The Role And Underlying Mechanism Of Microglia Mediated Inflammatory Response On Sleep Deprivation Aggravating Chronic Pain In Rats

Objective:The number of chronic pain patients in China is estimated over 300 millions,and it is increasing at a rate of 10 to 20 millions annually.Patients with chronic pain often co-exist with sleep disorders,while sleep disorders result in hyperalgesia and further aggravate chronic pain.Clinically,patients with such "sleep disorders" and "chronic pain" are difficult to treat,which become the family and social burdens.Therefore,it is of great significance to study the mechanism of sleep disorders aggravating chronic pain.It was found that the central nervous system(CNS)of patients with sleep disorders have obvious neuroinflammation with increased IL-1β and TNF-α levels.In the mean time,CNS inflammation is also underlying the mechanism of central sensitization of chronic pain.Therefore,we hypothesize that sleep disorders may aggravate chronic pain by enhancing the CNS inflammation.Microglia are immune cells in the CNS and are mainly involved in the regulation of inflammatory responses.The latest rat single-cell gene sequencing show that Toll-like receptor 4(TLR4)is highly expressed on the membrane of microglia.TLR4 mediates the activation of NLRP3 inflammasomes to increase the synthesis and secretion of IL-1β,TNF-α and other inflammatory factors involved in CNS inflammation.Ibuprofen is a classic non-steroidal anti-inflammatory drug that can penetrate the blood-brain barrier.Studies have reported that ibuprofen inhibits inflammation through TLR4 receptors and inhibits NLRP3 inflammasomes to exert anti-inflammatory and neuroprotective effects.However,it is unknown whether pro-inflammatory activation of microglia mediated by the TLR4/NLRP3/IL-1β signaling pathway is underlying the mechanism of sleep disorders aggravating chronic pain.Therefore,this study proposes the hypothesis that sleep disorders may enhance the microglia pro-inflammatory activation mediated by the TLR4/NLRP3/IL-1β signaling pathway,which would result in further CNS inflammation to contribute to central sensitization of chronic pain.Ibuprofen may inhibit the TLR4/NLRP3/IL-1β signaling pathway and then improve chronic pain behavior aggravation caused by sleep disorders.To verify the above hypothesis,we plan to establish an animal model combined chronic sleep deprivation(CSD)and chronic pain.We will use methods such as standard animal behavior testing measures to confirm that CSD would aggravate the chronic pain behaviour and use the molecular biology technology to reveal the involvement of TLR4/NLRP3/IL-1β signaling pathway mediates microglia M1 polarization.In addition,we will further explore the effect and the possible target of ibuprofen in the treatment of sleep disorders aggravating chronic pain.Chapter 1 CSD aggravated pain behavior and promoted microglia M1 polarization in chronic pain rats Materials and methods:Adult male Sprague-Dawley(SD)rats weighing about 200 g were used to establish an animal model of chronic pain with modified chronic constriction injury(m CCI).Three days after m CCI surgery,a modified multi-platform method was used for chronic sleep deprivation(CSD).Then,chronic sleep deprivation chronic pain(m CCI + CSD)rat model were established.To verify the success of model establishment,the rats were randomly divided into 4 groups(n = 6): sham operation group(Sham group),m CCI group,Sham + CSD group,m CCI + CSD group.The changes in paw-withdrawal mechanical threshold(PWMT)and paw-withdrawal pain threshold(PWTL)in rats at different time points were observed.And the forced swimming test(FST)immobility time,sucrose preference,body weight and coat score were observed.Molecular biology techniques were applied on the collected samples at certain time points.Protein expression of i NOS,NLRP3,ASC,Caspase-1(p20),IL-1β in the spinal cord(SC),hippocampus(Hip),prefrontal cortex(PFC),periaqueductal gray matter(PAG)were detected by Western blot.The activation of microglia in SC,Hip,PFC,and PAG were detected by IHC.The m RNA expression of microglia related cytokine(M1: CD68,i NOS,TLR4;M2: IL-4,CD206,Arg-1)were detected by RT-q PCR.The co-expression of i NOS+-Iba1+ and CD68+-Iba1+were detected by double-immunofluorescence labeling.Neuronal apoptosis was detected by TUNEL staining.The stages of neuronal apoptosis in the hippocampus was detected by FCM.Results:(1)CSD aggravated the pain behavior in chronic pain rats: compared with the Sham group,the PWMT and PWTL of rats in the m CCI group and Sham + CSD group were significantly lower.Compared with the m CCI group,the PWMT and PWTL of rats in the m CCI + CSD group were significantly lower,and the lowest was at 17 days after surgery.