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Excessive alcohol consumption is associated with spontaneous burning pain, hyperalgesia and allodynia. Although acetaldehyde has been implicated in the painful alcoholic neuropathy, the mechanism by which the ethanol metabolite causes pain symptoms is unknown. Acute ethanol ingestion caused delayed mechanical allodynia in mice. Inhibition of alcohol dehydrogenase (ADH) or deletion of transient receptor potential ankyrin 1 (TRPA1), a sensor for oxidative and carbonyl stress, prevented allodynia. Acetaldehyde generated by ADH in both liver and Schwann cells surrounding nociceptors was required for TRPA1-induced mechanical allodynia. Plp1-Cre;Trpa1fl/fl mice with a tamoxifen-inducible specific deletion of TRPA1 in Schwann cells revealed that channel activation by acetaldehyde in these cells initiates a NADPH oxidase-1 (NOX-1)-dependent production of hydrogen peroxide (H2O2) and 4-hydroxynonenal (4-HNE), which sustains allodynia by paracrine targeting of nociceptor TRPA1. Chronic ethanol ingestion caused prolonged mechanical allodynia and loss of intraepidermal small nerve fibers in WT mice. While Trpa1-/- or Plp1-Cre;Trpa1fl/fl mice did not develop mechanical allodynia, they did not show any protection from the small fiber neuropathy. Human Schwann cells express ADH/TRPA1/NOX1 and recapitulate the proalgesic functions of mouse Schwann cells. TRPA1 antagonists might attenuate some symptoms of alcohol-related pain.
Learn More >Transcripts of noxious stimulus-detecting TrpA1 channels are alternatively spliced. Despite the importance of nociception for survival, the in vivo significance of expressing different TrpA1 isoforms is largely unknown. Here, we develop a novel genetic approach to generate Drosophila knockin strains expressing single TrpA1 isoforms. Drosophila TrpA1 mediates heat and UVC-triggered nociception. We show that TrpA1-C and TrpA1-D, two alternative isoforms, are co-expressed in nociceptors. When examined in heterologous cells, both TrpA1-C and TrpA1-D are activated by heat and UVC. By contrast, analysis of knockin flies reveals the striking functional specificity; TrpA1-C mediates UVC-nociception, whereas TrpA1-D mediates heat-nociception. Therefore, in vivo functions of TrpA1-C and TrpA1-D are different from each other and are different from their in vitro properties. Our results indicate that a given sensory stimulus preferentially activates a single TrpA1 isoform in vivo and that polymodal nociception requires co-expression of TrpA1 isoforms, providing novel insights of how alternative splicing regulates nociception.
Learn More >Patients with irritable bowel syndrome (IBS) show pain hypersensitivity and smooth muscle hypercontractility in response to colorectal distension (CRD). Synaptic plasticity, a key process of memory formation, in the enteric nervous system may be a novel explanation. This study aimed to explore the regulatory role of ephrinB2/ephB2 in enteric synaptic plasticity and colonic hyperreactive motility in IBS. Postinfectious (PI)-IBS was induced by infection in rats. Isometric contractions of colonic circular muscle strips, particularly neural-mediated contractions, were recorded . Meanwhile, ephrinB2/ephB2-mediated enteric structural and functional synaptic plasticity were assessed in the colonic muscularis, indicating that ephrinB2 and ephB2 were located on enteric nerves and up-regulated in the colonic muscularis of PI-IBS rats. Colonic hypersensitivity to CRD and neural-mediated colonic hypercontractility were present in PI-IBS rats, which were correlated with increased levels of cellular homologous fos protein () and activity-regulated cystoskeleton-associated protein (arc), the synaptic plasticity-related immediate early genes, and were ameliorated by ephB2Fc (an ephB2 receptor blocker) or MK801 (an NMDA receptor inhibitor) exposure. EphrinB2/ephB2 facilitated synaptic sprouting and NMDA receptor-mediated synaptic potentiation in the colonic muscularis of PI-IBS rats and in the longitudinal muscle-myenteric plexus cultures, involving the Erk-MAPK and PI3K-protein kinase B pathways. In conclusion, ephrinB2/ephB2 promoted the synaptic sprouting and potentiation of myenteric nerves involved in persistent muscle hypercontractility and pain in PI-IBS. Hence, ephrinB2/ephB2 may be an emerging target for the treatment of IBS.-Zhang, L., Wang, R., Bai, T., Xiang, X., Qian, W., Song, J., Hou, X. EphrinB2/ephB2-mediated myenteric synaptic plasticity: mechanisms underlying the persistent muscle hypercontractility and pain in postinfectious IBS.
