Nerve growth factor mediates alterations of colonic sensitivity and mucosal barrier induced by neonatal stress in rats

GASTROENTEROLOGY 2004;127:524 –534

Nerve Growth Factor Mediates Alterations of Colonic Sensitivity and Mucosal Barrier Induced by Neonatal Stress in Rats FREDERICK BARREAU, CHRISTEL CARTIER, LAURENT FERRIER, JEAN FIORAMONTI, and LIONEL BUENO Neuro-Gastroenterology and Nutrition Unit, Institut National de la Recherche Agronomique, Toulouse, France

Background & Aims: Maternal deprivation (MD) increases nerve growth factor (NGF) expression and colonic mast cell density and alters visceral sensitivity. This study aimed to establish whether NGF overexpression induced by neonatal stress is involved in altered visceral sensitivity and gut mucosal integrity in adult rats. Methods: Male Wistar rat pups were either submitted to MD and treated with anti-NGF antibodies or left with their dam and treated daily with NGF. All rats were tested 10 weeks later for visceral sensitivity and 12 weeks later for gut permeability, myeloperoxidase activity, and mast cell numbers. Colonic NGF and NGF receptor expression were determined at 14 days and 12 weeks of age. To determine the involvement of colonic NGF overexpression and mast cell hyperplasia in visceral hyperalgesia induced by MD, neonatally deprived adult rats received anti-NGF antibodies or doxantrazole. Results: MD increased visceral sensitivity to rectal distention, gut permeability, colonic myeloperoxidase activity, and mast cell density, and anti-NGF antibodies abolished these effects. Neonatal daily treatment with NGF mimicked the alterations induced by MD on both rectal sensitivity and mucosal barrier. In deprived compared with nondeprived rats, colonic NGF immunostaining and NGF messenger RNA were increased at 14 days and 12 weeks. Overexpression of NGF receptor messenger RNA, present at 14 days, was not observed later. Moreover, adult deprived rats treated with doxantrazole or antiNGF antibodies exhibited normal gut permeability and visceral sensitivity to rectal distention. Conclusions: These data indicate that NGF triggers and maintains long-term alterations of visceral sensitivity and gut mucosal integrity induced by MD.

dverse events during childhood are considered potent stressors often associated in humans with gastrointestinal diseases such as Crohn’s disease1 or irritable bowel syndrome.2,3 The use of animal models of maternal deprivation (MD) has indicated the importance of neonatal stress in favoring the occurrence of gastrointestinal diseases in adults. For example, early maternal separation has been found to predispose to gastric erosions4 and colonic barrier dysfunction in response to a mild stress.5

A

As shown with Ussing chambers, neonatal stress increases colonic ion transport but does not modify colonic permeability at least in basal conditions and for the marker horseradish peroxidase.5 It has also been shown in rats that neonatal MD triggers long-term hypersensitivity to rectal distention (RD),6,7 which corresponds to the main pathophysiologic characteristic of irritable bowel syndrome in humans.8 Neonatal stress is known to increase nerve growth factor (NGF) expression in rat brain.9 –11 NGF is a member of the neurotrophin family involved in the development of numerous nerve functions such as proliferation,12 survival, and functionality of sensory nerves.13 NGF also participates in the development of hyperalgesia during inflammation.14,15 NGF is produced by sympathetic nerves,16 smooth muscle,16 and a variety of cell types such as mast cells,17 lymphocytes,18 and basophils18 and is involved physiologically in the development of the enteric nervous system.16 This neurotrophin exerts its effects through high-affinity tyrosine kinase receptor A (TrkA) receptors and low-affinity p75 neurotrophin receptor (P75NTR) receptors,19 both expressed by a variety of cell types including immune cells, mast cells, and basophils.18 NGF levels are substantially increased in inflamed tissue,20 and both NGF and TrkA receptor expression are enhanced in Crohn’s disease and ulcerative colitis.21 Systemic administration of NGF to rats or mice results in thermal and mechanical somatic hyperalgesia.14,15,22 Lamb et al.23 reported that administration of NGF induces gastric hypersensitivity to distention in rats. Moreover, neonatal daily administration of NGF induces thermal hyperalgesia in neonates and later in adult rats.24 Several mechanisms have been proposed to explain such long-lasting heat nociception in adult animals, such as long-lasting sensitization of A␦ fibers, Abbreviations used in this paper: MD, maternal deprivation; MPO, myeloperoxidase; NGF, nerve growth factor; P75NTR, p75 neurotrophin receptor; RD, rectal distention; TrkA, tyrosine kinase receptor A. © 2004 by the American Gastroenterological Association 0016-5085/04/$30.00 doi:10.1053/S0016-5085(04)00865-0

