Phenotype Change Of Microglia Contributes To Sleep Deprivation-Aggravated Anxiety/Depression Like Behaviors In Rat Model Of Neuropathic Pain

Background and objective:Neuropathic pain is a special type of chronic disease caused by damage or dysfunction of the central / peripheral nervous system,with main clinical symptoms of spontaneous pain,hyperalgesia,allodynia and paresthesia.Because of the pain is severe and long-lasting,patients with neuropathic pain often combined with sleep disorders,anxiety and depression.Neuropathic pain,sleep disorders,anxiety and depression affect each other,which seriously affects the daily life and social functions of patients.This is also one of the reasons for the poor treatment of patients with neuropathic pain,sleep disorders,anxiety/depression comorbidities.Therefore,it is important to investigate the relationship between sleep disorders and anxiety/depression in neuropathic pain,which is of great significance for the treatment of patients with clinical sleep disorders,pain,anxiety /depression.The inflammation of the central nervous system is one of the common mechanisms for the occurrence and development of central sensitization in chronic pain as well as sleep disorders,anxiety /depression.Animal studies have showen that acute sleep deprivation can activate microglia and increase IL-1β in hippocampus,causing short-term memory impairment.Imaging studies and autopsy analysis indicated that microglial activation may contribute to depression.In animal studies on sleep deprivation and neuropathic pain,it is found that P2X7 R activates NLRP3 inflammasome and promotes the secretion of proinflammatory cytokine IL-1β and induces the amplification of inflammatory response cascade.IL-1β is closely associated with mood and behavior disorders.M1/M2 phenotype changes of microglia play a key role in the outcome of Perkins’ disease and Alzheimer’s disease.Therefore,this study puts forward a scientific hypothesis: Acute sleep deprivation mediates microglial M1/M2 phenotype changes through the P2X7R-NLRP3inflammasome-IL-1β pathway to aggravate pain,anxiety and depression-like behaviors in rats with neuropathic pain.This study intends to establish a rat model of acute sleep deprivation and chronic constriction injury to explore the effect and mechanism of acute sleep deprivation on pain,anxiety and depression-like behaviors in CCI rats.To study the role of P2X7R-NLRP3 inflammasome-IL-1β signaling pathway in the phenotypic changes of microglia in the hippocampus and frontal cortex of CCI rats with acute sleep deprivation.venlafaxine and melatonin on anxiety-depressive behaviors in CCI rats with acute sleep deprivation,and its effect on P2X7R-NLRP3inflammasome-IL-1β pathway,microglia M1 phenotype changes and oxidative stress difference.To provide basic research basis and medication reference for the clinical treatment of patients comorbidities with neuropathic pain,sleep dysfunction,and anxiety and depression.Chapter 1: The influence of acute sleep deprivation on the pain,anxiety/depression-like behaviors of neuropathic pain rats and its relationship with the changes of microglia M1/M2 phenotype.Materials and Methods:Sprague Dawley rats were randomly divided into 4 groups(n = 24): Sham operation group(Sham);Sham operation + sleep deprivation group(Sham + SD);Chronic constriction injury group(CCI);Chronic constriction injury + sleep deprivation group(CCI + SD).CCI model was established.3 days after operation,rats were subjected to sleep deprivation with the method of water platform for 72 hours.Evaluate the pain,anxiety/depression-like behavior changes.Immunofluorescence staining is used to evaluate the effect of acute sleep deprivation on microglia activation,astrocyte activation and neuroapoptosis in the hippocampus and frontal cortex of SD-treated CCI rats.RT-PCR method was used to detect the mRNA expression changes of M1 phenotype pro-inflammatory cytokine,M2 phenotype anti-inflammatory cytokine and P2X7 R in the hippocampus and frontal cortex.Immunofluorescence staining is used to determine the cellular localization of P2X7 R in the central nervous system.Results:(1)SD-treated CCI rats’ right hindlimb mechanical and thermal hypersensitivity were significantly reduced.In the elevated plus maze test,forced swimming test,open field test,sucrose preference test,coat color evaluation and weight test,anxiety/depression like behaviors were significantly worsened.(2)The activation of microglia in the frontal cortex and hippocampus of SD-treated CCI rats increased.There were no significant increase of astrocyte activation and neuronal apoptosis in the hippocampus and frontal cortex at 7th day,astrocyte activation and neuronal apoptosis increased at 21 th day.(3)The expression of M1 type pro-inflammatory cytokines(IL-1β,TNF-α,CD68,i NOS)mRNA and the protein content of IL-1β and TNF-α in the hippocampus and frontal cortex of SD-treated CCI rats increased,M2 type anti-inflammatory cytokine(IL-4,Arg1,CD206,TGF-β)mRNA expression and IL-4,TGF-β protein content decreased;The M1/M2 phenotype changes of microglia in the frontal cortex and hippocampus are positively correlated with anxiety/depression like behaviors.