to sham values.
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(LLL) as previously described by Meer and colleagues (14) had similar hemodynamic variables and PaO2/FiO2 ratio values but significantly higher blood neutrophil counts (34 750T10 474/mm3 vs. 19 880T3641/mm3; P=0.0199) and bronchoalveolar lavage (BAL) neutrophil percentages (65% vs. 11%; PG0.0001). BAL cultures were negative in the sham animals and positive (9105 bacteria/mL) in 66% of the LI animals (PG0.0001). Repeated-measures analysis of variance was performed to compare four EVLP time points: before starting EVLP (24 hr after LI induction) then 1, 3, and 4 hr after starting EVLP. At the 4-hr time point, compared with the sham-EVLP group (saline instillation followed by EVLP), the LI-EVLP group (gastric acid instillation followed by EVLP) had significantly higher pulmonary vascular resistance (PVR) (P=0.0002) and significantly lower PaO2/FiO2 (PG0.0001). At the same 4-hr time point, the LI-Surf-EVLP group (gastric acid instillation followed by surfactant administration and EVLP) showed a return of PVR to sham-EVLP values and a significant improvement in PaO2/FiO2 compared with the LI-EVLP group (PG0.0001). However, PaO2/FiO2 values in the LI-SurfEVLP group remained lower than in the sham-EVLP group (P=0.0145) (Figs. 1 and 2). The percentage of neutrophils in BAL fluid after 4 hr of EVLP remained elevated in the LI-EVLP group compared with the sham-EVLP group (69% vs. 5%; P=0.0065). After surfactant lavage, the BAL neutrophil percentage remained elevated, similar to that in the LI-EVLP group (57% vs. 69%; P=NS) and higher than in the sham-EVLP group (P=0.0047). BAL-fluid bacteriologic cultures were negative in both sham groups and positive (9105) in 57% of LI animals and 60% of LI-EVLP animals (PG0.0001). Surfactant lavage failed to decrease the BAL infection rate (51%; P=NS). Bacteria found in BAL were Escherichia coli, Pseudomonas, and Aeromonas. On gross anatomy, LLLs were similar in the two sham groups. All right lower lobes (RLLs) were normal. The LLLs in the LI group showed a collapsed appearance that was not reversible by manual ventilation. After 4 hr of EVLP, the collapse was exacerbated, and both hemorrhage and edema were increased (Fig. 3).
FIGURE 3. Gross appearance of the left lower lobe in the LI-EVLP group.
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FIGURE 4. Semi-quantitative histologic damage severity score determined in the left lower lobe by an observer blinded to group assignment in the two sham groups and three lung-injured groups. *Sham versus LI, PG0.0001; U, LISurf-EVLP versus LI-EVLP, PG0.0001; $, LI-Surf-EVLP versus sham, PG0.0001. LI, lung injury; EVLP, ex vivo lung perfusion; Surf, surfactant.
The wet-to-dry ratio showed no significant differences across groups. The histologic injury severity scores were not significantly different across RLLs or between LLLs in the two sham groups (1.000T1.225 vs. 2.600T1.140, P=0.346). Compared with the sham group, the LI group had a significantly higher score (15.429T4.791 vs. 1.000T1.225; PG0.0001), and the score value did not improve with EVLP (12.400T2.408, P=0.061). Surfactant instillation immediately before EVLP was
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associated with significantly lower scores compared with EVLP alone (8.300T1.337 vs. 15.429T4.791; PG0.0001) (Fig. 4). In RLLs, the number of apoptotic cells per field as assessed by TUNEL was similar in all groups. In LLLs, there was a nonsignificant trend toward higher apoptotic cell counts in the LI group compared with the sham group (4.4 vs. 2.2, P=0.067). EVLP had no effect in sham animals (2.2 in the sham-EVLP group vs. 2.2 in the sham group; P=NS) but significantly increased the apoptotic cell count in injured lungs (LI-EVLP group, 8.2 vs. 4.4 in the LI group; P=0.004). Surfactant instillation (LI-Surf-EVLP group) decreased the apoptotic cell counts (3.3 vs. 8.2 in the LI-EVLP group; PG0.0001). Similarly, cytokine levels were comparable in all RLLs. In LLLs (Fig. 5), IL-10, INFF and TNF> levels were similar in all groups; whereas, compared with the sham group, IL-1A, IL-6, and IL-8 levels were increased in the LI group (P=0.0007, P=0.011, and P=0.0052, respectively), LI-EVLP group (PG0.0001, P=0.0006, and P=0.0106, respectively), and LI-SurfEVLP group (P=0.0005, P=0.0214, and P=0.0085, respectively). Only IL-6 values were significantly lower in the LI Surf-EVLP group compared with the LI-EVLP group (P=0.0414).
