- Email: [email protected]
Hydrogen inhalation protects against acute lung injury induced by hemorrhagic shock and resuscitation
Hydrogen inhalation protects against acute lung injury induced by hemorrhagic shock and resuscitation Keisuke Kohama, MD, PhD,a Hayato Yamashita, PhD,b Michiko Aoyama-Ishikawa, PhD,b Toru Takahashi, MD,c Timothy R. Billiar, MD,d Takeshi Nishimura, MD,a Joji Kotani, MD, PhD,a and Atsunori Nakao, MD, PhD,a Nishinomiya, Hyogo, and Okayama, Japan, and Pittsburgh, PA
Introduction. Hemorrhagic shock followed by fluid resuscitation (HS/R) triggers an inflammatory response and causes pulmonary inflammation that can lead to acute lung injury (ALI). Hydrogen, a therapeutic gas, has potent cytoprotective, antiinflammatory, and antioxidant effects. This study examined the effects of inhaled hydrogen on ALI caused by HS/R. Methods. Rats were subjected to hemorrhagic shock by withdrawing blood to lower blood pressure followed by resuscitation with shed blood and saline to restore blood pressure. After HS/R, the rats were maintained in a control gas of similar composition to room air or exposed to 1.3% hydrogen. Results. HS/R induced ALI, as demonstrated by significantly impaired gas exchange, congestion, edema, cellular infiltration, and hemorrhage in the lungs. Hydrogen inhalation mitigated lung injury after HS/R, as indicated by significantly improved gas exchange and reduced cellular infiltration and hemorrhage. Hydrogen inhalation did not affect hemodynamic status during HS/R. Exposure to 1.3% hydrogen significantly attenuated the upregulation of the messenger RNAs for several proinflammatory mediators induced by HS/R. Lipid peroxidation was reduced significantly in the presence of hydrogen, indicating antioxidant effects. Conclusion. Hydrogen, administered through inhalation, may exert potent therapeutic effects against ALI induced by HS/R and attenuate the activation of inflammatory cascades. (Surgery 2015;158:399-407.) From the Department of Emergency, Disaster and Critical Care Medicine,a Hyogo College of Medicine, Nishinomiya; the Kobe University Graduate School of Health Science,b Kobe, Hyogo; the Faculty of Health and Welfare Science,c Okayama Prefectural University, Okayama, Japan, and the Department of Surgery,d University of Pittsburgh, Pittsburgh, PA
HEMORRHAGE contributes to mortality after trauma accounting for 30–40% of deaths caused by trauma worldwide despite recent advances in resuscitation and critical care.1 Hemorrhagic shock followed by fluid resuscitation (HS/R) triggers an inflammatory response characterized by upregulation of proinflammatory cytokines and adhesion molecules and induces pulmonary inflammation that can lead to acute lung injury (ALI). ALI often predicates multiple organ failure and mortality.2 Thus, despite recent advances in intensive care, lung injury after HS/R is still Supported by JSPS KAKENHI Grant Number #25462840. Presented at the 10th Annual Academic Surgical Congress in Las Vegas, Nevada, February 3-5, 2015. Accepted for publication March 22, 2015. Reprint requests: Atsunori Nakao, MD, PhD, 1-1 Mukogawa, Nishinomiya, Hyogo, Japan. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2015.03.038
among the most common causes of death after trauma.3,4 Hydrogen is an important physiologic regulatory molecule and exerts antioxidant, antiinflammatory, and antiapoptotic protective effects on cells and organs.5 Because hydrogen can be administered via a ventilation circuit, inhaled hydrogen therapy is considered clinically applicable for patients with shock. Additionally, although hydrogen has therapeutic benefits in multiple organs, the lungs are an ideal target organ for hydrogen therapy, because inhalation is a straightforward delivery method. In fact, hydrogen treatment ameliorated lung injury in several model systems, including ventilatorinduced lung injury,6 ischemia–reperfusion injury during lung transplantation,7 and hyperoxic lung injury.8 Although the therapeutic efficacies of hydrogen have been studied, there is limited information on processes regulated by the hydrogen molecule. We hypothesized that, because of its antiinflammatory and antioxidant SURGERY 399
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properties, inhaled hydrogen therapy could ameliorate ALI after HS/R. METHODS Animals. Male Sprague-Dawley rats weighing 300–500 g (6–9 weeks old) were purchased from Clea Japan, Inc. (Tokyo, Japan) and were kept in individual stainless steel cages in a temperature, humidity, and light-controlled room (23 ± 38C; 55 ± 15%; 12-hour light–dark cycle) for 2–5 weeks before the experiments. During this period, all animals were provided standard food (AIN-93G diet; Oriental Kobo Corporation, Tokyo, Japan) and free access to water. All procedures involving rats were conducted in accordance with the guidelines of the Animal Care and Use Committees of the Hyogo College of Medicine and complied with the National Research Council’s Guide for the Humane Care and Use of Laboratory Animals. Animal model. Under general anesthesia with isoflurane, a catheter for monitoring arterial pressure was placed into the right femoral artery. To create a shock, blood was withdrawn through this catheter, and blood pressure was maintained at 30 ± 5 mmHg for 60 minutes. After 60 minutes, shed blood was reinfused (with saline if necessary for adequate volume restoration), the femoral artery was ligated, and the wound was closed (Fig 1). In sham control rats, we did not induce shock or resuscitate with fluid restoration; we simply placed the catheter, removed it 60 minutes later, ligated the femoral artery, and closed the wound. To maintain body temperature, 378C warming pads were used throughout this procedure. After the procedure, the rats were kept in an air/gas exposure box for 1–6 hours. Blood samples were collected. Rats were humanely killed 1, 3, or 6 hours after HS/R by isoflurane overdose (Fig 1). The lungs were excised and divided into 2 sections. The right lobe was snap frozen immediately with liquid nitrogen for further analysis. The left lobe of the lungs was used for histologic examination. Hydrogen treatment. For hydrogen gas treatment, cylinders with nitrogen-based, highpressure, premixed gases were purchased (Japan Fine Products, Kanagawa, Japan). The manufacturer confirmed the concentrations of H2 (1.3%), O2 (21%), and N2 (77.7%). In Japan, 1.3% is the highest concentration of H2 that can be mixed and bottled under high pressure with 21% oxygen for clinical use. As a control, additional N2 was administered instead of H2 (O2, 21%; N2, 79%). The premixed gases were delivered to the rats via a gas exposure chamber (Natsume Seisakusho
Fig 1. Experimental protocol. Blood was withdrawn, and blood pressure was maintained at 30 ± 5 mmHg for 60 minutes. After 60 minutes, shed blood was reinfused. Rats were then placed in an exposure chamber filled with either 1.3% H2 (H2, 1.3%; O2, 21%; N2, 77.7%) or with only oxygen and nitrogen (O2, 21%; N2, 79%). MABP, Mean arterial blood pressure.
