VOIE DE SIGNALISATION ET D'INTEGRATION DE LA DOULEUR VISCERALE Symposium Mayoly sur le Syndrome de l'Intestin Irritable. Organisateur: Pr. Boucekkine VOIE DE SIGNALISATION ET D'INTEGRATION DE LA DOULEUR VISCERALE L. Buéno Unité de Neurogastroentérologie INRA Toulouse, France Alger, 25 Avril 2010

Processing of Sensory Signals in Spinal Cord, Brain Stem, and Brain Gracilis and Cuneatus Brain Stem Dorsal column nociceptive pathway Lat. spinothalamic tract Spinal Cord Spinoreticular tract Dorsal Root Abdominal Viscus

Ascending Pain Pathways IBS - Ascending Visceral Pain Pathway Ascending Pain Pathways

Descending Pain Modulation ACC Thalamus PAG Locus coeruleus Caudal raphe nucleus Amygdala Noradrenergic Rostral ventral medulla Spinal visceral nociceptive transmission is subject to descending modulatory influences from supraspinal structures (e.g., periaqueductal gray, nucleus raphe magnus, locus ceruleus, nuclei reticularis gigantocellularis, and the ventrobasal complex of the thalamus). Descending modulation can be inhibitory, facilitatory or both depending on the context of the visceral stimulus or the intensity of the descending signal. Serotonergic, noradrenergic, dopaminergic and opioid projections are major components of descending modulatory pathways. The descending influence from the ventromedial medulla is mediated mainly by pathways traveling in the dorsolateral spinal cord and can be inhibitory or facilitatory based on stimulus intensity. In contrast, descending control from the thalamus is context-specific in that it may facilitate or inhibit spinal nociceptive processing depending upon the presence or absence of central sensitization. Aside from descending inhibitory influences, spinal visceral input is subject to tonically active supraspinal facilitating modulation, which may be a mechanism that enhances conscious perception of visceral sensations in the absence of noxious stimulation. Exaggeration of descending facilitating signals from the brain might also partly explain the visceral hypersensitivity that is found in a subset of IBS patients. 1. Zhuo M, Gebhart GF. Facilitation and attenuation of a visceral nociceptive reflex from the rostroventral medulla in the rat. Gastroenterology 2002;122:1007-1019. Serotonergic Test balloon Opioid Spinal cord Spinal afferent Rectosigmoid

IBS MCC pACC Brain Activation with Noxious Visceral Stimulation Prefrontal Cortex Cing Cx included MCC pACC Thalamus Pf, Re, Cl, Li Locus coeruleus Subnucleus reticularis dorsalis Spinal cord Lamina 1 IBS

Convergence of Somatic and Visceral Afferents in the Spinal Cord Brain Somatic afferent Dorsal root ganglion Spinothalamic tract Visceral afferent Gallbladder

Divergence of Somatic and Visceral Afferents in the Spinal Cord (Wolf et al. 1965)

(mechanosensitivity) Colonic Referred Pain (mechanosensitivity) Increased pain intensity

Mechanoreceptors of the Digestive tract - localisation Vascular (8/10 high threshold) Serosal (1/3 low threshold) Tonic & Phasic distensions Tonic Distension (act in series) Mucosal (8/10 low threshold) Phasic Distension (act in parallel)

Possible sites of alterations in gut sensitivity 1 Afferent nerve terminals Brain 2 Dorsal horn + - Raphe Cortex Limbic system 3 Brain integrative nuclei Nuclei 4 Diffuse noxious inhibitory system 5-HT 5-HT + - Thalamus 4 Gut Wall Dorsal root Ganglia 3 SPINAL + --- SP Nociceptive stimulus EAA CORD - DH PN + - + 2 1

