Hydrogen-rich Saline Reduces Delayed Neurologic Sequelae In Experimental Carbon Monoxide Toxicity

Background and objectiveCO is an invisible, colorless and odorless gas and is commonly viewed as a poison.occurs in nature as a product of oxidation or incomplete combustion of organic matter,suchas coal gas,liquefied petroleum gas and industrial gas. Carbon monoxide poison ing at highconcentrations is a major cause of illness and death, due to tissue hypoxia, with the CNS andthe heart being the most susceptible target organs due to their high oxygen needs. Delayedneuropsychological sequelae after acute carbon monoxide poisoning (DNS) is one of seriouscomplications of acute carbon monoxide poisoning. Which is characterized by dementia,psychiatric symptoms, even extrapyramidal symptoms, following a symptom-free interval ofweeks or longer after CO poisoning. A surprisingly high incidence of neuropsychologicsequelae was found. There is no ideal clinical treatment method. So it becomes a hot topic inclinical or laboratory research several years.It was unknown about mechanism of the delayed encephalopathy. Researchersconsidered that acute ischemia and hypoxia, cytotoxic injury, cell apoptosis, immunedysfunction, reperfusion injury and free radicals might be involved after carbon monoxidepoisoning. Among of them, oxygen free radicals and cell apoptosis played an important role.An amount of oxygen free radicals were produced during the process of hypoxia,reoxygenation and hyperbaric oxygen (HBO). And a high proportion of unsaturated fattyacids existed in the brain, which more easily induced neurons necrosis or apoptosis. Activatedmicroglia and high level of cytokines of brain were detected after CO poisoning. Immunedysfunction became the rational mechanism to explain symptom-free interval of a few weeksor longer after CO poisoning.Hydrogen is colorless,odorless,tasteless and reducing agent. Hydrogen has lowsolubility, and cannot easily been absorbed by human bodies. So hydrogen has beenconsidered one of physiological inert gases. However, in2007, Japanese scholars reportedthat inhalation of2%hydrogen gas procted against focal brain ischemia reperfusion injury inan animal model. Selective antioxidation is one possible mechanism of hydrogen treatment,Subsequently,our studies also proved its neuroprotective effects in a nlieonatal hypoxia-ischemia rat model by the way of intraperitoneal injection hydrogen saline. As a novel medical gas, hydrogen gas has potent antioxidant, anti-apoptotic and anti-inflammatory properties. Therefore, we supposed hydrogen saline could be used in thetreatment of DNS. The aim of this study is to examine to evaluate the feasibility and efficacyof hydrogen therapy for severe acute CO poisoning encephalopathy in an animal model. Andinvestigate its anti-apoptotic effects in cultured hippocampal neurons by hypoxia-reoxygena-tion.PartⅠA reliable method for the DNS model of rats after CO poisoningMethods:1. To establish the DNS model of rat with the method of acute static inhalation of COpoisoning:Rats were exposed to2000ppm (part per million) CO for40min and then3000ppm for up to20min. until they lost consciousness, then they were removed andregain consciousness.2. To observe the manifestation of rats exposed to CO poisoning.3. Morris Water maze testing was provided to test cognitive function differences in differenttimes between two groups.Results:1. Rats exhibited mild active and restless20min after CO poisoning, then became irritability,shortness of breath, cherry-red color of oral mucosa and the lower extremities after about30min. When concentration of CO was up to3000ppm, they experienced the period ofmanic, paralysis and coma.2. Maze measurement showed no significant difference between the group of CO poisonedrats and air exposed rats in the first6days,nevertheless,the escape latency of the COpoisoned rats became longer than the air exposed rats in the days of7,9after COpoisoning．PartⅡHydrogen-rich saline reduces delayed neurologic sequelae in experimental carbonmonoxide toxicityMethod:1. CO poisoning model was established.2. Morris Water maze testing was provided to test cognitive function differences amongdifference groups. 3.9days after CO poisoning, animals were sacrificed, with4%paraformaldehyde fixed, thenbrain segment was removed, Nissl staining was performed to indicate the morphologychanges in cerebral cortex and hippocampus.4. Oxidative stress markers such as MDA and SOD were examined in brain tissues.5. TUNEL staining was to detect apoptotic cell death caspase-3activity was also examined inthe cerebral cortex and hippocampus6. The activation of microglias was detected by immunostaining of Iba1, and MBPdegradation was also measured by immunohistochemistry.7. Concentrations of TNF-α, IL-1β and IL-6of the cerebral cortex and hippocampus weremeasured by ELISA.Results:1. Hydrogen-rich saline treatment could obviously improve decrements in learning functionafter CO poisoning by Morris Water maze testing.2. The number of Nissl staining positive cells of cortex and hippocampus in control animalswas lower than that in hydrogen saline treated animals.3. Compared to the control group, there is significantly decreased MDA content,8-OHdGlevels and increased serum SOD activity in hydrogen treated group.4. Relative to the control group. Hydrogen-rich saline treatment comparably reduced thepercent of TUNEL-positive cells and the activity of caspase-3.5. Hydrogen-rich saline treatment significantly surpressed the expression of microglia afterCO poisoning.and MBP degradation also decreased in hydrogen treated group.6. The levels of TNF-α, IL-1βand IL-6protein of the cerebral cortex and hippocampus weresignificantly reduced by hydrogen treatment.PartⅢ Anti-apoptotic effects of Hydrogen medium on hippocampal neurons byhypoxia-reoxygenation in RatsMethod:1. The culture of primary hippocampal neurons.2. To establish anoxia-reoxygenation injury model.neurons were treated with non-serumDMEM solution and then cultured in an incubator(37℃,95％N2,5％C02,oxygencontent under1%) for60min,10%normal rat serum was added before24hreoxygenation(5%C02,95%02)；3. The cultured hippocanpal neurons were identified by immunohistochernistry staining ofNSE and DAPI. 4. Cell viability change was detected by MTT assay among difference group.5. The content of MDA and activity of LDH were measured to evaluate the extent of neuronalinjury.6. TUNEL staining was to detect apoptotic cell death and caspase-3activity was alsoexamined in cultured hippocanpal neurons.7. The expression of Bcl-2and Bax protein was determined by western blotting．Results:1. Immunoreactive cells of neuron specific enolase (NSE) counterstained with DAPIamounted to94.2±2.1%2. Compared with control group, hydrogen therapy significantly elevated cell viability24hafter anoxia-reoxygenation injury.3. Hydrogen therapy suppressed the anoxia-reoxygenation injury characterized with the levelof MDA and LDH.4. Hydrogen therapy inhibited apoptosis and caspase-3activity of hippocampal neurons byhypoxia-reoxygenation in rats．5. Hydrogen therapy downregulated the expression of Bcl-2protein and upregulatedexpression of Bax protein.Conclusion:1. Hydrogen-rich saline significantly improved decrements in learning function in a rat modelof CO encephalopathy. This improvement maybe attributed to its antioxidant, anti-apoptotic and anti-inflammatory actions. The experiment provided a new approach for thetreatment of DNS.2. Hydrogen-rich medium prevented the apoptosis of hippocampal neurons by hypoxia-reoxygenation in rats at a cellular level.3. As a novel gas medicine, hydrogen gas has the characteristic of antioxidant,anti-inflammatory and anti-apoptotic effects.