Low-potassium Dextran Solution Research Articles

Custodiol-MP for ex vivo lung perfusion - A comparison in a porcine model of donation after circulatory determination of death.

Introduction:Ex vivo lung perfusion (EVLP) is an established technique to evaluate and eventually recondition lungs prior to transplantation. Custodiol-MP (C-MP) solution is a new solution, designed for clinical machine perfusion, that has been used for kidneys. The aim of this study was to compare the effects of EVLP with Custodiol-MP on lung functional outcomes to the gold standard of EVLP with Steen Solution™.Material and Methods:In a porcine EVLP model of DCDD (Donation after Circulatory Determination of Death), lungs were perfused with Steen Solution™ (SS, n = 7) or Custodiol-MP solution supplemented with 55 g/l albumin (C-MP, n = 8). Lungs were stored cold for 4 h in low potassium dextran solution and subsequently perfused ex vivo for 4 h. During EVLP pulmonary gas exchange, activities of lactate dehydrogenase (LDH) and alkaline phosphatase (AP) as well as levels of lactate in the perfusate were recorded hourly.Results:Oxygenation capacity differed significantly between groups (averaged over 4 h: SS 274 ± 178 mmHg; C-MP 284 ± 151 mmHg p = 0.025). Lactate dehydrogenase activities and lactate concentrations were significantly lower in Custodiol-MP perfused lungs.In a porcine model of DCDD with 4 h of EVLP the use of modified Custodiol-MP as perfusion solution was feasible. The use of C-MP showed at least comparable lung functional outcomes to the use of Steen SolutionTM. Furthermore C-MP perfusion resulted in significantly lower lactate dehydrogenase activity and lactate levels in the perfusate and higher oxygenation capacity.

Revisiting an Old Concept in a New Era: 36 Hour Lung Preservation Using 10ºC Static Cold Storage

Despite remote reports indicating 10°C to be the optimal temperature for cold static organ preservation (CSP), preservation of lungs in an ice cooler at 4°C remains the standard strategy in clinical transplantation. Here, we used a commercially available device that can keep a donor lung at 10°C conveniently and compared 10°C vs. 4°C during a prolonged period of CSP followed by functional and biological assessment of lung grafts. Lungs were procured from Yorkshire pigs (28-35kg), flushed with a low-potassium dextran solution and randomized into two groups (n=5 each): CSP at 4°C vs. CSP at 10°C (MyTemp Mini Incubator, Benchmark Scientific). After 36h of CSP, lungs were subjected to 12h of normothermic ex vivo lung perfusion (EVLP) with hourly functional assessment. Lung biopsies and perfusate samples were taken for biological and metabolic evaluation. During 12h of EVLP, lungs stored at 10°C presented superior physiological parameters compared to 4°C (Fig 1A). Reduced edema formation was seen as represented by a lower lung weight gain during EVLP (30 ± 34.1 vs. 201 ± 33.2 g, p = 0.0159). Metabolic profiles of the EVLP perfusate were significantly more favorable in the 10°C group compared to 4°C (Fig 1B). Furthermore, the 10°C group had significantly less inflammation demonstrated by lower levels of pro-inflammatory cytokines IL-1β (p = <0.0001, Fig 1C), and IL-8 (p = <0.0001, Fig 1C) in the EVLP perfusate. The levels of IL-6 were significantly lower in both EVLP perfusate (p = <0.0001, Fig 1C) and post-perfusion lung tissue (p = 0.0317). Immunohistochemical analysis for 8-Hydroxyguanosine demonstrated that lungs stored at 10°C tended to have less oxidative stress during CSP (p = 0.0952). With simple controllable refrigerators, 10°C preservation is easily achievable and provides significantly superior CSP in comparison to conventional 4°C storage. This could potentially remarkably improve the clinical and transportation logistics in lung transplantation.

Effects of immediate versus delayed ex-vivo lung perfusion in a porcine cardiac arrest donation model

