Sensitization of colonic nociceptors by IL-13 is dependent on JAK and p38 MAPK activity.

The effective management of visceral pain is a significant unmet clinical need for those affected by gastrointestinal diseases, such as inflammatory bowel disease (IBD). The rational design of novel analgesics requires a greater understanding of the mediators and mechanisms underpinning visceral pain. Interleukin-13 (IL-13) production by immune cells residing in the gut is elevated in IBD, and IL-13 appears to be important in the development of experimental colitis. Furthermore, receptors for IL-13 are expressed by neurons innervating the colon, though it is not known whether IL-13 plays any role in visceral nociception per se. To resolve this, we used Ca2+ imaging of cultured sensory neurons and ex vivo electrophysiological recording from the lumbar splanchnic nerve innervating the distal colon. Ca2+ imaging revealed the stimulation of small-diameter, capsaicin-sensitive sensory neurons by IL-13, indicating that IL-13 likely stimulates nociceptors. IL-13-evoked Ca2+ signals were attenuated by inhibition of Janus (JAK) and p38 kinases. In the lumbar splanchnic nerve, IL-13 did not elevate baseline firing, nor sensitize the response to capsaicin application, but did enhance the response to distention of the colon. In line with Ca2+ imaging experiments, IL-13-mediated sensitization of the afferent response to colon distention was blocked by inhibition of either JAK or p38 kinase signaling. Together, these data highlight a potential role for IL-13 in visceral nociception and implicate JAK and p38 kinases in pronociceptive signaling downstream of IL-13.

Sensitization of colonic nociceptors by TNFα is dependent on TNFR1 expression and p38 MAPK activity.

Visceral pain is a leading cause of morbidity in gastrointestinal diseases, which is exacerbated by the gut-related side-effects of many analgesics. New treatments are needed and further understanding of the mediators and mechanisms underpinning visceral nociception in disease states is required to facilitate this. The pro-inflammatory cytokine TNFα is linked to pain in both patients with inflammatory bowel disease and irritable bowel syndrome, and has been shown to sensitize colonic sensory neurons. Somatic, TNFα-triggered thermal and mechanical hypersensitivity is mediated by TRPV1 signalling and p38 MAPK activity respectively, downstream of TNFR1 receptor activation. We therefore hypothesized that TNFR1-evoked p38 MAPK activity may also be responsible for TNFα sensitization of colonic afferent responses to the TRPV1 agonist capsaicin, and noxious distension of the bowel. Using Ca2+ imaging of dorsal root ganglion sensory neurons, we observed TNFα-mediated increases in intracellular [Ca2+ ] and sensitization of capsaicin responses. The sensitizing effects of TNFα were dependent on TNFR1 expression and attenuated by p38 MAPK inhibition. Consistent with these findings, ex vivo colonic afferent fibre recordings demonstrated an enhanced response to noxious ramp distention of the bowel and bath application of capsaicin following TNFα pre-treatment. Responses were reversed by p38 MAPK inhibition and absent in tissue from TNFR1 knockout mice. Our findings demonstrate a contribution of TNFR1, p38 MAPK and TRPV1 to TNFα-induced sensitization of colonic afferents, highlighting the potential utility of these drug targets for the treatment of visceral pain in gastrointestinal disease. KEY POINTS: The pro-inflammatory cytokine TNFα is elevated in gastrointestinal disease and sensitizes colonic afferents via modulation of TRPA1 and NaV 1.8 activity. We further develop this understanding by demonstrating a role for p38 MAPK and TRPV1 in TNFα-mediated colonic afferent sensitization. Specifically, we show that: TNFα sensitizes sensory neurons and colonic afferents to the TRPV1 agonist capsaicin. TNFα-mediated sensitization of sensory neurons and colonic nociceptors is dependent on TNFR1 expression. TNFα sensitization of sensory neurons and colonic afferents to capsaicin and noxious ramp distension is abolished by inhibition of p38 MAPK. Collectively these data support the utility of targeting TNFα, TNFR1 and their downstream signalling via p38 MAPK for the treatment of visceral pain in gastrointestinal disease.

