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Alison Accarie / 2 ème année de thèse Directeur de thèse : Pr. Michel Dapoigny

9 ème symposium national du réseau Inserm de Recherche sur la Douleur Bordeaux 22-23 mars 2013. From the Poplar mecano-perception to visceral sensitivity in mice: role of ubiquitous transcription factor EGR-4?. Alison Accarie / 2 ème année de thèse

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Alison Accarie / 2 ème année de thèse Directeur de thèse : Pr. Michel Dapoigny

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  1. 9 ème symposium national du réseau Inserm de Recherche sur la Douleur Bordeaux 22-23 mars 2013 From the Poplar mecano-perception to visceral sensitivity in mice: role of ubiquitous transcription factor EGR-4? Alison Accarie / 2ème année de thèse Directeur de thèse : Pr. Michel Dapoigny Directeurs du laboratoire : Pr. Alain Eschalier et Pr. Denis Ardid UMR/INSERM 1107 Neuro Dol, Pharmacologie Fondamentale et Clinique de la douleur

  2. Introduction U1107 / Pharmacologie fondamentale et clinique de la douleur inadequate support of chronic pain Visceral pain Abdominal pain Colonic pain • First cause for gastroenterological consultation • Common symptoms of IBS et IBD (period of remission) IBS : irritable bowel syndrome IBD : intestinal bowel desease

  3. Introduction Treatment only for symptoms No real efficacity • need therapeutic innovation • exploration of associated factors such as hypersensitivity

  4. Introduction Colonic pain 2/3 of cases Colonic hypersensitivity • Exarcerbated response to painful stimulation or not • Peripheral sensitization (target organ)

  5. Introduction Colonic Hypersensitivity Different peripheral sensors Central integration

  6. Introduction Colonic mechanosensitivity: implication of ions channels Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010

  7. Introduction Colonic mechanosensitivity: implication of ionic channels Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010 ASICs ASICs

  8. Introduction Colonic mechanosensitivity: implication of ionic channels Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010 TRPV

  9. Introduction Colonic mechanosensitivity: implication of ionic channels Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010 TRPA1

  10. Introduction Colonic mechanosensitivity: implication of ionic channels Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010 Peripheral sensors? Not clearly defined

  11. Introduction Mecanoperception shared sensation in : Identification of a new transcription factor in case of mechanical stimulation in Arabidopsis & Poplar sp. (Equipe MECA, UMR PIAF Clermont-Ferrand) PtaZFP2

  12. Introduction Martin, 2009 Mechanosensor in plant genetic equivalent in animal PtaZFP2 EGR-4

  13. Introduction EGR Family: 4 subtypes (EGR-1) Transcription factor/ Early Gene Response Zinc fingers structure/ Same DNA binding sequence : GCC G/TGG GCG Largely expressed in periphery and central nervous system Description in cognitive process

  14. Introduction EGR-4 Only two major studies  Surexpression in somatosensory cortex after mechanical stimulation.  Implication in pro-inflammatory cytokines production in T-cells. PCR EGR-4 (Mack et al., 1995).

  15. Aim of the Study Involvement of EGR-4 transcription factor in visceral mechanosensation?

  16. Material and methods syringe and rectal probe • C57Bl6J Mice, 6-8 weeks old. • Colorectal distension with100 µL (water) during10 seconds. Viscero-motor response 20µl 40µl 80µl 100µl 60µl

  17. Materials and methods • samples at different time point after distension. • Colon

  18. Materials and methods • samples at different time point after distension. • Colon • DRG visceral (T10-13, L6 & S1)

  19. Materials and methods • samples at different time point after distension. • Colon • DRG visceral (T10-13, L6 & S1) • Spinal cord

  20. Expression of EGR-4 Euthanasia distension 10 min 15 min 30 min 45 min 1h 1h30 2h 5 min 0

  21. Expression of EGR-4 Euthanasia distension 10 min 15 min 30 min 45 min 1h 1h30 2h 5 min 0 ? Expression in colon, DRG and spinal cord  Western Blot

  22. Expression of EGR-4 in colon Expression of EGR-4 * p>0.05 *** p > 0.001 N = 6 N = 5 N = 5 N =6 Increase of total form of EGR-4 30 minutes after distension

  23. Expression of EGR-4 in DRG T11-13, L6 & S1 Expression of EGR-4 * p<0.05 N = 5 N = 3 N = 4 Increase of total form of EGR-4 45 minutes after distension

  24. Expression of EGR-4 in spinal cord Expression of EGR-4 *** p < 0.001 N = 4 N = 6 N =3 N = 4 Increase of total form of EGR-4 45 minutes after distension

  25. Conclusion • Development new tools (PCR, WB, HIC) distension 30 min 45 min 0 15 min Short Time : EGR-4 Colon, DRG & spinal cord

  26. Perspectives Future studies • Long time expression (WB) • mRNA expression studies (qPCR) • Identification of target genes (micro-array) • Female • Different models of colonic Hypersensitivity post-infectious neo natal stress

  27. Acknowledgement Monique Etienne Ludivine Boudieu Emilie Muller Agathe Gelot Frederic Carvalho Denis Ardid Thank you for your attention

  28. Protein expression : IHC Colon Euthanasia distension Fluorecsence intensity / surface unit 0 1h30 3h30 DRG noyau Spinal cord Fluorecsence intensity / surface unit Proprtion of immuno marqued nucleus neuron

  29. Localization of EGR-4 : IHC Colocalisation with sensitive fibers in DRG. Paraffine Anti-NGFI-C 1:500 Colocalisation with nervous fibers in colon

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