A Porcine Model to Study Ex Vivo Reconditioning of Injured Donor Lungs

Materials and Methods Male pigs (47 ± 8kg) were divided into three groups: LPS-group [LPS] ( n = 6) [instillation of lipopolysaccharides (15mg/lung)]; saline-group [SAL] ( n = 5) (50mL saline/lung); and sham-group [SHAM] ( n = 5). CT scans of the lungs were taken 17h before (T-17) and 31h after (T31) instillation. Broncho-alveolar lavage (BAL) was performed, and blood gases, hemodynamic, and aerodynamic parameters were measured at T 0 and T 50. Blood samples and temperature were taken at all time points. Pigs were sacrificed during cold pulmoplegia (T 50), and tissue samples were collected for histology. Wet lung weight was measured. Results Wet lung weight/body weight was higher in [LPS] versus [SAL] ( P < 0.05). Total BAL cells were higher in [LPS] versus [SAL] and [SHAM] at T 50 (left: P < 0.001 and P < 0.01; right: P < 0.01 and P < 0.001). More neutrophils were present in BAL of [LPS] at T 50 versus T 0 (left: P < 0.001; right: P < 0.01). [LPS] demonstrated more ground glass opacities (GGO) on CT at T 31 compared with [SAL] and [SHAM] ( P < 0.05). Histologically, more interstitial hemorrhage was observed in [LPS] versus [SAL] and [SHAM]( P < 0.01). Neutrophils in blood increased and lymphocytes decreased in [LPS] versus [SAL] ( P < 0.05). No differences were observed in hemodynamic and aerodynamic parameters and in saturation between groups at T 50. Conclusions LPS instillation caused inflammation with more cells in BAL, changes on CT, and histology. However, no physiologic changes occurred. Key Words lung transplantation lung donor shortage ex vivo lung perfusion lung injury Introduction Lung transplantation has become the standard treatment for well selected patients with end-stage lung failure. The number of lung transplantations performed worldwide increases each year with more patients referred as a result of a better post-transplant outcome [1] . The number of patients on the waiting list is therefore increasing just like waiting time and deaths on the waiting list [2] . Beside better donor management, many alternatives have been proposed to attenuate the problem of donor shortage, such as the use of non-heart-beating donors, extended criteria donors, and living (related) donors [3–11] . With standard criteria, only 15%–25% of cadaveric donors are suitable as lung donors [12, 13] . In a Californian analysis, more than 85% of the lungs were excluded [12] . Infection, aspiration, and sepsis were the reasons for rejection of the lungs in 24%. A recent study from our group demonstrated an increase in donor lung acceptance rate from 7% to 34% resulting from relaxing our lung donor criteria since the year 2000 [14] . Yet, abnormalities on chest X-ray remain an important reason for declining lungs in 30% of our donors. If the quality of donor lungs currently rejected could be improved after recovery prior to transplantation, more recipients could potentially be helped. Ex vivo reperfusion has been described as a technique to evaluate lungs after retrieval [15–18] . It is also hoped that donor lungs of inferior quality may be resuscitated with this novel technique prior to transplantation [19, 20] . A lung injury model is needed to further investigate mechanisms to improve injured lungs during ex vivo reperfusion. Endotoxins, which are glycolipids of the outer membrane of Gram-negative bacteria, were chosen to create an injury model with inflammation but without the risk of infection. To avoid systemic responses, the intra-tracheal way was used for administration of the toxic agent. The aim of the study was to develop a valid porcine lung injury model to investigate mechanisms for improvement of damaged donor lungs by ex vivo reperfusion prior to transplantation. Materials And Methods Animal Preparation All animals received human care in compliance with The Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 86-23, revised 1996). The study was approved by the Ethical Committee for Animal Research at the Katholieke Universiteit Leuven (P07 117). Anesthesia was induced with an intramuscular injection (0.2 mL/kg) of a mixture of zolazepam/tiletamine [Zoletil 100 (5 mL); Virbac s.a., Carros, France] and xylazine [Xyl-M 2% (100 mL); V.M.D.nv/sa, Arendonck, Belgium]. Animals were intubated with a 7.5 mm (internal diameter) cuffed endotracheal tube (Hi-contour; Mallinckrodt Medical, Athlone, Ireland), and anesthesia was maintained