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

Journal of Surgical Research 166, e175–e185 (2011) doi:10.1016/j.jss.2009.09.028

A Porcine Model to Study Ex Vivo Reconditioning of Injured Donor Lungs Caroline M. Meers, D.V.M.,*,1 Walter De Wever, M.D., Ph.D.,§ Eric Verbeken, M.D. Ph.D.,jj Shana Wauters, Ms.c.,* Robin Vos, M.D., Ph.D.,† Bart M. Vanaudenaerde, Ph.D.,† Ste´phanie I. De Vleeschauwer, D.V.M.,† Geert M. Verleden, M.D., Ph.D.,†,{ Toni E. Lerut, M.D., Ph.D.,‡ and Dirk Van Raemdonck, M.D., Ph.D.*,‡ *Laboratory of Experimental Thoracic Surgery, Katholieke Universiteit Leuven, Leuven, Belgium; †Laboratory of Pneumology, Katholieke Universiteit Leuven, Leuven, Belgium; ‡Department of Thoracic Surgery, University Hospital Gasthuisberg, Leuven, Belgium; §Department of Radiology, University Hospital Gasthuisberg, Leuven, Belgium; jjDepartment of Pathology, University Hospital Gasthuisberg, Leuven, Belgium; and {Department of Pneumology, University Hospital Gasthuisberg, Leuven, Belgium Submitted for publication June 23, 2009

Background. Brain death rapidly results in lung injury making many cadaveric donors unsuitable for lung transplantation. The aim of this study was to develop a porcine model of lung injury as a first step to study mechanisms to ameliorate the pretransplant graft quality during ex vivo perfusion. 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

1 To whom correspondence and reprint requests should be addressed at Laboratory of Experimental Thoracic Surgery, University Hospital Gasthuisberg,Herestraat 49, B-3000 Leuven, Belgium. E-mail: [email protected].

[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. Ó 2011 Elsevier Inc. All rights reserved.

Key Words: lung transplantation; lung shortage; ex vivo lung perfusion; lung injury.

donor

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%.

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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.

FIG. 1. Time outline in all three study groups. *Respiration and rectal temperature were assessed, and blood smears and serum were collected at T-17, T0, T31, and T50. Broncho-alveolar lavage was done and hemodynamic and aerodynamic parameters and oxygenation were measured at T0 and T50. CT-1 ¼ First chest CT-scan, CT-2 ¼ second chest CT-scan.

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.

MATERIALS AND METHODS Hemodynamic and Aerodynamic Parameters 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; Dra¨ger, Lu¨beck, Germany) at a tidal volume of 8-10 mL/kg body weight with an inspiratory oxygen fraction (FiO2) of 0.5 and a positive end-expiratory pressure (PEEP) of 5 cmH2O. Respiratory rate (RR) was adjusted to keep the end-tidal CO2 between 35 and 45 mmHg.

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.

Experimental Groups Sixteen specific pathogen free pigs (47 6 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

FIG. 2. A sketch of porcine lungs showing the tracheal bronchus on the right side (arrow); 10 mL of the LPS or SAL solution was administered in five different bronchi in each lung (black dots).

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TABLE 1 General parameters

Weight (kg) Interval CT1 - CT2 (h) Interval CT2 - moment of sacrifice (h) Interval instillation - moment of sacrifice (h)

[SAL] n¼5

[SHAM] n¼5

[LPS] n¼6

P-value

40.2 6 7.6 47.5 6 1.4 18.8 6 1.6 49.5 6 0.5

40.4 6 8.9 46.9 6 1.0 18.7 6 1.0 49.4 6 0.7

45.7 6 6.1 47.7 6 0.8 18.6 6 0.7 49.2 6 1.5

NS NS NS NS

[SAL] ¼ group instillation of saline solution; [SHAM] ¼ group without instillation; [LPS] ¼ group with instillation of lipopolysaccharides.

