Nerve and Vascular Biomarkers in Skin Biopsies Differentiate Painful From Painless Peripheral Neuropathy in Type 2 Diabetes.

Painful diabetic peripheral neuropathy can be intractable with a major impact, yet the underlying pain mechanisms remain uncertain. A range of neuronal and vascular biomarkers was investigated in painful diabetic peripheral neuropathy (painful-DPN) and painless-DPN and used to differentiate painful-DPN from painless-DPN. Skin biopsies were collected from 61 patients with type 2 diabetes (T2D), and 19 healthy volunteers (HV). All subjects underwent detailed clinical and neurophysiological assessments. Based on the neuropathy composite score of the lower limbs [NIS(LL)] plus seven tests, the T2D subjects were subsequently divided into three groups: painful-DPN (n = 23), painless-DPN (n = 19), and No-DPN (n = 19). All subjects underwent punch skin biopsy, and immunohistochemistry used to quantify total intraepidermal nerve fibers (IENF) with protein gene product 9.5 (PGP9.5), regenerating nerve fibers with growth-associated protein 43 (GAP43), peptidergic nerve fibers with calcitonin gene-related peptide (CGRP), and blood vessels with von Willebrand Factor (vWF). The results showed that IENF density was severely decreased (p < 0.001) in both DPN groups, with no differences for PGP9.5, GAP43, CGRP, or GAP43/PGP9.5 ratios. There was a significant increase in blood vessel (vWF) density in painless-DPN and No-DPN groups compared to the HV group, but this was markedly greater in the painful-DPN group, and significantly higher than in the painless-DPN group (p < 0.0001). The ratio of sub-epidermal nerve fiber (SENF) density of CGRP:vWF showed a significant decrease in painful-DPN vs. painless-DPN (p = 0.014). In patients with T2D with advanced DPN, increased dermal vasculature and its ratio to nociceptors may differentiate painful-DPN from painless-DPN. We hypothesized that hypoxia-induced increase of blood vessels, which secrete algogenic substances including nerve growth factor (NGF), may expose their associated nociceptor fibers to a relative excess of algogens, thus leading to painful-DPN.

Rational treatment of chemotherapy-induced peripheral neuropathy with capsaicin 8% patch: from pain relief towards 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.

Granulocyte-macrophage colony-stimulating factor receptor expression in clinical pain disorder tissues and role in neuronal sensitization.

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.

Glucagon-like peptide 1 receptor (GLP-1R) expression by nerve fibres in inflammatory bowel disease and functional effects in cultured neurons.

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.

Trench Foot or Non-Freezing Cold Injury As a Painful Vaso-Neuropathy: Clinical and Skin Biopsy Assessments.

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 < 0.0001), the remaining 3 patients had decreased nerve markers in subepidermis or foot skin. There were marked increases of all vascular markers (for vWF in calf skin, p < 0.0001), and increased sensory or regenerating SENF (for calf skin, GAP43, p = 0.002). TRPA1 (p = 0.0012) and P2X7 (p < 0.0001) were increased in basal keratinocytes. CONCLUSION: A range of skin biopsy markers and plantar nerve conduction studies are useful objective assessments for the diagnosis of peripheral neuropathy in NFCI. Our results suggest that an increase in blood vessels following tissue ischaemia/hypoxia could be associated with disproportionate and abnormal nerve fibres (irritable nociceptors), and may lead to NFCI as a "painful vaso-neuropathy."

Nociceptin/orphanin FQ receptor expression in clinical pain disorders and functional effects in cultured neurons.

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 µ-opioid receptor activation in cultured human and rat dorsal root ganglion (DRG) neurons. Quantification of NOP-positive nerve fibres within the bladder suburothelium revealed a remarkable several-fold increase in detrusor overactivity (P < 0.0001) and painful bladder syndrome patient specimens (P = 0.0014) compared with controls. In postmortem control human DRG, 75% to 80% of small/medium neurons (≤50 µm diameter) in the lumbar (somatic) and sacral (visceral) DRG were positive for NOP, and fewer large neurons; avulsion-injured cervical human DRG neurons showed similar numbers. NOP immunoreactivity was significantly decreased in injured peripheral nerves (P = 0.0004), and also in painful neuromas (P = 0.025). Calcium-imaging studies in cultured rat DRG neurons demonstrated dose-dependent inhibition of capsaicin responses in the presence of N/OFQ, with an IC50 of 8.6 pM. In cultured human DRG neurons, 32% inhibition of capsaicin responses was observed in the presence of 1 pM N/OFQ (P < 0.001). The maximum inhibition of capsaicin responses was greater with N/OFQ than µ-opioid receptor agonist DAMGO. Our findings highlight the potential of NOP agonists, particularly in urinary bladder overactivity and pain syndromes. The regulation of NOP expression in visceral and somatic sensory neurons by target-derived neurotrophic factors deserves further study, and the efficacy of NOP selective agonists in clinical trials.

Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT2R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies.

BACKGROUND: The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherally-restricted analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging. RESULTS: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine. CONCLUSION: The major AT2R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.

Increased levels of SV2A botulinum neurotoxin receptor in clinical sensory disorders and functional effects of botulinum toxins A and E in cultured human sensory neurons.

BACKGROUND: There is increasing evidence that botulinum neurotoxin A may affect sensory nociceptor fibers, but the expression of its receptors in clinical pain states, and its effects in human sensory neurons, are largely unknown. METHODS: We studied synaptic vesicle protein subtype SV2A, a receptor for botulinum neurotoxin A, by immunostaining in a range of clinical tissues, including human dorsal root ganglion sensory neurons, peripheral nerves, the urinary bladder, and the colon. We also determined the effects of botulinum neurotoxins A and E on localization of the capsaicin receptor, TRPV1, and functional sensitivity to capsaicin stimuli in cultured human dorsal root ganglion neurons. RESULTS: Image analysis showed that SV2A immunoreactive nerve fibers were increased in injured nerves proximal to the injury (P = 0.002), and in painful neuromas (P = 0.0027); the ratio of percentage area SV2A to neurofilaments (a structural marker) was increased proximal to injury (P = 0.0022) and in neuromas (P = 0.0001), indicating increased SV2A levels in injured nerve fibers. In the urinary bladder, SV2A nerve fibers were found in detrusor muscle and associated with blood vessels, with a significant increase in idiopathic detrusor over-activity (P = 0.002) and painful bladder syndrome (P = 0.0087). Colon biopsies showed numerous SV2A-positive nerve fibers, which were increased in quiescent inflammatory bowel disease with abdominal pain (P = 0.023), but not in inflammatory bowel disease without abdominal pain (P = 0.77) or in irritable bowel syndrome (P = 0.13). In vitro studies of botulinum neurotoxin A-treated and botulinum neurotoxin E-treated cultured human sensory neurons showed accumulation of cytoplasmic vesicles, neurite loss, and reduced immunofluorescence for the heat and capsaicin receptor, TRPV1. Functional effects included dose-related inhibition of capsaicin responses on calcium imaging after acute treatment with botulinum neurotoxins A and E. CONCLUSION: Differential levels of SV2A protein expression in clinical disorders may identify potential new targets for botulinum neurotoxin therapy. In vitro studies indicate that treatment with botulinum neurotoxins A and E may affect receptor expression and nociceptor function in sensory neurons.

Sodium channel Na v 1.7 immunoreactivity in painful human dental pulp and burning mouth syndrome.

BACKGROUND: Voltage gated sodium channels Na v 1.7 are involved in nociceptor nerve action potentials and are known to affect pain sensitivity in clinical genetic disorders. AIMS AND OBJECTIVES: To study Na v 1.7 levels in dental pulpitis pain, an inflammatory condition, and burning mouth syndrome (BMS), considered a neuropathic orofacial pain disorder. METHODS: Two 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 Na v 1.7 in dental pulp tissue samples from the dental pulpitis group, and tongue biopsies from the BMS group. RESULTS: There was a significantly increased visual intensity score for Na v 1.7 in nerve fibres in the painful dental pulp specimens, compared to controls. Image analysis showed a trend for an increase of the Na v 1.7 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 Na v 1.7 in the painful pulp group. Na v 1.7 immunoreactive fibres were seen in abundance in the sub-mucosal layer of tongue biopsies, with no significant difference between BMS and controls. CONCLUSION: Na v 1.7 sodium channel may play a significant role in inflammatory dental pain. Clinical trials with selective Na v 1.7 channel blockers should prioritize dental pulp pain rather than BMS.

Expression of the TRPV1 receptor differs in quiescent inflammatory bowel disease with or without abdominal pain.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 (p<0.0001) 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; p<0.0001) were significantly related to the abdominal pain score. CONCLUSIONS: 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.

Sensory purinergic receptor P2X3 is elevated in burning mouth syndrome.

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.

Increased cannabinoid receptor 1-immunoreactive nerve fibers in overactive and painful bladder disorders and their correlation with symptoms.

