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Keywords:

  • acid-sensing ion channel;
  • animal model;
  • colonic hypersensitivity;
  • nerve growth factor;
  • sensory neurons

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

Background

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with idiopathic colonic hypersensitivity (CHS). However, recent studies suggest that low-grade inflammation could underlie CHS in IBS. The pro-inflammatory mediator nerve growth factor (NGF) plays a key role in the sensitization of peripheral pain pathways and several studies have reported its contribution to visceral pain development. NGF modulates the expression of Acid-Sensing Ion Channels (ASICs), which are proton sensors involved in sensory neurons sensitization. This study examined the peripheral contribution of NGF and ASICs to IBS-like CHS induced by butyrate enemas in the rat colon.

Methods

Colorectal distension and immunohistochemical staining of sensory neurons were used to evaluate NGF and ASICs contribution to the development of butyrate-induced CHS.

Key Results

Systemic injection of anti-NGF antibodies or the ASICs inhibitor amiloride prevented the development of butyrate-induced CHS. A significant increase in NGF and ASIC1a protein expression levels was observed in sensory neurons of rats displaying butyrate-induced CHS. This increase was specific of small- and medium-diameter L1 + S1 sensory neurons, where ASIC1a was co-expressed with NGF or trkA in CGRP-immunoreactive somas. ASIC1a was also overexpressed in retrogradely labeled colon sensory neurons. Interestingly, anti-NGF antibody administration prevented ASIC1a overexpression in sensory neurons of butyrate-treated rats.

Conclusions & Inferences

Our data suggest that peripheral NGF and ASIC1a concomitantly contribute to the development of butyrate-induced CHS NGF-ASIC1a interplay may have a pivotal role in the sensitization of colonic sensory neurons and as such, could be considered as a potential new therapeutic target for IBS treatment.

Abbreviations
ASIC

acid sensing ion channel

CGRP

calcitonin-gene related peptide

CHS

colonic hypersensitivity

CRD

colorectal distension

DEG/ENaC

degenerin/epithelial sodium channel

DRG

dorsal root ganglia

IBS

irritable bowel syndrome

KO

knock-out

NGF

nerve growth factor

TrkA

receptor tyrosine kinase A

Key Messages
  • IBS is associated with idiopathic colonic hypersensitivity.
  • NGF is involved in visceral pain development, presumably via regulation of ion channels such as ASICs.
  • We tested the hypothesis that NGF contributes to colonic hypersensitivity via ASICs modulation.
  • The effect of NGF on ASICs expression and on colonic pain was assessed in rats with butyrate-induced colonic hypersensitivity.
  • Our results show that the development of butyrate-induced colonic hypersensitivity results from a NGF-dependent ASIC1a over expression in nociceptive colonic neurons.
  • NGF-ASIC1a interplay may have a pivotal role in the sensitization of colonic sensory neurons and should be considered as a potential new therapeutic target for IBS treatment.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

Chronic colonic hypersensitivity (CHS) is a key feature of irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, which strongly impairs patients’ quality of life.[1] Despite studies pointing to peripheral and central mechanisms of visceral afferent sensitization,[2, 3] CHS etiopathogenesis in IBS remains poorly understood.

Several mediators have been shown to sensitize visceral nociceptive afferents.[4] Among them, the nerve growth factor (NGF) plays a crucial role in the peripheral mechanisms leading to the development of inflammatory visceral hypersensitivity, as demonstrated in clinical and preclinical studies.[5-10] Exogenous administration of NGF[11, 12] or systemic blockade of NGF expression modulates colonic sensitivity in rodent models of inflammatory visceral pain.[13, 14] Nerve growth factor contribute to the sensitization of nociceptive neurons through the induction of neuroplastic changes that are notably characterized by an increased expression of proteins involved in nociception, such as neuropeptides, G protein-coupled receptors and voltage-gated ion channels.[15-17] Acid-Sensing Ion Channels (ASICs) are up-regulated by NGF.[18, 19] Acid-Sensing Ion Channels are part of the degenerin/epithelial sodium channel (DEG/ENaC) family and are composed of ASIC1 (ASIC1a and ASIC1b), ASIC2 (ASIC2a and ASIC2b), ASIC3 and ASIC4 subunits, assembling into homo- or heterodimers.[20-24] They are expressed both in the nervous system and in the gastrointestinal tract,[18, 25-27] where they contribute to nociceptive processes by sensing changes in proton concentration.[28-30] Protons are highly suspected of triggering pain and/or hypersensitivity through the activation of cationic currents in nociceptors.[31-34]

While IBS has been considered for years as a sine materia pathology, most recent insights suggest that low-grade inflammation could underlie its symptomatology (for reviews, see Refs. [35-37]) and NGF might play a critical role in this process.[5, 38] Therefore, we hypothesized that NGF could play a pro-nociceptive role in IBS pathophysiology by modulating ASICs. To test this hypothesis, we used a relevant rat model of IBS-like CHS induced by repeated butyrate colonic enemas, which combines a decrease in colorectal distension (CRD) pain thresholds and the absence of macroscopic or histological modifications of rat colonic mucosa.[39] Using this model, we examined (i) the peripheral contribution of NGF and ASICs to IBS-like CHS, (ii) changes in NGF and ASICs expression in dorsal root ganglia (DRG) sensory neurons of butyrate-treated rats and (iii) the effect of NGF blockade on ASICs expression in DRG sensory neurons. Our results suggest that butyrate-induced CHS could involve a NGF-mediated peripheral up-regulation of ASIC1a expression in colonic DRG sensory neurons, further sensitizing colonic nociceptors.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

All experiments were performed in accordance with the ethical guidelines of the International Association for the Study of Pain,[40] EU guidelines, the regulations of the French Agriculture and Forestry Ministry, and in agreement with local ethical committee (no's. B63-113.15, CE02-13, and CE03-13).