A novel, injury-free rodent model of vulnerability for assessment of acute and preventive therapies reveals temporal contributions of CGRP-receptor activation in migraine-like pain.

AIM: Development and characterization of a novel injury-free preclinical model of migraine-like pain allowing mechanistic assessment of both acute and preventive treatments. METHODS: A "two-hit" hyperalgesic priming strategy was used to induce vulnerability to a normally subthreshold challenge with umbellulone, a transient receptor potential ankyrin 1 (TRPA1) activator, in uninjured female and male C57BL/6 mice. Priming (i.e. the first hit) was induced by three consecutive daily episodes of restraint stress; repeated umbellulone was also evaluated for potential priming effects. Sixteen days after the first restraint stress, mice received inhalational umbellulone (i.e. the second hit) to elicit migraine-like pain. Medications currently used for acute or preventive migraine therapy including propranolol (a beta blocker) and sumatriptan (5HT1B/D agonist), as well as olcegepant, an experimental calcitonin gene related peptide (CGRP) receptor antagonist and nor-Binaltorphimine (nor-BNI), an experimental long-acting kappa opioid receptor (KOR) antagonist, were investigated for their efficacy to block priming and prevent or reverse umbellulone-induced allodynia in primed animals. To assess migraine-like pain, cutaneous allodynia was determined by responses to periorbital or hindpaw probing with von Frey filaments. RESULTS: Repeated restraint stress, but not umbellulone exposure, produced transient cutaneous allodynia that resolved within 16 d. Restraint stress produced long-lasting priming that persisted beyond 16 d, as demonstrated by reinstatement of cutaneous allodynia following inhalational umbellulone challenge. Pretreatment with propranolol or nor-BNI prior to restraint stress prevented both transient cutaneous allodynia and priming, demonstrated by a lack of umbellulone-induced cutaneous allodynia. Following establishment of restraint stress priming, olcegepant, but not propranolol or nor-BNI, prevented umbellulone-induced cutaneous allodynia. When administered 1 h after umbellulone, sumatriptan, but not olcegepant, reversed umbellulone-induced cutaneous allodynia in restraint stress-primed rats. CONCLUSION: We have developed a novel injury-free model with translational relevance that can be used to study mechanisms relevant to migraine-like pain and to evaluate novel acute or preventive treatments. Restraint stress priming induced a state of vulnerability to a subthreshold stimulus that has been referred to as "latent sensitization". The development of latent sensitization could be prevented by blockade of stress pathways with propranolol or with a kappa opioid receptor antagonist. Following establishment of latent sensitization, subthreshold stimulation with umbellulone reinstated cutaneous allodynia, likely from activation of meningeal TRPA1-expressing nociceptors. Accordingly, in restraint stress-primed animals, sumatriptan reversed umbellulone-induced cutaneous allodynia, supporting peripheral sites of action, while propranolol and nor-BNI were not effective. Surprisingly, olcegepant was effective in mice with latent sensitization when given prior to, but not after, umbellulone challenge, suggesting time-dependent contributions of calcitonin gene-related peptide receptor signaling in promoting migraine-like pain in this model. Activation of the calcitonin gene-related peptide receptor participates in initiating, but has a more limited role in maintaining, pain responses, supporting the efficacy of small molecule calcitonin gene-related peptide antagonists as preventive medications. Additionally, the effectiveness of sumatriptan in reversal of established pain thus suggests modulation of additional, non-calcitonin gene-related peptide receptor-mediated nociceptive mechanisms. Kappa opioid receptor antagonists may represent a novel preventive therapy for stress-related migraine.

The Dementias Platform UK (DPUK) Data Portal.

