Yugo Tanaka

Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo

Hyperoxic lung injury is a major concern in critically ill patients who receive high concentrations of oxygen to treat lung diseases. Successful abrogation of hyperoxic lung injury would have a huge impact on respiratory and critical care medicine. Hydrogen can be administered as a therapeutic medical gas. We recently demonstrated that inhaled hydrogen reduced transplant-induced lung injury and induced heme oxygenase (HO)-1. To determine whether hydrogen could reduce hyperoxic lung injury and investigate the underlying mechanisms, we randomly assigned rats to four experimental groups and administered the following gas mixtures for 60 h: 98% oxygen (hyperoxia), 2% nitrogen; 98% oxygen (hyperoxia), 2% hydrogen; 98% balanced air (normoxia), 2% nitrogen; and 98% balanced air (normoxia), 2% hydrogen. We examined lung function by blood gas analysis, extent of lung injury, and expression of HO-1. We also investigated the role of NF-E2-related factor (Nrf) 2, which regulates HO-1 expression, by examining the expression of Nrf2-dependent genes and the ability of hydrogen to reduce hyperoxic lung injury in Nrf2-deficient mice. Hydrogen treatment during exposure to hyperoxia significantly improved blood oxygenation, reduced inflammatory events, and induced HO-1 expression. Hydrogen did not mitigate hyperoxic lung injury or induce HO-1 in Nrf2-deficient mice. These findings indicate that hydrogen gas can ameliorate hyperoxic lung injury through induction of Nrf2-dependent genes, such as HO-1. The findings suggest a potentially novel and applicable solution to hyperoxic lung injury and provide new insight into the molecular mechanisms and actions of hydrogen.

Notch-Nrf2 Axis: Regulation of Nrf2 Gene Expression and Cytoprotection by Notch Signaling

ABSTRACT The Notch signaling pathway enables regulation and control of development, differentiation, and homeostasis through cell-cell communication. Our investigation shows that Notch signaling directly activates the Nrf2 stress adaptive response pathway through recruitment of the Notch intracellular domain (NICD) transcriptosome to a conserved Rbpjκ site in the promoter of Nrf2. Stimulation of Notch signaling through Notch ligand expression in cells and by overexpression of the NICD in RosaNICD/−::AlbCre mice in vivo induces expression of Nrf2 and its target genes. Continuous and transient NICD expression in the liver produces a Notch-dependent cytoprotective response through direct transcriptional activation of Nrf2 signaling to rescue mice from acute acetaminophen toxicity. This response can be reversed upon genetic disruption of Nrf2. Morphological studies showed that the characteristic phenotype of high-density intrahepatic bile ducts and enlarged liver in RosaNICD/−::AlbCre mice could be at least partially reversed after Nrf2 disruption. Furthermore, the liver and bile duct phenotypes could be recapitulated with constitutive activation of Nrf2 signaling in Keap1F/F::AlbCre mice. It appears that Notch-to-Nrf2 signaling is another important determinant in liver development and function and promotes cell-cell cytoprotective signaling responses.

A novel method of preserving cardiac grafts using a hydrogen-rich water bath

BACKGROUNDnExogenously administered hydrogen exerts cytoprotective effects through anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms in various disease settings, including organ transplantation. Our objective in this study was to evaluate the efficacy of a novel cold storage device equipped with a hydrogen-rich water bath.nnnMETHODSnThe study used an established rat heterotopic transplantation model. Syngeneic heart grafts from elderly donors (60- to 70-week-old Lewis rats) or allografts from adult donors (12-week-old Brown Norway rats) were exposed to prolonged cold preservation. The cardiac grafts were stored in plastic bags containing Celsior, which were immersed in the cold water bath equipped with an electrolyzer to saturate the water with hydrogen. The cardiac grafts then were heterotopically engrafted into Lewis rat recipients.nnnRESULTSnIn both experimental settings, serum troponin I and creatine phosphokinase were markedly elevated 3 hours after reperfusion in the control grafts without hydrogen treatment. The grafts exhibited prominent inflammatory responses, including neutrophil infiltration and the upregulation of messenger RNAs for pro-inflammatory cytokines and chemokines. Myocardial injury and inflammatory events were significantly attenuated by organ storage in the hydrogen-rich water bath. The grafts stored using the hydrogen-rich water bath also exhibited less mitochondrial damage and a higher adenosine triphosphate content.nnnCONCLUSIONSnHydrogen delivery to cardiac grafts during cold preservation using a novel hydrogen-supplemented water bath efficiently ameliorated myocardial injury due to cold ischemia and reperfusion. This new device to saturate organs with hydrogen during cold storage merits further investigation for possible therapeutic and preventative use during transplantation.

