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Purpose Chemotherapy-induced peripheral neuropathy (CIPN) with associated chronic pain is a common and disabling condition. Current treatments for neuropathic pain in CIPN are largely ineffective, with unfavorable side-effects. The capsaicin 8% patch (capsaicin 179 mg patch) is approved for the treatment of neuropathic pain: a single topical cutaneous application can produce effective pain relief for up to 12 weeks. We assessed the therapeutic potential of capsaicin 8% patch in patients with painful CIPN, and its mechanism of action. Patients and methods 16 patients with chronic painful CIPN (mean duration 2.5 years), in remission for cancer and not receiving chemotherapy, were treated with 30 min application of capsaicin 8% patch to the feet. Symptoms were monitored using the 11-point numerical pain rating scale (NPRS), and questionnaires. Investigations were performed at baseline and three months after patch application, including skin biopsies with a range of markers, and quantitative sensory testing (QST). Results Patients reported significant reduction in spontaneous pain (mean NPRS: -1.27; 95% CI 0.2409 to 2.301; p=0.02), touch-evoked pain (-1.823; p=0.03) and cold-evoked pain (-1.456; p=0.03). Short-Form McGill questionnaire showed a reduction in neuropathic (p=0.0007), continuous (p=0.01) and overall pain (p=0.004); Patient Global Impression of Change showed improvement (p=0.001). Baseline skin biopsies showed loss of intra-epidermal nerve fibers (IENF), and also of sub-epidermal nerve fibers quantified by image analysis. Post-patch application skin biopsies showed a significant increase towards normalization of intra-epidermal and sub-epidermal nerve fibers (for IENF: structural marker PGP9.5, p=0.009; heat receptor TRPV1, p=0.027; regenerating nerve marker GAP43, p=0.04). Epidermal levels of Nerve Growth Factor (NGF), Neurotrophin-3 (NT-3), and Langerhans cells were also normalized. QST remained unchanged and there were no systemic side-effects, as in previous studies. Conclusion Capsaicin 8% patch provides significant pain relief in CIPN, and may lead to regeneration and restoration of sensory nerve fibers ie, disease modification.

Purpose Chemotherapy-induced peripheral neuropathy (CIPN) with associated chronic pain is a common and disabling condition. Current treatments for neuropathic pain in CIPN are largely ineffective, with unfavorable side-effects. The capsaicin 8% patch (capsaicin 179 mg patch) is approved for the treatment of neuropathic pain: a single topical cutaneous application can produce effective pain relief for up to 12 weeks. We assessed the therapeutic potential of capsaicin 8% patch in patients with painful CIPN, and its mechanism of action. Patients and methods 16 patients with chronic painful CIPN (mean duration 2.5 years), in remission for cancer and not receiving chemotherapy, were treated with 30 min application of capsaicin 8% patch to the feet. Symptoms were monitored using the 11-point numerical pain rating scale (NPRS), and questionnaires. Investigations were performed at baseline and three months after patch application, including skin biopsies with a range of markers, and quantitative sensory testing (QST). Results Patients reported significant reduction in spontaneous pain (mean NPRS: −1.27; 95% CI 0.2409 to 2.301; p=0.02), touch-evoked pain (−1.823; p=0.03) and cold-evoked pain (−1.456; p=0.03). Short-Form McGill questionnaire showed a reduction in neuropathic (p=0.0007), continuous (p=0.01) and overall pain (p=0.004); Patient Global Impression of Change showed improvement (p=0.001). Baseline skin biopsies showed loss of intra-epidermal nerve fibers (IENF), and also of sub-epidermal nerve fibers quantified by image analysis. Post-patch application skin biopsies showed a significant increase towards normalization of intra-epidermal and sub-epidermal nerve fibers (for IENF: structural marker PGP9.5, p=0.009; heat receptor TRPV1, p=0.027; regenerating nerve marker GAP43, p=0.04). Epidermal levels of Nerve Growth Factor (NGF), Neurotrophin-3 (NT-3), and Langerhans cells were also normalized. QST remained unchanged and there were no systemic side-effects, as in previous studies. Conclusion Capsaicin 8% patch provides significant pain relief in CIPN, and may lead to regeneration and restoration of sensory nerve fibers ie, disease modification.

Supplemental Digital Content is Available in the Text., Introduction: Granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is highly expressed in peripheral macrophages and microglia, and is involved in arthritis and cancer pain in animal models. However, there is limited information on GM-CSFR expression in human central nervous system (CNS), peripheral nerves, or dorsal root ganglia (DRG), particularly in chronic pain conditions. Objectives: Immunohistochemistry was used to quantify GM-CSFR expression levels in human tissues, and functional sensory effects of GM-CSF were studied in cultured DRG neurons. Results: Granulocyte-macrophage colony-stimulating factor receptor was markedly increased in microglia at lesional sites of multiple sclerosis spinal cords (P = 0.01), which co-localised with macrophage marker CD68 (P = 0.009). In human DRG, GM-CSFR was expressed in a subset of small/medium diameter cells (30%) and few large cells (10%), with no significant change in avulsion-injured DRG. In peripheral nerves, there was a marked decrease in axonal GM-CSFR after chronic painful nerve injury (P = 0.004) and in painful neuromas (P = 0.0043); CD-68–positive macrophages were increased (P = 0.017) but did not appear to express GM-CSFR. Although control synovium showed absent GM-CSFR immunostaining, this was markedly increased in macrophages of painful osteoarthritis knee synovium. Granulocyte-macrophage colony-stimulating factor receptor was expressed in 17 ± 1.7% of small-/medium-sized cultured adult rat DRG neurons, and in 27 ± 3.3% of TRPV1-positive neurons. Granulocyte-macrophage colony-stimulating factor treatment sensitized capsaicin responses in vitro, which were diminished by p38 MAPK or TrkA inhibitors. Conclusion: Our findings support GM-CSFR as a therapeutic target for pain and hypersensitivity in clinical CNS and peripheral inflammatory conditions. Although GM-CSFR was decreased in chronic painful injured peripheral nerves, it could mediate CNS neuroinflammatory effects, which deserves study.