with 1% isoflurane (Forene; Abbott laboratories Ltd., Queensborough, Kent, UK). They were ventilated with a volume-controlled ventilator (Titus; Dräger, Lübeck, Germany) at a tidal volume of 8-10 mL/kg body weight with an inspiratory oxygen fraction (FiO 2 ) of 0.5 and a positive end-expiratory pressure (PEEP) of 5 cmH 2 O. Respiratory rate (RR) was adjusted to keep the end-tidal CO 2 between 35 and 45 mmHg. Experimental Groups Sixteen specific pathogen free pigs (47 ± 8 kg) were randomly assigned to three groups. General parameters, such as body weight and time intervals, were compared between the groups. The time outline of the experiments is shown in Fig. 1 . This time line was chosen for practical reasons as the CT scans could only be taken at certain time points. The time of sacrifice was chosen arbitrarily. In the study-group [LPS], 15 mg LPS (lipopolysaccharides from Escherichia coli 055:B5; Sigma-Aldrich, Bornem, Belgium) dissolved in 50 mL saline was instilled at T 0. In the placebo group [SAL], 50 mL saline solution was administered. In both groups, the instillation was carried out by means of a bronchoscope (FB 18BS, Pentax, Aartselaar, Belgium). In the sham-group [SHAM], only bronchoscopy was performed. A saline group was chosen to identify the possible impact of instillation of the carrier. The sham group was added to study the possible effect of bronchoscopy itself. At the moment of instillation, anesthetized animals were placed in the supine position. In [LPS] and in [SAL], 50 mL of the solution was delivered to each lung. The volume was equally administered in five different bronchi (10 mL each) ( Fig. 2 ). Thereafter, the animals were extubated and transferred to their cage for recovery. Respiration and Temperature At T 17, T 0, T 31, and T 50, respiratory rate of the animal was evaluated and rectal temperature was measured. Hemodynamic and Aerodynamic Parameters Several hemodynamic parameters were measured at T 0 [heart rate (HR)] and T 50 [HR, cardiac output (CO), pulmonary artery pressure (PAP), pulmonary vascular resistance (PVR), and mean arterial pressure (MAP)], as well as aerodynamic parameters [peak and plateau airway pressures (Ppeak, Pplat), dynamic lung compliance, and airway resistance (Raw)] at both time points. Blood Samples and Blood Smears Blood samples were taken at T 0 and T 50 to assess PO 2 /FiO 2 ratio. Serum was collected at T 17, T 0, T 31, and T 50 for measurement of C-reactive protein with a species specific acute phase protein assay (Tridelta Development Ltd., County Kildare, Ireland). At the same time points, blood smears were made to count white blood cells. Broncho-Alveolar Lavage (BAL) BAL with 2 × 30 mL saline solution was performed at T 0 and T 50 in the lingula of the left and in the middle lobe of the right lung. Returned fractions were mixed and centrifuged (3500 rpm, 10 min, 4 °C). The cell pellet was used for total and differential cell counts. Cell pellets were re-suspended in 1 mL saline for total cell count. Cells were stained with 0.4% trypan blue solution (Sigma-Aldrich, Bornem, Belgium) and counted in triplicate using a Bürker chamber (Superior; Marienfeld, Germany). A cytospin was made in a Shandon cytocentrifuge (Techgen, Zellik, Belgium) and stained with Diff-Quick (Dade Behring, Newark, NJ) to perform differential cell counts on at least 300 cells. CT-Scan Seventeen hours before (T 17) and 31 h (T 31) after the instillation, a chest CT-scan was taken in pigs sedated with an intramuscular injection (0.2 mL/kg) of zolazepam/tiletamine (Zoletil) and xylazine (Xyl-M). Scans were taken in both the ventral and dorsal position to exclude ground-glass opacities (GGO) attributable to physiologic fluid in the dependent parts of the lungs. All scans were evaluated and scored by a radiologist (WDW) blinded to the study protocol. Axial millimeter slices were performed and axial 5 mm reconstruction, both in lung window setting, was created and evaluated. Abnormalities on CT as GGO and consolidation were evaluated (global assessment). For more detailed assessment 10 mm slices (at five different levels, in left and right lung) were scored in each pig. A percentage of GGO/total lung tissue was calculated per lung and per slice. Lung Retrieval After animal preparation and intubation BAL was performed (T 50). Subsequently, anesthesia was maintained with 1% isoflurane, and animals were paralyzed with intermittent boli of 2 mg pancuronium bromide (Pavulon; N.V. Organon, Oss, The Netherlands). The neck was explored to expose the internal jugular vein and common carotid artery; 14 G catheters (Secalon T; Becton Dickinson Ltd., Singapore, Philippines) were inserted in the vein and artery to measure the central venous pressure (CVP) and mean arterial pressure (MAP), respectively. A 7.5 F pulmonary artery thermodilution catheter (Swan-Ganz; Edwards Lifesciences LLC, Irvine, CA) was inserted through the right external jugular vein to measure PAP and was connected to a cardiac output computer (Com-1; American Edwards Laboratories, Irvine, CA). The monitor was connected to a computer and hemodynamic and respiratory parameters were recorded with Datex-Ohmeda S/5 Collect (Datex-Ohmeda, Helsinki, Finland). Subsequently, a median sternotomy was performed and the thorax was entered. After removal of the thymus, the pleural cavities and pericardium were opened. Ligatures were placed around the cranial and caudal veins and the pulmonary artery trunk. Thereafter, sodium heparin 10,000 IU (heparine Rorer, 5000 IU/mL; Rhône-Poulene Rorer, Brussels, Belgium) was administered. A 24-Fr catheter (DLP Inc., Grand Rapids, MI) was inserted in the main pulmonary artery and secured with a purse-string in the right ventricle. All ligatures were tightened, and lungs were flushed in an anterograde way with a cold saline solution (50–100 mL/kg). Immediately after the start of pulmonary flush, left and right appendage were incised for venting. Thereafter, the lungs were excised and weighed. Tissue samples were taken, and lungs were dried in an oven (100 °C) for 48 h. Histology A piece of central and peripheral part of each lung was excised systematically in the same area and stored in 6% buffered formalin solution. Tissue samples were embedded in paraffin. Sections were prepared and stained with hematoxylin-eosin for histological analysis. A scoring system was developed by the pathologist (EV) blinded to the study protocol. A score was given between 0 (none) and 5 (severe) for three parameters (hemorrhage, edema, and inflammation) for each lung. Also, the extension of alveolar damage was scored from 0 (0% damage) to 5 (100% damage). The scores for left lung, right lung, and averages for left and right lung were compared between the groups. Statistics Data analysis was performed with GraphPad Prism 4 (San Diego, CA). One way analysis of variance (ANOVA) test was used to compare all groups. Differences between two study groups were analyzed using an unpaired t -test. Differences within each group at different time points were tested using a paired t -test. For detailed analysis of the CT scans, the Jonckheere-Terpstra test was used. A P value < 0.05 was considered as significant. All values are expressed as mean ± standard deviation. Results Experimental Groups No significant differences in general parameters were observed between the groups ( Table 1 ). Respiration, Temperature After instillation all animals in [LPS] developed dyspnea, tachypnea and signs of shock (lower body temperature and cold extremities). Twenty hours later, the temperature returned to nearly normal. All animals in [LPS] were apathetic and lacked appetite. Animals in [SAL] and [SHAM] did not show any clinical signs of respiratory distress. Hemodynamic and Aerodynamic Parameters and Gas Exchange No differences were observed in HR, CO, PAP, PVR, and MAP between groups at any time point and within groups at any time interval. Also, no differences were measured in Ppeak, Pplat, MAP, compliance, and Raw. Saturation was comparable between groups and time intervals ( Table 2 ). No significant differences in PO 2 /FiO 2 were found between T 0 and T5 0 in [LPS], [SAL], and [SHAM], and also not between groups at T 50. Serum Samples and Blood Smears No differences were seen in CRP levels between all groups. Cell counts in blood smears demonstrated an increase in neutrophils in [LPS] ( P < 0.05) between T 0 and T 31. More neutrophils ( P < 0.05) and less lymphocytes ( P < 0.05) were found in the blood at T 31 in [LPS] versus [SAL] and [SHAM] ( Table 3 ). Broncho-Alveolar Lavage Total Cells A significant increase in total cells was found at T 50 versus T 0 in BAL of left ( P < 0.001) and right lung ( P < 0.01) in [LPS] but not in [SHAM] and [SAL]. Total cell count was higher in [LPS] at T 50 compared with [SHAM] and [SAL] in both the left ( P < 0.01 and P < 0.001; respectively) and right lung ( P < 0.001 and P < 