The Dementias Platform UK Data Portal is a data repository facilitating access to data for 3 370 929 individuals in 42 cohorts. The Data Portal is an end-to-end data management solution providing a secure, fully auditable, remote access environment for the analysis of cohort data. All projects utilising the data are by default collaborations with the cohort research teams generating the data. The Data Portal uses UK Secure eResearch Platform infrastructure to provide three core utilities: data discovery, access, and analysis. These are delivered using a 7 layered architecture comprising: data ingestion, data curation, platform interoperability, data discovery, access brokerage, data analysis and knowledge preservation. Automated, streamlined, and standardised procedures reduce the administrative burden for all stakeholders, particularly for requests involving multiple independent datasets, where a single request may be forwarded to multiple data controllers. Researchers are provided with their own secure 'lab' using VMware which is accessed using two factor authentication. Over the last 2 years, 160 project proposals involving 579 individual cohort data access requests were received. These were received from 268 applicants spanning 72 institutions (56 academic, 13 commercial, 3 government) in 16 countries with 84 requests involving multiple cohorts. Projects are varied including multi-modal, machine learning, and Mendelian randomisation analyses. Data access is usually free at point of use although a small number of cohorts require a data access fee.

Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-fluorophenyl}acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain.

A novel series of EP4 agonists and antagonists have been identified, and then used to validate their potential in the treatment of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical models of pain, ultimately leading to the identification of the EP4 partial agonist GSK726701A.

Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins.

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.

Direct activation of KCC2 arrests benzodiazepine refractory status epilepticus and limits the subsequent neuronal injury in mice.

Hyperpolarizing GABAAR currents, the unitary events that underlie synaptic inhibition, are dependent upon efficient Cl- extrusion, a process that is facilitated by the neuronal specific K+/Cl- co-transporter KCC2. Its activity is also a determinant of the anticonvulsant efficacy of the canonical GABAAR-positive allosteric: benzodiazepines (BDZs). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to BDZ (BDZ-RSE). Here, we have identified small molecules that directly bind to and activate KCC2, which leads to reduced neuronal Cl- accumulation and excitability. KCC2 activation does not induce any overt effects on behavior but prevents the development of and terminates ongoing BDZ-RSE. In addition, KCC2 activation reduces neuronal cell death following BDZ-RSE. Collectively, these findings demonstrate that KCC2 activation is a promising strategy to terminate BDZ-resistant seizures and limit the associated neuronal injury.

A novel series of benzimidazole NR2B-selective NMDA receptor antagonists.

A series of novel benzimidazoles are discussed as NR2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists. High throughput screening (HTS) efforts identified a number of potent and selective NR2B antagonists such as 1. Exploration of the substituents around the core of this template identified a number of compounds with high potency for NR2B (pIC(50) >7) and good selectivity against the NR2A subunit (pIC(50) <4.3) as defined by FLIPR-Ca(2+) and radioligand binding studies. These agents offer potential for the development of therapeutics for a range of nervous system disorders including chronic pain, neurodegeneration, migraine and major depression.

Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain.

ATP is a known mediator of inflammatory and neuropathic pain. However, the mechanisms by which specific purinergic receptors contribute to chronic pain states are still poorly characterized. Here, we demonstrate that in response to peripheral nerve injury, P2X(4) receptors (P2X(4)R) are expressed de novo by activated microglia in the spinal cord. Using in vitro and in vivo models, we provide direct evidence that P2X(4)R stimulation leads to the release of BDNF from activated microglia and, most likely phosphorylation of the NR1 subunit of NMDA receptors in dorsal horn neurons of the spinal cord. Consistent with these findings, P2X4-deficient mice lack mechanical hyperalgesia induced by peripheral nerve injury and display impaired BDNF signaling in the spinal cord. Furthermore, ATP stimulation is unable to stimulate BDNF release from P2X(4)-deficient mice microglia in primary cultures. These results indicate that P2X(4)R contribute to chronic pain through a central inflammatory pathway. P2X(4)R might thus represent a potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain injury and neurodegenerative disorders.

