Animal Studies

Trigeminal neuralgia (TN) is the most common neuropathic pain in the facial area, for which the effective therapy is unavailable. Long non-coding RNA (lncRNA) such as lncRNA uc.48+ is involved in diabetic neuropathic pain and may affect purinergic signaling in ganglia of diabetic rats. In this research, chronic constriction injury of the infraorbital nerve (CCI-ION) was applied to establish a rat model of TN. Five days after local injection of siRNA targeting the lncRNA uc.48+ in trigeminal ganglia (TGs), the upregulated uc.48+ expression and the reduced mechanical withdrawal threshold (MWT) in the TN rats were significantly reversed. The expression of P2X receptor in TGs was increased in the TN group compared with the sham group, but uc.48+ siRNA treatment mitigated this effect. The phosphorylation of ERK1/2 in TGs of TN rats was significantly enhanced compared with the sham group, while uc.48+ siRNA treatment reversed this change. In addition, injection of the lncRNA uc.48+ overexpression plasmid in TGs of control rats significantly reduced the MWT but elevated the expression of P2X in TGs; the phosphorylation of ERK1/2 in TGs in these uc.48+-overexpressed rats was significantly higher, similar to the observations in rats of TN model. The interaction between uc.48+ and the P2X receptor was detected by RNA binding protein immunoprecipitation (RIP), indicating that P2X receptor could specifically bind to uc.48 + . In summary, knockdown of lncRNA uc.48+ by siRNA could inhibit transduction of TN signals, whereas uc.48+ overexpression promoted TN signal transduction. LncRNA uc.48+ may interact with P2X receptor to upregulate expression of P2X receptor and furthermore enhance the phosphorylation of ERK1/2 in TGs, thereby participating in pain transmission in TN.

The Tac4 gene-derived hemokinin-1 (HK-1) is present in pain-related regions and activates the tachykinin NK1 receptor, but with binding site and signaling pathways different from Substance P (SP). NK1 receptor is involved in nociception, but our earlier data showed that it has no role in chronic neuropathic hyperalgesia, similarly to SP. Furthermore, NK1 antagonists failed in clinical trials as analgesics due to still unknown reasons. Therefore, we investigated the role of HK-1 in pain conditions of distinct mechanisms using genetically modified mice. Chronic neuropathic mechanical and cold hyperalgesia after sciatic nerve ligation were determined by dynamic plantar aesthesiometry and withdrawal latency from icy water, motor coordination on the accelerating Rotarod. Peripheral nerve growth factor (NGF) production was measured by ELISA, neuronal and glia cell activation by immunohistochemistry in pain-related regions. Acute somatic and visceral chemonocifensive behaviors were assessed after intraplantar formalin or intraperitoneal acetic-acid injection, respectively. Resiniferatoxin-induced inflammatory mechanical and thermal hyperalgesia by aesthesiometry and increasing temperature hot plate. Chronic neuropathic mechanical and cold hypersensitivity were significantly decreased in HK-1 deficient mice. NGF level in the paw homogenates of intact mice were significantly lower in case of HK-1 deletion. However, it significantly increased under neuropathic condition in contrast to wildtype mice, where the higher basal concentration did not show any alterations. Microglia, but not astrocyte activation was observed 14 days after PSL in the ipsilateral spinal dorsal horn of WT, but not HK-1-deficient mice. However, under neuropathic conditions, the number of GFAP-positive astrocytes was significantly smaller in case of HK-1 deletion. Acute visceral, but not somatic nocifensive behavior, as well as neurogenic inflammatory mechanical and thermal hypersensitivity were significantly reduced by HK-1 deficiency similarly to NK1, but not to SP deletion. We provide evidence for pro-nociceptive role of HK-1, via NK1 receptor activation in acute inflammation models, but differently from SP-mediated actions. Identification of its targets and signaling can open new directions in pain research.

Pain is the main reason patients report Osteoarthritis (OA), yet current analgesics remain relatively ineffective. This study investigated both peripheral and central mechanisms that lead to the development of OA associated chronic pain.

The efficacy of mesenchymal stem cell (MSC) therapies is increasingly attributed to paracrine secretion, particularly exosomes. In this study, we investigated the role of MSC exosomes in the regulation of inflammatory response, nociceptive behaviour, and condylar cartilage and subchondral bone healing in an immunocompetent rat model of temporomandibular joint osteoarthritis (TMJ-OA). We observed that exosome-mediated repair of osteoarthritic TMJs was characterized by early suppression of pain and degeneration with reduced inflammation, followed by sustained proliferation and gradual improvements in matrix expression and subchondral bone architecture, leading to overall joint restoration and regeneration. Using chondrocyte cultures, we could attribute some of the cellular activities during exosome-mediated joint repair to adenosine activation of AKT, ERK and AMPK signalling. Specifically, MSC exosomes enhanced s-GAG synthesis impeded by IL-1β, and suppressed IL-1β-induced nitric oxide and MMP13 production. These effects were partially abrogated by inhibitors of adenosine receptor activation, AKT, ERK and AMPK phosphorylation. Together, our observations suggest that MSC exosomes promote TMJ repair and regeneration in OA through a well-orchestrated mechanism of action that involved multiple cellular processes to restore the matrix and overall joint homeostasis. This study demonstrates the translational potential of a cell-free ready-to-use exosome-based therapeutic for treating TMJ pain and degeneration.