Introduction Glucagon like-peptide 1 receptor (GLP-1R) agonists diminish appetite and may contribute to the weight loss in inflammatory bowel disease (IBD). Objectives The aim of this study was to determine, for the first time, the expression of GLP-1R by colon nerve fibres in patients with IBD, and functional effects of its agonists in cultured rat and human sensory neurons. Methods GLP-1R and other nerve markers were studied by immunohistochemistry in colon biopsies from patients with IBD (n = 16) and controls (n = 8), human dorsal root ganglia (DRG) tissue, and in GLP-1R transfected HEK293 cells. The morphological effects of incretin hormones oxyntomodulin, exendin-4 and glucagon were studied on neurite extension in cultured DRG neurons, and their functional effects on capsaicin and ATP signalling, using calcium imaging. Results Significantly increased numbers of colonic mucosal nerve fibres were observed in IBD biopsies expressing GLP-1R (p = 0.0013), the pan-neuronal marker PGP9.5 (p = 0.0008), and sensory neuropeptide CGRP (p = 0.0014). An increase of GLP-1R positive nerve fibres in IBD colon was confirmed with a different antibody to GLP-1R (p = 0.016). GLP-1R immunostaining was intensely positive in small and medium-sized neurons in human DRG, and in human and rat DRG cultured neurons. Co-localization of GLP-1R expression with neuronal markers in colon and DRG confirmed the neural expression of GLP-1R, and antibody specificity was confirmed in HEK293 cells transfected with the GLP-1R. Treatment with oxyntomodulin, exendin-4 and GLP-1 increased neurite length in cultured neurons compared with controls, but did not stimulate calcium influx directly, or affect capsaicin responses. However, exendin-4 significantly enhanced ATP responses in human DRG neurons. Conclusion Our results show that increased GLP-1R innervation in IBD bowel could mediate enhanced visceral afferent signalling, and provide a peripheral target for therapeutic intervention. The differential effect of GLP-1R agonists on capsaicin and ATP responses in neurons suggest they may not affect pain mechanisms mediated by the capsaicin receptor TRPV1, but may enhance the effects of purinergic agonists.

BACKGROUND: Trench foot, or non-freezing cold injury (NFCI), results from cold exposure of sufficient severity and duration above freezing point, with consequent sensory and vascular abnormalities which may persist for years. Based on observations of Trench foot in World War II, the condition was described as a vaso-neuropathy. While some reports have documented nerve damage after extreme cold exposure, sensory nerve fibres and vasculature have not been assessed with recent techniques in NFCI. OBJECTIVE: To assess patients with chronic sensory symptoms following cold exposure, in order to diagnose any underlying small fibre neuropathy, and provide insight into mechanisms of the persistent pain and cold hypersensitivity. METHODS: Thirty soldiers with cold exposure and persistent sensory symptoms (>4 months) were assessed with quantitative sensory testing, nerve conduction studies, and skin biopsies. Immunohistochemistry was used to assess intraepidermal (IENF) and subepidermal (SENF) nerve fibres with a range of markers, including the pan-neuronal marker protein gene product 9.5 (PGP 9.5), regenerating fibres with growth-associated protein 43 (GAP43), and nociceptor fibres with transient receptor potential cation channel subfamily V member 1 (TRPV1), sensory neuron-specific receptor (SNSR), and calcitonin gene-related peptide (CGRP). von Willebrand factor (vWF), endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF) were used for assessing blood vessels, and transient receptor potential cation channel, subfamily A member 1 (TRPA1) and P2X purinoceptor 7 (P2X7) for keratinocytes, which regulate nociceptors via release of nerve growth factor. RESULTS: Clinical examination showed pinprick sensation was abnormal in the feet of 20 patients (67%), and between 67 and 83% had abnormalities of thermal thresholds to the different modalities. 7 patients (23%) showed reduced sensory action potential amplitude of plantar nerves. 27 patients (90%) had decreased calf skin PGP 9.5 IENF (p

Background: The angiotensin II (AngII) receptor subtype 2 (AT2R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. Methods: We used immunostaining with characterized antibodies to study the localization of AT2R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. Results: AT2R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1R antagonist losartan had no effect on capsaicin responses. AT2R was localized in sensoryneuronsofhumanDRG,andnervefibresinperipheralnerves,skin, urinary bladder and bowel. A majority sub-population (60%) of small-/ medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. Conclusions: AT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.

The Nociceptin/Orphanin FQ peptide receptor (NOP), activated by its endogenous peptide ligand Nociceptin/Orphanin FQ (N/OFQ), exerts several effects including modulation of pain signalling. We have examined, for the first time, the tissue distribution of the NOP receptor in clinical visceral and somatic pain disorders by immunohistochemistry, and assessed functional effects of NOP and [micro] opioid receptor activation in cultured human and rat dorsal root ganglion (DRG) neurons. Quantification of NOP-positive nerve fibres within the bladder sub-urothelium revealed a remarkable several-fold increase in Detrusor Overactivity (p

The cannabinoid receptor CB1 is involved in modulation of neuronal hypersensitivity and pain. The aim of this study was to evaluate CB1 receptor levels for the first time in dental pain. A total of 19 patients due for molar extraction were divided into two groups, those with existing dental pain (n=9), and those with no history of pain (n=10). Immunohistochemistry and computer image analysis was used to evaluate CB1-positive nerve fibres in tooth pulp, with neurofilament-immunostaining as a structural nerve marker. CB1-immunoreactive nerve fibres were scattered throughout the tooth pulp and often seen in nerve bundles, but the fibres did not penetrate the subodontoblastic layer. There was no statistically significant change in the CB1 nerve fibre percentage area in the painful group compared to the non-painful group (p=0.146); the neurofilament fibres were significantly reduced in the painful group compared to the controls (p=0.028), but there was no difference in the ratio of CB1 to neurofilaments between the two groups. Thus, CB1 expression is maintained by nerve fibres in painful human dental pulp, and peripherally-restricted CB1 agonists currently in development may advance the treatment of dental pain.