Learn More >Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling pain condition which occurs as a consequence of cancer chemotherapy with anti-cancer agents like paclitaxel, oxaliplatin, etc. Despite immense research in the pathological pathways involved in CIPN, treatment options still remain limited. Recently, pathological involvement of Wnt signaling has been investigated in various neuropathic pain models, however there are no reports as yet on the role of Wnt signaling in CIPN. In the present study, we have investigated the neuroprotective effects of Wnt signaling inhibitors namely LGK974 (Porcupine inhibitor), NSC668036 (Disheveled inhibitor) and PNU76454 (β-catenin inhibitor) in paclitaxel-induced neuropathic pain. Paclitaxel (2 mg/kg, i. p.) was administered to male Sprague Dawley rats on four alternate days. After 21 days, paclitaxel-treated rats showed reduced behavioral pain thresholds (cold allodynia, heat & mechanical hyperalgesia) and nerve functions (nerve conduction velocity and nerve blood flow). Moreover, Wnt signaling proteins (Wnt3a, β-catenin, c-myc and Dvl1), inflammatory marker (matrix metalloproteinase 2) and endoplasmic reticulum stress marker (GRP78) were found to be upregulated in the sciatic nerves of paclitaxel-treated rats accompanied with loss of intraepidermal nerve fiber density as compared to the control rats. Intrathecal administration of Wnt inhibitors (each at dose of 10 and 30 μM) for three consecutive days to paclitaxel-treated rats, significantly improved behavioral pain thresholds and nerve functional parameters by inhibition of Wnt signaling, inflammation, endoplasmic reticulum stress and improvement of intraepidermal nerve fiber density. All these results suggested the neuroprotective potential of Wnt signaling inhibitors in CIPN.
Learn More >Chronic itch is one of the disturbing symptoms of inflammatory skin diseases. Kappa opioid receptor agonists are effective in suppressing scratching in mice against different pruritogens. Nalbuphine, a nonscheduled kappa opioid receptor agonist and mu opioid receptor antagonist, has been in clinical use for post-operative pain management since the 1980s and recently has been in clinical trials for chronic itch of prurigo nodularis (https://www.trevitherapeutics.com/nalbuphine). We studied whether nalbuphine is effective against chronic scratching induced by rostral neck application of 1-fluoro-2,4-dinitrobenzene (DNFB), an accepted mouse model of contact dermatitis to study pruritoceptive itch. Mice were treated once a week with either saline or nalbuphine 20 min before the third, fifth, seventh, and ninth sensitizations with DNFB and the number of scratching bouts was counted for 30 min. Skin samples from the neck of mice at week 4 were used to measure protein levels and mRNA expressions of chemokines and cytokines. Different sets of mice were used to study sedation and anhedonic-like behavior of nalbuphine. We found that: nalbuphine (a) antagonized scratching in a dose- and time-dependent manner without affecting locomotion, b) decreased IL-31, and increased anti-inflammatory IL-10, and c) induced more elevations in the levels of CCL2, CCL3, CCL12, CXCL1, CXCL2, CXCL9, CXCL10, IL-1β, IL-16, TIMP-1, M-CSF, TREM-1 and M1-type macrophages compared to saline. Increases in chemokines and cytokines and M1 macrophages by nalbuphine suggest an inflammatory phase of healing in damaged skin due to scratching. Our data indicate that nalbuphine is an effective antipruritic in murine model of pruritoceptive itch.
Learn More >Opioids are the most powerful analgesics available to date. However, they may also induce adverse effects including paradoxical opioid-induced hyperalgesia (OIH). A mechanism that might underlie OIH is the amplification of synaptic strength at spinal C-fibre synapses after withdrawal from systemic opioids such as remifentanil ("opioid-withdrawal-LTP").Here, we show that both, the induction as well as the maintenance of opioid-withdrawal-LTP were abolished by pharmacological blockade of spinal glial cells. In contrast, the blockade of TLR4 had no effect on the induction of opioid-withdrawal-LTP. D-serine, which may be released upon glial cell activation, was necessary for withdrawal-LTP. D-serine is the dominant co-agonist for neuronal NMDA-receptors, which are required for the amplification of synaptic strength upon remifentanil withdrawal.Unexpectedly, opioid-withdrawal-LTP was transferable via the cerebrospinal fluid between animals. This suggests that glial cell-derived mediators accumulate in the extracellular space and reach the cerebrospinal fluid at biologically active concentrations, thereby creating a soluble memory trace that is transferable to another animal ("transfer-LTP"). When we enzymatically degraded D-serine in the superfusate, LTP could no longer be transferred. Transfer-LTP was insensitive to pharmacological blockade of glial cells in the recipient animal, thus representing a rare form of glial-cell independent LTP in the spinal cord.
Learn More >Artemin is a neurotrophic factor that plays a crucial role in the regulation of neural development and regeneration and has also been implicated in the pathogenesis of inflammatory pain. The receptor for artemin, GFRα3, is expressed by sympathetic and nociceptive sensory neurons, including some that innervate the bone marrow, but it is unclear if it is also expressed in other cell types in the bone marrow. Our goal in the present study was to characterise the expression of GFRα3 in nonneuronal cells in the bone marrow. Immunohistochemical studies revealed that GFRα3-expressing cells in the bone marrow are spatially associated with blood vessels and are in intimate contact with nerve fibres. We used various combinations of markers to distinguish different cell types and found that the GFRα3-expressing cells expressed markers of nonmyelinating Schwann cells (e.g. GFAP, p75NTR, nestin). Analysis of bone marrow sections of Wnt1-reporter mice also demonstrated that they originate from the neural crest. Further characterisation using flow cytometry revealed that GFRα3 is expressed in a population of CD51Sca1PDGFRα cells, reinforcing the notion that they are neural crest-derived, nonmyelinating Schwann cells. In conclusion, there is a close association between peripheral nerve terminals and a population of nonneuronal cells that express GFRα3 in the bone marrow. The nonneuronal cells have characteristics consistent with a neural crest-derived, nonmyelinating Schwann cell phenotype. Our findings provide a better understanding of the expression pattern of GFRα3 in the bone marrow microenvironment.
