- Email: [email protected]
Ex vivo lung perfusion with adenosine A2A receptor agonist allows prolonged cold preservation of lungs donated after cardiac death
Accepted Manuscript Ex vivo lung perfusion with adenosine A2A receptor agonist allows prolonged cold preservation of donation after cardiac death donor lungs Cynthia E. Wagner, MD, Nicolas H. Pope, MD, Eric J. Charles, MD, Mary E. Huerter, MD, Ashish K. Sharma, MBBS, PhD, Morgan D. Salmon, PhD, Benjamin T. Carter, BS, Mark H. Stoler, MD, Christine L. Lau, MD, MBA, Victor E. Laubach, PhD, Irving L. Kron, MD PII:
S0022-5223(15)01293-3
DOI:
10.1016/j.jtcvs.2015.07.075
Reference:
YMTC 9779
To appear in:
The Journal of Thoracic and Cardiovascular Surgery
Received Date: 28 April 2015 Revised Date:
13 July 2015
Accepted Date: 19 July 2015
Please cite this article as: Wagner CE, Pope NH, Charles EJ, Huerter ME, Sharma AK, Salmon MD, Carter BT, Stoler MH, Lau CL, Laubach VE, Kron IL, Ex vivo lung perfusion with adenosine A2A receptor agonist allows prolonged cold preservation of donation after cardiac death donor lungs, The Journal of Thoracic and Cardiovascular Surgery (2015), doi: 10.1016/j.jtcvs.2015.07.075. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ex vivo lung perfusion with adenosine A2A receptor agonist allows prolonged cold preservation of donation after cardiac death donor lungs
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Nicolas H. Pope, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: T32 HL007849, R01 HL130053 Conflicts of interest: none
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Cynthia E. Wagner, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: T32 HL007849, R01 HL130053 Conflicts of interest: none
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Cynthia E. Wagner MD, Nicolas H. Pope MD, Eric J. Charles MD, Mary E. Huerter MD, Ashish K. Sharma MBBS, PhD, Morgan D. Salmon PhD, Benjamin T. Carter BS, Mark H. Stoler MD, Christine L. Lau MD, MBA, Victor E. Laubach PhD, Irving L. Kron MD
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Eric J. Charles, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: T32 HL007849, R01 HL130053 Conflicts of interest: none
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Mary E. Huerter, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none
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Ashish K. Sharma, MBBS, PhD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none Morgan D. Salmon, PhD University of Virginia Department of Surgery
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Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none
Mark H. Stoler, MD University of Virginia Department of Pathology Funding: none Conflicts of interest: none
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Christine L. Lau, MD, MBA University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none
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Benjamin T. Carter, BS University of Virginia School of Medicine Funding: R01 HL130053 Conflicts of interest: none
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Victor E. Laubach, PhD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none
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Irving L. Kron, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none PO Box 800679 Charlottesville, VA 22908 Phone: 434-924-2158 Fax: 434-982-3885 Email: [email protected]
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Abstract word count: 250 Article word count: 3500
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Abbreviations and Acronyms
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= adenosine A2A receptor = arterial blood gas = analysis of variance = A2AR agonist administration during EVLP = A2AR agonist administration during EVLP and reperfusion = bronchoalveolar lavage = bicinchoninic acid = bovine serum albumin = computed tomography = donation after brain death = donation after cardiac death = dimethyl sulfoxide = ethylenediaminetetraacetic acid = enzyme-linked immunosorbent assay = endotracheal tube = ex vivo lung perfusion = fraction of inspired oxygen = interferon = interleukin = ischemia-reperfusion injury = left atrium = left atrial appendage = left lower lobe = left upper lobe = millimeter of mercury = pulmonary artery = arterial oxygen partial pressure = phosphate buffered saline = positive end expiratory pressure = primary graft dysfunction = oxygen partial pressure = pulmonary vascular resistance = respiratory rate = tidal volume
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A2AR ABG ANOVA ATL-E ATL-E/R BAL BCA BSA CT DBD DCD DMSO EDTA ELISA ETT EVLP FiO2 IFN IL IRI LA LAA LLL LUL mmHg PA PaO2 PBS PEEP PGD PO2 PVR RR TV
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Objective: Ex vivo lung perfusion (EVLP) has been successful in the assessment of marginal donor lungs, including donation after cardiac death (DCD) donor lungs. EVLP also represents a unique platform for targeted drug delivery. We sought to determine if ischemia-reperfusion injury (IRI) would be decreased after transplantation of DCD donor lungs subjected to prolonged cold preservation and treated with an adenosine A2A receptor (A2AR) agonist during EVLP. Methods: Porcine DCD donor lungs were preserved at 4°C for 12 hours and underwent EVLP for 4 hours. Left lungs were then transplanted and reperfused for 4 hours. Three groups (n=4/group) were randomized according to treatment with the A2AR agonist ATL-1223 or the dimethyl sulfoxide (DMSO) vehicle: DMSO (infusion of DMSO during EVLP and reperfusion), ATL-E (infusion of ATL1223 during EVLP and DMSO during reperfusion), and ATL-E/R (infusion of ATL1223 during EVLP and reperfusion). Final PaO2/FiO2 ratios were determined from samples obtained from the left superior and inferior pulmonary veins. Results: Final PaO2/FiO2 ratios in the ATL-E/R group (430.1 ± 26.4 mmHg) were similar to final PaO2/FiO2 ratios in the ATL-E group (413.6 ± 18.8 mmHg), but both treated groups had significantly higher final PaO2/FiO2 ratios compared to the DMSO group (84.8 ± 17.7 mmHg). Low PO2 gradients during EVLP did not preclude superior postoperative oxygenation capacity. Conclusions: Following prolonged cold preservation, treatment of DCD donor lungs with an A2AR agonist during EVLP enabled PaO2/FiO2 ratios above 400 mmHg after transplantation in a preclinical porcine model. Pulmonary function during EVLP was not predictive of outcomes after transplantation.
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The number of patients on the waiting list continues to exceed the number
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of patients who undergo lung transplantation each year, though recent advances
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in donor selection and lung preservation have the potential to close this gap [1].
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Lungs are usually selected from donation after brain death (DBD) donors, yet
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only 15-20% of these lungs are deemed suitable and procured for transplantation
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after conservative evaluation. Marginal donor lungs, including those from
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donation after cardiac death (DCD) donors, represent an additional source for
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transplantation, but use of these lungs has been linked to increased mortality and
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primary graft dysfunction (PGD) in some studies [2], [3]. PGD is mediated by
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ischemia-reperfusion injury (IRI) and is associated with significant early and late
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mortality [4], [5] as well as progression to bronchiolitis obliterans syndrome and
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delayed graft failure [6], [7].
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Cold preservation of donor lungs reduces metabolic demand during
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ischemic storage but inhibits cellular regeneration that may be crucial before
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marginal donor lungs are transplanted. Normothermic ex vivo lung perfusion
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(EVLP) was initially developed to assess function of lungs from DCD donors prior
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to transplantation [8]. EVLP was subsequently modified to allow prolonged
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preservation of marginal donor lungs [9], and a pivotal clinical trial established
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non-inferior outcomes after transplantation of marginal donor lungs passively
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rehabilitated with EVLP compared to conventional donor lungs [10]. EVLP also
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offers a unique platform for targeted drug delivery to actively treat marginal donor
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lungs. We have previously demonstrated that the adenosine A2A receptor
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(A2AR) agonist ATL-1223 exerts anti-inflammatory effects and reduces IRI when
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administered to DCD donor lungs during EVLP in a porcine model of lung
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transplantation [11].
