Iain P. Chessell

Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain.

&NA; The P2X7 purinoceptor is a ligand‐gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)‐1β following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant‐induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro‐IL‐1β, and cytometric analysis of paw and systemic cytokines from knockout and wild‐type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL‐1β and IL‐10, with systemic reductions in adjuvant‐induced increases in IL‐6 and MCP‐1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients. We hypothesise that the P2X7 receptor, via regulation of mature IL‐1β production, plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential to deliver broad‐spectrum analgesia.

COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord

BackgroundWhile multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) are primarily inflammatory and degenerative disorders respectively, there is increasing evidence for shared cellular mechanisms that may affect disease progression, particularly glial responses. Cyclooxygenase 2 (COX-2) inhibition prolongs survival and cannabinoids ameliorate progression of clinical disease in animal models of ALS and MS respectively, but the mechanism is uncertain. Therefore, three key molecules known to be expressed in activated microglial cells/macrophages, COX-2, CB2 and P2X7, which plays a role in inflammatory cascades, were studied in MS and ALS post-mortem human spinal cord.MethodsFrozen human post mortem spinal cord specimens, controls (n = 12), ALS (n = 9) and MS (n = 19), were available for study by immunocytochemistry and Western blotting, using specific antibodies to COX-2, CB2 and P2X7, and markers of microglial cells/macrophages (CD 68, ferritin). In addition, autoradiography for peripheral benzodiazepine binding sites was performed on some spinal cord sections using [3H] (R)-PK11195, a marker of activated microglial cells/macrophages. Results of immunostaining and Western blotting were quantified by computerized image and optical density analysis respectively.ResultsIn control spinal cord, few small microglial cells/macrophages-like COX-2-immunoreactive cells, mostly bipolar with short processes, were scattered throughout the tissue, whilst MS and ALS specimens had significantly greater density of such cells with longer processes in affected regions, by image analysis. Inflammatory cell marker CD68-immunoreactivity, [3H] (R)-PK11195 autoradiography, and double-staining against ferritin confirmed increased production of COX-2 by activated microglial cells/macrophages. An expected 70-kDa band was seen by Western blotting which was significantly increased in MS spinal cord. There was good correlation between the COX-2 immunostaining and optical density of the COX-2 70-kDa band in the MS group (r = 0.89, P = 0.0011, n = 10). MS and ALS specimens also had significantly greater density of P2X7 and CB2-immunoreactive microglial cells/macrophages in affected regions.ConclusionIt is hypothesized that the known increase of lesion-associated extracellular ATP contributes via P2X7 activation to release IL-1 beta which in turn induces COX-2 and downstream pathogenic mediators. Selective CNS-penetrant COX-2 and P2X7 inhibitors and CB2 specific agonists deserve evaluation in the progression of MS and ALS.

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, P2X4 receptors (P2X4R) are expressed de novo by activated microglia in the spinal cord. Using in vitro and in vivo models, we provide direct evidence that P2X4R 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 P2X4-deficient mice microglia in primary cultures. These results indicate that P2X4R contribute to chronic pain through a central inflammatory pathway. P2X4R might thus represent a potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain injury and neurodegenerative disorders.

Psychophysical and functional imaging evidence supporting the presence of central sensitization in a cohort of osteoarthritis patients.

OBJECTIVE The groin pain experienced by patients with hip osteoarthritis (OA) is often accompanied by areas of referred pain and changes in skin sensitivity. We aimed to identify the supraspinal influences that underlie these clinical manifestations that we consider indicative of possible central sensitization. METHODS Twenty patients with hip OA awaiting joint replacement and displaying signs of referred pain were recruited into the study, together with age-matched controls. All subjects completed pain psychology questionnaires and underwent quantitative sensory testing (QST) in their area of referred pain. Twelve of 20 patients and their age- and sex-matched controls underwent functional magnetic resonance imaging (MRI) while the areas of referred pain were stimulated using cold stimuli (12 degrees C) and punctate stimuli (256 mN). The remaining 8 of 20 patients underwent punctate stimulation only. RESULTS Patients were found to have significantly lower threshold perception to punctate stimuli and were hyperalgesic to the noxious punctate stimulus in their areas of referred pain. Functional brain imaging illustrated significantly greater activation in the brainstem of OA patients in response to punctate stimulation of their referred pain areas compared with healthy controls, and the magnitude of this activation positively correlated with the extent of neuropathic-like elements to the patients pain, as indicated by the PainDETECT score. DISCUSSION Using psychophysical (QST) and brain imaging methods (functional MRI), we have identified increased activity with the periaqueductal grey matter associated with stimulation of the skin in referred pain areas of patients with hip OA. This offers a central target for analgesia aimed at improving the treatment of this largely peripheral disease.

The Voltage-Gated Sodium Channel Nav1.9 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, α) gene that encodes the voltage-gated sodium channel Nav1.9 to assess its contribution to pain. Nav1.9 is present in nociceptor sensory neurons that express TRPV1, bradykinin B2, and purinergic P2X3 receptors. In Nav1.9−/− 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 Nav1.9. Pain hypersensitivity elicited by intraplantar administration of prostaglandin E2, bradykinin, interleukin-1β, capsaicin, and P2X3 and P2Y receptor agonists, but not NGF, is either reduced or absent in Nav1.9−/− mice, whereas basal thermal and mechanical pain sensitivity is unchanged. Thermal, but not mechanical, hypersensitivity produced by peripheral inflammation (intraplanatar complete Freunds adjuvant) is substantially diminished in the null allele mutant mice, whereas hypersensitivity in two neuropathic pain models is unchanged in the Nav1.9−/− mice. Nav1.9 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.

