Steen Solution Research Articles

α1-Anti-trypsin improves function of porcine donor lungs during ex-vivo lung perfusion

Ex-vivo lung perfusion (EVLP), a technique for donor lung assessment, also represents a platform for donor lung repair and immunomodulation. α1-Anti-trypsin (A1AT), a medication used to treat emphysema in A1AT-deficient patients, has anti-inflammatory properties and has been shown to attenuate ischemia-reperfusion injury in rat and pig lung transplants. The objective of this study was to determine whether administration of A1AT during EVLP can improve donor lung quality after prolonged hypothermic preservation. Pig donor lungs were retrieved, preserved at 4°C for 24 hours, and then subjected to normothermic EVLP for 12 hours using the Toronto protocol. The treatment group (n = 6) received 3 mg/ml A1AT (Zemaira) in the EVLP perfusate, acellular Steen solution. The control group (n = 6) was perfused with Steen solution only. Physiologic functions and gas exchange were measured hourly. Pulmonary edema, lung injury, apoptosis and inflammatory mediators were evaluated in lung tissues and perfusate. A1AT treatment significantly reduced pulmonary arterial pressure, pulmonary vascular resistance and airway pressure changes from the baseline when compared with controls. A1AT treatment significantly improved both dynamic and static pulmonary compliance, and change in partial pressure of oxygen (ΔPO2) between the left atrium and the pulmonary artery. Furthermore, A1AT treatment also significantly reduced pulmonary edema (wet-to-dry ratio), pulmonary cell apoptosis and pro-inflammatory cytokine levels (interleukin-1α and -8) in the perfusate. Treatment of 24-hour-preserved pig donor lungs with A1AT during EVLP resulted in improved physiologic function, reduced pulmonary edema and inflammation and decreased cell death. Our findings suggest that treatment of donor lungs during EVLP with A1AT is a promising strategy to attenuate early lung injury and improve donor lung function before lung transplantation.

In vivo lung perfusion rehabilitates sepsis-induced lung injury

BackgroundSepsis is the leading cause of lung injury in adults and can lead to acute respiratory distress syndrome (ARDS). Using a novel technique of isolated in vivo lung perfusion (IVLP), we hypothesized that normothermic IVLP will improve oxygenation and compliance in a porcine model of sepsis-induced lung injury. MethodsMature adult swine (n = 8) were administered lipopolysaccharide (LPS; 50 μg/kg over 2 hours) via the external jugular vein, followed by sternotomy and central extracorporeal membrane oxygenation (ECMO) cannulation (right atrium to ascending aorta). The left pulmonary artery (inflow) and left superior and inferior pulmonary veins (outflow) were dissected out and cannulated to deliver isolated perfusion to the left lung. After 4 hours of normothermic IVLP with Steen solution, the left lung then underwent 4 hours of reperfusion after IVLP decannulation. Airway pressures and lung-specific pulmonary vein blood gases from the right lung (LPS control) and left lung (LPS + IVLP) of the same animal were compared. ResultsAll animals demonstrated a significant reduction in the ratio of partial pressure of oxygen in arterial blood (PaO2)/fraction of inspired oxygen (FiO2) (P/F ratio) and total lung compliance at 2 hours after the start of LPS infusion (mean, 469 ± 19.7 mm Hg vs 222.2 ± 21.4 mm Hg; P < .0001). After reperfusion, 6 animals (75%) exhibited improved lung function, allowing for ECMO decannulation. Lung-specific oxygenation was superior in the left lung after 4 hours of reperfusion (mean, 310.5 ± 54.7 mm Hg vs 201.1 ± 21.7 mm Hg; P = .01). Similarly, total lung compliance improved after IVLP of the left lung. The lung wet weight to dry weight ratio demonstrated reduced edema in rehabilitated left lungs (mean, 6.5 ± 0.3 vs 7.5 ± 0.4; P = .04). ConclusionsIVLP successfully rehabilitated LPS-injured lungs compared to ECMO support alone in this preclinical porcine model.