Co. Tokyo, Japan). Hydrogen or control gas (designated N2) was administered during shock and for 1, 3, or 6 hours before killing and tissue collection. The sham control rats were placed in the gas exposure chamber with control (N2) gas for 1 hour. Assessment of gas exchange function and blood lactate levels. Partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), and blood lactate levels were assessed by analysis of blood gases (iSTAT, Abbott Point Care Inc., Princeton, NJ) before killing. Blood gases were assessed on a fraction of inspired oxygen (FiO2) of 1.0 in blood drawn from the abdominal aorta 3 minutes after oxygen inspiration was initiated. Measurement of malondialdehyde. Lung tissues were harvested after 3 hours of gas exposure and kept at -808C until analysis. The tissue was homogenized, and tissue malondialdehyde (MDA) concentration was determined according to the manufacturer’s instructions (Kit MDA-586; Oxisresearch, Portland, OR). Histopathology. Lungs were fixed by inflation with buffered 4% paraformaldehyde for 24 hours. After embedding in paraffin, the sections were prepared and stained with hematoxylin and eosin. Polymorphonuclear neutrophils (PMNs) were stained using a naphthol AS-D chloroacetate esterase staining kit (Sigma Diagnostics) and identified by nuclear morphology stained in bright red. PMN were counted in 10 high-power fields (3400) per sample. ALI was scored with the samples’ identities masked according to previously described criteria,9 specifically (1) thickness of the alveolar wall, (2) infiltration or aggregation of neutrophils in air space, alveolar wall, or vessel wall,
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and (3) alveolar congestion. Each item was graded on a 4-point scale (0–3), with higher scores indicating more severe damage; the range of possible total ALI scores was 0–9. ALI scores were assessed in 10 high-power fields (3400) per sample. Immunohistochemical analysis. The slides were incubated with primary antibody (8-hydroxydeoxyguanosine [8-OHdG] monoclonal antibody [Nikken Seil, Shizuoka, Japan]) for 2 hours at room temperature. The washed samples were treated with the Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan) for 30 minutes at room temperature, followed by visualization with diaminobenzidine (liquid DAB + substrate chromogen system; Dako Japan, Tokyo, Japan), for 5 minutes, and counterstained with Mayer’s hematoxylin. Real-time reverse transcriptase polymerase chain reaction. Rat messenger RNAs (mRNA) were quantified in duplicate using SYBR Green 2-step, real-time reverse transcriptase polymerase chain reaction, as described previously.10-12 The following mRNAs were quantitated: rat tumor necrosis factor (TNF)-a, intercellular adhesion molecule (ICAM)-1, interleukin (IL)-1b, IL-6, inducible nitric oxide synthase (iNOS), chemokine (C-C motif) ligand 2 (CCL2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Optimized and validated primer sets were obtained from realtimeprimers.com (Elkins Park, PA). Western blot. Protein was extracted and divided into cytoplasmic and nuclear fractions using NEPER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc, Waltham, MA). Western blot was performed using anti-nuclear factor (NF)-kB p65 antibody (Cell Signaling Technology, Danvers, MA), anti-lamin (A/C) antibody (Cell Signaling Technology) and anti–b-actin antibody (Sigma-Aldrich, St. Louis, MO). Polyvinylidene difluoride membranes were incubated overnight at 48C with primary antibodies diluted in Can Get Signal immunoreaction enhancer solution 1 (Toyobo, Osaka, Japan), then with horseradish peroxidase-conjugated antimouse secondary antibodies diluted in Can Get Signal solution 2 (Toyobo) for 1 hour at room temperature. Antibody binding was detected using Enhanced Chemiluminescence Plus reagents (GE Healthcare, Buckinghamshire, UK) and the Optima Shot CL-420a image-capturing system (Wako, Osaka, Japan). The band intensities were quantified using NIH image analysis software. NF-kB DNA binding activity. NF-kB DNA binding activity was measured by electrophoretic mobility shirt assay (EMSA) using nuclear extracts from lung tissue and an NF-kB oligonucleotide
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(Promega, Madison, WI) based on the NF-kB sequence in the immunoglobulin light-chain enhancer as previously described.13,14 The band intensities were quantified using NIH image analysis software. Statistical analysis. Results are expressed as mean values ± standard error of the mean. Parametric data were analyzed with 1-way analysis of variance (ANOVA) followed by post hoc analysis with the Bonferroni correction. The lung injury score was analyzed with the Mann–Whitney U test with the Bonferroni correction. The cell count was analyzed using ANOVA with Tukey-Kramer methods. JMP version 8 (SAS Institute, Cary, NC) was used for all statistical analyses. RESULTS Hydrogen improved tissue oxygenation and reduced oxidative damage. HS/R impaired pulmonary function and resulted in a remarkable decrease in PO2 levels 3 hours after resuscitation (Fig 2, A). Treatment with hydrogen after HS/R improved blood oxygenation (Fig 2, A). Neither HS/R nor hydrogen inhalation altered PCO2 levels (Fig 2, B), and there was no difference in vital signs, including blood pressure and heart rate, between the groups (data not shown). Blood lactate levels increased notably after HS/R, and this increase was suppressed significantly in the rats treated with hydrogen (Fig 2, C). Blood pH and base excess were not different in any of the treatment groups (data not shown). Hydrogen inhalation reduced ALI and PMN infiltration. In histologic analysis, the lungs of rats exposed to HS/R showed marked cellular infiltration, edema in the interstitial area, and associated thickening of the alveolar septum. Treatment with inhaled hydrogen after HS/R reduced both edema and inflammatory cell infiltration (Fig 3, A-C). Although there was some suggestion that the wet/dry ratio, another indicator of altered lung permeability, was lower in H2-treated lungs than in control lungs after HS/R, this difference was not significant (data not shown). Damage, as indicated by the lung injury score, was significantly reduced in rats treated with H2 (4.5 ± 0.5 in the N2 group vs 3.3 ± 0.3 in the H2 group; P < .05; Fig 3, G). PMNs are the predominant infiltrating cells in the lung during hemorrhage shock-induced lung injury. Although there were scarce PMNs in the sham-treated lungs, massive neutrophil accumulation in the alveolar space, alveolar capillary congestion, and exudation in the lungs were seen in the rats subjected to HS/R and exposed to control gas
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Fig 2. Blood gas and lactate levels 3 hours after hemorrhagic shock followed by fluid resuscitation. A, Partial pressure of oxygen (PO2). B, Partial pressure of carbon dioxide (PCO2). C, Lactate levels. *P < .05 vs N2.