Learn More >P2Y purinergic receptors expressed in neurons and satellite glial cells (SGCs) of the trigeminal ganglion (TG) contributes to inflammatory and neuropathic pain. P2Y receptor expression is reported in the spinal cord, dorsal root ganglion (DRG), and TG. In present study, the role of P2Y receptor in the TG in inflammatory orofacial pain of Sprague-Dawley (SD) rats was investigated. Peripheral injection of complete Freund's adjuvant (CFA) induced mechanical hyperalgesia with the rapid upregulation of P2Y receptor, glial fibrillary acidic protein (GFAP), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), C-C chemokine CCL2, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phosphorylated p38 (p-p38) proteins in the TG. Furthermore, immunofluorescence staining confirmed the CFA-induced upregulation of P2Y receptor. Double immunostaining showed that P2Y receptor colocalized with glutamine synthetase (GS) and neuronal nuclei (NeuN). Finally, trigeminal injection of a selective antagonist (PPTN) of P2Y receptor attenuated CFA-induced mechanical hyperalgesia. PPTN also decreased the upregulation of the GFAP, IL-1β, TNF-α, CCL2, p-ERK1/2, and p-p38 proteins. Our findings showed that P2Y receptor in TG may contribute to orofacial inflammatory pain via regulating SGCs activation, releasing cytokines (IL-1β, TNF-α, and CCL2), and phosphorylating ERK1/2 and p38.
Learn More >Chronic pain is a worldwide major health problem and many pain-suffering patients are under opioid based therapy. Epidemiological data show that pain intensity correlates with the risk of misuse of prescription opioids, and other drugs of abuse including alcohol. This increased vulnerability to suffer Substance Use Disorders could be, in part, caused by functional changes that occur over the mesocorticolimbic system, a brain pathway involved in reward processing and addiction. Previous data in rats revealed that inflammatory pain desensitizes mu opioid receptors (MORs) in the ventral tegmental area (VTA). As a consequence, pain alters dopamine release in the nucleus accumbens (NAc) derived from MOR activation in the VTA and also increases intake of high doses of heroine. Given that the VTA neurons target different brain regions, in the present study we first analyzed changes induced by inflammatory pain in the MOR dependent activation pattern of the main VTA projecting areas. To do that, we administered two doses (7 or 14 ng) of DAMGO (MORs agonist) or artificial cerebrospinal fluid (aCSF) focally into the VTA of rats and measured the activation in projection areas by cFos immunohistochemistry. Our results show that focal injections of DAMGO in the VTA increases cFos expression in the majority of its projecting areas, namely NAc, basolateral amygdala (BLA), cingulate cortex (ACC) and bed nucleus of the stria terminalis (BNST), as compared to aCSF. Second, we analyzed whether inflammatory pain would affect to cFos expression using a group of rats injected with CFA in the hind paw. In this case, we found that cFos expression was not significantly different between DAMGO and aCSF administered rats in BLA, ACC and BNST. Our results confirm that inflammatory pain induces desensitization of VTA MORs in a region dependent manner which can be very relevant for addictive behaviours.
Learn More >Complex regional pain syndrome (CRPS) is a post-traumatic pain condition with an incompletely understood pathophysiological basis. Here, we have examined the cellular basis of pain in CRPS using behavioral and electrophysiological methods in mice treated with IgG from CRPS patients, in combination with a paw incision. Mice were subjected to a hind paw skin-muscle incision alone, or in combination with administration of IgG purified from either healthy control subjects (HC) or patients with persistent CRPS. Nociceptive function was examined behaviorally in vivo, and electrophysiologically in vitro using skin-nerve preparations to study the major classes of mechanosensitive single units. Administration of IgG from CRPS patients exacerbated and prolonged the post-surgical hypersensitivity to noxious mechanical, cold and heat stimulation, but did not influence tactile sensitivity following a paw incision. Studies of IgG preparations pooled from patient cohorts (n=26-27) show that pathological autoantibodies are present in the wider population of patients with persistent CRPS, and that patients with more severe pain have higher effective autoantibody titres than patients with moderate pain intensity. Electrophysiological investigation of skin-nerve preparations from mice treated with CRPS IgG from a single patient identified both a significantly increased evoked impulse activity in A- and C-nociceptors, and an increased spontaneous impulse rate in the intact saphenous nerve. Our results show that painful hypersensitivity in persistent CRPS is maintained by autoantibodies, which act by sensitizing A- and C-nociceptors.
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