Beware Cold Agglutinins in Organ Donors! Ex Vivo Lung Perfusion From an Uncontrolled Donation After Circulatory-Determination-of-Death Donor With a Cold Agglutinin: A Case Report

BackgroundWe began to recover lungs from uncontrolled donation after circulatory determination of death to assess for transplant suitability by means of ex vivo lung perfusion (EVLP) and computerized tomographic (CT) scan. Our first case had a cold agglutinin with an interesting outcome. Case reportA 60-year-old man collapsed at home and was pronounced dead by Emergency Medical Services personnel. Next-of-kin consented to lung retrieval, and the decedent was ventilated and transported. Lungs were flushed with cold Perfadex, removed, and stored cold. The lungs did not flush well. Medical history revealed a recent hemolytic anemia and a known cold agglutinin. Warm nonventilated ischemia time was 51 minutes. O2-ventilated ischemia time was 141 minutes. Total cold ischemia time was 6.5 hours. At cannulation for EVLP, established clots were retrieved from both pulmonary arteries. At initiation of EVLP with Steen solution, tiny red aggregates were observed initially. With warming, the aggregates disappeared and the perfusate became red. After 1 hour, EVLP was stopped because of florid pulmonary edema. The lungs were cooled to 20°C; tiny red aggregates formed again in the perfusate. Ex vivo CT scan showed areas of pulmonary edema and a pyramidal right middle lobe opacity. Dissection showed multiple pulmonary emboli—the likely cause of death. However, histology showed agglutinated red blood cells in the microvasculature in pre- and post-EVLP biopsies, which may have contributed to inadequate parenchymal preservation. ConclusionsOrgan donors with cold agglutinins may not be suitable owing to the impact of hypothermic preservation.

Cytoprotective and Antioxidant Effects of Steen Solution on Human Lung Spheroids and Human Endothelial Cells.

Respiratory diseases represent a major healthcare burden worldwide. Lung transplantation (LTx) is the "gold standard" for end-stage patients, strongly limited by shortage of available/suitable donor lungs. Normothermic ex vivo lung perfusion (EVLP) has significantly increased the number of lungs suitable for transplantation. Steen solution is used for EVLP, but the mechanisms involved in its beneficial properties remain to be clarified. We investigated the effects of Steen solution in an in vitro protocol of cold starvation and normothermic recovery on human lung spheroids, named pneumospheres (PSs), containing epithelial/basal cells, and on endothelial human umbilical vein endothelial cells (HUVEC). Steen solution significantly preserved the viability of PSs, reduced reactive oxygen species (ROS) release by PSs and HUVECs, decreased NADPH-oxidase (NOX) activity in PSs, and reduced inflammatory cytokines expression levels in HUVECs. Steen solution was able to specifically reduce NADPH oxidase 2 (NOX2) isoform activation, particularly in PSs, as detected by soluble-NOX2 peptide and p47-phosphorylation. Interestingly, a specific NOX2 inhibitor could partly mimic the pro-survival effect of Steen on PSs. We provide the first evidence that Steen solution can preserve lung epithelial/progenitor cells viability partially through NOX2 downregulation, and exert antioxidant effects on parenchymal cells, with consequent ROS reduction. These results suggest that NOX2 inhibition might be an additional strategy to reduce cellular damage during LTx procedures.

Ex Vivo Lung Perfusion Rehabilitates Sepsis-Induced Lung Injury

Sepsis is the number one cause of lung injury in adults. Exvivo lung perfusion (EVLP) is gaining clinical acceptance for donor lung evaluation and rehabilitation and may expand the use of marginal organs for transplantation. We hypothesized that 4 hours of normothermic EVLP would improve compliance and oxygenation in a porcine model of sepsis-induced lung injury. We used intravenous lipopolysaccharide (LPS) to induce a systemic inflammatory response in a porcine model of lung injury. Two groups of 4 animals each received a 2-hour infusion of LPS through the external jugular vein. Serial measurements of blood gases were performed every 30 minutes until the partial pressure of oxygen/fraction of inspired oxygen ratio dropped below 150 on two consecutive readings. Lungs were then randomized to treatment with 4 hours of normothermic EVLP with STEEN Solution (XVIVO Perfusion Inc, Englewood, CO) or 4 additional hours of invivo perfusion (control). Airway pressures and blood gases were recorded for calculation of dynamic lung compliance and partial pressure of oxygen/fraction of inspired oxygen ratios. EVLP was performed with hourly recruitment maneuvers and oxygen challenge. All animals reached a partial pressure of oxygen/fraction of inspired oxygen ratio of less than 150 mm Hg within 3 hours after start of the LPS infusion. Oxygenation and compliance in the control animals continued to decline during the 4-hour invivo perfusion period, and 3 of the 4 animals died of severe hypoxia within 4 hours. The EVLP group demonstrated significant improvements hour 1 to hour 4 in oxygenation (365.8 ± 53.0 vs 584.4 ± 21.0 mm Hg, p= 0.02) and dynamic compliance (9.0 ± 2.8 vs 15.0 ± 3.6, p= 0.02 mL/cm H2O). EVLP successfully rehabilitated LPS-induced lung injury in this preclinical porcine model and may thus provide a means to rehabilitate many types of acute lung injury.