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The purpose of this study was to determine if administration of ATL-1223 during EVLP would decrease IRI after transplantation of DCD donor lungs
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subjected to prolonged 12-hour cold preservation, and if further administration
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during reperfusion would augment this protection in a preclinical porcine model,
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with important implications to expand the donor lung pool by including DCD
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donors and allowing cross-country retrieval. We hypothesized that IRI would be
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reduced after transplantation of donor lungs treated with ATL-1223 during EVLP
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compared to lungs that received dimethyl sulfoxide (DMSO) vehicle, and that IRI
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would be further attenuated by continued administration of ATL-1223 to the
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recipient during reperfusion.
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Materials and Methods
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Animals
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This study was approved by the Institutional Animal Care and Use
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Committee at the University of Virginia. Animals received humane treatment in
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accordance with the “Guide for Care and Use of Laboratory Animals” from the
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National Institutes of Health.
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Study Design
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Mature domestic swine (19-35 kg) of both genders were randomized to 3
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different groups (n=4/group) based on treatment with the A2AR agonist ATL-
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1223. Donor swine underwent hypoxic arrest followed by 15 minutes of warm
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ischemia. Lungs were procured and preserved at 4°C for 12 hours before
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undergoing normothermic EVLP for 4 hours. Left lungs were then transplanted
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into size-matched recipients and reperfused for 4 hours. The DMSO group
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received an infusion of DMSO (vehicle) during EVLP and reperfusion. The ATL-E
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group received an infusion of ATL-1223 during EVLP and an infusion of DMSO
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during reperfusion, and the ATL-E/R group received an infusion of ATL-1223
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during EVLP and reperfusion.
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Donor Lung Procurement
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Donor animals were anesthetized, weighed, and prepped for a median sternotomy. Following induction, animals were intubated and maintained under
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general anesthesia with isoflurane. Animals were ventilated with 100% oxygen
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before initial donor arterial blood gas (ABG) analysis. Heparin was not
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administered to donor animals. The endotracheal tube (ETT) was clamped and
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donor animals underwent hypoxic arrest. Death was confirmed by cessation of
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electrocardiographic activity and absence of heart sounds. Animals were not
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touched for a 15-minute period of warm ischemia. Lungs were ventilated
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throughout the last 5 minutes of this time. A median sternotomy was performed.
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The main pulmonary artery (PA) was cannulated near the bifurcation with a
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cardioplegia cannula and 500 mcg prostaglandin E1 (Pfizer Inc) was
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administered. The main PA was clamped proximally, the left atrial appendage
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(LAA) was vented, and lungs were flushed with 1.5 L of 4°C Perfadex (XVIVO
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Perfusion, Englewood, CO). Topical cold slush was applied. At the completion of
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the flush, the heart and lungs were removed en bloc. A funnel-shaped cannula
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with a pressure port (XVIVO Perfusion, Englewood, CO) was sewn to the left
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atrial (LA) cuff with a running 4-0 prolene suture, a cannula with a pressure port
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(XVIVO Perfusion, Englewood, CO) was tied within the main PA, and a 7.0 mm
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ETT with the balloon removed was tied within the trachea. Lungs were inflated
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with gentle positive end expiratory pressure (PEEP) and the ETT was clamped. A
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500 ml retrograde flush with 4°C Perfadex was then performed. The lungs were
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submerged in 4°C Perfadex, surrounded by a double layer of cold slush, and
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stored at 4°C for 12 hours. Lungs were flushed antegrade with 500 ml 4°C
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Perfadex immediately prior to EVLP.
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Ex Vivo Lung Perfusion
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The EVLP circuit was primed with 2 L Steen solution (XVIVO Perfusion, Englewood, CO) supplemented with 10,000 units heparin, 500 mg
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methylprednisolone, and 500 mg cefazolin. Perfusate drained from the venous
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(LA) cannula into the venous reservoir and was warmed to 37°C, deoxygenated
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with a mixture of 86% nitrogen, 8% carbon dioxide, and 6% oxygen gas, and
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pumped into the arterial (PA) cannula (Medtronic BioMedicus centrifugal pump,
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Eden Prairie, MN). Lungs were placed within the EVLP chamber (XVIVO
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Perfusion, Englewood, CO), de-aired, and low flow (0.15 ml/min) through the
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lungs was established to initiate EVLP. The height of the venous reservoir was
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adjusted to maintain LA pressures between 0-5 mmHg. PA pressures were
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dependent on perfusate flow. Throughout the first 30 minutes of EVLP, flow was
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titrated up to 40% of estimated cardiac output (100 ml/kg/min) and perfusate was
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warmed to 37°C. After 30 minutes of EVLP, lungs were ventilated on room air
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(TV 10 ml/kg, RR 8 bpm, PEEP 5 cm H2O). EVLP was performed for 4 hours.
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Oxygenation was assessed every hour by increasing the FiO2 to 100% for 15
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minutes prior to PO2 analysis. Samples taken from the venous and arterial arms
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of the circuit allowed calculation of the oxygenation gradient across the
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pulmonary vasculature. Other parameters recorded every hour included LA
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pressure, PA pressure, peak airway pressure, mean airway pressure, and
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plateau pressure. The A2AR agonist ATL-1223 (Lewis and Clark
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Pharmaceuticals, Charlottesville, VA) was administered as an infusion (3.0
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ng/kg/min) throughout all 4 hours of EVLP. The same weight-based volume of
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DMSO was administered as an infusion throughout EVLP. At the conclusion of
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EVLP, donor lungs were flushed antegrade with 500 ml of 4°C Perfadex. Lungs
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were separated on the back table and the donor left lung was cooled with topical
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cold slush for 15 minutes prior to transplantation.
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Left Lung Transplantation
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Recipient animals were anesthetized, weighed, and prepped for a left thoracotomy. Animals were intubated and maintained under general anesthesia
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with isoflurane. A central venous line with a PA catheter, arterial line, and
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oxygenation saturation probe were placed. Initial recipient ABG analysis was
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performed on 100% oxygen and baseline vitals were recorded, including heart
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rate, blood pressure, PA pressure, and oxygenation saturation. A left
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thoracotomy was performed and 50 mg lidocaine were administered prior to
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dissection of the left hilum. Before clamping the left PA and ligating the left
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superior and inferior pulmonary veins, 5,000 units of heparin were given and
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allowed to circulate for 5 minutes. The left mainstem bronchus was clamped, the
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pneumonectomy was completed, and the donor lung was placed in the chest.
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The bronchial anastomosis was completed with a running 4-0 prolene suture and
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the PA anastomosis was then completed with a running 5-0 prolene suture. The
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recipient LAA was clamped, opened, and sewn to the donor LA cuff with a
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running 5-0 prolene suture. Clamps were removed to establish reperfusion and
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ventilation. ABG analysis was performed and vitals were recorded every 15-20
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minutes during all 4 hours of reperfusion. Animals were ventilated with 100%
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oxygen throughout reperfusion. Intermittent gentle PEEP was applied to reduce
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atelectasis. Acid-base status was optimized by adjusting respiratory rate at a
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constant tidal volume of 10 ml/kg. Intravenous fluids were given judiciously, and
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epinephrine was administered as needed. The A2AR agonist ATL-1223 was
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administered at 3.0 ng/kg/min or an equivalent weight-based volume of DMSO
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was given as an infusion throughout reperfusion. ABG samples were obtained
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from the left superior and inferior pulmonary veins to determine isolated
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oxygenation capacity of the transplanted left lung at the conclusion of
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reperfusion. Mean values were reported as final PaO2/FiO2 ratios. A final
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systemic ABG analysis was performed, the recipient was euthanized with
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Euthasol (Virbac AH, Inc), and the left lung was explanted.
