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Although lung transplant is a life-saving therapy for some patients, primary graft dysfunction (PGD) is a leading cause of mortality and morbidity soon after a transplant. Ischemia reperfusion injury is known to be one of the most critical factors in PGD development. PGD is by definition an acute lung injury syndrome that occurs during the first 3 d following lung transplantation. To successfully translate laboratory discoveries to clinical practice, a reliable and practical large animal model is critical. This protocol describes a surgical technique for swine lung transplantation and postoperative management for a further 3 d post transplant. The protocol includes the background and rationale, required supplies, and a detailed description of the donor operation, transplant surgery, postoperative care, and sacrifice surgery. A pig lung transplant model is reliably produced in which the recipients survive for 3 d post transplant. This 3-d survival model can be used by lung transplant researchers to assess the development of PGD and to test therapeutic strategies targeting PGD. In total, the protocol requires 5 h for the surgeries, plus ~2 h in total for the postoperative care.

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Neutrophil extracellular traps in ex vivo lung perfusion perfusate predict the clinical outcome of lung transplant recipients To the Editor: Lung transplantation is lifesaving for patients with end-stage lung diseases. However, a scarcity of donor lungs suitable for transplantation limits the number of procedures that can be performed. Furthermore, major obstacles to successful lung transplantation still exist; primary graft dysfunction (PGD), the leading cause of early morbidity and mortality post-lung transplant, develops in ∼15-30% of recipients [1]. Normothermic ex vivo lung perfusion (EVLP) maintains donor lungs in a physiological state through perfusion and ventilation, so that the lungs can be assessed, treated and accepted for transplantation, thereby increasing the donor pool and alleviating the severe shortage of viable donor lungs [2]. In addition to donor lung assessment, EVLP provides a unique platform to study cellular processes occurring in donor lungs. EVLP perfusate is a unique, noninvasive source of biological sampling, which offers valuable information about the state of the donor lung. Rapid, molecular diagnostic testing could be an important addition to assessment of donor lungs, and may help improve outcome [3-7]. Detection of injurious markers in EVLP perfusate may also help inform future targeted treatments for the lungs, which could be administered directly to the organ through the EVLP circuit. In order to develop a reliable molecular assessment of donor lungs, a stable, easily quantifiable biomarker that reflects donor lung injury is required. Neutrophil extracellular traps (NETs) are structures comprised of extracellular neutrophil DNA in a complex with antimicrobial granular proteins, such as neutrophil elastase (NE) and myeloperoxidase. Neutrophils form NETs in response to innate immune challenges. Although NETs are generally considered antibacterial, several recent studies suggest that the cytotoxicity of NETs can be highly detrimental to lung and other tissue through complement activation, exposed histone cytotoxicity, promotion of thrombosis, and propagation of neutrophil recruitment and activation [8-11]. NETs have been specifically implicated in a variety of lung disease and injury states, including ventilator-induced lung injury and PGD [12, 13]. NETs play an important role in lung injury, but their role in lung transplantation is largely unknown. As NETs are a quantifiable measure of the innate immune response to a variety of inflammatory stimuli, this study aimed to determine whether NETs are detectable in EVLP perfusate, indicating their production by neutrophils in donor lungs. Furthermore, this study assessed the ability of NETs to serve as a relevant biomarker of donor lung injury. We report that elevated NET levels in donor lung EVLP perfusate are associated with worse recipient outcomes. Written, informed consent was obtained, and this study was approved by the University Health Network Research Ethics Board. Clinical perfusate samples from consecutive EVLP cases that w...

Abbreviations: CBCT, C-arm cone-beam computed tomography; cDCD, controlled donation after circulatory death; Cdyn, dynamic compliance; CSP, cold static preservation; Csta, static compliance; DBD, donation after brain death; ELISA, enzyme-linked immunosorbent assay; EVLP, ex vivo lung perfusion; IL, interleukin; PawP, peak airway pressure; PEEP, positive end-expiratory pressure; Pplat, airway plateau pressure; PVR, pulmonary vascular resistance; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling; WDR, wet-to-dry weight ratio; WIT, warm ischemic time; WLST, withdrawal of life-sustaining therapy; ZO-1, Zonula occludens protein-1.A large proportion of controlled donation after circulatory death (cDCD) donor lungs are declined because cardiac arrest does not occur within a suitable time after the withdrawal of life-sustaining therapy. Improved strategies to preserve lungs after asystole may allow the recovery team to arrive after death actually occurs and enable the recovery of lungs from more cDCD donors. The aim of this study was to determine the effect of donor positioning on the quality of lung preservation after cardiac arrest in a cDCD model. Cardiac arrest was induced by withdrawal of ventilation under anesthesia in pigs. After asystole, animals were divided into 2 groups based on body positioning (supine or prone). All animals were subjected to 3 hours of warm ischemia. After the observation period, donor lungs were explanted and preserved at 4°C for 6 hours, followed by 6 hours of physiologic and biological lung assessment under normothermic ex vivo lung perfusion. Donor lungs from the prone group displayed significantly greater quality as reflected by better function during ex vivo lung perfusion, less edema formation, less cell death, and decreased inflammation compared with the supine group. A simple maneuver of donor prone positioning after cardiac arrest significantly improves lung graft preservation and function. K E Y W O R D Sanimal models: porcine, basic (laboratory) research/science, donors and donation: donation after circulatory death (DCD), donors and donation: extended criteria, lung transplantation/ pulmonology, organ perfusion and preservation, organ procurement, organ procurement and allocation, translational research/science

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