Insights into FcγR involvement in pain-like behavior induced by an RA-derived anti-modified protein autoantibody.

Joint pain is one of the most debilitating symptoms of rheumatoid arthritis (RA) and patients frequently rate improvements in pain management as their priority. RA is hallmarked by the presence of anti-modified protein autoantibodies (AMPA) against post-translationally modified citrullinated, carbamylated and acetylated proteins. It has been suggested that autoantibody-mediated processes represent distinct mechanisms contributing to pain in RA. In this study, we investigated the pronociceptive properties of monoclonal AMPA 1325:01B09 (B09 mAb) derived from the plasma cell of an RA patient. We found that B09 mAb induces pain-like behavior in mice that is not associated with any visual, histological or transcriptional signs of inflammation in the joints, and not alleviated by non-steroidal anti-inflammatory drugs (NSAIDs). Instead, we found that B09 mAb is retained in dorsal root ganglia (DRG) and alters the expression of several satellite glia cell (SGC), neuron and macrophage-related factors in DRGs. Using mice that lack activating FcγRs, we uncovered that FcγRs are critical for the development of B09-induced pain-like behavior, and partially drive the transcriptional changes in the DRGs. Finally, we observed that B09 mAb binds SGC in vitro and in combination with external stimuli like ATP enhances transcriptional changes and protein release of pronociceptive factors from SGCs. We propose that certain RA antibodies bind epitopes in the DRG, here on SGCs, form immune complexes and activate resident macrophages via FcγR cross-linking. Our work supports the growing notion that autoantibodies can alter nociceptor signaling via mechanisms that are at large independent of local inflammatory processes in the joint.

Docosahexaenoic acid and etanercept could reduce functional and metabolic alterations during collagen-induced arthritis in rats without any synergistic effect.

AIMS: Rheumatoid arthritis is an autoimmune disease which induces chronic inflammation and increases the risk for sarcopenia and metabolic abnormalities. Nutritional strategies using omega 3 polyunsaturated fatty acids could be proposed to alleviate inflammation and improve the maintenance of lean mass. Independently, pharmacological agents targeting key molecular regulators of the pathology such as TNF alpha could be proposed, but multiple therapies are frequently necessary increasing the risk for toxicity and adverse effects. The aim of the present study was to explore if the combination of an anti-TNF therapy (Etanercept) with dietary supplementation with omega 3 PUFA could prevent pain and metabolic effects of RA. MATERIALS AND METHODS: RA was induced using collagen-induced arthritis (CIA) in rats to explore of supplementation with docosahexaenoic acid, treatment with etanercept or their association could alleviate symptoms of RA (pain, dysmobility), sarcopenia and metabolic alterations. KEY FINDINGS: We observed that Etanercept had major benefits on pain and RA scoring index. However, DHA could reduce the impact on body composition and metabolic alterations. SIGNIFICANCE: This study revealed for the first time that nutritional supplementation with omega 3 fatty acid could reduce some symptoms of rheumatoid arthritis and be an effective preventive treatment in patients who do not need pharmacological therapy, but no sign of synergy with an anti-TNF agent was observed.

Identification of Lipid Biomarkers for Chronic Joint Pain Associated with Different Joint Diseases.

Lipids, especially lysophosphatidylcholine LPC16:0, have been shown to be involved in chronic joint pain through the activation of acid-sensing ion channels (ASIC3). The aim of the present study was to investigate the lipid contents of the synovial fluids from controls and patients suffering from chronic joint pain in order to identify characteristic lipid signatures associated with specific joint diseases. For this purpose, lipids were extracted from the synovial fluids and analyzed by mass spectrometry. Lipidomic analyses identified certain choline-containing lipid classes and molecular species as biomarkers of chronic joint pain, regardless of the pathology, with significantly higher levels detected in the patient samples. Moreover, correlations were observed between certain lipid levels and the type of joint pathologies. Interestingly, LPC16:0 levels appeared to correlate with the metabolic status of patients while other choline-containing lipids were more specifically associated with the inflammatory state. Overall, these data point at selective lipid species in synovial fluid as being strong predictors of specific joint pathologies which could help in the selection of the most adapted treatment.