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and/or up-regulation of the central connectivity of nociceptive afferences, or activation of skin mast cells. In agreement with this last hypothesis, it has been reported that NGF is a potent degranulating agent25 favoring mast cell proliferation26 and degranulation, which are both known to trigger visceral hypersensitivity. We have previously shown that neonatal MD triggers long-term hypersensitivity to RD,7 associated with increased mast cell numbers and altered gut permeability and immune response.27 We hypothesize that the release of NGF triggered by neonatal MD participates in the long-term alterations of visceral sensitivity and colonic mucosal integrity seen in adults. Consequently, this study was aimed to (1) evaluate in adult deprived rats the effects of neonatal treatment with anti-NGF antibodies on rectal sensitivity, gut paracellular permeability, colonic myeloperoxidase (MPO) activity, and mast cell numbers; (2) assess whether administration of NGF during the neonatal period reproduced the effects of MD; (3) determine whether neonatal MD is associated with altered expression of NGF and NGF receptors (P75NTR and TrkA) in colonic tissues in the short-term (14 days) and long-term (12 weeks); and (4) investigate whether increased NGF and mast cell density in the colonic wall induced by neonatal stress are involved in rectal hypersensitivity and in alterations of paracellular permeability observed in neonatally deprived adult rats.

Materials and Methods Animals Primiparous pregnant female Wistar rats were individually housed in standard polypropylene cages containing 2.5 cm of wood chip bedding material. Rats were kept in a constant-temperature room (23°C ⫾ 1°C) and maintained on a 12-hour light/dark cycle (lights on at 7 AM). Food (UAR pellets; Epinay, France) and water were available ad libitum. Mothers and their pups, as well as the young rats after weaning on day 22, were kept in the same conditions.

MD MD was performed according to a previously validated methodology.28 After delivery (day 1), litters were culled to 10 pups. MD was performed daily for 3 consecutive hours (from 9 AM to 12 PM), during which pups were removed from the home cage and kept in temperature-controlled cages at 28°C ⫾ 1°C; the bedding in these cages was changed every day. This procedure was applied between postnatal days 2 and 14. Control pups were left with their dam. From days 15 to 22, all control and maternally deprived pups were maintained with their dam. Weaning was performed on day 22, the siblings were sex matched, and males were selected. All experimental protocols described in this study were approved by the Local Animal Care and Use Committee of the Institut National de la

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Recherche Agronomique. Experiments were performed at 10 or 12 weeks of age.