Animals

Male Sprague-Dawley rats (Charles River, France) weighting 225–250 g were acclimatized to laboratory conditions for 1 week prior any experiment. Animals were housed 4 per cage on a 12-hours light/dark cycle with ad libitum access to food and water.

Butyrate-induced CHS

Colonic hypersensitivity was induced in rats by butyrate enemas in the distal colon as previously described.[39, 41-43] Briefly, colonic enemas were performed using a 2 mm Fogarty catheter introduced rectally at 7 cm from the anal margin. Each animal received six enemas of 1 mL of a 200 mM sodium n-butyrate diluted in saline (Sigma-Aldrich, Lyon, France, pH7), or saline (NaCl 0.9%, pH7). The six enemas were performed over 4 days, as follows: first enema in the evening (6 pm), then twice daily (8 am and 6 pm) for 2 days, and last enema in the morning (8 am).

Colorectal distention test

Experimental design

All experiments were performed on the last day of butyrate or saline enemas, allowing time for the development of a strong and persistent butyrate-induced CHS.[39, 42] Animals were randomly assigned to the different experimental groups using the Latin square block method, i.e. we assigned one animal per treatment group (butyrate or saline) to a single block, and were blind regarding group treatments. Each experimental series used a different set of animals.

Evaluation of colonic sensitivity

Colonic hypersensitivity was evaluated using the CRD test, which allows assessment of pharmacological drugs effects in vivo without requiring surgery (i.e. for electromyographic monitoring of abdominal muscle contraction), thus preserving visceral physiological status in animals. Mechanical distension during CRD test does not alter the colonic mucosa of distended-rats (Matricon et al.,[42] see Fig. S1). Colonic hypersensitivity was assessed in 24 h-fasted rats by measuring the intra-colonic pressure required to induce characteristic abdominal contractions.[44, 45] Distension probes were prepared using a 2 mm Fogarty catheter cut at 9 cm. A 2 cm length flexible latex balloon was ligated to the tip of the catheter. Following volatile anesthesia (2% isoflurane; Baxter, Maurepas, France) and feces removal, the probe was inserted intra-rectally at 7 cm from the anal margin, and the catheter taped to the base of the tail. After 5 min of recovery, rats were placed in the middle of a 40 × 40 cm polymethyl acrylate box and the catheter was connected to an electronic barostat apparatus (Synectics Visceral Stimulator, Medtronic, Boulogne-Billancourt, France). Balloon pressure was gradually increased from 0 to 80 mmHg during 10 min and pain behavior analyzed up to the cut-off pressure (80 mmHg). This procedure allows a consistent evaluation of CHS as previously shown.[39, 41-43, 46, 47] Typically, following an exploration phase, animals adopted a prostration behavior up to the appearance of characteristic abdominal contractions associated to an elevation of the posterior part of the animal's body.[44, 45] Pain threshold values were assessed by the same trained experimenters in order to limit inter-experimenter variability. Pain threshold was defined as the pressure necessary to induce the first long-lasting abdominal cramp observed, with rats staying prostrated and not resuming an exploratory behavior up to the cut-off signal. Animals displaying feces during the experiment were excluded from analysis, as feces could interfere with balloon volume.

In vivo treatments

Subchronic administration of anti-NGF antibodies

Three independent experiments were performed in order to investigate the effect of anti-NGF treatment on butyrate-induced CHS and on ASIC expression in the DRG of butyrate-treated rats.

Experiment 1: Assessment of the effect of anti-NGF antibodies treatment on CRD pain thresholds. Following each enema, butyrate- (n = 16) and saline-treated rats (n = 16) received one intraperitoneal injection of either an anti-NGF antibody (1 : 2000, 2 mL kg−1, Sigma-Aldrich, n = 8 per group) or a control isotype antibody (1 : 2000, 2 mL kg−1, Sigma-Aldrich, n = 8 per group) as previously described.[11, 12] Colorectal distension test was performed on the day of the last injection, the procedure starting at 1 pm. Since the last anti-NGF injection (and the last colon enema) is performed in the morning (8 am), the time lapse between the last anti-NGF injection and the CRD test is 5 h. This delay is consistent with the time course of NGF action on visceral pain, which lasts over 6 h[11, 12] and with in vivo antibodies half-life.[48-51]

Experiment 2: Assessment of the effect of anti-NGF antibodies treatment on ASIC1a expression by immunoblotting in L1 + S1 DRG. Following each enema, butyrate- (n = 10) and saline-treated rats (n = 10) received one intraperitoneal injection of either an anti-NGF antibody (1 : 2000, 2 mL kg−1, Sigma-Aldrich, n = 5 per group) or a control isotype antibody (1 : 2000, 2 mL kg−1, Sigma-Aldrich, n = 5 per group) as previously described.[11, 12] With respect to the time frame used for the CRD experiment, rats were sacrificed on the day of the last injection (the procedure starting at 1 pm) and lumbar L1 and sacral S1 DRG (L1 + S1), which contain the somas of colonic sensory neurons,[52-55] were dissected and processed for immunoblotting.

Experiment 3: Assessment of the effect of anti-NGF antibodies treatment on ASIC1a expression by immunohistochemistry in L1 + S1 DRG. Following each enema, butyrate- (n = 4) and saline-treated rats (n = 4) received one intraperitoneal injection of anti-NGF antibody (1 : 2000, 2 mL kg−1, Sigma-Aldrich) as previously described.[11, 12] Rats were sacrificed and DRG were dissected on the day of the last injection (the procedure starting at 1 pm) and processed for immunohistochemistry.