Recent studies show that P2X(3) may play a role in neuropathic pain, including orofacial pain. Burning mouth syndrome (BMS) is a chronic neuropathic pain condition affecting 0.6-12% of post-menopausal women in the Western world. This study evaluates, for the first time, P2X(3) immunoreactivity levels in lingual mucosa in BMS patients. Patients diagnosed with BMS (n=9) in accordance with International Association for the Study of Pain criteria and patients attending for wisdom tooth removal (n=10, controls), were involved in this study. A pain history and score was recorded on a visual analogue scale (VAS) prior to obtaining a lingual biopsy. Immunohistochemistry and image analysis were used to quantify submucosal nerve fibres expressing P2X(3) and the structural marker neurofilaments. P2X(3) positive fibres were significantly increased in BMS compared with controls (p=0.024). In contrast, neurofilament-staining fibres were reduced in BMS, and when expressed as a ratio of the neurofilament percentage area, there was a trend for an increase of P2X(3) positive fibres in the BMS group. Increased P2X(3) immunoreactivity in the trigeminal sensory system may play a role in the symptoms observed in BMS. P2X(3) may therefore be a therapeutic target for treating BMS and trigeminal neuropathic pain.

Transient receptor potential vanilloid type 1 (TRPV1) has been shown to play an important role in visceral hypersensitivity. A significant proportion of patients with inflammatory bowel disease (IBD) continue to complain of abdominal pain despite their disease being otherwise quiescent. We investigated TRPV1-immunoreactive fibres in rectosigmoid biopsies taken from such patients with correlation to abdominal pain severity.Rectosigmoid biopsies were collected from 20 patients with quiescent IBD fulfilling Rome II criteria for irritable bowel syndrome (IBS), from 20 asymptomatic patients with quiescent IBD and from 28 controls. Abdominal pain scores were recorded using a validated questionnaire. TRPV1- and neuronal marker protein gene product 9.5 (PGP 9.5)-expressing nerve fibres, and lymphocytes (CD3 and CD4) were quantified, following immunohistochemistry with specific antibodies. The biopsy findings were related to abdominal pain scores.A significant 3.9-fold increase in median number of TRPV1-immunoreactive fibres was found in biopsies from patients with quiescent IBD with IBS symptoms when compared with controls (p0.0001) and a 5-fold increase when compared with the asymptomatic quiescent IBD group (p=0.0003). In the IBD with IBS symptoms group, the total nerve fibres (PGP 9.5) did not differ from those in the asymptomatic IBD group (p=0.10) or the control group (p=0.33) and neither did the CD3 lymphocyte (asymptomatic IBD group p=0.17; controls p=0.88) or CD4 lymphocyte (asymptomatic IBD group p=0.39; controls p=0.97) counts. In stepwise multivariate linear regression analysis, only TRPV1-immunoreactive nerve fibres (R(2)=0.8; p0.0001) were significantly related to the abdominal pain score.Increased TRPV1 nerve fibres are seen in quiescent IBD with IBS-like symptoms together with a correlation to pain severity. TRPV1 may contribute to the pathophysiology of ongoing pain and visceral hypersensitivity in this group of patients, providing a potential therapeutic target.

Cannabinoid receptor 2 (CB2) agonists provide the potential for treating chronic pain states without CNS effects associated with CB1 receptor activation. Animal models suggest that they act mainly via non-neuronal cells, possibly inhibition of inflammatory cells in the periphery or CNS, or via release of beta-endorphin; however, the clinical relevance and mechanism of analgesic action is uncertain. Here, we demonstrate colocalisation of CB2 with CB1 and the capsaicin receptor TRPV1 in human dorsal root ganglion (DRG) sensory neurons and increased levels of CB2 receptors in human peripheral nerves after injury, particularly painful neuromas. In primary cultures of human DRG neurons, selective CB2 agonists blocked activation of inward cation currents and elevation of cytoplasmic Ca2+ in response to capsaicin. These inhibitory effects were reversed by GW818646X a CB2 antagonist, and 8-bromo cAMP, but not by SR141716 a CB1 antagonist, or naloxone. Thus CB2 receptor agonists functionally inhibited nociceptive signalling in human primary sensory neurons via a mechanism shared with opioids, of adenylyl cyclase inhibition, but not via mu-opioid receptors. We conclude that CB2 agonists deserve imminent clinical trials for nociceptive, inflammatory and neuropathic chronic pain, in which capsaicin or heat-activated responses via TRPV1 may provide a clinical marker.

JAM-C is an adhesion molecule that is expressed on cells within the vascular compartment and epithelial cells and, to date, has been largely studied in the context of inflammatory events. Using immunolabeling procedures in conjunction with confocal and electron microscopy, we show here that JAM-C is also expressed in peripheral nerves and that this expression is localized to Schwann cells at junctions between adjoining myelin end loops. Sciatic nerves from JAM-C–deficient [having the JAM-C gene knocked out (KO)] mice exhibited loss of integrity of the myelin sheath and defective nerve conduction as indicated by morphological and electrophysiological studies, respectively. In addition, behavioral tests showed motor abnormalities in the KO animals. JAM-C was also expressed in human sural nerves with an expression profile similar to that seen in mice. These results demonstrate that JAM-C is a component of the autotypic junctional attachments of Schwann cells and plays an important role in maintaining the integrity and function of myelinated peripheral nerves.

Painful bladder syndrome is a chronic, debilitating bladder hypersensitivity disorder characterized by urinary frequency, urgency and bladder pain without an identifiable cause. Recent advances in understanding the molecular basis of hypersensitivity provide an opportunity to advance the understanding of and treatment for painful bladder syndrome. We studied the heat and capsaicin receptor transient receptor potential vanilloid receptor subtype 1 in the bladder in patients with painful bladder syndrome and their relationship to pain symptoms.Bladder biopsies were obtained from 20 characterized subjects with painful bladder syndrome and 25 with asymptomatic microscopic hematuria as controls. Specimens were immunostained using specific antibodies to transient receptor potential vanilloid receptor subtype 1 and neurofilaments as a structural maker. Nerve fiber and urothelial staining were quantified with computerized image analysis. The results of immunohistochemistry were correlated with the pain score.There was a marked increase in suburothelial nerve fibers expressing transient receptor potential vanilloid receptor subtype 1 in painful bladder syndrome in comparison with that in controls (p0.0001). The ratio of transient receptor potential vanilloid receptor subtype 1 fibers to neurofilaments was also significantly increased in painful bladder syndrome, suggesting over expression of transient receptor potential vanilloid receptor subtype 1 (p0.0001). When all specimens studied were included, the pain score correlated significantly with the relative nerve fiber density of transient receptor potential vanilloid receptor subtype 1 in the suburothelium (r = 0.6862, p = 0.0002) as well as the ratio of transient receptor potential vanilloid receptor subtype 1 fibers to neurofilaments (r = 0.5554, p = 0.004). Urothelial transient receptor potential vanilloid receptor subtype 1 showed a tendency toward an increase in the painful bladder syndrome group but it did not achieve statistical significance. No correlation was found between transient receptor potential vanilloid receptor subtype 1 immunoreactivity of urothelium or neurofilament fibers and the pain score.This study shows increased transient receptor potential vanilloid receptor subtype 1 in nerve fibers of the bladder in painful bladder syndrome and a correlation of the pain score with the relative density of transient receptor potential vanilloid receptor subtype 1 nerve fibers. Transient receptor potential vanilloid receptor subtype 1 may have a role in the pathophysiology of painful bladder syndrome and it is a potential target for novel therapeutic agents.

