Prolonged Warm Ischemia Prevents Lung Allograft Acceptance in Lung Transplantation from Donation after Cardiac Death in the Mouse

Abstracts S143

3( 68) Pre- and Reconditioning of DCD Donor Lungs on Ex-Vivo Lung Perfusion by Methylprednisolone A. Martens ,1 D.E. Van Raemdonck,2 B.M. Vanaudenaerde,3 S.E. Verleden,3 G.M. Verleden,3 R. Vos,3 A.P. Neyrinck.1  1Cardiovascular Sciences Anesthesiology and Algology, KU Leuven, Leuven, Belgium; 2Department of Clinical and Experimental Medicine - Experimental Thoracic Surgery, KU Leuven, Leuven, Belgium; 3Department of Clinical and Experimental Medicine - Pneumology, KU Leuven, Leuven, Belgium. Purpose: Lung donation after circulatory death (DCD) is an accepted possibility to increase the number of transplantable organs. However, the role and timing of steroid administration in the DCD donor and during EVLP has not been thoroughly investigated. In addition, the importance of steroids during ex vivo lung perfusion (EVLP) is not clarified. We hypothesized that warm-ischemic related lung injury (90 min) can be critically modulated by administration of methylprednisolone (MP) prior to cardiac arrest and during EVLP. Methods: Domestic pigs were divided in 2 groups (n= 6/group). Following cardiac arrest in the donor, grafts were left untouched in the cadaver for 90 min. Graft function was then assessed during normothermic EVLP for 6 h with an acellular perfusate. In group 1 (MP) 500 mg methylprednisolone was given prior to the warm ischemia in the donor and during EVLP. In group 2 (CONT) no steroids were added. Physiological variables (PVR, PaO2/FiO2, Compl) were monitored and W/D was calculated. Broncho-alveolar lavage and tissue samples were collected. Data were analysed with a Mann-Whitney U test. Results: Physiological data at end of EVLP are presented in FIGURE 1 (boxplot). In the MP group, compliance at the end of EVLP was significantly better compared to the CONT group (p= 0.0260). Also W/D ratio was significantly better in de MP group compared to the CONT group (p= 0.0152). There was no statistical difference in PaO2/FiO2 and PVR between the groups. Conclusion: We have demonstrated that warm-ischemic related lung injury in DCD donation can be attenuated by steroid administration when given prior to switch-off and during EVLP. Future research should focus on the administration of MP in the donor or during EVLP alone. The ethical debate concerning pre-arrest donor treatment should be conducted and critical evaluation of current EVLP protocols may not be neglected. 3( 69) Cyclosporine A Can Mitigate Mitochondrial Dysfunction in Lung Grafts during Ex Vivo Lung Perfusion: A Potent Contributor for Better Preconditioning S. Haam , K. Noda, J. D’Cunha, J.D. Luketich, N. Shigemura.  Department of Cardiothoracic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA. Purpose: Despite its substantial benefits, the deleterious effects of ex vivo lung perfusion (EVLP) in particular activation of proinflammatory cascades and alteration of metabolic profiles in lung grafts on EVLP have been reported; however, their resolution remains to be defined. In this study, we

investigated the role of Cyclosporine A (CyA) which is well known as an immunosuppressant drug, in prohibiting mitochondrial permeability transition and subsequent proinflammatory changes in lung grafts, leading to their improved graft quality on EVLP. Methods: Using rat heart-lung blocks after 1 hour cold preservation, an acellular normothermic EVLP system was established for 4 hours. CyA was added into perfusate for 1 μ M as final content. The evaluation included the routine lung graft function, compliance (Cdyn), and vascular resistance (PVR) as well as biochemical markers in the perfusate at multiple time points. After 4 hours EVLP, their proinflammatory and metabolic profiles were also assessed in lung grafts, including tissue ATP levels, mitochondrial biogenesis and hypoxia inducible factor (HIF)-1α  expressions. Results: Lung grafts on EVLP with CyA exhibited significantly better functional and physiological parameters as compared to those without CyA (Fig.1A). CyA administration attenuated proinflammatory changes (Fig.1B) and prohibited glucose consumption during EVLP through mitigating mitochondrial dysfunction and promoting mitochondrial biogenesis in lung grafts (Fig.1C). CyA-treated lungs exhibited significantly attenuated expression of HIF-1α . Conclusion: CyA can be a potent contributor to better preconditioning lung grafts by protecting mitochondrial function during EVLP.