Ex-vivo lung perfusion is a promising tool to evaluate and recondition marginal donor lungs usually after a cold static preservation. The concept of continuous organ perfusion is supposed to reduce ischemic damage; however, the optimal perfusion protocol has not been established yet. The aim of this study was to compare immediate ex-vivo lung perfusion (I-EVLP) to delayed ex-vivo lung perfusion (D-EVLP) after a certain cold static preservation period on lung function in a large animal model. In a porcine model, lungs were procured after circulatory death and 60 min of no-touch warm ischemia. Lungs were preserved with single-flush cold low potassium dextran solution and prepared either for I-EVLP (n = 8) or stored cold for 9 h with subsequent D-EVLP (n = 8). Functional outcomes and morphology were compared during 4 h of ex-vivo lung perfusion, using STEEN SolutionTM as perfusion solution. Pulmonary functional data, perfusate activities of lactate dehydrogenase, alkaline phosphatase, and products of lipid peroxidation did not differ significantly. There was a trend toward lower wet-dry ratio (I-EVLP: 13.4 ± 2.9; D-EVLP: 9.1 ± 2.5) and higher ΔpO2 in D-EVLP group (I-EVLP: 209 ± 51.6 mmHg; D-EVLP: 236.3 ± 47.3 mmHg). In this donation-after-circulatory-death model, 9 h of cold static preservation followed by ex-vivo lung perfusion results in comparable pulmonary function to I-EVLP as indicated by oxygenation capacities and wet-dry ratio. Our findings indicate that prolonged cold static preservation prior to ex-vivo lung perfusion is as safe and effective as I-EVLP in the procurement of donor lungs.

Effect of hydrogen preconditioning during cold ischemia phase on activity of Nrf2 in rat pulmonary microvascular endothelial cells subjected to hypoxia-reoxygenation

Objective To evaluate the effect of hydrogen preconditioning during cold ischemia phase on the activity of nuclear factor erythroid 2-related factor 2 (Nrf2) in rat pulmonary microvascular endothelial cells (PMVECs) subjected to hypoxia-reoxygenation(H/R). Methods PMVECs were isolated from clean-grade male Sprague-Dawley rats, aged 2-3 weeks, using the tissue block adherence method and divided into 4 groups (n=25 each) using a random number table method: control group (group C), H/R group, oxygen group (O group) and hydrogen group (H group). Cells were incubated for 4 h with 4 ℃ low potassium dextransolution(LPD) pre-equilibrated with 95% oxygen and 5% carbondioxide to simulate the cold ischemia phase.LPD pre-balanced with 95% oxygen and 5% carbon dioxide was replaced with LPD, and then cells were incubated for 1 h at room temperature to simulate the lung transplantation period.LPD was rapidly replaced with 37 ℃ M199 complete culture solution, and cells were incubated in the mixture of 40% oxygen-5% carbondioxide-55% nitrogen to simulate the reperfusion period.In O and H groups, the cells were exposed to 40% oxygen-60% nitrogen and 3% hydrogen-40% oxygen-57% nitrogen during the cold ischemia period, respectively, and the gas mixture was replaced every 20 min.The cell culture fluid was collected 4 h later for determination of interleukin(IL)-6, IL-10 and tumor necrosis factor-alpha (TNF-α) concentrations (by enzyme-linked immunosorbent assay) and malondialdehyde (MDA) concentrations (by thiobarbituric acid method). The cytoplasm and nucleoproteins were extracted for measurement of Nrf2 expression(by Western blot) and cell apoptosis (by flow cytometry and TUNEL assay). The cell apoptosis rate was calculated. Results Compared with C group, the IL-6, TNF-α and MDA levels were significantly increased, the IL-10 level was decreased, the apoptosis rate was increased, and the expression of Nrf2 in nucleus was up-regulated in H/R group (P>0.05). Compared with H/R group, the IL-6, TNF-α and MDA levels were significantly decreased, the IL-10 level was increased, the apoptosis rate was decreased, and the Nrf2 expression in cytoplasm was down-regulated in O and H groups (P 0.05). Compared with O group, the IL-6, TNF-α and MDA levels were significantly decreased, the IL-10 level was increased, the apoptosis rate was decreased, the expression of Nrf2 in nucleus was up-regulated, and the expression of Nrf2 protein in cytoplasm was down-regulated in H group (P<0.05). Conclusion The mechanism by which hydrogen preconditioning during cold ischemia phase reduces H/R injury to rat PMVECs is related to activating Nrf2 and thus inhibiting oxidative stress. Key words: Hydrogen; Cold ischemia; Microvessels; Endothelial cells; Lung; NF-E2-related factor 2

Prediction of transplant outcome after 24-hour ex vivo lung perfusion using the Organ Care System in a porcine lung transplantation model.

