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Neuroprotective effects of methane-rich saline on experimental acute carbon monoxide toxicity
Journal of the Neurological Sciences 369 (2016) 361–367
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Neuroprotective effects of methane-rich saline on experimental acute carbon monoxide toxicity Meihua Shen a,c,1, Danfeng Fan b,1, Yu Zang d, Yan Chen c, Kaimin Zhu c, Zhonghai Cai c, Yueqin Liu c, Xuejun Sun e, Jiankang Liu a,⁎, Jianfeng Gong f,⁎ a Key Laboratory of Biomedical Information Engineering, Ministry of Education, Institute of Mitochondrial Biology and Medicine, Xi'an Jiaotong University, School of Life Science and Technology, Xi'an 710049, PR China b Department of Hyperbaric Oxygen, Navy General Hospital, No. 6, Fucheng Road, Beijing 100048, PR China c Department of Intensive Care Unit, Shanghai Provincial Corps Hospital, Chinese People's Armed Police Forces, 831 Hongxu Road, Shanghai 201103, PR China d Department of Neurology, The Affiliated Hospital of North China University of Science and Technology, No.57, Jianshe South Road, Tangshan, Hebei 063000, PR China e Department of Naval Aeromedicine, Faculty of Naval Medicine, Second Military Medical University, No.800, Xiangyin Road, Shanghai 200433, PR China f Department of General Surgery, The Sixth People's Hospital, Shanghai Jiao Tong University, No.600, Yishan Road, Shanghai 200233, PR China
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Article history: Received 1 July 2016 Received in revised form 29 July 2016 Accepted 24 August 2016 Available online 26 August 2016 Keywords: Methane Carbon monoxide poisoning Reactive oxygen species Antioxidant enzyme
a b s t r a c t Background: Methane has been reported to play a protective role in ischemia-reperfusion injury via anti-oxidation, anti-inflammatory and anti-apoptotic activities. This study was designed to determine the protective effects of methane-rich saline (MRS) on acute carbon monoxide (CO) poisoning. Methods: A total of 36 male Sprague-Dawley rats were randomly divided into 3 groups: sham group, CO group and MRS group. Acute CO poisoning was induced by exposing rats to 1000 ppm CO in air for 40 min and then to 3000 ppm CO for an additional 20 min until they lost consciousness. MRS at 10 ml/kg was intraperitoneally administered at 0 h, 8 h and 16 h after CO exposure. Rats were sacrificed 24 h after CO exposure. Brains were collected for Nissl staining. The cortex and hippocampus were separated for the detections of malondialdehyde (MDA), 3-nitrotyrosine (3-NT), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin1-β (IL-1β), interleukin-6 (IL-6) and superoxide dismutase (SOD) activities. Results: The results showed that MRS treatment improved neuronal injury, reduced MDA, 3-NT and 8-OHdG, and increased SOD activity of the hippocampus and cortex compared with normal saline-treated rats. In addition, MRS reduced the expression of TNF-α and IL-1β in the brain but had no effect on IL-6 expression. Conclusion: These findings suggest that MRS may protect the brain against acute CO poisoning-induced injury via its anti-oxidative and anti-inflammatory activities. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Acute carbon monoxide (CO) toxicity is a leading cause of gas poisoning-related deaths worldwide due to the increased use of carbonbased fuels [1]. It has been reported that CO poisoning is responsible for approximately 15,000 visits to emergency departments and nearly 500 deaths annually in the United States [1–3]. Acute CO poisoning
Abbreviations: ANOVA, analysis of variance; CO, carbon monoxide; COHb, carboxyhemoglobin; DNS, delayed neurological syndrome; HBO, hyperbaric oxygen; • OH, hydroxyl radical; IL-1β, interleukin 1 –β; IL-6, interleukin-6; MRS, methane-rich saline; MDA, Malondialdehyde; NS, normal saline; 3-NT, 3-nitrotyrosine; 8-OHdG, 8hydroxydeoxyguanosine; ROS, reactive oxygen species; SOD, superoxide dismutase; ONOO−, peroxynitrite; O− 2 , superoxide anion; TBA, thiobarbituric acid; TNF-α, tumor necrosis factor-α. ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Liu), [email protected] (J. Gong). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jns.2016.08.055 0022-510X/© 2016 Elsevier B.V. All rights reserved.
may produce severe brain damage, which can lead to high mortality and delayed neurological syndrome (DNS) [4]. Numerous studies have indicated that an increase in the production of reactive oxygen species (ROS) following CO poisoning is of crucial relevance to the pathophysiology of CO poisoning [5–7]. Although ROS play important roles in the clearance of invaded pathogens, they seem to produce substantial damage if they are produced in excess, which can lead to DNA strand breaks or to lipid and protein oxidation [8,9]. The brain is highly vulnerable to oxidative stress compared with other organs due to the high metabolic rate required to meet the energy consumption of the brain; and this high metabolic rate can lead to increased ROS production. When the defense is insufficient to scavenge these ROS, they may inevitably cause the oxidation of unsaturated fatty acids, which results in lipid peroxidation [10,11]. Enhanced ROS generation following brain insults, including cerebral ischemia/ hypoxia, brain trauma, and CO poisoning, may disrupt the balance between ROS generation and scavenging, which in turn accelerates neural injury, expands the injured area, and leads to poorer outcomes [5,12].
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Methane is the simplest aliphatic hydrocarbon and a major component of the natural gas used to heat homes and cook food. Certain microbes, called methanogens, may use carbon dioxide, acetate, or other small organic molecules as terminal electron acceptors under strictly anaerobic conditions and produce methane as a metabolic product [13]. Interestingly, it was reported that methane can be generated by rat liver mitochondria and formed from choline in the presence of hydrogen peroxide, catalyticiron, and ascorbic acid [14,15]. In addition, it has been shown that methane has anti-oxidative and anti-nitrosative activities in mesenteric ischemia-reperfusion injury [16]. In animal models, our team found that methane could protect the liver against ischemia-reperfusion injury [17] and the myocardium against myocardial infarction [18] via its antiapoptotic, anti-oxidative and anti-inflammatory activities. In addition, the protective effects of methane on diabetes mellitus were also found to be related to anti-inflammatory pathways [19]. Recently, it was reported that methane protected liver against Con A-induced injury through antiinflammatory and anti-oxidative pathways [20]. Our previous study showed methane-rich saline (MRS) was able to exert long term protection on the brain injury of rats after CO poisoning [21], but whether it protects the acute injury to the brain after CO poisoning is still unclear. This study was done to evaluate the protective effects of methanerich saline (MRS) on the brain injury secondary to acute CO poisoning and the potential protection mechanisms in a rat model. 2. Materials and methods All surgical procedures were approved by the Ethics Committee for Animal Experimentation and conducted according to the Guidelines
for Animal Experimentation of our institute. All efforts were made to minimize the number of animals used in this study, and every effort was taken to reduce animal suffering. 2.1. Animals and groups A total of 36 male Sprague-Dawley rats weighing 250–280 g were used in the present study. The animals were kept in a humidity- and temperature-controlled room with a 12-hour light/dark cycle and given ad libitum access to food and water. Animals were randomly distributed into three groups as follows: control group, CO poisoning plus normal saline (NS) (CO) group and CO poisoning plus MRS (CO + CH4) group (Fig. 1). 2.2. CO exposure and carboxyhemoglobin detection The establishment of acute CO poisoning in rats has been described previously [6]. Briefly, the rats were placed in a 7-L Plexiglass chamber and exposed to 1000 ppm CO (Shanghai Gas CO, China) at a rate of 4 L/min for 40 min, followed by 3000 ppm CO for another 20 min until they lost consciousness. Then, these rats were allowed to breathe fresh air and regain consciousness. The rats in the control group inhaled fresh air for 1 h. Hypothermia was avoided when poisoning was discontinued. Immediately after CO exposure, approximately 0.3 ml of whole blood was drawn for carboxyhemoglobin (COHb) assay after intraperitoneal anesthesia with 3% pentobarbital sodium (50 mg/kg). A Blood Gas Analyzer (Cobasb 221 system, Roche Diagnostics GmbH, Germany) was used for COHb detection.