0.01; respectively) ( Fig. 3 A and B). Differential Cell Count Differential cell counts in BAL of [LPS] are shown in Fig. 4 A and B for left and right lung, respectively. Both an increase in neutrophils ( P < 0.001 in left, P < 0.01 in right lung) and in lymphocytes ( P < 0.05 in left and right lung) was observed at T 50. In [SAL], an increase in neutrophils was found in the right lung ( P < 0.01) ( Fig. 4 D) whereas no significant changes were seen in the left lung of [SAL] ( Fig. 4 C) or in [SHAM] ( Fig. 4 E and F). At T 50, significant more neutrophils are present in BAL of [LPS] versus [SAL] and [SHAM] ( P < 0.0001 for left and right lung). Concerning lymphocytes, differences were found in [LPS] versus [SAL] and [SHAM] at T50 ( P < 0.05) in right lung only. Furthermore, a significant correlation could be demonstrated between the CT scores of GGO in the middle right lobe and the number of total cells ( P < 0.05; r = 0.63) as well as BAL neutrophils ( P < 0.05; r = 0.55) ( Fig. 5 A and B, respectively). No correlations were found for the left lung. Wet Lung Weight There was no significant difference in wet to dry weight ratio between groups. Wet lung weight/body weight ratio, however, was higher in [LPS] (12.8) versu s [SAL] (9.8) and [SHAM] (10.2) ( P < 0.05). Histology More interstitial hemorrhage was seen on histology in [LPS] versus [SAL] and [SHAM] ( P < 0.01 comparing average scores) ( Table 4 ). The area of alveolar damage was significant greater in left lung of [LPS] versus [SAL] and [SHAM]. No differences were found in scores of right lung and average score. No significant differences were observed in edema, inflammation and alveolar hemorrhage. Figure 6 illustrates the hemorrhage and inflammation in [LPS]. CT-Scan One pig in [SAL] showed a pneumothorax on CT-1. This pig was therefore excluded from the CT analysis. No differences between groups were seen on CT-scans at T 17. GGO were noticed in the basal parts of the lungs in all pigs. These opacities disappeared when the pig was placed in the ventral position, probably related to physiologic fluid accumulation in the dependent areas of the lung. Lung opacities due to hypoventilation were found in the posterior costodiaphragmatic sinus. Global assessment at T 31 showed more animals with diffuse GGO in [LPS] (4/6) compared with [SAL] (0/4) ( P < 0.05) and [SHAM] (0/5) ( P < 0.05). Also more consolidation was noticed in [LPS] (5/6) compared with [SAL] (0/4) ( P < 0.05) and [SHAM] (1/5) ( P < 0.05). For more detailed analysis of the scans, a mean score of the 10 slices was calculated for each pig. A significant difference was found between [LPS] versus [SAL] and [SHAM] ( P < 0.0001)( Fig. 7 ). CT scans pre- and post-instillation in each group are shown in Fig. 8 . Discussion This study demonstrated that LPS instillation results in lung injury, as reflected by an increase in total cells, neutrophils, and lymphocytes in BAL fluid in [LPS] compared with [SAL] and [SHAM]. The percentage of neutrophils in blood smears increased and lymphocytes decreased in [LPS] versus [SAL] and [SHAM]. More ground glass opacities were visible on chest CT, interstitial hemorrhage was seen on histology, and wet lung weight/body weight was higher in [LPS]. However, no physiologic changes were observed between groups. Lung transplantation is an effective treatment modality for patients suffering from any form of end-stage pulmonary disease. Enhanced post-transplant outcome leads to an increasing number of transplantations, more patients referred, and longer waiting lists. In addition, 10%–30% of the patients do not survive the waiting period. As only 15%–25% of (multi-)organ donors (MOD) have lungs suitable for transplantation when adhering to the standard donor criteria [12, 13] , it is necessary to liberalize the criteria. An acceptance rate of 34% was achieved by our group, resulting from adopting more liberal lung donor criteria since the year 2000 [21] . Yet, abnormalities on chest X-ray remain an important reason for declining lungs in 30% of our donors. Because of the current donor shortage, there is a need to expand the donor pool [7] . Organs from alternative donors, such as extended criteria donors [7, 22] , non-heart-beating donors [23–25] , and living donors [3] have been proposed to attenuate the donor shortage. Better donor management, including invasive monitoring, pressure support, antibiotic therapy, methylprednisolone, strict fluid management, physiotherapy, and