SDF1-CXCR4 Signaling Contributes To The Development And Maintenance Of Pathological Pain

Pathological pain including inflammatory pain and neuropathic pain,as a major challenge in clinical practice and basic science research,is the leading cause of adult disability worldwide which has a detrimental effect on function and quality of life.This results in a huge economic burden to the families and society.The efficacy of current treatment strategies using NSAIDS,opioids,antidepressant and anticonvulsants is unsatisfactory due to the extensive side effects observed in patients,which limited the clinical management of pathological pain.Thus,understanding the pathophysiological mechanisms of pathological pain and develop novel targeted analgesic drugs are in an urgent demand.Over the past few decades,some remarkable achievements have been made in exploring pain process.However,the underlying mechanisms of the initiation and maintenance of inflammatory and neuropathic pain remain far from clear.Recently,emerging evidence demonstrate that glial cells play a critical role in pain,both in the periphery nervous system(PNS)and in the central nervous system(CNS).Through increasing in number and releasing pro-inflammatory mediators,such as cytokines and chemokines,glial cells induce various neurobiological changes under pain conditions,including neuronal hyperexcitability,neurotoxicity and neuroinflammation,which result in the peripheral and central sensitization,finally contribute to the generation of pain.Stromal cell-derived factor 1(SDF1,also called CXCL12),as a member of the chemokine CXC family,is widely distributed in PNS and CNS.Through binding to its cognate receptor CXCR4,SDF1 modulates multiple activities of neurons and glial cells.Currently,accumulating behavioral pharmacological studies suggest that SDF1-CXCR4 signaling is associated with pain.However,these studies have not revealed the detailed mechanisms of SDF1-CXCR4 signaling involved in the development and maintenance of pathological pain due to the limited methods and pain models.Moreover,under different pain conditions,the effects of SDF1-CXCR4 signaling at different segments remain unknown.In the present study,we adopted three representative animal models of acute peripheral inflammatory pain,transition from acute to chronic pain state and chronic central neuropathic pain.Additionally,by utilizing a combination of molecular biology,behavioral pharmacology and electrophysiological recording,we detailedly investigated the role of SDF1-CXCR4 signaling in the development and maintenance of multiple pain conditions.The results are as follow: 1.SDF1–CXCR4 signaling contributes to the development and maintenance of acute inflammatory painUnder peripheral inflammatory pain state induced by intraplantar(i.pl.)bee venom(BV)injection,the expression levels of SDF1 and CXCR4 in the ipsilateral dorsal root ganglion(DRG)significantly increased.Double immunofluorescent staining showed that CXCR4 was localized in the soma of all(large,medium,and small)sized DRG neurons,including IB4-positive nonpeptidergic,subatance P(SP)-positive peptidergic and TRPV1-positive primary nociceptor neurons,while SDF1 was exclusively expressed in glial fibrillary acidic protein(GFAP)-positive satellite glial cells(SGCs).Whole cell patch clamp recording showed the excitability of medium-and small-sized DRG neurons were increased following i.pl.BV injection,reflecting as the increase in discharge frequency and the decrease in action potential(AP)rheobase.However,bath application with AMD3100,a potent and selective CXCR4 inhibitor,could reverse BVinduced hyperexcitability of medium-and small-sized DRG neurons.Further studies showed that the expression level of Nav1.8 and phosphorylated extracellular regulated protein kinases(p-ERK)in the DRG were also enhanced,and which were remarkably inhibited by i.pl.pre-injection of AMD3100 or U0126,a specific ERK inhibitor,at 10 min before BV injection.Likewise,intrathecal(i.t.)preinjection with U0126 also suppressed the up-regulation of Nav1.8 and p-ERK induced by i.pl.BV injection.At behavioral level,we demonstrated that BV-induced multiple pain-related behaviors,such as spontaneous pain,primary mechanical and thermal pain hypersensitivity,were prevented by blocking CXCR4 with AMD3100,but with the mirror-image thermal pain hypersensitivity being unaffected.Pre-injection with i.t.U0126 was sufficient to suppress all of BV-induced pain-related behaviors even the mirror-image thermal pain hypersensitivity.Finally,we found that i.pl.injection with A-803467,a specific Nav1.8 blocker,not only prevent BV-induced persistent spontaneous pain but also could reverse the primary mechanical and thermal pain hypersensitivity.2.SDF1-CXCR4 signaling contributes to the transition from acute to chronic pain stateBV i.pl.injection induced significant primary mechanical pain hypersensitivity which occurred pronouncedly at 2 h and then progressively restored to baseline value,with disappearing within 96 h,indicating i.pl.BV injection is a short-term acute inflammatory pain model.At 7 d after BV injection when the rats completely recovered from pain hypersensitivity,we injected prostaglandin E2(PGE2)into the same site as previous BV injection.We found PGE2 induced a prolonged mechanical hyperalgesia lasting >24 h,while PGE2 i.pl.injection only produced a transient(<4 h)mechanical hyperalgesia in saline-injected control rats,suggesting BV pre-injection can significantly enhance the pro-algesic effect of PGE2 which is called hyperalgesic priming(HP).Through extending the time window between BV and PGE2,we demonstrated that BV-induced HP persisted for at least 21 days.Both i.pl.AMD3100 pre-injection and intraganglionar CXCR4 siRNA pre-injection could significantly suppress BV-induced mechanical hyperalgesia and