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Cytokine Expression
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Lung tissue samples were obtained from the left lower lobe (LLL), frozen
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at -80°C, and homogenized using a gentleMACS tissue dissociator (Miltenyi
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Biotec, Auburn, CA) in a solution containing PBS, 0.5% BSA, and 2mM EDTA.
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Total protein concentration was determined using a BCA protein assay (Pierce,
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Rockford, IL). ELISAs were performed to determine expression of interferon-γ
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(IFN-γ) (RayBiotech, Inc., Norcross, GA) and interleukin 1β (IL-1β), interleukin 6
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(IL-6), and interleukin 8 (IL-8) (Abcam, Cambridge, MA). One sample in the ATL-
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E/R group had insufficient volume for the latter analyses. Cytokine
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concentrations were reported per 50 µg total protein in each sample.
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Pulmonary Edema
A bronchoalveolar lavage (BAL) of the left upper lobe (LUL) was
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performed with 50 ml normal saline. Total protein concentration was determined
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in BAL samples using a BCA protein assay as a measure of pulmonary edema.
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Lung tissue samples from the LUL (n=2/animal) and LLL (n=3/animal) were
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weighed immediately and again after a stable weight was achieved through
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desiccation in a vacuum oven. Wet-to-dry weight ratios were determined as an
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additional measure of pulmonary edema. The wet-to-dry weight ratio for each
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lung was calculated as the mean of the ratios from all 5 lung tissue samples.
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Histology
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was submerged in formalin overnight. Lung tissue samples from the LUL
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(n=2/animal) and LLL (n=3/animal) were placed in 70% ethanol, embedded in
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paraffin, and stained with hematoxylin and eosin. The lung injury severity score
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was determined by a pathologist blinded to treatment group. The score ranged
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from 0-9 and was a composite sum of 3 individual components, including number
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of neutrophils per high-powered field (score of 0, <5; 1, 6 to 10; 2, 11 to 20; and
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score of 3, >20), interstitial infiltration (score of 0, none; 1, minimal; 2, moderate;
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and 3, severe), and alveolar edema (score of 0, < 5%; 1, 6%-25%; 2, 26%-50%;
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and 3, > 50%). The lung injury severity score for each lung was calculated as the
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mean of the scores from all 5 lung tissue samples.
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Statistical Analysis
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Experimental methods were designed to test the null hypothesis that no
significant differences in lung IRI would be observed despite treatment with ATL-
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1223. Differences among groups were calculated using one-way ANOVA, and
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pairwise comparisons were made using Tukey’s multiple comparisons test
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(GraphPad Prism version 6.0e). An unpaired t test was used to calculate
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differences between time points. Data is expressed as the mean ± standard error
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of the mean. A p value less than 0.05 was considered statistically significant.
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Results
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Donor lung oxygenation during reperfusion
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There was no difference in mean initial PaO2/FiO2 ratios of donor animals
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among groups (Figure 1). However, after 4 hours of reperfusion, final PaO2/FiO2
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ratios of donor lungs in both groups treated with ATL-1223 (ATL-E: 413.6 ± 18.8
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mmHg; ATL-E/R: 430.1 ± 26.4 mmHg) were significantly higher than final
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PaO2/FiO2 ratios of control donor lungs (DMSO: 84.8 ± 17.7 mmHg) (p<0.0001).
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Further administration of ATL-1223 during reperfusion did not significantly
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increase final PaO2/FiO2 ratios in the ATL-E/R group compared to the ATL-E
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group (p=0.85) (Figure 1).
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Mean PaO2/FiO2 ratios of control donor lungs significantly worsened from baseline measurement in the donor animal to final assessment in the recipient.
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However, there was no significant difference between initial and final mean
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PaO2/FiO2 ratios of donor lungs in both groups treated with ATL-1223.
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Donor lung oxygenation during EVLP Overall, mean PO2 gradients decreased from the first hour to the fourth
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hour of EVLP in all groups (Figure 2). Trends in PO2 gradients of individual lungs
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were highly variable throughout EVLP and were frequently below the 350 mmHg
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threshold considered acceptable for clinical lung transplantation [10]. Mean final
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values decreased significantly in the DMSO group from 334.2 ± 16.7 mmHg after
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EVLP to 84.8 ± 17.7 mmHg after reperfusion (p<0.0001) and decreased only
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slightly in the ATL-E group from 431.7 ± 29.8 mmHg after EVLP to 413.6 ± 18.8
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mmHg after reperfusion. However, mean final values increased in the ATL-E/R
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group from 260.0 ± 75.3 mmHg after EVLP to 430.1 ± 26.4 mmHg after
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reperfusion (p=0.08).
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Physiologic parameters during EVLP
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Overall, mean values of peak airway pressure, dynamic compliance, and pulmonary vascular resistance (PVR) worsened throughout EVLP in all groups
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(Figure 3). Although certain individual parameters remained relatively stable or at
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times improved during EVLP, at least one of these three parameters in all donor
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lungs in all groups worsened by more than 15% between the 1st hour and 4th
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hour of EVLP (Figure 4), the threshold at which lungs would be deemed
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unsuitable for clinical lung transplantation [10].
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There were no differences among groups in mean percent change in peak airway pressure, dynamic compliance, or PVR throughout EVLP (Figure 4).
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Mean percent change in peak airway pressure and dynamic compliance were
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lowest in the DMSO group and highest in the ATL-E/R group. Mean percent
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change in PVR was highest in the ATL-E group.
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Cytokine Expression
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After 4 hours of reperfusion, there was a trend toward decreased
expression of proinflammatory cytokines (IFN-γ, IL-1β, IL-6, and IL-8) in the ATL-
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E and ATL-E/R groups compared to the DMSO group (Figure 5).
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Pulmonary Edema
There was no significant difference in pulmonary edema among groups as
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measured by total protein concentration in BAL fluid or mean wet-to-dry weight
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ratios (Figure 6).
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Histology
There was substantial variation in histologic appearance of lung injury
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within individual lung tissue sections. There was no difference in mean lung injury
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severity scores among groups (Figure 7), or in values of individual components
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of the overall score (neutrophil count, interstitial infiltration, and alveolar edema;
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not shown).
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Discussion
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This study demonstrates that PaO2/FiO2 ratios after transplantation of
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DCD donor lungs subjected to prolonged 12-hour cold preservation are
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significantly improved following treatment with an A2AR agonist during EVLP.
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Outcomes highlight the potential of EVLP with targeted A2AR agonist delivery to
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augment the available donor lung pool by increasing the use of marginal donor
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lungs, including DCD donor lungs, and by extending the traditionally recognized
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6-hour time constraint for cold preservation, thereby decreasing geographical
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restrictions of donor lung procurement. IRI manifests as PGD within the first 72 hours after lung transplantation.