Experimental Protocol Anti-NGF (fractionated antiserum against 2.5S-NGF purified from mouse submaxillary glands; Sigma Chemical Co., St Louis, MO) was developed in rabbit and stored as a lyophilized powder at a concentration of 33 mg/mL in 0.1 mL of deionized water. It is known that 1:2000 dilution of rabbit anti-NGF can detect 0.5 ␮g of 2.5S-NGF in a Western blot but does not react with contaminants present in a mouse submaxillary gland extract (Sigma Chemical Co.). A 1:27,000 dilution neutralized all actions of 2.5S-NGF on PC-12 cells. The 0.1 mL of anti-NGF solution from Sigma Chemical Co. was diluted in 2.7 mL of deionized water, and 10 ␮L of this final solution was administered intraperitoneally (IP) to the pups. In a first series of experiments, 6 groups of 10 rats were used. Groups 1–3 were submitted to MD (deprived rats). Group 1 was treated IP (10 ␮L) with anti-NGF antibodies 10 minutes before each session of separation. Group 2 received heat-inactivated NGF antibodies (boiled at 100°C for 30 minutes) in the same conditions. Group 3 received no injection. Rats in groups 4 – 6 were not separated from their dam (nondeprived rats). Group 4 was treated daily IP with NGF (1 mg 䡠 kg⫺1 䡠 day⫺1 in 10 ␮L) (Serotec, Varilhes, France) from postnatal day 2 to day 14. Group 5 received sterile saline (150 mmol/L NaCl) in the same conditions. Group 6 received no injection. All experiments were performed in a randomized manner. A last group of nondeprived rats (group 7) was treated with antibodies against NGF. Visceral sensitivity and gut permeability were studied at 10 and 12 weeks of age, respectively. After permeability measurements, the rats were killed and pieces of colons were harvested for determination of mucosal mast cell density and MPO activity. In a second set of experiments, 4 other groups of rats were used. Groups 8 and 9 were submitted to MD, and groups 10 and 11 remained with their dam. Groups 8 and 10 were killed on day 14, and groups 9 and 11 were killed at week 12. Pieces of distal colons were harvested for localization and expression of NGF assessed by immunohistochemistry. Messenger RNA (mRNA) expression of NGF, TrkA and P75NTR receptors was assessed by reverse-transcription polymerase chain reaction. In a third set of experiments, 8 other groups of rats were used to evaluate if mast cells are involved in visceral hypersensitivity and elevated gut paracellular permeability triggered by MD. Groups 12–15 were submitted to MD, and groups 16 –19 remained with their dam. Groups 12, 13, 16, and 17 were treated IP with 2 injections of doxantrazole, a mast cell stabilizer, at a dose of 5 mg/kg or its vehicle (200 ␮L 5% NaHCO3) administered 6 hours and 1 hour before either rectal sensitivity (groups 12 and 16) or gut permeability measurements (groups 13 and 17), respectively. Doxantrazole is an antiallergic drug that is reported to inhibit mast cell degran-

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ulation by elevating intracellular levels of adenosine 3⬘,5⬘cyclic monophosphate.29 Groups 14 and 18 were used to establish whether NGF is involved at adult age in colorectal hyperalgesia (groups 14 and 18) and increased gut paracellular permeability (groups 15 and 19) observed in deprived rats. Animals in groups 14, 15, 18, and 19 were treated IP with 2 injections of anti-NGF antibodies (400 ␮L), using the same timing as for doxantrazole.

Visceral Sensitivity Young adult rats were tested at 10 weeks of age. One week before assessment of visceral sensitivity, animals were surgically prepared for electromyography according to a previously described technique.28 Rats were anesthetized by IP administration of 0.6 mg/kg acepromazine (Calmivet; Vetoquinol, Lure, France) and 120 mg/kg ketamine (Imalgene 1000; Rhone-Me´ rieux, Lyon, France). Three groups of Ni-Cr wire electrodes (diameter, 0.08 mm) were implanted bilaterally in the abdominal external oblique musculature. Electrodes were exteriorized on the back of the neck and protected by a glass tube attached to the skin. The electrical activity of the abdominal striated muscles was recorded with an electroencephalograph machine (Mini VIII; Alvar, Paris, France) using a short time constant (0.03 seconds) to remove low-frequency signals (⬍3 Hz) and a paper speed of 3.6 cm/min. RD was performed as previously described.30 Rats were placed into plastic tunnels (diameter, 6 cm; length, 25 cm) in which they could not move, escape, or turn around. The rats were acclimated to this procedure for 3 days before RD to minimize stress reactions during experiments. The balloon (diameter, 2 mm; length, 2 cm) used for distentions was an arterial embolectomy probe (Fogarty; Edwards Laboratories Inc., Santa Ana, CA). RD was performed by insertion of the balloon into the rectum at 1 cm from the anus. The catheter was fixed to the tail with adhesive tape. The balloon was cumulatively inflated in stepwise volumes of 0.4 mL, from 0 to 1.2 mL, with each step of inflation lasting 5 minutes. As previously determined,29 volumes of 0.4, 0.8, and 1.2 mL correspond to pressures of 12.5, 24.8, and 39.5 mm Hg, respectively. The balloon was connected to a syringe filled with water at 37°C to avoid any effect of temperature. To detect any possible leakage, the volume of water introduced into the balloon was checked by complete removal at the end of the distention period.

Gut Paracellular Permeability The assessment of total gut permeability was performed by using 51Cr-EDTA as a selective marker of paracellular permeation of tight junctions. To determine total gut permeability, 0.7 ␮Ci of 51Cr-EDTA (Perkin Elmer Life Science, Paris, France) was diluted in 500 ␮L of saline and administered intragastrically using a gastric feeding tube. Rats were placed in metabolic cages for acclimatization (3 days), and then gut permeability was determined from urine collection for 24 hours. The radioactivity present in urine was measured with a gamma counter (Cobra II; Packard, Meriden, CT). Permeability to 51Cr-EDTA was expressed in percent of total administered radioactivity.