DISCUSSION In this study, exogenous surfactant therapy by direct instillation via a flexible bronchoscope followed by 4 hr of EVLP attenuated the lung damage present 24 hr after gastric-content instillation. Surfactant therapy returned PVR to normal values and significantly improved both the PaO2/FiO2 ratio and the histologic injuries. Without surfactant therapy, EVLP alone did not improve graft quality.
FIGURE 5. IL-1A (A), IL-6 (B), and IL-8 (C) levels in the left lower lobes from the two sham groups and three lung-injured groups. Compared with sham animals, IL-1A, IL-6, and IL-8 levels were increased in the LI group (*P=0.0007, *P=0.011, and *P=0.0052, respectively), LI-EVLP group (*PG0.0001, *P=0.0006, and *P=0.0106, respectively), and LI-Surf-EVLP group (*P=0.0005, *P=0.0214, and *P=0.0085, respectively). IL-6 values were significantly lower in the LI-Surf-EVLP group compared with the LI-EVLP group ($P=0.0414). LI, lung injury; EVLP, ex vivo lung perfusion; Surf, surfactant.
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Aspiration is one of the main factors that can make lungs unsuitable for transplantation. Gastric-content aspiration immediately before brain death is common either during vomiting when death is related to a neurologic event or during external cardiac massage for cardiopulmonary resuscitation. Organs are evaluated for transplantation after a few hours to several days of intensive care, at a time when blood gas values are usually acceptable. Aspiration may be suspected based on clinical history, chest imaging, or flexible bronchoscopy. Gastric fluid contains hydrochloric acid, food, pepsin, and other proteases that directly damage the alveolar-capillary membrane, causing severe LI. The resulting increased membrane permeability to serum proteins causes alveolar-space flooding by protein-rich fluid (15), alveolar hemorrhage, inhibition and degradation of surfactant, and ultimately, lung atelectasis (16). Currently, donor lungs with clinical evidence of aspiration on bronchoscopy are not used for transplantation. They represent 14% of lungs declined for transplantation, but this proportion is probably severely underestimated because not all declined lungs are evaluated (17). Ex vivo lung perfusion was first developed to assess lung function in nonYheart-beating donors before transplantation (18) and was then successfully extended to the reconditioning of donor lungs that were declined for transplantation, chiefly because of lung edema with PaO2/FiO2 impairment (4, 19). These preliminary promising results prompted experimental studies to identify means of reconditioning the lungs before transplantation (20). Ex vivo lung perfusion is now the most promising technique for overcoming the shortage of donor lungs (4), as lungs that would have been unsuitable can become suitable after EVLP reconditioning. Ex vivo lung perfusion has been used to treat LI induced by gastric-content instillation in animal models (13, 14). Inci and colleagues induced LI using a betaine-HCl/pepsin mixture (13). However, the use of gastric content is more reproducible, more stable, and more closely similar to the clinical situation in humans (14). Furthermore, in both previous studies, the animals were studied immediately after lung instillation, which did not replicate clinical practice (13, 14). We conducted our assessments 24 hr after instillation. Instillation was associated with lung infection and greater histologic damage severity. In another study, preemptive steroid treatment followed by EVLP improved lung function after gastric-content instillation (21). However, the treatment of established LI is more challenging. In pigs, BAL with diluted surfactant during EVLP improved the function of transplanted injured lungs (13). In our study, exogenous surfactant was administered undiluted, in both lungs, by instillation via a flexible bronchoscope. This method allowed us to remove food debris and to deliver a large surfactant dose (100 mL/Kg) equally to both lungs, thus protecting them from circulating inflammatory cytokines potentially released by the injured parenchyma. In contrast, Inci and colleagues (13) administered the surfactant by dilute lavage in a dose of 10 mL/Kg to achieve uniform delivery to both lungs (13). Furthermore, in contrast to Inci et al., we avoided cold storage of the lungs before EVLP to focus on aspiration-induced LI by minimizing ischemia-induced LI. These differences may explain the better histologic recovery in our study despite the greater LI severity. Interestingly, unlike previous reports demonstrating that EVLP for 2 hr started 2 hr after lung instillation of gastric