bronchoscopy with bronchial toilet, may also help to expand the lung donor pool [5, 8, 10, 11] . The aim of this study was to develop a valid large animal model of lung injury as a first step to investigate mechanisms of lung injury and possible methods to resuscitate damaged donor lungs by ex vivo reperfusion prior to transplantation [19] . Advantages of a porcine model are the feasibility to measure hemodynamic and aerodynamic parameters and the surgical comparability with human lungs. Ex vivo reperfusion is a well established technique used in our laboratory [18, 26, 27] . We and other groups have shown that ex vivo pig lung reperfusion is a reliable method to assess pulmonary graft function [18, 28] . Ex vivo reperfusion of human lungs is feasible and may be a useful technique to evaluate transplant suitability [29–31] , and to improve lungs of inferior quality prior to transplantation [20, 32] . Labor intensity and little availability of molecular and immunologic assays are disadvantages of the porcine model. Many animal models of acute lung injury have been described in the literature using different agents and techniques, such as oleic acid, endotoxin, acid aspiration, hyperoxia, saline lavages, sepsis, tracheal administration of bleomycin, and ischemia reperfusion injury. Each of these ALI models has its limitations, and one should therefore pay attention to choose a specific model to test the postulated hypothesis [33] . We believe that a LPS model is interesting to investigate as it mimics the inflammatory status of the donor lungs, without the risk of infection. LPS is a glycolipid of the outer membrane of Gram-negative bacteria, composed of a polar lipid head group (lipid A) and a chain of disaccharides [34] . In previous studies, LPS was administered intravenously to imitate acute respiratory distress syndrome (ARDS) in different species, such as domestic pigs, rats, and sheep [35–37] . Intratracheal instillation was used in Guinea pigs, mice, and rats [38, 39] . To our knowledge, there is only one study (Pompe et al. ) where LPS was administered intratracheally (through aerosol) in the pig [40] . These authors concluded that the absence of a systemic response offered the opportunity to investigate the effects of therapeutic interventions on pulmonary parameters. In contrast to our study, a large amount of LPS (119 ± 20 mg) was given continuously by nebulization until PO 2 dropped below 90 mmHg. This dose is expensive but also potentially harmful for investigators. In our model, on the other hand, we wanted to investigate changes in the hours following LPS administration. To avoid systemic responses, the intra-tracheal way was chosen for administration. Hemodynamic and aerodynamic parameters, CT scans, histologic, and physiologic parameters were compared. Based on preliminary experiments, a final dose of 15 mg LPS was chosen, and the technique was standardized with instillation of LPS in each lung administered in five different bronchi. We hypothesized that lung injury would be absent in [SHAM], low in [SAL], and marked in [LPS]. To visualize the injury chest, CT scans were taken. Some remarks, however, need to be addressed concerning the current study. There are little ELISA kits available for pigs. Therefore, CRP in blood was evaluated, as this is a clinically relevant and sensitive biomarker. Higher CRP levels in [LPS] compared with [SAL] and [SHAM] were expected; however, this could not be confirmed. On the other hand, at T 31, more neutrophils were observed in blood smears of [LPS]. A significant correlation between CT scores and cells was noticed in BAL in right but not in left lung, which we cannot explain, as the endotoxins were identically instilled in both bronchi. No problems were seen during the delivery of the endotoxins. A possible explication for the difference between both lungs could be that there was an overflow of solution from the left to the right side, resulting in more damage to the right lung. Unfortunately, this was not further investigated. No significant physiologic differences were observed between the study groups at any time point. An explanation for these negative findings could be that the lungs had already recovered at the moment of sacrifice. Therefore, in our opinion, no physiologic changes would have occurred if pigs were sacrificed later than 51 h after instillation. Possibly, differences in hemodynamic, aerodynamic, and oxygenation parameters