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The emerging role of inflammation in the initiation and maintenance of neuropathic pain has been confirmed. Previous studies have reported that miR138 has neuroprotective and anti-inflammatory effects in animal models of spinal cord injury and in human coronary artery endothelial cell injury, while its effect on neuropathic pain is still not known. As the mechanism of neuropathic pain remains unclear, we investigated whether miR138 is involved in the development of neuropathic pain and the role of miR138 in the modulation of inflammation in the spinal cord in a mouse model of neuropathic pain induced by spared sciatic nerve injury (SNI).
Learn More >We bilaterally injected 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle of rats and developed bilateral Parkinson's disease (PD) model rats in order to experimentally investigate the neural mechanisms underlying the alteration of nociception in the orofacial region of patients with PD. We explored the effects of dopamine depletion on nociception by investigating behavioral responses (face rubbing) triggered by subcutaneous administration of formalin into the vibrissa pad. We also assessed the number of c-Fos-immunoreactive (c-Fos-IR) cells in the superficial layers of the trigeminal spinal subnucleus caudalis (Vc). Subcutaneous formalin administration evoked a two-phase increase in face rubbing. We observed the first increase 0-5 min after formalin administration (first phase) and the second increase 10-60 min after administration (second phase). The number of face rubbing behaviors of 6OHDA-injected rats did not significantly change compared with saline-injected rats in both phases. Significant increase of c-Fos-IR cells in the Vc was found in 6-OHDA-injected rats after formalin administration compared with those in saline-injected rats after formalin administration. We also assessed expression of c-Fos-IR cells in the paraventricular nucleus (PVN), and significant decrease of c-Fos-IR cells in the PVN of 6-OHDA-injected rats was found. Taken together, these findings suggest that bilateral dopaminergic denervation evoked by 6-OHDA administration causes hyperalgesia in the trigeminal region and the PVN may be involved in the hyperalgesia.
Learn More >Orthodontic force produces mechanical irritation and inflammation in the periodontium, which is inevitably accompanied by pain. Despite its prevalence, treatment of orthodontic pain is ineffective. Elucidating underlying neural mechanisms is critical to improving the management of orthodontic pain. We have assessed the contribution of transient receptor potential vanilloid subtype 1 (TRPV1) and the TRPV1-expressing subset of nociceptive afferents to pain behaviors induced by orthodontic force in mice. Microfocus X-ray computed tomography analysis showed that application of an orthodontic force of 10 g to the maxillary first molar produced reliable tooth movement in mice. Mouse grimace scale (MGS) was evaluated as an indication of non-evoked spontaneous pain and bite force (BF) was measured for assessing bite-evoked nocifensive behaviors. Orthodontic force increased MGS and decreased BF, both of which were interpreted as increased levels of pain. These behaviors peaked at 1d and returned near to the sham level at 7d. Retrograde labeling and immunohistochemical assays showed TRPV1-expressing peptidergic afferents are abundantly projected to the periodontium. Direct injection of resiniferatoxin into trigeminal ganglia (TG) decreased TRPV1-expressing afferents by half in the targeted region of TG. The chemical ablation of TRPV1-expressing afferents significantly attenuated orthodontic pain behaviors assessed by MGS and BF. Consistently, the knockout of TRPV1 also attenuated orthodontic force-induced changes in MGS and BF. These results suggest that TRPV1 and TRPV1-expressing trigeminal nociceptors constitute a primary pathway mediating orthodontic pain behaviors in mice. This model will be useful for mechanistic studies on orthodontic pain aimed at developing novel approaches for painless orthodontics.
Learn More >Voltage-gated calcium channels (VGCCs) are important mediators of pain hypersensitivity during inflammatory states, but their role in sensory nerve growth remains underexplored. Here, we assess the role of the N-type calcium channel Cav2.2 in the complete Freund's adjuvant (CFA) model of inflammatory pain. We demonstrate with hybridization and immunoblotting, an increase in Cav2.2 expression after hind paw CFA injection in sensory neurons that respond to thermal stimuli, but not in two different mechanosensitive neuronal populations. Further, Cav2.2 upregulation post-CFA correlates with thermal but not mechanical hyperalgesia in behaving mice, and this hypersensitivity is blocked with a specific Cav2.2 inhibitor. Voltage clamp recordings reveal a significant increase in Cav2.2 currents post-CFA, while current clamp analyses demonstrate a significant increase in action potential frequency. Moreover, CFA-induced sensory nerve growth, which involves the extracellular signal-related kinase (ERK1/2) signaling pathway and likely contributes to inflammation-induced hyperalgesia, was blocked with the Cav2.2 inhibitor. Together, this work uncovers a role for Cav2.2 during inflammation, demonstrating that VGCC activity can promote thermal hyperalgesia through both changes in firing rates of sensory neurons as well as promotion of new neurite outgrowth.