We studied the cellular localization of muscarinic receptor subtypes 2 and 3 in the human bladder and related any changes in overactive and painful bladder syndromes to measures of clinical dysfunction.Bladder specimens obtained from patients with painful bladder syndrome (11), idiopathic detrusor overactivity (12) and from controls with asymptomatic microscopic hematuria (16) were immunostained using specific antibodies to muscarinic receptor subtypes 2 and 3, and to vimentin, which is a marker for myofibroblasts. Immunostaining results were quantified with computerized image analysis and correlated with clinical dysfunction using frequency and urgency scores.Muscarinic receptor subtype 2 and 3 immunoreactivity was observed in the urothelium, nerve fibers and detrusor layers. In addition, strong myofibroblast-like cell staining, similar to vimentin, was present in the suburothelial region and detrusor muscle. A significant increase in suburothelial myofibroblast-like muscarinic receptor subtype 2 immunoreactivity was seen in patients with painful bladder syndrome (p = 0.0062) and idiopathic detrusor overactivity (p = 0.0002), and in muscarinic receptor subtype 3 immunoreactivity in those with idiopathic detrusor overactivity (p = 0.0122) with a trend in painful bladder syndrome. Muscarinic receptor subtype 2 and 3 immunoreactivity significantly correlated with the urgency score (p = 0.0002 and 0.0206, respectively) and muscarinic receptor subtype 2 immunoreactivity correlated with the frequency score (p = 0.0029). No significant difference was seen in urothelial and detrusor muscarinic receptor subtypes 2 and 3 or vimentin immunostaining.To our knowledge this is the first study to show the cellular localization of muscarinic receptor subtypes 2 and 3 in the human bladder. The increase in muscarinic receptor subtypes 2 and 3 immunostaining in myofibroblast-like cells in clinical bladder syndromes and its correlation with clinical scores suggests a potential role in pathophysiological mechanisms and the therapeutic effect of anti-muscarinic agents.

The P2X(7) 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)-1beta 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-1beta, 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-1beta 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 P2X(7) receptor, via regulation of mature IL-1beta 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.

Objectives To study TRPV1 immunoreactivity in the urothelium of patients with neurogenic detrusor overactivity (NDO) before and after treatment with resiniferatoxin (RTX) and controls. Functional capsaicin TRPV1 receptors have been demonstrated in urothelial cells of rodent urinary bladder, and TRPV1-knockout mice exhibit diminished nitric oxide and stretch-evoked adenosine triphosphate release from urothelial cells. In patients with NDO, TRPV1 suburothelial nerve density is increased, which is reversed by successful treatment with intravesical RTX. However, the role of urothelial TRPV1 in human bladder disorders is unknown. Methods Flexible cystoscopic bladder biopsies were obtained from 14 patients with NDO before and after treatment with RTX and from 8 control patients. Using a specific antibody for immunostaining, TRPV1 immunoreactivity in the urothelium was quantified by image analysis. Results TRPV1 immunoreactivity was observed in basal and apical urothelial cells. Basal cell layer TRPV1 immunoreactivity was significantly increased in NDO compared with control bladders ( P = 0.003). In 5 patients who responded clinically to RTX, basal cell layer and total urothelial TRPV1 immunoreactivity decreased significantly after treatment ( P = 0.032 and P = 0.016, respectively). The decreases in the basal cell layer TRPV1 immunoreactivity after RTX were comparable to the decreases in suburothelial TRPV1 nerve fibers in the biopsies previously studied from the same patients. Conclusions Increased urothelial TRPV1 in patients with NDO may play a role in the pathophysiology, in concert with increased TRPV1 nerve fibers. Although it is not known whether similar pathogenic mechanisms are involved in the increase of urothelial and neuronal TRPV1, both may be targeted by successful RTX therapy.

Objectives: The ATP-gated purinergic receptor P2X3 is expressed by small diameter sensory neurons and has been identified in normal and neurogenic human bladder suburothelial fibres. Animal models have shown that ATP is released by the urothelium during bladder distension, suggesting a mechanosensory role for P2X3 receptors in normal bladder function. Successful treatment of spinal neurogenic detrusor overactivity (NDO) with intravesical resiniferatoxin (RTX), which partly acts on suburothelial C fibres, provides evidence for the emergence of a C fibre-mediated spinal reflex. The aim of this study was to investigate the possible role of P2X3-positive innervation in this pathological voiding reflex by comparing suburothelial P2X3 immunoreactivity of controls and in patients with NDO before and after intravesical RTX. Methods: Bladder biopsies were obtained from 8 controls and 20 patients with refractory NDO enrolled in a trial of intravesical RTX. P2X3 nerve fibre density and intensity were studied in the specimens by immunohistochemistry. Results: P2X3-IR nerve fibres were significantly increased in patients with NDO compared to controls ( p =0.014). Thirteen patients had pre- and post-RTX biopsies available for immunohistochemistry; 5 of them responded clinically and 8 were non-responders. In the 5 patients who responded to RTX, there was a significant decrease in P2X3-positive fibres ( p =0.032), whereas in non-responders, P2X3-IR nerve fibre density did not change significantly. Conclusions: In patients with NDO, the numbers of P2X3-IR nerve fibres were increased in the suburothelium. There was a significant decrease in P2X3 immunoreactivity in responders to RTX, indicating a potential pathophysiological role for the P2X3 expressing fibres.