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Colonic MPO Activity The activity of MPO, a marker of polymorphonuclear neutrophils, was assessed in colonic tissue according to Bradley et al.31 Samples of distal colon (1 cm) were suspended in a potassium phosphate buffer (50 mmol/L; pH 6) and homogenized in ice. Three cycles of freeze/thaw were undertaken. Suspensions were then centrifuged at 10,000g for 15 minutes at 4°C. Supernatants were eliminated and pellets resuspended in hexadecyltrimethylammonium bromide buffer (0.5% wt/ vol in 50 mmol/L potassium phosphate buffer; pH 6). These suspensions were sonicated on ice and centrifuged again at 10,000g for 15 minutes at 4°C. The supernatants obtained were diluted in potassium phosphate buffer (pH 6) containing 0.167 mg/mL of O-dianisidine dihydrochloride and 0.0005% of hydrogen peroxide. MPO from human neutrophils (0.1 U/100 ␮L) was used as a standard. The kinetic changes in absorbance at 450 nm, every 10 seconds over a 2-minute period, were recorded with a spectrophotometer. One unit of MPO activity was defined as the quantity of MPO degrading 1 ␮mol of hydrogen peroxide per minute at 25°C. Protein concentration was determined with a commercial kit of a modified method of Lowry (Detergent Compatible Assay; BioRad, Marnes la Coquette, France), and MPO activity was expressed as MPO units per gram of protein.

Colonic Mucosal Mast Cell Number A 2-cm-long portion of the distal colon was excised and washed in sterile saline. The collected fragments were fixed in Carnoy’s solution, embedded in paraffin blocks, and cut into 5-␮m sections. Transverse paraffin sections were stained with alcian blue/safranin. The number of mucosal mast cells per square millimeter of mucosa was evaluated with an image grabbing program and the image analysis software Optilab Pro 2.6.1 (Graftek, Voisins le Bretonneux, France).

Colonic NGF, P75NTR, and TrkA mRNA Expression Total mRNA from distal colonic tissue was isolated using Tri-reagent (Euromedex, Mundolsheim, France). One microgram of RNA sample was reverse transcripted into complementary DNA (cDNA) using 200 U of Moloney murine leukemia virus (Invitrogen, Cergy-Pontoise, France), random hexamers, and 2.5 mmol/L of each of the 4 deoxynucleotide triphosphates (Invitrogen) in a final reaction volume of 20 ␮L in the presence of 40 U/␮L of ribonuclease inhibitor (Invitrogen). Samples were incubated at 37°C for 50 minutes, followed by 15 minutes at 70°C to inactivate the enzyme. The samples were then stored at ⫺80°C until use. One microliter of the reverse-transcription reaction mixture was amplified by polymerase chain reaction using sense and antisense primers specific for the following: glyceraldehyde-3phosphate dehydrogenase, 5⬘-ATCACCATCTTCCAGGAGCG-3⬘ and 5⬘-TTCTGAGTGGCAGTGAGGGC-3⬘; NGF, 5⬘-TAGCGTAATGTCCATGTTGT-3⬘ and 5⬘-CCACACACTGACACTGTCA-3⬘; TrkA, 5⬘-GCCTTCGCCTCAACCAGCCCA-3⬘ and 5⬘-CTCTTGATGTGCTGTTAGTGT-3⬘; P75NTR, 5⬘-AGCCAACCAGACCGTGTGTG-3⬘ and 5⬘-

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TTGCAGCTGTTCCACCTCTT-3⬘. Polymerase chain reaction was performed in the presence of 1.25 U/reaction of AmpliTaq Gold DNA polymerase (Applied Biosystems, Courtaboeuf, France), 2.5 mmol/L of each of the 4 deoxynucleotide triphosphates, and 50 pmol of each sense and antisense primer in a final reaction volume of 50 ␮L. Amplification was performed by different number cycles consisting of denaturation for 1 minute at 94°C, primer annealing for 1 minute at 53°C, and primer extension for 1 minute at 72°C. Then, amplicons were stained with SYBR Gold (Molecular Probes, Leiden, The Netherlands) and separated by electrophoresis in 3% agarose gel for 1 hour at 100 V. The ratio between the amount of glyceraldehyde-3-phosphate dehydrogenase cDNA and NGF, P75NTR, and TrkA cDNA was calculated using an image analyzer (Quantity One software; Bio-Rad, Marnes la Coquette, France).

