Mechanical Allodynia In Bilateral Hindpaws Research Articles

CXCL13 contributes to chronic pain of a mouse model of CRPS-I via CXCR5-mediated NF-κB activation and pro-inflammatory cytokine production in spinal cord dorsal horn

BackgroundComplex regional pain syndrome type-I (CRPS-I) causes excruciating pain that affect patients’ life quality. However, the mechanisms underlying CRPS-I are incompletely understood, which hampers the development of target specific therapeutics.MethodsThe mouse chronic post-ischemic pain (CPIP) model was established to mimic CRPS-I. qPCR, Western blot, immunostaining, behavioral assay and pharmacological methods were used to study mechanisms underlying neuroinflammation and chronic pain in spinal cord dorsal horn (SCDH) of CPIP mice.ResultsCPIP mice developed robust and long-lasting mechanical allodynia in bilateral hindpaws. The expression of inflammatory chemokine CXCL13 and its receptor CXCR5 was significantly upregulated in ipsilateral SCDH of CPIP mice. Immunostaining revealed CXCL13 and CXCR5 was predominantly expressed in spinal neurons. Neutralization of spinal CXCL13 or genetic deletion of Cxcr5 (Cxcr5−/−) significantly reduced mechanical allodynia, as well as spinal glial cell overactivation and c-Fos activation in SCDH of CPIP mice. Mechanical pain causes affective disorder in CPIP mice, which was attenuated in Cxcr5−/− mice. Phosphorylated STAT3 co-expressed with CXCL13 in SCDH neurons and contributed to CXCL13 upregulation and mechanical allodynia in CPIP mice. CXCR5 coupled with NF-κB signaling in SCDH neurons to trigger pro-inflammatory cytokine gene Il6 upregulation, contributing to mechanical allodynia. Intrathecal CXCL13 injection produced mechanical allodynia via CXCR5-dependent NF-κB activation. Specific overexpression of CXCL13 in SCDH neurons is sufficient to induce persistent mechanical allodynia in naïve mice.ConclusionsThese results demonstrated a previously unidentified role of CXCL13/CXCR5 signaling in mediating spinal neuroinflammation and mechanical pain in an animal model of CRPS-I. Our work suggests that targeting CXCL13/CXCR5 pathway may lead to novel therapeutic approaches for CRPS-I.

The up-regulation of IL-6 in DRG and spinal dorsal horn contributes to neuropathic pain following L5 ventral root transection

Our previous works have shown that pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) plays an important role in neuropathic pain produced by lumber 5 ventral root transection (L5-VRT). In the present work we evaluate the role of interleukin-6 (IL-6), another key inflammatory cytokine, in the L5-VRT model. We found that IL-6 was up-regulated in the ipsilateral L4 and L5 dorsal root ganglian (DRG) neurons and in bilateral lumbar spinal cord following L5-VRT. Double immunofluorescence stainings revealed that in DRGs the increased immunoreactivity (IR) of IL-6 was almost restricted in neuronal cells, while in the spinal dorsal horn IL-6-IR up-regulated in both glial cells (astrocyte and microglia) and neurons. Intrathecal administration of IL-6 neutralizing antibody significantly delayed the induction of mechanical allodynia in bilateral hindpaws after L5-VRT. Furthermore, inhibition of TNF-α synthesis by intraperitoneal thalidomide prevented both mechanical allodynia and the up-regulation of IL-6 in DRGs following L5-VRT. These data suggested that the increased IL-6 in afferent neurons and spinal cord contribute to the development of neuropathic pain following motor fiber injury, and that TNF-α is responsible for the up-regulation of IL-6.

Peri-sciatic administration of recombinant rat IL-1β induces mechanical allodynia by activation of src-family kinases in spinal microglia in rats

Previous studies have shown that Interleukin-1 beta (IL-1β) is implicated in the modulation of pain sensitivity. In the present study, we found that a single peri-sciatic administration of rat recombinant IL-1β (rrIL-1β) at doses of 20 and 200pg (100, 1000ng/l, in 200μl volume) induced mechanical allodynia in bilateral hindpaws in rats, lasting for about 50days. No axonal or Schwann cell damage at the drug administration site was found following 1000ng/l rrIL-1β administration. The results of immunofluorescence showed that microglial cells in bilateral spinal dorsal horn were activated after peri-sciatic administration of rrIL-1β (1000ng/l). The immunoreactivity (IR) of Iba1 (a marker for microglia) and phosphorylated src-family kinases (p-SFKs) increased significantly in the ipsilateral and contralateral lumbar spinal dorsal horn on day 1 and day 3 after rrIL-1β administration, respectively. Double immunofluorescence staining revealed that the increased p-SFKs-IR was almost restricted within the microglia. Intrathecal delivery of minocycline (100μg in 10μl volume), a selective inhibitor of microglia, started 30min before rrIL-1β administration and once daily thereafter for 7days, blocked mechanical allodynia induced by rrIL-1β completely and inhibited the upregulation of p-SFKs. Intrathecal delivery of SFKs inhibitor PP2 (12μg in 10μl volume) also blocked mechanical allodynia induced by rrIL-1β completely. These data suggest that activation of SFKs in spinal microglia mediates mechanical allodynia induced by peri-sciatic administration of rrIL-1β.

Peri-sciatic administration of recombinant rat TNF-α induces mechanical allodynia via upregulation of TNF-α in dorsal root ganglia and in spinal dorsal horn: The role of NF-kappa B pathway

Previous studies have shown that tumor necrosis factor-alpha (TNF-α) and TNF receptor 1 (TNFR1) in dorsal root ganglia (DRG) and in spinal dorsal horn are upregulated after nerve injury and that many TNF-α-containing neurons overexpress TNFR1. In the present study, we found that peri-sciatic administration of rat recombinant TNF-α (rrTNF) at the concentrations of 10, 100 and 1000 pg/ml (daily for 2 days) induced mechanical allodynia in bilateral hindpaws, lasting for about 20 days. The immunoreactivity (IR) of TNF-α and TNFR1 in the ipsilateral (but not in the contralateral) L4 and L5 DRGs increased significantly on day 1 and day 3 after administration of rrTNF, respectively. Double immunofluorescence staining revealed that in DRGs the increased TNF-α-IR was mainly in neuronal cells and with a lesser extent in satellite glial cells, while the upregulation of TNFR1-IR was almost restricted at neuronal cells. TNF-α-IR but not TNFR1-IR also increased in bilateral lumbar spinal dorsal horn from day 3 to day 14, which was observed in astrocytes, microglias and neurons. In addition, a progressive infiltration of monocyte/macrophages and T lymphocytes in the ipsilateral L5 DRG and sciatic nerve was observed, starting on day 2 following administration of rrTNF. Intrathecal delivery of PDTC (8.2 ng in 10 μl volume), a nuclear factor-kappa B (NF-κB) inhibitor, 30 min before each rrTNF administration blocked mechanical allodynia completely and inhibited the upregulation of TNF-α-IR and TNFR1-IR substantially. The results suggest that peri-sciatic administration of rrTNF may induce mechanical allodynia by an autocrine mechanism via activation of the NF-κB pathway.