OBJECTIVE To compare PGP9.5 and transient receptor potential vanilloid receptor (TRPV1) suburothelial immunoreactivity between controls and patients with spinal neurogenic detrusor overactivity (NDO) before and after treatment with intravesical resiniferatoxin, as suburothelial PGP9.5-staining nerve fibres decrease in patients with spinal NDO who respond to intravesical capsaicin, and TRPV1 is present on these suburothelial nerve fibres in normal and overactive human urinary bladder. PATIENTS AND METHODS Patients with refractory NDO were enrolled in a prospective, randomized, parallel-group, double-blind, placebo-controlled trial using escalating doses of resiniferatoxin to a maximum of 1 µmol/L. Flexible cystoscopic bladder biopsies obtained at baseline, 4 weeks after each instillation and at the time of maximum clinical response were compared with biopsies taken from control subjects. Frozen sections were incubated with rabbit antibodies to TRPV1 and PGP9.5, and assessed using standard immunohistochemical methods. PGP9.5 nerve density was analysed using a nerve-counting graticule by an observer unaware of sample origin. Another two independent observers unaware of each other's results used a random grading scale to evaluate TRPV1 nerve fibre density and intensity. The immunohistochemistry results were compared with histology findings (haematoxylin-eosin), and the Mann–Whitney test used to assess any differences (P

OBJECTIVE To investigate endothelial nitric oxide synthase (eNOS) immunoreactivity in bladder biopsies from patients with neurogenic detrusor overactivity (NDO) before and after treatment with intravesical resiniferatoxin, and compare this with control material; the distribution of two other vascular markers, von Willebrand Factor (vWF) and the vascular endothelial growth factor (VEGF), was also studied. PATIENTS AND METHODS Flexible cystoscopic bladder biopsies from eight controls investigated for asymptomatic microhaematuria and 19 patients with refractory spinal NDO enrolled in a clinical trial of intravesical treatment with escalating doses of resiniferatoxin were immunostained with polyclonal rabbit antibodies for eNOS, vWF and VEGF. Fewer baseline NDO specimens (eight) were available for vWF and VEGF staining. Computerized image analysis was used to quantify immunoreactivity, and the Mann–Whitney test for statistical analysis. RESULTS eNOS immunoreactivity was found in the suburothelium and less often in the urothelium, with a distribution indicating a location in small blood vessels at the urothelium-suburothelium junction. Immunostaining for vWF showed a similar location. There was a trend to higher eNOS values before treatment in those responding than in those not responding to resiniferatoxin (P = 0.059), and a significant reduction in eNOS immunoreactivity after successful treatment (P = 0.016). VEGF staining was weaker but there was a significant increase in pretreatment biopsies of responders to resiniferatoxin (P = 0.048). Clinical and histopathology features were similar in both groups. CONCLUSIONS The trend for higher eNOS expression in patients with NDO who responded to resiniferatoxin suggests that increased vasculature or vasodilatation in the suburothelium may be necessary for successful intravesical treatment. Further studies with more patients are required to confirm this relationship and to examine the mechanisms underlying changes in vasculature with levels of bladder overactivity.

Objectives Acid-sensing ion channels (ASICs) are expressed by rat sensory neurons and may mediate pain associated with tissue acidosis after inflammation or injury. Our aim was to examine the molecular forms and localization of ASICs in human intestine and dorsal root ganglia using immunochemical techniques, and to measure the effects of inflammation and injury. Design and methods Inflamed Crohn's disease intestine and injured human dorsal root ganglia, with appropriate controls, were studied by Western blotting and immunohistochemistry, using specific affinity-purified ASIC antibodies. Results In the Western blot, there was a significant three-fold increase in the mean relative optical density of the ASIC-3 55-kDa band (but not ASIC-1 or ASIC-2) in full-thickness inflamed intestine, as well as in separated muscle and mucosal layers. There was a corresponding trend for an increased immunoreactive density and increased number of ASIC-3-positive neurons in the myenteric and sub-mucous plexus of inflamed intestine. In dorsal root ganglia, immunoreactivity for all ASICs was restricted to a sub-population (about 50%) of small-diameter (nociceptor) sensory neurons, and was generally less intense after injury. Conclusions Increased ASIC-3 in inflamed intestine suggests a role in pain or dysmotility, for which ASICs represent new therapeutic targets.

Objectives To determine the presence, distribution and molecular forms of the vanilloid receptor VR1, and confirm the presence and distribution of the ATP-gated ion channel P2X3 in the human urinary bladder. Materials and methods Normal urinary bladder tissues were obtained at postmortem from four subjects. Eight urinary bladder biopsies were also taken from patients with detrusor hyper-reflexia treated with intravesical resiniferatoxin. The specimens were studied using affinity-purified specific antibodies to VR1 and P2X3 by Western blotting and immunocytochemistry, and compared with immunostaining using antibodies to the pan-neuronal marker PGP 9.5 and Schwann cell marker S-100. Results VR1- and P2X3-immunoreactive fine nerve fibres were scattered throughout the suburothelium of the normal bladder and cystoscopic biopsies, and traversed the muscle layer. They had a similar distribution to PGP 9.5-immunoreactive fibres, but there were fewer, suggesting localization in subsets of axons. Western blot studies showed an expected 100-kDa VR1 protein and a P2X3-immunoreactive 66-kDa protein. Conclusion VR1 and P2X3 are present in the human urinary bladder and may contribute to distinct pathophysiological states of bladder overactivity, in accord with their differential expression in sensory neurones. Intravesical vanilloids act via VR1 and are effective in the treatment of detrusor hyper-reflexia. P2X3 may represent a selective therapeutic target for other causes of overactive bladder.

Two tetrodotoxin-resistant voltage-gated sodium channels, SNS/PN3 and SNS2/NaN, have been described recently in small-diameter sensory neurones of the rat, and play a key role in neuropathic pain. Using region-specific antibodies raised against different peptide sequences of their α subunits, we show by Western blot evidence for the presence of these channels in human nerves and sensory ganglia. The expected fully mature 260 kDa component of SNS/PN3 was noted in all injured nerve tissues obtained from adults; however, for SNS2/NaN, smaller bands were found, most likely arising from protein degradation. There was increased intensity of the SNS/PN3 260 kDa band in nerves proximal to the site of injury, whereas it was decreased distally, suggesting accumulation at sites of injury; all adult patients had a positive Tinel’s sign at the site of nerve injury, indicating mechanical hypersensitivity. Injured nerves from human neonates showed similar results for both channels, but neonate neuromas lacked the SNS2/NaN 180 kDa molecular form, which was strongly present in adult neuromas. The distribution of SNS/PN3 and SNS2/NaN sodium channels in injured human nerves indicates that they represent targets for novel analgesics, and could account for some differences in the development of neuropathic pain in infants.

