Localisation of SCN10A gene product Na(v)1.8 and novel pain-related ion channels in human heart.

We have shown that the gene SCN10A encoding the sodium channel Na(v)1.8 is a susceptibility factor for heart block and serious ventricular arrhythmia. Since Na(v)1.8 is known to be present in nerve fibres that mediate pain, it may be related to both cardiac pain and dysrhythmia. The localisation of Na(v)1.8 and other key nociceptive ion channels, including Na(v)1.7, Na(v)1.9, capsaicin receptor TRPV1, and purinergic receptor P2X(3), have not been reported in human heart. The aim of this study was to determine the distribution of Na(v)1.8, related sodium and other sensory channels in human cardiac tissue, and correlate their density with sympathetic nerves, regenerating nerves (GAP-43), and vascularity. Human heart atrial appendage tissues (n = 13) were collected during surgery for valve disease. Tissues were investigated by immunohistology using specific antibodies to Na(v)1.8 and other markers. Na(v)1.8 immunoreactivity was detected in nerve fibres and fascicles in the myocardium, often closely associated with small capillaries. Na(v)1.8 nerve fibres per mm(2) correlated significantly with vascular markers. Na(v)1.8-immunoreactivity was present also in cardiomyocytes with a similar distribution pattern to that seen with connexins, the specialised gap junction proteins of myocardial intercalated discs. Na(v)1.5-immunoreactivity was detected in cardiomyocytes but not in nerve fibres. Na(v)1.7, Na(v)1.9, TRPV1, P2X(3)/P2X(2), and GAP43 positive nerve fibres were relatively sparse, whereas sympathetic innervation and connexin43 were abundant. We conclude that sodium channel Na(v)1.8 is present in sensory nerves and cardiomyocytes of human heart. Na(v)1.8 and other pain channels provide new targets for the understanding and treatment of cardiac pain and dysrhythmia.

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.

Influence of pregnancy and labor on the occurrence of nerve fibers expressing the capsaicin receptor TRPV1 in human corpus and cervix uteri.

BACKGROUND: Cervical ripening is a prerequisite for a normal obstetrical outcome. This process, including labor, is a painful event that shares features with inflammatory reactions where peripheral nociceptive pathways are involved. The capsaicin and heat receptor TRPV1 is a key molecule in sensory nerves involved in peripheral nociception, but little is known regarding its role in the pregnant uterus. Therefore, the aim of this study was to investigate human corpus and cervix uteri during pregnancy and labor and non-pregnant controls for the presence of TRPV1. METHODS: We have investigated human uterine corpus and cervix biopsies at term pregnancy and parturition. Biopsies were taken from the upper edge of the hysterotomy during caesarean section at term (n = 8), in labor (n = 8) and from the corresponding area in the non-pregnant uterus after hysterectomy (n = 8). Cervical biopsies were obtained transvaginally from the anterior cervical lip. Serial frozen sections were examined immunohistochemically using specific antibodies to TRPV1 and nerve markers (neurofilaments/peripherin). RESULTS: In cervix uteri, TRPV1-immunoreactive fibers were scattered throughout the stroma and around blood vessels, and appeared more frequent in the sub-epithelium. Counts of TRPV1-immunoreactive nerve fibers were not significantly different between the three groups. In contrast, few TRPV1-immunoreactive fibers were found in nerve fascicles in the non-pregnant corpus, and none in the pregnant corpus. CONCLUSION: In this study, TRPV1 innervation in human uterus during pregnancy and labor is shown for the first time. During pregnancy and labor there was an almost complete disappearance of TRPV1 positive nerve fibers in the corpus. However, cervical innervation remained throughout pregnancy and labor. The difference in TRPV1 innervation between the corpus and the cervix is thus very marked. Our data suggest that TRPV1 may be involved in pain mechanisms associated with cervical ripening and labor. Furthermore, these data support the concept that cervix uteri may be the major site from which labor pain emanates. Our findings also support the possibility of developing alternative approaches to treat labor pain.

Increased nerve fiber expression of sensory sodium channels Nav1.7, Nav1.8, And Nav1.9 in rhinitis.

