Studies On Clinical Characteristics And Mechanisms Of Neuroprotection Induced By Hyperbaric Oxygen In Demyelination After Carbon Monoxide Poisoning

Carbon monoxide(CO)poisoning is one of the most common poisonings in daily life,which can result in certain complications,such as brain,heart,lung,and et al.The brain injuries after CO poisoning,in other words,neurologic and neuropsychological sequelae carry high incidence and fatal outcomes.Some researchers divided them into persistent neuropsychological sequelae and delayed neuropsychological sequelae.Many studies indicated that demyelination in white matter is the major pathological change.CO produces hypoxia by binding with hemoglobin,reducing the oxygen-carrying capacity of the blood and producing hypoxia in the tissues,which has been considered to be the major mechanism of CO toxicity.So far,CO exposure duration,age,hyperbaric oxygen therapy and genetic factors are possible factors that affect the prognosis of CO poisoning.There are some limitations in available studies,for example,small sample size,unknown proportion of suicidal patients,no severity grading,patients with loss of consciousness excluded,and so on.Oxygen administration,including hyperbaric oxygen is important method to treat CO poisoning,because it can accelerate the dissolution of carboxyhemoglobin(COHb)and ameliorate anoxia.As for hyperbaric oxygen,there are several arguments about its indications,therapy schedule,and termination standards.Some doctors think that in the emergency department,oxygen administration is needed until COHb is normal(<3%).However,“National guideline on clinical management of CO poisoning(Chinese Medical Association,2012,published in Chinese)” recommended for consecutive sessions of hyperbaric oxygen in one month.We demonstrated previously that hyperbaric oxygen therapy can ameliorate demyelination in white matter and activity of daily living of patients as well.In animal experiments of cerebral ischemia/reperfusion injury,repetitive hyperbaric oxygen therapies may accelerate remyelination and improve sensorimotor function.In adult mammals,Notch signaling pathway has important effects on both the regulation of neural stem cells and the differentiation of gliocytes.It is indicated that the activation of Notch signaling pathway is related to cerebral ischemia/reperfusion injury.It is unknown whether Notch signaling pathway could be activated by CO poisoning and the neuroprotection of hyperbaric oxygen is associated with Notch signaling pathway.The study was composed of clinical retrospective study,animal experiments and cell culture in vitro.1.Clinical retrospective studyThe couples,diagnosed with CO poisoning simultaneously,were included in our study.Their epidemiological characteristics,neuroimaging characteristics,and the factors affecting the prognosis were investigated.We demonstrated that demyelination in white matter is of great importance in brain injuries after CO poisoning.2.Animal experiments and cell culture in vitroIn experimental CO poisoning,we studied the characteristics of demyelination in white matter and the neuroprotection induced by hyperbaric oxygen.The changes of Notch signaling pathway were detected in order to verify the crosstalk between hyperbaric oxygen and Notch signaling pathway in brain injuries after CO poisoning.Through oligodendrocyte progenitor cells(OPCs)culture in vitro,the effect of CO poisoning on quantities,viability,and migration of OPCs were observed.We aimed to reveal the cell mechanisms of demyelination in white matter induced by CO poisoning.Part I Analysis of the factors affecting the prognosis of patients with CO poisoningObjectivesTo analyze the factors affecting the prognosis of patients with CO poisoning.Methods1.We conducted the retrospective cohort study of all patients presenting to Department of Hyperbaric Oxygen from January 2001 to March 2015.The couples were diagnosed with CO poisoning and included in our study.At least one of the members of the couple either presented with severe poisoning criteria or died following the CO poisoning.2.The couples were divided into two groups according to premenopausal or postmenopausal females.The prognosis after CO poisoning was compared between sex in total,premenopausal,and postmenopausal groups,including Glasgow Outcome Scale(GOS)scores,improvement rate and mortality rate.Multinomial ordinal logistic regression was conducted to determine the effects of sex and age on prognosis.Results1.A total of 66 couples diagnosed as CO poisoning were enrolled in this study.Under identical CO exposure the wives