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juice did not aggravate LI (14), we found that EVLP without surfactant worsened the LI. The use of homologous blood cells in the perfusate highlighted this aggravation in the shape of parenchymal hemorrhage (Fig. 3). This difference is probably ascribable to greater alveolar-capillary membrane deterioration 24 hr compared with 2 hr after aspiration. In keeping with this possibility, the diagnosis of aspiration in clinical practice is difficult early on but becomes much easier after 24 hr (22). Furthermore, our results highlight the key role for surfactant in the repair of aspiration-induced LI and the deleterious effect of EVLP. Whether surfactant instillation alone attenuates LI should be investigated. Our findings are consistent with a recent study showing improved function of acid-injured lungs treated with surfactant instillation immediately before transplantation (23). The proinflammatory cytokines IL-1>, IL-6, and IL8 were increased in injured lung tissue, consistent with the elevated levels of these cytokines in BAL fluid in an earlier study (13). In neither study did the cytokine levels decrease significantly with surfactant therapy, despite the clinical and histologic improvements (13). The mechanisms of cytokine elevation in aspiration-induced LI are unclear. In mouse lungs with ventilator-induced injury, inflammatory mediators were identified in the lung perfusate, which induced lung dysfunction when injected into normal lungs using an isolated perfused lung technique (24). This finding may explain the lung function impairments seen after reperfusion, despite the focal nature of the initial aspiration-induced LI, as well as the deterioration in injured lung function in our study after 4 hr of EVLP. The sustained elevations in inflammatory cytokines and neutrophils in BAL fluids despite surfactant therapy in the study by Inci and colleagues (13) and in the LLL tissue in the present study suggest a palliative effect of surfactant as opposed to a reparative effect on the alveolo-capillary membrane. However, surfactant therapy may prevent further deterioration of the membrane during reperfusion. In injured lung tissue, surfactant therapy also failed to decrease the levels of inflammatory mediators, despite improvements in lung function. These results argue against an active role for these mediators and are consistent with previous results showing no correlation between mediator levels and dysfunction severity (20, 25). Ex vivo lung perfusion is an important advance in lung transplantation. Although EVLP alone failed to improve LI because of gastric-acid aspiration, it may be useful for evaluating doubtful lungs (e.g., features of Mendelson’s syndrome without purulent secretions), identifying lungs unsuitable for transplantation, and testing treatments. Surfactant instillation combined with EVLP improved PaO2 and histology. Thus, surfactant therapy of marginal donor lungs may hold promise for increasing the number of lungs suitable for transplantation.
MATERIALS AND METHODS The study was approved by the Animal Care and Use Committee of the Paris Sud University.
Study Groups Thirty large white piglets weighing 23T2 kg were allocated at random to five groups of six animals each. Lung injury was induced by selectively infusing 1 mL/kg of autologous gastric content into the LLL under sterile bronchoscopic control. The lungs were harvested 24 hr later and studied (LI group) or subjected to either EVLP alone for 4 hr (LI-EVLP group) or surfactant lavage
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Khalife´-Hocquemiller et al.
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of the LLL followed immediately by EVLP for 4 hr (LI-Surf-EVLPgroup). These three LI groups were compared with two sham groups, in which saline was infused into the LLL instead of gastric juice; 24 hr later the lungs were studied in one group (sham group), whereas in the other, EVLP was given for 4 hr (sham-EVLP group). The EVLP circuit was prepared before harvesting to avoid lung ischemia. As soon as the lungs were connected to the circuit, surfactant was administered before starting EVLP.
Lung Aspiration Injury The animals were fed normally on the day before the study to ensure replication of clinical conditions. Under general anesthesia, after endotracheal intubation, a gastric tube was inserted and connected to a 50-mL syringe for gastric-content aspiration. The pH of the gastric juice was assessed and titrated to less than 4.0. The gastric tube was removed, and a sterile flexible bronchoscope was inserted for bronchial tree evaluation. The bronchoscope was then introduced selectively into the LLL and used for BAL followed by selective infusion of 1 mL/kg of autologous gastric content into the lower subsegmental branches of the LLL bronchus. In the sham groups, normal saline was used. Then, the animals were allowed to recover.