(962) - Cytokine Levels in Steen Solution Perfusate Increase During Ex-Vivo Lung Perfusion (EVLP) of Lungs from Conventional Donors (Conv) and Uncontrolled Donation After Circulatory Determination of Death Donors (uDCDDs)

(962) - Cytokine Levels in Steen Solution Perfusate Increase During Ex-Vivo Lung Perfusion (EVLP) of Lungs from Conventional Donors (Conv) and Uncontrolled Donation After Circulatory Determination of Death Donors (uDCDDs)

Ex Vivo Lung Perfusion Model to Study Pulmonary Tissue Extracellular Microvesicle Profiles

Extracellular microvesicles (EVs) are being increasingly studied for their diagnostic potential. We investigated the feasibility of studying the kinetics and tissue-specific profiles of pulmonary EVs in the context of exvivo lung perfusion (EVLP) used for salvaging marginal lungs for transplantation. Perfusate from six marginal donor lungs placed on EVLP was collected at the following time points: 0, 10, and 60 minutes after and after perfusate exchange with Steen Solution; 120 and 180 minutes. Three lungs were successfully recovered for transplantation (transplant group), and three were not recoverable (nontransplant group). Perfusate EVs were isolated using methods of size exclusion chromatography, ultrafiltration, and ultracentrifugation. EVs were analyzed on NanoSight nanoparticle detector for quantity, size distribution, and surface expression of pulmonary tissue-specific markers. EV cargoes were profiled using mass spectrometry, reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis. Time course analysis showed EV presence by 10-minute time point. EV median size was smaller in the transplant group (165 nm versus 212 nm, p= 0.04). EV cargo analysis on Western blot analysis, RT-PCR, and NanoSight showed contribution from monocytes (CD14), endothelium (platelet endothelial cell adhesion molecule 1), and pulmonary parenchyma (epithelial cell adhesion molecule) into the perfusate total EV pool. Mass spectrometry showed differences in the EV protein cargoes of the transplant group versus the nontransplant group. EVLP system provides a platform to understand the kinetics of pulmonary EVs in an isolated fashion. Donor lung recovery may be associated with changes in EV size distribution and proteomic profiles. Pulmonary tissue-specific EV profiling using the EVLP system may provide insights into EV contribution to pulmonary pathologic processes.

The development of ex vivo lung perfusion in lung transplantation

The shortage of donor lung remains one of the major problem for lung transplantation. With the development of modern lung preservation and repair technique, increasing marginal lung donors have been re-assessed and finally utilized for transplantation. The ex vivo lung perfusion technique (EVLP) was designed and has been developed for evaluation and repair of the lung. Nowadays, the indication of EVLP and the standard of qualified donor lung have reached a consensus according to the foreign publications. The EVLP system could be classified into three categories: the Toronto technique, the Lund technique and Hannover-Madrid technique. The major differences between the Toronto technique and the other two technique are the open left atrium status, the use of Steen solution mixed with erythrocyte and the perfusion at flows correspondent to 100% of the donor predicted cardiac output. With the accumulating experience, researchers have tried to imply some drugs in the circulation, modify the ventilation gas and delivery of adenoviral vector gene in order to improve the lung quality. But these are still in the research phase. Recently, the portable EVLP device has been developed and the lung preservation, assessment and repair could be conducted during transportation. So it could prolong the preservation time and expand the transportation distance of donor lung.