Single Subcutaneous Injection of Lysophosphatidyl-Choline Evokes ASIC3-Dependent Increases of Spinal Dorsal Horn Neuron Activity.

Lysophosphatidyl-choline (LPC), a member of the phospholipid family, is an emerging player in pain. It is known to modulate different pain-related ion channels, including Acid-Sensing Ion Channel 3 (ASIC3), a cationic channel mainly expressed in peripheral sensory neurons. LPC potentiates ASIC3 current evoked by mild acidifications, but can also activate the channel at physiological pH. Very recently, LPC has been associated to chronic pain in patients suffering from fibromyalgia or osteoarthritis. Accordingly, repetitive injections of LPC within mouse muscle or joint generate both persistent pain-like and anxiety-like behaviors in an ASIC3-dependent manner. LPC has also been reported to generate acute pain behaviors when injected intraplantarly in rodents. Here, we explore the mechanism of action of a single cutaneous injection of LPC by studying its effects on spinal dorsal horn neurons. We combine pharmacological, molecular and functional approaches including in vitro patch clamp recordings and in vivo recordings of spinal neuronal activity. We show that a single cutaneous injection of LPC exclusively affects the nociceptive pathway, inducing an ASIC3-dependent sensitization of nociceptive fibers that leads to hyperexcitabilities of both high threshold (HT) and wide dynamic range (WDR) spinal neurons. ASIC3 is involved in LPC-induced increase of WDR neuron's windup as well as in WDR and HT neuron's mechanical hypersensitivity, and it participates, together with TRPV1, to HT neuron's thermal hypersensitivity. The nociceptive input induced by a single LPC cutaneous rather induces short-term sensitization, contrary to previously described injections in muscle and joint. If the effects of peripheral LPC on nociceptive pathways appear to mainly depend on peripheral ASIC3 channels, their consequences on pain may also depend on the tissue injected. Our findings contribute to a better understanding of the nociceptive signaling pathway activated by peripheral LPC via ASIC3 channels, which is an important step regarding the ASIC3-dependent roles of this phospholipid in acute and chronic pain conditions.

Lysophosphatidylcholine 16:0 mediates chronic joint pain associated to rheumatic diseases through acid-sensing ion channel 3.

ABSTRACT: Rheumatic diseases are often associated to debilitating chronic pain, which remains difficult to treat and requires new therapeutic strategies. We had previously identified lysophosphatidylcholine (LPC) in the synovial fluids from few patients and shown its effect as a positive modulator of acid-sensing ion channel 3 (ASIC3) able to induce acute cutaneous pain in rodents. However, the possible involvement of LPC in chronic joint pain remained completely unknown. Here, we show, from 2 independent cohorts of patients with painful rheumatic diseases, that the synovial fluid levels of LPC are significantly elevated, especially the LPC16:0 species, compared with postmortem control subjects. Moreover, LPC16:0 levels correlated with pain outcomes in a cohort of osteoarthritis patients. However, LPC16:0 do not appear to be the hallmark of a particular joint disease because similar levels are found in the synovial fluids of a second cohort of patients with various rheumatic diseases. The mechanism of action was next explored by developing a pathology-derived rodent model. Intra-articular injections of LPC16:0 is a triggering factor of chronic joint pain in both male and female mice, ultimately leading to persistent pain and anxiety-like behaviors. All these effects are dependent on ASIC3 channels, which drive sufficient peripheral inputs to generate spinal sensitization processes. This study brings evidences from mouse and human supporting a role for LPC16:0 via ASIC3 channels in chronic pain arising from joints, with potential implications for pain management in osteoarthritis and possibly across other rheumatic diseases.

Tyrosine kinase type A-specific signalling pathways are critical for mechanical allodynia development and bone alterations in a mouse model of rheumatoid arthritis.

