from the poplar mecano-perception to visceral sensitivity in mice: role of ubiquitous transcription...
TRANSCRIPT
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
9 ème symposium national du réseau Inserm de Recherche sur la Douleur Bordeaux
22-23 mars 2013
U1107 / Pharmacologie fondamentale et clinique de la douleur
inadequate support of chronic pain
Introduction
Visceral pain
Abdominal pain
Colonic pain
• First cause for gastroenterological consultation
• Common symptoms of IBS et IBD (period of remission)
IBS : irritable bowel syndromeIBD : intestinal bowel desease
Treatment only for symptoms
No real efficacity
need therapeutic innovation
exploration of associated factors such as hypersensitivity
Introduction
Colonic hypersensitivity
Colonic pain
2/3 of cases
Exarcerbated response to painful stimulation or not
Peripheral sensitization (target organ)
Introduction
Central integration
Colonic Hypersensitivity Different peripheral sensors
Introduction
Colonic mechanosensitivity: implication of ions channels
Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010
Introduction
Colonic mechanosensitivity: implication of ionic channels
Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010
ASICs
ASICs
Introduction
Colonic mechanosensitivity: implication of ionic channels
Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010
TRPV
Introduction
Colonic mechanosensitivity: implication of ionic channels
Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010
TRPA1
Introduction
Colonic mechanosensitivity: implication of ionic channels
Stuart M. Brierley Autonomic Neuroscience: Basic and Clinical 2010
Peripheral sensors? Not clearly defined
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
Introduction
Martin, 2009
Mechanosensor in plant genetic equivalent in animal
PtaZFP2 EGR-4
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
Introduction
Surexpression in somatosensory cortex after mechanical stimulation.
(Mack et al., 1995).
PCR EGR-4
Implication in pro-inflammatory cytokines production in T-cells.
EGR-4
Only two major studies
Introduction
Involvement of EGR-4 transcription factor in
visceral mechanosensation?
Aim of the Study
C57Bl6J Mice, 6-8 weeks old.
Colorectal distension with100 µL (water) during10 seconds.
syringe and rectal probe
Material and methods
20µl 40µl 60µl 80µl 100µl
Viscero-motor response
Materials and methods
samples at different time point after distension.
Colon
Materials and methods
samples at different time point after distension.
Colon
DRG visceral (T10-13, L6 & S1)
Materials and methods
samples at different time point after distension.
Colon
DRG visceral (T10-13, L6 & S1)
Spinal cord
Expression of EGR-4
distension
Euthanasia
1h30 2h0 45 min30 min15 min10 min5 min 1h
Expression of EGR-4
1h30 2h0
distension
Western Blot
? Expression in colon, DRG and spinal cord
45 min30 min15 min
Euthanasia
10 min5 min 1h
Expression of EGR-4 in colon
Increase of total form of EGR-4 30 minutes after distension
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 45 minutes after distension
Expression of EGR-4
Expression of EGR-4 in DRG T11-13, L6 & S1
* p<0.05
N = 5 N = 3 N = 4
Increase of total form of EGR-4 45 minutes after distension
Expression of EGR-4
Expression of EGR-4 in spinal cord
*** p < 0.001
N = 4 N = 6 N =3 N = 4
Conclusion
Short Time :
EGR-4 Colon, DRG & spinal cord
Development new tools (PCR, WB, HIC)
0 45 min30 min15 min
distension
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
Thank you for your attention
Acknowledgement
Monique Etienne
Ludivine Boudieu
Emilie Muller
Agathe Gelot
Frederic Carvalho
Denis Ardid
Protein expression : IHC
Colon
Flu
orec
senc
e in
tens
ity /
sur
face
uni
t
Spinal cord
Flu
orec
senc
e in
tens
ity /
sur
face
uni
t
DRG noyau
Pro
prtio
n of
imm
uno
mar
qued
nuc
leus
ne
uron
distension
1h30 3h30
Euthanasia
0
Localization of EGR-4 : IHC
ParaffineAnti-NGFI-C 1:500
Colocalisation with sensitive fibers in DRG.Colocalisation with nervous fibers in colon