Spinal interleukin-17 promotes thermal hyperalgesia and NMDA NR1 phosphorylation in an inflammatory pain rat model.
ABSTRACT: It is known that interleukin-17 (IL-17) is associated with autoimmune disorders and that peripheral IL-17 plays a role in arthritis and neuropathic pain. The present study investigated the possibility of spinal cell expression of IL-17 during inflammatory pain and possible IL-17 involvement in such pain. Hyperalgesia was induced by injecting complete Freund adjuvant (CFA, 0.08mL, 40?g Mycobacterium tuberculosis) into one hind paw of the rat. Paw withdrawal latency (PWL) was tested before (-48h) and 2 and 24h after CFA injection to assess hyperalgesia. IL-17 antibody (0.2-2?g/rat) was given intrathecally (i.t.) 24h before CFA to block the action of basal IL-17 and 2h before each of 2 PWL tests to block CFA-induced IL-17. I.t. recombinant IL-17 (10-400ng per rat) was administered to naive rats to determine its effects on PWL and phosphorylated NR1 (p-NR1). p-NR1 modulates N-methyl-d-aspartate receptor (NMDAR) activity to facilitate pain. Spinal cords were removed for IL-17 immunostaining, double immunostaining of IL-17/cell markers and IL-17 receptor A (IL-17RA)/NR1, for Western blot testing of IL-17, p-NR1, IL-17RA, and GFAP, for in situ IL-17RA hybridization, and for real time polymerase chain reaction of IL-17RA. The data reveal that IL-17 is up-regulated in activated and nonactivated astrocytes; that IL-17RA is localized in NR1-immunoreactive neurons and up-regulated; and that IL-17 antibody at 2?g/rat significantly increased PWL (P<.05) and decreased p-NR1 and IL-17RA compared to control in CFA- and IL-17-injected rats. The results suggest that spinal IL-17 is produced by astrocytes and enhances p-NR1 to facilitate pain.
Project description:<h4>Background</h4>This study aims to identify that intrathecal (i.t.) injection of dexmedetomidine (Dex) and ropivacaine (Ropi) induces synergistic analgesia on chronic inflammatory pain and is accompanied with corresponding "neuron-astrocytic" alterations.<h4>Methods</h4>Male, adult Sprague-Dawley rats were randomly divided into sham, control and i.t. medication groups. The analgesia profiles of i.t. Dex, Ropi, and their combination detected by Hargreaves heat test were investigated on the subcutaneous (s.c.) injection of complete Freund adjuvant (CFA) induced chronic pain in rat and their synergistic analgesia was confirmed by using isobolographic analysis. During consecutive daily administration, pain behavior was daily recorded, and immunohistochemical staining was applied to investigate the number of Fos-immunoreactive (Fos-ir) neurons on hour 2 and day 1, 3 and 7, and the expression of glial fibrillary acidic protein (GFAP) within the spinal dorsal horn (SDH) on day 1, 3, 5 and 7 after s.c. injection of CFA, respectively, and then Western blot to examine spinal GFAP and ?-actin levels on day 3 and 7.<h4>Results</h4>i.t. Dex or Ropi displayed a short-term analgesia in a dose-dependent manner, and consecutive daily administrations of their combination showed synergistic analgesia and remarkably down-regulated neuronal and astrocytic activations indicated by decreases in the number of Fos-ir neurons and the GFAP expression within the SDH, respectively.<h4>Conclusion</h4>i.t. co-delivery of Dex and Ropi shows synergistic analgesia on the chronic inflammatory pain, in which spinal "neuron-astrocytic activation" mechanism may play an important role.
Project description:Following peripheral inflammation, NMDA receptor (NMDAR) activation in spinal cord dorsal horn neurons facilitates the generation of pain in response to low threshold inputs (allodynia) and signals the phosphorylation of protein kinase C (pPKC) and extracellular signal-regulated kinase 2 (pERK2). Intraplantar complete Freund's adjuvant (CFA) induces inflammatory nociception (allodynic pain) at 24 hours (h) with a concurrent increase in neuronal pPKCgamma and pERK2 but not glial pERK2. These effects are attenuated in a spatial knockout of the NMDAR (NR1 KO) confined to SCDH neurons. Although glia and proinflammatory cytokines are implicated in the maintenance of inflammatory pain and neuronal activation, the role of NMDARs and neuronal-glial-cytokine interactions that initiate and maintain inflammatory pain are not well defined. In the maintenance phase of inflammatory pain at 96h after CFA the NR1 KO mice are no longer protected from allodynia and the SCDH expression of pPKCgamma and pERK2 are increased. At 96h the expression of the proinflammatory cytokine, IL-1beta, and pERK2 are increased in astrocytes. Intrathecal IL-1 receptor antagonist (IL-1ra), acting on neuronal IL-1 receptors, completely reverses the allodynia at 96h after CFA. Deletion of NMDAR-dependent signaling in neurons protects against early CFA-induced allodynia. Subsequent NMDAR-independent signaling that involves neuronal expression of pPKCgamma and the induction of pERK2 and IL-1beta in activated astrocytes contributes to the emergence of NMDAR-independent inflammatory pain behavior at 96h after CFA. Effective reduction of the initiation and maintenance of inflammatory pain requires targeting the neuron-astrocyte-cytokine interactions revealed in these studies.