ABSTRACT: Rheumatoid arthritis is frequently associated with chronic pain that still remains difficult to treat. Targeting nerve growth factor (NGF) seems very effective to reduce pain in at least osteoarthritis and chronic low back pain but leads to some potential adverse events. Our aim was to better understand the involvement of the intracellular signalling pathways activated by NGF through its specific tyrosine kinase type A (TrkA) receptor in the pathophysiology of rheumatoid arthritis using the complete Freund adjuvant model in our knock-in TrkA/C mice. Our multimodal study demonstrated that knock-in TrkA/C mice exhibited a specific decrease of mechanical allodynia, weight-bearing deficit, peptidergic (CGRP+) and sympathetic (TH+) peripheral nerve sprouting in the joints, a reduction in osteoclast activity and bone resorption markers, and a decrease of CD68-positive cells in the joint with no apparent changes in joint inflammation compared with wild-type mice after arthritis. Finally, transcriptomic analysis shows several differences in dorsal root ganglion mRNA expression of putative mechanotransducers, such as acid-sensing ionic channel 3 and TWIK-related arachidonic acid activated K+ channel, as well as intracellular pathways, such as c-Jun, in the joint or dorsal root ganglia. These results suggest that TrkA-specific intracellular signalling pathways are specifically involved in mechanical hypersensitivity and bone alterations after arthritis using TrkA/C mice.

Antibody-induced pain-like behavior and bone erosion: links to subclinical inflammation, osteoclast activity, and acid-sensing ion channel 3-dependent sensitization.

ABSTRACT: Several bone conditions, eg, bone cancer, osteoporosis, and rheumatoid arthritis (RA), are associated with a risk of developing persistent pain. Increased osteoclast activity is often the hallmark of these bony pathologies and not only leads to bone remodeling but is also a source of pronociceptive factors that sensitize the bone-innervating nociceptors. Although historically bone loss in RA has been believed to be a consequence of inflammation, both bone erosion and pain can occur years before the symptom onset. Here, we have addressed the disconnection between inflammation, pain, and bone erosion by using a combination of 2 monoclonal antibodies isolated from B cells of patients with RA. We have found that mice injected with B02/B09 monoclonal antibodies (mAbs) developed a long-lasting mechanical hypersensitivity that was accompanied by bone erosion in the absence of joint edema or synovitis. Intriguingly, we have noted a lack of analgesic effect of naproxen and a moderate elevation of few inflammatory factors in the ankle joints suggesting that B02/B09-induced pain-like behavior does not depend on inflammatory processes. By contrast, we found that inhibiting osteoclast activity and acid-sensing ion channel 3 signaling prevented the development of B02/B09-mediated mechanical hypersensitivity. Moreover, we have identified secretory phospholipase A2 and lysophosphatidylcholine 16:0 as critical components of B02/B09-induced pain-like behavior and shown that treatment with a secretory phospholipase A2 inhibitor reversed B02/B09-induced mechanical hypersensitivity and bone erosion. Taken together, our study suggests a potential link between bone erosion and pain in a state of subclinical inflammation and offers a step forward in understanding the mechanisms of bone pain in diseases such as RA.

c-Jun/p38MAPK/ASIC3 pathways specifically activated by nerve growth factor through TrkA are crucial for mechanical allodynia development.

Mechanical allodynia is a cardinal sign of several inflammatory pain disorders where nerve growth factor, a prototypic neurotrophin, plays a crucial role by binding to TrkA receptors. Here, we took the advantage of our generated knock-in mouse model expressing a chimeric TrkA/TrkC receptor that seems to not specifically develop mechanical allodynia after inflammation, to identify the TrkA downstream pathways involved in this phenomenon. We confirmed and extended that disrupting TrkA-specific pathways leads to a specific deficit in mechanical hypersensitivity development after somatic (systemic nerve growth factor administration and paw incision) and, to a lesser extent, visceral injuries. Despite a deficit in thin, mainly peptidergic, fibre innervation in TrkAC mice, thermal hyperalgesia development was not different from WT mice. Inflammatory reaction (oedema, IL-6 content), pain behaviours after intraplantar capsaicin, as well as TRPV1 calcium imaging response of dorsal root ganglion neurons were similar between TrkAC and WT mice. This deficiency in mechanical allodynia development in TrkAC mice is likely due to the alteration of the expression of different TrkA transduction pathways (ie, Akt, p38 MAPK, and c-Jun) especially p38 MAPK, in the dorsal root ganglion cell bodies, ultimately leading to an alteration of at least, ASIC3 channel overexpression, known to participate in nociceptor mechanosensory function.