(N2) for 3 hours after resuscitation. On the other hand, the rats exposed to 1.3% hydrogen for 3 hours had reduced neutrophil infiltration into the alveolar space after HS/R (Fig 3, D-F, H). Hydrogen inhalation reduced lipid peroxidation. Lipid peroxidation was determined by measurement of tissue MDA levels, because MDA is a toxic metabolite of oxidation-induced lipid peroxidation. HS/R increased MDA levels in the lungs. Hydrogen treatment reduced this increase significantly, demonstrating antioxidant effects of hydrogen in the lungs when administered after HS/R (Fig 4, A). To further evaluate the role of hydrogen as an antioxidant, we stained lung tissue for 8-OHdG, another marker of oxidative injury. Although few 8-OHdG–positive cells were noted in the sham lung, increased 8-OHdG–positive cells were seen in the epithelial cells of the control lung subjected to HS/R. Hydrogen inhalation remarkably reduced the number of stained cells for this oxidative injury marker (Fig 4, B-D). Hydrogen inhalation decreased the upregulation of mRNAs for inflammatory mediators. Three hours after HS/R, the mRNAs for TNF-a, IL-1b,
IL-6, ICAM-1, iNOS, and CCL2 were upregulated significantly compared with sham-operated animals. However, exposure to 1.3% H2 after HS/R significantly reduced the peak expression of the transcripts for these inflammatory mediators (Fig 5). Hydrogen inhalation inhibited activation of the NF-kB signaling pathway. The NF-kB signaling pathway is activated by HS/R, resulting in upregulation of inflammatory mediators.15 Consistent with this known role of NF-kB, Western blots demonstrated that activated p65 protein was increased in the nucleus 6 hours after HS/R (Fig 6, A, B). NFkB DNA-binding activity was also increased in the lung tissue 6 hours after HS/R. Treatment with inhaled hydrogen significantly reduced the level of activated p65. Similarly, hydrogen treatment reduced NF-kB DNA-binding activity in the lung tissue 6 hours after HS/R as compared with the nitrogen-treated controls (Fig 6, C, D). DISCUSSION In the present study, we demonstrated that inhalation of 1.3% hydrogen significantly reduced lung injury after HS/R. Additionally, hydrogen
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Fig 3. Histopathologic changes after hemorrhagic shock followed by fluid resuscitation (HS/R). A-C; ALI score were assessed by HE staining (X400 magnification). Black triangles showed hyperplasia of alveolar septum. A, Shamoperated animals. Normal alveolar structure. No hyperemia, neutrophil infiltration, or interstitial edema was observed. B, HS/R with N2. Deformed alveolar structures. Collapsed alveoli, loss of integrity, and alveolar capillary congestion were observed (3 hours after resuscitation). C, HS/R followed by H2 treatment. Significantly less alveolar edema was observed (black arrowheads; 3 hours after resuscitation). D-F; The number of neutrophils were assessed by naphthol AS-D chloroacetate esterase staining (X400 magnification). Black arrows showed neutrophils. D, Neutrophil staining in sham operated animals. E, Neutrophil staining in lungs from the HS/R group excised 3 hours after resuscitation. The number of neutrophils in the tissue is increased markedly. F, Neutrophil staining in lungs from the HS/R + H2 group, neutrophil accumulation and the alveolar–capillary exudate were reduced compared with the N2 control. Black arrows indicate positively stained polymorphonuclear neutrophils. Representative images are shown. G, Lung injury score (n = 6; *P < .05 vs N2). H, Neutrophil staining as indicated by number of naphthol AS-D chloroacetate esterase-positive cells in ten 3400 microscopy fields (n = 6; *P < .05 vs N2). ALI, Acute lung injury.
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Fig 4. A, Tissue malondialdehyde (MDA) levels (n = 6; *P < .05 vs N2). B, 8-Hydroxy-deoxyguanosine (8-OHdG) staining in sham-operated animals. C, 8-OHdG–positive cells in the epithelium of the lung 3 hours after hemorrhagic shock followed by fluid resuscitation (HS/R). D, Immunostaining for 8-OHdG in lungs from the HS/R + H2 group showed fewer positive cells than the HS/R lung. Representative images are shown. Black arrows show 8-OHdG positive cells.
administration did not affect the hemodynamic status during HS/R. Adjunctive therapy with inhaled hydrogen is promising and might be reasonable for lung disease as hydrogen can be easily delivered through the ventilation circuit and, thus, is straightforward therapeutic option.6-8,16 In this study, we demonstrate an application of inhaled hydrogen therapy with the potential to improve acute care medicine. Other reports have also shown promising uses of hydrogen in the field of acute care medicine. Hayashida et al17 reported that H2 inhalation, when commenced at the start of hyperoxic cardiopulmonary resuscitation, significantly improves brain and cardiac function using a rat model of cardiac arrest. The same investigators demonstrated the effectiveness of this therapeutic approach when H2 inhalation was commenced upon the return of spontaneous circulation under
normoxic conditions, either alone or in combination with targeted temperature management.18 Hemorrhage results in both circulatory and inflammatory disturbances.1 Decreased macrovascular perfusion after hemorrhage leads to systemic hypotension and decreased end-organ perfusion, which ultimately results in decreased perfusion in the capillaries. Simultaneously, hemorrhage triggers the immune system and increases inflammation through secondary messenger modulation, changes in gene expression and neutrophil activation.1,19 The expression of proinflammatory and immunoregulatory cytokines rapidly increases in the lungs after hemorrhage, and resuscitation after hemorrhagic shock causes a systemic inflammatory response. In particular, TNF-a is among the most well-characterized inflammatory and cardiodepressant factors contributing to cardiovascular shock in hemorrhage and trauma.20 Blood loss also
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Fig 5. Levels of messenger RNAs for proinflammatory mediators in the lungs. Levels of tumor necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6, and intercellular adhesion molecule (ICAM)-1, inducible nitric oxide synthase (iNOS), and chemokine (C-C motif) ligand 2 (CCL2) were quantitated 1 and 3 hours after HS/R (n = 6; *P < .05 vs N2). GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.