Subchronic administration of NGF

To investigate the effect of NGF on ASIC1a expression in sensory neurons, saline-treated rats (n = 12) were subchronically injected with NGF (10 ng in 0.1% BSA, 1 mL kg−1, Sigma-Aldrich, n = 6) or its vehicle (n = 6) as previously described[11, 12] on a time frame similar to the one used for subchronic treatment with anti-NGF antibodies. Rats were sacrificed and L1 + S1 DRG were dissected on the day of the last injection (the procedure starting at 1 pm) and processed for immunoblotting.

Administration of the ASICs inhibitor amiloride

To investigate ASICs contribution to butyrate-induced CHS, butyrate-treated rats (n = 8) were injected intravenously with amiloride (1, 3 or 6 mM, Tocris, Bristol, UK) or saline (100 μL per 100 g) 10 min before CRD test. Behavioral assessment of amiloride effects was performed following administration at in vivo doses (e.g. 1 mM corresponding to a commonly used dose of 0.25 μg kg−1),[56-59] in order to avoid smooth muscle relaxation,[56, 57, 59] which could interfere with CRD test.

Analysis of ASICs mRNA expression

mRNA extraction

Molecular analysis of ASICs mRNA expression used a new set of rats. Lumbosacral L1 + S1 DRG were rapidly dissected on ice from butyrate- or saline-treated rats (n = 5), frozen in liquid nitrogen and preserved at −80 °C. Total mRNA was extracted using a phenol/chloroform method and quantified using a spectrophotometer at 260 nm. mRNA purity and integrity were confirmed by electrophoresis in a 2% agarose gel containing ethidium bromide.

RT-PCR experiments

Total mRNA (2 μg) was reverse-transcribed using the First-Strand cDNA Synthesis Kit (GE Healthcare, Amersham, UK). Semi-quantitative PCR analysis was performed in the linear cDNA amplification phase using the housekeeping ribosomal gene L32 as a reference. All primers are listed in Table 1. After migration in a 2%-agarose gel containing ethidium bromide, the intensity of the PCR product was measured using Kodak Digital Science 1D Image Analysis Software (Kodak, Maisons-Alfort, France). All experiments were performed in duplicate.

Table 1. Sequence of primers for rat mRNA quantification
NamesForward primerReverse primerReference
  1. a

    Waldmann et al. ([23]) Nature 386:173–177.

  2. b

    Chen et al. ([21]) Proc Natl Acad Sci USA 95:10240–10245.

  3. c

    Waldmann et al. (1996) J Biol Chem 271:10433–10436.

  4. d

    Lingueglia et al. ([22]) J Biol Chem 272:29778–29783.

  5. e

    Waldmann et al. ([23]) J Biol Chem 272:20975–20978.

  6. f

    Bonnefont et al. (2007) Mol Pharmacol 71:407–415.

ASIC1a5′-ACA GAT GGC TGA TGA AAA GCA G-3′5′-CAT GGT AAC AGC ATT GCA GGT GC-3′ a
ASIC1b5′-ATG CCG TGC GGT TGT CCC-3′5′-CAT GGT AAC AGC ATT GCA GGT GC-3′ b
ASIC2a5′-CAA CCT ACA GAT TCC CGA CCC G-3′5′-CGA GTC CCA TCT CTG AGG ACC GG-3′ c
ASIC2b5′-CTG CCT TCA TGG ACC GTT TG-3′5′-CGA GTC CCA TCT CTG AGG ACC GG-3′ d
ASIC35′-CCC AGA CCC AGA CCC AGC CCT CC-3′5′-CTG TTC CAG AAA TAC CCC AGG AC-3′ e
L325′-GTG AAG CCC AAG ATC GTC AA-3′5′-TTG GTG ACT CTG ATG GCC AG-3′ f

Analysis of ASIC1a protein expression

Immunoblotting was performed using a mini-gel apparatus and binding was revealed by electrochemiluminescence using the Pierce ECL kits (Pierce, Brebieres, France). Lumbosacral L1 + S1 DRG from butyrate- or saline-treated rats injected with anti-NGF or a control isotype antibody (see Effect of subchronic administration of anti-NGF antibodies: Experiment 2) or from saline-treated rats injected with NGF or its vehicle (see Effect of subchronic administration of NGF) were rapidly removed and dissected on ice and homogenized in 300 μL of ice-cold lysis buffer containing 20 μM leupeptin and 100 IU mL−1 aprotinin (Sigma-Aldrich). The protein concentration of tissue lysates was determined with the BCA protein assay kit from Interchim (Montluçon, France). Proteins were separated using 4–10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (BioTrace, VWR, Fontenay-sous-Bois, France). Membranes were blocked for 1 h at room temperature (RT) in Tris Phosphate Buffer containing 0.1% Tween (TBS-T) and 5% of non-fat dry milk, and incubated overnight at 4 °C with the appropriate antibodies. A rabbit polyclonal anti-ASIC1a antibody (Alpha Diagnostics International, San Antonio, TX, USA) and a mouse monoclonal anti-β-actin antibody (Lab Vision, Brebieres, France) were used at 1 : 1000 dilutions. Blots were washed three times in TBS-T and incubated for 1 h at RT with horseradish peroxidase-conjugated secondary antibodies (Pierce). The signal was developed in ECL solution (SuperSignal West Pico or Femto chemiluminescent substrate) for 5 min, and exposed on Kodak BioMax hyperfilms (Amersham Biosciences, Buc, France) for 10 s–10 min. The intensity of the immunoreactive bands was quantified using Image J software (NIH) and normalized to β-actin levels and a loading calibrator. Proteins levels are expressed as a percentage of β-actin levels following standardization to the calibrator. All experiments were performed in duplicate.