Abstract 12560: Effect of Somah Solution on Hypoxia/Reoxygenation Induced Coronary Artery Endothelial Injury: A Comparison to Current Ex-Vivo Heart Perfusates

Introduction: Ex-Vivo Heart Perfusion (EVHP) has been proposed to expand cardiac donation by enabling resuscitation and preservation of hearts donated after circulatory death. Hypoxia/reoxygenation endothelial injury affects organ viability and patient’s outcomes. STEEN Solution is accepted as the primary component for perfusion of other solid organs. However, there is lack of evidence to determine the optimal perfusate composition to maintain cardiac viability during longer periods of perfusion. Somah solution has been proposed as a novel cardioprotective solution optimized to meet cardiac metabolism and confer enhanced protective effects against reperfusion injury. Our aim was to compare the effects of STEEN and Somah on endothelial reoxygenation injury in vitro. Methods: Human coronary artery endothelial cells were exposed to 16 hours of hypoxia in culture medium followed by 16 hours of reoxygenation treated with Celsior, STEEN or Somah to simulate the EVHP setting. Endothelial activation markers’ gene expression was assessed through qRT-PCR; supernatant was collected to quantify endothelin-1 (ET-1) with ELISA; and quantitative neutrophil adhesion assay was performed to evaluate endothelial function. Results: Treatment with Somah demonstrated decreased expression of cell surface adhesion molecules E-Selectin (Me 0.133±0.12, So -0.073±0.34, ST 1.624±0.46, Ce 0.999±0.45 fold-change from hypoxia; p=0.002) and VCAM-1 (Me -0.004±0.20, So -0.363±0.43, ST 0.428±0.39, Ce 0.597±0.13 fold-change from hypoxia; p=0.023), and vasoactive molecule MCP-1 (Me 0.225±0.72, So -0.510±0.70, ST 1.500±0.49, Ce 0.829±0.28 fold-change from hypoxia; p=0.015). Celsior and Somah reduced cell secretion of ET-1 (Me 395 pg/ml, So 20 pg/ml, ST 448 pg/ml, Ce 16 pg/ml; n=4, p&lt;0.05). Reoxygenation injury increased neutrophil adhesion to endothelial cells; there was a non-statistically significant trend towards protection against this injury with Somah. Conclusions: Somah exerts protective effect against reoxygenation injury when compared to STEEN in vitro. It may have utility in enhancing cardiac organ preservation during EVHP. Future studies in a pre-clinical setting should be conducted to address this question.

Attenuation of Pulmonary Ischemia-Reperfusion Injury by Adenosine A2B Receptor Antagonism

Ischemia-reperfusion injury (IRI) is a major source of morbidity and mortality after lung transplantation. We previously demonstrated a proinflammatory role of adenosine A2B receptor (A2BR) in lung IR injury. The current study tests the hypothesis that A2BR antagonism is protective of ischemic lungs after invivo reperfusion or exvivo lung perfusion (EVLP). Mice underwent lung IR with or without administration of ATL802, a selective A2BR antagonist. A murine model of EVLP was also used to evaluate rehabilitation of donation after circulatory death (DCD) lungs. DCD lungs underwent ischemia, cold preservation, and EVLP with Steen solution with or without ATL802. A549 human type 2 alveolar epithelial cells were exposed to hypoxia-reoxygenation (HR) (3 hours/1 hour) with or without ATL802 treatment. Cytokines were measured in bronchoalveolar lavage (BAL) fluid and culture media by enzyme-linked immunoassay (ELISA). After IR, ATL802 treatment significantly improved lung function (increased pulmonary compliance and reduced airway resistance and pulmonary arterypressure) and significantly attenuated proinflammatory cytokine production, neutrophil infiltration, vascular permeability, and edema. ATL802 also significantly improved the function of DCD lungs after EVLP(increased compliance and reduced pulmonary artery pressure). After HR, A549 cells exhibited robust production of interleukin (IL)-8, a potent neutrophil chemokine, which was significantly attenuated by ATL802. These results demonstrate that A2BR antagonism attenuates lung IRI and augments reconditioning of DCD lungs by EVLP. The protective effects of ATL802 may involve targeting A2BRs onalveolar epithelial cells to prevent IL-8 production. A2BR may be a novel therapeutic target for mitigating IRI to increase the success of lung transplantation.

Ex Vivo Perfusion With Adenosine A2A Receptor Agonist Enhances Rehabilitation of Murine Donor Lungs After Circulatory Death.