Ex vivo lung perfusion (EVLP) has become routine practice in lung transplantation. Still, running periods exceeding 12hours have not been undertaken clinically to date, and it remains unclear how the perfusion solution for extended running periods should be composed and which parameters may predict outcomes. Twenty-four porcine lungs underwent EVLP for 24hours using the Organ Care System (OCS). Lungs were ventilated and perfused with STEEN's solution enriched with erythrocytes (n=8), acellular STEEN's solution (n=8), or low-potassium dextran (LPD) solution enriched with erythrocytes (n=8). After 24hours, the left lungs were transplanted into recipient pigs. After clamping of the contralateral lung, the recipients were observed for 6hours. The most favorable outcome was observed in organs utilizing STEEN solution enriched with erythrocytes as perfusate, whereas the least favorable outcome was seen with LPD solution enriched with erythrocytes for perfusion. Animals surviving the observation period showed lower peak airway pressure (PAWP) and pulmonary vascular resistance (PVR) during OCS preservation. The results suggest that transplantation of lungs following 24hours of EVLP is feasible but dependent on the composition of the perfusate. PAWP and PVR during EVLP are early and late predictors of transplant outcome, respectively.

One-year experience with ex vivo lung perfusion: Preliminary results from a single center.

To enlarge the donor pool for lung transplantation, an increasing number of extended criteria donor lungs are used. However, in more than 50% of multi-organ donors the lungs are not used. Ex vivo lung perfusion offers a unique possibility to evaluate and eventually recondition the injured donor lungs. The aim of our study was to assess the enlargement of the donor pool and the outcome with extended criteria donor lungs after ex vivo lung perfusion. Data were prospectively collected in our lung transplant database. We compared the results of lung transplants after ex vivo lung perfusion with those after conventional cold static preservation. In total, 11 extended criteria donor lungs processed with ex vivo lung perfusion and 41 cold static preservation lungs transplanted consecutively between May 2016 and May 2017 were evaluated. Normothermic ex vivo lung perfusion was performed according to the Toronto protocol for 4 h. Cold static preservation lungs were stored in low-potassium dextran solution. Ex vivo lung perfusion lungs before procurement had significantly lower PaO2/FiO2 (P/F) ratios and more X-ray abnormalities. There were no statistically significant differences for pre-donation ventilation time, smoking history, or sex. After reconditioning with ex vivo lung perfusion, 9 out of 11 processed lungs were considered suitable and successfully transplanted. The mean postoperative ventilation time and in-hospital stay were not significantly different in ex vivo lung perfusion and cold static preservation recipients. Ex vivo lung perfusion can safely be used in the evaluation of lungs initially considered not suitable for transplantation. The primary outcome was not negatively affected and normothermic ex vivo lung perfusion is a useful tool to increase the usage of potentially transplantable lungs.

Improved metabolism and redox state with a novel preservation solution: implications for donor lungs after cardiac death (DCD).

Lungs donated after cardiac death (DCD) are an underutilized resource for a dwindling donor lung transplant pool. Our study investigates the potential of a novel preservation solution, Somah, to better preserve statically stored DCD lungs, for an extended time period, when compared to low-potassium dextran solution (LPD). We hypothesize that Somah is a metabolically superior organ preservation solution for hypothermic statically stored porcine DCD lungs, possibly improving lung transplant outcomes. Porcine DCD lungs (n = 3 per group) were flushed with and submerged in cold preservation solution. The lungs were stored up to 12 h, and samples were taken from lung tissue and the preservation medium throughout. Metabolomic and redox potential were analyzed using high performance liquid chromatography, mass spectrometry, and RedoxSYS®, comparing substrate and pathway utilization in both preservation solutions. Glutathione reduction was seen in Somah but not in LPD during preservation. Carnitine, carnosine, and n-acetylcarnosine levels were elevated in the Somah medium compared with LPD throughout. Biopsies of Somah exposed lungs demonstrated similar trends after 2 h, up to 12 h. Adenosine gradually decreased in Somah medium over 12 h, but not in LPD. An inversely proportional increase in inosine was found in Somah. Higher oxidative stress levels were measured in LPD. Our study suggests suboptimal metabolic preservation in lungs stored in LPD. LPD had poor antioxidant potential, cytoprotection, and an insufficient redox potential. These findings may have immediate clinical implications for human organs; however, further investigation is needed to evaluate DCD lung preservation in Somah as a viable option for transplant.