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PGD severity is determined by oxygenation capacity and the radiographic
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appearance of pulmonary edema within the transplanted lung(s), with grade 0
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PGD occurring in the presence of PaO2/FiO2 ratios greater than 300 mmHg and
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normal lung fields and grades 1-3 PGD occurring in the presence of diffuse
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pulmonary infiltrates and PaO2/FiO2 ratios greater than 300 mmHg, between
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200-300 mmHg, and less than 200 mmHg, respectively [12], [13]. PGD severity
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correlates with outcomes after lung transplantation. Patients who develop grade
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3 PGD have significantly increased operative mortality [14] and higher rates of
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late mortality and impaired pulmonary function compared to patients with grade 0
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or 1 PGD [15]. In this study, final PaO2/FiO2 ratios of the transplanted left lungs
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from all animals in the ATL-E and ATL-E/R groups were above 300 mmHg, while
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final PaO2/FiO2 ratios of the transplanted left lungs from all animals in the DMSO
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group were below 200 mmHg. Although radiographic imaging was not performed
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in this study, there was no difference in pulmonary edema among groups. Thus,
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administration of ATL-1223 to donor lungs during EVLP appears to have reduced
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IRI from grade 3 PGD to grade 1 PGD. These results are similar to prior porcine
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models of lung transplantation demonstrating a reduction in IRI after A2AR
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agonist treatment during EVLP [11], [16] or during reperfusion [17], [18].
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Further administration of ATL-1223 during reperfusion did not significantly decrease IRI beyond that achieved by administration of ATL-1223 during EVLP
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alone, since PaO2/FiO2 ratios from the ATL-E/R and ATL-E groups were not
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different. This finding suggests that the inflammatory cascade mediating IRI is
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initiated by resident immune cells within the donor lung that are inactivated by
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ATL-1223 during EVLP, and that this inhibition persists during reperfusion.
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Previous studies have shown distinct populations of cells to be important at
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different stages of lung IRI: resident macrophages and invariant natural killer T
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cells in the donor lung in an early phase and circulating neutrophils in the
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recipient in a later phase [19], [20], [21]. In prior animal models of lung IRI,
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administration of an A2AR agonist to the donor animal before lung procurement
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decreased IRI to a similar extent as administration of the A2AR agonist during
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reperfusion, suggesting that both donor and recipient populations of immune
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cells are important in the development of lung IRI [18], [22]. In a rabbit model of
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lung IRI, pretreatment of the donor with the A2AR agonist resulted in a significant
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decrease in the number of pulmonary macrophages during reperfusion compared
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to treatment of the recipient at a later time [22]. Taken together, these studies
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support a role for early activation of resident immune cells in the donor lung in
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the initiation of IRI. EVLP provides an ideal platform for isolated drug delivery to
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inactivate these cells in marginal donor lungs, thereby avoiding side effects
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associated with systemic therapy in the donor or recipient.
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The accuracy of ex vivo evaluation in predicting lung function after transplantation has been shown to depend on physiologic parameters (peak
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airway pressure, dynamic compliance, and PVR) more than on PO2 gradients
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during EVLP [23]. In a previous clinical trial, marginal donor lungs rehabilitated
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with EVLP were transplanted if physiologic parameters did not deteriorate by
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more than 15% and if PO2 gradients were greater than 350 mmHg during 4
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hours of EVLP [10]. No single lung in the present study met these inclusion
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criteria after 4 hours of EVLP. Physiologic parameters and oxygenation capacity
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during EVLP were frequently superior in the DMSO group, yet these lungs were
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associated with severe IRI in the recipient. Conversely, lungs treated with ATL-
467
1223 often had inferior pulmonary function during EVLP, yet had mean
468
PaO2/FiO2 ratios greater than 400 mmHg after 4 hours of reperfusion.
469
Therefore, neither physiologic parameters nor PO2 gradients during EVLP were
470
predictive of lung function during reperfusion in this preclinical porcine model.
471
Future studies will require identification of more reliable markers of lung function
472
during EVLP that correlate with pulmonary function in the recipient before lungs
473
can be safely transplanted clinically.
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A related limitation of this study is the lack of data to explain how ATL-
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1223 administration promoted superior final PaO2/FiO2 ratios in the treated
476
groups. There was a trend toward decreased proinflammatory cytokine
477
expression in these groups, and statistical significance may have been achieved
478
with more animals per group. It is unclear why there was no difference in
479
pulmonary edema or histologic appearance of lung injury among groups. There
480
was considerable variation within individual lung tissue sections, which made
481
calculation of a single representative lung injury severity score somewhat
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challenging, and this score may not accurately reflect differences in IRI among
483
groups. Instead, the most established and clinically applicable measure of IRI is
484
the PaO2/FiO2 ratio after transplantation, which was significantly increased in
485
recipients of lungs treated with ATL-1223 during EVLP.
486
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Clinically, potential lung donors are evaluated with imaging modalities,
including CT and bronchoscopy, before a decision is made regarding donor lung
488
eligibility. Imaging of porcine donor lungs was not performed prior to procurement
489
in this study. Therefore, it was not possible to screen donor lungs for intrinsic
490
pulmonary disease. There were several instances in which it became
491
increasingly difficult to ventilate donor lungs during reperfusion in all groups due
492
to severe baseline pulmonary disease, and these animals were not included in
493
the final analysis. This demonstrates that pulmonary injury within a subset of
494
donor lungs is beyond rehabilitation with targeted ATL-1223 treatment during
495
EVLP. Moreover, severe IRI occurred in all control donor lungs without apparent
496
pulmonary disease. These findings show that EVLP alone without concomitant
497
ATL-1223 treatment is unable to prevent IRI after transplantation of DCD donor
498
lungs subjected to prolonged 12-hour cold preservation. Quantifying a threshold
499
of irreversible donor lung injury will be an important component of clinical
500
translation of animal models of EVLP.
501
Conclusions
502
EVLP with targeted A2AR agonist treatment attenuates IRI after transplantation
503
of DCD donor lungs subjected to prolonged 12-hour cold preservation in a
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preclinical porcine model. Pulmonary function during EVLP was not predictive of
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oxygenation capacity during reperfusion, as donor lungs that would have been
506
rejected clinically after EVLP had postoperative PaO2/FiO2 ratios above 400
507
mmHg if treated with an A2AR agonist during EVLP. Translation of this study
508
may significantly expand the donor lung pool.
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Acknowledgements
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The authors wish to thank Dr. Curtis Tribble and Dr. Benjamin Kozower for their
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mentorship and Sheila Hammond, Tony Herring, and Cindy Dodson for their
513
technical assistance.
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Gazoni LM, Laubach VE, Mulloy DP, Bellizzi A, Unger EB, Linden J,
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627 628 629 630
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Sato M, Waddell TK, Liu M, Slutsky AS, Keshavjee S, Physiologic
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Figure Legends
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Figure 1. Comparison of initial and final PaO2/FiO2 ratios among groups. Initial
653
donor PaO2/FiO2 ratios were obtained before procurement. Final PaO2/FiO2
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ratios of donor lungs were determined from samples obtained from the left
655
superior and inferior pulmonary veins after 4 hours of reperfusion. *p < 0.0001 vs.
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DMSO.
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Figure 2. Trends in PO2 gradients throughout 4 hours of EVLP in DMSO, ATL-E,
659
and ATL-E/R groups. The red line represents the 350 mmHg threshold for clinical
660
lung transplantation.
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Figure 3. Changes in physiologic parameters throughout 4 hours of EVLP. Peak
663
airway pressure, dynamic compliance, and pulmonary vascular resistance in the
664
DMSO, ATL-E, and ATL-E/R groups.
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Figure 4. Percent change in peak airway pressure, dynamic compliance, and
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pulmonary vascular resistance between the 1st hour and the 4th hour of EVLP
668
among groups. The red line represents the 15% threshold for clinical lung
669
transplantation.
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Figure 5. Proinflammatory cytokine levels in lung tissue after 4 hours of
672
reperfusion.
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Figure 6. Pulmonary edema after 4 hours of reperfusion as measured by total
675
protein concentration in BAL fluid and lung wet-to-dry weight ratios.