simulates a-adrenergic signaling resulting in NFkB–mediated elevations in pulmonary cytokines.21,22 Treatment of mice subjected to experimentally induced hemorrhage with an aadrenergic receptor antagonist prevented increases in the mRNAs for IL-1b and TNF-a, prevented increased expression of the IL-1b protein, and reduced activation of NF-kB.21,22 Our study of hydrogen treatment after HS/R showed similar modulation of the mRNAs for IL-1b and TNF-a and decreased activation of NF-kB. Although the molecular mechanisms of protective effects of hydrogen in this study are not completely
understood, our results strongly suggest a role for NF-kB–mediated pathways. Also, our results indicate that endogenous nitric oxide might not play a key role of the antiinflammatory effects afforded by hydrogen, despite the fact that NO is an important regulator of the inflammatory response after hemorrhagic shock.21,22 One finding of our study was that hydrogen treatment reduced the elevation in lung tissue MDA levels typically seen after HS/R. This suggests that hydrogen mitigates oxidative damage caused by HS/R. Excessive reactive oxygen species generated from blood clots are features of ALI
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Fig 6. A, Subcellular localization of p65 6 hours after hemorrhagic shock followed by fluid resuscitation. Western blot of nuclear and cytoplasmic extracts. Loading controls demonstrate similar total protein levels per lane. Data are representative of 3 independent experiments. B, Histogram of western blot band intensity (n = 3 for each group; *P < .05). C, electrophoretic mobility shirt assay (EMSA) for nuclear factor-kB. Data are representative of 3 independent experiments. D, Histogram of EMSA band intensity (n = 3 for each group; *P < .05).
pathophysiology.23 We speculate that the attenuation of epithelial cell damage and lung edema in the lung via the reduction of potent reactive oxygen species may be at least partially responsible for the improvement of lung function. Lung injury after HS/R is a dynamic process, and inflammation and lung function undoubtedly change over time. One limitation of our study is the lack of analysis of long-term outcomes after hydrogen treatment for HS/R. Although early changes will likely hint at mechanisms involved, further study with an emphasis on lung function and systemic inflammatory events in the late phase after HS/R is needed. Additionally, from a practical standpoint of effective translational research, solvated hydrogen may be safer
to use and transport than hydrogen gas.24,25 Moreover, use of an injectable fluid saturated with hydrogen gives rise to immensely different ethical and safety considerations than inhaled hydrogen. We are performing ongoing studies aimed at determining the efficacies of soluble hydrogen in an injectable fluid. In conclusion, using a rat HS/R model, this study demonstrated that hydrogen, administered through inhalation, may exert potent therapeutic effects against ALI induced by HS/R and attenuate the activation of inflammatory cascades. Thus, hydrogen treatment of ventilated patients may yield a novel, clinically feasible therapy that would be easy to incorporate without alteration of interventions.
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The authors thank Shannon Wyszomierski, PhD, for editing this manuscript. REFERENCES 1. Santry HP, Alam HB. Fluid resuscitation: past, present, and the future. Shock 2010;33:229-41. 2. Jarrar D, Chaudry IH, Wang P. Organ dysfunction following hemorrhage and sepsis: mechanisms and therapeutic approaches (Review). Int J Mol Med 1999;4:575-83. 3. Ciesla DJ, Moore EE, Johnson JL, Burch JM, Cothren CC, Sauaia A. The role of the lung in postinjury multiple organ failure. Surgery 2005;138:749-57. 4. Rushing GD, Britt LD. Reperfusion injury after hemorrhage: a collective review. Ann Surg 2008;247:929-37. 5. Huang CS, Kawamura T, Toyoda Y, Nakao A. Recent advances in hydrogen research as a therapeutic medical gas. Free Radic Res 2010;44:971-82. 6. Huang CS, Kawamura T, Lee S, Tochigi N, Shigemura N, Buchholz BM, et al. Hydrogen inhalation ameliorates ventilator-induced lung injury. Crit Care 2010;14:R234. 7. Kawamura T, Huang CS, Tochigi N, Lee S, Shigemura N, Billiar TR, et al. Inhaled hydrogen gas therapy for prevention of lung transplant-induced ischemia/reperfusion injury in rats. Transplantation 2010;90:1344-51. 8. Kawamura T, Wakabayashi N, Shigemura N, Huang CS, Masutani K, Tanaka Y, et al. Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo. Am J Physiol Lung Cell Mol Physiol 2013;304:L646-56. 9. Kohama K, Nakao A, Terashima M, Aoyama-Ishikawa M, Shimizu T, Harada D, et al. Supplementation of parenteral nutrition with fish oil attenuates acute lung injury in a rat model. J Clin Biochem Nutr 2014;54:116-21. 10. Nakao A, Kimizuka K, Stolz DB, Neto JS, Kaizu T, Choi AM, et al. Carbon monoxide inhalation protects rat intestinal grafts from ischemia/reperfusion injury. Am J Pathol 2003;163:1587-98. 11. Kohmoto J, Nakao A, Kaizu T, Tsung A, Ikeda A, Tomiyama K, et al. Low-dose carbon monoxide inhalation prevents ischemia/reperfusion injury of transplanted rat lung grafts. Surgery 2006;140:179-85. 12. Itoh T, Itoh A, Pleasure D. Bcl-2-related protein family gene expression during oligodendroglial differentiation. J Neurochem 2003;85:1500-12. 13. Nakao A, Otterbein LE, Overhaus M, Sarady JK, Tsung A, Kimizuka K, et al. Biliverdin protects the functional integrity of a transplanted syngeneic small bowel. Gastroenterology 2004;127:595-606.