Project description:Spinal cord glia (microglia and astrocytes) contribute to enhanced pain states. One model that has been used to study this phenomenon is intrathecal (i.t.) administration of gp120, an envelope glycoprotein of HIV-1 known to activate spinal cord glia and thereby induce low-threshold mechanical allodynia, a pain symptom where normally innocuous (non-painful) stimuli are perceived as painful. Previous studies have shown that i.t. gp120-induced allodynia is mediated via the release of the glial pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF), and interleukin-1beta (IL-1). As we have recently reported that i.t. gp120 induces the release of interleukin-6 (IL-6), in addition to IL-1 and TNF, the present study tested whether this IL-6 release in spinal cord contributes to gp120-induced mechanical allodynia and/or to gp120-induced increases in TNF and IL-1. An i.t. anti-rat IL-6 neutralizing antibody was used to block IL-6 actions upon its release by i.t. gp120. This IL-6 blockade abolished gp120-induced mechanical allodynia. While the literature predominantly documents the cascade of pro-inflammatory cytokines as beginning with TNF, followed by the stimulation of IL-1, and finally TNF plus IL-1 stimulating the release of IL-6, the present findings indicate that a blockade of IL-6 inhibits the gp120-induced elevations of TNF, IL-1, and IL-6 mRNA in dorsal spinal cord, elevation of IL-1 protein in lumbar dorsal spinal cord, and TNF and IL-1 protein release into the surrounding lumbosacral cerebrospinal fluid. These results would suggest that IL-6 induces pain facilitation, and may do so in part by stimulating the production and release of other pro-inflammatory cytokines.
Project description:<h4>Background and purpose</h4>Cannabinoid CB? receptor activation by selective agonists has been shown to produce analgesic effects in preclinical models of inflammatory and neuropathic pain. However, mechanisms underlying CB?-mediated analgesic effects remain largely unknown. The present study was conducted to elucidate the CB? receptor expression in 'pain relevant' tissues and the potential sites of action of CB? agonism in rats.<h4>Experimental approach</h4>Expression of cannabinoid receptor mRNA was evaluated by quantitative RT-PCR in dorsal root ganglia (DRGs), spinal cords, paws and several brain regions of sham, chronic inflammatory pain (CFA) and neuropathic pain (spinal nerve ligation, SNL) rats. The sites of CB? mediated antinociception were evaluated in vivo following intra-DRG, intrathecal (i.t.) or intraplantar (i.paw) administration of potent CB?-selective agonists A-836339 and AM1241.<h4>Key results</h4>CB? receptor gene expression was significantly up-regulated in DRGs (SNL and CFA), spinal cords (SNL) or paws (CFA) ipsilateral to injury under inflammatory and neuropathic pain conditions. Systemic A-836339 and AM1241 produced dose-dependent efficacy in both inflammatory and neuropathic pain models. Local administration of CB? agonists also produced significant analgesic effects in SNL (intra-DRG and i.t.) and CFA (intra-DRG) pain models. In contrast to A-836339, i.paw administration of AM-1241 dose-relatedly reversed the CFA-induced thermal hyperalgesia, suggesting that different mechanisms may be contributing to its in vivo properties.<h4>Conclusions and implications</h4>These results demonstrate that both DRG and spinal cord are important sites contributing to CB? receptor-mediated analgesia and that the changes in CB? receptor expression play a crucial role for the sites of action in regulating pain perception.