Pyridine-3-carboxamides as novel CB(2) agonists for analgesia.

We describe herein the medicinal chemistry approach which led to the discovery of a novel pyridine-3-carboxamide series of CB(2) receptor agonists. The SAR of this new template was evaluated and culminated in the identification of analogue 14a which demonstrated efficacy in an in vivo model of inflammatory pain.

Identification of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidinyl] amines and ethers as potent and selective cyclooxygenase-2 inhibitors.

A novel series of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidine-based cyclooxygenase-2 (COX-2) inhibitors, which have a different arrangement of substituents compared to the more common 1,2-diarylheterocycle based molecules, have been discovered. For example, 2-(butyloxy)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine (47), a member of the 2-pyrimidinyl ether series, has been shown to be a potent and selective inhibitor with a favourable pharmacokinetic profile, high brain penetration and good efficacy in rat models of hypersensitivity.

Noninvasive Mechanical Joint Loading as an Alternative Model for Osteoarthritic Pain.

OBJECTIVE: Mechanisms responsible for osteoarthritic (OA) pain remain poorly understood, and current analgesic therapies are often insufficient. This study was undertaken to characterize and pharmacologically test the pain phenotype of a noninvasive mechanical joint loading model of OA, thus providing an alternative murine model for OA pain. METHODS: The right knees of 12-week-old male C57BL/6 mice were loaded at 9N or 11N (40 cycles, 3 times per week for 2 weeks). Behavioral measurements of limb disuse and mechanical and thermal hypersensitivity were acquired before mechanical joint loading and monitored for 6 weeks postloading. The severity of articular cartilage lesions was determined postmortem with the Osteoarthritis Research Society International scoring system. To assess efficacy of various treatments for pain, 9N-loaded mice were treated for 4 weeks with diclofenac (10 mg/kg), gabapentin (100 mg/kg), or anti-nerve growth factor (anti-NGF) (3 mg/kg). RESULTS: Mechanical hypersensitivity and weight bearing worsened significantly in 9N-loaded mice (n = 8) and 11N-loaded mice (n = 8) 2 weeks postloading, compared to baseline values and nonloaded controls. Maximum OA scores of ipsilateral knees confirmed increased cartilage lesions in 9N-loaded mice (mean ± SEM 2.8 ± 0.2; P < 0.001) and 11N-loaded mice (5.3 ± 0.3; P < 0.001), compared to nonloaded controls (1.0 ± 0.0). Gabapentin and diclofenac restored pain behaviors to baseline values after 2 weeks of daily treatment, and gabapentin was more effective than diclofenac. A single injection of anti-NGF alleviated nociception 2 days after treatment and remained effective for 2 weeks, with a second dose inducing stronger and more prolonged analgesia. CONCLUSION: Our findings show that mechanical joint loading induces OA lesions in mice and a robust pain phenotype that can be reversed using analgesics known to alleviate OA pain in patients. This establishes the use of mechanical joint loading as an alternative model for the study of OA pain.

The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity.

We used a mouse with deletion of exons 4, 5, and 6 of the SCN11A (sodium channel, voltage-gated, type XI, alpha) gene that encodes the voltage-gated sodium channel Na(v)1.9 to assess its contribution to pain. Na(v)1.9 is present in nociceptor sensory neurons that express TRPV1, bradykinin B2, and purinergic P2X3 receptors. In Na(v)1.9-/- mice, the non-inactivating persistent tetrodotoxin-resistant sodium TTXr-Per current is absent, whereas TTXr-Slow is unchanged. TTXs currents are unaffected by the mutation of Na(v)1.9. Pain hypersensitivity elicited by intraplantar administration of prostaglandin E2, bradykinin, interleukin-1beta, capsaicin, and P2X3 and P2Y receptor agonists, but not NGF, is either reduced or absent in Na(v)1.9-/- mice, whereas basal thermal and mechanical pain sensitivity is unchanged. Thermal, but not mechanical, hypersensitivity produced by peripheral inflammation (intraplanatar complete Freund's adjuvant) is substantially diminished in the null allele mutant mice, whereas hypersensitivity in two neuropathic pain models is unchanged in the Na(v)1.9-/- mice. Na(v)1.9 is, we conclude, an effector of the hypersensitivity produced by multiple inflammatory mediators on nociceptor peripheral terminals and therefore plays a key role in mediating peripheral sensitization.