INTRODUCTION: Voltage-gated sodium channels Nav1.7, Nav1.8, and Nav1.9 are involved in nerve action potentials and have been proposed to underlie neuronal hypersensitivity. We have therefore studied their levels in allergic and nonallergic rhinitis. MATERIALS AND METHODS: Inferior turbinate biopsies from 50 patients (n = 18 controls, n = 20 allergic, and n = 12 nonallergic rhinitis) were studied by immunohistology using antibodies to Nav1.7, Nav1.8, and Nav1.9, the structural nerve marker (protein gene product [PGP]9.5), nerve growth factor (NGF), mast cells (c-kit), macrophages (CD68), and T cells (CD3). Sodium channel-positive nerve fibers were counted per millimeter length of subepithelium, and immunoreactivity for inflammatory cell markers PGP9.5 and NGF were image analyzed. RESULTS: All three sodium channel-immunoreactive nerve fiber numbers were significantly increased in allergic (Nav1.7, P = .0004; Nav1.8, P = .028; Nav1.9, P = .02) and nonallergic (Nav1.7, P = .006; Nav1.8, P = .019; Nav1.9, P = .0037) rhinitis. There was a significant increase of subepithelial innervation (PGP9.5, P = .01) and epithelial NGF immunoreactivity (P = .03) in nonallergic rhinitis, comparable with our previous report in allergic rhinitis. Inflammatory cell markers were significantly increased in allergic (mast cells, P = .06; macrophages, P = .044; T cells, P = .007) but not nonallergic rhinitis. CONCLUSION: The increased levels of sensory sodium channels in allergic and nonallergic rhinitis may contribute to the hypersensitive state, irrespective of the degree of active inflammation. Selective blockers of these sodium channels, administered topically, may have therapeutic potential in rhinitis.

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.

Use of the novel Contact Heat Evoked Potential Stimulator (CHEPS) for the assessment of small fibre neuropathy: correlations with skin flare responses and intra-epidermal nerve fibre counts.

BACKGROUND: The Contact Heat Evoked Potential Stimulator (CHEPS) rapidly stimulates cutaneous small nerve fibres, and resulting evoked potentials can be recorded from the scalp. We have studied patients with symptoms of sensory neuropathy and controls using CHEPS, and validated the findings using other objective measures of small nerve fibres i.e. the histamine-induced skin flare response and intra-epidermal fibres (IEF), and also quantitative sensory testing (QST), a subjective measure. METHODS: In patients with symptoms of sensory neuropathy (n = 41) and healthy controls (n = 9) we performed clinical examination, QST (monofilament, vibration and thermal perception thresholds), nerve conduction studies, histamine-induced skin flares and CHEPS. Skin punch biopsies were immunostained using standard ABC immunoperoxidase for the nerve marker PGP 9.5 or the heat and capsaicin receptor TRPV1. Immunoreactive IEF were counted per length of tissue section and epidermal thickness recorded. RESULTS: Amplitudes of Adelta evoked potentials (muV) following face, arm or leg stimulation were reduced in patients (e.g. for the leg: mean +/- SEM - controls 11.7 +/- 1.95, patients 3.63 +/- 0.85, p = 0.0032). Patients showed reduced leg skin flare responses, which correlated with Adelta amplitudes (rs = 0.40, p = 0.010). In patient leg skin biopsies, PGP 9.5- and TRPV1-immunoreactive IEF were reduced and correlated with Adelta amplitudes (PGP 9.5, rs = 0.51, p = 0.0006; TRPV1, rs = 0.48, p = 0.0012). CONCLUSION: CHEPS appears a sensitive measure, with abnormalities observed in some symptomatic patients who did not have significant IEF loss and/or QST abnormalities. Some of the latter patients may have early small fibre dysfunction or ion channelopathy. CHEPS provides a clinically practical, non-invasive and objective measure, and can be a useful additional tool for the assessment of sensory small fibre neuropathy. Although further evaluation is required, the technique shows potential clinical utility to differentiate neuropathy from other chronic pain states, and provide a biomarker for analgesic development.

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

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

Neuronal markers in allergic rhinitis: expression and correlation with sensory testing.