Project description:It has been reported that remarkable and sustained activation of astrocytes and/or microglia occurs in cancer induced pain (CIP), which is different from neuropathic and inflammatory pain. The present study was designed to investigate the role of spinal Toll-like receptor 4 (TLR4) induced glial neuroinflammation in cancer induced pain using a modified rat model of bone cancer. The rat model of CIP consisted of unilateral intra-tibial injection with Walker 256 mammary gland carcinoma. Nine days after Walker 256 inoculation, a robust activation of both astrocytes and microglia in bilateral spinal dorsal horn was observed together with significant bilateral mechanical allodynia. This neuroinflammation was characterized by enhanced immunostaining of both glial fibrillary acidic protein (GFAP, astrocyte marker) and OX-42 (microglia marker), and an elevated level of IL-1?, IL-6 and TNF-? mRNA. I.t. administration of fluorocitrate (an inhibitor of glial metabolism, 1 nmol) or minocycline (an inhibitor of microglia, 100 ?g) has significant anti-allodynic effects on day 12 after Walker 256 inoculation. Naloxone (a nonstereoselective TLR4 signaling blocker, 60 ?g, i.t.) also significantly alleviated mechanical allodynia and simultaneously blocked the increased inflammatory cytokine mRNA. The results suggested that spinal TLR4 might play an important role in the sustained glial activation that critically contributed to the robust and sustained spinal neuroinflammation in CIP. This result could potentially help clinicians and researchers to better understand the mechanism of complicated cancer pain.
Project description:Cytokines such as interleukins are known to be involved in the development of neuropathic pain through activation of neuroglia. However, the role of chemokine (C-C motif) ligand 1 (CCL-1), a well-characterized chemokine secreted by activated T cells, in the nociceptive transmission remains unclear. We found that CCL-1 was upregulated in the spinal dorsal horn after partial sciatic nerve ligation. Therefore, we examined actions of recombinant CCL-1 on behavioural pain score, synaptic transmission, glial cell function and cytokine production in the spinal dorsal horn. Here we show that CCL-1 is one of the key mediators involved in the development of neuropathic pain. Expression of CCL-1 mRNA was mainly detected in the ipsilateral dorsal root ganglion, and the expression of specific CCL-1 receptor CCR-8 was upregulated in the superficial dorsal horn. Increased expression of CCR-8 was observed not only in neurons but also in microglia and astrocytes in the ipsilateral side. Recombinant CCL-1 injected intrathecally (i.t.) to naive mice induced allodynia, which was prevented by the supplemental addition of N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801. Patch-clamp recordings from spinal cord slices revealed that application of CCL-1 transiently enhanced excitatory synaptic transmission in the substantia gelatinosa (lamina II). In the long term, i.t. injection of CCL-1 induced phosphorylation of NMDA receptor subunit, NR1 and NR2B, in the spinal cord. Injection of CCL-1 also upregulated mRNA level of glial cell markers and proinflammatory cytokines (IL-1?, TNF-? and IL-6). The tactile allodynia induced by nerve ligation was attenuated by prophylactic and chronic administration of neutralizing antibody against CCL-1 and by knocking down of CCR-8. Our results indicate that CCL-1 is one of the key molecules in pathogenesis, and CCL-1/CCR-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain.
Project description:BACKGROUND AND PURPOSE: Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. EXPERIMENTAL APPROACH: We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. KEY RESULTS: Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. CONCLUSIONS AND IMPLICATIONS: The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.
Project description:Studies show that spinal (intrathecal; i.t.) interleukin-10 (IL-10) gene therapy reverses neuropathic pain in animal models, and co-administration with the mannose receptor (MR; CD206) ligand d-mannose (DM) greatly improves therapeutic efficacy. However, the actions of endogenous IL-10 may be required for enduring pain control observed following i.t. IL-10 gene therapy, potentially narrowing the application of this non-viral transgene delivery approach. Here, we show that i.t. application of naked plasmid DNA expressing the IL-10 transgene co-injected with DM (DM/pDNA-IL-10) for the treatment of peripheral neuropathic pain in IL-10 deficient (IL-10 KO) mice results in a profound and prolonged bilateral pain suppression. Neuropathic pain is induced by unilateral sciatic chronic constriction injury (CCI), and while enduring relief of light touch sensitivity (mechanical allodynia) in both wild type (WT) and IL-10 KO mice was observed following DM/pDNA-IL-10 co-therapy, transient reversal from allodynia was observed following i.t. DM alone. In stably pain-relieved IL-10 KO mice given DM/pDNA-IL-10, mRNA for the IL-10 transgene is detected in the cauda equina and ipsilateral dorsal root ganglia (DRG), but not the lumbar spinal cord. Further, DM/pDNA-IL-10 application increases anti-inflammatory TGF-?1 and decreases pro-inflammatory TNF mRNA in the ipsilateral DRG compared to allodynic controls. Additionally, DM/pDNA-IL-10 treated mice exhibit decreased spinal pro-inflammatory mRNA expression for TNF, CCL2 (MCP-1), and for the microglial-specific marker TMEM119. Similarly, DM/pDNA-IL-10 treatment decreases immunoreactivity for the astrocyte activation marker GFAP in lumbar spinal cord dorsal horn. Despite transient reversal and early return to allodynia in DM-treated mice, lumbar spinal cord revealed elevated TNF, CCL2 and TMEM119 mRNA levels. Both MR (CD206) and IL-10 receptor mRNAs are increased in the DRG following CCI manipulation independent of injection treatment, suggesting that pathological conditions stimulate upregulation and availability of relevant receptors in critical anatomical regions required for the therapeutic actions of the DM/pDNA-IL-10 co-therapy. Taken together, the current report demonstrates that non-viral DM/pDNA-IL-10 gene therapy does not require endogenous IL-10 for enduring relief of peripheral neuropathic pain and does not require direct contact with the spinal cord dorsal horn for robust and enduring relief of neuropathic pain. Spinal non-viral DM/pDNA-IL-10 co-therapy may offer a framework for the development of non-viral gene therapeutic approaches for other diseases of the central nervous system.