Discovery of 2-[(2,4-dichlorophenyl)amino]-N-[(tetrahydro- 2H-pyran-4-yl)methyl]-4-(trifluoromethyl)- 5-pyrimidinecarboxamide, a selective CB2 receptor agonist for the treatment of inflammatory pain.

Selective CB2 receptor agonists are promising potential therapeutic agents for the treatment of inflammatory and neuropathic pain. A focused screen identified a pyrimidine ester as a partial agonist at the CB2 receptor with micromolar potency. Subsequent lead optimization identified 35, GW842166X, as the optimal compound in the series. 35 has an oral ED50 of 0.1 mg/kg in the rat FCA model of inflammatory pain and was selected as a clinical candidate for this indication.

Pressure application measurement (PAM): a novel behavioural technique for measuring hypersensitivity in a rat model of joint pain.

Chronic joint pain affects physical well being and can lead to severe psychological and social problems, therefore successful long-term management is highly sought-after. No current behavioural measures of pain used in pre-clinical models mimic the clinical dolorimeter, which provides an objective measure of joint hypersensitivity. In this study we aim to use a novel behavioural readout alongside an established measure to mimic the multifactorial measurements taken in the clinic. Using the pressure application measurement (PAM) device a gradually increasing squeeze was applied across the knee joint of rats until the animal gave an indication of pain or discomfort. PAM and the incapacitance tester were used to detect joint hypersensitivity in a well-established rodent model of adjuvant-induced arthritis. Subsequently, the analgesic effects of prednisolone (1, 3 or 10 mg kg(-1)), morphine (3 mg kg(-1)) and celecoxib (15 mg kg(-1)) were assessed. Both PAM and the incapacitance tester detected a reversal of hypersensitivity 1h post-drug administration. Furthermore, the two readouts were highly correlated, and power analysis indicated that PAM was highly reproducible. In conclusion, PAM provides a novel, accurate behavioural tool for detecting a primary mechanical hypersensitivity in a rat model of chronic inflammatory joint pain.

Changes in dorsal root ganglion CGRP expression in a chronic inflammatory model of the rat knee joint: differential modulation by rofecoxib and paracetamol.

Neuropeptide-expressing small diameter sensory neurones are thought to be vital in generating inflammatory hyperalgesic responses. Within the dorsal root ganglion (DRG), both the levels of the neuropeptide calcitonin gene-related peptide (CGRP) and the numbers of CGRP-immunoreactive (CGRP-IR) DRG neurones have been shown to increase in a number of acute adjuvant-induced inflammatory pain models. The aim of this study was to look specifically at changes in numbers of CGRP-IR DRG neurones in a chronic model of inflammatory joint pain following complete Freund's adjuvant (CFA) injection into the rat knee. In this model, there were significant increases in the number of ipsilateral CGRP-IR small DRG neurones at days 1, 16 and 35 following intra-articular CFA, compared to saline-injected sham animals. This correlated with the behavioural readouts of hypersensitivity and knee joint inflammation at the same time points. There was also a significant increase in the number of ipsilateral CGRP-IR medium DRG neurones and contralateral CGRP-IR small DRG neurones at day 1. Following dosing of CFA-injected rats with rofecoxib (Vioxx) or paracetamol, there was a significant decrease in the number of ipsilateral CGRP-IR small and medium DRG neurones in rofecoxib- but not paracetamol-treated rats. These data also correlated with behavioural readouts where hypersensitivity and knee joint inflammation were significantly reduced by rofecoxib but not paracetamol treatment. In conclusion, these data show that changes in ipsilateral CGRP expression within small DRG neurones are consistent with behavioural readouts in both time course, rofecoxib and paracetamol studies in this model of chronic inflammatory pain.