Fig. 1. Flow chart of the study.
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2.3. Preparation of MRS Purified methane was dissolved into NS for 2 h under 0.6 MPa pressure. MRS was administered by peritoneal injection (10 ml/kg) immediately and repeated again at 8 h and 16 h after CO insult.
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test for multiple comparisons. A value of P b 0.05 was considered statistically significant. Statistical analysis was performed with SPSS version 13.0 (SPSS Inc., Chicago, USA). 3. Results
2.4. Nissl staining and cell counting
3.1. CO exposure and COHb
Animals were anesthetized with 2% pentobarbital sodium (50 mg/kg) and then perfused through the left ventricle with 200 ml of ice-cold 0.1 mol/l phosphate buffered saline (PBS) followed by 400 ml of 4% paraformaldehyde in 0.1 mol/l PBS (pH 7.4). The brains were removed and fixed for two days. After dehydration in the graded ethanol and xylene, the brain sections were embedded in paraffin. The tissues were cut into 4 μm sections, which were then dewaxed and rehydrated according to standard protocols. These sections were stained in 1% toluidine blue at 50 °C for 5 min. After rinsing with double distilled water, the sections were dehydrated in increasing concentrations of ethanol transparentized in xylene, mounted with permount cover slip and observed under a light microscope. Cells with Nissl substance in the cytoplasm, loose chromatin and prominent nucleoli were considered normal neurons, and damaged neurons were characterized by the loss of Nissl substance, cavitation around nucleus and the presence of pyknotic homogenousnuclei. The cortex and CA1 region of hippocampus were photographed in each section, and viable neurons with Nissl body were counted by two investigators who were blind to the grouping in this study, and consensus was achieved. Data are represented as the number of cells per mm2.
The free-moving rats in the chamber became slightly agitated after 30 min of exposure to 1000 ppm CO; they then calmed down and breathed deeply approximately 10 min later. The CO concentration was elevated to 3000 ppm until the animals lost consciousness, accompanied by limb paralysis, negative righting reflex and no response to stimulation in the following 20 ± 3 min. The COHb was increased to a near-lethal level (54.8% ± 4.9%) in poisoned rats, whereas it was only 0.5% ± 0.2% in the control group.
2.5. Measurements of superoxide dismutase activity and malondialdehyde content
3.2. Nissl staining Fig. 2 shows representative photographs from Nissl staining of the cortex and the CA1 region of the hippocampus. A large number of dark, pyknotic neurons was noted in the cortex and CA1 region of COpoisoned rats that were treated with normal saline (CO group) (B1– 4). However, more Nissl stained cells (C1–4) were observed in CO-poisoned rats treated with MRS (CO + CH4 group) (cortex: 50.08 ± 2.56 cells/mm2 vs. 36.83 ± 3.76 cells/mm2, P b 0.05; hippocampus: 82.67 ± 2.62 cells/mm2 vs. 69.92 ± 4.71 cells/mm2, P b 0.05). Of note, the number of Nissl cells in CO + CH4 group was still lower than in control group at both hippocampus and cortex. This suggests that MRS fails to completely reverse the damage of CO poisoning to the neurons. 3.3. MDA content of the brain
The samples were homogenized in cold saline with a weight-to-volume ratio of 1:9. The homogenate was centrifuged at 2000 rpm for 10 min at 4 °C, and the supernatant was assayed for protein concentration by Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). The treated samples were removed to a cuvette for the next assay. The measurements of superoxide dismutase (SOD) activity and malonaldehyde (MDA) contents in the tissue homogenate were performed according to the manufacturer's instruction (Jianchen Biological Institute, China). SOD activity was measured following the reduction of nitrite by a xanthine-xanthine oxidase system, which is an O–2 nerator. One unit of SOD was defined as the amount that shows 50% inhibition. The activity was expressed as U/mg-protein. MDA content was measured through the thiobarbituric acid (TBA) reaction with the absorbance at 535 nm was determined and used to estimate the MDA level which was expressed as nmol/mg protein. 2.6. Detection of tumor necrosis factor-α, interleukin-1β, interleukin-6, 8hydroxyguanine, 3-nitrotyrosine Brain tissues were collected, weighed, washed in normal saline and then homogenized immediately in 10 volumes of normal saline at 4 °C. After centrifugation, supernatants were collected and stored at −80 °C. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, 3-nitrotyrosine (3-NT), and 8-hydroxyguanine (8-OHdG) were measured using a commercial enzyme-linked immunosorbent assay kit (ELISA kit) (CUSABIOBIOTECHCO., Ltd., Wuhan, China) according to the manufacturer's instructions and were quantified using commercial kits with a microplate reader at 450 nm. 2.7. Statistical analysis All quantitative data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed with one-way analysis of variance (ANOVA), followed by a Student-Newman-Keuls (SNK)
MDA is a marker of lipid peroxidation, and its activity in the brain was measured as a marker of oxidative stress. MDA was significantly increased in the brain at 24 h after CO exposure compared to control group (Fig. 3; cortex: 3.4615 ± 0.4133 nmol/mg prot vs. 2.4320 ± 0.9807 nmol/mg prot, P b 0.01; hippocampus: 4.7308 ± 1.5868 vs. 2.2792 ± 0.4370, P b 0.01). In contrast, MRS dramatically suppressed the production of MDA in the cortex (2.5937 ± 0.3914) and hippocampus (2.7854 ± 0.8236) (P b 0.05 vs. CO group). However, there was no significant difference in the MDA content between control group and CO + CH4 group in bother cortex and hippocampus although it was higher in CO + CH4 group. 3.4. 3-NT content of the brain 3-NT is a product of tyrosine nitration that is mediated by reactive nitrogen species. 3-NT in the brain increased 24 h after CO insult as compared to control group (cortex: 0.3447 ± 0.0799 ng/mg prot vs. 0.3186 ± 0.1205 ng/mg prot, P N 0.05; hippocampus: 0.5079 ± 0.0992 ng/mg prot vs. 0.3133 ± 0.0549 ng/mg prot, P b 0.01). However, MRS suppressed 3-NT production in the cortex (0.3107 ± 0.0608 ng/mg prot, P N 0.05), and hippocampus (0.3893 ± 0.0788 ng/mg prot, P b 0.05) as compared to CO group. Significant difference in 3-NT content was only observed in the hippocampus between control group and CO + CH4 group (P b 0.05). 3.5. 8-OHdG level of the brain As shown in Figs. 3, 24 h after CO poisoning, the level of 8-OHdG, as a marker of oxidative DNA damage, was determined in the cortex and hippocampus. 8-OHdG content of the hippocampus was higher in CO group than in control group (cortex: 2.6614 ± 0.2912 ng/mg prot vs. 2.6043 ± 0.7032 ng/mg prot, P N 0.05; hippocampus: 4.7495 ± 0.4869 ng/mg prot vs. 4.0968 ± 0.5315 ng/mg prot, P b 0.05). MRS
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Fig. 2. Nissl staining of cortex and hippocampus and cell count at 24 h after CO poisoning. (A) Nissl staining. Cortex and hippocampus in each group are shown at a magnification of ×200. Some neurons in the cortex of both CO + saline (CO) and CO + methane (CH4) groups and in the CA1 region of the CO + saline group shrank, had enlarged intracellular space, and were dark in color, and some neurons even disappeared. More viable neurons were found in both the cortex and CA1 sector of CO + CH4 as compared to CO + saline group. (B) Cell counting. The number of Nissl-stained cells in the cortex and hippocampus of the CO + saline group was lower than that of the CO + CH4 group (*P b 0.05; **P b 0.01).