Ex vivo lung perfusion (EVLP) enables assessment and rehabilitation of marginal donor lungs before transplantation. We previously demonstrated that adenosine A2A receptor (A2AR) agonism attenuates lung ischemia-reperfusion injury. The current study utilizes a novel murine EVLP model to test the hypothesis that A2AR agonist enhances EVLP-mediated rehabilitation of donation after circulatory death (DCD) lungs. Mice underwent euthanasia and 60 minutes warm ischemia, and lungs were flushed with Perfadex and underwent cold static preservation (CSP, 60 minutes). Three groups were studied: no EVLP (CSP), EVLP with Steen solution for 60 minutes (EVLP), and EVLP with Steen solution supplemented with ATL1223, a selective A2AR agonist (EVLP + ATL1223). Lung function, wet/dry weight, cytokines and neutrophil numbers were measured. Microarrays were performed using the Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Ex vivo lung perfusion significantly improved lung function versus CSP, which was further, significantly improved by EVLP + ATL1223. Lung edema, cytokines, and neutrophil counts were reduced after EVLP and further, significantly reduced after EVLP + ATL1223. Gene array analysis revealed differential expression of 1594 genes after EVLP, which comprise canonical pathways involved in inflammation and innate immunity including IL-1, IL-8, IL-6, and IL-17 signaling. Several pathways were uniquely regulated by EVLP + ATL1223 including the downregulation of genes involved in IL-1 signaling, such as ADCY9, ECSIT, IRAK1, MAPK12, and TOLLIP. Ex vivo lung perfusion modulates proinflammatory genes and reduces pulmonary dysfunction, edema, and inflammation in DCD lungs, which are further reduced by A2AR agonism. This murine EVLP model provides a novel platform to study rehabilitative mechanisms of DCD lungs.

Experimental Ex Vivo Lung Perfusion with Sevoflurane: Effect on donors after circulatory death (DCD) lung grafts in a rodent model.

Ischemia-reperfusion injury is a key mechanism of graft damage during lung transplantation, which could be targeted by therapies applied during ex-vivo lung perfusion (EVLP). The inhalational anaesthetic Sevoflurane was found to protect to some degree against ischemia-reperfusion injury1. In this experimental study we wanted to determine the therapeutic potential of volatile Sevoflurane added to the perfusate during EVLP of damaged lung grafts. Two groups of 6 Sprague-Dawley rats were used. After cardiac arrest and a warm ischemic time of 1 hour the lungs were flushed with cold Perfadex®, harvested and kept for 2 hours at 4°C. Normothermic EVLP during 3 hours was performed using a customized circuit primed either with Steen solution® only (control group) or supplemented within the first 30 minutes of EVLP with a gas mixture containing 2% of sevoflurane (treatment group). Differential oxygen partial pressures in the perfusate (ΔvapO2), pulmonary artery pressure (PAP), vascular resistance (PVR), lung compliance (LC), peak airway pressure (PAWP) and lung weight gain (WG) were measured. At the end of EVLP, protein and lactate dehydrogenase (LDH) levels were determined in bronchoalveolar lavage (BAL) and interleukin-6 (IL-6) and protein carbonyl (index of oxidative stress) were measured in lung tissue. Normothermic EVLP of lungs harvested after warm ischemia resulted in markedly declined ΔvapO2, LC, and elevated PAP, PVR, PAWP, WG. In contrast, lungs treated with Sevoflurane during EVLP displayed significantly improved LC, reduced weight gain and significantly lower LDH and protein carbonyl levels as compared to controls. PAWP and IL-6 were diminished in treated lungs, but were not statistically different to controls. The intravascular administration of sevoflurane during EVLP reduces oxidative stress and improves the functional status of DCD rat lungs in this experimental setting.

