Chemokine CCL7 mediates trigeminal neuropathic pain via CCR2/CCR3-ERK pathway in the trigeminal ganglion of mice.

BACKGROUND: Chemokine-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. The chemokine CC motif ligand 7 (CCL7) and its receptor CCR2 have been reported to contribute to neuropathic pain via astrocyte-microglial interaction in the spinal cord. Whether CCL7 in the trigeminal ganglion (TG) involves in trigeminal neuropathic pain and the involved mechanism remain largely unknown. METHODS: The partial infraorbital nerve transection (pIONT) was used to induce trigeminal neuropathic pain in mice. The expression of Ccl7, Ccr1, Ccr2, and Ccr3 was examined by real-time quantitative polymerase chain reaction. The distribution of CCL7, CCR2, and CCR3 was detected by immunofluorescence double-staining. The activation of extracellular signal-regulated kinase (ERK) was examined by Western blot and immunofluorescence. The effect of CCL7 on neuronal excitability was tested by whole-cell patch clamp recording. The effect of selective antagonists for CCR1, CCR2, and CCR3 on pain hypersensitivity was checked by behavioral testing. RESULTS: Ccl7 was persistently increased in neurons of TG after pIONT, and specific inhibition of CCL7 in the TG effectively relieved pIONT-induced orofacial mechanical allodynia. Intra-TG injection of recombinant CCL7 induced mechanical allodynia and increased the phosphorylation of ERK in the TG. Incubation of CCL7 with TG neurons also dose-dependently enhanced the neuronal excitability. Furthermore, pIONT increased the expression of CCL7 receptors Ccr1, Ccr2, and Ccr3. The intra-TG injection of the specific antagonist of CCR2 or CCR3 but not of CCR1 alleviated pIONT-induced orofacial mechanical allodynia and reduced ERK activation. Immunostaining showed that CCR2 and CCR3 are expressed in TG neurons, and CCL7-induced hyperexcitability of TG neurons was decreased by antagonists of CCR2 or CCR3. CONCLUSION: CCL7 activates ERK in TG neurons via CCR2 and CCR3 to enhance neuronal excitability, which contributes to the maintenance of trigeminal neuropathic pain. CCL7-CCR2/CCR3-ERK pathway may be potential targets for treating trigeminal neuropathic pain.

TRAF6 Contributes to CFA-Induced Spinal Microglial Activation and Chronic Inflammatory Pain in Mice.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been reported to be expressed in spinal astrocytes and is involved in neuropathic pain. In this study, we investigated the role and mechanism of TRAF6 in complete Freund's adjuvant (CFA)-evoked chronic inflammatory hypersensitivity and the effect of docosahexaenoic acid (DHA) on TRAF6 expression and inflammatory pain. We found that TRAF6 was dominantly increased in microglia at the spinal level after intraplantar injection of CFA. Intrathecal TRAF6 siRNA alleviated CFA-triggered allodynia and reversed the upregulation of IBA-1 (microglia marker). In addition, intrathecal administration of DHA inhibited CFA-induced upregulation of TRAF6 and IBA-1 in the spinal cord and attenuated CFA-evoked mechanical allodynia. Furthermore, DHA prevented lipopolysaccharide (LPS)-caused increase of TRAF6 and IBA-1 in both BV2 cell line and primary cultured microglia. Finally, intrathecal DHA reduced LPS-induced upregulation of spinal TRAF6 and IBA-1, and alleviated LPS-induced mechanical allodynia. Our findings indicate that TRAF6 contributes to pain hypersensitivity via regulating microglial activation in the spinal dorsal horn. Direct inhibition of TRAF6 by siRNA or indirect inhibition by DHA may have therapeutic effects on chronic inflammatory pain.

Cytochrome P450 26A1 Contributes to the Maintenance of Neuropathic Pain.

