Low-potassium Dextran Research Articles

A novel rat lung transplantation model using venoarterial extracorporeal membrane oxygenation support

ObjectivesExtracorporeal membrane oxygenation (ECMO) has become an important life support technique during lung transplantation. We aimed to develop a rat model for lung transplantation using venoarterial (VA) ECMO support. MethodsAdult male Sprague-Dawley rats weighing 400 to 450 g were used in this study. ECMO circuits were created by obtaining venous access from the femoral vein with subsequent extracorporeal oxygen exchange, which was then returned to the circulatory system through the left carotid artery (ie, VA-ECMO). Simultaneously, the donor lungs were retrieved and immersed in cold, low-potassium dextran lung preservation solution. Orthotopic left lung transplantation supported by VA-ECMO was performed. Thereafter, a respiratory failure rat model was constructed using ventilation with a hypoxic and hypercapnic gas mixture, consisting of 6% oxygen, 8% carbon dioxide, and 86% nitrogen, before lung transplantation. Similarly, left lung transplantation supported by VA-ECMO was performed in rats with respiratory failure. Arterial blood gas levels were measured at designated time points throughout the experiment. ResultsWe found that VA-ECMO provided sufficient oxygenation and carbon dioxide removal to allow for smooth left lung transplantation in healthy rats and those with respiratory failure. ConclusionsWe established a rat model for lung transplantation using VA-ECMO. Left lung transplantation using VA-ECMO support is also feasible and safe in rat models of respiratory failure. These models provide efficient and economical models for translational medicine for lung transplantation using ECMO. Moreover, it will be invaluable to evaluate the physiological and pathophysiological roles of ECMO during lung transplantation.

Thyroid hormone protects human lung epithelial cells from cold preservation and warm reperfusion-induced injury

BackgroundCellular stress associated with static-cold storage (SCS) and warm reperfusion of donor lungs can contribute to ischemia–reperfusion (IR) injury during transplantation. Adding cytoprotective agents to the preservation solution may be conducive to reducing graft deterioration and improving post-transplant outcomes.MethodsSCS and warm reperfusion were simulated in human lung epithelial cells (BEAS-2B) by exposing cells to low potassium dextran glucose solution at 4 °C for different periods and then switching back to serum-containing culture medium at 37 °C. Transcriptomic analysis was used to explore potential cytoprotective agents. Based on its results, cell viability, caspase activity, cell morphology, mitochondrial function, and inflammatory gene expression were examined under simulated IR conditions with or without thyroid hormones (THs).ResultsAfter 18 h SCS followed by 2 h warm reperfusion, genes related to inflammation and cell death were upregulated, and genes related to protein synthesis and metabolism were downregulated in BEAS-2B cells, which closely mirrored gene profiles found in thyroid glands of mice with congenital hypothyroidism. The addition of THs (T3 or T4) to the preservation solution increases cell viability, inhibits activation of caspase 3, 8 and 9, preserves cell morphology, enhances mitochondrial membrane potential, reduces mitochondrial superoxide production, and suppresses inflammatory gene expression.ConclusionAdding THs to lung preservation solutions may protect lung cells during SCS by promoting mitochondrial function, reducing apoptosis, and inhibiting pro-inflammatory pathways. Further in vivo testing is warranted to determine the potential clinical application of adding THs as therapeutics in lung preservation solutions.

The Advent of Semi-Elective Lung Transplantation-Prolonged Static Cold Storage at 10°C.

Since the early days of clinical lung transplantation the preservation of donor organs has become a fairly standardized procedure and most centers do follow similar processes. This includes the use of low-potassium high dextran flush solutions and static cold storage (SCS) in a cooler filled with ice. Depending on the length of SCS, organs usually arrive at the recipient hospital at a temperature of 0°C-4°C. The question of the optimal storage temperature for donor lung preservation has been revisited as data from large animal experiments demonstrated that organs stored at 10°C experience less mitochondrial damage. Thus, prolonged cold ischemic times can be better tolerated at 10°C-even in pre-damaged organs. The clinical applicability of these findings was demonstrated in an international multi-center observational study including three high-volume lung transplant centers. Total clinical preservation times of up to 24hrs have been successfully achieved in organs stored at 10°C without hampering primary organ function and short-term outcomes. Currently, a randomized-controlled trial (RCT) is recruiting patients with the aim to compare standard SCS on ice with prolonged SCS protocol at 10°C. If, as anticipated, this RCT confirms data from previous studies, lung transplantation could indeed become a semi-elective procedure.