STUDY ON THE PROTECTION OF ISOLATED RABBIT'S PULMONARY ISCHEMIA REPERFUSION INJURY BY ADDING TANSHINONE IIA INTO LOW POTASSIUM DEXTRAN SOLUTION

Purpose: This study investigates the protective effect of Tanshinone IIA (Tan IIA) on isolated rabbit's pulmonary ischemia reperfusion injury (IRI) and discusses its potential for the treatment of microcirculation disorders. Methods: We chose isolated rabbit's IRI as our experimental models, and randomly divided 24 New Zealand rabbits into two groups. For the control group (L), the lungs were perfused with Low Potassium Dextran (LPD) solution under 4[Formula: see text]C, and then it was preserved under 10[Formula: see text]C. For the experimental group (LT), the lungs were perfused and preserved with LPD[Formula: see text]Tan solution using the same approach. After the lungs have been preserved for 18[Formula: see text]h, we again perfused it for another 30 min. After reperfusion was completed, we determined the contents of SOD, NO, and MDA immediately. The wet to dry weight ratio (W/[Formula: see text] was then obtained. Finally, we use a combination of light microscopy and transmission electron microscopy to observe the ultra-structural changes of lung tissues. Results: We discovered that the contents of SOD and NO in group LT were higher than that in group L ([Formula: see text]). However, the MDA and the W/D values in group LT were lower than that in group L ([Formula: see text] or [Formula: see text]). We also observed that part of the tissues has hyperemia, edema, and congestion. Under the light microscopy, the structures of the pulmonary alveoli, bronchi, and capillaries were complete. But under high-power field, group L had a more obvious phenomenon of alveolar epithelium and capillary epithelium cells swelling and hypertrophy with respect to group LT. It could be observed that in the partial tissues, erythrocytes exuded from alveoli and the interval tends to widen in the alveoli. Under transmission electron microscopy, group L is observed to have mitochondria swelling, vacuolization, and even autosome edge accumulation. Microvillus of cell Type II significantly decreased or disappeared. Blood–air barrier swelled severely and a portion of it was cracked. The phenomena above were obviously reduced for group LT. Conclusion: Our experiments confirmed that adding Tan IIA into LPD solution can reduce the isolated rabbit's IRI effectively.

Effects of exogenous hydrogen sulfide on nitric oxide and nitric oxide synthase during lung ischemia-reperfusion in a rat model of lung transplantation

Objective To evaluate the effects of exogenous hydrogen sulfide (H2S) on nitric oxide (NO) and nitric oxide synthase (NOS) during lung ischemia-reperfusion in a rat model of lung transplantation.Methods Forty pathogen-free male Sprague-Dawley rats,weighing 250-350 g,aged 2-3 months,were used as donor and recipient rats in this study.The animals were randomly divided into 4 groups (n =10 each):sham operation group (group S); lung transplantation group (group L); lung transplantation + H2S synthesis-CSE inhibitor D,L-propargylgylcine (PPG) group (group PPG); lung transplantation + exogenous H2S donor NaHS group (group NaHS).The donor lungs were removed from live donor rats and placed in low potassium dextran solution at 4 ℃.Orthotopic left lung transplantation was performed using modified three-cuff technique and the time for cold ischemia was 50-70 min.In PPG and NaHS groups,PPG 37.5 mg/kg and NaHS 14 μmol/kg were injected intraperitoneally at 5 min before opening of the hilum of the transplanted lung.The implanted donor lungs were ventilated and reperfused.The donor lungs were removed after being implanted,ventilated and reperfused for 2 h for microscopic examination and for determination of wet to dry lung weight (W/D) ratio and the levels of malondialdehyde (MDA),myeloperoxidase (MPO),inducible NOS (iNOS),endothelial NOS (eNOS),and NO.Results Compared with group S,W/D ratio and the levels of MDA,iNOS,NO and MPO were significantly increased,and the content of eNOS was decreased in group L,and no significant change was found in the parameters mentioned above in group NaHS.Compared with group L,W/D ratio and the levels of MDA,iNOS,NO and MPO were significantly increased,and the content of eNOS was decreased in group PPG,and W/D ratio and the levels of MDA,iNOS,NO and MPO were decreased,and the content of eNOS was increased in group NaHS.The pathological changes of lungs were significantly attenuated in group NaHS as compared with group L.Conclusion Exogenous H2S can attenuate the lung ischemia-reperfusion injury in a rat model of lung transplantation,and decreased iNOS activity and increased eNOS activity are involved in the mechanism. Key words: Hydrogen sulfide; Lung transplantation ; Reperfusion injury ; Nitric oxide ; Nitric oxide synthase

Optimal lung inflation techniques in a rat lung transplantation model: a revisit.