Blood Samples and Blood Smears Blood samples were taken at T 0 and T 50 to assess PO2/FiO2 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 3 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 Bu¨rker 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

TABLE 2 Hemodynamic, aerodynamic parameters and saturation atT0 and T50 [SAL]

[SHAM]

[LPS]

Parameter

T0

T 50

T0

T 50

T0

T 50

Heart rate (/min) Cardiac output (L/min) PAP (mm Hg) PVR (dynes 3 sec 3 cm5) MAP (mm Hg) Ppeak (cm H2O) Pplat (cm H2O) Pmean (cm H2O) compliance (mL/cm H2O) Raw (cm H2O/L/s) SaO2 (%)

77 6 15 / / / / 23 6 3 16 6 3 11 6 3 36 6 11 13 6 1 95 6 2

94 6 14 361 22 6 6 300 6 196 67 6 24 27 6 9 18 6 4 11 6 2 32 6 12 11 6 1 94 6 2

102 6 11 / / / / 18 6 3 14 6 3 961 43 6 15 962 96 6 4

88 6 12 562 18 6 3 162 6 44 65 6 22 19 6 1 14 6 1 861 32 6 4 10 6 1 97 6 2

82 6 15 / / / / 21 6 4 15 6 3 10 6 2 38 6 8 10 6 1 96 6 2

79 6 11 361 19 6 3 222 6 103 69 6 11 22 6 3 17 6 2 10 6 2 31 6 5 11 6 2 96 6 2

No significant differences were observed between groups at any time point and within groups between time intervals. PAP ¼ pulmonary artery pressure; PVR ¼ pulmonary vascular resistance; MAP ¼ mean arterial pressure; Ppeak ¼ peak airway pressure ¼ Pplat plateau airway pressure; Pmean ¼ mean airway pressure; Raw ¼ airway resistance; SAO2 ¼ oxygen saturation. [SAL] ¼ group with instillation of saline solution - [SHAM] ¼ group without instillation; [LPS] ¼ group with instillation of lipopolysaccharides.

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TABLE 3

Histology

Percentage of white blood cells in blood smears at T31

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.

Cells

[SAL]

[SHAM]

[LPS]

Neutrophils Lymphocytes Monocytes Eosinophils Basophils

26.4 6 5.9 63.6 6 4.7 9.8 6 4.3 0.0 6 0.0 0.2 6 0.4

34.8 6 9.8 58.6 6 11.4 6.4 6 4.2 0.0 6 0.0 0.2 6 0.4

55.5 6 21.5* 38.2 6 21.6* 6.3 6 3.1 0.0 6 0.0 0.0 6 0.0

[SAL] ¼ group with instillation of saline solution; [SHAM] ¼ group without instillation; [LPS] ¼ group with instillation of lipopolysaccharides. * P < 0.05 for [LPS] versus [SAL] and [SHAM] (ANOVA).

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; Rhoˆne-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.

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 6 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 PO2/FiO2 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

FIG. 3. Total cell count in BAL at T0 and T50. (A) Left lung. (B) Right lung. **P < 0.01; ***P < 0.001 between T0 and T50. yy P < 0.01; yyyP < 0.001 between groups.

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).

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FIG. 4. Differential cell counts in the three study groups. (A) [LPS] left lung. (B) [LPS] right lung. (C) [SAL]-left lung; (D) [SAL]-right lung; (E) [SHAM] left lung. (F) [SHAM] right lung. *P < 0.05; **P < 0.01; ***P < 0.001 between T0 and T50.

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. 3A and B). Differential Cell Count Differential cell counts in BAL of [LPS] are shown in Fig. 4A 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. 4D) whereas no significant

changes were seen in the left lung of [SAL] (Fig. 4C) or in [SHAM] (Fig. 4E 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 ¼ 0.63) as well as BAL neutrophils (P < 0.05; r ¼ 0.55) (Fig. 5A 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.

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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. FIG. 5. (A) Correlation between CT score of ground glass opacities in right middle lobe and total cells in BAL. (B) Correlation between CT score of ground glass opacities in right middle lobe and neutrophils in BAL.