could have been observed when measured earlier after instillation, since dyspnea and tachypnea were observed in the early phase of the experiment. During ex vivo lung perfusion, aerodynamic and hemodynamic parameters can be easily evaluated. Therefore, the goal was to create an injury model in which the functioning of the lung is impaired, so improvements during resuscitation can be noticed by evaluating the physiologic parameters. Another possible limitation of this study is the lack of repetitive chest CT scans in paralyzed and intubated animals. It would have been interesting to take serial chest CT scans at 1, 2, 4, and 8 h after instillation. This was not possible because of practical reasons. Further studies are needed to develop a model of ALI that enables studying differences in physiologic parameters before and after treatment during ex vivo reperfusion. A higher dose of LPS or instillation with another toxic agent, such as gastric juice could be other options. The Zurich group recently reported that BAL with diluted surfactant during ex vivo perfusion improved graft function of pig lungs injured by gastric acid aspiration [41] . Our experiments will be continued using gastric juice to cause lung injury. The interval between administration of the toxic agent and evaluation will be shortened in order to procure the lungs in the early phase of lung damage. In conclusion, LPS instillation caused lung injury reflected by increased total cells and inflammatory cells in BAL, ground glass opacities on CT and hemorrhage on histology. No changes in physiologic parameters could be observed, questioning the clinical usefulness of this model to investigate lung reconditioning prior to transplantation. Therefore, further studies may be needed with shorter time intervals after instillation, or other doses or agents, to establish a useful model to study treatment of injured lungs with ex vivo reperfusion. Acknowledgments The authors thank Walter Coudijzer for his help in performing CT scans, and Hans Scheers for statistical analysis. This study was supported by grants from the Fund for Research-Flanders (FWO) (G.0576.06). DVR is a senior investigator of the FWO (G.3C04.99). BMV is a senior research fellow and RV is a research fellow of the FWO. References 1 J.D. Christie L.B. Edwards P. Aurora Registry of the International Society for Heart and Lung Transplantation: Twenty-fifth official adult lung and heart/lung transplantation report–2008 J Heart Lung Transplant 27 2008 957 2 E.A. Pomfret R.S. Sung J. Allan Solving the organ shortage crisis: The 7th annual American Society of Transplant Surgeons' State-of-the-Art Winter Symposium Am J Transplant 8 2008 745 3 H. Date M. Yamane S. Toyooka Current status and potential of living-donor lobar lung transplantation Front Biosci 13 2008 1433 4 D.G. de Antonio R. Marcos R. Laporta Results of clinical lung transplant from uncontrolled non-heart-beating donors J Heart Lung Transplant 26 2007 529 5 E. Gabbay T.J. Williams A.P. Griffiths Maximizing the utilization of donor organs offered for lung transplantation Am J Respir Crit Care Med 160 1999 265 6 D.P. Mason L. Thuita J.M. Alster Should lung transplantation be performed using donation after cardiac death? The United States experience J Thorac Cardiovasc Surg 136 2008 1061 7 J.B. Orens A. Boehler M. De Perrot A review of lung transplant donor acceptability criteria J Heart Lung Transplant 22 2003 1183 8 M. Straznicka D.M. Follette M.D. Eisner Aggressive management of lung donors classified as unacceptable: Excellent recipient survival one year after transplantation J Thorac Cardiovasc Surg 124 2002 250 9 D. Van Raemdonck A. Neyrinck G.M. Verleden Lung donor selection and management Proc Am Thorac Soc 6 2009 28 10 R.V. Venkateswaran V.B. Patchell I.C. Wilson Early donor management increases the retrieval rate of lungs for transplantation Ann Thorac Surg 85 2008 278 11 D.R. Wheeldon C.D. Potter A. Oduro Transforming the “unacceptable” donor: Outcomes from the adoption of a standardized donor management technique J Heart Lung Transplant 14 1995 734 12 L.B. Ware Y. Wang X. Fang Assessment of lungs rejected for transplantation and implications for donor selection Lancet 360 2002 619 13 K. Hornby H. Ross S. Keshavjee Nonutilization of hearts and lungs after consent for donation: A Canadian multicenter study Can J Anaesth 53 2006 831 14 C. Meers D. Van Raemdonck F. Van Gelder Change in donor profile influenced the