Hydrogen‐supplemented drinking water protects cardiac allografts from inflammation‐associated deterioration

Recent evidence suggests that molecular hydrogen has therapeutic value for disease states that involve inflammation. We hypothesized that drinking hydrogen‐rich water (HW) daily would protect cardiac and aortic allograft recipients from inflammation‐associated deterioration. Heterotopic heart transplantation with short‐course tacrolimus immunosuppression and orthotopic aortic transplantation were performed in allogeneic rat strains. HW was generated either by bubbling hydrogen gas through tap water (Bu‐HW) or via chemical reaction using a magnesium stick [Mgu2003+u20032H2O → Mg (OH)2u2003+u2003H2] immersed in tap water (Mg‐HW). Recipients were given either regular water (RW), Mg‐HW, Bu‐HW, or Mg‐HW that had been subsequently degassed (DW). Graft survival was assessed by daily palpation for a heartbeat. Drinking Mg‐HW or Bu‐HW was remarkably effective in prolonging heart graft survival and reducing intimal hyperplasia in transplanted aortas as compared with grafts treated with RW or DW. Furthermore, T cell proliferation was significantly inhibited in the presence of hydrogen in vitro, accompanied by less production of interleukin‐2 and interferon‐γ. Hydrogen treatment was also associated with increased graft ATP levels and increased activity of the enzymes in mitochondrial respiratory chain. Drinking HW prolongs survival of cardiac allografts and reduces intimal hyperplasia of aortic allografts.

A Novel Dual Ex Vivo Lung Perfusion Technique Improves Immediate Outcomes in an Experimental Model of Lung Transplantation

The lungs are dually perfused by the pulmonary artery and the bronchial arteries. This study aimed to test the feasibility of dual‐perfusion techniques with the bronchial artery circulation and pulmonary artery circulation synchronously perfused using ex vivo lung perfusion (EVLP) and evaluate the effects of dual‐perfusion on posttransplant lung graft function. Using rat heart‐lung blocks, we developed a dual‐perfusion EVLP circuit (dual‐EVLP), and compared cellular metabolism, expression of inflammatory mediators, and posttransplant graft function in lung allografts maintained with dual‐EVLP, standard‐EVLP, or cold static preservation. The microvasculature in lung grafts after transplant was objectively evaluated using microcomputed tomography angiography. Lung grafts subjected to dual‐EVLP exhibited significantly better lung graft function with reduced proinflammatory profiles and more mitochondrial biogenesis, leading to better posttransplant function and compliance, as compared with standard‐EVLP or static cold preservation. Interestingly, lung grafts maintained on dual‐EVLP exhibited remarkably increased microvasculature and perfusion as compared with lungs maintained on standard‐EVLP. Our results suggest that lung grafts can be perfused and preserved using dual‐perfusion EVLP techniques that contribute to better graft function by reducing proinflammatory profiles and activating mitochondrial respiration. Dual‐EVLP also yields better posttransplant graft function through increased microvasculature and better perfusion of the lung grafts after transplantation.

Successful prolonged ex vivo lung perfusion for graft preservation in rats

OBJECTIVESnEx 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.nnnMETHODSnThe 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).nnnRESULTSnR-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.nnnCONCLUSIONSnOur 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.

Preservation solution supplemented with biliverdin prevents lung cold ischaemia/reperfusion injury