BackgroundVoltage gated sodium channels Nav1.7 are involved in nociceptor nerve action potentials and are known to affect pain sensitivity in clinical genetic disorders.Aims and ObjectivesTo study Nav1.7 levels in dental pulpitis pain, an inflammatory condition, and burning mouth syndrome (BMS), considered a neuropathic orofacial pain disorder.MethodsTwo groups of patients were recruited for this study. One group consisted of patients with dental pulpitis pain (n = 5) and controls (n = 12), and the other patients with BMS (n = 7) and controls (n = 10). BMS patients were diagnosed according to the International Association for the Study of Pain criteria; a pain history was collected, including the visual analogue scale (VAS). Immunohistochemistry with visual intensity and computer image analysis were used to evaluate levels of Nav1.7 in dental pulp tissue samples from the dental pulpitis group, and tongue biopsies from the BMS group.ResultsThere was a significantly increased visual intensity score for Nav1.7 in nerve fibres in the painful dental pulp specimens, compared to controls. Image analysis showed a trend for an increase of the Nav1.7 immunoreactive % area in the painful pulp group, but this was not statistically significant. When expressed as a ratio of the neurofilament % area, there was a strong trend for an increase of Nav1.7 in the painful pulp group. Nav1.7 immunoreactive fibres were seen in abundance in the sub-mucosal layer of tongue biopsies, with no significant difference between BMS and controls.ConclusionNav1.7 sodium channel may play a significant role in inflammatory dental pain. Clinical trials with selective Nav1.7 channel blockers should prioritise dental pulp pain rather than BMS.

Objective: The capsaicin receptor TRPV1 (transient receptor potential vanilloid type-1) may play an important role in visceral pain and hypersensitivity states. In irritable bowel syndrome (IBS), abdominal pain is a common and distressing symptom where the pathophysiology is still not clearly defined. TRPV1-immunoreactive nerve fibres were investigated in colonic biopsies from patients with IBS, and this was related to abdominal pain. Methods: Rectosigmoid biopsies were collected from 23 IBS patients fulfilling Rome II criteria, and from 22 controls. Abdominal pain scores were recorded using a validated questionnaire. TRPV1-, substance P- and neuronal marker protein gene product (PGP) 9.5-expressing nerve fibres, mast cells (c-kit) and lymphocytes (CD3 and CD4) were quantified, following immunohistochemistry with specific antibodies. The biopsy findings were related to the abdominal pain scores. Results: A significant 3.5-fold increase in median numbers of TRPV1-immunoreactive fibres was found in biopsies from IBS patients compared with controls (p

During an oral glucose tolerance test (oGTT) and an isoglycaemic intravenous glucose infusion, blood glucose and the responses of insulin and glucose-dependent insulinotropic polypeptide (GIP) were measured in six healthy volunteers. On a subsequent occasion a constant infusion of human synthetic GIP (2 pmol kg-1 min-1 for 30 min and 0.5 pmol kg-1 min-1 for another 30 min was given to each subject, again with a simultaneous infusion of glucose to maintain isoglycaemia to the oGTT. During the oGTT, plasma GIP concentrations rose from 92pM18 pmol 1-1 to 257 pM42 pmol 1-1 60 min after ingestion of glucose (meanpMSEM). When glucose was administered intravenously plasma GIP levels did not rise significantly over basal. The infusion of hGIP mimicked the physiological plasma GIP response after oral glucose during the first 60 min of the study. Plasma insulin concentrations were significantly lower between 45 and 60 min than during the oGTT (438 pM 67 vs. 200pM48 pmol l-1p

Peptide histidine valine (PHV) is a 42 amino acid polypeptide closely related to the neuropeptides VIP, PHI and PHM. We have performed a placebo-controlled, double-blind study to assess the hypothesis that the cardiovascular response to PHV infusion may be mediated via the sympathetic nervous system. Four subjects received atenolol or matched placebo 90 min prior to a controlled incremental infusion of PHV, with monitoring of heart rate, blood pressure and skin temperature. Following placebo all subjects showed a dose-related increase in heart rate and skin temperature with no effect on blood pressure during PHV infusion. β-Blockade had no effect on skin temperature response. Pre-treatment with atenolol reduced the resting blood pressure and the maximum heart rate achieved, but did not affect the percentage increase in heart rate during PHV infusion. This suggests that the action of PHV does not involve β-receptors. The lack of effect of PHV infusion on blood pressure, despite tachycardia and marked cutaneous vasodilatation, implies that PHV has a different effect on the resistance vessels from that of other peptides such as VIP.

Tumors associated with the Verner Morrison syndrome secrete peptide histidine methionine, its C-terminally extended variant peptide histidine valine, and vasoactive intestinal peptide. There is evidence that vasoactive intestinal peptide mediates diarrhea, but recent evidence suggested that peptide histidine methionine and peptide histidine valine may be at least as important. Infusion of vasoactive intestinal peptide, peptide histidine methionine, and peptide histidine valine into patients with ileostomies produced mean plateau plasma levels of 163, 1301, and 2106 pM, respectively, which are within the range seen in the Verner Morrison syndrome. Vasoactive intestinal peptide produced an integrated ileal output of 174 (53) g (mean [SEM]), compared with only 20 (7) g with peptide histidine methionine and 10 (3) g with peptide histidine valine. These results suggest that vasoactive intestinal peptide is substantially more important than peptide histidine methionine or peptide histidine valine in mediating diarrhea in the Verner Morrison syndrome.