slightly reduce the 8-OHdG content in the cortex (2.6712 ± 0.4046 ng/ mg prot, P N 0.05) and hippocampus (4.5898 ± 0.5686 ng/mg prot, P N 0.05). 8-OHdG content of the hippocampus in the CO + CH4 group was still significantly higher than in control group, and there was no marked difference in the cortex between them. 3.6. Inflammatory factors The expression of TNF-α, IL-1β and IL-6 in the brain was measured 24 h after CO exposure (Fig. 4). The expression of TNF-α in the cortex and hippocampus was significantly higher in CO group than in control group (cortex: 35.8145 ± 7.3932 pg/mg prot vs. 26.7288 ± 8.1849 pg/mg prot, P b 0.05; hippocampus: 43.5858 ± 14.4229 vs. 28.5550 ± 5.4425 pg/mg prot, P b 0.01). The expression of IL-1β in the cortex and hippocampus was significantly higher in CO group than in control group (cortex: 134.8508 ± 14.9319 pg/mg prot vs. 74.4456 ± 23.3022 pg/mg prot, P b 0.01; hippocampus: 204.1105 ± 54.2337 pg/mg prot vs. 116.7567 ± 17.1759 pg/mg prot, P b 0.05). However, MRS significantly reduced the expression of TNF-α and IL-1β in the cortex (26.3004 ± 2.1105 pg/mg prot and 86.2382 ± 8.2103 pg/ mg prot) and hippocampus (28.5217 ± 10.2874 pg/mg prot and 122.8932 ± 20.8232 pg/mg prot) (P b 0.05) as compared to CO group. The IL-6 content increased significantly after CO poisoning in the hippocampus, but not in the cortex. MRS failed to significantly reduced IL-6 content in both cortex and hippocampus (P N 0.05). There were no marked differences in the inflammation related cytokines between control group and CO + CH4 group (except for IL-6 of the hippocampus) although these cytokines were still higher in CO + CH4 group than in control group. 3.7. SOD activity of the brain The SOD activity of the brain was measured 24 h after CO insult (Fig. 5). The SOD activity of the cortex and hippocampus was significantly lower in
CO group than in control group (cortex: 22.20 ± 7.73 U/mg prot vs. 57.65 ± 12.84 U/mg prot, P b 0.01; hippocampus: 108.91 ± 9.66 U/mg prot vs. 131.03 ± 17.92 U/mg prot, P b 0.05). MRS significantly increased SOD activity of the cortex (39.19 ± 8.55 U/mg prot vs. 22.20 ± 7.73 U/mg prot, P b 0.05) and hippocampus (137.82 ± 19.77 U/mg prot vs. 108.91 ± 9.66 U/mg prot; P b 0.01) as compared to CO group. SOD activity of the hippocampus was comparable between control group and CO + CH4 group, but SOD activity of the cortex was still significantly lower in CO + CH4 group than in control group (P b 0.05). 4. Discussion In the present study, we used a rat model to investigate the protective effects of intraperitoneal MRS on the brain injury secondary to CO poisoning. Our findings indicated that MRS was able to protect the brain against CO poisoning-induced injury. Our previous studies using animal models demonstrated that MRS exerted therapeutic effects on both liver ischemia reperfusion injury [17] and myocardial infarction [18] via its anti-oxidative, anti-inflammatory and anti-apoptotic activities. Moreover, our previous study also confirmed MRS was able to exert long term protection against CO poisoning induced injury to the rat brain (9 days after injury). The present study was undertaken to investigate the short term protection of MRS on the CO poisoning. Our results also showed that MRS could protect the neurons in the cortex and hippocampus against CO poisoning-induced injury by decreasing cerebral lipid peroxidation, attenuating DNA oxidative stress in neurons, increasing SOD activity, and reducing inflammation in a rat model that underwent acute CO poisoning. Oxidative stress is the process of cellular injury caused by the excess production of ROS. ROS have been considered essential signaling molecules in cells. However, when the production of ROS overwhelms the scavenging process, excess ROS may exert toxic effects and cause damage to all cellular components, including lipids, proteins and DNA, ultimately leading to cell death. The brain is highly vulnerable to
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Fig. 3. Levels of MDA, 3-NT and 8-OH-dG in the cortex and hippocampus. (A,B) MDA and SOD levels, (C,D) 3-NT level, (E,F) 8-OH-dG level. The levels of MDA, 3-NT and 8-OH-dG were determined by ELISA. Data are expressed as mean ± SD. (*P b 0.05; **P b 0.01).