Structural and functional alterations in the retrosplenial cortex following neuropathic pain.

Human and animal imaging studies demonstrated that chronic pain profoundly alters the structure and the functionality of several brain regions. In this article, we conducted a longitudinal and multimodal study to assess how chronic pain affects the brain. Using the spared nerve injury model which promotes both long-lasting mechanical and thermal allodynia/hyperalgesia but also pain-associated comorbidities, we showed that neuropathic pain deeply modified the intrinsic organization of the brain functional network 1 and 2 months after injury. We found that both functional metrics and connectivity of the part A of the retrosplenial granular cortex (RSgA) were significantly correlated with the development of neuropathic pain behaviours. In addition, we found that the functional RSgA connectivity to the subiculum and the prelimbic system are significantly increased in spared nerve injury animals and correlated with peripheral pain thresholds. These brain regions were previously linked to the development of comorbidities associated with neuropathic pain. Using a voxel-based morphometry approach, we showed that neuropathic pain induced a significant increase of the gray matter concentration within the RSgA, associated with a significant activation of both astrocytes and microglial cells. Together, functional and morphological imaging metrics of the RSgA could be used as a predictive biomarker of neuropathic pain.

Acid-Sensing Ion Channel 1a in the amygdala is involved in pain and anxiety-related behaviours associated with arthritis.

Chronic pain is associated with anxiety and depression episodes. The amygdala plays a key role in the relationship between emotional responses and chronic pain. Here, we investigated the role of Acid-Sensing Ion Channels 1a within the basolateral amygdala (BLA), in pain and associated anxiety in a rat model of monoarthritis (MoAr). Administration within the BLA of PcTx1 or mambalgin-1, two specific inhibitors of ASIC1a-containing channels significantly inhibited pain and anxiety-related behaviours in MoAr rats. The effect of PcTx1 was correlated with a reduction of c-Fos expression in the BLA. We examined the expression profile of ASICs and other genes in the amygdala in MoAr and sham animals, and found no variation of the expression of ASIC1a, which was confirmed at the protein level. However, an increase in the BLA of MoAr rats of both PI3Kinase mRNA and the phosphorylated form of Akt, along with Bdnf mRNA, suggest that the BDNF/PI3-kinase/Akt pathway might regulate ASIC1a in BLA neurons as demonstrated in spinal sensitisation phenomenon. We also observed changes in several kinase mRNAs expression (PICK1, Sgk1) that are potentially involved in ASIC1a regulation. These results show a crucial role of ASIC1a channels in the BLA in pain and anxiety-related behaviours during arthritis.

Agomelatine: a new opportunity to reduce neuropathic pain-preclinical evidence.

Antidepressants are first-line treatments of neuropathic pain but not all these drugs are really effective. Agomelatine is an antidepressant with a novel mode of action, acting as an MT1/MT2 melatonergic receptor agonist and a 5-HT2C receptor antagonist that involves indirect norepinephrine release. Melatonin, serotonin, and norepinephrine have been involved in the pathophysiology of neuropathic pain. Yet, no study has been conducted to determine agomelatine effects on neuropathic pain in animal models. Using 3 rat models of neuropathic pain of toxic (oxaliplatin/OXA), metabolic (streptozocin/STZ), and traumatic (sciatic nerve ligation/CCI [chronic constriction nerve injury]) etiologies, we investigated the antihypersensitivity effect of acute and repeated agomelatine administration. We then determined the influence of melatonergic, 5-HT2C, α-2 and ß-1/2 adrenergic receptor antagonists in the antihypersensitivity effect of agomelatine. The effect of the combination of agomelatine + gabapentin was evaluated using an isobolographic approach. In STZ and CCI models, single doses of agomelatine significantly and dose dependently reduced mechanical hypersensitivity. After daily administrations for 2 weeks, this effect was confirmed in the CCI model and agomelatine also displayed a marked antihypersensitivity effect in the OXA model. The antihypersensitivity effect of agomelatine involved melatonergic, 5-HT2C, and α-2 adrenergic receptors but not beta adrenoceptors. The isobolographic analysis demonstrated that the combination of agomelatine + gabapentin had additive effects. Agomelatine exerts a clear-cut antihypersensitivity effect in 3 different neuropathic pain models. Its effect is mediated by melatonergic and 5-HT2C receptors and, although agomelatine has no affinity, also by α-2 adrenergic receptors. Finally, agomelatine combined with gabapentin produces an additive antihypersensitivity effect.