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14. Nakao A, Schmidt J, Harada T, Tsung A, Stoffels B, Cruz RJ Jr, et al. A single intraperitoneal dose of carbon monoxidesaturated ringer’s lactate solution ameliorates postoperative ileus in mice. J Pharmacol Exp Ther 2006;319:1265-75. 15. Fan J, Ye RD, Malik AB. Transcriptional mechanisms of acute lung injury. Am J Physiol Lung Cell Mol Physiol 2001;281:L1037-50. 16. Nakao A, Sugimoto R, Billiar TR, McCurry KR. Therapeutic antioxidant medical gas. J Clin Biochem Nutr 2009;44: 1-13. 17. Hayashida K, Sano M, Kamimura N, Yokota T, Suzuki M, Maekawa Y, et al. H gas improves functional outcome after cardiac arrest to an extent comparable to therapeutic hypothermia in a rat model. J Am Heart Assoc 2012;1:e003459. 18. Hayashida K, Sano M, Kamimura N, Yokota T, Suzuki M, Ohta S, et al. Hydrogen inhalation during normoxic resuscitation improves neurological outcome in a rat model of cardiac arrest, independent of targeted temperature management. Circulation 2014;130:2173-80. 19. Sato H, Tanaka T, Kasai K, Kita T, Tanaka N. Role of p38 mitogen-activated protein kinase on renal dysfunction after hemorrhagic shock in rats. Shock 2005;24:488-94. 20. Tracey KJ, Cerami A. Tumor necrosis factor: an updated review of its biology. Crit Care Med 1993;21(Suppl 10): S415-22. 21. Le Tulzo Y, Shenkar R, Kaneko D, Moine P, Fantuzzi G, Dinarello CA, et al. Hemorrhage increases cytokine expression in lung mononuclear cells in mice: involvement of catecholamines in nuclear factor-kappaB regulation and cytokine expression. J Clin Invest 1997;99:1516-24. 22. Hierholzer C, Harbrecht B, Menezes JM, Kane J MacMicking J, Nathan CF, et al. Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock. J Exp Med 1998;187:917-28. 23. Mondrinos MJ, Zhang T, Sun S, Kennedy PA, King DJ, Wolfson MR, et al. Pulmonary endothelial protein kinase C-delta (PKCdelta) regulates neutrophil migration in acute lung inflammation. Am J Pathol 2014;184:200-13. 24. Kang KM, Kang YN, Choi IB, Gu Y, Kawamura T, Toyoda Y, et al. Effects of drinking hydrogen-rich water on the quality of life of patients treated with radiotherapy for liver tumors. Med Gas Res 2011;1:11. 25. Buchholz BM, Masutani K, Kawamura T, Peng X, Toyoda Y, Billiar TR, et al. Hydrogen-enriched preservation protects the isogeneic intestinal graft and amends recipient gastric function during transplantation. Transplantation 2011;92: 985-92.
Introduction. Hemorrhagic shock followed by fluid resuscitation (HS/R) triggers an inflammatory response and causes pulmonary inflammation that can lead to acute lung injury (ALI). Hydrogen, a therapeutic gas, has potent cytoprotective, antiinflammatory, and antioxidant effects. This study examined the effects of inhaled hydrogen on ALI caused by HS/R. Methods. Rats were subjected to hemorrhagic shock by withdrawing blood to lower blood pressure followed by resuscitation with shed blood and saline to restore blood pressure. After HS/R, the rats were maintained in a control gas of similar composition to room air or exposed to 1.3% hydrogen. Results. HS/R induced ALI, as demonstrated by significantly impaired gas exchange, congestion, edema, cellular infiltration, and hemorrhage in the lungs. Hydrogen inhalation mitigated lung injury after HS/R, as indicated by significantly improved gas exchange and reduced cellular infiltration and hemorrhage. Hydrogen inhalation did not affect hemodynamic status during HS/R. Exposure to 1.3% hydrogen significantly attenuated the upregulation of the messenger RNAs for several proinflammatory mediators induced by HS/R. Lipid peroxidation was reduced significantly in the presence of hydrogen, indicating antioxidant effects. Conclusion. Hydrogen, administered through inhalation, may exert potent therapeutic effects against ALI induced by HS/R and attenuate the activation of inflammatory cascades. (Surgery 2015;158:399-407.) From the Department of Emergency, Disaster and Critical Care Medicine,a Hyogo College of Medicine, Nishinomiya; the Kobe University Graduate School of Health Science,b Kobe, Hyogo; the Faculty of Health and Welfare Science,c Okayama Prefectural University, Okayama, Japan, and the Department of Surgery,d University of Pittsburgh, Pittsburgh, PA
HEMORRHAGE contributes to mortality after trauma accounting for 30–40% of deaths caused by trauma worldwide despite recent advances in resuscitation and critical care.1 Hemorrhagic shock followed by fluid resuscitation (HS/R) triggers an inflammatory response characterized by upregulation of proinflammatory cytokines and adhesion molecules and induces pulmonary inflammation that can lead to acute lung injury (ALI). ALI often predicates multiple organ failure and mortality.2 Thus, despite recent advances in intensive care, lung injury after HS/R is still Supported by JSPS KAKENHI Grant Number #25462840. Presented at the 10th Annual Academic Surgical Congress in Las Vegas, Nevada, February 3-5, 2015. Accepted for publication March 22, 2015. Reprint requests: Atsunori Nakao, MD, PhD, 1-1 Mukogawa, Nishinomiya, Hyogo, Japan. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2015.03.038
among the most common causes of death after trauma.3,4 Hydrogen is an important physiologic regulatory molecule and exerts antioxidant, antiinflammatory, and antiapoptotic protective effects on cells and organs.5 Because hydrogen can be administered via a ventilation circuit, inhaled hydrogen therapy is considered clinically applicable for patients with shock. Additionally, although hydrogen has therapeutic benefits in multiple organs, the lungs are an ideal target organ for hydrogen therapy, because inhalation is a straightforward delivery method. In fact, hydrogen treatment ameliorated lung injury in several model systems, including ventilatorinduced lung injury,6 ischemia–reperfusion injury during lung transplantation,7 and hyperoxic lung injury.8 Although the therapeutic efficacies of hydrogen have been studied, there is limited information on processes regulated by the hydrogen molecule. We hypothesized that, because of its antiinflammatory and antioxidant SURGERY 399