oxidative stress compared with other organs due to the high metabolic rate required to meet the energy consumption of the brain; this also leads to an increase in ROS generation. Numerous studies have indicated that ROS and the subsequent lipid peroxidation are the major contributors to brain injury following CO exposure ± reoxygenation [6,22,23]. Cell membrane lipids are the major targets of ROS [24]. Increases in lipid peroxidation products such as MDA are markers of lipid damage. In addition, DNA is a major target of ROS, and 8-OHdG has served as a marker of DNA damage caused by hydroxyl free radicals [25]. In our study, increases in MDA, 3-NT and 8-OHdG were observed in rat brain after CO poisoning, and intraperitoneal MRS following CO exposure decreased the MDA, 3-NT and 8-OHdG in the cortex and hippocampus. Endogenous antioxidants are important participants in the anti-oxidative defense system and can significantly inhibit or delay the oxidative process [26]. Anti-oxidases are important for protection from CO exposure. Treatment with SOD immediately after CO poisoning has been found to suppress lipid peroxidation [27]. Therefore, an increase in anti-oxidase activity implies an elevation of the anti-oxidative effects. In the present study, MRS induced an increase in SOD activity in the brain, indicating an increase in anti-oxidative capability. The mechanism of protection against SOD most likely occurs as follows: O–2 can undergo dismutation spontaneously or via the enzyme mediated catalysis
(SOD) into hydrogen peroxide (H2O2), or it can react with nitric oxide (NO•) to form toxic ONOO− [28]. CO exposure may also cause inflammation, which is believed to play an important role in the pathogenesis of CO poisoning-induced encephalopathy [29,30]. In our study, compared to saline-treated rats, MRS significantly inhibited the expression of pro-inflammatory cytokines IL-1β and TNF-α in the cortex and hippocampus. Recently, one new theory about the catecholamine crisis proposed it as a novel mechanism underlying the pathogenesis of acute CO poisoning [31]. Microglial activation has been shown to be involved in the progression of brain injury in a variety of pathophysiological processes [32]. This is required to be further studied. Currently, numerous strategies have been applied to treat CO poisoning; among these, hyperbaric oxygen (HBO) therapy is the only method that has been widely accepted and shows good therapeutic efficacy. However, there are still some controversies over HBO treatment for CO poisoning because of its adverse effects, such as the augmented formation of ROS [33,34]. Methane can be produced by carbohydrate fermentation, during which CO2 is reduced to methane via the anaerobic metabolism of methanogenic microorganisms [35]. Methane is then absorbed into the circulation; thus, it is intrinsically nontoxic in vivo. Currently, the detection of exhaled methane has been used in the diagnosis of some gastrointestinal diseases [36]. It has been proposed
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Fig. 4. Levels of TNF-α, IL-1β and IL-6 in the cortex and hippocampus. (A,B) TNF-α level, (C,D) IL-1β level, (E,F) IL-6 level. The levels of TNF-α, IL-1β and IL-6 were determined by ELISA. Data are expressed as mean ± SD. (*P b 0.05; **P b 0.01).
that methane may be the fourth gasotransmitter [37] in mammalians, in addition to NO, CO and hydrogen sulfide (H2S). Some investigators hypothesize that methane might confer an effect on membrane channels,
accumulate transiently at cell membrane interfaces to change the physicochemical properties or the in situ functionality of proteins embedded within this environment or be catalyzed by potential oxygenase using
Fig. 5. Levels of SOD in cortex and hippocampus at 24 h after CO poisoning. Data are expressed as the mean ± SD. (*P b 0.05; **P b 0.01).
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methane as a substrate [38]. More studies are required whether there are these mechanisms underlying the therapeutic effects of methane. In conclusion, our findings indicate that methane may clinically protect the brain against acute CO poisoning induced injury and that the neuroprotective effects of MRS are probably mediated by its anti-oxidative and anti-inflammatory activities. In future studies, it will be necessary to elucidate how methane exerts its protective effects and which signaling pathway(s) are involved in the protective effects of methane on CO poisoning. Conflict of interest statement None declared. Acknowledgement This work was supported by the Natural Science Foundation of Shanghai (No. 14ZR1449500) and the National Nature Science Foundation of China (No. 81401855, 81371316). References [1] J.A. Raub, M. Mathieu-Nolf, N.B. Hampson, S.R. Thom, Carbon monoxide poisoning-a public health perspective, Toxicology 145 (1) (2000) 1–14. [2] M. Braubach, A. Algoet, M. Beaton, S. Lauriou, M.E. Héroux, M. Krzyzanowski, Mortality associated with exposure to carbon monoxide in WHO European Member States, Indoor Air 23 (2) (2013) 115–125. [3] Centers for Disease Control and Prevention (CDC), Carbon monoxide exposuresUnited States, 2000–2009, MMWR Morb. Mortal. Wkly Rep. 60 (30) (2011) 1014–1017. [4] H.J. Bidmon, J. Wu, I. Buchkremer-Ratzmann, B. Mayer, O.W. Witte, K. Zilles, Transient changes in the presence of nitric oxide synthases and nitrotyrosine immunoreactivity after focal cortical lesions, Neuroscience 82 (2) (1998) 377–395. [5] S. Hara, T. Mukai, K. Kurosaki, F. Kuriiwa, T. Endo, Characterization of hydroxyl radical generation in the striatum of free-moving rats due to carbon monoxide poisoning, as determined by in vivo microdialysis, Brain Res. 1016 (2) (2004) 281–284. [6] S.R. Thom, Carbon-monoxide-mediated brain lipid peroxidation in the rat, J. Appl. Physiol. (1985) 68 (3) (1990) 997–1003. [7] A. Ernst, J.D. Zibrak, Carbon monoxide poisoning, N. Engl. J. Med. 339 (22) (1998) 1603–1608. [8] D.A. Linseman, Targeting oxidative stress for neuroprotection, Antioxid. Redox Signal. 11 (3) (2009) 421–424. [9] M. Klein, U. Koedel, H.W. Pfister, Oxidative stress in pneumococcal meningitis: a future target for adjunctive therapy? Prog. Neurobiol. 80 (6) (2006) 269–280. [10] P.H. Evans, Free radicals in brain metabolism and pathology, Br. Med. Bull. 49 (3) (1993) 577–587. [11] R.J. Reiter, Oxidative processes and antioxidative defense mechanisms in the aging brain, FASEB J. 9 (7) (1995) 526–533. [12] A. Lewen, P. Matz, P.H. Chan, Free radical pathways in CNS injury, J. Neurotrauma 17 (10) (2000) 871–890. [13] A.P. Liou, M. Paziuk, J.M. Luevano Jr., S. Machineni, P.J. Turnbaugh, L.M. Kaplan, Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity, Sci. Transl. Med. 5 (178) (2013), 178ra41. [14] M. Ghyczy, C. Torday, J. Kaszaki, A. Szabó, M. Czóbel, M. Boros, Hypoxia-induced generation of methane in mitochondria and eukaryotic cells: an alternative approach to methanogenesis, Cell. Physiol. Biochem. 21 (1–3) (2008) 251–258. [15] M. Ghyczy, C. Torday, M. Boros, Simultaneous generation of methane, carbon dioxide, and carbon monoxide from choline and ascorbic acid: a defensive mechanism against reductive stress? FASEB J. 17 (9) (2003) 1124–1126.