Immunohistochemical detection of NGF and ASIC1 proteins

At all stages of immunohistochemical experiments, samples from both butyrate- and saline-treated animals were processed concomitantly to ensure similar conditions of dissection, preparation and analysis. All slices processed for immunohistochemistry carried the two conditions in a randomized fashion (butyrate on top and saline on bottom, or vice-versa), to ensure that the samples were processed similarly. NGF and ASIC1 specific detection by the anti-NGF and the anti-ASIC1 antibody respectively was supported by the observation of a sole and specific Western Blot band and the absence of immunostaining in negative controls (omitting primary or secondary antibodies).

Lumbosacral L1 + S1 DRG from butyrate- or saline-treated rats injected with anti-NGF or a control isotype antibody (see Effect of subchronic administration of anti-NGF antibodies: Experiment 3) were dissected, fixed with 4% paraformaldehyde (PFA) in Phosphate Buffer Saline (PBS), pH 7.4, and embedded in paraffin. Five micrometer-thick sections were mounted, deparaffinized and processed for 20 min in a 10 mM citrate bath at 100 °C. After washing with TBS-T, endogenous peroxidase activity was inhibited with 3% H2O2 for 10 min. Sections were then incubated with anti-ASIC1a (1 : 100, Alpha Diagnostic International), anti-ASIC1b (1 : 2000, AbCys, Paris, France) or antibodies overnight at 4 °C. Then, sections were washed in TBS-T and processed with the Dako streptavidin-biotin-peroxidase kit (Dako, Les Ulis, France). Binding was revealed using 3,3′-diaminobenzidine (DAB). Immunohistochemistry of NGF used a different set of rats: butyrate- or saline-treated rats (n = 5) were subjected to the same immunohistochemical procedure using an anti-NGF antibody (1 : 600, AbCys). Slides were viewed and imaged with an Eclipse E800 microscope equipped with a digital camera (Nikon, Champigny Sur Marne, France). For all analyses, the staining intensity level was corrected across images by applying the ‘white blank’ tool of the acquisition software (Nikon Lucia G), to adjust the staining intensity to the background. Assessment of NGF expression was semi-quantitative. Nerve growth factor expression was semi-quantified by measuring the fractional area immunostained. For ASIC1a/b quantification, total and ASIC1a/b-immunoreactive (IR) cells were counted. Consecutive DRG slices were never used for neuronal counts in order to avoid double counting. Nociceptive neurons in the DRG have small soma sizes (<500 μm2) while other sensory and motor neurons have medium (500–1000 μm2) to large soma (>1000 μm2).[26, 60, 61] For this reason, cross-sectional areas of ASIC1a/b-IR cells were measured with the Lucia G image analysis software (Nikon), in order to determine the type of fibers (nociceptive or not) expressing ASIC1a/b.

ASIC1a immunohistochemical detection in retrogradely labeled DRG colonic neurons

Retrograde labeling of DRG colonic neurons

ASIC1a immunohistochemical detection in retrograde-labeled DRG colonic neurons used a new set of rats. Butyrate- or saline-treated rats (n = 5) were anesthetized with a ketamine (100 mg kg−1; Imalgène®1000, Merial, Lyon, France)/xylazine (10 mg kg−1; Rompun 2%, Bayer-Pharma, Loos, France) mixture in saline. A 1 cm abdominal incision was made to access the distal colon, and 4 injections of 5 μL of a 3%-Fluorogold™ (Sigma-Aldrich) solution were performed using a 25 μL Hamilton syringe. The abdominal incision was sutured, antibiotic (Aureomycine Monot 3% pomade, Merck, Semoy, France) was applied, and the animals allowed recovering.

ASIC1a immunohistochemical detection

Seven days following colonic Fluorogold™ injections, immunohistochemistry was performed on lumbosacral L1 + S1 DRG. DRG were fixed 2 h in a 4% PFA in PBS solution (pH 7.4) at 4 °C and embedded in paraffin. Five micrometer-thick sections were deparaffinized and incubated in a 5% donkey serum blocking solution in TBS-T for 1 h at RT. Sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-ASIC1a antibody (1 : 500, Alpha Diagnostic International) that was revealed by a Cy3-conjugated donkey anti-rabbit secondary antibody (1 : 500, Jackson Laboratories, Suffolk, UK) for 2 h at RT. Images were acquired using a Zeiss microscope equipped for epifluorescence. ASIC1a-IR, determined in arbitrary units of gray levels per area unit, was quantified in Fluorogold™-positive DRG neurons only (n = 125 per group, saline vs butyrate) using Lucia G image analysis software (Nikon). Each Fluorogold™-positive DRG neuron was manually outlined in order to determine the diameter and area of ASIC1a-IR fibers. ASIC1a expression was represented as the ratio between ASIC1a-IR and DRG neuron areas.

Confocal imaging of NGF-ASIC1a-CGRP and trkA-ASIC1a-CGRP triple immunolabeling in DRG sensory neurons

Confocal imaging used a new set of rats. Butyrate- or saline-treated rats (n = 2) were deeply anesthetized with sodium pentobarbital (60 mg kg−1, CEVA® Santé Animale, Libourne, France) and perfused transcardiacally with a 4 °C solution of 4%-PFA in PBS (pH 7.4). Lumbosacral L1 + S1 DRGs were embedded in TissuTek (Sakura Fineteck, Alphen aan den Rijn, the Netherlands) and snap frozen in liquid nitrogen. 10 μm-thick cryostat sections were rinsed in PBS for 3 × 10mn, incubated in a 5% donkey serum in PBS-T blocking solution for 1 h at RT, and overnight at 4 °C in PBS-T containing a rabbit polyclonal anti-ASIC1a antibody (1 : 50, Alpha Diagnostic International), a mouse monoclonal anti-CGRP antibody (1 : 50, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and a goat polyclonal anti-NGF antibody (1 : 60, Sigma-Aldrich) or a goat polyclonal anti-trkA antibody (1 : 50, Santa Cruz Biotechnologies). Primary antibodies were revealed by incubating sections in PBS-T containing Cy3-conjugated donkey anti-rabbit, FITC-conjugated donkey anti-goat or Cy5-conjugated donkey anti-mouse secondary antibodies (all diluted 1 : 400, Jackson Laboratories) 2 h at RT. Images acquisition was performed using a Zeiss LSM 510 confocal microscope (40× objective, numerical aperture 1.3). Background staining provided by secondary antibodies was determined by omitting primary antibodies and was subtracted from complete primary + secondary immunohistochemical detection.