Colonic NGF Immunohistochemistry A 2-cm-long portion of the colon was excised and washed in sterile saline. The collected fragments were fixed in Duboscq-Brazil solution for 24 hours, dehydrated in ethanol solution, embedded in paraffin blocks, and cut into 5-␮m sections. After hydration of transverse paraffin sections, picric acid was removed by NH4OH solution and endogenous peroxidases were inhibited by a methanol solution containing 2% H2O2. Then, sections were blocked with phosphate-buffered saline/Tween 20 (0.01%)/bovine serum albumin (0.1%) for 10 minutes and incubated for 90 minutes at room temperature with anti-NGF antibody (Chemicon International, Hampshire, England). After washing in phosphate-buffered saline/ Tween 20 (0.01%), peroxidase-labeled anti-rabbit antibody (DAKO Envision System Dakocytomation, Trappes, France) was added for 30 minutes. Peroxidase activity was determined by addition of 3,3⬘-diaminobenzidine (DAB Kit; ICN Pharmaceuticals, Costa Mesa, CA), and colonic sections were dehydrated and mounted into DePeX (VWR, Pessac, France).

Image Analysis Microscopy and analysis were performed on a blinded basis using Nikon Microphot-SG (Champigny sur Marne, France) linked by means of a Neotech Image Grabber (Graftek, Mirmande, France) to a Power Macintosh 8100 computer loaded with Optilab Pro 2.6.1 software. NGF expression was quantified with dedicated software measuring the total number of gray levels of peroxidized 3,3⬘-diaminobenzidine per square micrometer of colonic mucosa or muscularis or per colonic mucosal mast cell. Ten areas of mucosa or muscularis and 10 mast cells were analyzed in each sample, and the mean value was calculated.

Statistical Analysis Data were analyzed by one-way analysis of variance followed by the nonparametric Tukey test where relevant. Values are expressed as mean ⫾ SEM. To determine the effect of neonatal MD on colonic NGF, TrkA, and P75NTR receptors, Student t test was used to

Figure 1. (A) Effect of neonatal treatment with active and inactivated anti-NGF antibodies on visceral sensitivity in 10-week-old rats previously submitted to neonatal MD (deprived rats) and (B) effect of neonatal NGF treatment in nondeprived rats. Values are means ⫾ SEM (n ⫽ 10). *P ⬍ 0.05, significantly different from controls in the same graph; †P ⬍ 0.05, significantly different from corresponding nondeprived controls in B.

analyze the differences. Differences were considered significant for P ⬍ 0.05.

Results Treatments During the Neonatal Period Visceral sensitivity to RD. Gradual RD increased the frequency of abdominal contractions in a distention volume– dependent manner. In nondeprived rats, a volume of 0.8 mL was the threshold at which RD significantly increased (P ⬍ 0.05) the number of abdominal contractions compared with the predistention period. In deprived rats that received no injection, this threshold was significantly reduced (P ⬍ 0.05) to 0.4 mL (Figure 1). Compared with nondeprived controls, neonatal MD significantly increased (P ⬍ 0.05) the number of abdom-

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Colonic MPO activity. In nondeprived control

Figure 2. (A) Effect of neonatal treatment with anti-NGF antibodies on gut paracellular permeability in 12-week-old rats previously submitted to neonatal MD (deprived rats) and (B) effect of neonatal NGF treatment in nondeprived rats. Values are means ⫾ SEM (n ⫽ 10). *P ⬍ 0.05, significantly different from controls in the same graph; †P ⬍ 0.05, significantly different from nondeprived controls in B.