3( 70) Prolonged Warm Ischemia Prevents Lung Allograft Acceptance in Lung Transplantation from Donation after Cardiac Death in the Mouse Y. Hirano ,1 S. Sugimoto,1 T. Kurosaki,2 S. Otani,2 K. Miyoshi,1 M. Yamane,1 T. Oto,2 S. Miyoshi.1  1Department of General Thoracic Surgery, Okayama University Hospital, Okayama City, Japan; 2Organ Transplant Center, Okayama University Hospital, Okayama City, Japan. Purpose: Donation after cardiac death (DCD) has been introduced worldwide as a potential strategy to increase the donor pool in lung transplantation (LT). In DCD donors, warm ischemia inevitably occurs after cardiac arrest and may cause ischemia-reperfusion injury (IRI). Recently, extended cold preservation time of lungs has been shown to enhance IRI and lead to acute rejection in the model of murine LT, suggesting a link between innate and adaptive immune responses after LT. However, the effect of warm IRI on alloimmunity in LT remains unknown. Our study aimed to examine the effect of warm IRI on lung allograft rejection in a model of murine LT from DCD. Methods: LT following DCD were performed in the Balb/c >  B6 combination after 0 (WIT0 group), 15 (WIT15 group) or 60 (WIT60 group) minutes of warm ischemic time. Balb/c >  B6 lung recipients were treated with 250 ug of anti-CD40 ligand and 200 ug of CTLA4Ig to promote acceptance. Seven days after LT, lung allografts were examined histologically, and lung graft lymphocytic infiltrates were analyzed by flow cytometry. Results: In WIT0 group, LT recipients continued to accept their lungs. In WIT15 group, B6 recipients of Balb/c grafts had minimal inflammation, and there were nearly equal numbers of CD8+ and CD4+ T cells in these grafts (the ratio of intragraft CD8+ to CD4+ T-cells =  1.0 ± 0.5). In contrast, in WIT60 group, B6 recipients of Balb/c grafts had evidence of acute rejection with extensive perivascular and interstitial lymphocytic infiltrates, and CD8+ T cells outnumbered CD4+ T cells (the ratio of intragraft CD8+ to CD4+ T-cells =  2.6 ± 0.2). Moreover, among graft-infiltrating T lymphocytes, the percentages of IFN-γ  producing CD8+ T cells in WIT60 group were higher than those in WIT15 group. Conclusion: Our results suggest that enhanced warm IRI abrogates lung allograft acceptance in LT from DCD. In clinical settings, to minimize the

S144

The Journal of Heart and Lung Transplantation, Vol 35, No 4S, April 2016

warm ischemic time after cardiac arrest may contribute to the improvement of long-term survival after LT from DCD. 3( 71) Protective Effect of Imatinib on Ischemia-Reperfusion Injury in Rat Lung S. Tanaka , T.F. Chen-Yoshikawa, T. Menju, M. Saito, A. Takahagi, E. Miyamoto, M. Takahashi, K. Ohata, T. Kondo, K. Hijiya, H. Motoyama, A. Aoyama, H. Date.  Department of Thoracic Surgery, Kyoto University, Graduate School of Medicine, Kyoto, Japan. Purpose: Ischemia-reperfusion injury (IRI) remains a significant complication after lung transplantation. Endothelial damage contributes to its development. Imatinib has recently been reported to regulate vascular permeability by protecting endothelium via inhibition of Abl and Abl-related gene (Arg) kinases under various permeability-inducing conditions. We hypothesized that imatinib could have a protective effect against IRI. Methods: Heparinized rat underwent left thoracotomy, and the left hilum was clamped for 90 minutes, followed by reperfusion for 120 minutes (rat hilar clamp model). Imatinib mesylate (50 mg/kg) and the solvent were intraperitoneally administered 20 min before ischemia in the Imatinib group and the Vehicle group, respectively (n= 7, each). After reperfusion, lung mechanics, lung wet-to-dry weight ratio (W/D), and histological findings were obtained. The expression of vascular-endothelial cadherin (VEC) and the phosphorylation of CrkL, an exclusive target of Abl/Arg, were evaluated by Western blot analysis using lung tissue lysates. The cytokine levels in lung tissue were measured by ELISA. Results: In the Imatinib group, lung mechanics were significantly improved (P< 0.05). W/D was lower (P= 0.01) and perivascular edema was ameliorated on histology. The number of neutrophils infiltrated into the lung was significantly decreased (P= 0.02). The expression of VEC was maintained and pCrkL was inhibited by imatinib. The IL-10 level was significantly higher in the Imatinib group (P= 0.008). Conclusion: In a rat IRI model, imatinib inhibited Abl/Arg in lung tissue, maintained endothelial junction with attenuated lung edema, increased antiinflammatory cytokine, and showed less neutrophil infiltration. Imatinib demonstrated anti-permeability and anti-inflammatory effects in IRI; thus, it can be a novel treatment strategy.