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Neuroprotective effects of methane-rich saline on experimental acute carbon monoxide toxicity Meihua Shen a,c,1, Danfeng Fan b,1, Yu Zang d, Yan Chen c, Kaimin Zhu c, Zhonghai Cai c, Yueqin Liu c, Xuejun Sun e, Jiankang Liu a,⁎, Jianfeng Gong f,⁎ a Key Laboratory of Biomedical Information Engineering, Ministry of Education, Institute of Mitochondrial Biology and Medicine, Xi'an Jiaotong University, School of Life Science and Technology, Xi'an 710049, PR China b Department of Hyperbaric Oxygen, Navy General Hospital, No. 6, Fucheng Road, Beijing 100048, PR China c Department of Intensive Care Unit, Shanghai Provincial Corps Hospital, Chinese People's Armed Police Forces, 831 Hongxu Road, Shanghai 201103, PR China d Department of Neurology, The Affiliated Hospital of North China University of Science and Technology, No.57, Jianshe South Road, Tangshan, Hebei 063000, PR China e Department of Naval Aeromedicine, Faculty of Naval Medicine, Second Military Medical University, No.800, Xiangyin Road, Shanghai 200433, PR China f Department of General Surgery, The Sixth People's Hospital, Shanghai Jiao Tong University, No.600, Yishan Road, Shanghai 200233, PR China
a r t i c l e
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Article history: Received 1 July 2016 Received in revised form 29 July 2016 Accepted 24 August 2016 Available online 26 August 2016 Keywords: Methane Carbon monoxide poisoning Reactive oxygen species Antioxidant enzyme
a b s t r a c t Background: Methane has been reported to play a protective role in ischemia-reperfusion injury via anti-oxidation, anti-inflammatory and anti-apoptotic activities. This study was designed to determine the protective effects of methane-rich saline (MRS) on acute carbon monoxide (CO) poisoning. Methods: A total of 36 male Sprague-Dawley rats were randomly divided into 3 groups: sham group, CO group and MRS group. Acute CO poisoning was induced by exposing rats to 1000 ppm CO in air for 40 min and then to 3000 ppm CO for an additional 20 min until they lost consciousness. MRS at 10 ml/kg was intraperitoneally administered at 0 h, 8 h and 16 h after CO exposure. Rats were sacrificed 24 h after CO exposure. Brains were collected for Nissl staining. The cortex and hippocampus were separated for the detections of malondialdehyde (MDA), 3-nitrotyrosine (3-NT), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin1-β (IL-1β), interleukin-6 (IL-6) and superoxide dismutase (SOD) activities. Results: The results showed that MRS treatment improved neuronal injury, reduced MDA, 3-NT and 8-OHdG, and increased SOD activity of the hippocampus and cortex compared with normal saline-treated rats. In addition, MRS reduced the expression of TNF-α and IL-1β in the brain but had no effect on IL-6 expression. Conclusion: These findings suggest that MRS may protect the brain against acute CO poisoning-induced injury via its anti-oxidative and anti-inflammatory activities. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Acute carbon monoxide (CO) toxicity is a leading cause of gas poisoning-related deaths worldwide due to the increased use of carbonbased fuels [1]. It has been reported that CO poisoning is responsible for approximately 15,000 visits to emergency departments and nearly 500 deaths annually in the United States [1–3]. Acute CO poisoning
Abbreviations: ANOVA, analysis of variance; CO, carbon monoxide; COHb, carboxyhemoglobin; DNS, delayed neurological syndrome; HBO, hyperbaric oxygen; • OH, hydroxyl radical; IL-1β, interleukin 1 –β; IL-6, interleukin-6; MRS, methane-rich saline; MDA, Malondialdehyde; NS, normal saline; 3-NT, 3-nitrotyrosine; 8-OHdG, 8hydroxydeoxyguanosine; ROS, reactive oxygen species; SOD, superoxide dismutase; ONOO−, peroxynitrite; O− 2 , superoxide anion; TBA, thiobarbituric acid; TNF-α, tumor necrosis factor-α. ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Liu), [email protected] (J. Gong). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jns.2016.08.055 0022-510X/© 2016 Elsevier B.V. All rights reserved.
may produce severe brain damage, which can lead to high mortality and delayed neurological syndrome (DNS) [4]. Numerous studies have indicated that an increase in the production of reactive oxygen species (ROS) following CO poisoning is of crucial relevance to the pathophysiology of CO poisoning [5–7]. Although ROS play important roles in the clearance of invaded pathogens, they seem to produce substantial damage if they are produced in excess, which can lead to DNA strand breaks or to lipid and protein oxidation [8,9]. The brain is highly vulnerable to oxidative stress compared with other organs due to the high metabolic rate required to meet the energy consumption of the brain; and this high metabolic rate can lead to increased ROS production. When the defense is insufficient to scavenge these ROS, they may inevitably cause the oxidation of unsaturated fatty acids, which results in lipid peroxidation [10,11]. Enhanced ROS generation following brain insults, including cerebral ischemia/ hypoxia, brain trauma, and CO poisoning, may disrupt the balance between ROS generation and scavenging, which in turn accelerates neural injury, expands the injured area, and leads to poorer outcomes [5,12].
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Methane is the simplest aliphatic hydrocarbon and a major component of the natural gas used to heat homes and cook food. Certain microbes, called methanogens, may use carbon dioxide, acetate, or other small organic molecules as terminal electron acceptors under strictly anaerobic conditions and produce methane as a metabolic product [13]. Interestingly, it was reported that methane can be generated by rat liver mitochondria and formed from choline in the presence of hydrogen peroxide, catalyticiron, and ascorbic acid [14,15]. In addition, it has been shown that methane has anti-oxidative and anti-nitrosative activities in mesenteric ischemia-reperfusion injury [16]. In animal models, our team found that methane could protect the liver against ischemia-reperfusion injury [17] and the myocardium against myocardial infarction [18] via its antiapoptotic, anti-oxidative and anti-inflammatory activities. In addition, the protective effects of methane on diabetes mellitus were also found to be related to anti-inflammatory pathways [19]. Recently, it was reported that methane protected liver against Con A-induced injury through antiinflammatory and anti-oxidative pathways [20]. Our previous study showed methane-rich saline (MRS) was able to exert long term protection on the brain injury of rats after CO poisoning [21], but whether it protects the acute injury to the brain after CO poisoning is still unclear. This study was done to evaluate the protective effects of methanerich saline (MRS) on the brain injury secondary to acute CO poisoning and the potential protection mechanisms in a rat model. 2. Materials and methods All surgical procedures were approved by the Ethics Committee for Animal Experimentation and conducted according to the Guidelines
for Animal Experimentation of our institute. All efforts were made to minimize the number of animals used in this study, and every effort was taken to reduce animal suffering. 2.1. Animals and groups A total of 36 male Sprague-Dawley rats weighing 250–280 g were used in the present study. The animals were kept in a humidity- and temperature-controlled room with a 12-hour light/dark cycle and given ad libitum access to food and water. Animals were randomly distributed into three groups as follows: control group, CO poisoning plus normal saline (NS) (CO) group and CO poisoning plus MRS (CO + CH4) group (Fig. 1). 2.2. CO exposure and carboxyhemoglobin detection The establishment of acute CO poisoning in rats has been described previously [6]. Briefly, the rats were placed in a 7-L Plexiglass chamber and exposed to 1000 ppm CO (Shanghai Gas CO, China) at a rate of 4 L/min for 40 min, followed by 3000 ppm CO for another 20 min until they lost consciousness. Then, these rats were allowed to breathe fresh air and regain consciousness. The rats in the control group inhaled fresh air for 1 h. Hypothermia was avoided when poisoning was discontinued. Immediately after CO exposure, approximately 0.3 ml of whole blood was drawn for carboxyhemoglobin (COHb) assay after intraperitoneal anesthesia with 3% pentobarbital sodium (50 mg/kg). A Blood Gas Analyzer (Cobasb 221 system, Roche Diagnostics GmbH, Germany) was used for COHb detection.