(2)CSD aggravated the depressive behavior in chronic pain rats: compared with the m CCI group,rats in the m CCI + CSD group had a longer forced swimming immobility time,a lower sucrose preference,a lighter body weight,and a lower core score after surgery.(3)CSD increased the inflammatory response and microglia M1 polarization of the CNS in chronic pain rats: Western blot showed that compared with the m CCI group,the protein expression of i NOS,NLRP3,ASC,Caspase-1(p20),IL-1β in the four brain regions of SC,Hip,PFC,and PAG were significantly higher in the m CCI + CSD group,the same is microglia M1 polarization.In m CCI + CSD group,the protein expression of i NOS,NLRP3,ASC,Caspase-1(p20)and IL-1β in the Hip area of rats were significantly higher than that of the SC,PFC,and PAG areas,and the percentage of CD68~+-Iba1~+ was significantly higher than that of the SC,PFC,and PAG areas.Therefore,the hippocampus subregions were selected for further observation.(4)CSD aggravated inflammatory factor protein expression levels,M1 related cytokine m RNA expression levels,and microglia M1 polarization of the hippocampus in chronic pain rats: Western blot and RT-q PCR showed that compared with the m CCI group,the protein expression of i NOS,NLRP3,ASC,Caspase-1(p20),IL-1β in the hippocampus DG,CA1,CA3 and m RNA expression of the M1 related cytokine(CD68,i NOS,TLR4)were significantly higher in the m CCI + CSD group.The higher expression indicated that all subregions of the hippocampus are involved in the important regulatory mechanism of CSD aggravating chronic pain behavior.Immunofluorescence staining showed that compared with the m CCI group,the number of microglia Iba1~+ cells in the Hip area,the percentage of i NOS~+-Iba1~+/Iba1~+ and CD68~+-Iba1~+/Iba1~+ were significantly higher,and the number of cell branches was significantly less in the m CCI + CSD group.(5)CSD promoted neuronal apoptosis of the hippocampus in chronic pain rats:TUNEL staining and FCM showed that compared with the m CCI group,the number of neuronal apoptosis in the hippocampus of rats was significantly more,and the early,late neuronal apoptosis percentage were significantly higher in the m CCI + CSD group.Chapter 2 TLR4/NLRP3/IL-1β signaling pathway mediated microglia M1 polarization and participated in CSD aggravating pain behavior in chronic pain ratsMaterials and Methods:The m CCI + CSD rat modeling were established the same as described previously.In this chapter,TAK-242 and MCC950 were selected to down-regulate the expression of TLR4 and NLRP3 respectively in the m CCI + CSD modeling group for observation.Rats in the m CCI + CSD + TAK-242 group and m CCI +CSD + MCC950 group were intraperitoneally injected with TAK-242(3 mg/kg)and MCC950(1 mg/kg)daily following sleep deprivation for 14 days.The other rats were injected with an equal volume of normal saline at the same time point.The pain and depressive behavior of the rats were observed.Protein expression of i NOS,NLRP3,ASC,Caspase-1(p20),IL-1β and m RNA expression of CD68,i NOS,TLR4 were detected by Western blot and RT-q PCR.The co-expression of microglia and TLR4 was detected by double-immunofluorescence labeling.Neuronal apoptosis was detected by TUNEL staining.The stages of neuronal apoptosis in the hippocampus was detected by FCM.Results:(1)TAK-242 and MCC950 improved the pain behavior in CSD chronic pain rats: compared with the m CCI + CSD group,the PWMT and PWTL of rats in the m CCI + CSD + TAK-242 and m CCI + CSD + MCC950 groups were significantly higher.(2)TAK-242 and MCC950 improved the depressive behavior in CSD chronic pain rats: compared with the m CCI + CSD group,rats in the m CCI + CSD +TAK-242 group and m CCI + CSD + MCC950 group had a shorter forced swimming immobility time,a higher sucrose preference,a heavier weight,and a higher core score after surgery.