Yiangos Yiangou

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

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

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

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

Vanilloid receptor 1 immunoreactivity in inflamed human bowel

Vanilloid receptor 1 (VR1) is expressed by sensory neurons. Once activated, these neurons evoke the sensation of burning pain and release neuropeptides that induce neurogenic inflammation. We used immunoblotting and immunostaining to estimate the density of VR1 in colonic tissues of patients with inflammatory bowel disease and of controls. Our study results indicate that VR1 immunoreactivity is greatly increased in colonic nerve fibres of patients with active inflammatory bowel disease. Thus, the discovery of new drugs that can bind the VR1 receptor, or antagonise endogenous inflammatory substances that activate this receptor, could lead to new therapies for pain and dysmotility.

Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain

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<0.0001). Substance P-immunoreactive fibres (p = 0.01), total nerve fibres (PGP9.5) (p = 0.002), mast cells (c-kit) (p = 0.02) and lymphocytes (CD3) (p = 0.03) were also significantly increased in the IBS group. In multivariate regression analysis, only TRPV1-immuno-reactive fibres (p = 0.005) and mast cells (p = 0.008) were significantly related to the abdominal pain score. Conclusions: Increased TRPV1 nerve fibres are observed in IBS, together with a low-grade inflammatory response. The increased TRPV1 nerve fibres may contribute to visceral hypersensitivity and pain in IBS, and provide a novel therapeutic target.

Parallel changes in bladder suburothelial vanilloid receptor TRPV1 and pan‐neuronal marker PGP9.5 immunoreactivity in patients with neurogenic detrusor overactivity after intravesical resiniferatoxin treatment

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.

Capsaicin receptor VR1 and ATP-gated ion channel P2X3 in human urinary bladder.

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.

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

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

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

BackgroundThe 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.MethodsBladder 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.ResultsTRPM8-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.ConclusionThis 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.

Increased vanilloid receptor VR1 innervation in vulvodynia

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<0.002). VR1 expression was found to be significantly increased when the percentage area immunostained was expressed as a ratio of VR1 to PGP 9.5, a pan‐neuronal marker (P=0.01). VR1‐positive fine epidermal fibres also appeared to be increased in vulvodynia tissues, by inspection. Fibres immunoreactive to the voltage‐gated sodium channel SNS1/PN3 (Nav1.8), also expressed by nociceptors, were relatively scarce in both vulvodynia and control tissues. We hypothesize that increased expression of VR1 by nociceptors could mediate some of the symptoms in vulvodynia, for which systemic or topical specific VR1 antagonists may provide novel treatment.

ATP-gated ion channel P2X3 is increased in human inflammatory bowel disease

P2X3 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, P2X3 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, P2X3‐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 P2X3‐immunoreactive neurones was significantly increased in the myenteric plexus of inflamed colon compared to controls (n=13; P=0.01). In humans, unlike rodents, P2X3 is thus not restricted to sensory neurones. Increased P2X3 in inflamed intestine suggests a potential role in dysmotility and pain, for which it represents a new therapeutic target.