percentage of organs transplanted from multiple organ donors Transplant Proc 41 2009 572 15 J. Yeung M. Cypel T.K. Waddel Update on donor assessment, resuscitation and acceptance criteria including novel techniques—non-heart-beating donor lung retrieval and ex vivo donor lung perfusion Thorac Surg Clin 19 2009 261 16 F. Rega N.C. Jannis G.M. Verleden Should we ventilate or cool the pulmonary graft inside the non-heart-beating donor? J Heart Lung Transplant 22 2003 1226 17 F. Rega A.P. Neyrinck G.M. Verleden How long can we preserve the pulmonary graft inside the non-heart-beating donor? Ann Thorac Surg 77 2004 438 18 F. Rega N.C. Jannis G.M. Verleden Long-term preservation with interim evaluation of lungs from a non-heart-beating donor after a warm ischemic interval of 90 minutes Ann Surg 238 2003 782 19 M. Cypel J.C. Yeung S. Hirayama Technique for prolonged normothermic ex vivo lung perfusion J Heart Lung Transplant 27 2008 1319 20 R. Ingemansson A. Eyjolfsson L. Mared Clinical transplantation of initially rejected donor lungs after reconditioning ex vivo Ann Thorac Surg 87 2009 255 21 C. Meers D. Van Raemdonck G.M. Verleden Whence the lungs? A review of current lung donor profile and acceptance rate Interactive Cardiovasc Thorac Surg 7 Suppl 2 2008 S161 22 G.M. Abouna The use of marginal-suboptimal donor organs: A practical solution for organ shortage Ann Transplant 9 2004 62 23 T.M. Egan Non-heart-beating donors in thoracic transplantation J Heart Lung Transplant 23 2004 3 24 S. Steen Q. Liao P.N. Wierup Transplantation of lungs from non-heart-beating donors after functional assessment ex vivo Ann Thorac Surg 76 2003 244 25 D. Van Raemdonck F.R. Rega A.P. Neyrinck Non-heart-beating donors Semin Thorac Cardiovasc Surg 16 2004 309 26 C. Van De Wauwer A.P. Neyrinck N. Geudens Retrograde flush following topical cooling is superior to preserve the non-heart-beating donor lung Eur J Cardiothorac Surg 31 2007 1125 27 A. Neyrinck C. Van De Wauwer N. Geudens Comparative study of donor lung injury in heart-beating versus non-heart-beating donors Eur J Cardiothorac Surg 30 2006 628 28 M.E. Erasmus M.H. Fernhout J.M. Elstrodt Normothermic ex vivo lung perfusion of non-heart-beating donor lungs in pigs: From pretransplant function analysis towards a 6-h machine preservation Transpl Int 19 2006 589 29 T.M. Egan J.A. Haithcock W.A. Nicotra Ex vivo evaluation of human lungs for transplant suitability Ann Thorac Surg 81 2006 1205 30 A. Neyrinck F. Rega N. Jannis Ex vivo reperfusion of human lungs declined for transplantation: A novel approach to alleviate donor organ shortage? J Heart Lung Transplant [23] 2004 S172 173 31 P. Wierup A. Haraldsson F. Nilsson Ex vivo evaluation of nonacceptable donor lungs Ann Thorac Surg 81 2006 460 32 S. Steen R. Ingemansson L. Eriksson First human transplantation of a nonacceptable donor lung after reconditioning ex vivo Ann Thorac Surg 83 2007 2191 33 G. Matute-Bello C.W. Frevert T.R. Martin Animal models of acute lung injury Am J Physiol Lung Cell Mol Physiol 295 2008 L379 L399 34 C.R. Raetz R.J. Ulevitch S.D. Wright Gram-negative endotoxin: An extraordinary lipid with profound effects on eukaryotic signal transduction FASEB J 5 1991 2652 35 A. Jakubowski N. Maksimovich R. Olszanecki S-nitroso human serum albumin given after LPS challenge reduces acute lung injury and prolongs survival in a rat model of endotoxemia Naunyn Schmiedebergs Arch Pharmacol 379 2008 281 36 V. Kuklin M. Kirov M. Sovershaev Tezosentan-induced attenuation of lung injury in endotoxemic sheep is associated with reduced activation of protein kinase C Crit Care 9 2005 211 37 R. Schmidhammer E. Wassermann P. Germann Infusion of increasing doses of endotoxin induces progressive acute lung injury but prevents early pulmonary hypertension in pigs Shock 25 2006 389 38 Y. Wu M. Singer F. Thouron Effect of surfactant on pulmonary expression of type IIA PLA(2) in an animal model of acute lung injury Am J Physiol Lung Cell Mol Physiol 282 2002 743 39 S. Garantziotis S.M. Palmer L.D. Snyder Alloimmune lung injury induced by local innate immune activation through inhaled lipopolysaccharide Transplantation 84 2007 1012 40 J.C. Pompe J. Kesecioglu H.A. Bruining Nebulization of endotoxin during mechanical ventilation: An experimental model of ARDS in pigs Adv Exp Med Biol 388 1996 599 41 I. Inci L. Ampollini S. Arni Ex vivo reconditioning of marginal donor lungs injured by acid aspiration J Heart Lung Transplant 27 2008 1229