Localisation and modulation of prostanoid receptors EP1 and EP4 in the rat chronic constriction injury model of neuropathic pain.

Immunohistochemistry was used to examine the expression of prostaglandin E(2) receptors EP1 and EP4 in sciatic nerves from the rat chronic constriction injury (CCI) model of neuropathic pain. At 21 days post-surgery the CCI rats had developed mechanical hyperalgesia on the operated side, and quantitative image analysis showed a highly significant doubling of the area occupied by EP1- and EP4-positive pixels in sections from CCI nerves when compared to sham-operated controls. Co-localisation studies with the marker ED1 revealed that 73% of the EP1-positive cells and 54% of the EP4-positive cells in the injured nerves represented infiltrating macrophages. Cells negative for ED1 and positive for either EP1 or EP4 were characterised as Schwann cells from their morphology and expression of myelin basic protein and S100 antigens. Similar EP1- and EP4-positive Schwann cell profiles were observed in sections of uninjured control nerves. Low levels of EP receptor expression were found in neurofilament-immunostained axons, but no consistent differences were observed in the levels of axonal EP staining between CCI and control tissue. These data provide further evidence of the importance of prostaglandins in the pathogenesis of neuropathic pain, and suggest that not only infiltrating macrophages but also Schwann cells may be involved in the modulation of these mediators in response to nerve injury.

Voltage-gated ion channel Nav1.7 innervation in patients with idiopathic rectal hypersensitivity and paroxysmal extreme pain disorder (familial rectal pain).

Faecal urgency and incontinence with rectal hypersensitivity is a chronic, unexplained condition that is difficult to treat. The aim of this study was to determine if there was an altered level of the voltage gated tetrodotoxin-sensitive (TTX-s) sodium channel Na(v)1.7 in rectal sensory fibres, since this channel has been implicated in clinical nociceptive disorders. Full thickness rectal biopsies from patients with physiologically characterised rectal hypersensitivity (n=7) were compared with control tissues (n=10). Formalin fixed specimens were studied by immunohistochemistry using affinity purified antibodies to Na(v)1.7 and the pan-neuronal structural marker PGP9.5, and the immunoreactive nerve fibres quantified by computerised image analysis. In rectal hypersensitivity, Na(v)1.7 immunoreactive nerve fibres were significantly increased in mucosal (P=0.0004), sub-mucosal (P=0.019), and muscle layers (P=0.0076), while PGP9.5 immunoreactive nerve fibres were increased significantly only in the mucosa (P=0.04); ratios of Na(v)1.7:PGP9.5 showed a significant increase in all layers, suggesting increased expression of Na(v)1.7, and nerve sprouting in the mucosa. The cause of this increase remains uncertain, but may be due to increase of nerve growth factor (NGF), which regulates the expression of both Na(v)1.7 and TRPV1, which we have previously reported to be increased in this condition. In paroxysmal extreme pain disorder (familial rectal pain), where the gene that encodes Na(v)1.7 is mutated, Na(v)1.7 protein was undetectable in the rectum (n=2), which suggests reduced Na(v)1.7 immunoreactivity or expression. Drugs that target Na(v)1.7-expressing nerve terminals may be useful for treating rectal hypersensitivity, and combining these with TRPV1 antagonists may enhance efficacy.

Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy.