Neuromedin-N, a hexapeptide recently isolated and purified from porcine spinal cord, has close sequence homology with the C-terminal region of the tridecapeptide neurotensin. Both peptides have a remarkably similar peripheral distribution. Little is known of the biological activity of neuromedin-N. Neurotensin and peptide histidine methionine are known to stimulate net fluid secretion into rat small intestine. We have therefore tested the effect of neuromedin-N and the hexapeptide neurotensin-(8–13), the smallest fully active analogue of neurotensin in this system, compared with that of neurotensin and peptide histidine methionine. All four peptides reduced net absorption in low doses and caused net secretion in larger doses. However, whereas peptide histidine methionine was active in all areas of the small intestine, neurotensin, neurotensin-(8–13) and neuromedin-N were inactive in the duodenum. In the post-duodenal areas neurotensin was approximately 7 times more active than peptide histidine methionine, 21 times more potent than neuromedin-N and 33 times more potent than neurotensin-(8–13).

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.

Doppler is conducted in POTS patients with family history of MALS and persistent gastric symptoms. Peak systolic velocity (PSV), End Diastolic velocity (EDV) is calculated in all the patients. PSV greater than 200 cm/s, EDV greater than 55 cm/s are suggestive of celiac artery stenosis associated with MALS. Results: Out of 93 patients, 95% patients are Females (n = 88, age 28.88 ± 9.36), 5% patients are Males (n = 5, age 25.83 ± 6.19). PSV (cm/sec) 223.84 ± 109.52, EDV (cm/sec) 71.07 ± 54.46, PSV (neutral) 179.77 ± 83.01, PSV Expiration 218.58 ± 109.79. PSV (cm/sec) N 200 in 51 patients (55%), EDV (cm/sec) N55 in 50 patients (54%), PSV Expiration (cm/sec) N 200 in 52 patients (56%). PSV (Cm/sec) N300 in 18 Patients. Conclusion: Our research study demonstrated that higher percentages of POTS patients (N50%) are found to have MALS. MALS was found to be more common in females POTS Patients. There appears to be tremendous improvement in symptoms of two POTS patients after surgery for MALS.

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

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.

Background Recent studies show that inflammatory processes may contribute to neuropathic pain. Cyclooxygenase-2 (Cox-2) is an inducible enzyme responsible for production of prostanoids, which may sensitise sensory neurones via the EP1 receptor. We have recently reported that while macrophages infiltrate injured nerves within days of injury, they express increased Cox-2-immunoreactivity (Cox-2-IR) from 2 to 3 weeks after injury. We have now investigated the time course of EP1 and Cox-2 changes in injured human nerves and dorsal root ganglia (DRG), and the chronic constriction nerve injury (CCI) model in the rat. Methods Tissue sections were immunostained with specific antibodies to EP1, Cox-2, CD68 (human macrophage marker) or OX42 (rat microglial marker), and neurofilaments (NF), prior to image analysis, from the following: human brachial plexus nerves (21 to 196 days post-injury), painful neuromas (9 days to 12 years post-injury), avulsion injured DRG, control nerves and DRG, and rat CCI model tissues. EP1 and NF-immunoreactive nerve fibres were quantified by image analysis. Results EP1:NF ratio was significantly increased in human brachial plexus nerve fibres, both proximal and distal to injury, in comparison with uninjured nerves. Sensory neurones in injured human DRG showed a significant acute increase of EP1-IR intensity. While there was a rapid increase in EP1-fibres and CD-68 positive macrophages, Cox-2 increase was apparent later, but was persistent in human painful neuromas for years. A similar time-course of changes was found in the rat CCI model with the above markers, both in the injured nerves and ipsilateral dorsal spinal cord. Conclusion Different stages of infiltration and activation of macrophages may be observed in the peripheral and central nervous system following peripheral nerve injury. EP1 receptor level increase in sensory neurones, and macrophage infiltration, appears to precede increased Cox-2 expression by macrophages. However, other methods for detecting Cox-2 levels and activity are required. EP1 antagonists may show therapeutic effects in acute and chronic neuropathic pain, in addition to inflammatory pain.

Background: The tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 (SNS1/PN3) is expressed by nociceptors and may play a role in pain states. Methods: Using specific antibodies for immunohistochemistry, we studied Nav1.8 – immunoreactivity in human dental pulp in relation to the neuronal marker neurofilament. Human tooth pulp was extracted from teeth harvested from a total of twenty-two patients (fourteen without dental pain, eight patients with dental pain). Results: Fibres immunoreactive for Nav1.8, were significantly increased on image analysis in the painful group: median (range) Nav1.8 to Neurofilament % area ratio, non-painful 0.059 (0.006–0.24), painful 0.265 (0.13–0.5), P = 0.0019. Conclusion: Nav1.8 sodium channels may thus represent a therapeutic target in trigeminal nerve pain states.

To investigate the levels of the capsaicin or vanilloid receptor-1 (VR1) and the ATP-gated purinoceptor P2X3 in painful and nonpainful human tooth pulps.Immunohistochemistry with specific antibodies and image analysis was used to quantify VR1- and P2X3-positive nerve fibers in painful (n = 13) and nonpainful (n = 33) human tooth pulps, and VR1 immunoreactivity was compared with immunoreactivity for the structural neuronal marker peripherin.Strong VR1-like immunoreactivity was documented for the first time in dental pulp neurons. Weaker P2X3-like immunoreactivity was also detected in fewer nerve fibers. The ratio of VR1 to peripherin immunoreactivity was not significantly different between nonpainful and painful tissues (mean +/- SE% area of VR1 to peripherin; nonpainful 53.4 +/- 4.7%, n = 33; pulpitis 35.1 +/- 7.1%, n = 13; P = .07).The presence of VR1 and P2X3 in fibers of human tooth pulp suggest that they may play a role in perception of dental pain, but further studies, including quantitation of their ligands, are necessary to elucidate any role they may play in pathophysiologic states.

Root avulsion from the spinal cord occurs in brachial plexus lesions. It is the practice to repair such injuries by transferring an intact neighbouring nerve to the distal stump of the damaged nerve; avulsed dorsal root ganglia (DRG) are removed to enable nerve transfer. Such avulsed adult human cervical DRG ( [Formula: see text] ) obtained at surgery were compared to controls, for the first time, using GeneChip oligonucleotide arrays. We report 91 genes whose expression levels are clearly altered by the injury. This first study provides a global assessment of the molecular events or "gene switches" as a consequence of DRG injuries, as the tissues represent a wide range of surgical delay, from 1 to 100 days. A number of these genes are novel with respect to sensory ganglia, while others are known to be involved in neurotransmission, trophism, cytokine functions, signal transduction, myelination, transcription regulation, and apoptosis. Cluster analysis showed that genes involved in the same functional groups are largely positioned close to each other. This study represents an important step in identifying new genes and molecular mechanisms in human DRG, with potential therapeutic relevance for nerve repair and relief of chronic neuropathic pain.