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properties, inhaled hydrogen therapy could ameliorate ALI after HS/R. METHODS Animals. Male Sprague-Dawley rats weighing 300–500 g (6–9 weeks old) were purchased from Clea Japan, Inc. (Tokyo, Japan) and were kept in individual stainless steel cages in a temperature, humidity, and light-controlled room (23 ± 38C; 55 ± 15%; 12-hour light–dark cycle) for 2–5 weeks before the experiments. During this period, all animals were provided standard food (AIN-93G diet; Oriental Kobo Corporation, Tokyo, Japan) and free access to water. All procedures involving rats were conducted in accordance with the guidelines of the Animal Care and Use Committees of the Hyogo College of Medicine and complied with the National Research Council’s Guide for the Humane Care and Use of Laboratory Animals. Animal model. Under general anesthesia with isoflurane, a catheter for monitoring arterial pressure was placed into the right femoral artery. To create a shock, blood was withdrawn through this catheter, and blood pressure was maintained at 30 ± 5 mmHg for 60 minutes. After 60 minutes, shed blood was reinfused (with saline if necessary for adequate volume restoration), the femoral artery was ligated, and the wound was closed (Fig 1). In sham control rats, we did not induce shock or resuscitate with fluid restoration; we simply placed the catheter, removed it 60 minutes later, ligated the femoral artery, and closed the wound. To maintain body temperature, 378C warming pads were used throughout this procedure. After the procedure, the rats were kept in an air/gas exposure box for 1–6 hours. Blood samples were collected. Rats were humanely killed 1, 3, or 6 hours after HS/R by isoflurane overdose (Fig 1). The lungs were excised and divided into 2 sections. The right lobe was snap frozen immediately with liquid nitrogen for further analysis. The left lobe of the lungs was used for histologic examination. Hydrogen treatment. For hydrogen gas treatment, cylinders with nitrogen-based, highpressure, premixed gases were purchased (Japan Fine Products, Kanagawa, Japan). The manufacturer confirmed the concentrations of H2 (1.3%), O2 (21%), and N2 (77.7%). In Japan, 1.3% is the highest concentration of H2 that can be mixed and bottled under high pressure with 21% oxygen for clinical use. As a control, additional N2 was administered instead of H2 (O2, 21%; N2, 79%). The premixed gases were delivered to the rats via a gas exposure chamber (Natsume Seisakusho
Fig 1. Experimental protocol. Blood was withdrawn, and blood pressure was maintained at 30 ± 5 mmHg for 60 minutes. After 60 minutes, shed blood was reinfused. Rats were then placed in an exposure chamber filled with either 1.3% H2 (H2, 1.3%; O2, 21%; N2, 77.7%) or with only oxygen and nitrogen (O2, 21%; N2, 79%). MABP, Mean arterial blood pressure.
Co. Tokyo, Japan). Hydrogen or control gas (designated N2) was administered during shock and for 1, 3, or 6 hours before killing and tissue collection. The sham control rats were placed in the gas exposure chamber with control (N2) gas for 1 hour. Assessment of gas exchange function and blood lactate levels. Partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), and blood lactate levels were assessed by analysis of blood gases (iSTAT, Abbott Point Care Inc., Princeton, NJ) before killing. Blood gases were assessed on a fraction of inspired oxygen (FiO2) of 1.0 in blood drawn from the abdominal aorta 3 minutes after oxygen inspiration was initiated. Measurement of malondialdehyde. Lung tissues were harvested after 3 hours of gas exposure and kept at -808C until analysis. The tissue was homogenized, and tissue malondialdehyde (MDA) concentration was determined according to the manufacturer’s instructions (Kit MDA-586; Oxisresearch, Portland, OR). Histopathology. Lungs were fixed by inflation with buffered 4% paraformaldehyde for 24 hours. After embedding in paraffin, the sections were prepared and stained with hematoxylin and eosin. Polymorphonuclear neutrophils (PMNs) were stained using a naphthol AS-D chloroacetate esterase staining kit (Sigma Diagnostics) and identified by nuclear morphology stained in bright red. PMN were counted in 10 high-power fields (3400) per sample. ALI was scored with the samples’ identities masked according to previously described criteria,9 specifically (1) thickness of the alveolar wall, (2) infiltration or aggregation of neutrophils in air space, alveolar wall, or vessel wall,
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and (3) alveolar congestion. Each item was graded on a 4-point scale (0–3), with higher scores indicating more severe damage; the range of possible total ALI scores was 0–9. ALI scores were assessed in 10 high-power fields (3400) per sample. Immunohistochemical analysis. The slides were incubated with primary antibody (8-hydroxydeoxyguanosine [8-OHdG] monoclonal antibody [Nikken Seil, Shizuoka, Japan]) for 2 hours at room temperature. The washed samples were treated with the Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan) for 30 minutes at room temperature, followed by visualization with diaminobenzidine (liquid DAB + substrate chromogen system; Dako Japan, Tokyo, Japan), for 5 minutes, and counterstained with Mayer’s hematoxylin. Real-time reverse transcriptase polymerase chain reaction. Rat messenger RNAs (mRNA) were quantified in duplicate using SYBR Green 2-step, real-time reverse transcriptase polymerase chain reaction, as described previously.10-12 The following mRNAs were quantitated: rat tumor necrosis factor (TNF)-a, intercellular adhesion molecule (ICAM)-1, interleukin (IL)-1b, IL-6, inducible nitric oxide synthase (iNOS), chemokine (C-C motif) ligand 2 (CCL2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Optimized and validated primer sets were obtained from realtimeprimers.com (Elkins Park, PA). Western blot. Protein was extracted and divided into cytoplasmic and nuclear fractions using NEPER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc, Waltham, MA). Western blot was performed using anti-nuclear factor (NF)-kB p65 antibody (Cell Signaling Technology, Danvers, MA), anti-lamin (A/C) antibody (Cell Signaling Technology) and anti–b-actin antibody (Sigma-Aldrich, St. Louis, MO). Polyvinylidene difluoride membranes were incubated overnight at 48C with primary antibodies diluted in Can Get Signal immunoreaction enhancer solution 1 (Toyobo, Osaka, Japan), then with horseradish peroxidase-conjugated antimouse secondary antibodies diluted in Can Get Signal solution 2 (Toyobo) for 1 hour at room temperature. Antibody binding was detected using Enhanced Chemiluminescence Plus reagents (GE Healthcare, Buckinghamshire, UK) and the Optima Shot CL-420a image-capturing system (Wako, Osaka, Japan). The band intensities were quantified using NIH image analysis software. NF-kB DNA binding activity. NF-kB DNA binding activity was measured by electrophoretic mobility shirt assay (EMSA) using nuclear extracts from lung tissue and an NF-kB oligonucleotide