Most of the experimental work assessing optimal lung inflation during lung graft preservation was performed in the late 1990s. Since that time, lung preservation before transplantation has been more standardized, and the optimal lung inflation techniques used during lung preservation in the current clinical setting remain undefined. Nonetheless, lung inflation during storage may play a pivotal role in optimal lung preservation. Lewis rat lungs were perfused with and stored in cold, low-potassium dextran solution (Perfadex, Vitrolife, Göteborg, Sweden) for 6 hours at different levels of lung inflation (25, 50, 75, or 100% of vital capacity [VC]). Orthotopic left lung transplantation using cuff techniques was performed in syngeneic Lewis rats. Posttransplant allograft function, expression of proinflammatory mediators, and expression of lung surfactants were evaluated. Lungs inflated to 75 or 100% VC showed a significantly better oxygenation in blood gas analysis than lungs inflated to 25 or 50% VC. The levels of mRNAs for tumor necrosis factor-α, pro-interleukin-1β, intracellular adhesion molecule 1 were attenuated in lungs inflated to 75 or 100% VC as compared with deflated lungs, suggesting reduced ischemia/reperfusion injury. In addition, transmission electron microscopy demonstrated better preserved lung surfactants in the alveolar space in the lungs inflated to 75 or 100% VC. Inflating lungs to 75 or 100% VC during preservation may be beneficial and result in better posttransplant allograft function through attenuated reperfusion injury and better preserved lung surfactants.

Successful prolonged ex vivo lung perfusion for graft preservation in rats

Ex vivo lung perfusion (EVLP) strategies represent a new frontier in lung transplantation technology, and there have been many clinical studies of EVLP in lung transplantation. The establishment of a reliable EVLP model in small animals is crucial to facilitating translational research using an EVLP strategy. The main objective of this study was to develop a reproducible rat EVLP (R-EVLP) model that enables prolonged evaluation of the explanted lung during EVLP and successful transplantation after EVLP. The donor heart-lung blocks were procured with cold low-potassium dextran solution and immersed in the solution for 1 h at 4 °C. And then, the heart-lung blocks were flushed retrogradely and warmed up to 37 °C in a circuit perfused antegradely with acellular perfusate. The perfusate was deoxygenated with a gas mixture (6% O2, 8% CO2, 86% N2). The perfusion flow was maintained at 20% of the entire cardiac output. At 37 °C, the lungs were mechanically ventilated and perfusion continued for 4 h. Every hour, the perfused lung was evaluated for gas exchange, dynamic lung compliance (Cdyn) and pulmonary vascular resistance (PVR). R-EVLP was performed for 4 h. Pulmonary oxygenation ability (pO2/pCO2) was stable for 4 h during EVLP. It was noted that Cdyn and PVR were also stable. After 4 h of EVLP, pO2 was 303 ± 19 mmHg, pCO2 was 39.6 ± 1.2 mmHg, PVR was 1.75 ± 0.10 mmHg/ml/min and Cdyn was 0.37 ± 0.03 ml/cmH2O. Lungs that were transplanted after 2 h of R-EVLP resulted in significantly better post-transplant oxygenation and compliance when compared with those after standard cold static preservation. Our R-EVLP model maintained stable lung oxygenation, compliance and vascular resistance for up to 4 h of perfusion duration. This reliable model should facilitate further advancement of experimental work using EVLP.

Evaluation of Liver Preservation Solutions by Using Rats Transgenic for Luciferase

IntroductionThe solution in which graft tissue is stored (that is, preservation solution) is an important component of liver transplantation technology. Its protective effect is induced by substances in the solution, including radical scavengers, buffers, and energy-giving substances. New preservation solutions have proven to be effective in preventing organ damage during cold ischemia and in extending the time limits for storage. AimThis study determined the relationship between luminescence intensity and content of adenosine triphosphate (ATP) in liver tissue and proposes a new ex vivo screening system that uses Lewis rats transgenic for luciferase for evaluating the effectiveness of preservation solutions. MethodsSamples (diameter, 2 mm) of liver were obtained from transgenic rats. The viability of these tissues after storage for as long as 6 hours in University of Wisconsin (UW) solution, extracellular trehalose solution of Kyoto, Euro-Collins (EC) solution, histidine–tryptophan–ketoflutarate solution, low potassium dextran solution, or normal saline was assessed by determining ATP content and luminescence intensity. ResultsLuminescence had a linear relationship (R = 0.88) with ATP levels. Regardless of the preservation solution used, the luminescence intensities of the liver tissue chips decreased linearly with time especially through a short span of time (0 to 2 hours; R2 = 0.58–1.0). The luminescence of liver chip tissues maintained long term (2 to 6 hours) in UW solution tended to be higher than those of tissues stored in other solutions (P < .05; 6 hours). On the basis of luminescence intensity, EC might be preferable to the other solutions tested for ultra-short–term storage (0.5 to 2 hours). ConclusionOur model, which combines the use of the bioimaging system and Lewis rats transgenic for luciferase, effectively assessed the viability of liver tissue samples. We believe that this ex vivo screening system will be an effective tool for evaluating preservation solutions for liver grafts.