Then, we measured the hydrogen concentration in the left lung using a sensor gas chromatograph (N=  3 per group). In the second experiment, lung ischemia-reperfusion injury was induced by occlusion of the left pulmonary hilum for 1 h, followed by reperfusion for 3 h. During the ischemic period, the left thoracic cavity was filled with either normal saline or hydrogen-rich saline. After reperfusion, we assessed lung function, histological changes, and cytokine production (N=  5-7 per group). Results: Storing lungs in hydrogen-rich saline resulted in an elevated hydrogen concentration in the lung (6.9±2.9 μ mol/1g lung, Figure B). After ischemia-reperfusion injury, pulmonary function (pulmonary compliance, and oxygenation levels) was significantly better in the hydrogen-rich saline group than in the normal saline group (P < 0.05). Similarly, pro-inflammatory cytokine levels (interleukin-1β  and interleukin-6) in the left lung were significantly lower in the hydrogen-rich saline group than in the normal saline group (P <  0.05, Figure C). Conclusion: Storage in hydrogen-rich saline delivered hydrogen into the lung. In addition, using this method during an ischemic period attenuated lung ischemia-reperfusion injury.

3( 73) MHC-Inbred CLAWN Miniature Swine as Preclinical Large Animal Model for Lung Transplantation H. Sahara , M. Sekijima, T. Iwanaga, Y. Ichinari, A. Shimizu, K. Yamada.  Division of Organ Replacement and Xenotransplantation Surgery, Center for Advanced Biomedical Science and Swine Research, Kagoshima University, Kagoshima, Japan.

3( 72) Lung Storage in Hydrogen-Rich Saline Attenuates Lung IschemiaReperfusion Injury M. Takahashi , T.F. Chen-Yoshikawa, M. Saito, S. Tanaka, E. Miyamoto, A. Takahagi, K. Ohata, T. Kondo, H. Motoyama, K. Hijiya, A. Aoyama, H. Date.  Graduate School of Medicine, Kyoto University, Kyoto, Japan. Purpose: The anti-oxidant effects of hydrogen-rich solution have been reported in several studies examining solid organ transplantation. In this study, we evaluated the therapeutic efficacy of storing lungs in hydrogen-rich saline on lung ischemia-reperfusion injury. Methods: Rats were divided into three groups: 1.sham, 2.normal saline, and 3.hydrogen-rich saline. In the first experiment, the left thoracic cavity was filled with either normal saline or hydrogen-rich saline for 1h (Figure A).

Purpose: We have previously demonstrated that 12-days of high-dose of tacrolimus facilitates tolerance induction of fully MHC-mismatched (m/m) lungs in MGH miniature swine. Recently, we developed a line of Japanese MHC-inbred CLAWN miniature swine. To clarify that CLAWN swine represent a highly-valuable preclinical model of transplantation, we established several models of lung transplantation. Methods: Five CLAWN swine received fully MHC-m/m lungs with the same 12-days of tacrolimus (35-45 ng/ml) as MGH study (Group 1). Four CLAWN swine received MHC-matched lungs to investigate the influence of MHC disparity on allograft survival (Group 2). To investigate whether brain death (BD) or cardiac death (DCD) affects the graft survival, three recipients were transplanted with fully MHC-m/m lung from BD donors induced by subdural balloon and subsequently ventilated for 6 h before procurement (Group 3) and six recipients were transplanted with MHC-matched DCD lungs induced by venous injection of potassium chloride and allowed for 90 min before procurement (Group 4). Graft function was monitored by histology and X-rays. Results: All fully MHC-m/m grafts was rejected within 63 days with diffuse mononuclear infiltrates associated with intra-alveolar hemorrhage and capillary congestion, consistent with severe cellular and antibody-mediated rejection (Group 1). In contrast, all MHC-matched lung was uniformly accepted over one year with occasional presence of minimum acute cellular rejection (Group 2; the fourth animal is ongoing >  120 days). Rejection of pulmonary allografts was accelerated (< 35 days) by BD in Group 3 with accelerated