A Novel Tissue Preservation and Transport Solution as a Substitute for Formalin

Formalin, a common laboratory fixative, is a type 1 carcinogen; a biohazard with risks, environmental, disposal, and legal costs; and a chemical modifier of protein epitopes in tissues. A less-toxic tissue preservation method is therefore badly needed. We have developed a novel tissue preservation medium, Amber, composed of low-potassium dextran glucose, 10% honey, and 1% coconut oil. This study investigates Amber as compared with formalin with respect to the following aspects: (1) histologic preservation, (2) epitope integrity with immunohistochemistry (IHC) and immunofluorescence (IF), and (3) integrity of tissue RNA. Rat and human lung, liver, kidney, and heart tissues were collected and stored for 24 hours at 4 °C in Amber or formalin. The tissues were evaluated with hematoxylin and eosin; IHC: thyroid transcription factor, muscle-specific actin, hepatocyte-specific antigen, and common acute lymphoblastic leukemia antigen; and IF: VE-cadherin, vimentin, and muscle-specific actin. RNA quality upon extraction was also assessed. Amber demonstrated superior and/or noninferior performance in rat and human tissue evaluation with respect to standard techniques of histology, IHC, IF, and extracted RNA quality. Amber maintains high-quality morphology without compromising the ability to perform IHC and nucleic acid extraction. As such, Amber could be a safer and superior substitute to formalin for clinical tissue preservation for contemporary pathological examination.

Time to Try Something New: Establishing Rat Models of Lung Transplantation Under ECMO Support

<h3>Purpose</h3> Extracorporeal membrane oxygenation (ECMO) has emerged as an important preoperative, perioperative, and postoperative life-support requisite in lung transplantation. Therefore, we aimed to develop rat models of lung transplantation under veno-arterial (VA) and veno-venous (VV) ECMO support. <h3>Methods</h3> Adult male Sprague-Dawley rats weighing 400-450g (recipients, n=10; donors, n=10) were used. The ECMO circuits were initiated by venous drainage from the femoral vein with extracorporeal oxygen exchange and returned to the blood circulation through the right carotid artery (VA-ECMO) or external jugular vein (VV-ECMO). Before ECMO implantation, 5ml heparinized blood from the donor rat was drawn into tubes. Simultaneously, donor lungs were obtained after pulmonary arterial perfusion with low potassium dextran lung preservation solution. ECMO-supported orthotopic left lung transplantation was performed by using cuff technique. Arterial blood gases were measured at designated intervals throughout the experiments. <h3>Results</h3> We found that VA- and VV-ECMO provided sufficient oxygenation to support a smooth left single lung transplantation procedure. The intraoperative hemodynamic and blood gas parameters were maintained stable. <h3>Conclusion</h3> In this study, we first established rat models of lung transplantation under VA and VV ECMO support. This model provides an efficient, economical, and feasible platform for studying lung transplantation with ECMO. This model can be preclinically applied to unravel significant insights in support of further translational models in large animal and human lung transplantation.

Using nutrient-rich solutions and adding multiple cytoprotective agents as new strategies to develop lung preservation solutions.