Learn More >Pain is the most severe and common symptom of endometriosis. Its underlying pathogenetic mechanism is poorly understood. Nerve sensitization is a particular research challenge, due to the limitations of general endometriosis models and sampling nerve tissue from patients. The chemokine fractalkine (FKN) has been demonstrated to play a key role in various forms of neuropathic pain, while its role in endometriotic pain is unknown. Our study was designed to explore the function of FKN in the development and maintenance of peripheral hyperalgesia and central sensitization in endometriosis using a novel endometriosis animal model developed in our laboratory. After modeling, behavioral tests were carried out and the optimal time for molecular changes was obtained. We extracted ectopic tissues and L4-6 spinal cords to detect peripheral and central roles for FKN, respectively. To assess morphologic characteristics of endometriosis-like lesions-as well as expression and location of FKN/CX3CR1-we performed H&E staining, immunostaining, and western blotting analyses. Furthermore, inhibition of FKN expression in the spinal cord was achieved by intrathecal administration of an FKN-neutralizing antibody to demonstrate its function. Our results showed that implanted autologous uterine tissue around the sciatic nerve induced endometriosis-like lesions and produced mechanical hyperalgesia and allodynia. FKN was highly expressed on macrophages, whereas its receptor CX3CR1 was overexpressed in the myelin sheath of sciatic nerve fibers. Overexpressed FKN was also observed in neurons. CX3CR1/pp38-MAPK was upregulated in activated microglia in the spinal dorsal horn. Intrathecal administration of FKN-neutralizing antibody not only reversed the established mechanical hyperalgesia and allodynia, but also inhibited the expression of CX3CR1/pp38-MAPK in activated microglia, which was essential for the persistence of central sensitization. We concluded that the FKN/CX3CR1 signaling pathway might be one of the mechanisms of peripheral hyperalgesia in endometriosis, which requires further studies. Spinal FKN is important for the development and maintenance of central sensitization in endometriosis, and it may further serve as a novel therapeutic target to relieve persistent pain associated with endometriosis.
Learn More >Osteoarthritis (OA) is a degenerative disease that induces pain, cartilage deformation, and joint inflammation. Mesenchymal stem cells (MSCs) are potential therapeutic agents for treatment of OA. However, MSC therapy can cause excessive inflammation. Signal transducer and activator of transcription 3 (STAT3) modulates secretion of many proinflammatory cytokines. Experimental OA was induced by intra-articular (IA) injection of monosodium iodoacetate (MIA) to the right knee of rats. MSCs from OA patients (OA-MSCs) were treated with STA21, a small molecule that blocks STAT3 signaling, by IA or intravenous (IV) injection after MIA injection. Pain severity was quantified by assessment of secondary tactile allodynia using the von Frey assessment test. Cartilage degradation was measured by microcomputed tomography image analysis, histological analysis, and the Mankin score. Protein and gene expression was evaluated by enzyme-linked immunosorbent assay, immunohistochemistry, and real-time polymerase chain reaction. MSCs increased production of proinflammatory cytokines under inflammatory conditions. STA21 significantly decreased expression of these proinflammatory molecules via inhibition of STAT3 activity but increased gene expression of molecules related to migration potential and immunomodulation in OA-MSCs. STAT3-inhibited OA-MSCs administrated by IV or IA injection decreased pain severity and cartilage damage in rats with MIA-induced OA rats by decreasing proinflammatory cytokines in the joints. Combined IA and IV-injected STAT3-inhibited OA-MSCs had an additive effect of pain relief in MIA-induced OA rats. STAT3 inhibition may optimize the therapeutic activities of MSCs for treating OA by attenuating pain and progression of MIA by inhibiting inflammation and cartilage damage.