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

BackgroundTransient 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.MethodsWe 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.ResultsTRPV1 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.ConclusionThe 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.

An fMRI study of cerebral processing of brush-evoked allodynia in neuropathic pain patients.

Previous human imaging studies have revealed a network of brain regions involved in the processing of allodynic pain; this includes prefrontal areas, insula, cingulate cortex, primary and secondary somatosensory cortices and parietal association areas. In this study, the neural correlates of the perceived intensity of allodynic pain in neuropathic pain patients were investigated. In eight patients, dynamic mechanical allodynia was provoked and brain responses recorded using functional magnetic resonance imaging (fMRI). Voxels in which the magnitude of fMRI signal correlated linearly with the ratings of allodynic pain across the group were determined in a whole brain analysis using a general linear model. To ensure that activation reflected only allodynic pain ratings, a nuisance variable containing ratings of ongoing pain was included in the analysis. We found that the magnitude of activation in the caudal anterior insula (cAI) correlates with the perceived intensity of allodynic pain across subjects, independent of the level of ongoing pain. However, the peak of activation in the allodynic condition was located in the rostral portion (rAI). This matches the representation of other clinical pain syndromes, confirmed by a literature review. In contrast, experimental pain in healthy volunteers resides predominantly in the cAI, as shown by the same literature review. Taken together, our data and the literature review suggest a functional segregation of anterior insular cortex.

The putative cannabinoid receptor GPR55 plays a role in mechanical hyperalgesia associated with inflammatory and neuropathic pain

Abstract It has been postulated that the G protein‐coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB1 and CB2 receptors are effective analgesic and anti‐inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well‐validated GPR55 tool compounds, a GPR55 knockout (GPR55−/−) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55−/− mice revealed no obvious primary differences, compared with wild‐type (GPR55+/+) littermates. GPR55−/− mice were then tested in the models of adjuvant‐induced inflammation and partial nerve ligation. Following intraplantar administration of Freund’s complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55−/− mice up to 14 days post‐injection. Cytokine profiling experiments showed that at 14 days post‐FCA injection there were increased levels of IL‐4, IL‐10, IFNγ and GM‐CSF in paws from the FCA‐injected GPR55−/− mice when compared with the FCA‐injected GPR55+/+ mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55−/− mice. In the model of neuropathic hypersensitivity, GPR55−/− mice also failed to develop mechanical hyperalgesia up to 28 days post‐ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.

Burning mouth syndrome as a trigeminal small fibre neuropathy: Increased heat and capsaicin receptor TRPV1 in nerve fibres correlates with pain score.

Burning mouth syndrome (BMS) is often an idiopathic chronic and intractable pain condition, affecting 1.5-5.5% of middle-aged and elderly women. We have studied the heat and capsaicin receptor TRPV1, and its regulator nerve growth factor (NGF), in BMS. Patients with BMS (n=10) and controls (n=10) were assessed for baseline and post-topical capsaicin pain scores, and their tongue biopsies immunostained for TRPV1, NGF, and structural nerve markers neurofilament and peripherin. Nerve fibres penetrating the epithelium were less abundant in BMS (p<0.0001), indicating a small fibre neuropathy. TRPV1-positive fibres were overall significantly increased in BMS (p=0.0011), as were NGF fibres (p<0.0001) and basal epithelial cell NGF staining (p<0.0147). There was a significant correlation between the baseline pain score and TRPV1 (p=0.0143) and NGF fibres (p=0.0252). A significant correlation was observed between baseline and post-capsaicin pain (p=0.0006). Selective TRPV1 and NGF blockers may provide a new therapy for BMS.

TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons

TRPA1 is a receptor expressed by sensory neurons, that is activated by low temperature (<17 degrees C) and plant derivatives such as cinnamaldehyde and isoeugenol, to elicit sensations including pain. Using immunohistochemistry, we have, for the first time, localised TRPA1 in human DRG neurons, spinal cord motoneurones and nerve roots, peripheral nerves, intestinal myenteric plexus neurones, and skin basal keratinocytes. TRPA1 co-localised with a subset of hDRG neurons positive for TRPV1, the heat and capsaicin receptor. The number of small/medium TRPA1 positive neurons (< or =50 microm) was increased after hDRG avulsion injury [percentage of cells, median (range): controls 16.5 (7-23); injured 46 (34-55); P<0.005], but the number of large TRPA1 neurons was unchanged [control 19.5 (13-31); injured 21 (11-35)]. Similar TRPA1 changes were observed in cultured hDRG neurons, after exposure to a combination of key neurotrophic factors NGF, GDNF and NT-3 (NTFs) in vitro. We used calcium imaging to examine responses of HEK cells transfected with hTRPA1 cDNA, and of human and rat DRG neurons cultured with or without added NTFs, to cinnamaldehyde (CA) and isoeugenol (IE). Exposure to NTFs in vitro sensitized cultured human sensory neuronal responses to CA; repeated CA exposure produced desensitisation. In rDRG neurons, low (225 microM) CA preincubation enhanced capsaicin responses, while high (450 microM and 2mM) CA caused inhibition which was partially reversed in the presence of 8 bromo cAMP, indicating receptor dephosphorylation. While TRPA1 localisation is more widespread than TRPV1, it represents a promising novel drug target for the treatment of chronic pain and hypersensitivity.