Wet lung weight/body weight ratio, however, was higher in [LPS] (12.8) versus [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

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

TABLE 4 Histological scoring of left and right lung at T50 [SAL]

Area of alveolar damage Interstitial hemorrhage Alveolar hemorrhage Inflammation (pmn) Edema

[SHAM]

[LPS]

Left

Right

Average

Left

Right

Average

Left

Right

Average

2.1 6 0.4 0.6 6 0.4 0.3 6 0.4 1.1 6 0.8 1.4 6 0.2

2.2 6 0.7 0.2 6 0.2 0.1 6 0.2 0.7 6 0.5 0.7 6 0.8

22 6 0.5 0.4 6 0.1 0.2 6 0.2 0.9 6 0.7 1.5 6 0.9

3.0 6 0.8 0.3 6 0.4 0.3 6 0.4 0.7 6 0.8 1.2 6 1.1

2.9 6 1.1 0.2 6 0.3 0.2 6 0.2 0.9 6 0.4 1.1 6 0.8

3.0 6 0.8 0.2 6 0.3 0.3 6 0.3 0.8 6 0.6 1.2 6 0.8

3.1 6 0.7* 1.2 6 0.5* 0.4 6 0.4 1.8 6 0.5 1.8 6 0.8

2.6 6 0.9 0.5 6 0.2* 0.2 6 0.3 0.5 6 0.3 0.9 6 0.4

2.9 6 0.5 0.9 6 0.3** 0.3 6 0.2 1.1 6 0.3 1.4 6 0.5

[SAL] ¼ group with instillation of saline solution; [SHAM] ¼ group without instillation; [LPS] ¼ group with instillation of lipopolysaccharides; pmn ¼ polymorpho-nuclear cells. * P < 0.05, ** P < 0.01 for [LPS] versus [SAL] and [SHAM] (ANOVA).

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FIG. 6. Hematoxylin-eosin-stained lung biopsies in [LPS] (A) (B), [SAL] (C) (D), [SHAM] (E) (F). Original magnification 350 in (A), (C), (D); 3400 in (B), (E), (F). Hemorrhage [circles in (A), (B)] and inflammation [arrows in (B)] were seen in [LPS].

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

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FIG. 7. Column bars for average score for GGO.

donors, such as extended criteria donors [7, 22], nonheart-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 6 20 mg) was given continuously by nebulization until PO2 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,

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FIG. 8. CT scans in three study groups pre- and post-instillation. (A) [LPS] pre-, (B) [LPS] post-, (C) [SAL] pre, (D) [SAL] post-, (E) [SHAM] pre-, (F) [SHAM] post-instillation. Ground glass opacities due to LPS instillation (B), which were absent in [SAL] (D) and [SHAM] (F).

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

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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. Christie JD, Edwards LB, Aurora P, et al. 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 2008;27:957. 2. Pomfret EA, Sung RS, Allan J, et al. Solving the organ shortage crisis: The 7th annual American Society of Transplant Surgeons’ State-of-the-Art Winter Symposium. Am J Transplant 2008; 8:745. 3. Date H, Yamane M, Toyooka S, et al. Current status and potential of living-donor lobar lung transplantation. Front Biosci 2008;13:1433. 4. de Antonio DG, Marcos R, Laporta R, et al. Results of clinical lung transplant from uncontrolled non-heart-beating donors. J Heart Lung Transplant 2007;26:529. 5. Gabbay E, Williams TJ, Griffiths AP, et al. Maximizing the utilization of donor organs offered for lung transplantation. Am J Respir Crit Care Med 1999;160:265. 6. Mason DP, Thuita L, Alster JM, et al. Should lung transplantation be performed using donation after cardiac death? The United States experience. J Thorac Cardiovasc Surg 2008; 136:1061. 7. Orens JB, Boehler A, De Perrot M, et al. A review of lung transplant donor acceptability criteria. J Heart Lung Transplant 2003;22:1183. 8. Straznicka M, Follette DM, Eisner MD, et al. Aggressive management of lung donors classified as unacceptable: Excellent recipient survival one year after transplantation. J Thorac Cardiovasc Surg 2002;124:250. 9. Van Raemdonck D, Neyrinck A, Verleden GM, et al. Lung donor selection and management. Proc Am Thorac Soc 2009;6:28. 10. Venkateswaran RV, Patchell VB, Wilson IC, et al. Early donor management increases the retrieval rate of lungs for transplantation. Ann Thorac Surg 2008;85:278. 11. Wheeldon DR, Potter CD, Oduro A, et al. Transforming the ‘‘unacceptable’’ donor: Outcomes from the adoption of a standardized donor management technique. J Heart Lung Transplant 1995; 14:734. 12. Ware LB, Wang Y, Fang X, et al. Assessment of lungs rejected for transplantation and implications for donor selection. Lancet 2002;360:619.