Commonly, donor lungs are preserved with low-potassium dextran glucose solution at low temperature. We hypothesized that adding nutrients and/or cytoprotective agents to preservation solutions improves donor lung quality. Human lung epithelial cells and human pulmonary microvascular endothelial cells cultured at 37°C with serum containing medium were switched to designated testing solutions at 4°C with 50% O2 for different cold ischemic time, followed by switching back to serum containing culture medium at 37°C to simulate reperfusion. We found that bicarbonate buffer system should be avoided in preservation solution. When pH was maintained at physiological levels, cell culture media showed better cell survival than in low-potassium dextran glucose solution. Phosphate-buffered cell culture media were further improved by adding colloid dextran 40. When rat donor lungs were preserved at 4°C for 24 h, phosphate-buffered Roswell Park Memorial Institute-1640 medium [RPMI-1640(p)] plus dextran 40 or adding cytoprotective agents (alpha 1 antitrypsin, raffinose, and glutathione) to low-potassium dextran glucose solution prevented alveolar wall swelling, apoptosis, activation of endothelial cells, and cellular edema. Using nutrient-rich solution and/or adding multiple cytoprotective agents is a new direction for designing and developing organ preservation solutions. Cell culture model, as a screening tool, reduces the use of animals and provides potential underlying mechanisms.

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.

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.

Profiling Proteinic Changes Induced by Vildagliptin Treatment in a Mouse Lung Transplantation Model: The Role of Kininogen-1.

BACKGROUND This study investigated the protective effects of pharmaceutical CD26/dipeptidylpeptidase-4 (CD26/DPP-4) inhibitor in lung transplantation (LTx). Changes in protein expression associated with the treatment were screened and identified to evaluate the role of kininogen-1 in early-term ischemia/reperfusion (I/R) injury after LTx. MATERIAL AND METHODS Orthotopic single LTx was performed in syngeneic C57BL/6 mice, with a pharmaceutical CD26/DPP-4 inhibitor (vildagliptin, subcutaneous injection, 10 mg/kg, every 12 h) administered to the investigational group. All donors were perfused and preserved with low potassium dextran (LPD). Grafts were harvested at 60 h post-transplantation after 8 h of cold ischemia. Myeloperoxidase activity and wet/dry weight ratio were measured, followed by histopathological examination. Proteins were separated, analyzed, and identified using proteomics and database searches. The target proteins were validated by Western blot. Immunohistochemical studies were performed in the same lung specimen locus. RESULTS Investigational group (IN) versus control group (CON) comparison showed decreased myeloperoxidase enzymatic activity, as well as decreased edema and interstitial-alveolar inflammation. Proteomics results revealed 78 spots with significant differences in abundance between the 2 groups. Fifteen proteins were identified. Kininogen-1 was up-regulated in CON and down-regulated in IN, with contrasting results for the heat shock protein 70. Immunohistochemical results revealed significantly different staining with kininogen-1 in alveolar macrophages and inflammatory cells. CONCLUSIONS Combined vildagliptin and LPD significantly ameliorated I/R injury after LTx. This treatment may change local pulmonary protein levels. Moreover, proper application of proteins such as kininogen-1 may enhance the protective effects against I/R injury during transplantation.

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.

Mesenchymal stem cells attenuate ischemia-reperfusion injury after prolonged cold ischemia in a mouse model of lung transplantation: a preliminary study.

Mesenchymal stem cells (MSCs) suppress inflammation and immune responses. We conducted this study to find out if MSCs attenuate ischemia-reperfusion injury in a mouse model of lung transplantation. C57BL/6J mouse lungs perfused with low-potassium dextran glucose solution were preserved at 4°C for 18h. Human MSCs were slowly injected into the left pulmonary artery of the lung grafts, and orthotopic left lung transplantation was then performed. The lung isografts were reperfused for 6h, and bronchoalveolar lavage fluid (BALF) from the left lung graft was collected. We measured the protein concentration, cell count, and proinflammatory cytokine concentrations in the BALF. The protein concentration and cell count in the BALF were significantly lower in the MSC-administered grafts than in the PBS-administered controls. Concentrations of proinflammatory cytokines, including IL-1β, IL-17A, and TNF-α, in BALF tended to be lower in the MSC-administered grafts than in the controls, but the difference was not significant. The pre-transplant administration of MSCs via the pulmonary artery of the lung graft attenuated ischemia-reperfusion injury after prolonged cold ischemia in this mouse model of lung transplantation.

Reduction of ischaemia-reperfusion injury in a rat lung transplantation model by low-concentration GV1001.

