Study On The Mechanism Of Fractalkine/CX3CR1 In Both Peripheral Hyperalgesia And Central Sensitization In Endometriotic Pain

BackgroundEndometriosis(EMs)is a common chronic inflammatory gynecologic disorder that is characterized by the growth of endometrial epithelial and stromal cells outside the uterus.It affects 6%-10%of women of reproductive age.The common symptoms of EMs are pain and subfertility.More than 80%of EMs patients suffer from chronic pain.Pain symptoms associated with endometriosis include dysmenorrhea,dyspareunia,dysuria,abdominal pain and non-menstruation-related chronic pelvic pain.As one of the most common and serious symptoms of EMs,chronic pain profoundly affects women’s quality of life,social relationship and mental health.Recently,increasing evidence of animal model experiments and clinical data suggested that ectopic endometrium growth could induce inflammatory microenvironment and increase neuroangiogenesis/sensory nerve fibers and so on,which probably provide various potential theoretical basis for explaining the multifarious pain problems.However,the pathogenesis of pain associated with EMs is still unclear.The pathogenic factors of endometriotic pain are known to be multivariate and complex,so its occurrence and development is due to the multiple factors interaction rather than the single effect.In-depth study on the interaction of inflammation,peripheral nerve fibers and central nervous system will contribute to the fully recognizing and understanding the potential mechanism of endometriotic pain.Recent research data indicates that endometriotic pain is a type of inflammatory and neuropathic pain.During ectopic endometrial growth,large numbers of inflammatory cells like macrophages,mastocytes,and neutrophils are recruited to secrete a variety of pro-inflammatory/inflammatory cytokines and contributed to the inflammation microenvironment.Macrophages are the major source of pro-inflammatory cytokines/chemokines,which play a role in the development of EMs.The majorities of these molecules have been demonstrated to be associated with the development of pain.Endometriotic lesions also exhibit increased neuroangiogenesis,which could be attributed to localized inflammation,and promote the interaction between inflammatory microenvironment and nerve fibers.While,inflammatory molecules and neuropeptides might directly activate or sensitize sprouting sensory fibers in the tissues or excite nociceptive neurons,which resulted in pain and/or mechanical hypersensitivity/allodynia.Prolonged exposure of sensory neurons to inflammatory mediators would then lead to central sensitization and cause estrogen-independent pain,as observed in some patients with endometriosis and persistent pelvic pain.The level of pain may not be associated with severity,implant position or surrounding environmental conditions of EMs.However,the concrete mechanism of cytokines/chemokines directly function on nerve fibers in endometriotic pain is rarely reported.Spinal microglia were thought to be the indicators of central sensitization in neuropathic pain.They were the first to become activated by peripheral nociceptive signals,and could remain active for several weeks.Cytokines——including fractalkine(FKN)——were thought to be associated with microglia activation.FKN is a member of the chemokine family that consists principally of secreted proinflammatory molecules,and acts by binding to a unique receptor,CX3CR1,expressed on corresponding cells.The chemokines widely expressed in the periphery and central nervous system(CNS)have two forms:a membrane-bound protein and soluble forms(sFKN),each of which mediates different biologic functions.In the periphery,predominant FKN-expressing cells are endothelial and epithelial cells.Membrane-bound FKN serves as an adhesion molecule to promote adhesion of inflammatory cells,while sFKN is a potent chemoattractant for the recruitment of inflammatory cells during chronic inflammation.In the CNS,FKN-expressing cells are restricted to the intrinsic neurons and sensory afferents,whereas the FKN receptor(CX3CR1)is expressed exclusively in the spinal microglia.Although increased levels of FKN were found in the peritoneal fluid of women with endometriosis,the mechanism of FKN/CX3CR1 signaling pathway on endometriotic pain remains unclear.Many clinical cases of endometriosis in the abdominopelvic cavity were reported to directly affect nerves(including the pudendal,obturator,femoral,and sciatic)or spinal roots.Due to the limitations of general endometriosis animal models and sampling nerve system from clinical EMs patients,it is extremely