Animal Studies

NADPH oxidase (NOX2) is an enzyme that induces reactive oxygen species (ROS) and serves as a switch between the pro-inflammatory and neurorestorative microglial/macrophage phenotypes; such changes play an important role in neuropathic pain and motor dysfunction. Increased NOX2 expression after spinal cord injury (SCI) has been reported, and inhibition of NOX2 improves motor function. However, the underlying mechanisms of NOX2 in post-traumatic pain and motor deficit remain unexplored. In the present study, we report that depletion of NOX2 (NOX2) or inhibition of NOX2 using NOX2ds-tat significantly reduced mechanical/thermal cutaneous hypersensitivity and motor dysfunction after moderate contusion SCI at T10 in male mice. Western blot (WB, 3 mm lesion area) and immunohistochemistry (IHC) showed that SCI elevates NOX2 expression predominantly in microglia/macrophages up to 8 weeks post-injury. Deletion of NOX2 significantly reduced CD11b/CD45F4/80 macrophage infiltration at 24h post-injury detected by flow cytometry and 8-OHG ROS production at 8 weeks post-injury by IHC in both lesion area and lumbar enlargement. NOX2 deficiency also altered microglial/macrophage pro-inflammatory and anti-inflammatory balance towards the neurorestorative response. WB analysis showed robust increase of Arginase-1 and YM1 proteins in NOX2 mice. Furthermore, qPCR analysis showed significant up-regulation of anti-inflammatory cytokine IL-10 levels in NOX2 mice, associated with reduced microRNA-155 expression. These findings were confirmed in CD11b microglia/macrophages isolated from spinal cord at 3 days post-injury. Taken together, our data suggest an important role for IL-10/miR-155 pathway in regulating NOX2-mediated SCI-dysfunction. Thus, specific targeting of NOX2 may provide an effective strategy for treating neurological dysfunction in SCI patients.

The accurate diagnosis and treatment of pain is dependent upon the knowledge of variables that might alter this response. Some of these variables are the locality of the noxious stimulus, the sex of the individual, and the presence of chronic diseases. Among these chronic diseases, hypertension is considered a serious and silent disease that has been associated with hypoalgesia. The main goal of this study was to evaluate the potential nociceptive differences in spontaneously hypertensive rats (SHR) regarding the locality of the stimulus, the temporomandibular joint or paw, the sex, and the role of ovarian hormones in a model of mechanical nociception (Von Frey test) or formalin-induced inflammatory nociception. Our results indicate that SHR has lower orofacial mechanical nociception beyond the lower mechanical nociception in the paw compared to WKY rats. In a model of formalin-induced inflammatory nociception, SHR also has a decreased nociception compared to normotensive rats. We also sought to evaluate the influence of sex and ovarian hormones on orofacial mechanical nociception in SHR. We observed that female SHR has higher mechanical nociception than male SHR only in the paw, but it has higher formalin-induced orofacial nociception than male SHR. Moreover, the absence of ovarian hormones caused an increase in mean arterial pressure and a decrease in paw nociception in female SHR.

Sphingosine-1-phosphate (S1P) receptor 1 subtype (S1PR1) activation by its ligand S1P in the dorsal horn of the spinal cord (DH-SC) causes mechano-hypersensitivity. The cellular and molecular pathways remain poorly understood. We now report that activation of S1PR1 with intrathecal injection of the highly selective S1PR1 agonist SEW2871 led to the development of mechano-allodynia by activating the nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome (increased expression of NLRP3, cleaved caspase 1 and mature interleukin (IL)-1β) in the DH-SC. The functional S1PR1 antagonist FTY720 blocked NLRP3 activation and IL-1β production. Moreover, inhibiting IL-10 signaling with an intrathecal injection of an anti-IL-10 antibody attenuated the beneficial effects exerted by FTY720. This suggests that disrupting S1PR1 signaling engages beneficial IL-10-dependent pathways. Noteworthy, we found that mice with astrocyte-specific deletions of S1pr1 did not develop mechano-allodynia following intrathecal injection of SEW2871 and exhibited reduced levels of cleaved caspase 1; identifying astrocytes as a key cellular locus for S1PR1 activity. Our findings provide novel mechanistic insights on how S1PR1 activation in the spinal cord contributes to the development of nociception while identifying the cellular substrate for these activities. PERSPECTIVE: This is the first study to link the activation of NLRP3 and IL-1β signaling in the spinal cord and S1PR1 signaling in astrocytes to the development of S1PR1-evoked mechano-allodynia. These findings provide critical basic science insights to support the development of therapies targeted toward S1PR1.

Long-term stress was suggested to cause visceral hypersensitivity and promote functional gastrointestinal disorders (FGIDs). Some brain regions such as the anterior cingulate cortex (ACC) may play an important role for generating visceral hypersensitivity; however, its molecular mechanisms are not clear. This study aimed to explore the role of 5-HT1A receptors (HTR1As) in activating ACC and corresponding mechanism, in stress-induced visceral hyperalgesia rats.

Painful diabetic neuropathy (PDN) is a severely debilitating chronic pain syndrome. Spinal chemokine CXCL13 and its receptor CXCR5 were recently demonstrated to play a pivotal role in the pathogenesis of chronic pain induced by peripheral tissue inflammation or nerve injury. In this study we investigated whether CXCL13/CXCR5 mediates PDN and the underlying spinal mechanisms. We used the db/db type 2 diabetes mice, which showed obvious hyperglycemia and obese, long-term mechanical allodynia, and increased expression of CXCL13, CXCR5 as well as pro-inflammatory cytokines TNF-α and IL-6 in the spinal cord. Furthermore, in the spinal cord of db/db mice there is significantly increased gliosis and upregulated phosphorylation of cell signaling kinases, including pERK, pAKT and pSTAT3. Mechanical allodynia and upregulated pERK, pAKT and pSTAT3 as well as production of TNF-α and IL-6 were all attenuated by the noncompetitive NMDA receptor antagonist MK-801. If spinal giving U0126 (a selective MEK inhibitor) or AG490 (a Janus kinase (JAK) -STAT inhibitor) to db/db mice, both of them can decrease the mechanical allodynia, but only inhibit pERK (by U0126) or pSTAT3 (by AG490) respectively. Acute administration of CXCL13 in C57BL/6J mice resulted in exacerbated thermal hyperalgesia and mechanical allodynia, activation of the pERK, pAKT and pSTAT3 pathways and increased production of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6), which were all attenuated by knocking out of Cxcr5. In all, our work showed that chemokine CXCL13 and its receptor CXCR5 in spinal cord contribute to the pathogenesis of PDN and may help develop potential novel therapeutic approaches for patients afflicted with PDN.