The cytochrome P450 proteins (CYP450s) have been implicated in catalyzing numerous important biological reactions and contribute to a variety of diseases. CYP26A1, a member of the CYP450 family, carries out the oxidative metabolism of retinoic acid (RA), the active metabolite of vitamin A. Here we report that CYP26A1 was dramatically upregulated in the spinal cord after spinal nerve ligation (SNL). CYP26A1 was mainly expressed in spinal neurons and astrocytes. HPLC analysis displayed that the content of all-trans-RA (at-RA), the substrate of CYP26A1, was reduced in the spinal cord on day 7 after SNL. Inhibition of CYP26A1 by siRNA or inhibition of CYP26A1-mediated at-RA catabolism by talarozole relieved the SNL-induced mechanical allodynia during the maintenance phase of neuropathic pain. Talarozole also reduced SNL-induced glial activation and proinflammatory cytokine production but increased anti-inflammatory cytokine (IL-10) production. The RA receptors RARα, RXRß, and RXRγ were expressed in spinal neurons and glial cells. The promoter of Il-10 has several binding sites for RA receptors, and at-RA directly increased Il-10 mRNA expression in vitro. Finally, intrathecal IL-10 attenuated SNL-induced neuropathic pain and reduced the activation of astrocytes and microglia. Collectively, the inhibition of CYP26A1-mediated at-RA catabolism alleviates SNL-induced neuropathic pain by promoting the expression of IL-10 and suppressing glial activation. CYP26A1 may be a potential therapeutic target for the treatment of neuropathic pain.

TFAP2A is involved in neuropathic pain by regulating Grin1 expression in glial cells of the dorsal root ganglion.

Neuropathic pain is a highly prevalent and refractory condition, yet its mechanism remains poorly understood. While NR1, the essential subunit of NMDA receptors, has long been recognized for its pivotal role in nociceptive transmission, its involvement in presynaptic stimulation is incompletely elucidated. Transcription factors can regulate the expression of both pro-nociceptive and analgesic factors. Our study shows that transcription factor TFAP2A was up-regulated in the dorsal root ganglion (DRG) neurons, satellite glial cells (SGCs), and Schwann cells following spinal nerve ligation (SNL). Intrathecal injection of siRNA targeting Tfap2a immediately or 7 days after SNL effectively alleviated SNL-induced pain hypersensitivity and reduced Tfap2a expression levels. Bioinformatics analysis revealed that TFAP2A may regulate the expression of the Grin1 gene, which encodes NR1. Dual-luciferase reporter assays confirmed TFAP2A's positive regulation of Grin1 expression. Notably, both Tfap2a and Grin1 were expressed in the primary SGCs and upregulated by lipopolysaccharides. The expression of Grin1 was also down-regulated in the DRG following Tfap2a knockdown. Furthermore, intrathecal injection of siRNA targeting Grin1 immediately or 7 days post-SNL effectively alleviated SNL-induced mechanical allodynia and thermal hyperalgesia. Finally, intrathecal Tfap2a siRNA alleviated SNL-induced neuronal hypersensitivity, and incubation of primary SGCs with Tfap2a siRNA decreased NMDA-induced upregulation of proinflammatory cytokines. Collectively, our study reveals the role of TFAP2A-Grin1 in regulating neuropathic pain in peripheral glia, offering a new strategy for the development of novel analgesics.

Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons.

Neuropathic pain is a debilitating chronic condition that lacks effective treatment. The role of cytokine- and chemokine-mediated neuroinflammation in its pathogenesis has been well documented. Follistatin (FST) is a secreted protein known to antagonize the biological activity of cytokines in the transforming growth factor-ß (TGF-ß) superfamily. The involvement of FST in neuropathic pain and the underlying mechanism remain largely unknown. Here, we report that FST was up-regulated in A-fiber sensory neurons after spinal nerve ligation (SNL) in mice. Inhibition or deletion of FST alleviated neuropathic pain and reduced the nociceptive neuron hyperexcitability induced by SNL. Conversely, intrathecal or intraplantar injection of recombinant FST, or overexpression of FST in the dorsal root ganglion (DRG) neurons, induced pain hypersensitivity. Furthermore, exogenous FST increased neuronal excitability in nociceptive neurons. The biolayer interferometry (BLI) assay and coimmunoprecipitation (co-IP) demonstrated direct binding of FST to the insulin-like growth factor-1 receptor (IGF1R), and IGF1R inhibition reduced FST-induced activation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), as well as neuronal hyperexcitability. Further co-IP analysis revealed that the N-terminal domain of FST exhibits the highest affinity for IGF1R, and blocking this interaction with a peptide derived from FST attenuated Nav1.7-mediated neuronal hyperexcitability and neuropathic pain after SNL. In addition, FST enhanced neuronal excitability in human DRG neurons through IGF1R. Collectively, our findings suggest that FST, released from A-fiber neurons, enhances Nav1.7-mediated hyperexcitability of nociceptive neurons by binding to IGF1R, making it a potential target for neuropathic pain treatment.

CXCL10 and CXCR3 in the Trigeminal Ganglion Contribute to Trigeminal Neuropathic Pain in Mice.