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(Promega, Madison, WI) based on the NF-kB sequence in the immunoglobulin light-chain enhancer as previously described.13,14 The band intensities were quantified using NIH image analysis software. Statistical analysis. Results are expressed as mean values ± standard error of the mean. Parametric data were analyzed with 1-way analysis of variance (ANOVA) followed by post hoc analysis with the Bonferroni correction. The lung injury score was analyzed with the Mann–Whitney U test with the Bonferroni correction. The cell count was analyzed using ANOVA with Tukey-Kramer methods. JMP version 8 (SAS Institute, Cary, NC) was used for all statistical analyses. RESULTS Hydrogen improved tissue oxygenation and reduced oxidative damage. HS/R impaired pulmonary function and resulted in a remarkable decrease in PO2 levels 3 hours after resuscitation (Fig 2, A). Treatment with hydrogen after HS/R improved blood oxygenation (Fig 2, A). Neither HS/R nor hydrogen inhalation altered PCO2 levels (Fig 2, B), and there was no difference in vital signs, including blood pressure and heart rate, between the groups (data not shown). Blood lactate levels increased notably after HS/R, and this increase was suppressed significantly in the rats treated with hydrogen (Fig 2, C). Blood pH and base excess were not different in any of the treatment groups (data not shown). Hydrogen inhalation reduced ALI and PMN infiltration. In histologic analysis, the lungs of rats exposed to HS/R showed marked cellular infiltration, edema in the interstitial area, and associated thickening of the alveolar septum. Treatment with inhaled hydrogen after HS/R reduced both edema and inflammatory cell infiltration (Fig 3, A-C). Although there was some suggestion that the wet/dry ratio, another indicator of altered lung permeability, was lower in H2-treated lungs than in control lungs after HS/R, this difference was not significant (data not shown). Damage, as indicated by the lung injury score, was significantly reduced in rats treated with H2 (4.5 ± 0.5 in the N2 group vs 3.3 ± 0.3 in the H2 group; P < .05; Fig 3, G). PMNs are the predominant infiltrating cells in the lung during hemorrhage shock-induced lung injury. Although there were scarce PMNs in the sham-treated lungs, massive neutrophil accumulation in the alveolar space, alveolar capillary congestion, and exudation in the lungs were seen in the rats subjected to HS/R and exposed to control gas
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Fig 2. Blood gas and lactate levels 3 hours after hemorrhagic shock followed by fluid resuscitation. A, Partial pressure of oxygen (PO2). B, Partial pressure of carbon dioxide (PCO2). C, Lactate levels. *P < .05 vs N2.
(N2) for 3 hours after resuscitation. On the other hand, the rats exposed to 1.3% hydrogen for 3 hours had reduced neutrophil infiltration into the alveolar space after HS/R (Fig 3, D-F, H). Hydrogen inhalation reduced lipid peroxidation. Lipid peroxidation was determined by measurement of tissue MDA levels, because MDA is a toxic metabolite of oxidation-induced lipid peroxidation. HS/R increased MDA levels in the lungs. Hydrogen treatment reduced this increase significantly, demonstrating antioxidant effects of hydrogen in the lungs when administered after HS/R (Fig 4, A). To further evaluate the role of hydrogen as an antioxidant, we stained lung tissue for 8-OHdG, another marker of oxidative injury. Although few 8-OHdG–positive cells were noted in the sham lung, increased 8-OHdG–positive cells were seen in the epithelial cells of the control lung subjected to HS/R. Hydrogen inhalation remarkably reduced the number of stained cells for this oxidative injury marker (Fig 4, B-D). Hydrogen inhalation decreased the upregulation of mRNAs for inflammatory mediators. Three hours after HS/R, the mRNAs for TNF-a, IL-1b,
IL-6, ICAM-1, iNOS, and CCL2 were upregulated significantly compared with sham-operated animals. However, exposure to 1.3% H2 after HS/R significantly reduced the peak expression of the transcripts for these inflammatory mediators (Fig 5). Hydrogen inhalation inhibited activation of the NF-kB signaling pathway. The NF-kB signaling pathway is activated by HS/R, resulting in upregulation of inflammatory mediators.15 Consistent with this known role of NF-kB, Western blots demonstrated that activated p65 protein was increased in the nucleus 6 hours after HS/R (Fig 6, A, B). NFkB DNA-binding activity was also increased in the lung tissue 6 hours after HS/R. Treatment with inhaled hydrogen significantly reduced the level of activated p65. Similarly, hydrogen treatment reduced NF-kB DNA-binding activity in the lung tissue 6 hours after HS/R as compared with the nitrogen-treated controls (Fig 6, C, D). DISCUSSION In the present study, we demonstrated that inhalation of 1.3% hydrogen significantly reduced lung injury after HS/R. Additionally, hydrogen
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Fig 3. Histopathologic changes after hemorrhagic shock followed by fluid resuscitation (HS/R). A-C; ALI score were assessed by HE staining (X400 magnification). Black triangles showed hyperplasia of alveolar septum. A, Shamoperated animals. Normal alveolar structure. No hyperemia, neutrophil infiltration, or interstitial edema was observed. B, HS/R with N2. Deformed alveolar structures. Collapsed alveoli, loss of integrity, and alveolar capillary congestion were observed (3 hours after resuscitation). C, HS/R followed by H2 treatment. Significantly less alveolar edema was observed (black arrowheads; 3 hours after resuscitation). D-F; The number of neutrophils were assessed by naphthol AS-D chloroacetate esterase staining (X400 magnification). Black arrows showed neutrophils. D, Neutrophil staining in sham operated animals. E, Neutrophil staining in lungs from the HS/R group excised 3 hours after resuscitation. The number of neutrophils in the tissue is increased markedly. F, Neutrophil staining in lungs from the HS/R + H2 group, neutrophil accumulation and the alveolar–capillary exudate were reduced compared with the N2 control. Black arrows indicate positively stained polymorphonuclear neutrophils. Representative images are shown. G, Lung injury score (n = 6; *P < .05 vs N2). H, Neutrophil staining as indicated by number of naphthol AS-D chloroacetate esterase-positive cells in ten 3400 microscopy fields (n = 6; *P < .05 vs N2). ALI, Acute lung injury.