Spinal Gating for Three Classes of Visceral Nociceptors (Low, High and Silent) Account for Normal Regulatory Functions, Acute and Chronic Pain Synaptic terminal, second order, active neuron Synaptic terminal, spinal and inactive neuron This slide illustrates the concept that changes in levels of firing in low- and high-threshold hold afferent and silent afferents underlie enteric sensory neurophysiology. Low-threshold afferents accounts for the sensory information necessary for central regulation of autonomic functions and for non-painful sensations from the digestive tract. Information coded by the low-threshold afferents is relayed by second order spinal neurons to processing centers in the brain. Normal sensory function without pain is associated with activation of these afferents. Stimulation of sufficient intensity (e.g., distension or strong muscular contaction) activates both low- and high-threshold mechanosensitive afferents and the second order spinal neurons that relay the information from both kinds of receptors to processing centers in the brain. The recruitment of second order high-threshold pathways is interpreted by the brain and relayed to consciousness as acute pain of variable intensity depending on the frequency of firing in the combined pathways. Mediators released by inflammatory/immune cells sensitize both low- and high-threshold afferents to fire at higher frequency than normal in response to a given stimulus strength. Mediators associated with inflammation also sensitize normally silent afferents. This leads to increased intensity of activity transmitted by spinal pathways to processing centers in the brain that may be projected to consciousness as chronic abdominal pain. 1. Modeled after: Cervero F, Janig W. Visceral nociceptors: a new world order? Trends Neurosci 1992; 15:374-378. Threshold Threshold Threshold Low High Silent Low High Silent Low High Silent Normal sensation (No pain) High intensity (Acute pain) Inflammation (Chronic pain)

Postsynaptic receptors at dorsal horn level: - NMDA, AMPA - NK1, NK2, NK3 - CGRP1, SST3 - 5-HT1, 5-HT3 - opioid (µ,d,k) - a2 adrenergic - GABAA Presynaptic receptors at dorsal horn level: - CCK - GABAA - 5-HT3 - µ,d,k - a2 adrenergic Mediators contained in visceral primary afferents : - glutamate, aspartate - SP, NKA, NKB - CGRP - somatostatin - VIP, galanin (Bueno et al. 1996)

Receptors at nerve terminals of nociceptive fibers as putative targets to reduce inflammation-induced hyperalgesia Other putative targets (spinal cord level) TRPV1….5 mGluR1,5 Sub.P CB1, CB2 2 opioid Nociceptive terminal NK1,2,3 CCK1 and….. others H+ ATP NGF IL-1ß PGE2 Hist. Tryp. 5-HT Pressure Heat Bradykinin Other putative targets (periphery) Tissue damage Mast cell LIF IL6 TNFa CRF NGF Proteases CRF-R1, CRF-R2 PAR1, PAR2 TrkA, p75

Role of Mast cell in mucosal inflammation-induced visceral hyperalgesia Stress Mast cell CRF Degranulation minutes Allergy Histamine Leukotrienes Cytokines* proteases NGF IgE, SP,5-HT PAF, ATP Inflammation hours GM-CSF, IL-1 Secretion Infection NGF, NPY Cytokines* Chemokines Sympathetic activation * Cytokines: TNFa, IL-3, IL-5, IL-4, IL-13, IL-10, GM-CSF (adapted from Shakoory et al,2004, Penicci et al.2003)

Endothelium/ Epithelium Mast cell Eosinophil/ neutrophils BIDIRECTIONAL REGULATION OF MAST-CELL-EOSINOPHIL FUNCTION SP SCF Endothelium/ Epithelium IL-4 PAR2 tryptase PAR2 Mast cell Cytokines chymase Eotaxin PAR2 SCF Cytokines GM-CSF Chemokines IL-1 / TNF-a CCR3 Eosinophil/ neutrophils Adapted from Shakoory et al. 2004

P P P P ROLE OF MAST CELLS IN VISCERAL PAIN: (degranulation in response to mechanical stimuli) Sensitization Density Normal barosensitivity Normal barosensitivity P P Post-infection Pain Post-stress Pain P P MC degranulation MC degranulation

Role of mast cell in favorising increase in colonic gut permeability and subsequent increase in visceral sensitivity to distension STRESS CRF Mast cell activation 5-HT, tryptase NGF activation Visceral hyperalgesia Cytokines Chemokines Inflammatory mediators sensory nerve terminal sensitization T-lymphocyte Neutrophils TJ OPENING IFN  MLC MLCK MLCP (Ait-Belgnaoui et al. Pain 2005)