Learn More >Enhancement of endocannabinoid signaling has emerged as an attractive strategy for the treatment of pain. In addition to the well-characterized hydrolytic pathways, cyclooxygenase-2 (COX-2) mediated oxygenation is thought to be an alternative route for endocannabinoid metabolism and therefore provides a new avenue for drug intervention. In this study, we examined the therapeutic effect of indomethacin morpholinamide (IMMA), a novel substrate-selective COX-2 inhibitor, in the chronic constriction injury (CCI) mouse model. Treatment with IMMA significantly alleviated hyperalgesia and mechanical allodynia demonstrated by increased thermal withdrawal latency in Hargreaves test and tactile thresholds in Von Frey test. Accumulation of astrocytes and microglia in spinal cord dorsal horn and infiltration of macrophages into the dorsal root ganglion and sciatic nerve were reduced by drug treatment. Co-administration of the CB2 receptor antagonist, but not the CB1 receptor antagonist partially reversed the inhibitory effect of IMMA on pain sensitivity and inflammatory infiltrates. IMMA downregulated the mRNA expression of TNF-α and IL-1β and the production of IL-6 and MCP-1 proteins in the ipsilateral sciatic nerve. The enhanced NF-κB DNA binding activity in the CCI mouse dorsal spinal cord was also significantly reduced, suggesting that inactivation of NF-κB contributes to the anti-inflammatory property of IMMA. However, different from the previous reports showing that IMMA can increase endocannabinoids without interfering with arachidonic acid metabolism, treatment with IMMA failed to elevate the endogenous levels of AEA and 2-AG, but significantly reduced the production of prostaglandin E (PGE). Furthermore, the mRNA expression of enzymes involved in PGE production, COX-2 and prostaglandin E synthase 2 in the ipsilateral sciatic nerve was also suppressed by IMMA treatment. Taken together, these results suggested that IMMA might exert anti-nociceptive effects through multiple mechanisms which include, but are not limited to, CB2 receptor activation and reduced PGE production.
Learn More >Neuropathic pain often arises from damage to peripheral nerves and is difficult to treat. Activation of opioid receptors in peripheral sensory neurons is devoid of respiratory depression, sedation, nausea, and addiction mediated in the brain, and ameliorates neuropathic pain in animal models. Mechanisms of peripheral opioid analgesia have therefore gained interest, but the role of G protein-coupled inwardly rectifying potassium (Kir3) channels, important regulators of neuronal excitability, remains unclear. Whereas functional Kir3 channels have been detected in dorsal root ganglion (DRG) neurons in rats, some studies question their contribution to opioid analgesia in inflammatory pain models in mice. However, neuropathic pain can be diminished by activation of peripheral opioid receptors in mouse models. Therefore, here we investigated effects of the selective μ-opioid receptor (MOR) agonist [D-Ala, -Me-Phe, Gly-ol]-enkephalin (DAMGO) on potassium conductance in DRG neurons upon a chronic constriction injury (CCI) of the sciatic nerve in mice. For verification, we also tested human embryonic kidney (HEK) 293 cells transfected with MOR and Kir3.2. Using patch clamp, we recorded currents at -80 mV and applied voltage ramps in high extracellular potassium concentrations, which are a highly sensitive measures of Kir3 channel activity. We found a significantly higher rate of HEK cells responding with potassium channel blocker barium-sensitive inward current (233 ± 51 pA) to DAMGO application in transfected than in untransfected group, which confirms successful recordings of inward currents through Kir3.2 channels. Interestingly, DAMGO induced similar inward currents (178 ± 36-207 ± 56 pA) in 15-20% of recorded DRG neurons from naïve mice and in 4-27% of DRG neurons from mice exposed to CCI, measured in voltage clamp or voltage ramp modes. DAMGO-induced currents in naïve and CCI groups were reversed by barium and a more selective Kir3 channel blocker tertiapin-Q. These data indicate the coupling of Kir3 channels with MOR in mouse peripheral sensory neuron cell bodies, which was unchanged after CCI. A comparative analysis of opioid-induced potassium conductance at the axonal injury site and peripheral terminals of DRG neurons could clarify the role of Kir3 channel-MOR interactions in peripheral nerve injury and opioid analgesia.
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