Alternative solution for ex vivo lung perfusion, experimental study on donated human lungs non-accepted for transplantation

To evaluate a new perfusate solution to be used for ex vivo lung perfusion. Randomized experimental study using lungs from rejected brain-dead donors harvested and submitted to 1 hour of ex vivo lung perfusion (EVLP) using mainstream solution or the alternative. From 16 lungs blocs tested, we found no difference on weight after EVLP: Steen group (SG) = 1,097±526g; Alternative Perfusion Solution (APS) = 743±248g, p=0.163. Edema formation, assessed by Wet/dry weigh ratio, was statistically higher on the Alternative Perfusion Solution group (APS = 3.63 ± 1.26; SG = 2.06 ± 0.28; p = 0.009). No difference on PaO2 after EVLP (SG = 498±37.53mmHg; APS = 521±55.43mmHg, p=0.348, nor on histological analyses: pulmonary injury score: SG = 4.38±1.51; APS = 4.50±1.77, p=0.881; apoptotic cells count after perfusion: SG = 2.4 ± 2.0 cells/mm2; APS = 4.8 ± 6.9 cells/mm2; p = 0.361). The ex vivo lung perfusion using the alternative perfusion solution showed no functional or histological differences, except for a higher edema formation, from the EVLP using Steen Solution(r) on lungs from rejected brain-dead donors.

(727) - The Impact of Perfusate Oxygenation During Ex Vivo Lung Perfusion on Post-transplant Outcomes: Deoxygenated vs. Fully-Oxygenated

Purpose: The metabolic circumstances in lung grafts on ex vivo lung perfu- sion (EVLP) are compromised as compared with those in lungs in vivo as described in previous studies. While the current EVLP protocol suggests using deoxygenated perfusate with acellular solution, little evidence supports that is the optimal condition. This study aimed to evaluate the quality of lung grafts in the different modes of oxygenated perfusate during EVLP and to define their impact on posttransplant functional outcomes. Methods: Using rat heart-lung blocks after 1 hr cold preservation, the EVLP sys- tem was established with Steen solution at 37°C for 4 hrs. We compared the results of four different modes of perfusate O2 during EVLP: 6%, 40%, 60% and 100%. The evaluation included lung graft function, compliance (Cdyn), and vascular resistance (PVR) as well as circulating glucose, lactate levels and other biochemi-

(731) - Mitochondrial Calcium: The Missing Link Between Hypoxia and Quality in Lung Grafts During Ex Vivo Lung Perfusion

(731) - Mitochondrial Calcium: The Missing Link Between Hypoxia and Quality in Lung Grafts During Ex Vivo Lung Perfusion

Novel thermographic detection of regional malperfusion caused by a thrombosis duringex vivolung perfusion

Although ex vivo lung perfusion (EVLP) has been clinically applied as a novel rig to evaluate marginal donor lungs, no parameters have been reported to objectively detect regional lung damage during EVLP. The aim of this study was to investigate whether regional donor lung malperfusion-related damage caused by a thrombus could be detected by thermography during EVLP. Lewis rats were divided into two groups: the Thrombosis group and the Control group (n = 6 in each group). All rats were heparinized and the lungs were flushed with 20 ml of Steen solution. In the Thrombosis group, a 30-mg artificial thrombus was inserted into the left main pulmonary artery. All the lungs were perfused and ventilated using the EVLP system. Perfusion flow was increased every 2 min up to 10 ml/min. The lungs were evaluated by collecting thermographical and physiological data during EVLP. Pulmonary artery pressure was higher and lung compliance was lower in the Thrombosis group compared with those in the Control group (P = 0.0005 and <0.0001, respectively). Macroscopically, no differences were seen between the perfused area and the malperfused area, whereas significant differences were detected between them by thermography. The surface temperature of both lungs in the Control group and the right lungs in the Thrombosis group rose with increasing perfusion flow, whereas the surface temperature of the left lungs in the Thrombosis group did not rise (P < 0.0001). Although physiological data could possibly imply the existence of thrombi in the Thrombosis group, it could not reveal which area was obstructed by thrombi; however, thermography could detect a malperfused region. Thermographical evaluation may become a promising strategy to detect regional damage in donor lungs.