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Figure 7. (Top) Representative lung histology (hematoxylin and eosin stain at
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20X magnification) after 4 hours of reperfusion in DMSO, ATL-E, and ATL-E/R
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groups. (Bottom) Comparison of mean lung injury severity scores among groups
680
as determined from histology.
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S0022-5223(15)01293-3
DOI:
10.1016/j.jtcvs.2015.07.075
Reference:
YMTC 9779
To appear in:
The Journal of Thoracic and Cardiovascular Surgery
Received Date: 28 April 2015 Revised Date:
13 July 2015
Accepted Date: 19 July 2015
Please cite this article as: Wagner CE, Pope NH, Charles EJ, Huerter ME, Sharma AK, Salmon MD, Carter BT, Stoler MH, Lau CL, Laubach VE, Kron IL, Ex vivo lung perfusion with adenosine A2A receptor agonist allows prolonged cold preservation of donation after cardiac death donor lungs, The Journal of Thoracic and Cardiovascular Surgery (2015), doi: 10.1016/j.jtcvs.2015.07.075. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ex vivo lung perfusion with adenosine A2A receptor agonist allows prolonged cold preservation of donation after cardiac death donor lungs
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Nicolas H. Pope, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: T32 HL007849, R01 HL130053 Conflicts of interest: none
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Cynthia E. Wagner, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: T32 HL007849, R01 HL130053 Conflicts of interest: none
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Cynthia E. Wagner MD, Nicolas H. Pope MD, Eric J. Charles MD, Mary E. Huerter MD, Ashish K. Sharma MBBS, PhD, Morgan D. Salmon PhD, Benjamin T. Carter BS, Mark H. Stoler MD, Christine L. Lau MD, MBA, Victor E. Laubach PhD, Irving L. Kron MD
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Eric J. Charles, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: T32 HL007849, R01 HL130053 Conflicts of interest: none
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Mary E. Huerter, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none
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Ashish K. Sharma, MBBS, PhD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none Morgan D. Salmon, PhD University of Virginia Department of Surgery
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Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none
Mark H. Stoler, MD University of Virginia Department of Pathology Funding: none Conflicts of interest: none
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Christine L. Lau, MD, MBA University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none
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Benjamin T. Carter, BS University of Virginia School of Medicine Funding: R01 HL130053 Conflicts of interest: none
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Victor E. Laubach, PhD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none
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Irving L. Kron, MD University of Virginia Department of Surgery Division of Thoracic and Cardiovascular Surgery Funding: R01 HL130053 Conflicts of interest: none PO Box 800679 Charlottesville, VA 22908 Phone: 434-924-2158 Fax: 434-982-3885 Email: [email protected]
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Abstract word count: 250 Article word count: 3500
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Abbreviations and Acronyms
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= adenosine A2A receptor = arterial blood gas = analysis of variance = A2AR agonist administration during EVLP = A2AR agonist administration during EVLP and reperfusion = bronchoalveolar lavage = bicinchoninic acid = bovine serum albumin = computed tomography = donation after brain death = donation after cardiac death = dimethyl sulfoxide = ethylenediaminetetraacetic acid = enzyme-linked immunosorbent assay = endotracheal tube = ex vivo lung perfusion = fraction of inspired oxygen = interferon = interleukin = ischemia-reperfusion injury = left atrium = left atrial appendage = left lower lobe = left upper lobe = millimeter of mercury = pulmonary artery = arterial oxygen partial pressure = phosphate buffered saline = positive end expiratory pressure = primary graft dysfunction = oxygen partial pressure = pulmonary vascular resistance = respiratory rate = tidal volume
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A2AR ABG ANOVA ATL-E ATL-E/R BAL BCA BSA CT DBD DCD DMSO EDTA ELISA ETT EVLP FiO2 IFN IL IRI LA LAA LLL LUL mmHg PA PaO2 PBS PEEP PGD PO2 PVR RR TV
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Objective: Ex vivo lung perfusion (EVLP) has been successful in the assessment of marginal donor lungs, including donation after cardiac death (DCD) donor lungs. EVLP also represents a unique platform for targeted drug delivery. We sought to determine if ischemia-reperfusion injury (IRI) would be decreased after transplantation of DCD donor lungs subjected to prolonged cold preservation and treated with an adenosine A2A receptor (A2AR) agonist during EVLP. Methods: Porcine DCD donor lungs were preserved at 4°C for 12 hours and underwent EVLP for 4 hours. Left lungs were then transplanted and reperfused for 4 hours. Three groups (n=4/group) were randomized according to treatment with the A2AR agonist ATL-1223 or the dimethyl sulfoxide (DMSO) vehicle: DMSO (infusion of DMSO during EVLP and reperfusion), ATL-E (infusion of ATL1223 during EVLP and DMSO during reperfusion), and ATL-E/R (infusion of ATL1223 during EVLP and reperfusion). Final PaO2/FiO2 ratios were determined from samples obtained from the left superior and inferior pulmonary veins. Results: Final PaO2/FiO2 ratios in the ATL-E/R group (430.1 ± 26.4 mmHg) were similar to final PaO2/FiO2 ratios in the ATL-E group (413.6 ± 18.8 mmHg), but both treated groups had significantly higher final PaO2/FiO2 ratios compared to the DMSO group (84.8 ± 17.7 mmHg). Low PO2 gradients during EVLP did not preclude superior postoperative oxygenation capacity. Conclusions: Following prolonged cold preservation, treatment of DCD donor lungs with an A2AR agonist during EVLP enabled PaO2/FiO2 ratios above 400 mmHg after transplantation in a preclinical porcine model. Pulmonary function during EVLP was not predictive of outcomes after transplantation.
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The number of patients on the waiting list continues to exceed the number
185
of patients who undergo lung transplantation each year, though recent advances
186
in donor selection and lung preservation have the potential to close this gap [1].
187
Lungs are usually selected from donation after brain death (DBD) donors, yet
188
only 15-20% of these lungs are deemed suitable and procured for transplantation
189
after conservative evaluation. Marginal donor lungs, including those from
190
donation after cardiac death (DCD) donors, represent an additional source for
191
transplantation, but use of these lungs has been linked to increased mortality and
192
primary graft dysfunction (PGD) in some studies [2], [3]. PGD is mediated by
193
ischemia-reperfusion injury (IRI) and is associated with significant early and late
194
mortality [4], [5] as well as progression to bronchiolitis obliterans syndrome and
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delayed graft failure [6], [7].
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ischemic storage but inhibits cellular regeneration that may be crucial before
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marginal donor lungs are transplanted. Normothermic ex vivo lung perfusion
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(EVLP) was initially developed to assess function of lungs from DCD donors prior
200
to transplantation [8]. EVLP was subsequently modified to allow prolonged
201
preservation of marginal donor lungs [9], and a pivotal clinical trial established
202
non-inferior outcomes after transplantation of marginal donor lungs passively
203
rehabilitated with EVLP compared to conventional donor lungs [10]. EVLP also
204
offers a unique platform for targeted drug delivery to actively treat marginal donor
205
lungs. We have previously demonstrated that the adenosine A2A receptor
206
(A2AR) agonist ATL-1223 exerts anti-inflammatory effects and reduces IRI when
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administered to DCD donor lungs during EVLP in a porcine model of lung
208
transplantation [11].
209
The purpose of this study was to determine if administration of ATL-1223 during EVLP would decrease IRI after transplantation of DCD donor lungs
211
subjected to prolonged 12-hour cold preservation, and if further administration
212
during reperfusion would augment this protection in a preclinical porcine model,
213
with important implications to expand the donor lung pool by including DCD
214
donors and allowing cross-country retrieval. We hypothesized that IRI would be
215
reduced after transplantation of donor lungs treated with ATL-1223 during EVLP
216
compared to lungs that received dimethyl sulfoxide (DMSO) vehicle, and that IRI
217
would be further attenuated by continued administration of ATL-1223 to the
218
recipient during reperfusion.