Statistical analysis

Results are expressed as mean ± SEM. Colorectal distension pain thresholds values were analyzed using a two-way anova followed by a Bonferroni post-hoc test to compare treatment groups. Molecular biological and immunohistochemical data were analyzed using a one-way anova followed by a Student-Neuman-Keuls post-hoc test. Differences were considered significant for P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

NGF contribution to butyrate-induced colonic hypersensitivity

To assess the peripheral contribution of NGF to IBS-like CHS, we administered anti-NGF antibodies, which do not cross the brain-blood barrier,[62] or non-specific isotypes (IgG) to butyrate- (CHS model) or saline-treated rats (control). In animals receiving IgG, CRD pain thresholds of butyrate-treated rats were significantly lower than those treated with saline (42 ± 4 mmHg vs 57 ± 4 mmHg, respectively; P < 0.05; Fig. 1A), reflecting the expected butyrate-induced CHS.[39, 41-43] In contrast, in animals receiving anti-NGF antibodies, butyrate had no effect on CRD pain threshold (55 ± 6 mmHg vs 56 ± 5 mmHg for butyrate- vs saline-treated animals, respectively; P > 0.05; Fig. 1A), suggesting that peripheral NGF contributes to the development of butyrate-induced CHS.

image

Figure 1. Nerve growth factor (NGF) involvement in butyrate-induced colonic hypersensitivity. (A) Butyrate enemas decreased colorectal distension pain thresholds compared to saline enemas, reflecting butyrate-induced colonic hypersensitivity (CHS). Butyrate-induced CHS was reversed by concomitant administration of anti-NGF antibodies, but not of non-specific IgG isotypes. n = 8 per group; *P < 0.05 saline vs butyrate. (B) Increased NGF-immunoreactive fractional area in L1 + S1 DRG neurons of butyrate-treated rats. White stars indicate neurons with low NGF-immunoreactivity compared to background; black stars indicate neurons with high NGF-immunoreactivity compared to background. Scale = 100 μm; n = 4 per group; *P < 0.05.

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We then investigated whether this contribution could arise from an increased expression of NGF at peripheral level. Accordingly, we semi-quantified NGF expression in L1 + S1 DRG neurons, known to innervate the distal colon[52-55] and to be activated by CRD,[42, 63-65] of butyrate and saline-treated rats. We observed a significant increase in NGF expression levels in lumbosacral DRG neurons of butyrate-treated rats when compared with saline-treated controls (27 ± 7 vs 54 ± 4, respectively; P < 0.05, Fig. 1B). Therefore, these data suggest that butyrate-mediated CHS could be partially mediated by a peripheral NGF-dependent mechanism occurring in lumbosacral L1 + S1 DRG neurons.

ASIC1a contribution to butyrate-induced colonic hypersensitivity

Acid-Sensing Ion Channels contribute to nociceptive processes by sensing protons[66, 67] and have been shown to participate at peripheral level to CHS of inflammatory origin.[68, 69] To assess whether peripheral ASICs could participate to CHS in our model, we first investigated the effect of amiloride, a non-selective ASICs antagonist, on CRD pain thresholds in butyrate-treated rats. The mean CRD pain threshold of butyrate-treated animals receiving an intravenous injection of amiloride at the concentration of 6 mM was significantly increased and higher than that of butyrate-treated animals receiving the vehicle solution (65 ± 6 mmHg vs 42 ± 2 mmHg, respectively; P < 0.05; Fig. 2A). The transient receptor vanilloid1 (TRPV1) is another channel involved in acid sensing by sensory neurons[70-73] and TRPV1 has been shown to be modulated by NGF[74, 75] and to participate in visceral sensitization.[5, 31, 55, 76, 77] To rule out the possibility that TRPV1 might contribute to CHS development in our model, CRD was performed in butyrate-treated animals injected with the TRPV1 antagonist capsazepine. No effect was observed on CRD pain thresholds (Fig. S1). These data suggest that ASICs expressed at the periphery could play a role in the development of butyrate-induced CHS.

image

Figure 2. ASICs involvement in butyrate-induced colonic hypersensitivity. (A) Dose-effect of amiloride (1, 3 and 6 mM, i.v.) on colonic hypersensitivity (CHS) in butyrate-treated rats. Butyrate-treated rats injected with vehicle had decreased colorectal distension pain thresholds compared to saline-treated rats (dashed line). Butyrate-induced CHS was reversed with the dose of 6 mM amiloride n = 8 per group; *P < 0.05. (B) Increased ASIC1a and ASIC1b mRNA levels in lumbosacral DRG neurons of butyrate-treated rats compared to saline-treated rats. No change was found when assessing ASIC2a, ASIC2b and ASIC3 mRNA levels. The intensity of ASIC PCR products was normalized to that of the ribosomal mRNA L32. All experiments were performed in duplicate. n = 5 per group **P < 0.01, ***P < 0.001.

