Non–coding RNAs affect various cellular processes through interaction with DNA, RNA and protein. Accordingly, non–coding RNAs, microRNAs and more recently long non–coding RNAs, have been shown to be involved in pain disorders, including neuropathic pain. MicroRNAs inhibit translational step of gene expression and dysregulation of microRNAs underlies the neuropathic pain. On the other hand, lncRNAs regulate diverse steps of gene expression, including epigenetic modulation, transcription, alternative splicing and translation, although a role of lncRNAs in the pain disorders remain poorly understood. Interestingly, a part of non–coding RNAs are released to extracellular space and mediate a cell–cell communication. Extracellular microRNAs are shown to modulate nociceptive transmission. Furthermore, extracellular non–coding RNAs are expected as a specific biomarker for neuronal damage or pain in the blood. In this review, we summarize current insights into non–coding RNA significance in the neuropathic pain.

There is much evidence showing that a group of neuropeptides produced in the hypo­thalamus, oxytocin and orexins, inhibit nociceptive transmission in the rat spinal dorsal horn. In order to reveal cellular mechanisms underlying this antinociception, we examined how oxytocin, orexins A and B affect spontaneous synaptic transmission in rat spinal lamina II (substantia gelatinosa; SG) neurons, which play a pivotal role in regulating nociceptive transmission. The experiments were performed by applying the blind whole–cell patch–clamp technique to SG neurons in adult rat spinal cord slices. Bath–applied oxytocin unaffected glutamatergic spontaneous excitatory transmission while producing an inward current at −70 mV (membrane depolarization) and enhancing both GABAergic and glycinergic spontaneous inhibitory transmissions in >70% of the neurons tested. The depolarization, and increased GABAergic and glycinergic spontaneous inhibitory postsynaptic current (sIPSC) frequencies were concentration–dependent with half–maximal effective concentration (EC₅₀) values of 0.022, 0.024 and 0.038 µM, respectively. On the other hand, orexins A and B produced an inward current at −70 mV and/or increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) without changing its amplitude in some 70% of the neurons examined. EC₅₀ values for orexin A in their effects were 0.0045 and 0.030 µM, respectively; those for orexin B were 0.020 and 0.039 µM, respectively. EC₅₀ value for orexin B in producing inward current was similar to that of oxytocin while being four–fold larger than that of orexin A; EC₅₀ value for orexin B in increasing sEPSC frequency was comparable to orexin A’s one and also to oxytocin’s ones for sIPSC frequency increase. Like oxytocin, orexin A enhanced both GABAergic and glycinergic transmissions in >50% of the neurons tested, whereas orexin B facilitated glycinergic but not GABAergic transmission in the majority (about 70%) of neurons tested. Inhibitory transmission enhancements produced by oxytocin, orexins A and B dis­appeared in the presence of the voltage–gated Na⁺–channel blocker tetrodotoxin. Oxytocin activities were mimicked by an oxytocin–receptor agonist TGOT and were inhibited by an oxytocin–receptor antagonist dVOT, indicating an activation of oxytocin receptors. Orexin A activities were inhibited by an orexin–1 receptor antagonist (SB334867) but not an orexin–2 receptor antagonist (JNJ10397049) while orexin B activities were inhibited by JNJ10397049 but not SB334867, indicating that orexins A and B activities are mediated by orexin–1 and –2 receptors, respectively. It is concluded that oxytocin, orexins A and B increase neuronal activity through membrane depolari­zation and/or increased _L–glutamate release from nerve terminals, by activating their specific receptors, which in turn results in GABAergic and/or glycinergic spontaneous inhibitory transmission enhancements, a possible mechanism for antinociception.

Interstitial cystitis ⁄ painful bladder syndrome (IC ⁄ PBS) is a chronic bladder disorder accompanied by urinary dysfunction and bladder pain. The bladder pain is often resistant to current analgesics, such as amitriptyline and gabapentin, and decreases the quality of life for patients with IC ⁄ PBS. Novel analgesics with greater efficacy are urgently required; however, the pain mechanism in IC ⁄ PBS is not fully understood. Transient receptor potential vanilloid–4 (TRPV4) is expressed in the bladder epithelium to detect the mechanical stimulation associated with cell swelling and shear stress. Some reports have shown the involvement of TRPV4 in urinary dysfunction in rodent models, but little is known about bladder pain. Therefore, we investigated whether TRPV4 could be involved in the bladder pain induced by cyclophosphamide (CYP) in mice. Repeated intraperitoneal injection of CYP at 150 mg/kg for 4 days produced mild edema with some infiltration of inflammatory cells in the bladder, and persist­ent mechanical hypersensitivity in the lower abdomen of mice. The phosphorylated TRPV4 was significantly increased in the bladder of chronic cystitis mice, although the level of TRPV4 mRNA and the distribution of TRPV4 were not changed in the bladder compared with vehicle–injected mice. The gene depletion of TRPV4 completely prevented mechanical hypersensitivity in chronic cystitis mice. In addition, oral administration of the TRPV4 antagonist, GSK2193874, inhibited mecha­nical hyper­sensitivity in these mice. These results show that the TRPV4 antagonis­t may become a therapeutic option for bladder pain in patients with IC ⁄ PBS.