had higher GOS scores(P=0.012)and improvement rate(P=0.013).In premenopausal group,the wives’ GOS scores(P=0.023)and improvement rate(P=0.035)were better than their husbands,but in postmenopausal group,there was no obvious difference.About mortality rate,there was no statistical significance in present study.2.Sex was related to the severity classification and the females had milder classifications in 24 hours(odds ratio [OR]=2.968,P=0.010).3.Sex and age were related to the GOS scores of 1 month after CO poisoning.The females(OR=0.485,P=0.034)or patients younger than 40 years(OR=5.760,P<0.001)had higher GOS scores.4.As the patients diagnosed as mild or moderate poisoning were excluded,age was still related to the GOS scores of 1 month(OR=5.714;P=0.001),but sex was not related to the GOS scores(P=0.158).Conclusions1.Age and sex are possible factors that affect the prognosis of CO poisoning.2.Females show an advantage over their male spouses by severity of poisoning and prognosis after CO poisoning,especially in premenopausal stage.However,the females’ advantage is lost in patients with severe poisoning.Part II Study on the epidemiological and neuroimaging characteristics of patients with neurologic and neuropsychological sequelae after CO poisoningObjectives1.To analyze the epidemiological and neuroimaging characteristics of patients with neurologic and neuropsychological sequelae after CO poisoning.2.To discuss the crosstalk between damage in the globus pallidus or white matter and the prognosis of patients with neurologic and neuropsychological sequelae after CO poisoning.Methods1.We conducted the retrospective cohort study of all patients presenting to Department of Hyperbaric Oxygen from January 2001 to March 2015.The couples were diagnosed with CO poisoning and included in our study.2.The couples diagnosed as CO poisoning were enrolled in this study.The couples were divided into two groups according to premenopausal or postmenopausal females.The incidence rates of neurologic and neuropsychological sequelae after CO poisoning were compared between sex in total,premenopausal,and postmenopausal groups.The effect of sex on prognosis of patients with neurologic and neuropsychological sequelae was analyzed.The neuroimaging characteristics were compared and the crosstalk between damage in the globus pallidus or white matter and the prognosis were studied.Results1.According to the including patients with different severity classifications in acute phase,the incidence rates of neurologic and neuropsychological sequelae after CO poisoning were from 39.88% to 64.95%.About incidence rates in different groups,there was no statistical significance between females and males.2.Sex was not related to the GOS scores of 1 month after CO poisoning.3.The neuroimaging characteristics included damage in the white matter alone,basal ganglia alone or both of them.The incidence rate of damage in the basal ganglia was much higher in patients with persistent neuropsychological sequelae than that of in patients with delayed neuropsychological sequelae(P<0.01).The incidence rate of damage in the white matter(86.44%)was very high in patients with neurologic and neuropsychological sequelae,which was two times higher than that of damage in the basal ganglia.The damage regions were not related to the GOS scores of 1 month after CO poisoning.Conclusions1.The incidence rate of neurologic and neuropsychological sequelae after CO poisoning is very high.There are significant differences according to different severity classifications in acute phase.2.Demyelination in white matter is the major neuroimaging change.Part III Effect of CO poisoning on demyelination in white matter and the neuroprotection induced by hyperbaric oxygen in miceObjectivesTo observe the characteristics of demyelination in white matter and the neuroprotection induced by hyperbaric oxygen in experimental CO poisoning.Methods1.The male C57 BL/6 mice were randomly divided into three groups(n=6).(1)Control group(sham group): The mice inhaled air only.(2)CO poisoning group: The mice inhaled CO gas according to CO exposure protocol only.