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To investigate which ASIC subtypes could be involved in this mechanism, we used semi-quantitative RT-PCR in L1 + S1 DRG to detect potential changes in ASIC subunits mRNA levels induced by butyrate treatment. Our results showed increased ASIC1a (0.99 ± 0.14 vs 1.73 ± 0.35, respectively; 74.7% increase, P < 0.001) and ASIC1b (1.22 ± 0.16 vs 1.81 ± 0.19, respectively; 48.4% increase, P < 0.01) mRNA levels in L1 + S1 DRG of butyrate-treated rats when compared to saline-treated rats (Fig. 2B). In contrast, no change was detected for ASIC2a (0.99 ± 0.05 vs 1 ± 0.06), ASIC2b (0.97 ± 0.11 vs 0.81 ± 0.07) or ASIC3 (1 ± 0.08 vs 0.91 ± 0.03) subunits (Fig. 2B). We thus focused our attention on ASIC1a and ASIC1b subunits in the following experiments.

To confirm ASIC1a and ASIC1b increased levels in butyrate-treated rats, we quantified their protein expression using immunohistochemistry in L1 + S1 lumbosacral DRG neurons. Compared to rats receiving saline, the proportion of ASIC1a-IR neurons was significantly higher in L1 + S1 DRG neurons of butyrate-treated rats (0.42 ± 0.03 vs 0.53 ± 0.03, respectively; 26% increase, P < 0.05, Fig. 3A), but, importantly, not in thoracic DRG neurons (Fig. S2), which do not innervate the lower part of the gastrointestinal tract[52, 53] and were used as a negative control. In contrast, the proportion of ASIC1b-IR neurons did not change significantly in L1 + S1 (0.38 ± 0.02 vs 0.49 ± 0.04, P = 0.06, Fig. 3B). Interestingly, we found that the increase in ASIC1a-IR neuron number was specific of small-diameter neurons (cross-sectional area up to 500 μm2) whose proportion increased (0.27 ± 0.03 vs 0.41 ± 0.03 saline- vs butyrate-treated; 52% increase, P < 0.05, Fig. 3A), as shown by cross-sectional measures of ASIC1a-IR cell somas.

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Figure 3. Increased density of ASIC1a expressing neurons in L1 + S1 DRG of butyrate-treated rats. Quantification of ASIC1a- (A) and ASIC1b- (B) immunoreactive neurons in L1 + S1 DRG. The percentage of ASIC1a-IR neurons was significantly increased in butyrate-treated rats when compared to saline-treated rats. This increase was specific of small-diameter neurons (cross-sectional area <500 μm2). In contrast, no statistical difference in the percentage of ASIC1b-IR neurons could be evidenced between butyrate- and saline-treated rats. At least 250 cells were measured for each group. Scale = 100 μm; n = 4 per group; *P < 0.05.

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To finally confirm the specificity of ASIC1a involvement in butyrate-induced CHS, ASIC1a protein expression was quantified in retrogradely labeled colon neurons of L1 + S1 DRG. A significant increase in ASIC1a immunofluorescence level was observed in L1 + S1 sensory neurons of butyrate-treated rats when compared to saline controls (2.78 ± 0.13 vs 3.72 ± 0.15 ASIC1a-IR/m2, respectively; 34% increase, P < 0.01, Fig. 4D). Moreover, this increase was specific of small- (3.95 ± 0.20 vs 5.22 ± 0.22 ASIC1a-IR/m2, saline- vs butyrate-treated; 32% increase, P < 0.01) and medium- (1.99 ± 0.08 vs 2.32 ± 0.06 ASIC1a-IR/m2, saline- vs butyrate-treated; 17% increase, P < 0.01) diameter neurons (Fig. 4D).

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Figure 4. Increased ASIC1a protein expression in retrogradely labeled colon neurons of butyrate-treated rats. Quantification of ASIC1a protein immunofluorescence was performed in colonic L1 + S1 neurons retrogradely labeled by fluorogold. (A–C) Examples of colonic S1 DRG neurons stained for ASIC1a in butyrate- and saline-treated rats. White boxes in column (A) are enlarged in columns (B and C). (D) In L1 + S1 DRG, the mean ASIC1a immunofluorescence density of fluorogold-labeled neurons was significantly increased in butyrate-treated rats when compared to saline-treated rats. This increase was observed in small- and medium-diameter neurons (cross-sectional area <1000 μm2). At least 250 cells were measured for each group. Scale = 25 μm; n = 5 per group; **P < 0.01.

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Altogether, these results show that ASIC1a expression was specifically increased in colonic L1 + S1 DRG neurons following butyrate treatment, suggesting their contribution to butyrate-mediated CHS.

ASIC1a overexpression in lumbosacral DRG neurons is NGF-dependent

Our results show that butyrate treatment is associated with an increase in NGF and ASIC1a expression levels in L1 + S1 DRG neurons. To investigate whether NGF mediates ASIC1a overexpression in sensory DRG neurons, we first assessed ASIC1a co-expression with NGF or its specific receptor trkA in small-diameter L1 + S1 lumbosacral DRG neurons expressing Calcitonin Gene Related Peptide (CGRP), a marker of nociceptive neurons. Using ASIC1a-NGF-CGRP and ASIC1a-trkA-CGRP triple immunolabeling and confocal microscopy, we observed a strong overlap of ASIC1a staining with NGF or trkA staining in CGRP-positive DRG neurons of both saline- and butyrate-treated rats (Fig. 5).

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Figure 5. Colocalization of ASIC1a and nerve growth factor (NGF) or its receptor trkA in CGRP-positive L1 + S1 DRG neurons of butyrate-treated rats. Examples of L1 + S1 DRG neurons stained with a triple labeling for ASIC1a + NGF + CGRP or ASIC1a + trkA + CGRP in butyrate- (A and C) and saline-treated rats (B and D). ASIC1a (red) was frequently co-expressed with NGF or trkA (green) in sensory DRG neurons expressing CGRP (white). Insets in the top row indicate neurons imaged at higher magnification below. Arrows indicate triple-labeled neurons. Scale bar = 50 μm (top row) and 25 μm (subsequent rows).

