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Étude de l'implication du nerve growth factor (NGF) et des acid-sensing ion channels (ASIC) dans l'hyper-sensibilité colique induite par le butyrate chez le rat

J. Matricon 1
1 Pharmacologie fondamentale et clinique de la douleur
Neuro-Dol - Neuro-Dol, UdA - Université d'Auvergne - Clermont-Ferrand I, INSERM - Institut National de la Santé et de la Recherche Médicale : U766
Abstract : Functional gastrointestinal disorders are characterized by abdominal pain and discomfort that affect the quality of life of many patients worldwide. Irritable bowel syndrome (IBS), the most common functional gastrointestinal disorder, is characterized by intractable visceral hypersensitivity in absence of any structural or biochemical abnormality of the colon. The lack of understanding regarding its precise pathophysiology makes the identification of new drug targets difficult. This issue is a public healthcare stake since functional gastrointestinal disorders, and especially IBS, affect 20% of the western population. Animal models are an useful way of testing etiological hypotheses on the mechanisms of visceral pain provided by clinical studies. We thus used an animal model of IBS, the butyrate model, which has been developped in the lab with respect to clinical data reporting a higher colonic concentration of butyrate, a short chain fatty acid, in patients with IBS. This model consists of six intracolonic enemas of butyrate 200mM, performed in four days, which induce a colonic hypersensitivity (CHS) in the rat starting from the last enema. Butyrate-induced CHS can be assessed using the colorectal distention (CRD) test. This CHS is persistent and is not associated with mucosal alterations what is relevant for the clinical caracteristics of IBS. Face validity, predicitive validity and construct validity of the butyrate model have been explored and we now aimed at decyphering the peripheral and central mechanisms by which butyrate induces CHS. Preliminary results had shown that a neonatal treatment with capsaicin, which destroys nociceptive peptidergic C-fibres, prevented the development of butyrate-induced CHS in adult rats. Colonic type C afferences thus convey the nociceptive message induced by butyrate. The main goal of our work was to determine what mechanisms underly sensitization of these afferences by butyrate. We hypothesized that nerve growth factor (NGF), a neurotrophin involved in nociception and in neurogenic inflammation of enteric nerves in IBS, could contribute to sensitization of the colon. Involvement of NGF in CHS We have shown that repeated administrations of anti-NGF antibodies (i.p. route) prevented the butyrate-induced CHS, assessed by the CRD test. Moreover, we have shown with immuno-histo-chemistry (IHC) that NGF was expressed in the colon of rats treated with butyrate but its expression was not increased. On the other hand, NGF was overexpressed in dorsal root ganglia (DRG) innervating the colon. These results suggested that NGF is involved in butyrate-induced CHS and that it could exert its effect mainly in the peripheral nervous system (DRG). The NGF is indeed involved in the development of hyperalgia by inducing the expression of molecules playing a key role in nociception such as ion channels. We have focused on acid-sensing ion channels (ASIC) and transient receptor potential vannilloid1 (TRPV1) according to numerous papers reporting their involvement in visceral pain and their modulation by the NGF. Involvement of ASIC in CHS We have shown that administration of amiloride (ASIC antagonist, i.v. route) but not of capsazepine (TRPV1 antagonist, i.p. route) prevented butyrate-induced CHS, assessed with the CRD test. Butyrate-induced CHS thus involves an ASIC-dependent TRPV1-independent mechanism. Furthermore, we have shown with semi-quantitative RT-PCR that the expression level of ASIC1a and ASIC1b mRNA was increased in DRG of rats treated with butyrate. This increase was correlated to an increase in the expression of the ASIC1A protein in DRG, quantified with IHC. By determinating the proportion of ASIC1A immuno-reactive (IR) neurons, we have shown that this overexpression was limited to small-diameter neurons. These results indicated that the channel ASIC1A was involved in butyrate-induced CHS. We then wanted to know if, according to the bibliography, ASIC1A overexpression could be consecutive to that of NGF. Modulation of ASIC1A expression by NGF in CHS We first have shown with confocal microscopy that NGF and its high affinity receptor (trkA) were colocalized with ASIC1A in sensory neurons and especially in those expressing calcitonin gene-related peptide. We then have shown using IHC and Western blot that ASIC1A overexpression in DRG was prevented by blocking NGF with repeated administrations of anti-NGF (i.p. route). These results suggested that ASIC1A overexpression was NGF-dependent. We concluded that NGF and ASIC1A intervene in DRG (and likely in nociceptive sensory neurons) where they contribute to sensitization phenomena leading to butyrate-induced CHS. However, we did not report any variation in the expression of NGF and ASIC1A in the colon. This result suggested that these molecules are involved rather in the presynaptic element of the first central nociceptive synapse where they could enhance the synaptic transmission than in the free synaptic endings of the colonic wall. We thus have hypothesized that butyrate-induced CHS was associated to central sensitization of the spinal cord. Central sensitization in CHS We have used the Fos method, a marker for neuronal activity, in order to evaluate the spinal excitability in rats treated with butyrate. The study of spinal expression of Fos protein in rats challenged with repeated noxious CRD showed that butyrate rats presented an increase in the density of Fos-IR neurons in the superficial layers of the thoracic dorsal spinal cord in comparison with control rats. Butyrate-induced CHS was characterised by a specific recruitment of the thoracic spinal segments T10-T11-T12 in which we found a neuronal hyperresponsivness to CRD. Moreover, in absence of colonic stimulation, butyrate-treated rats presented a basal hyperactivity in these segments. As the spinal ASIC1A channel contributes to ion currents evoked by painful stimulations, we wanted to know if this spinal hyperactivity could be consecutive to an increased synaptic transmission involving ASIC1A. We have shown that PcTx1 administration (ASIC1A antagonist, i.t. route) prevented the butyrate-induced CHS, assessed with the CRD test. This result suggested that ASIC1A channels contribute to central mechanisms of CHS in the butyrate model. We have also shown with RT-PCR and Western blot that ASIC1A was overexpressed in the spinal cord of rats treated with butyrate. Moreover, it was expressed in spinal neurons activated by CRD where it was colocalized with Fos. ASIC1A could thus participate to the transmission of the nociceptive visceral message in the dorsal spinal cord. The expression level of ASIC2a and ASIC2b mRNA was also found increased in the spinal cord using RT-PCR what suggested that the spinal hyperexcitability in the butyrate model could result from an increase in ion conductances generated by the activation of heteromeric channels type ASIC1/ASIC2. Similarly to the peripheral mechanisms of butyrate-induced CHS, the overexpression of ASIC1A could be NGF-dependent. Our results have indeed shown that NGF was expressed in the spinal cord and that systemic administration of anti-NGF antibodies prevented the overexpression of ASIC1A mRNA and protein in butyrate rats. To conclude, this work suggest that NGF and ASIC1A play a key role in the development of visceral pain by contributing to peripheral and central sensitization. Improving the understanding of NGF-ASIC1A interplay could lead to new lines of research in the treatment of visceral pain of unknown etiology such as CHS in IBS, based on strategies aiming the potentiating effect of NGF on ASIC.
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Contributor : Julien Matricon <>
Submitted on : Monday, May 3, 2010 - 2:21:37 PM
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J. Matricon. Étude de l'implication du nerve growth factor (NGF) et des acid-sensing ion channels (ASIC) dans l'hyper-sensibilité colique induite par le butyrate chez le rat. Sciences du Vivant [q-bio]. Université d'Auvergne - Clermont-Ferrand I, 2010. Français. ⟨tel-00480055⟩

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