HP.Single i.pl.injection with SDF1 was also sufficient to produce does-dependent acute mechanical hyperalgesia which occured at 0.5 h and resolved within 72 h.At 5 d after SDF1 injection,PGE2 i.pl.injection at the same site also produced a prolonged mechanical hyperalgesia lasting >24 h,indicating single SDF1 injection was able to establish HP.Similar to BV,SDF1-induced mechanical hyperalgesia and HP were also completely prevented by blocking CXCR4 at peripheral site or knocking down CXCR4 at DRG level.Immunoblotting showed that SDF1 i.pl.injection significantly induced upregulation of p-ERK and p-AKT in DRG,suggesting ERK and PI3K-AKT signaling were activated by SDF1.Pre-injection with i.pl.U0126 and LY294002,a specific PI3 KAKT signaling inhibitor,dramatically prevented SDF1-induced mechanical hyperalgesia and HP.Moreover,pre-injection with translation inhibitors,temsirolimus and cordycepin,also efficiently suppressed mechanical hyperalgesia and HP induced by SDF1 i.pl.injection,suggesting local protein translation is required in the induction of mechanical hyperalgesia and HP.Finally,we demonstrated that knocking down CXCR4 with intraganglionar CXCR4 siRNA injection not only prevent the occurrence but also reverse the maintenance of complete Freund’s adjuvant(CFA)-induced chronic inflammatory pain and spared nerve injury(SNI)-induced chronic neuropathic pain.3.SDF1-CXCR4 signaling plays a critical role in the development and maintenance of central post-stroke painInjection IV collagenase into ventral posterior lateral nucleus(VPL)of thalamus resulted in significant bilateral mechanical hyperalgesia,which were identified on day 7 post-injection and remained unchanged until day 28 post-injection,suggesting a experimental central post stroke pain(CPSP)model was successfully established.Immunofluorescence staining and immunoblotting showed that microglia and astrocytes were persistently activated in peri-thalamic lesion site.The expression level of Iba-1 and GFAP was substantially increased on day 3 after collagenase injection,reached peak on day 7 and remained unchanged until day 28.To investigate the roles of microglial and astrocytic activation in the CPSP,we injection microglial inhibitor minocycline and astrocytic inhibitor fluorocitrate into VPL at 10 d after collagenase injection founding that the bilateral mechanical hyperalgesia was reversed.The antiallodynic effect of minocycline reached peak at 6 h after injection and maintained at a significant level for at least 7 days,while the anti-allodynic effect of fluorocitrate only last for 3 days.We also identified that intra-VPL SDF1 injection was sufficient to evoke mechanical hyperalgesia in bilateral hindpaws,and pre-injection with AMD3100 or minocycline or fluorocitrate could prevent SDF1-induced mechanical hyperalgesia,suggesting SDF1-CXCR4 signaling induced pain hypersensitivity via activating glial cells.The expression level of SDF1 in peri-thalamic lesion sites was also persistent enhanced over the whole observed time.Linear correlation analysis revealed that the expression level of SDF1 was highly correlated with the amplitude of pain hypersensitivity,suggesting the important role of SDF1 in CPSP.Next,we sought to examine the expression of CXCR4 in peri-thalamic lesion sites founding it was also upregulated and which co-localized with Neun,Iba-1 and GFAP,indicating neurons and microglia as well as astrocytes expressed CXCR4.Injection AMD3100 into thalamus obviously prevented the activation of microglia and astrocytes in peri-thalamic lesion sites induced by collagenase injection.Moreover,thalamic hemorrhage-induced up-regulation of TNF-α,IL-6 and IL-1β were suppressed by AMD3100 pre-injection.Likewise,neuronal activity marked by c-Fos immunoreactivity was reduced by AMD3100.Behavioral assay demonstrated that intra-thalamus AMD3100 injection not only prevented but also reverse CPSP.The HIF-1α expression in peri-thalamic lesion sites was increased transiently which reached peak on day 3 and last for 7 days.Intra-thalamus injection with YC-1,a specific HIF-1α inhibitor,could only prevent the development of CPSP without effect on the maintenance of CPSP.Concomitantly,thalamic hemorrhage-induced upregulation of SDF1,CXCR4 and Iba-1 as well as GFAP were suppressed by YC-1 intrathalamus injection.Similar to collagenase,intra-thalamus injection with Cocl2,a wellknown HIF-1α inducer,was also able to increase the expression of HIF-1α,SDF1,CXCR4,Iba-1 and GFAP which were inhibited by pre-injection with AMD3100 and YC-1.Furthermore,intra-thalamus injection of CoCl2 induced bilateral mechanical pain hypersensitivity that could be blocked by pre-treatment of AMD3100 through intra-thalamic microinjection.Conclusions: I.Under peripheral inflammatory pain state,the expression of SDF1 was dramatically increased in the SGCs.Through binding to CXCR4 up-regulated in primary nociceptor neurons,SDF1 activated intracellular ERK signaling and induced Nav1.8 up-regulation.Thus,intraganglionar inflammatory microenvironment was established by SDF1-CXCR4 signaling which contributed to the maintenance of neuronal hyperexcitability and multiple inflammatory pain-related behaviors.II.Through intracellular ERK and PI3K-AKT signaling mediated protein translation,peripheral SDF1-CXCR4 signaling induces the transition from acute to chronic pain state and finally contributes to the induction and maintenance of chronic pain.III.At the early stage of thalamic hemorrhage,HIF-1α was activated abundantly in the peri-thalamic lesion sites and which evoked the up-regulation of SDF1 and CXCR4.At the late stage of thalamic hemorrhage,SDF1-CXCR4 signaling mediated microglialastrocytic-neuronal interactions which promoted pro-inflammatory cytokines release and neuronal hyperexcitability,finally contributed to the maintenance of CPSP.