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Knowing that NGF can modulate the expression of several ion channels involved in pain mechanisms,[16] we then analyzed ASIC1a protein expression in L1 + S1 DRG neurons of saline-treated rats following subchronic treatment with NGF. We found that NGF administration resulted in an increase of ASIC1a protein (1.06 ± 0.31 vs 4.85 ± 0.40, vehicle- vs NGF-injected; 358% increase, P < 0.001, Fig. 6).

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Figure 6. Nerve growth factor (NGF) treatment induced ASIC1a protein overexpression in L1 + S1 DRG neurons of butyrate-treated rats. Western blot analyzes showed that subchronic injections of NGF induced ASIC1a protein overexpression in saline-treated rats compared to rats injected with vehicle. All experiments were performed in duplicate. n = 6 per group; *P < 0.001 NGF vs vehicle.

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We also analyzed ASIC1a protein expression in L1 + S1 DRG neurons of butyrate-treated rats following subchronic administration of anti-NGF antibodies or IgG. While an expected increase (1.95 ± 0.14 vs 2.63 ± 0.20, saline- vs butyrate-treated; 32% increase, P < 0.05, Fig. 7A) in ASIC1a protein levels was observed in butyrate-treated animals receiving IgG, anti-NGF treatment prevented this up-regulation (2.63 ± 0.20 vs 1.43 ± 0.17, butyrate-treated rats, anti-NGF vs IgG, P < 0.05, Fig. 7A). We further showed no difference in the proportion of ASIC1a-IR neurons (all size: 44.87 ± 1.31 vs 45.08 ± 1.59, saline- vs butyrate-treated, Fig. 7B) in L1 + S1 DRG of butyrate- and saline-treated rats receiving anti-NGF antibodies, regardless of DRG cell bodies diameter.

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Figure 7. Anti-nerve growth factor (NGF) antibody treatment prevented ASIC1a protein overexpression in L1 + S1 DRG neurons of butyrate-treated rats. (A) Western blot analyzes showed that injections of non-specific IgG isotypes had no impact on increased ASIC1a expression in butyrate-treated rats. In contrast, ASIC1a protein expression level in butyrate-treated rats receiving anti-NGF injections was normalized to the level observed in saline-treated rats injected with IgG isotypes. (B) Quantification of ASIC1a immunoreactive neurons in L1 + S1 DRG showed no difference between butyrate- and saline-treated rats after anti-NGF antibodies injections. All experiments were performed in duplicate. n = 5 per group; *P < 0.05 saline vs butyrate, #P < 0.05 non-specific IgG isotypes vs anti-NGF antibodies.

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In conclusion, our data support the hypothesis that an NGF-dependent ASIC1a overexpression in small L1 + S1 sensory colonic DRG neurons underlies the development of butyrate-induced CHS.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

Irritable bowel syndrome is a visceral pain syndrome of multifactorial origin, which involves both central and peripheral mechanisms.[78] Its pathophysiology is notably characterized by abdominal pain or discomfort in the absence of tissue injury. While colonic hypersensitivity is often proposed as a biological marker for IBS,[79] with patients displaying lower sensory thresholds to colorectal balloon distension, studies have failed so far to find clear neuroanatomical or neuromolecular basis of these sensory alterations. At the periphery, CHS could arise from primary afferent fibers sensitization.[3, 80, 81] It has been suggested that peripheral sensitization of colonic afferents might involve NGF following neuro-immune interactions between enteric neurons and mast cells.[82] Indeed, during colonic inflammation, NGF stimulates neuropeptides expression and immune cells activity to mediate neurogenic inflammation.[17, 83, 84] While no evident macroscopic signs of inflammation could be associated with IBS, recent findings suggest that low-grade inflammation could contribute to its pathophysiology,[35-37] in which NGF could play a significant role.[5, 38]

To test the hypothesis of NGF involvement in IBS pathophysiology, we used a clinically relevant rat model of IBS, which combines CHS induced by colonic butyrate enemas and absence of macroscopic or histological modifications of colonic mucosa.[39] We showed in this model that peripheral neutralization of NGF prevented the development of CHS through modulation of ASIC1a expression in nociceptive peptidergic neurons innervating the colon. Specifically, our data showed that NGF expression is increased in lumbosacral L1 + S1 DRG of rats with butyrate-induced non-inflammatory CHS. Peripheral neutralization of NGF with anti-NGF antibodies administered systemically, which fail to cross the brain-blood barrier,[62] blocked this CHS, suggesting that CHS induced in our model depends on NGF-dependent peripheral mechanisms. Nerve growth factor was previously involved in peripheral sensitization under inflammatory conditions, including the development of inflammatory CHS induced by tri-nitrobenzene sulfonic acid (TNBS).[11, 12] However, our study is the first to report NGF involvement in a non-inflammatory model.