Osteoarthritis of the knee (knee OA) is a common disease in the elderly, and the chief complaint of these patients is knee pain. Knee OA pain reduces the activities of daily living and the quality of life in these patients. The degree of pain does not correlate with radiographic grades, and the intense wear of the cartilage does not neces­sarily mean intense pain. However, the cellular mechanism of chronic pain in this disease has been unclear. This study aimed to investigate the effects of the transient receptor potential vanilloid 1 (TRPV1) on weight–bearing pain–related behaviors in knee OA model rats using the CatWalk system. To generate the knee OA model, monosodium iodoacetate (MIA) was injected in the right knee joint of male adult Sprague–Dawley rats. Four weeks after the MIA injection, we used the knee OA rats for the behavioral study. Using the CatWalk system, we investigated the effects of the intra–articular injection of the TRPV1 selective agonist capsaicin, TRPV1 selective antagonist capsazepine, and saline on the nociceptive behaviors of the rats with knee OA at 1, 2, 7, and 10 days after the intra–articular injection. The rats treated with intra–articular capsazepine injection showed significantly greater improvement in swing speed, a pain–related behavioral parameter of the CatWalk system, than those treated with capsaicin on day 2 after injection. The maximum contact area on day 10 showed significantly greater improvement in the capsazepine group than in the capsaicin group. These results suggest that TRPV1 is an important contributor to weight–bearing pain–related behavior of patients with knee OA. However, in this study, these effects were observed at later time points. Thus, TRPV1 could be related to not only the peripheral nociceptive pain mechanism, but also to the secondary pain mechanism.

Introduction: In our clinical situation, pain is evaluated by the subjective complaints reported by patients because an objective measurement is still unrealizable. As an alter­native, we usually evaluate pain–related behaviour to analyze animal pain. However, the phenomenon of analyzing pain–related behaviour is limited because some amounts of stimulations are required to be provided first. At present, the opinion that pain–related behaviour is not a response to pain but a mere reflex still cannot be completely disproved. Moreover, as observed with chronic pain patients, pain triggered by some kind of stimuli is less important than pain experienced continuous­ly in a given period from our group’s clinical data. This means that the common pain study using the general way of evaluation cannot contribute to new analgesics’ discovery which patients are eager to await. The goal of this study is to evaluate rodents’ spontaneous pain objectively without any stimulation by high resolutio­n MRI.

Methods: All surgical and experimental protocols were approved by the Institutional Animal Care and Use Committee of Osaka university graduate school of medicine. 31 male Wister rats were anesthetized with sodium pentobarbital (60 mg/kg i.p.) and sevoflurane (2 – 3%). An infra–orbital nerve (ION) loose ligation model was made by the procedure that two nylon (5–0) ligatures (2 mm apart) were loosely tied around the ION. Sham–operation was performed only by cutting the skin on the face and suturing in the same way as the procedure of ION loose ligation model. The scanning schedule was set 3 weeks after surgery and 3 days before the scanning day, 5 mg/kg manganese was injected intraperitoneally for three days. MRI measurements were performed with an 11.7 T MR scanner (AVANCE 500WB, Bruker BioSpin MRI GmbH, Ettlingen, Germany), under isoflurane anesthesia (2 – 2.5%), T1 weighted MR–images were acquired with a spin echo sequence. All the images were standardized and divided into 52 areas automatically and uptakes of manganese were analyzed. Tukey–Kramer multiple comparison test and correlation analyses were performed using JMP12.0.0 software.

Results & Discussion: There was no difference in manganese uptake data in medulla and both sides of thalamus region between sham, and nerve–injured groups. Correlation coefficients against medulla were different from both side of thalamus and midbrain respectively between nerve–injured group and sham–operated group. Correlation coefficients against thalamus were also different from the same side of insula cortex and motor cortex. Nerve injured animals showed significantly stronger correlation to each region (p<0.001). However, sham–operated animals didn’t show the same pattern correlation as with the injured animals.

Conclusion: The uptake pattern difference between nerve–injured rats and sham–operated rats indicated the system change in a brain by surgical procedure which can evoke chronic neuropathic pain. This method could successfully illustrate rodents’ spontaneous pain objectively. MEMRI (Manganese–Enhanced MRI) is a potentially excellent way to evaluate rodents’ pain without any stimulations under anesthesia during scanning because manganese takes a long time to be discharged by animals. By using this method, experimenters’ bias can be completely excluded. To evaluate pain in animals without awake scanning condition is another merit for pain research.