difficult and challenging to study the interaction mechanism between EMs inflammation and the nervous system.,Herein we planned to explore the functional roles of FKN/CX3CR1 signaling pathway in contributing to endometriotic pain using a novel rat model of sciatic endometriosis that we originally described in a previous publication.PART I:Establishment of sciatic endometriosis rat modelObjective:1.To discuss the method and technical point of the sciatic EMs rat model,and provide a suitable model basis for the theoretical study on the interaction between inflammation and nervous system.2.To evaluate pain behavior changes and discuss the feasibility of the sciatic EMs rat model on researching the mechanism of endometriotic pain.3.According to pain behavior curve,we aimed to explore the optimal time point for the steady pain state and molecular biological changes in the sciatic EMs rat mode.Materials&Methods:Female,24,Sprague Dawley(SD)rats,Specific Pathogen Free(SPF),weighing 200-280g were used to establish the sciatic EMs rat model.Before modeling,pain behavior baseline of all the rats were detected by the electronic Von Frey test,tactile allodynia test and acetone test.Rats were randomly divided into 3 groups:endometrial graft group(Endo,n=8),fat graft group(Fat,n=8)and normal control group(Naive,n=8).In the endometrial graft group,the autologous endometrium was transplanted to surround the sciatic nerve in the middle and upper femoral——sciatic EMs rat model;in the fat graft group,the adipose tissue around the uterus was used to wrapping the sciatic nerve in the middle and upper femoral;the normal control group were without any treatment.On day 1,3,5,7,14,21,28(post-operative day,POD)after transplantation,the electronic Von Frey test,tactile allodynia test and acetone test were used to monitor the pain behavior changes of the ipsilateral and contralateral hind paw,respectively.The pain behavior curve was used to estimate the earliest and optimal time point for the successful establishment of painful state,which was used for the verification of subsequent molecular biology.Based on the optimal time point determined by pain behavior curve-POD21,48,SPF,SD female rats were selected and randomly allocated to the above 3 groups,16 rats/group,which were used to observe the general and pathological characteristics of the sciatic EMs rat model.Before collecting,all the rats were performed pain behavior test.Then,each group of rats was randomly divided into 2 groups,8 rats/group,which were prepared for Western blot and paraffin embedding,respectively.On POD21,the general features of graft tissues in vivo were evaluated with anatomic microscope.The histopathological characteristics of graft tissues were demonstrated using hematoxylin-eosin staining(H&E staining)to estimate whether the sciatic EMs rat model was successfully established on POD21.The remaining fresh tissues and paraffin blocks were used for the PART Ⅱ and PART Ⅲ.Results:1.Pain behavior testThe baseline threshold of rats in each group were kept identical.Electronic Von Frey test showed that rats with uterine graft developed significantly increased ipsilateral mechanical hypersensitivity since POD3,and kept continuously increasing compared to the contralateral hind paw and the control groups until the end of the experiment(P<0.05).Tactile allodynia test showed that rats in endometrial graft group developed significantly increased ipsilateral light touch-induced tactile allodynia from POD5 to POD28 compared to the contralateral hind paw and the control groups(P<0.01).Acetone test showed that,since POD7,rats with uterine graft developed significantly increased ipsilateral cold allodynia,and kept continuously increasing compared to the contralateral hind paw and the control groups until the end of the test(P<0.001).However,all the data showed that there was no statistically significant difference between the fat graft group and the normal control group(P>0.05).Also,there were no pain behavior changes in the contralateral hind paw of each group(P>0.05).The pain behavior curve showed that pain reached its peak on POD21,suggesting that POD21 was the optimal time for the steady painful state in the sciatic EMs rat model.2.The general and histopathological featuresGeneral observation results showed that ectopic endometrial tissue formed a single cyst wrapping around the sciatic nerve in the endometrium graft group on POD21,about 6±1.18mm in diameter.The fat graft group had no morphological changes.H&E staining of the sciatic nerve cross-sections showed that uterine grafts exhibited typical glandular formations