A whole blood–based perfusate provides superior preservation of myocardial function during ex vivo heart perfusion

Ex vivo heart perfusion (EVHP) provides the opportunity to resuscitate unused donor organs and facilitates assessments of myocardial function that are required to demonstrate organ viability before transplantation. We sought to evaluate the effect of different oxygen carriers on the preservation of myocardial function during EVHP. Twenty-seven pig hearts were perfused ex vivo in a normothermic beating state for 6 hours and transitioned into working mode for assessments after 1 (T1), 3 (T3), and 5 (T5) hours. Hearts were allocated to 4 groups according to the perfusate composition. Red blood cell concentrate (RBC, n = 6), whole blood (RBC+Plasma, n = 6), an acellular hemoglobin-based oxygen carrier (HBOC, n = 8), or HBOC plus plasma (HBOC+Plasma, n = 7) were added to STEEN Solution (XVIVO Perfusion, Goteborg, Sweden) to achieve a perfusate hemoglobin concentration of 40 g/liter. The perfusate composition affected the preservation of systolic (T5 dP/dtmax: RBC+Plasma = 903 ± 99, RBC = 771 ± 77, HBOC+Plasma = 691 ± 82, HBOC = 563 ± 52 mm Hg/sec; p = 0.047) and diastolic (T5 dP/dtmin: RBC+Plasma = -574 ± 48, RBC = -492 ± 63, HBOC+Plasma = -326 ± 32, HBOC = -268 ± 22 mm Hg/sec; p < 0.001) function, and the development of myocardial edema (weight gain: RBC+Plasma = 6.6 ± 0.9, RBC = 6.6 ± 1.2, HBOC+Plasma = 9.8 ± 1.7, HBOC = 16.3 ± 1.9 g/hour; p < 0.001) during EVHP. RBC+Plasma hearts exhibited less histologic evidence of myocyte damage (injury score: RBC+Plasma = 0.0 ± 0.0, RBC = 0.8 ± 0.3, HBOC+Plasma = 2.6 ± 0.2, HBOC = 1.75 ± 0.4; p < 0.001) and less troponin-I release (troponin-I fold-change T1-T5: RBC+Plasma = 7.0 ± 1.7, RBC = 13.1 ± 1.6, HBOC+Plasma = 20.5 ± 1.1, HBOC = 16.7 ± 5.8; p < 0.001). Oxidative stress was minimized by the addition of plasma to RBC and HBOC hearts (oxidized phosphatidylcholine compound fold-change T1-T5: RBC+Plasma = 1.83 ± 0.20 vs RBC = 2.31 ± 0.20, p < 0.001; HBOC+Plasma = 1.23 ± 0.17 vs HBOC = 2.80 ± 0.28, p < 0.001). A whole blood-based perfusate (RBC+Plasma) minimizes injury and provides superior preservation of myocardial function during EVHP. The beneficial effect of plasma on the preservation of myocardial function requires further investigation.

284 * HAEMOFILTRATION IN EX-VIVO LUNG PERFUSION: A STUDY IN EXPERIMENTALLY-INDUCED PULMONARY OEDEMA

Objectives: Ex-vivo lung perfusion (EVLP) could potentially reduce pulmonary oedema. In a pig model with induced pulmonary oedema, we evaluated the effect of haemofiltration (HF) during EVLP on lung function, perfusate oncotic pressure and lung weight. Methods: In anaesthetised pigs (n = 14), pulmonary oedema was induced by an inflatable balloon in the left atrium for 2 h, combined with crystalloid infusion (20 ml/kg). The lungs were harvested and kept cold for 2 h. The lungs were randomised to EVLP, with (HF group, n = 7) or without (control group, n = 7) a haemofilter. EVLP was performed with cellular perfusate at a haematocrit of 10–15%. Oncotic pressure, lung performance and weight were measured before and after 180 min of EVLP reconditioning ± HF. Results: After induction of oedema in vivo, PaO2/FiO2 and compliance decreased by 63% and 16%, respectively. PaO2/FiO2 was considerably improved at first evaluation ex-vivo. HF increased oncotic pressure by 43% and decreased lung weight by 15%, with negligible effects on these variables in the control group. Compliance decreased in both groups during reconditioning, although less so in the HF group (P < 0.05). PaO2/FiO2, shunt fraction and oxygen saturation remained unchanged in both groups. Pulmonary flow index decreased in both groups, partially reversed by nitroglycerin. Conclusion: In this lung oedema model, EVLP reconditioning with the hyperoncotic Steen solution did not affect the degree of lung oedema. HF during EVLP increased perfusate oncotic pressure, decreased lung weight with beneficial effects on compliance, but did not improve lung oxygenation capacity.

The Effects of Hydrogen Gas Inhalation On Non-Heart-Beating Donor Lung During Ex Vivo Lung Perfusion.