(3)Hyperbaric oxygen group: After inhaling CO gas,the mice received three sessions of hyperbaric oxygen therapy once a day.2.Right after CO exposure,the concentration of COHb was detected to judge the severity.The body weight was monitored everyday for one week.Morris Water maze testing was performed to test cognitive function.At the nineth day after CO exposure,the mice were sacrificed to detect demyelination in the corpus callosum,the white matter beside the lateral ventricle,and the hippocampus.Immunofluorescence staining with FluoroMyelinTM Green and double-immunostaining with both Anti-MBP antibody and Anti-Neurofilament antibody were performed.The ultrastructural changes of myelin sheath after CO poisoning in the mouse corpus callosum were observed by the electron microscope.Results1.At the seventh day after CO poisoning,the increasing range of body weight was significantly reduced in the CO poisoning group,compared with that in the control group(P<0.05).In contrast,the increasing range of body weight was significantly improved after hyperbaric oxygen therapy(P<0.05).2.In the CO poisoning group,the escape latency was significantly longer,compared with that in the control group(P<0.05).In contrast,the escape latency was significantly shorter after hyperbaric oxygen therapy(P<0.05).In the CO poisoning group,the number of passing over times was significantly decreased,compared with that in the control group(P<0.05).In contrast,the number of passing over times was significantly improved after hyperbaric oxygen therapy(P<0.05).3.Demyelination after CO poisoning in the mouse corpus callosum was observed by staining with FluoroMyelinTM Green.In the CO poisoning group,the fluorescence intensity and the density of the corpus callosum were decreased,compared with that in the control group(P<0.05).In contrast,the fluorescence intensity and the density of the corpus callosum were ameliorated after hyperbaric oxygen therapy(P<0.05).4.Double-immunostaining was performed with immunofluorescence.In the CO poisoning group,the fluorescence intensity and the density of myelin sheath in the corpus callosum and the white matter beside the lateral ventricle were decreased,compared with those in the control group.In contrast,the fluorescence intensity and the density were ameliorated after hyperbaric oxygen therapy.However,there was no significant difference in the fluorescence intensity and the density of myelin sheath in the hippocampus among the three groups.There was no significant difference in the fluorescence intensity of the axons among the three groups.5.In the CO poisoning group,the number of myelinated nerve fiber was obviously reduced and the structure of myelin damaged,compared with those in the control group.In the hyperbaric oxygen group,the aboving changes were significantly inhibited.Conclusions1.CO poisoning results in reduced body weight,decrements in learning and memory function,and demyelination.In the corpus callosum and the white matter beside the lateral ventricle,demyelination is obvious without the axons damage.However,there is no significant demyelination in the hippocampus.In the corpus callosum,the number of myelinated nerve fiber was obviously reduced and the structure of myelin damaged.2.Hyperbaric oxygen possesses neuroprotection against brain injuries after CO poisoning.Part IV Study on the mechanisms of the neuroprotection induced by hyperbaric oxygen in CO poisoningObjectives1.To detect the changes of Notch signaling pathway and verify the crosstalk between the neuroprotection induced by hyperbaric oxygen and Notch signaling pathway in brain injuries after CO poisoning.2.To observe the effect of CO poisoning on quantities,viability,and migration of OPCs.Methods1.The male C57 BL/6 mice were randomly divided into three groups according to Part III.Western blotting was performed to detect the difference of proteins level of Notch1 and Hes5.RT-PCR was performed to detect the difference of mRNA level of Notch1 and Hes5.2.The purified OPCs were cultured and divided into two groups.(1)Control group: The OPCs were cultured in 5% CO2 and 95% air.(2)CO poisoning group: The OPCs were cultured in 5% CO2,1%CO,and 94% air.3.The MTT assay was performed to detect the difference of the viability of OPCs between the two groups.The migration of OPCs was observed by Transwell method.Results1.In the CO poisoning group,both the protein level and genes expression of Notch1 and Hes5 were increased,compared with those in the control group(P<0.05).Hyperbaric oxygen therapy significantly suppressed them(P<0.05).2.In the CO poisoning group,the optical density and the count of migrated cells were decreased,compared with those in the control group(P<0.05).Conclusions1.Notch signaling pathway can be activated by CO poisoning,while hyperbaric oxygen can suppress it,which is probably the mechanism of the neuroprotection induced by hyperbaric oxygen in CO poisoning.2.CO poisoning suppresses the viability and migration of OPCs,which is probably the cell mechanism of demyelination in white matter induced by CO poisoning.