Lung ischaemia-reperfusion (IR) injury is one of the major complications following lung transplantation. The novel peptide GV1001, which is derived from human telomerase reverse transcriptase, has been reported to possess both antitumour and anti-inflammatory effects. In this study, we focused on the anti-inflammatory effects of GV1001 to investigate the IR injury prevention effect of GV1001 in a rat lung transplantation model. An orthotopic left lung transplantation rat model was established using the modified cuff technique. We applied 50 ml of normal saline (control), Perfadex (low-potassium standard dextran containing perfusion solution), Perfadex with 5 mg GV1001 (5-mg GV, low concentration) and Perfadex with 50 mg GV1001 (50-mg GV, high concentration) as both flushing and preservation solutions. The left lung was stored in the same solution as the flushing solution at 4°C for 3 h. After transplantation, the recipient rats were monitored for 3 h. Arterial blood gas analysis (ABGA), bronchoalveolar lavage (BAL) analysis, wet/dry ratio, histological analysis, apoptotic cell analysis and cytokine [tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6)] analysis were performed to determine the reduction or prevention effect of GV1001 regarding lung IR injury. Compared with the control group, the neutrophil count in BAL, reperfusion oedema and cytokine (TNF-α, IL-6) levels of the transplanted lung were significantly decreased in the 5-mg GV group. Compared with the Perfadex group (16.85 ± 2.43), the neutrophil count in BAL was also significantly decreased in the 5-mg GV group (5.39 ± 0.81) (P< 0.001). In addition, the cytokine (TNF-α, IL-6) levels of the transplanted lung were also significantly decreased in the 5-mg GV group (41.99 ± 12.79, 1069.74 ± 98.48 pg/ml) compared with the Perfadex group (90.73 ± 23.87, 2051.92 ± 243.57 pg/ml) (P < 0.05 and P < 0.001, respectively). However, the 50-mg GV group showed less effect than the 5-mg GV group. Adding a low concentration of GV1001 to the lung preservation solution (Perfadex) provided potential protective effects against IR injury after lung transplantation in rats. Therefore, GV1001 should be considered as a promising anti-inflammatory agent for IR injury.

(467) - Polymerized Human Serum Albumin as an Osmotic Molecule to Support Ex-Vivo Lung Perfusion

Ex vivo lung perfusion (EVLP) is an integral method of organ preservation that could expand the donor pool by allowing organ assessment and recovery before transplantation. The composition of the perfusion solution is crucial in maintaining and improving the function of the organ during EVLP. This study was performed to compare the outcomes of EVLP using perfusate supplemented with polymeric human serum albumin (polyHSA) versus human serum albumin (HSA) using a small animal model. Rat heart-lung blocks were procured, cold flushed with low potassium dextran glucose solution, and divided into two treatment groups: normothermic EVLP using modified cell culture media supplemented with either 4% HSA or 4% polyHSA (n=6, each). The deoxygenated solution (6% O2, 8%CO2, 86% N2) was at 20% cardiac output for 120 minutes at 37oC. The lungs were evaluated using oxygen production, lung compliance, pulmonary vascular resistance (PVR), wet-to dry ratio and lung weight. Standard statistical methods were used. PolyHSA concentrations were formulated and perfused in comparison to weight by volume protein concentration. At 2 hours of normothermic EVLP the type of perfusion solution (HSA or polyHSA) significantly impacted the end organ metrics. Oxygen production, lung compliance, and PVR were comparable between the cohorts (p>0.05) at 2 hours of EVLP. The HSA group showed a significant increase in lung weight and wet to dry ration, when compared to the polyHSA cohort (both p< 0.05, figure). In a small animal model of normothermic EVLP a polyHSA-supplemented perfusate demonstrated significantly less edema, as compared to HSA. The solution oncotic pressure and physical properties of the molecule can have a direct impact on tissue edema and lung weight that develops with EVLP. This observation demonstrates the importance of refining preservation / perfusion solutions and the need to optimize the perfusate used in EVLP with use of polyHSA to limit pulmonary edema.