Influence of extract of Ginkgo biloba leaves tablets on the aquaporin-1 expression in isolated lung ischemia reperfusion

Background The extract of Ginkgo biloba leaves tablets, ginaton, is widely used in treating ischemic cerebrovascular disease in the clinic. This study aimed to investigate the expression of aquaporin-1 (AQP-1) in rat lung with ischemia/ reperfusion injury after pretreatment with ginaton, and whether the pretreatment with ginaton reduces the acute lung injury caused by ischemia/reperfusion injury. Methods Adult Wistar rats were divided into two groups. Some rats were used as donors (n=20), the others as recipients (n=20). Left lungs of donor rats were used for the isolated lung reperfusion model, which perfused only with low potassium dextran (LPD) solution as group A (n=10); the others were pretreated with ginaton before reperfusion as group C (n=10). Right lung of donor rat without any treatment was used as a control group (group B and group D, n=10 for each group). After the model was established, the expression of AQP-1 in the lung tissues was examined by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction. Results Immunohistochemical examination revealed that AQP-1 was expressed in endothelia. Immunoblotting demonstrated that the relative gray values of AQP-1 protein in groups A and C were 0.65±0.06, 0.88±0.11, respectively. The relative gray values of the mRNA expression in groups A and C were 0.30±0.08, 0.49±0.11, respectively. The expression of AQP-1 protein and mRNA in group C was significantly higher than in group A (P &lt;0. 05). Conclusion The pretreatment with ginaton can reduce the acute lung injury caused by ischemia/reperfusion.

MAPK mediates inflammatory response and cell death in rat pulmonary microvascular endothelial cells in an ischemia–reperfusion model of lung transplantation

Hypoxia-reoxygenation of cultured macrovascular endothelial cells is used to study ischemia-reperfusion (IR)-related cellular and molecular changes; however, these models do not accurately depict events in pulmonary microvascular endothelial cells (PMVECs) during conventional lung retrieval and transplantation. We used rat PMVECs in a new non-hypoxic cell-based lung transplantation model to assess these events. To simulate cold storage, rat PMVECs were preserved in 95% O2-5% CO2 at 4°C for 6 hours in low-potassium dextran solution. Dishes were warmed for 1 hour to room temperature for simulating implantation. Medium was added at 37°C in 50% O2-5% CO2-45% N2 to simulate reperfusion. Additional PMVECs were transfected with siRNA targeting mitogen-activated protein kinases (MAPKs) and then subjected to simulated IR. MAPKs and NF-κB were activated during simulated reperfusion, and AP-1 was activated during ischemia and reperfusion. Increased malondialdehyde levels were found during cold ischemia, and apoptosis and production of IL-1β, IL-6, and TNF-α were observed during reperfusion. Silencing of MAPKs attenuated oxidative stress, inflammation and apoptosis. Silencing of JNK and p38 decreased NF-κB phosphorylation and increased inhibitor of NF-κB (IκB)α levels. Knockdown of ERK1/2 increased NF-κB phosphorylation but had no effect on IκBα expression. Silencing of JNK and ERK1/2, but not p38, decreased AP-1 phosphorylation. Exposing rat PMVECs to simulated non-hypoxic IR caused lipid peroxidation, inflammation and apoptosis, which required MAPK-mediated NF-κB and AP-1 activation and distinct regulation of MAPKs by these 2 transcription factors. This model could be used to uncouple mechanisms of IR and evaluate potential therapeutics in alleviating IR injury.