(4)P2X7R mRNA expression and protein content in hippocampus and frontal cortex of SD-treated CCI rats increased significantly.P2X7 R is mainly located in microglia in the central nervous system;the increase of M1 phenotype of microglia is related to the increase of P2X7 R expression.Chapter 2: The molecular mechanism of the P2X7R-NLRP3inflammasome-IL-1β pathway involved in the regulation of the M1/M2 phenotype of microglia Materials and Methods:SD rats were used to prepare SD-treated CCI rat model.During sleep deprivation,P2X7 R blocker A438079 and NLRP3 blocker CY-09 were injected intraperitoneally for bi-level block intervention.RT-PCR and Western blot were used to detect P2X7 R,NLRP3,ASC,Caspase-1 and IL-1βmRNA and protein expression changes in the rat hippocampus and frontal cortex.Immunofluorescence staining was used to analyze the M1/M2 phenotype and cell morphology of microglia.ELISA was used to detect the changes of M1 pro-inflammatory cytokines(IL-1β,TNF-α)and M2 anti-inflammatory cytokines(IL-4,TGF-β)in microglia.Western blot was used to detect the changes in the expression of Bcl-2 and Bax proteins in the hippocampus and frontal cortex,Neu N immunofluorescence staining and TUNEL staining were used to observe nerve apoptosis changes in the hippocampus and frontal cortex of rats.Results:(1)The P2X7R-NLRP3 inflammasome-IL-1β pathway was involved in the regulation of pain,anxiety/depression like behaviors in SD-treated CCI rats.A438079 and CY-09 can significantly improved the pain and anxiety/depression like behaviors in acute sleep deprivation CCI rats.(2)The expression of P2X7 R,NLRP3,ASC,Caspase-1 and IL-1β mRNA and protein in the hippocampus and frontal cortex of rats of CCI+SD group increased significantly;A438079 and CY-09 reduced P2X7 R,NLRP3,ASC,and Caspase-1And IL-1β mRNA and protein expression.(3)A438079 and CY-09 reduced the expression of IL-1β,TNF-α and increased the expression of IL-4,TGF-β in the hippocampus and frontal cortex of SD-treated CCI rats.A438079 and CY-09 increased the number and percentage of microglia CD206+ /Iba-1 +,decreased the number and percentage of microglia CD206 + /Iba-1 +in the hippocampus and frontal cortex of SD-treated CCI rats,inhibited M1 phenotypic changes and promote M2 phenotype changes.(4)A438079 and CY-09 reduced the expression of Bax protein and increased the expression of Bcl-2 protein in the hippocampus and frontal cortex of SD-treated CCI rats,reduced the neuronal apoptosis in the hippocampus and frontal cortex of SD-treated CCI rats.Chapter 3: Effects of venlafaxine and melatonin on anxiety/depression-like behaviors in CCI rats with acute sleep deprivation and its mechanism Materials and Methods:SD rats were used to prepare SD-treated CCI rat model,during sleep deprivation,venlafaxine 30 mg/kg or melatonin 150 mg/kg was given for intervention.Behavioral tests were used to evaluate the effects of venlafaxine and melatonin on the pain and anxiety/depression like behaviors of SD-treated CCI rats.RT-PCR,Western blot and ELISA were used to detect P2X7 R,NLRP3,ASC,Caspase-1,IL-1β and TNF-α mRNA and protein expression changes in the hippocampus and frontal cortex of SD-treated CCI rats,and compared the effects of venlafaxine and melatonin on the above cytokines.Immunofluorescence staining was used to observe the morphological changes of microglia,and analyzed the correlation between the phenotypic changes of microglia M1 and the expression of IL-1β and the effects of venlafaxine and melatonin on it.MDA and SOD detection kits were used to detect the contents of MDA and SOD in the hippocampus and frontal cortex of SD-treated CCI rats and the effects of venlafaxine and melatonin on it.Results:(1)Venlafaxine and melatonin improved the pain,anxiety/depression like behaviors in CCI rats with acute sleep deprivation,and the effect of venlafaxine is better than melatonin.(2)Venlafaxine and melatonin significantly reduced the mRNA and protein expression of P2X7 R,NLRP3,ASC,Caspase-1,TNF-α and IL-1β in the frontal cortex and hippocampus of SD-treated CCI rats.Venlafaxine was better than melatonin.(3)Venlafaxine and melatonin significantly inhibited the activation of microglia and the expression of IL-1β in the hippocampus and frontal cortex of SD-treated CCI rats.The inhibitory effect of venlafaxine on the activation of microglia and the release of IL-1β was significantly stronger than that of melatonin.The proportion of Iba-1 and IL-1β-positive cells was positively correlated.(4)In acute sleep deprivation CCI rats,MDA increased in the frontal cortex and hippocampus,while SOD decreased.Venlafaxine had a significantly better inhibitory effect on oxidative stress after neuroinflammation in SD-treated CCI rats than melatonin.Conclusion:Acute sleep deprivation modulates the M1/M2 phenotype changes of microglia through the P2X7R-NLRP3 inflammasome-IL-1β pathway and aggravates pain,anxiety/depression like behaviors in CCI rats with neuropathic pain.Venlafaxine and melatonin reducing microglia neuroinflammation and oxidative stress improve the pain,anxiety and depression-like behaviors of CCI rats with acute sleep deprivation,and the effect of venlafaxine is better than that of melatonin.