Preparation for EVLP Heart-lung block harvesting was performed as described elsewhere (13). Autologous blood was collected (about 700 mL) to be added to the priming solution. A cannula with a lateral hole allowing pressure monitoring was sewn onto the PA trunk, and the lungs were flushed in situ with 1 L cold (+4-C) extracellular Celsior solution (Genzyme, Limoges, France) in the antegrade direction through the pulmonary arterial trunk. Retrograde flush was performed through the pulmonary veins. The heart-lung block was removed with the lungs inflated, following a standardized procedure. A cuffed endotracheal tube measuring 6.5 to 7.0 mm in inner diameter (Portex tracheal tube, Smiths, Hythe, Kent, UK) was inserted into the trachea and secured firmly using umbilical tape. A similar cannula was inserted through the left ventricle into the left atrium and secured by a purse suture around the inlet port. As soon as the heart-lung block was harvested and prepared, it was connected to the EVLP machine to avoid warm ischemia.
EVLP Circuit (Sorin Group, Clamart, France) All procedures were done under sterile conditions as previously described (13). Ex vivo lung perfusion duration was 4 hr. Circuit priming was initiated with 1 L of acellular normothermic perfusate (Steen Solution, Vitrolife AB, Kungsbacka, Sweden). Then, donor blood was added to obtain 10% to 20% hematocrit after cell-saver washing (Dideco, Electa concept, Sorins, Italy). We added 10 IU of insulin and 10 000 IU of sodium heparin. After deairing of the circuit, EVLP was started at low flow (100 mL/min) in normothermia. Ex vivo lung perfusion output was adjusted to maintain pulmonary artery pressure (PAP) between 15 and 20 mm Hg. Left atrial pressure was kept between 0 and 2 mm Hg by adjusting the height of the reservoir. Then, the deoxygenation system was connected and adapted to PaCO2.
centrifuged (1500/min, +4-C, 3 min), and the supernatant was stained with hematoxylin-eosin. At the end of the experiments, biopsy specimens weighing 300 to 500 mg were taken from the left and right lower lobes, from the same predefined areas, regardless of gross appearance. Fixed lung sections were processed using standard histologic techniques and embedded in paraffin. Acute LI was scored by a pathologist (P.D.), who was blinded to group assignment and used a semiquantitative damage scale based on hemorrhage, edema, atelectasis, and neutrophil and macrophage infiltration in air-spaces or vessel walls, as described by Mikawa and colleagues (26); as well as on necrosis and lymphocyte infiltration. Each of these features was scored from 0 to 4, and the item scores were then summed to obtain the damage severity score. Apoptotic cells were detected using the DeadEnd colorimetric TUNEL system kit (Promega, Madison, WI, USA) on the LLL as specified by the manufacturer. Stained cells were counted by evaluating 20 fields per sample, without knowledge of group assignment, and the mean of the 20 values was computed. The mean number of apoptotic cells per field was computed. The wet-to-dry ratio was determined for LLL and RLL samples harvested at the same level. Lung tissue samples were taken for assays of proinflammatory cytokines (IL-1A, TNF>, IL-6, IL-8, and IFNF). The samples were homogenized, sonicated, and centrifuged. Supernatants were evaluated in duplicate using specific enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. Protein content was determined using Bradford’s method. The final cytokine levels were expressed as pg/mL/Kg total protein.
Statistical Analysis Statistical analyses were performed using Statview Software version 5 (Abacus Concept, Berkeley, CA). Data were described as meanTSD. Values obtained at four time points during 4 hr of ex vivo evaluation were compared across groups using repeated-measures analysis of variance followed by Fisher’s test. The paired t test was used to compare prereperfusion and postreperfusion samples. For histologic scores, the nonparametric MannWhitney test was used. PG0.05 was considered significant.
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2. 3. 4. 5.
6.
Surfactant Administration Immediately before lung perfusion initiation by the EVLP pump, undiluted exogenous surfactant (Curosurf, Chiesi Farmaceutici, Parma, Italy) in a dose of 100 mg/kg was instilled into both lungs in equal amounts via a flexible bronchoscope introduced down to each main bronchus.
7.
Measurements
9.
Hemodynamic variables and blood gases were measured before gastriccontent instillation then 1 and 24 hr later. During EVLP, PAP, pulmonary artery flow, and left atrial pressure (LAP) were monitored continuously, and blood gases were measured hourly. Pulmonary vascular resistance was calculated as PAP-LAP/flow. Leukocyte counts were determined before gastric-content instillation then 1 and 24 hr later. Bronchoalveolar lavage was performed by injecting 20 mL of normal saline solution via a flexible bronchoscope introduced into the LLL before and 24 hr after LI induction. For each BAL, the aspirated residue was
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