Effect of Warm Perfusion of Donor Lungs Using the OCS on Immune Mediator Release during Preservation

Purpose Warm perfusion of donor lungs for transplantation (Tx) using Organ Care System® (OCS) may provide superior preservation compared to standard cold preservation (SOC). Release and composition of donor-derived soluble immune mediators (IM), triggering allorecognition and inflammation after Tx, might be differentially affected. We analyzed 55 soluble IM in the respective preservation solutions (PS), as well as in peripheral blood (PB) of the recipient. Methods and Materials IM were quantified at protein level (pg/ml) by Luminex-based multiplex-technology in PS at the end of cold storage (SOC, n=11) or warm preservation in OCS (n=13), either based on low potassium dextrane solution. Differences were assessed using Mann-Whitney-U and ROC / AUC statistics. Results Unexpectedly huge differences were found between concentrations in PS, extrapolating to several μg for large volume of PS (1.0– 1.5l). We focus on those 20 factors displaying highest statistical differences with higher release in OCS: IL-6 p Conclusions Donor-derived IM remained low in SOC, whereas OCS potentially depleted them from the organ with accumulation in PS. This ‘dialysis’ effect may indicate anti-inflammatory properties. Further studies on clinical outcomes of patients transplanted with OCS vs. SOC preserved lungs are warranted.

Endobronchial Perfluorocarbon Reduces Inflammatory Activity before and after Lung Transplantation in an Animal Experimental Model

Background. The aim of this study was to evaluate the use of liquid perfluorocarbon (PFC) as an adjuvant substance for lung preservation and assess its role in pulmonary protection after transplantation. Methods. Seventy-two rat lungs were flushed with low-potassium dextran (LPD) solution and randomized into three main groups: control with LPD alone and experimental with 3 (PFC3) and 7 mL/kg (PFC7) of endobronchial PFC instilled just after harvest. Each group was divided into four subgroups according to preservation time (3, 6, 12, and 24 hours). Afterwards, we performed lung transplantation using rat lungs preserved for 12 hours with LPD alone or with 7 mL/kg of endobronchial PFC. Results. There was a significant increase in oxidative stress in the control group at 6 h of cold ischemic time compared with the PFC3 and PFC7 groups. The apoptotic activity and NF-κB expression were significantly higher in the control group compared with the PFC groups at 3, 12, and 24 h of cold preservation. After transplantation, the NF-κB, iNOS, and nitrotyrosine expression as well as caspase 3 activity were significantly lower in the PFC groups. Conclusion. The use of endobronchial PFC as an adjuvant to the current preservation strategy improved graft viability.

Prophylactic Surfactant Inhalation Attenuates Warm Ischemic Injury in Rat Lungs from Donation after Cardiac Death Donors

Background: To deal with the shortage of donor organs in lung transplantation, donation after cardiac death (DCD) has been introduced worldwide as one of the potential strategies to increase the donor pool. Considering ischemia-reperfusion injury in solid organ transplantation from DCD donors, it is very important to alleviate graft injury during warm ischemia. It was reported that surfactant function deteriorated with increasing warm ischemic time, resulting in graft injury. Therefore, we investigated the effect of surfactant inhalation against warm ischemic injury in rat lungs from DCD donors. In this experiment, only ventilation was initiated after warm ischemia, therefore, warm ischemic injury was assessed from the view of hypoxia-reoxygenation injury. Methods/Materials: Male Lewis rats were used. One hundred and ten minutes after cardiac arrest induced by ventricular fibrillation, the lungs were initiated to ventilate and flushed by Low Potassium Dextran solution. Thereafter, heart and lung block was isolated, and ex vivo lung ventilation (EVLV) was conducted. Rats were randomly allocated to 2 groups, the CTRL and SURF groups (n = 5 each). In the SURF group, surfactant was inhaled for 3 min, while nothing was inhaled in the CTRL group. Just after this inhalation period (total warm ischemic time: 135 min), dynamic compliance and airway resistance were evaluated on time throughout 60 min of ventilation. After 60 min of EVLV, lung tissue levels of ATP, ADP, and AMP were measured by high-performance liquid chromatography: messenger RNA (mRNA) expression of NO synthase (neuronal, inducible, endothelial), caspase-3, Bax, and Bcl-2 were measured by real-time RT-PCR: Hematoxylin-Eosin (H-E) staining, immunohistochemical staining for 8-hydroxy-2′-deoxyguanosine (8-OHdG), and in situ apoptosis detection (TUNEL assay) were examined. Results: During 60 min of ventilation, surfactant inhalation significantly increased dynamic compliance (p < 0.01), and decreased airway resistance (p < 0.001). Surfactant inhalation effectively increased lung tissue levels of ATP (p < 0.05). After 60 min of ventilation, mRNA levels of NO synthase (neuronal, inducible, endothelial) and caspase-3 were significantly lower, and mRNA levels of Bcl-2 were significantly higher in the SURF group than those in the CTRL group. In the histologic examination with H-E staining, both the CTRL and SURF groups showed no differences with relatively normal structure. However, in 8-OHdG stain and in TUNEL assay, there were fewer positive cells in the SURF group than those in the CTRL group. Conclusion: We confirmed that surfactant inhalation in the last phase of warm ischemia mitigated warm ischemic injury in an isolated rat lung ventilation model. Reduction of oxidative stress and inhibition of apoptosis might contribute to the protection of the warm ischemic lungs in our study.