Project description:Mas oncogene-related gene (Mrg) receptors are exclusively distributed in small-sized neurons in trigeminal and dorsal root ganglia (DRG). We investigated the effects of MrgC receptor activation on inflammatory hyperalgesia and its mechanisms.A selective MrgC receptor agonist, bovine adrenal medulla peptide 8-22 (BAM8-22) or melanocyte-stimulating hormone (MSH) or the ?-opioid receptor (MOR) antagonist CTAP was administered intrathecally (i.t.) in rats injected with complete Freund's adjuvant (CFA) in one hindpaw. Thermal and mechanical nociceptive responses were assessed. Neurochemicals were measured by immunocytochemistry, Western blot, ELISA and RT-PCR.CFA injection increased mRNA for MrgC receptors in lumbar DRG. BAM8-22 or MSH, given i.t., generated instant short and delayed long-lasting attenuations of CFA-induced thermal hyperalgesia, but not mechanical allodynia. These effects were associated with decreased up-regulation of neuronal NOS (nNOS), CGRP and c-Fos expression in the spinal dorsal horn and/or DRG. However, i.t. administration of CTAP blocked the induction by BAM8-22 of delayed anti-hyperalgesia and inhibition of nNOS and CGRP expression in DRG. BAM8-22 also increased mRNA for MORs and pro-opiomelanocortin, along with ?-endorphin content in the lumbar spinal cord and/or DRG. MrgC receptors and nNOS were co-localized in DRG neurons.Activation of MrgC receptors suppressed up-regulation of pronociceptive mediators and consequently inhibited inflammatory pain, because of the activation of up-regulated MrgC receptors and subsequent endogenous activity at MORs. The uniquely distributed MrgC receptors could be a novel target for relieving inflammatory pain.
Project description:BACKGROUND:Neuropathic pain belongs to chronic pain and is caused by the primary dysfunction of the somatosensory nervous system. Long noncoding RNAs (lncRNAs) have been reported to regulate neuronal functions and play significant roles in neuropathic pain. DLEU1 has been indicated to have close relationship with neuropathic pain. Therefore, our study focused on the significant role of DLEU1 in neuropathic pain rat models. METHODS:We first constructed a chronic constrictive injury (CCI) rat model. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were employed to evaluate hypersensitivity in neuropathic pain. RT-qPCR was performed to analyze the expression of target genes. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the concentrations of interleukin-6 (IL-6), tumor necrosis factor-? (TNF-?) and IL-1?. The underlying mechanisms of DLEU1 were investigated using western blot and luciferase reporter assays. RESULTS:Our findings showed that DLEU1 was upregulated in CCI rats. DLEU1 knockdown reduced the concentrations of IL-6, IL-1? and TNF-? in CCI rats, suggesting that neuroinflammation was inhibited by DLEU1 knockdown. Besides, knockdown of DLEU1 inhibited neuropathic pain behaviors. Moreover, it was confirmed that DLEU1 bound with miR-133a-3p and negatively regulated its expression. SRPK1 was the downstream target of miR-133a-3p. DLEU1 competitively bound with miR-133a-3p to upregulate SRPK1. Finally, rescue assays revealed that SRPK1 overexpression rescued the suppressive effects of silenced DLEU1 on hypersensitivity in neuropathic pain and inflammation of spinal cord in CCI rats. CONCLUSION:DLEU1 regulated inflammation of the spinal cord and mediated hypersensitivity in neuropathic pain in CCI rats by binding with miR-133a-3p to upregulate SRPK1 expression.