INTRODUCTION: Although the role of immunoglobulin E-mediated hypersensitivity reactions in allergic rhinitis is well known, the relative contribution of sensory nerves to the symptoms of rhinitis is uncertain. This study looked at the level of specific neuronal markers including the nerve marker protein gene product 9.5 (PGP 9.5), sensory and autonomic neuropeptides, the capsaicin/heat receptor TRPV1, and nerve growth factor (NGF) in patients with allergic rhinitis and controls and their correlation with nasal sensitivity. MATERIALS AND METHODS: Forty patients (23 controls, 17 rhinitis) having nasal surgery were recruited. Nasal sensitivity was tested using graded monofilaments. Inferior turbinate biopsies were collected and studied using immunohistology, with measurement of nerve fibers by direct observation or computerized image analysis. RESULTS: Nerve fibers (PGP 9.5) in the epithelium, subepithelium, and glandular/vascular regions were significantly increased in allergic rhinitis (P=.037, <.01, and .04, respectively), as were subepithelial and glandular/vascular fibers immunoreactive for neuropeptide substance P (P=.04 subepithelium; .02 glandular/vascular) and neuropeptide tyrosine (P<.01 glandular/vascular), markers for sensory and sympathetic nerves, respectively. TRPV1 epithelial fiber counts were higher in rhinitis, but this was not statistically significant. Epithelial NGF immunoreactivity (% area) was significantly increased in rhinitis (P=.027). Nasal sensitivity was correlated significantly with PGP 9.5 subepithelial innervation (control touch P=.023, irritation P=.046; rhinitis touch P=.042, irritation P=.043). A correlation was also observed between epithelial NGF and subepithelial PGP 9.5 innervation, which included all subjects (P=.044). CONCLUSION: The increased number and specific phenotypical changes of sensory nerves may play a role in nasal hypersensitivity and provide new targets for the treatment of rhinitis.

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.

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.

Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human breast pain.

BACKGROUND: Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood. METHODS: Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF). RESULTS: TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness (TRPV1 fibres / mm epidermis, median [range] - no pain group, n = 8, 0.69 [0-1.27]; pain group, n = 10, 2.15 [0.77-4.38]; p = 0.0009). Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] - no pain group, n = 6, 0.75 [0-2]; pain group, n = 11, 2 123, p = 0.008; TRPV4, median [range] - no pain group, n = 6, [0-1]; pain group, n = 11, 1 [0.5-2], p = 0.014). CONCLUSION: Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study.

Expression of the sodium channel beta3 subunit in injured human sensory neurons.

Voltage-gated sodium channel alpha-subunits play a key role in pain pathophysiology, and are modulated by beta-subunits. We previously reported that beta1- and beta2-subunits were decreased in human sensory neurons after spinal root avulsion injury. We have now detected, by immunohistochemistry, beta3-subunits in 82% of small/medium and 67% of large diameter sensory neurons in intact human dorsal root ganglia: 54% of beta3 small/medium neurons were NGF receptor trkA negative. Unlike beta1- and beta2, beta3-immunoreactivity did not decrease after avulsion injury, and the beta3:neurofilament ratio was significantly increased in proximal injured human nerves. beta3-subunit expression may thus be regulated differently from beta1, beta2 and Nav1.8. Targeting beta3 interactions with key alpha-subunits, particularly Nav1.3 and Nav1.8, may provide novel selective analgesics.

Decreased potassium channel IK1 and its regulator neurotrophin-3 (NT-3) in inflamed human bowel.

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.

Immunohistochemical detection of the calcitonin receptor-like receptor protein in the microvasculature of rat endothelium.

Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.

Increased capsaicin receptor TRPV1 nerve fibres in the inflamed human oesophagus.