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Materials and Methods
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Animals
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This study was approved by the Institutional Animal Care and Use
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Committee at the University of Virginia. Animals received humane treatment in
223
accordance with the “Guide for Care and Use of Laboratory Animals” from the
224
National Institutes of Health.
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Study Design
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Mature domestic swine (19-35 kg) of both genders were randomized to 3
227
different groups (n=4/group) based on treatment with the A2AR agonist ATL-
228
1223. Donor swine underwent hypoxic arrest followed by 15 minutes of warm
229
ischemia. Lungs were procured and preserved at 4°C for 12 hours before
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undergoing normothermic EVLP for 4 hours. Left lungs were then transplanted
231
into size-matched recipients and reperfused for 4 hours. The DMSO group
232
received an infusion of DMSO (vehicle) during EVLP and reperfusion. The ATL-E
233
group received an infusion of ATL-1223 during EVLP and an infusion of DMSO
234
during reperfusion, and the ATL-E/R group received an infusion of ATL-1223
235
during EVLP and reperfusion.
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Donor Lung Procurement
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Donor animals were anesthetized, weighed, and prepped for a median sternotomy. Following induction, animals were intubated and maintained under
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general anesthesia with isoflurane. Animals were ventilated with 100% oxygen
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before initial donor arterial blood gas (ABG) analysis. Heparin was not
241
administered to donor animals. The endotracheal tube (ETT) was clamped and
242
donor animals underwent hypoxic arrest. Death was confirmed by cessation of
243
electrocardiographic activity and absence of heart sounds. Animals were not
244
touched for a 15-minute period of warm ischemia. Lungs were ventilated
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throughout the last 5 minutes of this time. A median sternotomy was performed.
246
The main pulmonary artery (PA) was cannulated near the bifurcation with a
247
cardioplegia cannula and 500 mcg prostaglandin E1 (Pfizer Inc) was
248
administered. The main PA was clamped proximally, the left atrial appendage
249
(LAA) was vented, and lungs were flushed with 1.5 L of 4°C Perfadex (XVIVO
250
Perfusion, Englewood, CO). Topical cold slush was applied. At the completion of
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the flush, the heart and lungs were removed en bloc. A funnel-shaped cannula
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with a pressure port (XVIVO Perfusion, Englewood, CO) was sewn to the left
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atrial (LA) cuff with a running 4-0 prolene suture, a cannula with a pressure port
254
(XVIVO Perfusion, Englewood, CO) was tied within the main PA, and a 7.0 mm
255
ETT with the balloon removed was tied within the trachea. Lungs were inflated
256
with gentle positive end expiratory pressure (PEEP) and the ETT was clamped. A
257
500 ml retrograde flush with 4°C Perfadex was then performed. The lungs were
258
submerged in 4°C Perfadex, surrounded by a double layer of cold slush, and
259
stored at 4°C for 12 hours. Lungs were flushed antegrade with 500 ml 4°C
260
Perfadex immediately prior to EVLP.
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Ex Vivo Lung Perfusion
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The EVLP circuit was primed with 2 L Steen solution (XVIVO Perfusion, Englewood, CO) supplemented with 10,000 units heparin, 500 mg
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methylprednisolone, and 500 mg cefazolin. Perfusate drained from the venous
265
(LA) cannula into the venous reservoir and was warmed to 37°C, deoxygenated
266
with a mixture of 86% nitrogen, 8% carbon dioxide, and 6% oxygen gas, and
267
pumped into the arterial (PA) cannula (Medtronic BioMedicus centrifugal pump,
268
Eden Prairie, MN). Lungs were placed within the EVLP chamber (XVIVO
269
Perfusion, Englewood, CO), de-aired, and low flow (0.15 ml/min) through the
270
lungs was established to initiate EVLP. The height of the venous reservoir was
271
adjusted to maintain LA pressures between 0-5 mmHg. PA pressures were
272
dependent on perfusate flow. Throughout the first 30 minutes of EVLP, flow was
273
titrated up to 40% of estimated cardiac output (100 ml/kg/min) and perfusate was
274
warmed to 37°C. After 30 minutes of EVLP, lungs were ventilated on room air
275
(TV 10 ml/kg, RR 8 bpm, PEEP 5 cm H2O). EVLP was performed for 4 hours.
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Oxygenation was assessed every hour by increasing the FiO2 to 100% for 15
277
minutes prior to PO2 analysis. Samples taken from the venous and arterial arms
278
of the circuit allowed calculation of the oxygenation gradient across the
279
pulmonary vasculature. Other parameters recorded every hour included LA
280
pressure, PA pressure, peak airway pressure, mean airway pressure, and
281
plateau pressure. The A2AR agonist ATL-1223 (Lewis and Clark
282
Pharmaceuticals, Charlottesville, VA) was administered as an infusion (3.0
283
ng/kg/min) throughout all 4 hours of EVLP. The same weight-based volume of
284
DMSO was administered as an infusion throughout EVLP. At the conclusion of
285
EVLP, donor lungs were flushed antegrade with 500 ml of 4°C Perfadex. Lungs
286
were separated on the back table and the donor left lung was cooled with topical
287
cold slush for 15 minutes prior to transplantation.
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Left Lung Transplantation
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Recipient animals were anesthetized, weighed, and prepped for a left thoracotomy. Animals were intubated and maintained under general anesthesia
291
with isoflurane. A central venous line with a PA catheter, arterial line, and
292
oxygenation saturation probe were placed. Initial recipient ABG analysis was
293
performed on 100% oxygen and baseline vitals were recorded, including heart
294
rate, blood pressure, PA pressure, and oxygenation saturation. A left
295
thoracotomy was performed and 50 mg lidocaine were administered prior to
296
dissection of the left hilum. Before clamping the left PA and ligating the left
297
superior and inferior pulmonary veins, 5,000 units of heparin were given and
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allowed to circulate for 5 minutes. The left mainstem bronchus was clamped, the
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pneumonectomy was completed, and the donor lung was placed in the chest.
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The bronchial anastomosis was completed with a running 4-0 prolene suture and
301
the PA anastomosis was then completed with a running 5-0 prolene suture. The
302
recipient LAA was clamped, opened, and sewn to the donor LA cuff with a
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running 5-0 prolene suture. Clamps were removed to establish reperfusion and
304
ventilation. ABG analysis was performed and vitals were recorded every 15-20
305
minutes during all 4 hours of reperfusion. Animals were ventilated with 100%
306
oxygen throughout reperfusion. Intermittent gentle PEEP was applied to reduce
307
atelectasis. Acid-base status was optimized by adjusting respiratory rate at a
308
constant tidal volume of 10 ml/kg. Intravenous fluids were given judiciously, and
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epinephrine was administered as needed. The A2AR agonist ATL-1223 was
310
administered at 3.0 ng/kg/min or an equivalent weight-based volume of DMSO
311
was given as an infusion throughout reperfusion. ABG samples were obtained
312
from the left superior and inferior pulmonary veins to determine isolated
313
oxygenation capacity of the transplanted left lung at the conclusion of
314
reperfusion. Mean values were reported as final PaO2/FiO2 ratios. A final
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systemic ABG analysis was performed, the recipient was euthanized with
316
Euthasol (Virbac AH, Inc), and the left lung was explanted.