Cholinergic Neurotransmission in the Posterior Insular Cortex Is Altered in Preclinical Models of Neuropathic Pain: Key Role of Muscarinic M2 Receptors in Donepezil-Induced Antinociception.

Neuropathic pain is one of the most debilitating pain conditions, yet no therapeutic strategy has been really effective for its treatment. Hence, a better understanding of its pathophysiological mechanisms is necessary to identify new pharmacological targets. Here, we report important metabolic variations in brain areas involved in pain processing in a rat model of oxaliplatin-induced neuropathy using HRMAS (1)H-NMR spectroscopy. An increased concentration of choline has been evidenced in the posterior insular cortex (pIC) of neuropathic animal, which was significantly correlated with animals' pain thresholds. The screening of 34 genes mRNA involved in the pIC cholinergic system showed an increased expression of the high-affinity choline transporter and especially the muscarinic M2 receptors, which was confirmed by Western blot analysis in oxaliplatin-treated rats and the spared nerve injury model (SNI). Furthermore, pharmacological activation of M2 receptors in the pIC using oxotremorine completely reversed oxaliplatin-induced mechanical allodynia. Consistently, systemic treatment with donepezil, a centrally active acetylcholinesterase inhibitor, prevented and reversed oxaliplatin-induced cold and mechanical allodynia as well as social interaction impairment. Intracerebral microdialysis revealed a lower level of acetylcholine in the pIC of oxaliplatin-treated rats, which was significantly increased by donepezil. Finally, the analgesic effect of donepezil was markedly reduced by a microinjection of the M2 antagonist, methoctramine, within the pIC, in both oxaliplatin-treated rats and spared nerve injury rats. These findings highlight the crucial role of cortical cholinergic neurotransmission as a critical mechanism of neuropathic pain, and suggest that targeting insular M2 receptors using central cholinomimetics could be used for neuropathic pain treatment. SIGNIFICANCE STATEMENT: Our study describes a decrease in cholinergic neurotransmission in the posterior insular cortex in neuropathic pain condition and the involvement of M2 receptors. Targeting these cortical muscarinic M2 receptors using central cholinomimetics could be an effective therapy for neuropathic pain treatment.

99mTc-NTP 15-5 Imaging for Cartilage Involvement in Experimental Rheumatoid Arthritis: Comparison with Routinely Used Molecular Imaging Methods and Sensitivity to Chronic Nonsteroidal Antiinflammatory Drug Treatment.

UNLABELLED: This study determined, using the intraarticular complete Freund adjuvant arthritis mice model, whether the radiotracer (99m)Tc-N-(triethylammonium)-3-propyl-[15]ane-N5 ((99m)Tc-NTP 15-5) targeting proteoglycans has a pathophysiologic validity for in vivo imaging of rheumatoid arthritis (RA) and its response to chronic nonsteroidal antiinflammatory drugs. METHODS: We investigated the time course of cartilage remodeling by (99m)Tc-NTP 15-5 scintigraphy, bone damages by (99m)Tc-hydroxymethylene diphosphonate imaging, inflammation by (18)F-FDG PET, and joint proteoglycan content and pain behavior in animals, without and with meloxicam treatment. Paw circumference, thermal pain behavior, and histology as well as proteoglycan content of the whole joint were determined. RESULTS: (99m)Tc-NTP 15-5 showed specific tracer accumulation within RA joints, with a significant increase in scintigraphic ratio observed in RA versus shams from day 3 to day 28. (18)F-FDG evidenced uptake in RA joints from day 15 to day 29. Animals treated with meloxicam (5 mg/kg) exhibited a dose-dependent decrease in both (99m)Tc-NTP 15-5 and (18)F-FDG uptake ratios versus saline-treated animals. (99m)Tc-hydroxymethylene diphosphonate bone scans were only positive at day 14 in RA versus shams, with a significant effect of meloxicam. An increase in proteoglycans of RA joint and thermal pain behavior were observed and were dose-dependently reduced by meloxicam. CONCLUSION: These experimental results bring data in favor of the (99m)Tc-NTP 15-5 radiotracer for assessing, in vivo, cartilage remodeling in RA that could be used to monitor therapy.