Glycine receptors (GlyRs) are pentameric proteins that consist of α (α1-α4) subunits and/or β subunit. In the spinal cord of adult animals, the majority of inhibitory glycinergic neurotransmission is mediated by α1 subunit-containing GlyRs. The reduced glycinergic inhibition (disinhibition) is proposed to increase the excitabilities and spontaneous activities of spinal nociceptive neurons during pathological pain. However, the molecular mechanisms by which peripheral lesions impair GlyRs-α1-mediated synaptic inhibition remain largely unknown. Here we found that activity-dependent ubiquitination of GlyRs-α1 subunit might contribute to glycinergic disinhibition after peripheral inflammation. Our data showed that HUWE1 (HECT, UBA, WWE domain containing 1), an E3 ubiquitin ligase, located at spinal synapses and specifically interacted with GlyRs-α1 subunit. By ubiquitinating GlyRs-α1, HUWE1 reduced the surface expression of GlyRs-α1 through endocytic pathway. In the dorsal horn of Complete Freund's Adjuvant-injected mice, shRNA-mediated knockdown of HUWE1 blunted GlyRs-α1 ubiquitination, potentiated glycinergic synaptic transmission and attenuated inflammatory pain. These data implicated that ubiquitin modification of GlyRs-α1 represented an important way for peripheral inflammation to reduce spinal glycinergic inhibition and that interference with HUWE1 activity generated analgesic action by resuming GlyRs-α1-mediated synaptic transmission.

Chronic pain is associated with neuroplastic changes in the amygdala that may promote hyper-responsiveness to mechanical and thermal stimuli (allodynia and hyperalgesia) and/or enhance emotional and affective consequences of pain. Stress promotes dynorphin-mediated signaling at the kappa opioid receptor (KOR) in the amygdala and mechanical hypersensitivity in rodent models of functional pain. Here, we tested the hypothesis that KOR circuits in the central nucleus of the amygdala (CeA) undergo neuroplasticity in chronic neuropathic pain resulting in increased sensory and affective pain responses. After spinal nerve ligation (SNL) injury in rats, pretreatment with a long-acting KOR antagonist, nor-binaltorphimine (nor-BNI), subcutaneously or through microinjection into the right CeA, prevented conditioned place preference (CPP) to intravenous gabapentin, suggesting that nor-BNI eliminated the aversiveness of ongoing pain. By contrast, systemic or intra-CeA administration of nor-BNI had no effect on tactile allodynia in SNL animals. Using whole-cell patch-clamp electrophysiology, we found that nor-BNI decreased synaptically evoked spiking of CeA neurons in brain slices from SNL but not sham rats. This effect was mediated through increased inhibitory postsynaptic currents, suggesting tonic disinhibition of CeA output neurons due to increased KOR activity as a possible mechanism promoting ongoing aversive aspects of neuropathic pain. Interestingly, this mechanism is not involved in SNL-induced mechanical allodynia. Kappa opioid receptor antagonists may therefore represent novel therapies for neuropathic pain by targeting aversive aspects of ongoing pain while preserving protective functions of acute pain.

Stability of local medial prefrontal cortex (mPFC) network activity is believed to be critical for sustaining cognitive processes such as working memory (WM) and decision making. Dysfunction of the mPFC has been identified as a leading cause to WM deficits in several chronic pain conditions; however, the underlying mechanisms remain largely undetermined. Here, to address this issue, we implanted multichannel arrays of electrodes in the prelimbic region of the mPFC and recorded the neuronal activity during a food-reinforced delayed nonmatch to sample (DNMS) task of spatial WM. In addition, we used an optogenetic technique to selectively suppress the activity of excitatory pyramidal neurons that are considered the neuronal substrate for memory retention during the delay period of the behavioral task. Within-subject behavioral performance and pattern of neuronal activity were assessed after the onset of persistent pain using the spared nerve injury model of peripheral neuropathy. Our results show that the nerve lesion caused a disruption in WM and prelimbic spike activity and that this disruption was reversed by the selective inhibition of prelimbic glutamatergic pyramidal neurons during the delay period of the WM task. In spared nerve injury animals, photoinhibition of excitatory neurons improved the performance level and restored neural activity to a similar profile observed in the control animals. In addition, we found that selective inhibition of excitatory neurons does not produce antinociceptive effects. Together, our findings suggest that disruption of balance in local prelimbic networks may be crucial for the neurological and cognitive deficits observed during painful syndromes.