OBJECTIVESnBiliverdin (BV), one of the byproducts of heme catalysis through the heme oxygenase system, is a known scavenger of the reactive oxygen species. We hypothesized that adding BV to the perfusate and cold storage solution could protect rat lung grafts from oxidative injuries via its antioxidant efficacies.nnnMETHODSnOrthotopic left lung transplantation was performed in a syngenic Lewis-to-Lewis rat combination under 100% oxygen. Grafts were preserved in low-potassium dextran (LPD; Perfadex) at 4°C for 6 h with or without supplementation of 1 or 10 μM of BV into LPD.nnnRESULTSnProlonged cold storage and reperfusion resulted in a considerable deterioration of graft functions associated with massive apoptosis in the grafts after reperfusion. The untreated grafts exhibited the early up-regulations of mRNA for inflammatory mediators and an increase in a marker of lipid peroxidation, showing oxidative injuries. Although BV supplementation of LPD at a lower concentration (1 μM) did not improve the graft gas exchange, the grafts treated with BV (10 μM) showed a significant improvement of oxygenation and less inflammatory responses as well as reduced lipid peroxidation and apoptosis. Although the rapid activations of mitogen-activated protein kinases (MAPKs) were seen 30 min after reperfusion in the grafts stored in control LPD, BV treatment significantly reduced phosphorylated-MAPK protein expression.nnnCONCLUSIONSnThis study demonstrates that the exposure of the lung grafts to BV during cold storage can impart potent cytoprotective effects to lung cold ischaemia/reperfusion injury and significantly improve the lung graft function following extended cold preservation and transplantation by the mechanism of a reduction in oxidative injury and following inflammatory events.

Profiling molecular changes induced by hydrogen treatment of lung allografts prior to procurement

BACKGROUNDnWe previously demonstrated that donor treatment with inhaled hydrogen protects lung grafts from cold ischemia/reperfusion (I/R) injury during lung transplantation. To elucidate the mechanisms underlying hydrogens protective effects, we conducted a gene array analysis to identify changes in gene expression associated with hydrogen treatment.nnnMETHODSnDonor rats were exposed to mechanical ventilation with 98% oxygen and 2% nitrogen or 2% hydrogen for 3 h before harvest; lung grafts were stored for 4h in cold Perfadex. Affymetrix gene array analysis of mRNA transcripts was performed on the lung tissue prior to implantation.nnnRESULTSnPretreatment of donor lungs with hydrogen altered the expression of 229 genes represented on the array (182 upregulated; 47 downregulated). Hydrogen treatment induced several lung surfactant-related genes, ATP synthase genes and stress-response genes. The intracellular surfactant pool, tissue adenosine triphosphate (ATP) levels and heat shock protein 70 (HSP70) expression increased in the hydrogen-treated grafts. Hydrogen treatment also induced the transcription factors C/EBPα and C/EBPβ, which are known regulators of surfactant-related genes.nnnCONCLUSIONnDonor ventilation with hydrogen significantly increases expression of surfactant-related molecules, ATP synthases and stress-response molecules in lung grafts. The induction of these molecules may underlie hydrogens protective effects against I/R injury during transplantation.

Adenosine injection prior to cardioplegia enhances preservation of senescent hearts in rat heterotopic heart transplantation

OBJECTIVESnAdvanced donor age is one of the risk factors for graft failure and is the leading cause of early death after heart transplantation. Better myocardial preservation methods should reduce graft failure. The purpose of this study was to determine if adenosine, which is known to enhance cardioplegic protection, enhances myocardial preservation during heart transplantation using older donors.nnnMETHODSnWe used a rat heterotopic heart transplantation model with Lewis rats that were at least 60 weeks old as donors. We injected saline (control) or adenosine (0.1 or 0.2 mg/kg) before cardioplegia, perfused with cold Celsior and stored the hearts in Celsior for 6 h at 4°C. The grafts were transplanted into syngenic, 12-16-week old recipients, and blood and tissue were collected 3 h after reperfusion.nnnRESULTSnBolus injection of adenosine led to faster mechanical arrest after perfusion with Celsior and faster reanimation after reperfusion compared with controls. Adenosine treatment significantly reduced myocardial injury, as indicated by serum troponin I and creatine phosphokinase levels. The mRNAs for inflammatory cytokines were markedly increased in the control grafts, but were less upregulated in the grafts treated with adenosine. The grafts treated with adenosine also exhibited less mitochondrial damage, fewer infiltrating cells and a higher adenosine triphosphate content.nnnCONCLUSIONSnAdenosine injection prior to perfusion of cardioplegia significantly reduced cold ischaemia/reperfusion injury in cardiac grafts from older donors and improved the stores of cellular energy after reperfusion. This procurement protocol may be clinically feasible and should be considered in the clinical setting, particularly for older donors.

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

BACKGROUNDnMost 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.nnnMATERIALS AND METHODSnLewis 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.nnnRESULTSnLungs 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.nnnCONCLUSIONSnInflating 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.