PURPOSE: Trigeminal neuropathic pain is very common clinically, but effective treatments are lacking. Chemokines and their receptors have been implicated in the pathogenesis of chronic pain. This study explored the role of the chemokine CXCL10 and its receptor, CXCR3, in trigeminal neuropathic pain in mice. MATERIALS AND METHODS: Trigeminal neuropathic pain was established by partial infraorbital nerve ligation (pIONL) in wild-type and Cxcr3 -/- mice. Facial mechanical allodynia was evaluated by behavioral testing. A lentivirus containing Cxcr3 shRNA (LV-Cxcr3 shRNA) was microinjected into the trigeminal ganglion (TG) to knock down Cxcr3 expression. Quantitative polymerase chain reaction assays and immunofluorescence staining were used to examine Cxcl10/Cxcr3 mRNA expression and protein distribution. Western blotting was performed to examine activation of extracellular signal-regulated kinase (ERK) and AKT in the TG. Intra-TG injection of an AKT inhibitor was performed to examine the role of AKT in trigeminal neuropathic pain. RESULTS: pIONL induced persistent trigeminal neuropathic pain, which was alleviated in Cxcr3 -/- mice. Intra-TG injection of LV-Cxcr3 shRNA attenuated pIONL-induced mechanical allodynia. Furthermore, pIONL increased the expression of CXCR3 and its major ligand, CXCL10, in TG neurons. Intra-TG injection of CXCL10 induced pain hypersensitivity in wild-type mice but not in Cxcr3 -/- mice. CXCL10 also induced activation of ERK and AKT in the TG of wild-type mice. Finally, pIONL-induced activation of ERK and AKT was reduced in Cxcr3 -/- mice. Intra-TG injection of the AKT inhibitor alleviated pIONL-induced mechanical allodynia in WT mice but not in Cxcr3 -/- mice. CONCLUSION: CXCL10 acts on CXCR3 to induce ERK and AKT activation in TG neurons and contributes to the maintenance of trigeminal neuropathic pain.

CXCL10/CXCR3 Signaling in the DRG Exacerbates Neuropathic Pain in Mice.

Chemokines and receptors have been implicated in the pathogenesis of chronic pain. Here, we report that spinal nerve ligation (SNL) increased CXCR3 expression in dorsal root ganglion (DRG) neurons, and intra-DRG injection of Cxcr3 shRNA attenuated the SNL-induced mechanical allodynia and heat hyperalgesia. SNL also increased the mRNA levels of CXCL9, CXCL10, and CXCL11, whereas only CXCL10 increased the number of action potentials (APs) in DRG neurons. Furthermore, in Cxcr3-/- mice, CXCL10 did not increase the number of APs, and the SNL-induced increase of the numbers of APs in DRG neurons was reduced. Finally, CXCL10 induced the activation of p38 and ERK in ND7-23 neuronal cells and DRG neurons. Pretreatment of DRG neurons with the P38 inhibitor SB203580 decreased the number of APs induced by CXCL10. Our data indicate that CXCR3, activated by CXCL10, mediates p38 and ERK activation in DRG neurons and enhances neuronal excitability, which contributes to the maintenance of neuropathic pain.

Immunohistochemical demonstration of airway epithelial cell markers of guinea pig.

The guinea pig (Cavea porcellus) is a mammalian non-rodent species in the Caviidae family. The sensitivity of the respiratory system and the susceptibility to infectious diseases allows the guinea pig to be a useful model for both infectious and non-infectious lung diseases such as asthma and tuberculosis. In this report, we demonstrated for the first time, the major cell types and composition in the guinea pig airway epithelium, using cell type-specific markers by immunohistochemical staining using the commercial available immunological reagents that cross-react with guinea pig. Our results revealed the availability of antibodies cross-reacting with airway epithelial cell types of basal, non-ciliated columnar, ciliated, Clara, goblet and alveolar type II cells, as well as those cells expressing Mucin 5AC, Mucin 2, Aquaporin 4 and Calcitonin Gene Related Peptide. The distribution of these various cell types were quantified in the guinea pig airway by immunohistochemical staining and were comparable with morphometric studies using an electron microscopy assay. Moreover, this study also demonstrated that goblet cells are the main secretory cell type in the guinea pig's airway, distinguishing this species from rats and mice. These results provide useful information for the understanding of airway epithelial cell biology and mechanisms of epithelial-immune integration in guinea pig models.