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Fig 4. A, Tissue malondialdehyde (MDA) levels (n = 6; *P < .05 vs N2). B, 8-Hydroxy-deoxyguanosine (8-OHdG) staining in sham-operated animals. C, 8-OHdG–positive cells in the epithelium of the lung 3 hours after hemorrhagic shock followed by fluid resuscitation (HS/R). D, Immunostaining for 8-OHdG in lungs from the HS/R + H2 group showed fewer positive cells than the HS/R lung. Representative images are shown. Black arrows show 8-OHdG positive cells.
administration did not affect the hemodynamic status during HS/R. Adjunctive therapy with inhaled hydrogen is promising and might be reasonable for lung disease as hydrogen can be easily delivered through the ventilation circuit and, thus, is straightforward therapeutic option.6-8,16 In this study, we demonstrate an application of inhaled hydrogen therapy with the potential to improve acute care medicine. Other reports have also shown promising uses of hydrogen in the field of acute care medicine. Hayashida et al17 reported that H2 inhalation, when commenced at the start of hyperoxic cardiopulmonary resuscitation, significantly improves brain and cardiac function using a rat model of cardiac arrest. The same investigators demonstrated the effectiveness of this therapeutic approach when H2 inhalation was commenced upon the return of spontaneous circulation under
normoxic conditions, either alone or in combination with targeted temperature management.18 Hemorrhage results in both circulatory and inflammatory disturbances.1 Decreased macrovascular perfusion after hemorrhage leads to systemic hypotension and decreased end-organ perfusion, which ultimately results in decreased perfusion in the capillaries. Simultaneously, hemorrhage triggers the immune system and increases inflammation through secondary messenger modulation, changes in gene expression and neutrophil activation.1,19 The expression of proinflammatory and immunoregulatory cytokines rapidly increases in the lungs after hemorrhage, and resuscitation after hemorrhagic shock causes a systemic inflammatory response. In particular, TNF-a is among the most well-characterized inflammatory and cardiodepressant factors contributing to cardiovascular shock in hemorrhage and trauma.20 Blood loss also
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Fig 5. Levels of messenger RNAs for proinflammatory mediators in the lungs. Levels of tumor necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6, and intercellular adhesion molecule (ICAM)-1, inducible nitric oxide synthase (iNOS), and chemokine (C-C motif) ligand 2 (CCL2) were quantitated 1 and 3 hours after HS/R (n = 6; *P < .05 vs N2). GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.
simulates a-adrenergic signaling resulting in NFkB–mediated elevations in pulmonary cytokines.21,22 Treatment of mice subjected to experimentally induced hemorrhage with an aadrenergic receptor antagonist prevented increases in the mRNAs for IL-1b and TNF-a, prevented increased expression of the IL-1b protein, and reduced activation of NF-kB.21,22 Our study of hydrogen treatment after HS/R showed similar modulation of the mRNAs for IL-1b and TNF-a and decreased activation of NF-kB. Although the molecular mechanisms of protective effects of hydrogen in this study are not completely
understood, our results strongly suggest a role for NF-kB–mediated pathways. Also, our results indicate that endogenous nitric oxide might not play a key role of the antiinflammatory effects afforded by hydrogen, despite the fact that NO is an important regulator of the inflammatory response after hemorrhagic shock.21,22 One finding of our study was that hydrogen treatment reduced the elevation in lung tissue MDA levels typically seen after HS/R. This suggests that hydrogen mitigates oxidative damage caused by HS/R. Excessive reactive oxygen species generated from blood clots are features of ALI
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Fig 6. A, Subcellular localization of p65 6 hours after hemorrhagic shock followed by fluid resuscitation. Western blot of nuclear and cytoplasmic extracts. Loading controls demonstrate similar total protein levels per lane. Data are representative of 3 independent experiments. B, Histogram of western blot band intensity (n = 3 for each group; *P < .05). C, electrophoretic mobility shirt assay (EMSA) for nuclear factor-kB. Data are representative of 3 independent experiments. D, Histogram of EMSA band intensity (n = 3 for each group; *P < .05).
pathophysiology.23 We speculate that the attenuation of epithelial cell damage and lung edema in the lung via the reduction of potent reactive oxygen species may be at least partially responsible for the improvement of lung function. Lung injury after HS/R is a dynamic process, and inflammation and lung function undoubtedly change over time. One limitation of our study is the lack of analysis of long-term outcomes after hydrogen treatment for HS/R. Although early changes will likely hint at mechanisms involved, further study with an emphasis on lung function and systemic inflammatory events in the late phase after HS/R is needed. Additionally, from a practical standpoint of effective translational research, solvated hydrogen may be safer
to use and transport than hydrogen gas.24,25 Moreover, use of an injectable fluid saturated with hydrogen gives rise to immensely different ethical and safety considerations than inhaled hydrogen. We are performing ongoing studies aimed at determining the efficacies of soluble hydrogen in an injectable fluid. In conclusion, using a rat HS/R model, this study demonstrated that hydrogen, administered through inhalation, may exert potent therapeutic effects against ALI induced by HS/R and attenuate the activation of inflammatory cascades. Thus, hydrogen treatment of ventilated patients may yield a novel, clinically feasible therapy that would be easy to incorporate without alteration of interventions.
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