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

We 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 hydrogen’s protective effects, we conducted a gene array analysis to identify changes in gene expression associated with hydrogen treatment. Donor rats were exposed to mechanical ventilation with 98% oxygen and 2% nitrogen or 2% hydrogen for 3h 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. Pretreatment 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. Donor 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 hydrogen’s protective effects against I/R injury 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 [Mg + 2H(2) O → Mg (OH)(2) + H(2) ] 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 method of preserving cardiac grafts using a hydrogen-rich water bath

Background: Exogenously 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. Methods: The 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. Results: In 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. Conclusions: Hydrogen 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 gas reduces hyperoxic lung injury via the Nrf2 pathway

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 4 experimental groups and administered the following gas mixtures for 60 hours: 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.

Hydrogen Preconditioning During Ex Vivo Lung Perfusion Improves the Quality of Lung Grafts in Rats

Background: Although the benefits of ex vivo lung perfusion (EVLP) have been globally advocated, the potentially deleterious effects of applying EVLP, in particular activation of proinflammatory cascades and alteration of metabolic profiles, are rarely discussed. This study examined proinflammatory events and metabolic profiles in lung grafts on EVLP and tested whether preconditioning lung grafts with inhaled hydrogen, a potent, cytoprotective gaseous signaling molecule, would alter the lungs’ response to EVLP. Methods: Rat heart-lung blocks were mounted on an acellular normothermic EVLP system for 4 hr and ventilated with air or air supplemented with 2% hydrogen. Arterial and airway pressures were monitored continuously; perfusate was sampled hourly to examine oxygenation. After EVLP, the lung grafts were transplanted orthotopically into syngeneic rats, and lung function was examined. Results: Placing lung grafts on EVLP resulted in significant upregulation of the messenger RNAs for several proinflammatory cytokines, higher glucose consumption, and increased lactate production. Hydrogen administration attenuated proinflammatory changes during EVLP through upregulation of the heme oxygenase-1. Hydrogen administration also promoted mitochondrial biogenesis and significantly decreased lactate production. Additionally, in the hydrogen-treated lungs, the expression of hypoxia-inducible factor-1 was significantly attenuated during EVLP. These effects were maintained throughout EVLP and led to better posttransplant lung graft function in the recipients of hydrogen-treated lungs. Conclusions: Lung grafts on EVLP exhibited prominent proinflammatory changes and compromised metabolic profiles. Preconditioning lung grafts using inhaled hydrogen attenuated these proinflammatory changes, promoted mitochondrial biogenesis in the lungs throughout the procedure, and resulted in better posttransplant graft function.