with columnar epithelial cells and hemosiderin-laden macrophages,accompanied by large numbers of inflammatory cells around the adjacent endometrial stroma and sciatic nerve,which were consistent with the pathological characteristics of human EMs.No inflammatory cells infiltration was observed in either the two control groups or contralateral hind paw.Conclusions:1.The sciatic EMs could induce mechanical hyperalgesia and allodynia and have almost the same typical general and pathological characteristics as human EMs.To some extent,it could simulate clinical diseases.2.The sciatic EMs could recruit large numbers of inflammatory cells to infiltrate into the nerve fiber bundles,which provides the foundation for studying the interaction between inflammation and nerve fibers.PART Ⅱ:Study on the mechanism of Fractalkine/CX3CR1 in peripheral hyperalgesia of endometriotic painObjective:1.To detect the expression of FKN/CX3CR1 and macrophages in ectopic endometrium,normal endometrium and sciatic nerve.2.To analyze the correlation between FKN/CX3CR1 expression in sciatic nerve and EMs pain severity.3.To explore the potential mechanism of FKN/CX3CR1 signaling pathway in the interaction between inflammation and nerve fibers of endometriotic pain.Materials&Methods:The remaining protein tissues and wax blocks from PART I were used for the mechanism study of PART II.Immunohistochemical staining was used to detect the localization and expression of FKN/CX3CR1 and Iba1(a marker for activated microglia/macrophage)in the ectopic endometrium,normal endometrium,and sciatic nerve.The correlation coefficients of FKN/CX3CR1 expression in sciatic nerve and EMs pain severity were analyzed by nonparametric rank sum test.Western blot was performed to assess the quantitative expression of FKN/CX3CR1 protein and distinguish membrane-bound and soluble forms FKN.The co-localization and expression of FKN and Ibal,CX3CR1 and PGP9.5(a marker for nerve fibers),CX3CR1 and MBP(a marker for myelin sheath)were detected by immunofluorescence.The co-localization and expression of FKN,CX3CR1,MBP,Ibal and DAPI were tested by multiplex immunohistochemistry.Results:1.Expression of FKN/CX3CR1 and macrophagesImmunohistochemical staining showed that FKN/CX3CR1 expression and macrophages was significantly increased in the ectopic endometrium compared with normal endometrium(P<0.01).FKN(P<0.001),CX3CR1(P<0.01)and Ibal expression(macrophages,P<0.001)in the sciatic nerve of the endometrial graft group were significantly higher than the control groups.Western blot showed that the protein of FKN/CX3CR1 and Ibal in the graft tissues was significantly increased in the endometrial graft group compared to the control groups(all P<0.01).The level of membrane-bound FKN was higher than soluble forms in the endometrial graft group.2.Correlation between FKN/CX3CR1 expression and pain severityNon-parametric rank sum test showed that FKN/CX3CR1 expression in the sciatic nerve was negatively correlated with pain threshold,so it was positively associated with pain severity.3.The potential mechanism of FKN/CX3CR1 signaling pathway between inflammation and nerve fibers in EMsImmunofluorescence staining showed that in the sciatic nerve of the endometrial graft group,FKN-positive activated macrophages and CX3CR1-positive myelinated nerve fibers was significantly increased compared to the two control groups(P<0.001).Multiplex immunohistochemistry suggested that the expression of FKN-positive activated macrophages and CX3CR1-positive myelinated nerve fibers in the sciatic nerve were overlapped,and the co-localization of FKN,Iba1,CX3CR1,MBP and DAPI were significantly higher than those in the fat graft group(P<0.05)and the normal control group(P<0.01).Conclusions:1.EMs increases the FKN/CX3CR1 expression in the nerve fibers,accompanied by the bulk of macrophages infiltration and their expressions are positively correlated with pain severity,indicating that FKN/CX3CR1 signaling pathway might be involved in the interaction between inflammation and nerve fibers in the development of endometriotic pain.2.In the nerve fibers of ectopic lesions,FKN expressed on activated macrophages bind to CX3CR1 expressed on Schwann cells to trigger the macrophage-nerve crosstalk,which contribute to peripheral hyperalgesia in EMs.PART III:Study on the mechanism of Fractalkine/CX3CR1 in the endometriotic pain conduction and central sensitizationObjective:1.To investigate the potential role of dorsal root ganglion(DRG)FKN/CX3CR1 in the endometriotic pain conduction.2.To investigate the mechanism of spinal FKN/CX3CR1 in the development of central