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Cytokine Expression
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Lung tissue samples were obtained from the left lower lobe (LLL), frozen
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at -80°C, and homogenized using a gentleMACS tissue dissociator (Miltenyi
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Biotec, Auburn, CA) in a solution containing PBS, 0.5% BSA, and 2mM EDTA.
321
Total protein concentration was determined using a BCA protein assay (Pierce,
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Rockford, IL). ELISAs were performed to determine expression of interferon-γ
323
(IFN-γ) (RayBiotech, Inc., Norcross, GA) and interleukin 1β (IL-1β), interleukin 6
324
(IL-6), and interleukin 8 (IL-8) (Abcam, Cambridge, MA). One sample in the ATL-
325
E/R group had insufficient volume for the latter analyses. Cytokine
326
concentrations were reported per 50 µg total protein in each sample.
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Pulmonary Edema
A bronchoalveolar lavage (BAL) of the left upper lobe (LUL) was
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performed with 50 ml normal saline. Total protein concentration was determined
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in BAL samples using a BCA protein assay as a measure of pulmonary edema.
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Lung tissue samples from the LUL (n=2/animal) and LLL (n=3/animal) were
332
weighed immediately and again after a stable weight was achieved through
333
desiccation in a vacuum oven. Wet-to-dry weight ratios were determined as an
334
additional measure of pulmonary edema. The wet-to-dry weight ratio for each
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lung was calculated as the mean of the ratios from all 5 lung tissue samples.
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Histology
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was submerged in formalin overnight. Lung tissue samples from the LUL
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(n=2/animal) and LLL (n=3/animal) were placed in 70% ethanol, embedded in
340
paraffin, and stained with hematoxylin and eosin. The lung injury severity score
341
was determined by a pathologist blinded to treatment group. The score ranged
342
from 0-9 and was a composite sum of 3 individual components, including number
343
of neutrophils per high-powered field (score of 0, <5; 1, 6 to 10; 2, 11 to 20; and
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score of 3, >20), interstitial infiltration (score of 0, none; 1, minimal; 2, moderate;
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and 3, severe), and alveolar edema (score of 0, < 5%; 1, 6%-25%; 2, 26%-50%;
346
and 3, > 50%). The lung injury severity score for each lung was calculated as the
347
mean of the scores from all 5 lung tissue samples.
348
Statistical Analysis
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Experimental methods were designed to test the null hypothesis that no
significant differences in lung IRI would be observed despite treatment with ATL-
351
1223. Differences among groups were calculated using one-way ANOVA, and
352
pairwise comparisons were made using Tukey’s multiple comparisons test
353
(GraphPad Prism version 6.0e). An unpaired t test was used to calculate
354
differences between time points. Data is expressed as the mean ± standard error
355
of the mean. A p value less than 0.05 was considered statistically significant.
356
Results
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Donor lung oxygenation during reperfusion
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There was no difference in mean initial PaO2/FiO2 ratios of donor animals
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among groups (Figure 1). However, after 4 hours of reperfusion, final PaO2/FiO2
360
ratios of donor lungs in both groups treated with ATL-1223 (ATL-E: 413.6 ± 18.8
361
mmHg; ATL-E/R: 430.1 ± 26.4 mmHg) were significantly higher than final
362
PaO2/FiO2 ratios of control donor lungs (DMSO: 84.8 ± 17.7 mmHg) (p<0.0001).
363
Further administration of ATL-1223 during reperfusion did not significantly
364
increase final PaO2/FiO2 ratios in the ATL-E/R group compared to the ATL-E
365
group (p=0.85) (Figure 1).
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Mean PaO2/FiO2 ratios of control donor lungs significantly worsened from baseline measurement in the donor animal to final assessment in the recipient.
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However, there was no significant difference between initial and final mean
369
PaO2/FiO2 ratios of donor lungs in both groups treated with ATL-1223.
370
Donor lung oxygenation during EVLP Overall, mean PO2 gradients decreased from the first hour to the fourth
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hour of EVLP in all groups (Figure 2). Trends in PO2 gradients of individual lungs
373
were highly variable throughout EVLP and were frequently below the 350 mmHg
374
threshold considered acceptable for clinical lung transplantation [10]. Mean final
375
values decreased significantly in the DMSO group from 334.2 ± 16.7 mmHg after
376
EVLP to 84.8 ± 17.7 mmHg after reperfusion (p<0.0001) and decreased only
377
slightly in the ATL-E group from 431.7 ± 29.8 mmHg after EVLP to 413.6 ± 18.8
378
mmHg after reperfusion. However, mean final values increased in the ATL-E/R
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group from 260.0 ± 75.3 mmHg after EVLP to 430.1 ± 26.4 mmHg after
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reperfusion (p=0.08).
381
Physiologic parameters during EVLP
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Overall, mean values of peak airway pressure, dynamic compliance, and pulmonary vascular resistance (PVR) worsened throughout EVLP in all groups
384
(Figure 3). Although certain individual parameters remained relatively stable or at
385
times improved during EVLP, at least one of these three parameters in all donor
386
lungs in all groups worsened by more than 15% between the 1st hour and 4th
387
hour of EVLP (Figure 4), the threshold at which lungs would be deemed
388
unsuitable for clinical lung transplantation [10].
389 390
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There were no differences among groups in mean percent change in peak airway pressure, dynamic compliance, or PVR throughout EVLP (Figure 4).
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Mean percent change in peak airway pressure and dynamic compliance were
392
lowest in the DMSO group and highest in the ATL-E/R group. Mean percent
393
change in PVR was highest in the ATL-E group.
394
Cytokine Expression
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After 4 hours of reperfusion, there was a trend toward decreased
expression of proinflammatory cytokines (IFN-γ, IL-1β, IL-6, and IL-8) in the ATL-
397
E and ATL-E/R groups compared to the DMSO group (Figure 5).
398
Pulmonary Edema
There was no significant difference in pulmonary edema among groups as
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measured by total protein concentration in BAL fluid or mean wet-to-dry weight
401
ratios (Figure 6).
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Histology
There was substantial variation in histologic appearance of lung injury
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within individual lung tissue sections. There was no difference in mean lung injury
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severity scores among groups (Figure 7), or in values of individual components
406
of the overall score (neutrophil count, interstitial infiltration, and alveolar edema;
407
not shown).
408
Discussion
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This study demonstrates that PaO2/FiO2 ratios after transplantation of
410
DCD donor lungs subjected to prolonged 12-hour cold preservation are
411
significantly improved following treatment with an A2AR agonist during EVLP.
412
Outcomes highlight the potential of EVLP with targeted A2AR agonist delivery to
413
augment the available donor lung pool by increasing the use of marginal donor
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lungs, including DCD donor lungs, and by extending the traditionally recognized
415
6-hour time constraint for cold preservation, thereby decreasing geographical
416
restrictions of donor lung procurement. IRI manifests as PGD within the first 72 hours after lung transplantation.
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PGD severity is determined by oxygenation capacity and the radiographic
419
appearance of pulmonary edema within the transplanted lung(s), with grade 0
420
PGD occurring in the presence of PaO2/FiO2 ratios greater than 300 mmHg and
421
normal lung fields and grades 1-3 PGD occurring in the presence of diffuse
422
pulmonary infiltrates and PaO2/FiO2 ratios greater than 300 mmHg, between
423
200-300 mmHg, and less than 200 mmHg, respectively [12], [13]. PGD severity
424
correlates with outcomes after lung transplantation. Patients who develop grade
425
3 PGD have significantly increased operative mortality [14] and higher rates of
426
late mortality and impaired pulmonary function compared to patients with grade 0
427
or 1 PGD [15]. In this study, final PaO2/FiO2 ratios of the transplanted left lungs
428
from all animals in the ATL-E and ATL-E/R groups were above 300 mmHg, while
429
final PaO2/FiO2 ratios of the transplanted left lungs from all animals in the DMSO
430
group were below 200 mmHg. Although radiographic imaging was not performed
431
in this study, there was no difference in pulmonary edema among groups. Thus,
432
administration of ATL-1223 to donor lungs during EVLP appears to have reduced
433
IRI from grade 3 PGD to grade 1 PGD. These results are similar to prior porcine
434
models of lung transplantation demonstrating a reduction in IRI after A2AR
435
agonist treatment during EVLP [11], [16] or during reperfusion [17], [18].