Fig. 1. Flow chart of the study.
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2.3. Preparation of MRS Purified methane was dissolved into NS for 2 h under 0.6 MPa pressure. MRS was administered by peritoneal injection (10 ml/kg) immediately and repeated again at 8 h and 16 h after CO insult.
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test for multiple comparisons. A value of P b 0.05 was considered statistically significant. Statistical analysis was performed with SPSS version 13.0 (SPSS Inc., Chicago, USA). 3. Results
2.4. Nissl staining and cell counting
3.1. CO exposure and COHb
Animals were anesthetized with 2% pentobarbital sodium (50 mg/kg) and then perfused through the left ventricle with 200 ml of ice-cold 0.1 mol/l phosphate buffered saline (PBS) followed by 400 ml of 4% paraformaldehyde in 0.1 mol/l PBS (pH 7.4). The brains were removed and fixed for two days. After dehydration in the graded ethanol and xylene, the brain sections were embedded in paraffin. The tissues were cut into 4 μm sections, which were then dewaxed and rehydrated according to standard protocols. These sections were stained in 1% toluidine blue at 50 °C for 5 min. After rinsing with double distilled water, the sections were dehydrated in increasing concentrations of ethanol transparentized in xylene, mounted with permount cover slip and observed under a light microscope. Cells with Nissl substance in the cytoplasm, loose chromatin and prominent nucleoli were considered normal neurons, and damaged neurons were characterized by the loss of Nissl substance, cavitation around nucleus and the presence of pyknotic homogenousnuclei. The cortex and CA1 region of hippocampus were photographed in each section, and viable neurons with Nissl body were counted by two investigators who were blind to the grouping in this study, and consensus was achieved. Data are represented as the number of cells per mm2.
The free-moving rats in the chamber became slightly agitated after 30 min of exposure to 1000 ppm CO; they then calmed down and breathed deeply approximately 10 min later. The CO concentration was elevated to 3000 ppm until the animals lost consciousness, accompanied by limb paralysis, negative righting reflex and no response to stimulation in the following 20 ± 3 min. The COHb was increased to a near-lethal level (54.8% ± 4.9%) in poisoned rats, whereas it was only 0.5% ± 0.2% in the control group.
2.5. Measurements of superoxide dismutase activity and malondialdehyde content
3.2. Nissl staining Fig. 2 shows representative photographs from Nissl staining of the cortex and the CA1 region of the hippocampus. A large number of dark, pyknotic neurons was noted in the cortex and CA1 region of COpoisoned rats that were treated with normal saline (CO group) (B1– 4). However, more Nissl stained cells (C1–4) were observed in CO-poisoned rats treated with MRS (CO + CH4 group) (cortex: 50.08 ± 2.56 cells/mm2 vs. 36.83 ± 3.76 cells/mm2, P b 0.05; hippocampus: 82.67 ± 2.62 cells/mm2 vs. 69.92 ± 4.71 cells/mm2, P b 0.05). Of note, the number of Nissl cells in CO + CH4 group was still lower than in control group at both hippocampus and cortex. This suggests that MRS fails to completely reverse the damage of CO poisoning to the neurons. 3.3. MDA content of the brain
The samples were homogenized in cold saline with a weight-to-volume ratio of 1:9. The homogenate was centrifuged at 2000 rpm for 10 min at 4 °C, and the supernatant was assayed for protein concentration by Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). The treated samples were removed to a cuvette for the next assay. The measurements of superoxide dismutase (SOD) activity and malonaldehyde (MDA) contents in the tissue homogenate were performed according to the manufacturer's instruction (Jianchen Biological Institute, China). SOD activity was measured following the reduction of nitrite by a xanthine-xanthine oxidase system, which is an O–2 nerator. One unit of SOD was defined as the amount that shows 50% inhibition. The activity was expressed as U/mg-protein. MDA content was measured through the thiobarbituric acid (TBA) reaction with the absorbance at 535 nm was determined and used to estimate the MDA level which was expressed as nmol/mg protein. 2.6. Detection of tumor necrosis factor-α, interleukin-1β, interleukin-6, 8hydroxyguanine, 3-nitrotyrosine Brain tissues were collected, weighed, washed in normal saline and then homogenized immediately in 10 volumes of normal saline at 4 °C. After centrifugation, supernatants were collected and stored at −80 °C. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, 3-nitrotyrosine (3-NT), and 8-hydroxyguanine (8-OHdG) were measured using a commercial enzyme-linked immunosorbent assay kit (ELISA kit) (CUSABIOBIOTECHCO., Ltd., Wuhan, China) according to the manufacturer's instructions and were quantified using commercial kits with a microplate reader at 450 nm. 2.7. Statistical analysis All quantitative data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed with one-way analysis of variance (ANOVA), followed by a Student-Newman-Keuls (SNK)
MDA is a marker of lipid peroxidation, and its activity in the brain was measured as a marker of oxidative stress. MDA was significantly increased in the brain at 24 h after CO exposure compared to control group (Fig. 3; cortex: 3.4615 ± 0.4133 nmol/mg prot vs. 2.4320 ± 0.9807 nmol/mg prot, P b 0.01; hippocampus: 4.7308 ± 1.5868 vs. 2.2792 ± 0.4370, P b 0.01). In contrast, MRS dramatically suppressed the production of MDA in the cortex (2.5937 ± 0.3914) and hippocampus (2.7854 ± 0.8236) (P b 0.05 vs. CO group). However, there was no significant difference in the MDA content between control group and CO + CH4 group in bother cortex and hippocampus although it was higher in CO + CH4 group. 3.4. 3-NT content of the brain 3-NT is a product of tyrosine nitration that is mediated by reactive nitrogen species. 3-NT in the brain increased 24 h after CO insult as compared to control group (cortex: 0.3447 ± 0.0799 ng/mg prot vs. 0.3186 ± 0.1205 ng/mg prot, P N 0.05; hippocampus: 0.5079 ± 0.0992 ng/mg prot vs. 0.3133 ± 0.0549 ng/mg prot, P b 0.01). However, MRS suppressed 3-NT production in the cortex (0.3107 ± 0.0608 ng/mg prot, P N 0.05), and hippocampus (0.3893 ± 0.0788 ng/mg prot, P b 0.05) as compared to CO group. Significant difference in 3-NT content was only observed in the hippocampus between control group and CO + CH4 group (P b 0.05). 3.5. 8-OHdG level of the brain As shown in Figs. 3, 24 h after CO poisoning, the level of 8-OHdG, as a marker of oxidative DNA damage, was determined in the cortex and hippocampus. 8-OHdG content of the hippocampus was higher in CO group than in control group (cortex: 2.6614 ± 0.2912 ng/mg prot vs. 2.6043 ± 0.7032 ng/mg prot, P N 0.05; hippocampus: 4.7495 ± 0.4869 ng/mg prot vs. 4.0968 ± 0.5315 ng/mg prot, P b 0.05). MRS
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Fig. 2. Nissl staining of cortex and hippocampus and cell count at 24 h after CO poisoning. (A) Nissl staining. Cortex and hippocampus in each group are shown at a magnification of ×200. Some neurons in the cortex of both CO + saline (CO) and CO + methane (CH4) groups and in the CA1 region of the CO + saline group shrank, had enlarged intracellular space, and were dark in color, and some neurons even disappeared. More viable neurons were found in both the cortex and CA1 sector of CO + CH4 as compared to CO + saline group. (B) Cell counting. The number of Nissl-stained cells in the cortex and hippocampus of the CO + saline group was lower than that of the CO + CH4 group (*P b 0.05; **P b 0.01).