(3)TAK-242 and MCC950 reduced inflammatory factor protein expression levels,M1 related cytokine m RNA expression levels,and microglia M1 polarization in CSD chronic pain rats: Western blot and RT-q PCR results showed that compared with the m CCI + CSD group,the protein expression levels of i NOS,NLRP3,ASC,Caspase-1(p20),IL-1β,and the m RNA expression levels of TLR4,CD68,i NOS in the m CCI + CSD + TAK-242 group and m CCI + CSD + MCC950 group were significantly lower.Double-immunofluorescence labeling of TLR4~+-Iba1~+ showed that compared with the m CCI + CSD group,the m CCI + CSD + TAK-242 and m CCI + CSD+ MCC950 groups had a lower percentage of co-expression.(4)TAK-242 and MCC950 reduced neuronal apoptosis in CSD chronic pain rats: TUNEL staining and FCM showed that compared with m CCI + CSD group,the number of neuronal apoptosis and the early,late neuronal apoptosis percentages in the hippocampus were significantly lower in m CCI + CSD +TAK-242 group and m CCI + CSD + MCC950 group.Chapter 3: Ibuprofen improved the pain behavior in CSD chronic pain rats by reducing microglia neuroinflammation through inhibiting the TLR4/NLRP3/IL-1β signaling pathwayMaterials and methods:The m CCI +CSD rat modeling was established the same as described previously.In this chapter,rats in the m CCI + CSD + ibuprofen group were intragastric administrated with ibuprofen(30 mg/kg)daily following sleep deprivation for 14 days.The other rats were given an equal volume of normal saline at the same time points.The pain and depressive behavior of the rats were observed.Protein expression of i NOS,NLRP3,ASC,Caspase-1(p20),IL-1β and m RNA expression of CD68,i NOS,TLR4 were detected by Western blot and RT-q PCR.The co-expression changes of microglia and CD68 was detected by double-immunofluorescence labeling.Neuronal apoptosis was detected by TUNEL staining.The stages of neuronal apoptosis in the hippocampus was detected by FCM.Results:(1)Ibuprofen reduced the pain behavior in CSD chronic pain rats: compared with the m CCI + CSD group,rats in the m CCI + CSD + ibuprofen group had a significantly higher PWMT and PWTL after surgery.Compared with the m CCI group,rats in the m CCI + CSD + ibuprofen group had a significantly higher PWMT and PWTL after surgery.(2)Ibuprofen improved the depressive behavior in CSD chronic pain rats:compared with the m CCI + CSD group,the m CCI + CSD + ibuprofen group had a shorter forced swimming immobility time,a higher sucrose preference,a heavier weight,and a higher core score after surgery.(3)Ibuprofen reduced inflammatory factor protein expression levels,M1-related cytokine m RNA expression levels,and microglia M1 polarization in CSD chronic pain rats: Western blot and RT-q PCR showed that compared with the m CCI + CSD group,the protein expression of i NOS,NLRP3,ASC,Caspase-1(p20),IL-1β and the m RNAof TLR4,CD68,i NOS in the m CCI + CSD + ibuprofen group were significantly lower.Double-immunofluorescence labeling of CD68+-Iba1+ showed that compared with the m CCI + CSD group,the percentage of CD68+-Iba1+ in the m CCI +CSD + ibuprofen was significantly lower.(4)Ibuprofen reduced neuronal apoptosis in CSD chronic pain rats: TUNEL staining and FCM showed that compared with m CCI + CSD group,the number of neuronal apoptosis and the early,late neuronal apoptosis percentages in the hippocampus were significantly lower in m CCI + CSD +ibuprofen group.Conclusion:(1)Chronic sleep deprivation aggravates the pain behavior in chronic pain rats through the TLR4/NLRP3/IL-1β signaling pathway,which mediates the microglia M1 polarization,promotes the CNS inflammation,and neuronal apoptosis.(2)TAK-242 and MCC950 down-regulates TLR4/NLRP3/IL-1β signaling pathway,both of which can reduce the microglia M1 polarization and the release of pro-inflammatory factors,and improve the pain behavior in CSD chronic pain rats.(3)Ibuprofen improves the pain behavior in CSD chronic pain rats by reducing microglia neuroinflammation through inhibiting the TLR4/NLRP3/IL-1βsignaling pathway.(4)Microglia M1 polarization is an important mechanism for chronic sleep deprivation aggravating chronic pain in rats.Inhibiting microglia M1 polarization to reduce CNS inflammation may be a key target for treating sleep disorders and chronic pain comorbidity.