Vincent Cibert-Goton

EphrinB2 induces tyrosine phosphorylation of NR2B via Src-family kinases during inflammatory hyperalgesia

In recent years a role for EphB receptor tyrosine kinases and their ephrinB ligands in activity-dependent synaptic plasticity in the CNS has been identified. The aim of the present study was to test the hypothesis that EphB receptor activation in the adult rat spinal cord is involved in synaptic plasticity and processing of nociceptive inputs, through modulation of the function of the glutamate ionotropic receptor NMDA (N-methyl-D-aspartate). In particular, EphB receptor activation would induce phosphorylation of the NR2B subunit of the NMDA receptor by a Src family non-receptor tyrosine kinase. Intrathecal administration of ephrinB2-Fc in adult rats, which can bind to and activate EphB receptors and induce behavioral thermal hyperalgesia, led to NR2B tyrosine phosphorylation, which could be blocked by the Src family kinase inhibitor PP2. Furthermore animals pre-treated with PP2 did not develop behavioral thermal hyperalgesia following EphrinB2-Fc administration, suggesting that this pathway is functionally significant. Indeed, EphB1-Fc administration, which competes with the endogenous receptor for ephrinB2 binding and prevents behavioral allodynia and hyperalgesia in the carrageenan model of inflammation, also inhibited NR2B phosphorylation in this model. Taken together these findings support the hypothesis that EphB–ephrinB interactions play an important role in NMDA-dependent, activity-dependent synaptic plasticity in the adult spinal cord, inducing the phosphorylation of the NR2B subunit of the receptor via Src family kinases, thus contributing to chronic pain states.

Multiple roles for NaV1.9 in the activation of visceral afferents by noxious inflammatory, mechanical, and human disease-derived stimuli.

&NA; NaV1.9 regulates normal colonic afferent mechanosensation and is required for hypersensitivity to noxious inflammatory mediators and those derived from inflammatory bowel disease tissues. &NA; Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage‐gated sodium channel subtype NaV1.9 is expressed in half of gut‐projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9−/− mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus–response function to ramp colonic distension. By contrast, responses to rapid, high‐intensity phasic distension of the colon are initially unaffected; however, run‐down of responses to repeat phasic distension were exacerbated in NaV1.9−/− afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9−/− mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high‐value target for development of visceral analgesics.

P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors

Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Nav1.9−/− mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease. SIGNIFICANCE STATEMENT Chronic visceral pain is a debilitating symptom of many gastrointestinal disorders. The activation of pain-sensing nerves located in the bowel wall and their sensitization to physiological stimuli, including bowel movements, underpins the development of such pain, and is associated with mediators released during disease. This work addresses the unstudied role of purine and pyrimidine nucleotides in modulating colonic nociceptors via P2Y receptors using a combination of electrophysiological recordings from human ex vivo samples and a detailed functional study in the mouse. This is the first report to identify colonic purinergic signaling as a function of P2Y receptor activation, in addition to established P2X receptor activity, and the results contribute to our understanding of the development of visceral pain during gastrointestinal disease.

Involvement of EphB1 Receptors Signalling in Models of Inflammatory and Neuropathic Pain

EphB receptors tyrosine kinases and ephrinB ligands were first identified as guidance molecules involved in the establishment of topographical mapping and connectivity in the nervous system during development. Later in development and into adulthood their primary role would switch from guidance to activity-dependent modulation of synaptic efficacy. In sensory systems, they play a role in both the onset of inflammatory and neuropathic pain, and in the establishment of central sensitisation, an NMDA-mediated form of synaptic plasticity thought to underlie most forms of chronic pain. We studied wild type and EphB1 knockout mice in a range of inflammatory and neuropathic pain models to determine 1), whether EphB1 expression is necessary for the onset and/or maintenance of persistent pain, regardless of origin; 2), whether in these models cellular and molecular changes, e.g. phosphorylation of the NR2B subunit of the NMDA receptor, increased c-fos expression or microglial activation, associated with the onset of pain, are affected by the lack of functional EphB1 receptors. Differences in phenotype were examined behaviourally, anatomically, biochemically and electrophysiologically. Our results establish firstly, that functional EphB1 receptors are not essential for the development of normal nociception, thermal or mechanical sensitivity. Secondly, they demonstrate a widespread involvement of EphB1 receptors in chronic pain. NR2B phosphorylation, c-fos expression and microglial activation are all reduced in EphB1 knockout mice. This last finding is intriguing, since microglial activation is supposedly triggered directly by primary afferents, therefore it was not expected to be affected. Interestingly, in some models of long-term pain (days), mechanical and thermal hyperalgesia develop both in wild type and EphB1 knockout mice, but recovery is faster in the latter, indicating that in particular models these receptors are required for the maintenance, rather than the onset of, thermal and mechanical hypersensitivity. This potentially makes them an attractive target for analgesic strategies.

Visceral and somatic pain modalities reveal NaV1.7‐independent visceral nociceptive pathways

Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.

Ex vivo study of human visceral nociceptors

Objective The development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human tissue that could aid future target identification and drug evaluation. Design Electrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV4) modulation on mechanical responses. Results Two distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (−20.8±6.9%, n=6, p<0.05), a treatment for IBS, or application of HC067047 (−34.9±10.0%, n=7, p<0.05), a TRPV4 antagonist, highlighting the utility of the preparation to examine the mechanistic action of existing drugs or novel analgesics. Repeated application of bradykinin or ATP produced consistent afferent responses following desensitisation to the first application, demonstrating their utility as test stimuli to evaluate analgesic activity. Conclusions Functionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.

Perfusion assessment in rat spinal cord tissue using photoplethysmography and laser Doppler flux measurements

Abstract. Animal models are widely used to investigate the pathological mechanisms of spinal cord injury (SCI), most commonly in rats. It is well known that compromised blood flow caused by mechanical disruption of the vasculature can produce irreversible damage and cell death in hypoperfused tissue regions and spinal cord tissue is particularly susceptible to such damage. A fiberoptic photoplethysmography (PPG) probe and instrumentation system were used to investigate the practical considerations of making measurements from rat spinal cord and to assess its suitability for use in SCI models. Experiments to assess the regional perfusion of exposed spinal cord in anesthetized adult rats using both PPG and laser Doppler flowmetry (LDF) were performed. It was found that signals could be obtained reliably from all subjects, although considerable intersite and intersubject variability was seen in the PPG signal amplitude compared to LDF. We present results from 30 measurements in five subjects, the two methods are compared, and practical application to SCI animal models is discussed.

The extracellular matrix glycoprotein tenascin‐X regulates peripheral sensory and motor neurones

Tenascin‐X (TNX) is an extracellular matrix glycoprotein with anti‐adhesive properties in skin and joints. Here we report the novel finding that TNX is expressed in human and mouse gut tissue where it is exclusive to specific subpopulations of neurones. Our studies with TNX‐deficient mice show impaired defecation and neural control of distal colonic motility that can be rescued with a 5‐HT4 receptor agonist. However, colonic secretion is unchanged. They are also susceptible to internal rectal intussusception. Colonic afferent sensitivity is increased in TNX‐deficient mice. Correspondingly, there is increased density of and sensitivity of putative nociceptive fibres in TNX‐deficient mucosa. A group of TNX‐deficient patients report symptoms highly consistent with those in the mouse model. These findings suggest TNX plays entirely different roles in gut to non‐visceral tissues – firstly a role in enteric motor neurones and secondly a role influencing nociceptive sensory neurones Studying further the mechanisms by which TNX influences neuronal function will lead to new targets for future treatment.