slightly reduce the 8-OHdG content in the cortex (2.6712 ± 0.4046 ng/ mg prot, P N 0.05) and hippocampus (4.5898 ± 0.5686 ng/mg prot, P N 0.05). 8-OHdG content of the hippocampus in the CO + CH4 group was still significantly higher than in control group, and there was no marked difference in the cortex between them. 3.6. Inflammatory factors The expression of TNF-α, IL-1β and IL-6 in the brain was measured 24 h after CO exposure (Fig. 4). The expression of TNF-α in the cortex and hippocampus was significantly higher in CO group than in control group (cortex: 35.8145 ± 7.3932 pg/mg prot vs. 26.7288 ± 8.1849 pg/mg prot, P b 0.05; hippocampus: 43.5858 ± 14.4229 vs. 28.5550 ± 5.4425 pg/mg prot, P b 0.01). The expression of IL-1β in the cortex and hippocampus was significantly higher in CO group than in control group (cortex: 134.8508 ± 14.9319 pg/mg prot vs. 74.4456 ± 23.3022 pg/mg prot, P b 0.01; hippocampus: 204.1105 ± 54.2337 pg/mg prot vs. 116.7567 ± 17.1759 pg/mg prot, P b 0.05). However, MRS significantly reduced the expression of TNF-α and IL-1β in the cortex (26.3004 ± 2.1105 pg/mg prot and 86.2382 ± 8.2103 pg/ mg prot) and hippocampus (28.5217 ± 10.2874 pg/mg prot and 122.8932 ± 20.8232 pg/mg prot) (P b 0.05) as compared to CO group. The IL-6 content increased significantly after CO poisoning in the hippocampus, but not in the cortex. MRS failed to significantly reduced IL-6 content in both cortex and hippocampus (P N 0.05). There were no marked differences in the inflammation related cytokines between control group and CO + CH4 group (except for IL-6 of the hippocampus) although these cytokines were still higher in CO + CH4 group than in control group. 3.7. SOD activity of the brain The SOD activity of the brain was measured 24 h after CO insult (Fig. 5). The SOD activity of the cortex and hippocampus was significantly lower in
CO group than in control group (cortex: 22.20 ± 7.73 U/mg prot vs. 57.65 ± 12.84 U/mg prot, P b 0.01; hippocampus: 108.91 ± 9.66 U/mg prot vs. 131.03 ± 17.92 U/mg prot, P b 0.05). MRS significantly increased SOD activity of the cortex (39.19 ± 8.55 U/mg prot vs. 22.20 ± 7.73 U/mg prot, P b 0.05) and hippocampus (137.82 ± 19.77 U/mg prot vs. 108.91 ± 9.66 U/mg prot; P b 0.01) as compared to CO group. SOD activity of the hippocampus was comparable between control group and CO + CH4 group, but SOD activity of the cortex was still significantly lower in CO + CH4 group than in control group (P b 0.05). 4. Discussion In the present study, we used a rat model to investigate the protective effects of intraperitoneal MRS on the brain injury secondary to CO poisoning. Our findings indicated that MRS was able to protect the brain against CO poisoning-induced injury. Our previous studies using animal models demonstrated that MRS exerted therapeutic effects on both liver ischemia reperfusion injury [17] and myocardial infarction [18] via its anti-oxidative, anti-inflammatory and anti-apoptotic activities. Moreover, our previous study also confirmed MRS was able to exert long term protection against CO poisoning induced injury to the rat brain (9 days after injury). The present study was undertaken to investigate the short term protection of MRS on the CO poisoning. Our results also showed that MRS could protect the neurons in the cortex and hippocampus against CO poisoning-induced injury by decreasing cerebral lipid peroxidation, attenuating DNA oxidative stress in neurons, increasing SOD activity, and reducing inflammation in a rat model that underwent acute CO poisoning. Oxidative stress is the process of cellular injury caused by the excess production of ROS. ROS have been considered essential signaling molecules in cells. However, when the production of ROS overwhelms the scavenging process, excess ROS may exert toxic effects and cause damage to all cellular components, including lipids, proteins and DNA, ultimately leading to cell death. The brain is highly vulnerable to
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Fig. 3. Levels of MDA, 3-NT and 8-OH-dG in the cortex and hippocampus. (A,B) MDA and SOD levels, (C,D) 3-NT level, (E,F) 8-OH-dG level. The levels of MDA, 3-NT and 8-OH-dG were determined by ELISA. Data are expressed as mean ± SD. (*P b 0.05; **P b 0.01).