Repetitive Stimulation Sensitizes the Spinal Cord Dorsal root ganglion Wind-up Sensitized spinal circuits Repeated balloon distention Mechanosensitive afferent Repeated inflation of a balloon to noxious levels in the recto-sigmoid region leads to lowered perception thresholds for discomfort and pain in IBS patients. This phenomenon is believed to be due to centrally mediated hyperalgesia. Centrally mediated hyperalgesia reflects hyperexcitability of second order sensory neurons in the dorsal horn of the spinal cord. Under conditions of severe and persistent nociceptive stimulation, small-diameter, nonmyelinated C fibers fire repetitively and their input progressively increases responses of second order dorsal horn neurons. “Wind-up” of the second order neurons is generated by the release of the neurotransmitter glutamate from the C fibers. Noxious stimulation of the type applied in balloon distention studies in IBS patients can produce long-term changes in the excitability of dorsal horn neurons. This phenomenon is called central sensitization to distinguish it from the sensitization that occurs at the endings of the sensory neurons in the intestinal wall during release of inflammatory mediators. Long-lasting changes in the excitability of dorsal horn neurons impart a memory for nociceptive input from C fibers. Upregulation in the expression of neuropeptides and neurotransmitters and their receptors presumably underlie the phenomenon of central sensitization. Elevated excitability in nociceptive dorsal horn neurons underlies spinally mediated hyperalgesia, which is termed central sensitization to distinguish it from sensitization that occurs at nociceptive terminals in the periphery. In conditions of severe tissue injury and persistent injury, nociceptive C fibers fire repetitively and the excitability of the second order neurons in the dorsal horn increases progressively in response to the elevated synaptic input. This effect is sometimes called wind-up and reflects the synaptic release of glutamate from the incoming C fibers and activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors expressed by the second order neurons. One piece of suggestive evidence for wind-up hypersensitivity is the finding that second order neurons in the dorsal horn show induction of the early gene c-fos in response to noxious balloon distention of the colon in rats (96). These long-lasting changes in the excitability of dorsal horn neurons are like a memory imprint of the nociceptive input. Accumulating evidence suggests that the spinal wind-up phenomenon may be operational and be an underlying factor in the intestinal hypersensitivity associated with IBS. 1. Basbaum AI, Jessell TM. The perception of pain. In: Principles of neuroscience 4th ed. Kandel ER, Schwartz JH, Jessell TM (eds.) New York: McGraw-Hill 2000:472-491.   2. Munakata J, Naliboff B, Harraf F, Kodner A, Lembo T, Chang L, Silverman DHS, Mayer EA. Repetitive sigmoid stimulation induces rectal hyperalgesia in patients with irritable bowel syndrome. Gastroenterology 1997;112:55-63. 3. Traub RJ, Pechman P, Iadarola MJ, Gebhart GF. Fos-like proteins in the lumbosacral spinal cord following noxious and non-noxious colorectal distention in the rat. Pain 1992;49:393-403.

MODULATION OF NEURONAL NOCICEPTIVE TRANSMISSION AT SPINAL CORD LEVEL BY GLIAL CELLS (from Svensson et al. 2010)

? ? GLIAL CELL NEUROMEDIATORS INVOLVED IN NOCICEPTIVE TRANSMISSION AT SPINAL CORD LEVEL ? ? (from Svensson et al. 2010)

SPINAL MICROGLIA ACTIVATION IN STRESS -INDUCED VISCERAL HYPERALGESIA Bradesi et al. 2009

SPINAL MICROGLIA ACTIVATION IN “NARCOTIC BOWEL SYNDROME” Agostini et al. 2010 (in press)

CONCLUSIONS La douleur d'origine digestive est le plus souvent associée à une sensibilisation des mécano-récepteurs pariétaux principalement par les produits de dégranulation des mastocytes. La présence d'une micro-inflammation associée à une redistribution des terminaisons est un facteur majeur de l'hypersensibilité viscérale observée dans le SII. La douleur viscérale chronique est déclenchée par à une hypersensibilité locale mais facilité et entretenue par une facilitation et amplification spinale des messages nociceptifs Outre les facteurs locaux, les cellules gliales et en particulier la microglie participent à la facilitation spinale de cette transmission nociceptive