BACKGROUND: Transient receptor potential (TRP) receptors expressed by primary sensory neurons mediate thermosensitivity, and may play a role in sensory pathophysiology. We previously reported that human dorsal root ganglion (DRG) sensory neurons co-expressed TRPV1 and TRPV3, and that these were increased in injured human DRG. Related receptors TRPV4, activated by warmth and eicosanoids, and TRPM8, activated by cool and menthol, have been characterised in pre-clinical models. However, the role of TRPs in common clinical sensory neuropathies needs to be established. METHODS: We have studied TRPV1, TRPV3, TRPV4, and TRPM8 in nerves (n = 14) and skin from patients with nerve injury, avulsed dorsal root ganglia (DRG) (n = 11), injured spinal nerve roots (n = 9), diabetic neuropathy skin (n = 8), non-diabetic neuropathic nerve biopsies (n = 6), their respective control tissues, and human post mortem spinal cord, using immunohistological methods. RESULTS: TRPV1 and TRPV3 were significantly increased in injured brachial plexus nerves, and TRPV1 in hypersensitive skin after nerve repair, whilst TRPV4 was unchanged. TRPM8 was detected in a few medium diameter DRG neurons, and was unchanged in DRG after avulsion injury, but was reduced in axons and myelin in injured nerves. In diabetic neuropathy skin, TRPV1 expressing sub- and intra-epidermal fibres were decreased, as was expression in surviving fibres. TRPV1 was also decreased in non-diabetic neuropathic nerves. Immunoreactivity for TRPV3 was detected in basal keratinocytes, with a significant decrease of TRPV3 in diabetic skin. TRPV1-immunoreactive nerves were present in injured dorsal spinal roots and dorsal horn of control spinal cord, but not in ventral roots, while TRPV3 and TRPV4 were detected in spinal cord motor neurons. CONCLUSION: The accumulation of TRPV1 and TRPV3 in peripheral nerves after injury, in spared axons, matches our previously reported changes in avulsed DRG. Reduction of TRPV1 levels in nerve fibres in diabetic neuropathy skin may result from the known decrease of nerve growth factor (NGF) levels. The role of TRPs in keratinocytes is unknown, but a relationship to changes in NGF levels, which is produced by keratinocytes, deserves investigation. TRPV1 represents a more selective therapeutic target than other TRPs for pain and hypersensitivity, particularly in post-traumatic neuropathy.

Investigation into the role of P2X(3)/P2X(2/3) receptors in neuropathic pain following chronic constriction injury in the rat: an electrophysiological study.

1. Two P2X(3)/P2X(2/3) receptor antagonists with different potencies were profiled electrophysiologically in a rat model of nerve injury. 2. A-317491 has poor CNS penetrance (blood:brain, 1:<0.05), and was therefore administered intravenously in chronic constriction injury (CCI)- and sham-operated rats to study the involvement of P2X(3) subunit-containing receptors in the periphery in neuropathic pain. A-317491 and Compound A were administered topically to the spinal cord to investigate the central contribution. 3. There were no significant inhibitory effects of A-317491 intravenous (i.v.) seen in sham-operated animals compared to vehicle controls. In CCI-operated animals, there were significant inhibitory effects of 3 mg kg(-1) A-317491 i.v. on C fibre-evoked responses, and with 10 mg kg(-1) A-317491 i.v. on A delta and C fibre-evoked responses. No significant effects of A-317491 were observed after topical application to the spinal cord. In contrast, when Compound A was administered spinally in CCI animals, there was a decrease in A delta and C fibre-evoked responses, and wind up. 4. These changes indicate that A-317491 has a selective effect on neuronal responses in CCI animals compared to sham, demonstrating an increased involvement of P2X(3)/P2X(2/3) receptors in sensory signalling following nerve injury. In addition, the more potent antagonist Compound A was effective spinally, unmasking a potential central role of P2X(3)/P2X(2/3) receptors at this site post nerve injury. These data support a role for P2X(3)/P2X(2/3) antagonists in the modulation of neuropathic pain.