inal contractions for all volumes of RD applied. This effect was abolished by treatment with anti-NGF antibodies and remained unchanged when the pups were treated with inactivated anti-NGF antibodies (Figure 1A). Compared with controls, which were not separated and did not receive any injection, IP administration of saline from day 2 to day 14 did not modify the abdominal response regardless of the volume of RD. In contrast, NGF (1 mg 䡠 kg⫺1 䡠 day⫺1 IP) significantly increased (P ⬍ 0.05) the number of abdominal contractions for the volumes of 0.8 mL and 1.2 mL (Figure 1B). For these 2 volumes, the number of abdominal contractions after NGF treatment was similar (P ⬎ 0.05) to that observed in deprived rats receiving no injection or inactivated anti-NGF antibodies. Moreover, nondeprived rats that received anti-NGF antibodies from postnatal day 2 to day 14 did not exhibit any change in colonic visceral sensitivity compared with nondeprived rats receiving no injection (data not shown). Gut paracellular permeability. In nondeprived control rats, 2.2% ⫾ 0.2% of orally administered 51CrEDTA was excreted in urine over 24 hours. This value was greater after neonatal MD (4.3% ⫾ 0.3%; P ⬍ 0.05) (Figure 2). The increase in gut paracellular permeability induced by neonatal MD was suppressed by anti-NGF treatment (2.3% ⫾ 0.2 %; P ⬍ 0.05), whereas inactivated anti-NGF antibodies had no effect (4.1% ⫾ 0.3 %) (Figure 2A). IP treatment with saline did not modify (P ⬎ 0.05) the permeability observed in nondeprived rats, which received no injection. In contrast, NGF treatment significantly increased this control permeability (4.1% ⫾ 0.3% vs. 2.2% ⫾ 0.2 %; P ⬍ 0.05) (Figure 2B). The permeability value after NGF treatment in nondeprived rats was similar (P ⬎ 0.05) to that observed in deprived rats in the absence of treatment (Figure 2).

rats, colonic MPO activity was 128 ⫾ 22 U/g protein. This activity was strongly enhanced by neonatal MD (426 ⫾ 69 U/g protein; P ⬍ 0.05) (Figure 3). The increase in colonic MPO activity was suppressed by neonatal anti-NGF treatment (137 ⫾ 24 U/g protein), whereas inactivated anti-NGF treatment had no effect (330 ⫾ 59 U/g protein) (Figure 3A). Treatment with saline did not affect colonic MPO activity in nondeprived rats (112 ⫾ 27 vs. 128 ⫾ 22 U/g protein; P ⬎ 0.05). In contrast, colonic MPO activity was significantly enhanced by neonatal NGF treatment (349 ⫾ 62 U/g protein) (Figure 3B) and was similar (P ⬎ 0.05) to that observed in nontreated deprived rats (Figure 3). Colonic mast cell density. In 14-day-old rats, neonatal MD triggered colonic mucosal mast cell hyperplasia compared with nondeprived control rats (30 ⫾ 3 vs. 13 ⫾ 4 cells/mm2; P ⬍ 0.05) (Figure 4A). In nondeprived control rats at 12 weeks, the number of colonic mucosal mast cells was 61 ⫾ 5 cells/mm2. Neonatal MD significantly increased this number to 101 ⫾ 13 cells/mm2 (P ⬍ 0.05) (Figure 4). This increase was prevented by neonatal anti-NGF treatment (52 ⫾ 3 cells/mm2; P ⬍ 0.05), whereas inactivated anti-NGF treatment had no effect (90 ⫾ 2 cells/mm2; P ⬎ 0.05) (Figure 4B). IP treatment with saline did not affect the number of colonic mucosal mast cells (56 ⫾ 4 vs. 61 ⫾ 5 cells/mm2; P ⬎ 0.05) (Figure 4C). This number was significantly enhanced by neonatal NGF treatment (93 ⫾ 7 cells/ mm2; P ⬎ 0.05) (Figure 4C). Colonic NGF immunohistochemistry. In 14-dayold rats, immediately after the last session of MD, NGF labeling was significantly increased (P ⬍ 0.05) in colonic mucosa and in muscular layers compared with nondeprived rats (Figure 5 and Table 1). At 12 weeks, the

Figure 3. (A) Effect of neonatal treatment with anti-NGF antibodies on colonic MPO activity in 12-week-old rats previously submitted to neonatal MD (deprived rats) and (B) effect of neonatal NGF treatment in nondeprived rats. Values are means ⫾ SEM (n ⫽ 10). *P ⬍ 0.05, significantly different from controls in the same graph; †P ⬍ 0.05, significantly different from nondeprived controls in B.