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Further administration of ATL-1223 during reperfusion did not significantly decrease IRI beyond that achieved by administration of ATL-1223 during EVLP
438
alone, since PaO2/FiO2 ratios from the ATL-E/R and ATL-E groups were not
439
different. This finding suggests that the inflammatory cascade mediating IRI is
440
initiated by resident immune cells within the donor lung that are inactivated by
441
ATL-1223 during EVLP, and that this inhibition persists during reperfusion.
442
Previous studies have shown distinct populations of cells to be important at
443
different stages of lung IRI: resident macrophages and invariant natural killer T
444
cells in the donor lung in an early phase and circulating neutrophils in the
445
recipient in a later phase [19], [20], [21]. In prior animal models of lung IRI,
446
administration of an A2AR agonist to the donor animal before lung procurement
447
decreased IRI to a similar extent as administration of the A2AR agonist during
448
reperfusion, suggesting that both donor and recipient populations of immune
449
cells are important in the development of lung IRI [18], [22]. In a rabbit model of
450
lung IRI, pretreatment of the donor with the A2AR agonist resulted in a significant
451
decrease in the number of pulmonary macrophages during reperfusion compared
452
to treatment of the recipient at a later time [22]. Taken together, these studies
453
support a role for early activation of resident immune cells in the donor lung in
454
the initiation of IRI. EVLP provides an ideal platform for isolated drug delivery to
455
inactivate these cells in marginal donor lungs, thereby avoiding side effects
456
associated with systemic therapy in the donor or recipient.
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The accuracy of ex vivo evaluation in predicting lung function after transplantation has been shown to depend on physiologic parameters (peak
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airway pressure, dynamic compliance, and PVR) more than on PO2 gradients
460
during EVLP [23]. In a previous clinical trial, marginal donor lungs rehabilitated
461
with EVLP were transplanted if physiologic parameters did not deteriorate by
462
more than 15% and if PO2 gradients were greater than 350 mmHg during 4
463
hours of EVLP [10]. No single lung in the present study met these inclusion
464
criteria after 4 hours of EVLP. Physiologic parameters and oxygenation capacity
465
during EVLP were frequently superior in the DMSO group, yet these lungs were
466
associated with severe IRI in the recipient. Conversely, lungs treated with ATL-
467
1223 often had inferior pulmonary function during EVLP, yet had mean
468
PaO2/FiO2 ratios greater than 400 mmHg after 4 hours of reperfusion.
469
Therefore, neither physiologic parameters nor PO2 gradients during EVLP were
470
predictive of lung function during reperfusion in this preclinical porcine model.
471
Future studies will require identification of more reliable markers of lung function
472
during EVLP that correlate with pulmonary function in the recipient before lungs
473
can be safely transplanted clinically.
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A related limitation of this study is the lack of data to explain how ATL-
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1223 administration promoted superior final PaO2/FiO2 ratios in the treated
476
groups. There was a trend toward decreased proinflammatory cytokine
477
expression in these groups, and statistical significance may have been achieved
478
with more animals per group. It is unclear why there was no difference in
479
pulmonary edema or histologic appearance of lung injury among groups. There
480
was considerable variation within individual lung tissue sections, which made
481
calculation of a single representative lung injury severity score somewhat
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challenging, and this score may not accurately reflect differences in IRI among
483
groups. Instead, the most established and clinically applicable measure of IRI is
484
the PaO2/FiO2 ratio after transplantation, which was significantly increased in
485
recipients of lungs treated with ATL-1223 during EVLP.
486
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Clinically, potential lung donors are evaluated with imaging modalities,
including CT and bronchoscopy, before a decision is made regarding donor lung
488
eligibility. Imaging of porcine donor lungs was not performed prior to procurement
489
in this study. Therefore, it was not possible to screen donor lungs for intrinsic
490
pulmonary disease. There were several instances in which it became
491
increasingly difficult to ventilate donor lungs during reperfusion in all groups due
492
to severe baseline pulmonary disease, and these animals were not included in
493
the final analysis. This demonstrates that pulmonary injury within a subset of
494
donor lungs is beyond rehabilitation with targeted ATL-1223 treatment during
495
EVLP. Moreover, severe IRI occurred in all control donor lungs without apparent
496
pulmonary disease. These findings show that EVLP alone without concomitant
497
ATL-1223 treatment is unable to prevent IRI after transplantation of DCD donor
498
lungs subjected to prolonged 12-hour cold preservation. Quantifying a threshold
499
of irreversible donor lung injury will be an important component of clinical
500
translation of animal models of EVLP.
501
Conclusions
502
EVLP with targeted A2AR agonist treatment attenuates IRI after transplantation
503
of DCD donor lungs subjected to prolonged 12-hour cold preservation in a
504
preclinical porcine model. Pulmonary function during EVLP was not predictive of
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oxygenation capacity during reperfusion, as donor lungs that would have been
506
rejected clinically after EVLP had postoperative PaO2/FiO2 ratios above 400
507
mmHg if treated with an A2AR agonist during EVLP. Translation of this study
508
may significantly expand the donor lung pool.
509
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Acknowledgements
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The authors wish to thank Dr. Curtis Tribble and Dr. Benjamin Kozower for their
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mentorship and Sheila Hammond, Tony Herring, and Cindy Dodson for their
513
technical assistance.
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Figure Legends
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Figure 1. Comparison of initial and final PaO2/FiO2 ratios among groups. Initial
653
donor PaO2/FiO2 ratios were obtained before procurement. Final PaO2/FiO2
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ratios of donor lungs were determined from samples obtained from the left
655
superior and inferior pulmonary veins after 4 hours of reperfusion. *p < 0.0001 vs.
656
DMSO.
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Figure 2. Trends in PO2 gradients throughout 4 hours of EVLP in DMSO, ATL-E,
659
and ATL-E/R groups. The red line represents the 350 mmHg threshold for clinical
660
lung transplantation.
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Figure 3. Changes in physiologic parameters throughout 4 hours of EVLP. Peak
663
airway pressure, dynamic compliance, and pulmonary vascular resistance in the
664
DMSO, ATL-E, and ATL-E/R groups.
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Figure 4. Percent change in peak airway pressure, dynamic compliance, and
667
pulmonary vascular resistance between the 1st hour and the 4th hour of EVLP
668
among groups. The red line represents the 15% threshold for clinical lung
669
transplantation.
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Figure 5. Proinflammatory cytokine levels in lung tissue after 4 hours of
672
reperfusion.
673
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Figure 6. Pulmonary edema after 4 hours of reperfusion as measured by total
675
protein concentration in BAL fluid and lung wet-to-dry weight ratios.
676
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Figure 7. (Top) Representative lung histology (hematoxylin and eosin stain at
678
20X magnification) after 4 hours of reperfusion in DMSO, ATL-E, and ATL-E/R
679
groups. (Bottom) Comparison of mean lung injury severity scores among groups
680
as determined from histology.
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