OBJECTIVE: To study the expression of cannabinoid receptor 1 (CB1) in human urinary bladder hypersensitivity and overactivity disorders, and correlate changes with symptoms. Cannabinoid receptor agonists have been shown to modulate urinary bladder contractility and reduce pain after bladder inflammation; their clinical efficacy on lower urinary tract symptoms was demonstrated in the Cannabinoids in Multiple Sclerosis study. METHODS: Bladder tissue specimens were obtained from patients with painful bladder syndrome (PBS, n=13), idiopathic detrusor overactivity (IDO, n=14), and from controls with asymptomatic microscopic hematuria (n=16). The severity of symptoms was assessed using the Pelvic Pain and Urgency/Frequency Questionnaire. Pain score was also recorded on a visual analogue scale. Specimens were immunostained using specific antibodies to CB1 and to neurofilaments as a structural maker. Detrusor and suburothelial nerve fiber density was quantified with a visual grading scale. The immunohistochemistry results were correlated with "Pain, Frequency, and Urgency" scores. RESULTS: CB1-immunoreative nerve fibers were significantly increased in the suburothelium of PBS (P=.0123) and IDO (P=.0013) specimens, and in detrusor layer in IDO (P=.0003), as compared with controls. CB1-immunoreactive suburothelial nerve fiber density correlated significantly with pain scores (Visual Analogue Scale) in PBS (r=.6878, P=.0347) and urgency scores in IDO (r=.6623, P=.0027). Neurofilaments-immunoreactive suburothelial nerve fibers were significantly increased in PBS (P=.019) and IDO (P=.05). CONCLUSIONS: The results of this study suggest that increased nerve fibers, which express CB1, may be related to bladder pain in PBS and urgency in IDO. Our findings support clinical trials of CB1 agonists in bladder disorders.

Cannabinoid receptor CB2 localisation and agonist-mediated inhibition of capsaicin responses in human sensory neurons.

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.

Expression and function of junctional adhesion molecule-C in myelinated peripheral nerves.

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.

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.

Transient receptor potential vanilloid receptor subtype 1 in painful bladder syndrome and its correlation with pain.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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 (p <0.0001). 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 (p <0.0001). 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. CONCLUSIONS: 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.

Localization of M2 and M3 muscarinic receptors in human bladder disorders and their clinical correlations.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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

BACKGROUND: While 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. METHODS: Frozen 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. RESULTS: In 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. CONCLUSION: It 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.

Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations.

BACKGROUND: The recent identification of the cold-menthol sensory receptor (TRPM8; CMR1), provides us with an opportunity to advance our understanding of its role in the pathophysiology of bladder dysfunction, and its potential mediation of the bladder cooling reflex. In this study, we report the distribution of the cool and menthol receptor TRPM8 in the urinary bladder in patients with overactive and painful bladder syndromes, and its relationship with clinical symptoms. METHODS: Bladder specimens obtained from patients with painful bladder syndrome (PBS, n = 16), idiopathic detrusor overactivity (IDO, n = 14), and asymptomatic microscopic hematuria (controls, n = 17), were immunostained using specific antibodies to TRPM8; nerve fibre and urothelial immunostaining were analysed using fibre counts and computerized image analysis respectively. The results of immunohistochemistry were compared between the groups and correlated with the Pain, Frequency and Urgency scores. RESULTS: TRPM8-immunoreactive staining was observed in the urothelium and nerve fibres scattered in the suburothelium. The nerve fibre staining was seen in fine-calibre axons and thick (myelinated) fibres. There was marked increase of TRPM8-immunoreactive nerve fibres in IDO (P = 0.0249) and PBS (P < 0.0001) specimens, compared with controls. A significantly higher number of TRPM8-immunoreactive axons were also seen in the IDO (P = 0.0246) and PBS (P < 0.0001) groups. Urothelial TRPM8 and TRPM8-immunoreactive thick myelinated fibres appeared unchanged in IDO and PBS. The relative density of TRPM8-immunoreactive nerve fibres significantly correlated with the Frequency (r = 0.5487, P = 0.0004) and Pain (r = 0.6582, P < 0.0001) scores, but not Urgency score. CONCLUSION: This study demonstrates increased TRPM8 in nerve fibres of overactive and painful bladders, and its relationship with clinical symptoms. TRPM8 may play a role in the symptomatology and pathophysiology of these disorders, and may provide an additional target for future overactive and painful bladder pharmacotherapy.

Prostanoid receptor EP1 and Cox-2 in injured human nerves and a rat model of nerve injury: a time-course study.

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.