Emerging evidence indicates that CXCL12/ CXCR4 signaling is involved in chronic pain. However, few studies have systemically assessed its role in direct nerve injury-induced neuropathic pain and the underlying mechanism. Here, we determined that spared nerve injury (SNI) increased the expression of CXCL12 and its cognate receptor CXCR4 in lumbar 5 dorsal root ganglia (DRG) neurons and satellite glial cells. SNI also induced longlasting upregulation of CXCL12 and CXCR4 in the ipsilateral L4-5 spinal cord dorsal horn, characterized by CXCL12 expression in neurons and microglia, and CXCR4 expression in neurons and astrocytes. Moreover, SNIinduced a sustained increase in TNF-a expression in the DRG and spinal cord. Intraperitoneal injection (i.p.) of the TNF-a synthesis inhibitor thalidomide reduced the SNI-induced mechanical hypersensitivity and inhibited the expression of CXCL12 in the DRG and spinal cord. Intrathecal injection (i.t.) of the CXCR4 antagonist AMD3100, both 30 min before and 7 days after SNI, reduced the behavioral signs of allodynia. Rats given an i.t. or i.p. bolus of AMD3100 on day 8 of SNI exhibited attenuated abnormal pain behaviors. The neuropathic pain established following SNI was also impaired by i.t. administration of a CXCL12-neutralizing antibody. Moreover, repetitive i.t. AMD3100 administration prevented the activation of ERK in the spinal cord. The mechanical hypersensitivity induced in naïve rats by i.t. CXCL12 was alleviated by pretreatment with the MEK inhibitor PD98059. Collectively, our results revealed that TNF-a might mediate the upregulation of CXCL12 in the DRG and spinal cord following SNI, and that CXCL12/CXCR4 signaling via ERK activation contributes to the development and maintenance of neuropathic pain.

Nuclear factor kappa B (NF-κB) in the spinal cord is involved in pro-infl ammatory cytokine-mediated pain facilitation. However, the role of NF-κB activation in chronic morphine-induced analgesic tolerance and the underlying mechanisms remain unclear. In the present study, we found that the level of phosphorylated NF-κB p65 (p-p65) was increased in the dorsal horn of the lumbar 4-6 segments after intrathecal administration of morphine for 7 consecutive days, and the p-p65 was co-localized with neurons and astrocytes. The expression of TNF-α and IL-1β was also increased in the same area. In addition, pretreatment with pyrrolidinedithiocarbamate (PDTC) or SN50, inhibitors of NF-κB, prevented the development of morphine analgesic tolerance and alleviated morphine withdrawal-induced allodynia and hyperalgesia. The increase in TNF-α and IL-1β expression induced by chronic morphine exposure was also partially blocked by PDTC pretreatment. In another experiment, rats receiving PDTC or SN50 beginning on day 7 of morphine injection showed partial recovery of the anti-nociceptive effects of morphine and attenuation of the withdrawal-induced abnormal pain. Meanwhile, intrathecal pretreatment with lipopolysaccharide from Rhodobacter sphae roides, an antagonist of toll-like receptor 4 (TLR4), blocked the activation of NF-κB, and prevented the development of morphine tolerance and withdrawalinduced abnormal pain. These data indicated that TLR4-mediated NF-κB activation in the spinal cord is involved in the development and maintenance of morphine analgesic tolerance and withdrawalinduced pain hypersensitivity.

BackgroundIt has been demonstrated that upregulation of CXCL12 and CXCR4 in spinal cord involves in the pathogenesis of neuropathic, inflammatory, and cancer pain. However, whether CXCL12/CXCR4 signaling contributes to postsurgical pain remains unknown. The aim of the present study is to investigate the role of CXCL12/CXCR4 signaling in the genesis of postsurgical pain and the underlying mechanism.ResultsPlantar incision in rat hind paw resulted in increased expressions of CXCL12 and CXCR4 in spinal dorsal horn. Double immunofluorescence staining revealed that CXCL12 expressed in neurons and astrocytes, and CXCR4 exclusively co-localized with neuronal cells. Prior administration of AMD3100, a specific antagonist of CXCR4, or CXCL12 neutralizing antibody, intrathecally attenuated plantar incision-induced mechanical allodynia and thermal hyperalgesia. Plantar incision also augmented the phosphorylation of NF-κB p65 in spinal cord. Pre intrathecal (i.t.) injection of PDTC, a specific NF-κB activation inhibitor, alleviated plantar incision-induced postsurgical pain and reduced the expression of CXCL12 in spinal cord. Correlated with the upregulation of CXCL12 and CXCR4, plantar incision also resulted in an increased phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in spinal cord. Prior i.t. administration of AMD3100 prevented extracellular signal-regulated kinase, but not Akt, activation in spinal cord. Rats when given a repetitive i.t. PD98059, a specific extracellular signal-regulated kinase inhibitor, started 30 min before surgery also ameliorate plantar incision-induced mechanical and thermal pain hypersensitivity.ConclusionOur results suggests that plantar incision-induced activation of NF-κB signaling may mediate upregulation of CXCL12 in spinal cord, and CXCL12/CXCR4 signaling via extracellular signal-regulated kinase activation contributes to the genesis of postsurgical pain.

Analgesic tolerance and hyperalgesia hinder the long-term utility of opioids. We examined whether spinal high mobility group box 1 (HMGB1) is involved in morphine tolerance and its underlying mechanisms by using a model of repeated intrathecal (i.t.) injections of morphine. The results showed that chronic i.t. morphine exposure led to increased expression of HMGB1, Toll-like receptor 4 (TLR4), and receptor for advanced glycation end products (RAGE) and their mRNAs in the dorsal horn. Morphine challenge also promoted HMGB1 expression and release in cultured spinal neurons, but these effects were inhibited by TAK-242, naloxone (antagonists of TLR4), and TLR4 siRNA. Intrathecal coadministration of morphine with TAK-242 or PDTC (inhibitor of NF-κB activation) also reduced HMGB1 expression in the spinal cord. Repeated i.t. coinjections of morphine with glycyrrhizin (GL, an HMGB1 inhibitor) or HMGB1 siRNA prevented reduction of the maximal possible analgesic effect (MPAE) of morphine and alleviated morphine withdrawal-induced hyperalgesia. The established morphine tolerance and hyperalgesia were partially reversed when i.t. injections of GL or HMGB1 antibody started at day 7 of morphine injection. Repeated i.t. injections of morphine with HMGB1 siRNA inhibited the activation of NF-κB, but not that of JNK and p38. A single i.t. injection of HMGB1 in naïve rats caused pain-related hypersensitivity and reduction in MPAE. Moreover, phosphorylated NF-κB p65, TNF-α, and IL-1β levels in the dorsal horn were upregulated following this treatment, but this upregulation was prevented by coinjection with TAK-242. Together, these results suggest that morphine-mediated upregulation of spinal HMGB1 contributes to analgesic tolerance and hyperalgesia via activation of TLR4/NF-κB signaling, and the HMGB1 inhibitor might be a promising adjuvant to morphine in the treatment of intractable pain in the clinic.

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