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Figure 4. Mast cell density on the last day of MD in (A) 14-day-old pups and (B) 12-week-old adult rats after anti-NGF treatment in deprived rats and (C) NGF treatment in nondeprived rats. Values are means ⫾ SEM (n ⫽ 10). *P ⬍ 0.05, significantly different from controls in the same graph; †P ⬍ 0.05, significantly different from nondeprived controls in B.

increased NGF labeling in colonic mucosa and in the muscular layer persisted in deprived rats compared with nondeprived rats (Figure 5 and Table 1). In 14-day-old rats, NGF immunoreactivity was very low on mast cells and did not permit comparison between deprived and nondeprived rats. In 12-week-old adult rats, an intense and similar NGF labeling of mast cells was observed in both deprived and nondeprived rats (P ⬎ 0.05; Table 1). Because the number of mast cells was higher in deprived rats, total NGF of mast cell origin was also higher in these animals. Colonic NGF, TrkA, and P75NTR mRNA expression. At 14 days of age, immediately after the last

session of MD, we observed a significant increase (P ⬎ 0.05) in colonic NGF, TrkA receptor, and P75NTR receptor mRNA expression (Table 2). At 12 weeks of age, a significant increase (P ⬍ 0.05) of colonic NGF mRNA expression persisted in deprived rats compared with nondeprived rats. However, no significant difference (P ⬎ 0.05) in mRNA expression of TrkA and P75NTR receptors was observed between the 2 groups. Treatments in Adults Visceral sensitivity to distention. Acute treatment of adult deprived rats with anti-NGF antibodies suppressed, for all volumes of distention, the hypersensitivity induced by neonatal MD (Figure 6A). Anti-NGF antibodies did not change rectal sensitivity in nondeprived rats (Figure 6A). Similarly, treatment with the mast cell stabilizer doxantrazole in deprived rats restored a normal rectal sensitivity for all volumes of distention when compared with nondeprived rats (Figure 6B). When injected in nondeprived rats, doxantrazole did not change rectal sensitivity (Figure 6B). Gut paracellular permeability. In nondeprived control rats, gut paracellular permeability was not mod-

ified by treatment with anti-NGF antibodies compared with its vehicle (2.1% ⫾ 0.2% vs. 2.2% ⫾ 0.2%; P ⬎ 0.05). In deprived animals, the increase in gut permeability was abolished by treatment with anti-NGF antibodies (2.6% ⫾ 0.2% vs. 4.1% ⫾ 0.3%; P ⬎ 0.05) (Figure 7A). Similarly, mast cell stabilization by doxantrazole in adult rats abolished the increase in paracellular permeability induced by MD (Figure 7B). Doxantrazole administered to adult nondeprived rats had no effect.

Discussion This study provides new evidence that NGF plays a major role in triggering (neonatal period) and maintaining long-term visceral hyperalgesia and alteration of the colonic epithelial barrier in neonatally stressed rats by MD. We also show that mast cell degranulation participates in the mucosal and sensitivity alterations induced by neonatal MD. The first part related to the role of NGF in neonatal stress is supported by our data showing that the effects of neonatal separation are suppressed by treatment of pups with anti-NGF antibodies and are mimicked by neonatal NGF treatment. In agreement with these findings, we also show that neonatal MD increases NGF and its mRNA level in peripheral tissue (colonic) at 14 days and later in life (12 weeks of age). These alterations of NGF mRNA expression are associated with an enhanced expression of TrkA and P75NTR receptor mRNA in colonic tissue in MD pups but not in adult rats. Moreover, in neonatally deprived adult rats, we also show that acute anti-NGF antibodies and doxantrazole suppressed rectal hypersensitivity and elevated gut paracellular permeability. Neonatal stress has already been shown to induce visceral hypersensitivity to RD.6,7 Moreover, neonatal treatment with NGF promotes thermal hyperalgesia in

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Table 1. Effect of Neonatal MD on Colonic NGF Immunohistochemistry in Pups (After the Last Session of MD) and in Adult Rats Pups (14 days old)

Mucosa Muscular layers Mast cells

Adults (12 weeks old)

Control

Deprived

Control

Deprived

0.20 ⫾ 0.03 0.28 ⫾ 0.03

0.30 ⫾ 0.04a 0.38 ⫾ 0.02a

0.45 ⫾ 0.04 0.48 ⫾ 0.09

0.60 ⫾ 0.02a 0.72 ⫾ 0.03a

ND

ND

1.20 ⫾ 0.12

1.10 ⫾ 0.25

NOTE. Results were obtained from at least 6 different rats from each group (control and deprived). Data (mean ⫾ SEM) are expressed as the total number of gray levels per square micrometer of colonic mucosa or muscular layers or per mucosal mast cell. ND, not determined. aP ⬍ 0.05 from control (Student t test).