Purpose: The shortage of donor lung is a limiting factor in lung transplantation (LTx). In order to resolve this problem, non-heart-beating donor (NHBD) lungs are evaluated in ex vivo lung perfusion (EVLP) system prior to LTx. In this study, the effects of hydrogen gas inhalation on NHBD lungs were investigated during EVLP. Methods: Ten 40kg female pigs were randomly divided into control (n=5) and study group (n=5). After cardiac arrest and one hour of warm ischemic injury, the lungs were harvested after flushing with Perfadex solution, and the lungs assessed in the EVLP system for 4 hours. Steen solution was used as perfusate. During EVLP, the lungs were ventilated in the control group with room air (0.2 of FiO2), and with 2% hydrogen gas in the study group. Oxygen capacity, pulmonary vascular resistance and peak airway pressure were measured every hour for 4 hours during EVLP. After the EVLP, lung specimens were excised for measurement of p38, JNK, and ERK 1/2 phosphorylation, and the expression of IL-1ß, IL-6, IL-8, and TNF-α. Pathologic evaluation was performed using lung injury severity score after H & E stain, and wet/dry ratio was examined. Results: Oxygen capacity in the study group was higher than the control group but did not show a significant difference (p = 0.0862). However, pulmonary vascular resistance (p = 0.0111) and peak airway pressure (p = 0.0189) were significantly lower in the study group. The extents of phosphorylation of p38, JNK, and ERK 1/2 were lower in the study group, but did not show statistically significant difference. The expression of IL-1ß (p = 0.0317), IL-6 (p = 0.0159), IL-8 (p = 0.0195), and TNF-α (p = 0.0159) were all significantly lower in the study group. Lung injury score (p = 0.0358) and wet/dry ratio (p = 0.040) were also lower in the study group.Conclusion: Inhalation of hydrogen gas in NHBD lungs during EVLP may improve the function of damaged lungs, and might possibly increase the utilization of NHBD lungs.

(115) - The Effects of Hydrogen Inhalation on Non-Beating Heart Donor Lung During Ex Vivo Lung Perfusion

Lung transplantation (LTx) is an effective treatment for end-stage lung disease. However, the shortage of donor lung causes the death of recipients in waiting list. In order to resolve the shortage of donor lung, non-beating heart donor (NBHD) lungs are used and ex vivo lung perfusion (EVLP) is performed in clinical setting. In this study, the effects of hydrogen gas known as antioxidant and anti-inflammatory gas on NBHD lungs were investigated during EVLP. Ten 40kg female pigs were randomly divided into control (group I, n=5) and study group (group II, n=5). The atrial fibrillation was induced by 9V electric shock and one hour of warm ischemic injury was induced by leaving them without any treatments after cardiac arrest. After flush using Perfadex solution, the harvested lungs were applied to EVLP system for 4 hours. Steen solution was used as perfusate. During EVLP, the lungs were ventilated with room air (0.2 of FiO2) in control group and 2% hydrogen gas in study group. Oxygen capacity ((PaO2- pulmonary artery of the left atrium PaO2) / FiO2), pulmonary vascular resistance (Pulmonary artery pressure-left atrium pressure) X 80/ pulmonary artery flow) and peak airway pressure were measured every hour for 4 hours of EVLP. After the EVLP, lung specimens were excised for measurement of the extent of p38, JNK, and ERK 1/2 phosphorylation and the expression of IL-1ß, IL-6, IL-8, and TNF-α. Pathologic evaluation was performed using lung injury severity score after H & E stain. Wet/dry ratio was examined. Oxygen capacity in study group was higher than in control group, which did not show a significant difference (p = 0.0862). However, pulmonary vascular resistance (p = 0.0111) and peak airway pressure (p = 0.0189) were significantly lower in study group. There was a tendency that the extents of phosphorylation of p38, JNK, and ERK 1/2 were lower in study group, but it was not statistically significant. The expressions of IL-1ß (p = 0.0317), IL-6 (p = 0.0159), IL-8 (p = 0.0195), and TNF-α (p = 0.0159) were all significantly lower in study group. Lung injury score (p = 0.0358) and wet/dry ratio (p = 0.040) were also lower in study group. The inhalation of hydrogen gas during EVLP improved the function of NBHD lung and it might increase the utilization of NHBD lung with warm ischemic injury.