Monoarthritis-induced emotional and cognitive impairments in rats are sensitive to low systemic doses or intra-amygdala injections of morphine.

Chronic pain is a multidimensional experience that not only includes changes in nociception but also impairments in emotional and cognitive functions, not often taken into account in preclinical research. The present study investigated emotional and cognitive impairments in an animal model of persistent inflammatory pain as well as the involvement of the basolateral complex (BLC) of the amygdala in these components. Monoarthritis was induced by intra-articular injection of complete Freund׳s adjuvant. Mechanical hypersensitivity, anxiety and depressive-like behaviours as well as cognitive capacities were assessed using several tests, such as von Frey, social interaction, open field, saccharin preference, spatial and social recognition memory tests. The effects of morphine administered systemically or into the BLC of the amygdala were also studied. Monoarthritic rats exhibited mechanical hypersensitivity, anxiety and depressive-like behaviours as well as cognitive impairments. Whereas low systemic doses and intra-BLC infusion of morphine failed to reduce mechanical hypersensitivity, they reversed monoarthritis-induced anxiety-like behaviours and cognitive impairments. Our findings further support a crucial role of amygdala in the effect of morphine on emotional/cognitive components of pain and not on mechanical hypersensitivity. Finally, our study highlights the interest of a multi-behavioural approach in the assessment of pain and the analgesic effect of drugs.

Molecular mechanisms underlying the enhanced analgesic effect of oxycodone compared to morphine in chemotherapy-induced neuropathic pain.

Oxycodone is a µ-opioid receptor agonist, used for the treatment of a large variety of painful disorders. Several studies have reported that oxycodone is a more potent pain reliever than morphine, and that it improves the quality of life of patients. However, the neurobiological mechanisms underlying the therapeutic action of these two opioids are only partially understood. The aim of this study was to define the molecular changes underlying the long-lasting analgesic effects of oxycodone and morphine in an animal model of peripheral neuropathy induced by a chemotherapic agent, vincristine. Using a behavioural approach, we show that oxycodone maintains an optimal analgesic effect after chronic treatment, whereas the effect of morphine dies down. In addition, using DNA microarray technology on dorsal root ganglia, we provide evidence that the long-term analgesic effect of oxycodone is due to an up-regulation in GABAB receptor expression in sensory neurons. These receptors are transported to their central terminals within the dorsal horn, and subsequently reinforce a presynaptic inhibition, since only the long-lasting (and not acute) anti-hyperalgesic effect of oxycodone was abolished by intrathecal administration of a GABAB receptor antagonist; in contrast, the morphine effect was unaffected. Our study demonstrates that the GABAB receptor is functionally required for the alleviating effect of oxycodone in neuropathic pain condition, thus providing new insight into the molecular mechanisms underlying the sustained analgesic action of oxycodone.

Specific involvement of atypical PKCζ/PKMζ in spinal persistent nociceptive processing following peripheral inflammation in rat.

BACKGROUND: Central sensitization requires the activation of various intracellular signalling pathways within spinal dorsal horn neurons, leading to a lowering of activation threshold and enhanced responsiveness of these cells. Such plasticity contributes to the manifestation of chronic pain states and displays a number of features of long-term potentiation (LTP), a ubiquitous neuronal mechanism of increased synaptic strength. Here we describe the role of a novel pathway involving atypical PKCζ/PKMζ in persistent spinal nociceptive processing, previously implicated in the maintenance of late-phase LTP. RESULTS: Using both behavioral tests and in vivo electrophysiology in rats, we show that inhibition of this pathway, via spinal delivery of a myristoylated protein kinase C-ζ pseudo-substrate inhibitor, reduces both pain-related behaviors and the activity of deep dorsal horn wide dynamic range neurons (WDRs) following formalin administration. In addition, Complete Freund's Adjuvant (CFA)-induced mechanical and thermal hypersensitivity was also reduced by inhibition of PKCζ/PKMζ activity. Importantly, this inhibition did not affect acute pain or locomotor behavior in normal rats and interestingly, did not inhibited mechanical allodynia and hyperalgesia in neuropathic rats. Pain-related behaviors in both inflammatory models coincided with increased phosphorylation of PKCζ/PKMζ in dorsal horn neurons, specifically PKMζ phosphorylation in formalin rats. Finally, inhibition of PKCζ/PKMζ activity decreased the expression of Fos in response to formalin and CFA in both superficial and deep laminae of the dorsal horn. CONCLUSIONS: These results suggest that PKCζ, especially PKMζ isoform, is a significant factor involved in spinal persistent nociceptive processing, specifically, the manifestation of chronic pain states following peripheral inflammation.