sensitization in EMs.Materials&Methods:The L4-L6 spinal cord wax mass and fresh tissue collected from the PART I was used to detect whether spinal FKN/CX3CR1/P38-MAPK was associated with endometriotic pain by immunohistochemical staining and Western blot.Female,132,SD rats,SPF,weighing about 200-280g,were performed intrathecal catheter implantation.After 1 week,rats with neuromuscular disorders and emaciation were eliminated,then the remaining 120 rats were randomly divided into 5 groups:normal control group,endometrial graft + intrathecal injection of IgG group,fat graft+ intrathecal injection of IgG group,endometrial graft + intrathecal injection of Anti-FKN group,fat graft + intrathecal injection of Anti-FKN group,24/group.Each group were randomly assigned for pain behavior tests(n=8),Western blot(n=8)and paraffin embedding(n=8).FKN-neutralizing antibody and IgG were injected on POD 3,7,14,21.The long-term anti-nociceptive effect of FKN-neutralizing antibody was assessed on POD 1,3,5,7,14,21;and the acute effect of FKN-neutralizing antibody was assessed by electronic von Frey analysis at 0 h,0.5 h,1h,and 3 h after injection on POD 3,7,14,21.Electronic von Frey was tested for all the rats on POD21 sampling.The expression and localization of FKN/CX3CR1 in L4-L6 DRG were detected by immunohistochemical staining and mRNA in situ hybridization.Western blot was used to detect the protein levels of FKN/CX3CR1,P38-MAPK and pP38-MAPK in L4-L6 spinal cord.The co-localization and expression of FKN and NeuN(a neuronal marker),CX3CR1 and Ibal,pP38-MAPK and Ibal in the dorsal horn of L4-L6 spinal cord were assessed by immunofluorescence.Results:1.Spinal FKN/CX3CR1/P38-MAPK/microglia was related to endometriotic pain Immunohistochemical and immunofluorescent results showed that FKN/CX3CR1 expression and the total number of microglia(OX-42,a marker for both rest and activated microglia)in the L4-L6 dorsal horn were significantly increased in the endometrial graft group compared with the fat graft group and the normal control group,but there was no statistical significance between the two control groups(P>0.05).Western blot results showed that P38-MAPK protein phosphorylation level of L4-L6 spinal cord in the endometrial graft group was significantly higher than the fat graft group(P<0.05)and the normal control group(P<0.01),but there was no statistical significance between the two control groups(P>0.05).2.Pain behavior testThe long-term pain behavior test showed that the intrathecal administration of FKN-neutralizing antibody markedly reduced mechanical sensitivity and allodynia between POD7 and POD21.Electronic von Frey showed that rats in the endometriosis plus FKN antibody group,the paw withdrawal threshold(PWT)to mechanical stimulation began to return toward baseline on days 3,7,14,and 21 after injection.On POD 7,14,21,the PWT of Oh injection was no significant difference with pain behavior baseline.Single injection of FKN neutralizing antibody might bring a long-term effect on pain relief.3.Expression of FKN/CX3CR1 in DRGImmunohistochemical staining showed that FKN expression was primarily expressed in neuronal cells while intrathecal administration of FKN-neutralizing antibody decreased FKN expression in the endometrial graft + intrathecal injection of Anti-FKN group.Soluble FKN was mainly observed in the nerve fibers around DRG neurons in the endometrial graft + intrathecal injection of IgG group rather than other groups.In situ hybridization staining showed that CX3CR1 mRNA was overexpressed in the peri-neuronal cells in the endometrial graft + intrathecal injection of IgG group while inhibition FKN also decreased CX3CR1 mRNA expression in DRG.4.Spinal FKN/CX3CR1 in the mechanism of central sensitizationWestern blot showed that FKN/CX3CR1 and pP38-MAPK in L4-L6 spinal cord were significantly down regulated after receiving FKN-neutralizing antibody.Immunofluorescence staining showed that FKN was mainly located on neuron in the in the spinal dorsal horn.The CX3CR1-positive and pP38-MAPK-positive microglia in the dorsal horn was reduced by treatment with neutralizing antibody on POD 21.Conclusions:1.DRG and spinal FKN/CX3CR1 is associated with central sensitization,and inhibition of spinal FKN can alleviate EMs-induced mechanical hypersensitivity and allodynia,even reverse the development of pain.2.FKN/CX3CR1 signaling pathway may mediate DRG neuron-glial cell interaction to participate in EMs pain conduction.3.Spinal FKN/CX3CR1/P38-MAPK signaling pathway contributes to the development of central sensitization mediates in EMs via neuron-microglia interactions.