Calcium-activated potassium currents of intermediate conductance (IK1) have been described in the rodent enteric nervous system, where they may regulate afterhyperpolarisation of intrinsic primary afferent neurons. Using specific antibodies for immuno-cytochemistry, we now report IK1-like immunoreactivity for the first time in enteric neurons of human colon, and a significant decrease of IK1-positive cells in myenteric plexus in inflamed colon from patients with Crohn's disease and ulcerative colitis (p = 0.031). Neurotrophin-3 (NT-3), which regulates IK1 expression, was also observed in fewer neurons of the myenteric ganglia in Crohn's bowel (p = 0.048), and in inflamed colonic extracts by Western blotting (p = 0.004); the numbers of neurons expressing the NT-3 high affinity receptor trk C were unchanged. Our findings may explain the diarrhoea and colicky abdominal pain produced by inflammatory bowel disease, and by IK1-blocking pyridine drugs prescribed for neuromuscular disorders.

Vulvodynia is characterised by painful burning sensation, allodynia and hyperalgesia in the region of the vulval vestibulus. While in many patients the cause of vulvodynia remains uncertain, we and others have previously shown increased intraepithelial and papillary innervation in vulvodynia. The vanilloid receptor VR1 (TRPV1) is expressed by nociceptors, and is triggered by capsaicin, noxious heat, protons, and chemicals produced during inflammation. In the present study we show increased papillary VR1 fibres by immunostaining and image analysis in vulvodynia tissues compared to controls (p

Precursors of NGF have been shown to be the predominant forms of this neurotrophin in human brain and peripheral tissues, and proNGF has been shown recently to preferentially bind p75NTR with high affinity. In our studies of human and rat skin and nerve extracts, a 53 kDa band was detected by Western blot using antibodies against rhNGF or prepro-NGF (-91 to -60), that could correspond to a previously described modified prepro-NGF-like molecule. The relative optical intensity of the 53-kDa bands was markedly reduced in skin extracts from patients with subclinical diabetic neuropathy (diabetic: 1.5, 1.0-8.0, n = 6; controls: 52.0, 23.0-85.0, n = 6, p = 0.0022) but was increased in extracts of inflamed colon from patients with Crohn's disease (inflamed: median 12.0, range 0.1-12.0, n = 11: controls: 0.1, 0.1-3.0, n = 8, p = 0.0055). Antibodies to both rhNGF and prepro-NGF immunostained basal keratinocytes in tissue sections of normal human and rat skin showed accumulation of immunoreactivity in nerve fibers distal to sciatic nerve ligation in rats. Prepro-NGF antibody immunostained rat large/medium sensory neurons, whereas only small sensory neurons were stained with antibodies to mature rhNGF, suggesting that prepro-NGF may be preferentially taken up and transported by p75NTR. The different molecular forms derived from prepro-NGF are likely to be of importance in sensory mechanisms, and deserve further investigation.

P2X(3) is a novel ATP-gated cation channel that is selectively expressed by small-diameter sensory neurones in rodents, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. We have studied, for the first time, P2X(3) immunoreactivity in human inflammatory bowel disease, using Western blotting and immunohistochemistry. A major 66-kDa specific protein was found by Western blotting in all colon extracts. In the inflamed group there was a significant two-fold increase in the relative optical density of the 66-kDa band (21.2 +/- 3.1; n=8) compared to controls (11.4 +/- 3.7; n=8; P=0.009). In the control colon, P2X(3)-immunoreactive neurones were scattered throughout the myenteric and submucosal plexuses, with some neurones showing immunopositive axons/dendrites. The pattern of immunostaining was similar to the neuronal marker peripherin. In general, the intensity of the staining was greater in myenteric than submucosal neurones. The number of P2X(3)-immunoreactive neurones was significantly increased in the myenteric plexus of inflamed colon compared to controls (n=13; P=0.01). In humans, unlike rodents, P2X(3) is thus not restricted to sensory neurones. Increased P2X(3) in inflamed intestine suggests a potential role in dysmotility and pain, for which it represents a new therapeutic target.

Nerve injury, particularly of the brachial plexus, may result in lifelong disability and chronic pain, despite technically excellent reconstructive surgery. Studies of molecular changes in injured nerves may identify new treatments to enhance the success of nerve repair, such as with recombinant human neurotrophic factors. Interleukin-6 (IL-6) is a member of the neuropoietic cytokine family that includes ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), and oncostatin M. As there is increasing evidence of a neurotrophic role for IL-6 in animal models of nerve injury and inflammation,1 2 we have studied, for the first time in humans, IL-6 protein in injured and control peripheral nerve and dorsal root ganglia, using specific immunoassay, immunocytochemistry, and western blotting. We report a most remarkable increase of IL-6 concentrations in acutely avulsed dorsal root ganglia and injured nerves. Proximal and distal injured nerve segments were obtained from six adult patients with traction brachial plexus injury, ranging from 2 weeks to 10 weeks after trauma. Injured dorsal root ganglia were collected from seven adult patients with brachial spinal root avulsion injuries (central axotomy), ranging from 3 days to 15 months after trauma. Tissue removal was a necessary part …

The ATP-gated cation channel P2X3 is expressed selectively by rat sensory neurones, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. However, the distribution of this channel in human sensory neurons is not known. Using a specific antibody, we have demonstrated intense P2X3 immunoreactivity within a subset (60%) of small/medium diameter sensory neurones and fine nerve fibres in intact post-mortem human dorsal root ganglia (DRG). Co-localization studies showed < 15% overlap with the trkA immunostaining in DRG, indicating that P2X3 was expressed predominantly in sensory neurons that are also isolectin B4 positive. There was a significant decrease in numbers of P2X3-like immunoreactive neurons in human DRG after central axotomy (to 36%), similar to the decrease in rat DRG after peripheral axotomy. However, Western blotting demonstrated a specific 66 kDa band in human DRG and peripheral organs, including intestine, where histochemistry showed P2X3 immunoreactivity in myenteric plexus neurons. Thus P2X3 antagonists may be analgesic, but are unlikely to have a selective effect on pain in humans.