Perturbing PSD-95 interactions with NR2B-subtype receptors attenuates spinal nociceptive plasticity and neuropathic pain.

Peripheral inflammation or nerve injury induces a primary afferent barrage into the spinal cord, which can cause N-methyl D-aspartate (NMDA) receptor-dependent alterations in the responses of dorsal horn sensory neurons to subsequent afferent inputs. This plasticity, such as "wind-up" and central sensitization, contributes to the hyperexcitability of dorsal horn neurons and increased pain-related behavior in animal models, as well as clinical signs of chronic pain in humans, hyperalgesia and allodynia. Binding of NMDA receptor subunits by the scaffolding protein postsynaptic density protein-95 (PSD-95) can facilitate downstream intracellular signaling and modulate receptor stability, contributing to synaptic plasticity. Here, we show that spinal delivery of the mimetic peptide Tat-NR2B9c disrupts the interaction between PSD-95 and NR2B subunits in the dorsal horn and selectively reduces NMDA receptor-dependent events including wind-up of spinal sensory neurons, and both persistent formalin-induced neuronal activity and pain-related behaviors, attributed to central sensitization. Furthermore, a single intrathecal injection of Tat-NR2B9c in rats with established nerve injury-induced pain attenuates behavioral signs of mechanical and cold hypersensitivity, with no effect on locomotor performance. Thus, uncoupling of PSD-95 from spinal NR2B-containing NMDA receptors may prevent the neuronal plasticity involved in chronic pain and may be a successful analgesic therapy, reducing side effects associated with receptor blockade.

Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats.

Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain.

Ultraviolet-B-induced mechanical hyperalgesia: A role for peripheral sensitisation.

Ultraviolet (UV) induced cutaneous inflammation is emerging as a model of pain with a novel sensory phenotype. A UVB dose of 1000mJ/cm2 produces a highly significant thermal and mechanical hypersensitivity. Here we examined the properties and mechanisms of such hyperalgesia in rats. Significantly, the mechanical hyperalgesia (with approximately 60% change in withdrawal thresholds) was restricted to the lesion site with no changes in mechanical threshold in adjacent non-irradiated skin (i.e. no secondary hypersensitivity), suggesting a peripheral mechanism. Consistent with this, we found that primary mechanical hypersensitivity showed no significant changes after intrathecal treatment with 10microg of the NMDA-receptor antagonist MK-801. Using an in vitro skin-nerve preparation, in the presence and absence of UVB-inflammation, suprathreshold responses to skin displacement stimuli of 6-768microm of 103 peripheral nociceptors were recorded. At the peak of UVB-induced hyperalgesia we observed that mechanical response properties of Adelta-nociceptors recorded from UVB-inflamed skin (n=19) were significantly diminished, by approximately 50%, compared to those recorded from naïve skin (n=13). The mechanical response properties of heat-sensitive C-nociceptors were unchanged while their heat responses were significantly increased, by approximately 75%, in UVB-inflamed (n=26) compared to naïve skin (n=12). Heat-insensitive C-nociceptors, however, demonstrated significantly enhanced (by approximately 60%) response properties to mechanical stimulation in UVB-inflamed (n=21) compared to naïve skin (n=12). Notably alteration in mechanical responses of Adelta- and heat-insensitive C-nociceptors were particular to stronger stimuli. Spontaneous activity was not induced by this dose of UVB. We conclude that UVB-induced mechanical hyperalgesia may be explained by a net shift in peripheral nociceptor response properties.