Extension of donor lung preservation with hypothermic storage after normothermic ex vivo lung perfusion

Ex vivo lung perfusion (EVLP) allows normothermic evaluation and treatment of donor lungs not currently acceptable for transplant and improves organ use. Donor lungs undergo a period of cold preservation before (cold ischemic time [CIT]-1) and after (CIT-2) EVLP. We investigated the effect of an extended CIT-2 on lung function after transplantation. Explanted pig lungs, preserved in low-potassium dextran flush (Perfadex) at 4°C for 10 hours, were subjected to 6 hours of EVLP. They were subsequently allocated to 2 groups: short CIT-2 (CIT-2 = 2 hours; n = 5), and long CIT-2 (CIT-2 = 10 hours; n = 5). In a control group (n = 6), explanted lungs were placed in cold static preservation for 24 hours without EVLP. After the total preservation period, the left lung was transplanted in all groups. After 4 hours of reperfusion, oxygenation function, acute lung injury score, inflammatory markers, and cell death pathway markers were similar between short and long CIT-2 groups. Both EVLP groups fared significantly better than the control group in oxygenation function (p < 0.05). The intervention of EVLP improved lung function after transplantation, and this was not affected by a prolonged cold static preservation time after EVLP. These results provide the basis for a practical prolonged lung preservation strategy using a combination of cold and warm preservation techniques, which may improve lung transplantation logistics and outcomes.

Re: Retrogradein situversus antegrade pulmonary preservation in clinical lung transplantation: a single-centre experience

Historically, grafts were preserved by means of autoperfusion with extracorporeal circulation, topical cooling or donor core cooling. In the 1980s, the standard lung preservation method was single pulmonary artery flush perfusion, using modified Euro-Collins solution at 4°C [1]. However, reperfusion injury remained a significant factor for morbidity and early mortality after lung transplant. Since the early years of lung preservation strategies, some advances have emerged in transplant teams, such as adding prostaglandin E2 to prevent counteract temperature and potassium-induced vasoconstriction. The use of low potassium dextran glucose solutions shows better overall lung function, superior early oxygenation, higher lung compliance, lower incidence of severe primary graft dysfunction and 30-day mortality [2]. As Gohrbandt et al. point out, the route to administer the preservation solution may also have an impact on early lung graft performance [3]. Anterograde flush initiating the infusion through the pulmonary artery with pulmonary vein drainage was the traditional approach for many years [4]. This technique, however, presents a number of limitations. Flushing the pulmonary artery leaves the bronchial tissue to be preserved by topical cooling alone, raising the complication from the bronchial suture. Also, clots and fat or brain tissue emboli, especially in donors with major trauma and multiple bone fractures, may hamper uniform flushing and cooling and, therefore, impair graft function upon reperfusion. Adding a retrograde flush, whereby the preservation solution is administered via the left atrial appendage or four pulmonary veins into the pulmonary venous system and drained through the pulmonary artery, has the potential to flush not only pulmonary vessels, but also the bronchial network, contributing to the adequate perfusion of the tracheobronchial submucosa tissue, and also limiting the effect of hypothermic pulmonary arterial vasoconstriction. On the other hand, less ventilated areas, such as the posterior segments of lower lobes, are better flushed. Also residual blood and harmful constituents and any possible microthrombi that may obstruct the pulmonary vessels are washed out. The evidence from clinical series of this technique is limited [5]; authors such as Gohrbandt et al. show us that there are no clinical benefits of retrograde lung preservation alone and that it may cause or prolong mechanical ventilation and primary graft dysfunction at 72 h and prolong ICU stay [3]. On the other hand, some evidence has been published, showing that biphasic lung preservation is not only clinically feasible [6], but also related with better early oxygenation ratio, lower mean airway pressure and chest radiography score [7, 8]. Many transplant teams have now adopted a biphasic technique with an antegrade flush through the pulmonary artery followed by a retrograde flush through each of the pulmonary veins after heart excision with the lung still ventilated inside the cadaver. However, there is lack of controlled randomized studies.

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