Mechanisms of primary afferents sensitization mediated by NGF are notably associated to an increased membrane expression of several ion channels expressed on nociceptors, such as ASIC3[18, 19] and TRPV1,[75] which are known to contribute to inflammatory colonic pain.[32] Accordingly, we hypothesized that these channels could be involved downstream of NGF and mediate CHS in our model. We found that blocking ASICs with amiloride improved butyrate-mediated CHS. However, administration of capsazepine, a specific TRPV1 antagonist, had no effect on CRD thresholds of butyrate-treated rats. This result suggests that TRPV1 involvement may not be relevant in non-inflammatory CHS. Due to amiloride poor specificity, molecular investigation of butyrate-mediated changes in ASICs expression was then performed and showed an overexpression of ASIC1a mRNA in L1 + S1 DRG. On the other hand, ASIC1b, ASIC2a/b and ASIC3 mRNA expression levels were not altered in our model. In contrast to the contribution of these ASIC subunits to inflammatory visceral pain,[32, 34, 69, 85] these results suggest a specific role of ASIC1a in non-inflammatory conditions. However, it is noteworthy that functional ASICs assemble in homo- and hetero-dimers,[72, 86] ASIC1/3 and ASIC2b/3 hetero-dimers being preponderant in sensory neurons.[87, 88] As such, ASIC2b and ASIC3 subunits could have a modulatory role by modifying the electrical or gating properties of ASIC1a.[89] We rather support a specific role of ASIC1a homomeric channels in CHS development as we previously showed their central contribution to butyrate-mediated CHS.[42] Nonetheless, further experiments are needed to rule out the possibility of an involvement of functional ASIC1a heterodimers in butyrate-induced CHS. Subsequent assessment of ASIC1a protein expression confirmed ASIC1a overexpression in L1 + S1 retrogradely labeled colonic DRG neurons of butyrate-treated rats when compared with saline-treated controls. Overall, our results indicate a critical role of ASIC1a in butyrate-induced CHS. ASIC1a was previously shown to mediate acidosis-related hyperalgesia in muscle[90-92] and osteo-articular inflammation.[93-95] Here, we demonstrate for the first time a peripheral involvement of ASIC1a in visceral pain. Previous studies focused on ASICs contribution to visceral pain showed ASIC2 or ASIC3 involvement in inflammatory CHS but failed to demonstrate a role for ASIC1a,[32, 69] further suggesting that the role of ASIC1a in visceral pain is restricted to non-inflammatory conditions.

ASIC1a overexpression was mostly specific of small-diameter DRG cell bodies innervating the lower part of the digestive tract and expressing CGRP, likely to represent colon nociceptive neurons.[52, 53] Nerve growth factor, which drives phenotypic changes in nociceptors by enhancing release of pro-inflammatory mediators,[16, 17, 96] contributes to inflammatory CHS development by enhancing CGRP release through peptidergic C-fibers.[9, 97] In addition, Chan et al. ([5]) have demonstrated that rectal hypersensitivity reported by IBS patients involves NGF-dependent mechanisms that contribute to an increased CGRP expression in human submucosal neurons.[5] In our model, neonatal capsaicin or CGRP antagonist injections have been shown to prevent butyrate-induced CHS development in adult rats[39] and we observed that NGF was consistently present in DRG neurons expressing CGRP. These observations suggest that increased NGF levels in nociceptive peptidergic C-fibers leads to butyrate-induced CHS, presumably by a process involving upregulation of ASIC1a expression. Accordingly, we demonstrated that subchronic NGF treatment can induce ASIC1a protein overexpression in L1 + S1 DRG neurons and that ASIC1a protein overexpression in L1 + S1 DRG neurons of butyrate-treated animals was prevented by anti-NGF antibodies administration. These results support the view of an NGF-dependent ASIC1a modulation in butyrate-induced CHS, which may involve the activation of the NGF high-affinity receptor trkA and associated downstream signaling pathways, similarly to what has been shown in vitro for TRPV1 and the NGF-trkA-PI3K-MAPK system.[98] Mamet et al. ([19]) have found that NGF can activate the NGF-p75-JNK-p38-MAPK pathway to enhance ASIC3 gene transcription in inflammatory conditions.[19] Nerve growth factor upregulates ASIC3 expression and increases its activity, leading to an enhanced excitability of small-diameter sensory neurons.[18, 19] As confocal microscopy indicates a strong co-localization of NGF, trkA and ASIC1a in peptidergic CGRP-positive C-fibers, we hypothesize that a similar mechanism involving ASIC1a underlies butyrate-induced CHS development. Additional experiments are required to decipher the downstream signaling pathways in our model and to determine whether NGF acts via trkA to enhance the sensitivity of colonic afferent fibers by up-regulating the expression/activity of ASIC1a.

In summary, our data provide new insights into the pathophysiological mechanisms of IBS-like CHS in our model, indicating that NGF can sensitize colonic nociceptive afferents through specific ASIC1a activation in non-or low-grade inflammatory conditions. These findings identify the interplay between NGF and ASIC1a as a potential new drug targeted mechanism for IBS treatment.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

JM received financial support from the Ministère de l'Education Nationale, de la Recherche et des Technologies. We thank Dr. David Barrière for his careful and critical reading of the manuscript. We thank Dr. Xavier Pichon and Dr. Damien Brosson, who helped for immunoblotting experiments. We also thank the anonymous reviewers for their invaluable comments. English language editing was performed by American Journal Expert (USA).

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

This study was supported by an ATC (Action Thematique Concertee, Nutrition 2002, No. ASE2128CSA) and an ANR (Agence Nationale pour la Recherche) grant for scientific research (ANR-09-MNPS-037-01 VISCERALGY Canaux ioniques: cibles du traitement de la douleur viscérale).

Author Contribution

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

JM performed immunoblotting and immunohistochemical experiments, associated analyses and wrote the manuscript; EM and MM performed retrograde labeling of colonic neurons; EM and ME contributed to immunohistochemical experiments and analyses; JM, AG and SB performed CRD experiments; JB and SB performed RT-PCR experiments and analyses; AA contributed to immunoblotting experiments and analyses; JM and EM performed confocal imaging and analyses; DA and AG designed the study and supervised the project; All authors contributed to data discussion and reviewed the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Conflicts of Interest
  10. Author Contribution
  11. References
  12. Supporting Information
FilenameFormatSizeDescription
nmo12199-sup-0001-FigureS1.jpgimage/jpg1194KFigure S1. Effect of capsazepine in the CRD test.
nmo12199-sup-0002-FigureS2.jpgimage/jpg1266KFigure S2. ASIC1a expression in non-colonic sensory neurons.

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