Effects And Mechanisms Of Orexin A On Cognitive Impairment In Mice Induced By Chronic Intermittent Hypoxia

Part One The Effect of Orexin A on the Cognitive Function Induced by Chronic Intermittent Hypoxia in MiceObjective:To investigate the effect and mechanism of Orexin A（OXA）on cognitive function induced by chronic intermittent hypoxia（CIH）in mice.Methods:32 adult C57BL/6 male mice were randomly divided into four groups:normoxia control（NC）+normal saline（NS）,NC+OXA,CIH+NS and CIH+OXA group（8 mice per group）.Mice were exposed to intermittent hypoxia with normal atmospheric pressure 8 hours per day for 4 consecutive weeks to establish a CIH mice model.OXA（3nMol）was injected into the right lateral ventricles of the mice using micro-injection system.The water maze test was used to assess spatial memory abilities of the mice.Immunofluorescence staining was performed to investigate the expression of OXA and c-Fos in the lateral hypothalamus（LH）.The changes of apoptosis and oxidative stress in hippocampus were measured using TUNEL,immunohistochemistry,western blot and biochemical analysis respectively.Results:Behavioral tests of water maze revealed that CIH significantly increased the escape latency and time of arriving platform,which were obviously reversed by OXA treatment.Similarly,OXA treatment significantly increased the times of platform crossing and detention time in the target quadrant,of which were significant lower in CIH+NS group than that in NC+NS group and NC+OXA group.CIH decreased the number of OXA+neuron but increased the percentage of c-Fos+/OXA+neuron in LH.Furthermore,we found that CIH induced obvious apoptosis and oxidative stress in the hippocampus while micro-injection of OXA could attenuate the oxidative stress and apoptosis.Conclusions:CIH could induce cognitive impairment in mice,but OXA might improve cognitive impairment induced by CIH in mice through inhibiting hippocampal apoptosis and oxidative stress.Part Two PKCα/ERK1/2 Signaling Pathway Involved in the Regulation of Orexin A on Cognitive Impairment in Mice induced by Chronic Intermittent HypoxiaObjective:To determine whether PKCα/ERK1/2 signaling pathway was involved in the regulation of OXA on cognitive impairment in CIH mice.Methods:Male C57BL/6 mice（25-30g）were randomly divided into the following groups.The group that received micro-injection of OXA was same as Part one.Mice that received micro-injection of the Orexin A receptor（OX1R）antagonist SB334867（SB）were randomly divided into four groups:NC+DMSO group,NC+SB group,CIH+DMSO group and CIH+SB group（8 mice per group）.The CIH mice model was established successfully same as Part one.SB（30nMol）was injected into the right ventricle of mice using micro-injection system for 5 consecutive days.The mRNA and protein levels of OX1R,OX2R,PKCα,p-ERK1/2 and total ERK1/2 in hippocampus were detected by real-time PCR and western blot respectively.Results:CIH down-regulated the OX1R mRNA and protein levels in mouse hippocampus,but had no significant effect on the OX2R mRNA and protein levels.Micro-injection of OXA attenuated the decrease of OX1R mRNA and protein levels induced by CIH.Similarly,the PKCαmRNA and protein levels,as well as p-ERK1/2protein levels in the hippocampus were decreased in CIH mice compared to NC mice,but there was no significant difference of total ERK1/2 protein levels.Micro-injection of OXA significantly increased the PKCαmRNA and protein levels and upregulated the phosphorylation of ERK1/2 protein in the hippocampus of CIH mice.Micro-injection of SB had no obvious effect on the PKCαmRNA and protein levels and p-ERK1/2 protein levels in the hippocampus of CIH mice.However,SB treatment inhibited the PKCαmRNA and protein levels,as well as p-ERK1/2 protein levels in NC+SB group.Conclusions:CIH inhibited the expression of OX1R but not OX2R in the hippocampus of mice,the downstream PKCα/ERK1/2 signaling pathway of OX1R was involved in the regulation of OXA on cognitive impairment of the mice induced by CIH.Part Three Relationship between Daytime or Nighttime Orexin A Levels and Cognitive Function in Patients with Obstructive Sleep ApneaObjective:This study aimed to evaluate associations between daytime or nighttime OXA level and cognitive function in OSA patients,and the diagnostic value of plasma OXA in cognitive impairment of OSA.Methods:The subjects were suspected OSA patients who visited to Renmin Hospital of Wuhan University.All subjects were required to complete overnight polysomnography（PSG）,Montreal Cognitive Function Assessment（MoCA）,and Epworth Sleepiness Scale（ESS）.According to apnea hyponea index（AHI）,the subjects were divided into control group（AHI<5 events/hour）and OSA group（AHI≥5 events/hour）.The venous blood were collected both in daytime and at night,and the plasma OXA levels were measured by ELISA.Spearman correlation coefficient was used to evaluate the correlations between daytime or nighttime plasma OXA and MoCA score,sleep related parameters and ESS respectively.ROC curves were used to evaluate the diagnostic value of plasma OXA levels in assessing cognitive impairment in OSA patients.Results:A total of 106 subjects finally met the inclusion criteria for this study including 28 in the normal control group and 78 in the OSA group.In this study,MoCA score and plasma OXA concentration in OSA group were significantly lower than those of the normal control group.The daytime plasma OXA in the normal control group was significantly higher than that at nighttime,but the circadian rhythm of OXA was not obvious in the OSA group.In terms of cognition,only patients with moderate to severe OSA showed decreased cognitive scores.Compared with OSA patients without cognitive impairment,OSA patients with cognitive impairment had significantly lower levels of OXA both in the daytime and nighttime.There was a positive correlation between daytime plasma OXA concentration and MoCA score in OSA patients with cognitive impairment.However,there was no significant correlation between nocturnal plasma OXA concentration and MoCA score.In addition,both daytime and nighttime plasma OXA levels in patients with OSA were positively correlated with MoCA scores and nadir SaO₂,but were negatively correlated with AHI,ODI,and TS90%,and there is not any correlations with ESS scores were found.The optimal threshold of daytime plasma OXA to diagnose OSA with cognitive impairment was 49.34 pg/ml,with a sensitivity of 80.0%and a specificity of 74.3%.Conclusions:Plasma OXA level might be related to the cognitive function and severity of OSA.The decreased plasma OXA levels in daytime might have a certain diagnostic value for cognitive impairment in OSA patients.