Normothermic perfusion of donor lungs for preservation and assessment with the Organ Care System Lung before bilateral transplantation: a pilot study of 12 patients

Cold flush and static cold storage is the standard preservation technique for donor lungs before transplantations. Several research groups have assessed normothermic perfusion of donor lungs but all devices investigated were non-portable. We report first-in-man experience of the portable Organ Care System (OCS) Lung device for concomitant preservation, assessment, and transport of donor lungs. Between Feb 18, and July 1, 2011, 12 patients were transplanted at two academic lung transplantation centres in Hanover, Germany and Madrid, Spain. Lungs were perfused with low-potassium dextran solution, explanted, immediately connected to the OCS Lung, perfused with Steen's solution supplemented with two red-cell concentrates. We assessed donor and recipient characteristics and monitored extended criteria donor lung scores; primary graft dysfunction scores at 0, 24, 48, and 72 h; time on mechanical ventilation after surgery; length of stays in hospital and the intensive-care unit after surgery; blood gases; and survival of grafts and patients. Eight donors were female and four were male (mean age 44·5 years, range 14-72). Seven recipients were female and five were male (mean age 50·0 years, range 31-59). The preharvest donor ratio of partial pressure of oxyen (PaO(2)) to fractional concentration of oxygen in inspired air (F(I)O(2)) was 463·9 (SD 91·4). The final ratio of PaO(2) to F(I)O(2) measured with the OCS Lung was 471·58 (127·9). The difference between these ratios was not significant (p=0·72). All grafts and patients survived to 30 days; all recipients recovered and were discharged from hospital. Lungs can be safely preserved with the OCS Lung, resulting in complete organ use and successful transplantation in our series of high-risk recipients. In November, 2011, we began recruitment for a prospective, randomised, multicentre trial (INSPIRE) to compare preservation with OCS Lung with standard cold storage. TransMedics and German Federal Ministry of Education and Research.

Modelo experimental de perfusão pulmonar ex vivo em ratos: avaliação histopatológica e de apoptose celular em pulmões preservados com solução de baixo potássio dextrana vs. solução histidina-triptofano-cetoglutarato

To compare histopathological findings and the degree of apoptosis among rat lungs preserved with low-potassium dextran (LPD) solution, histidine-tryptophan-ketoglutarate (HTK) solution, or normal saline (NS) at two ischemia periods (6 h and 12 h) using an experimental rat model of ex vivo lung perfusion. Sixty Wistar rats were anesthetized, randomized, and submitted to antegrade perfusion via pulmonary artery with one of the preservation solutions. Following en bloc extraction, the heart-lung blocks were preserved for 6 h or 12 h at 4 ºC and then reperfused with homologous blood for 60 min in an ex vivo lung perfusion system. At the end of the reperfusion, fragments of the middle lobe were extracted and processed for histopathological examination. The parameters evaluated were congestion, alveolar edema, alveolar hemorrhage, inflammatory infiltrate, and interstitial infiltrate. The degree of apoptosis was assessed using the TdT-mediated dUTP nick end labeling method. The histopathological examination showed that all of the lungs preserved with NS presented alveolar edema after 12 h of ischemia. There were no statistically significant differences among the groups in terms of the degree of apoptosis. In this study, the histopathological and apoptosis findings were similar with the use of either LPD or HTK solutions, whereas the occurrence of edema was significantly more common with the use of NS.

Successful lung autotransplantation for central non-small-cell lung cancer: report of a case

Non-small-cell lung cancer (NSCLC) confined to the lung is generally treated by surgical resection. The extent of resection is determined by the location of the tumor and the patient's pulmonary function. This report presents a successful lung autotransplantation in a man with NSCLC that could not tolerate pneumonectomy or sleeve lobectomy. Right upper and middle bilobectomies were performed, the right lower lobe was resected and retrograde perfusion of Raffinose low-potassium dextran solution (4 °C) was administered. The isolated lower lobe was reimplanted by anastomosis of the bronchus, pulmonary artery, and vein. The patient was cancer-free 1 year after the surgery. Lung autotransplantation can therefore be successfully performed for selected patients with central NSCLC.