13. Hornby K, Ross H, Keshavjee S, et al. Nonutilization of hearts and lungs after consent for donation: A Canadian multicenter study. Can J Anaesth 2006;53:831. 14. Meers C, Van Raemdonck D, Van Gelder F, et al. Change in donor profile influenced the percentage of organs transplanted from multiple organ donors. Transplant Proc 2009;41:572. 15. Yeung J, Cypel M, Waddel TK, et al. 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 2009;19:261. 16. Rega F, Jannis NC, Verleden GM, et al. Should we ventilate or cool the pulmonary graft inside the non-heart-beating donor? J Heart Lung Transplant 2003;22:1226. 17. Rega F, Neyrinck AP, Verleden GM, et al. How long can we preserve the pulmonary graft inside the non-heart-beating donor? Ann Thorac Surg 2004;77:438. 18. Rega F, Jannis NC, Verleden GM, et al. Long-term preservation with interim evaluation of lungs from a non-heart-beating donor after a warm ischemic interval of 90 minutes. Ann Surg 2003; 238:782. 19. Cypel M, Yeung JC, Hirayama S, et al. Technique for prolonged normothermic ex vivo lung perfusion. J Heart Lung Transplant 2008;27:1319. 20. 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. 21. Meers C, Van Raemdonck D, Verleden GM. Whence the lungs? A review of current lung donor profile and acceptance rate. Interactive Cardiovasc Thorac Surg 2008;7(Suppl 2):S161. 22. Abouna GM. The use of marginal-suboptimal donor organs: A practical solution for organ shortage. Ann Transplant 2004; 9:62. 23. Egan TM. Non-heart-beating donors in thoracic transplantation. J Heart Lung Transplant 2004;23:3. 24. Steen S, Liao Q, Wierup PN, et al. Transplantation of lungs from non-heart-beating donors after functional assessment ex vivo. Ann Thorac Surg 2003;76:244. 25. Van Raemdonck D, Rega FR, Neyrinck AP, et al. Non-heartbeating donors. Semin Thorac Cardiovasc Surg 2004;16:309. 26. Van De Wauwer C, Neyrinck AP, Geudens N, et al. Retrograde flush following topical cooling is superior to preserve the nonheart-beating donor lung. Eur J Cardiothorac Surg 2007; 31:1125. 27. Neyrinck A, Van De Wauwer C, Geudens N, et al. Comparative study of donor lung injury in heart-beating versus non-heartbeating donors. Eur J Cardiothorac Surg 2006;30:628. 28. Erasmus ME, Fernhout MH, Elstrodt JM, et al. 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 2006;19:589. 29. Egan TM, Haithcock JA, Nicotra WA, et al. Ex vivo evaluation of human lungs for transplant suitability. Ann Thorac Surg 2006; 81:1205. 30. Neyrinck A, Rega F, Jannis N. 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. Wierup P, Haraldsson A, Nilsson F, et al. Ex vivo evaluation of nonacceptable donor lungs. Ann Thorac Surg 2006;81:460. 32. 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. 33. Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung injury. Am J Physiol Lung Cell Mol Physiol 2008; 295:L379–99. 34. Raetz CR, Ulevitch RJ, Wright SD, et al. Gram-negative endotoxin: An extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB J 1991;5:2652.

MEERS ET AL.: PORCINE LUNG INJURY MODEL 35. Jakubowski A, Maksimovich N, Olszanecki R, et al. 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 2008;379:281. 36. Kuklin V, Kirov M, Sovershaev M, et al. Tezosentan-induced attenuation of lung injury in endotoxemic sheep is associated with reduced activation of protein kinase C. Crit Care 2005; 9:211. 37. Schmidhammer R, Wassermann E, Germann P, et al. Infusion of increasing doses of endotoxin induces progressive acute lung injury but prevents early pulmonary hypertension in pigs. Shock 2006;25:389.

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38. Wu Y, Singer M, Thouron F, et al. 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 2002;282:743. 39. Garantziotis S, Palmer SM, Snyder LD, et al. Alloimmune lung injury induced by local innate immune activation through inhaled lipopolysaccharide. Transplantation 2007;84:1012. 40. Pompe JC, Kesecioglu J, Bruining HA, et al. Nebulization of endotoxin during mechanical ventilation: An experimental model of ARDS in pigs. Adv Exp Med Biol 1996;388:599. 41. 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.