oxidative stress compared with other organs due to the high metabolic rate required to meet the energy consumption of the brain; this also leads to an increase in ROS generation. Numerous studies have indicated that ROS and the subsequent lipid peroxidation are the major contributors to brain injury following CO exposure ± reoxygenation [6,22,23]. Cell membrane lipids are the major targets of ROS [24]. Increases in lipid peroxidation products such as MDA are markers of lipid damage. In addition, DNA is a major target of ROS, and 8-OHdG has served as a marker of DNA damage caused by hydroxyl free radicals [25]. In our study, increases in MDA, 3-NT and 8-OHdG were observed in rat brain after CO poisoning, and intraperitoneal MRS following CO exposure decreased the MDA, 3-NT and 8-OHdG in the cortex and hippocampus. Endogenous antioxidants are important participants in the anti-oxidative defense system and can significantly inhibit or delay the oxidative process [26]. Anti-oxidases are important for protection from CO exposure. Treatment with SOD immediately after CO poisoning has been found to suppress lipid peroxidation [27]. Therefore, an increase in anti-oxidase activity implies an elevation of the anti-oxidative effects. In the present study, MRS induced an increase in SOD activity in the brain, indicating an increase in anti-oxidative capability. The mechanism of protection against SOD most likely occurs as follows: O–2 can undergo dismutation spontaneously or via the enzyme mediated catalysis
(SOD) into hydrogen peroxide (H2O2), or it can react with nitric oxide (NO•) to form toxic ONOO− [28]. CO exposure may also cause inflammation, which is believed to play an important role in the pathogenesis of CO poisoning-induced encephalopathy [29,30]. In our study, compared to saline-treated rats, MRS significantly inhibited the expression of pro-inflammatory cytokines IL-1β and TNF-α in the cortex and hippocampus. Recently, one new theory about the catecholamine crisis proposed it as a novel mechanism underlying the pathogenesis of acute CO poisoning [31]. Microglial activation has been shown to be involved in the progression of brain injury in a variety of pathophysiological processes [32]. This is required to be further studied. Currently, numerous strategies have been applied to treat CO poisoning; among these, hyperbaric oxygen (HBO) therapy is the only method that has been widely accepted and shows good therapeutic efficacy. However, there are still some controversies over HBO treatment for CO poisoning because of its adverse effects, such as the augmented formation of ROS [33,34]. Methane can be produced by carbohydrate fermentation, during which CO2 is reduced to methane via the anaerobic metabolism of methanogenic microorganisms [35]. Methane is then absorbed into the circulation; thus, it is intrinsically nontoxic in vivo. Currently, the detection of exhaled methane has been used in the diagnosis of some gastrointestinal diseases [36]. It has been proposed
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Fig. 4. Levels of TNF-α, IL-1β and IL-6 in the cortex and hippocampus. (A,B) TNF-α level, (C,D) IL-1β level, (E,F) IL-6 level. The levels of TNF-α, IL-1β and IL-6 were determined by ELISA. Data are expressed as mean ± SD. (*P b 0.05; **P b 0.01).
that methane may be the fourth gasotransmitter [37] in mammalians, in addition to NO, CO and hydrogen sulfide (H2S). Some investigators hypothesize that methane might confer an effect on membrane channels,
accumulate transiently at cell membrane interfaces to change the physicochemical properties or the in situ functionality of proteins embedded within this environment or be catalyzed by potential oxygenase using
Fig. 5. Levels of SOD in cortex and hippocampus at 24 h after CO poisoning. Data are expressed as the mean ± SD. (*P b 0.05; **P b 0.01).
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methane as a substrate [38]. More studies are required whether there are these mechanisms underlying the therapeutic effects of methane. In conclusion, our findings indicate that methane may clinically protect the brain against acute CO poisoning induced injury and that the neuroprotective effects of MRS are probably mediated by its anti-oxidative and anti-inflammatory activities. In future studies, it will be necessary to elucidate how methane exerts its protective effects and which signaling pathway(s) are involved in the protective effects of methane on CO poisoning. Conflict of interest statement None declared. Acknowledgement This work was supported by the Natural Science Foundation of Shanghai (No. 14ZR1449500) and the National Nature Science Foundation of China (No. 81401855, 81371316). References [1] J.A. Raub, M. Mathieu-Nolf, N.B. Hampson, S.R. Thom, Carbon monoxide poisoning-a public health perspective, Toxicology 145 (1) (2000) 1–14. [2] M. Braubach, A. Algoet, M. Beaton, S. Lauriou, M.E. Héroux, M. Krzyzanowski, Mortality associated with exposure to carbon monoxide in WHO European Member States, Indoor Air 23 (2) (2013) 115–125. [3] Centers for Disease Control and Prevention (CDC), Carbon monoxide exposuresUnited States, 2000–2009, MMWR Morb. Mortal. Wkly Rep. 60 (30) (2011) 1014–1017. [4] H.J. Bidmon, J. Wu, I. Buchkremer-Ratzmann, B. Mayer, O.W. Witte, K. Zilles, Transient changes in the presence of nitric oxide synthases and nitrotyrosine immunoreactivity after focal cortical lesions, Neuroscience 82 (2) (1998) 377–395. [5] S. Hara, T. Mukai, K. Kurosaki, F. Kuriiwa, T. Endo, Characterization of hydroxyl radical generation in the striatum of free-moving rats due to carbon monoxide poisoning, as determined by in vivo microdialysis, Brain Res. 1016 (2) (2004) 281–284. [6] S.R. Thom, Carbon-monoxide-mediated brain lipid peroxidation in the rat, J. Appl. Physiol. (1985) 68 (3) (1990) 997–1003. [7] A. Ernst, J.D. Zibrak, Carbon monoxide poisoning, N. Engl. J. Med. 339 (22) (1998) 1603–1608. [8] D.A. Linseman, Targeting oxidative stress for neuroprotection, Antioxid. Redox Signal. 11 (3) (2009) 421–424. [9] M. Klein, U. Koedel, H.W. Pfister, Oxidative stress in pneumococcal meningitis: a future target for adjunctive therapy? Prog. Neurobiol. 80 (6) (2006) 269–280. [10] P.H. Evans, Free radicals in brain metabolism and pathology, Br. Med. Bull. 49 (3) (1993) 577–587. [11] R.J. Reiter, Oxidative processes and antioxidative defense mechanisms in the aging brain, FASEB J. 9 (7) (1995) 526–533. [12] A. Lewen, P. Matz, P.H. Chan, Free radical pathways in CNS injury, J. Neurotrauma 17 (10) (2000) 871–890. [13] A.P. Liou, M. Paziuk, J.M. Luevano Jr., S. Machineni, P.J. Turnbaugh, L.M. Kaplan, Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity, Sci. Transl. Med. 5 (178) (2013), 178ra41. [14] M. Ghyczy, C. Torday, J. Kaszaki, A. Szabó, M. Czóbel, M. Boros, Hypoxia-induced generation of methane in mitochondria and eukaryotic cells: an alternative approach to methanogenesis, Cell. Physiol. Biochem. 21 (1–3) (2008) 251–258. [15] M. Ghyczy, C. Torday, M. Boros, Simultaneous generation of methane, carbon dioxide, and carbon monoxide from choline and ascorbic acid: a defensive mechanism against reductive stress? FASEB J. 17 (9) (2003) 1124–1126.
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