BACKGROUND: Gastro-oesophageal reflux disease (GORD) patients commonly describe symptoms of heartburn and chest pain. The capsaicin receptor vanilloid receptor 1 (TRPV1) (VR1) is a cation channel expressed by sensory neurones and activated by heat, acid pH and ethanol, which may trigger burning pain. AIM: To study the distribution of TRPV1-expressing nerve fibres in oesophageal mucosal biopsies from patients with symptomatic oesophagitis and in control subjects. METHODS: Biopsies were taken at gastroscopy from the distal oesophagus of seven symptomatic oesophagitis patients and seven asymptomatic patients undergoing investigation for iron-deficiency anaemia. These biopsies were studied by immunohistochemistry using affinity-purified antibodies to TRPV1 and to the neuronal marker peripherin. The density of oesophageal epithelial TRPV1 innervation was assessed by calculating the proportion of papillae in each oesophageal epithelium biopsy specimen containing TRPV1-immunoreactive fibres. RESULTS: TRPV1-immunoreactive nerves were distributed within the lamina propria in healthy subjects and in oesophagitis patients. The percentage of papillae positive for TRPV1 was elevated in oesophagitis patients compared with controls. Peripherin fibre density was not significantly different between the groups. CONCLUSIONS: TRPV1-immunoreactive sensory nerve fibres are expressed in human oesophageal mucosa both in health and in disease. Increased TRPV1 expression in the inflamed oesophagus may mediate the heartburn in oesophagitis, and TRPV1 blockers may provide novel treatment.

The time course of CO2 laser-evoked responses and of skin nerve fibre markers after topical capsaicin in human volunteers.

OBJECTIVE: To assess the temporal relationship between skin nerve denervation and regeneration (dermal and intra-epidermal fibres, IENF) and functional changes (CO(2) laser-evoked potentials, LEPs, and quantitative sensory tests, QST) after topical cutaneous application of capsaicin. METHODS: Capsaicin (0.075%) was applied to the lateral calf for three consecutive days. QST, LEPs and skin biopsies were performed at baseline and time intervals up to 54days post-capsaicin treatment. Biopsies were immunostained with antibodies for PGP9.5, TRPV1, and GAP-43 (marker of regenerating nerve fibres), and analyzed for IENFs and dermal innervation (for GAP-43). RESULTS: At 1day post-capsaicin, cutaneous thermal sensitivity was reduced, as were LEPs. PGP9.5, TRPV1, and GAP-43 immunoreactive-nerve fibres were almost completely absent. By Day 12, LEPs had fully recovered, but PGP9.5 and TRPV1 IENF continued to be significantly decreased 54days post-capsaicin. In contrast, dermal GAP-43 immunoreactivity closely matched recovery of LEPs. CONCLUSIONS: A good correlation was observed between LEPs and GAP-43 staining, in contrast to PGP9.5 and TRPV1. Laser stimulation is a non-invasive and sensitive method for assessing the initial IENF loss, and regenerating nerve fibres. SIGNIFICANCE: Assessing skin biopsies by PGP9.5 immunostaining alone may miss significant diagnostic and prognostic information regarding regenerating nerve fibres, if other approaches are neglected, e.g. LEPs or GAP-43 immunostaining.

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.

Cyclooxygenase-2 (Cox-2) in injured human nerve and a rat model of nerve injury.

Inflammation associated with nerve injury produces a number of pathogenic chemical mediators of which prostanoids are a potent component. Cyclooxygenases (Cox-1 and Cox-2) are the enzymes responsible for prostanoid production. We have investigated Cox-2 immunoreactivity (Cox-2-IR) and glial activation in human injured (n = 16) and uninjured (n = 8) nerves and in the chronic constriction injury (CCI) model of nerve injury in the rat, using immunohistological and autoradiographic methods. Tissues were immunostained with antibodies to Cox-2, CD-68 (human macrophage marker), OX42 (rat microglial marker), or incubated with tritiated PK11195 (marker of glial activation), prior to image analysis. In human nerves, Cox-2-IR was detected in cells with morphology and distribution similar to macrophages/microglia - these were increased significantly in human nerve proximal to injury (p < 0.002), reaching a peak at 4-6 weeks after injury. In the rat CCI model, at 40 days after injury, microglia-like cells with Cox-2-IR were increased significantly in the injured nerve (p < 0.004) and ipsilateral dorsal spinal cord (p < 0.008). PK11195-binding results were similar for Cox-2-IR in chronic injured human nerve and rat tissues. These findings suggest that Cox-2-immunoreactive cells could play a role in processes associated with Wallerian degeneration, nerve regeneration, and the development of persistent pain. Selection of patients 4-6 weeks after nerve injury would be more likely to show any efficacy of Cox-2 inhibitors.

Molecular forms of NGF in human and rat neuropathic tissues: decreased NGF precursor-like immunoreactivity in human diabetic skin.

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.