Figure 5. Effects of neonatal MD on colonic NGF expression in 14day-old pups and 12-week-old adult rats. NGF immunoreactivity (brown) in colonic sections of (A1 and A2) nondeprived and (B1 and B2) deprived rats at 14 days and (C1 and C2) nondeprived and (D1 and D2) deprived rats at 12 weeks. A1 , B1 , C1 , and D1: bar ⫽ 100 ␮m; A2 , B2 , C2 , and D2: bar ⫽ 50 ␮m.

neonates that is still present in adults, suggesting that NGF may trigger long-term alterations in nociceptive signal processing.24 Several mechanisms may explain this somatic and/or visceral hypersensitivity. First, NGF has been reported to degranulate and to increase the number of mucosal mast cells,26 2 factors involved in the genesis of visceral hypersensitivity.32,33 Administration of NGF in neonates induces a hyperplasia of both mucosal and connective tissue mast cells,26 and psychological stress increases mast cell density in some thalamic nuclei.34 Here, we report that both administration of NGF in pups and neonatal stress increase the number of colonic mucosal mast cells that may be involved in the visceral hyperalgesia observed in adult rats. Indeed, in neonatally deprived adult rats, doxantrazole restores a normal abdominal response to RD. Second, several studies have reported that, among neurotrophins, NGF is mainly involved in neuronal proliferation,12 survival, and functionality of sensory

nerves.13 For example, NGF promotes neurite outgrowth in guinea pig myenteric plexus ganglia35 and enhances neurite arborization of sensory neurons.36 We can thus hypothesize that, in our model, NGF favors both arborization of sensory neurons and neurite outgrowth in myenteric plexus ganglia within the gut that may generate long-term visceral hypersensitivity. Third, NGF has been described to participate in the regulation of the expression of numerous peptides in the central nervous system. Among these peptides, NGF increases the expression of the nociceptive peptides substance P,37,38 neurokinin A,39 and calcitonin gene-related peptide,40 which are involved in the regulation of visceral sensitivity.41 For example, Kamp et al. reported that activation of spinal NK1 and NK2 receptors, presumably by their endogenous ligands (substance P and neurokinin A, respectively), maintains visceral hyperalgesia.42 Thus, we can hypothesize that, in our model, NGF induces both calcitonin gene-related peptide and substance P overexpression in dorsal root ganglia or in primary afferent neurons, which may generate long-term visceral hypersensitivity. In Crohn’s disease and ulcerative colitis, 2 disorders in which visceral hypersensitivity occurs, NGF and TrkA Table 2. Effect of Neonatal Stress on Colonic NGF, TrkA, and P75NTR mRNA Expression Pups (14 days old)

NGF TrkA P75NTR

Adults (12 weeks old)

Control

Deprived

Control

Deprived

0.40 ⫾ 0.07 0.10 ⫾ 0.02 0.25 ⫾ 0.02

0.63 ⫾ 0.08a 0.26 ⫾ 0.05a 0.43 ⫾ 0.06a

0.92 ⫾ 0.15 0.74 ⫾ 0.10 1.13 ⫾ 0.37

1.63 ⫾ 0.24a 1.05 ⫾ 0.21 1.49 ⫾ 0.80

NOTE. Results were obtained from at least 6 different samples, and data (mean ⫾ SEM) are expressed as the ratio of NGF, TrKa, and P75NTR to glyceraldehyde-3-phosphate dehydrogenase. aP ⬍ 0.05 from control (Student t test).

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neonatally deprived adult rats similar to that observed in nondeprived rats. This indicates that NGF overexpression observed in adult deprived rats is involved in visceral hypersensitivity to RD. This new result extends to a chronic low level of inflammation, or proinflammatory state, with recent data showing that NGF may participate in visceral hyperalgesia induced by acute inflammation.45,46 The effects of NGF on nociception in adult rats can be mediated by several pathways including peripheral and central mechanisms, such as long-lasting sensitization of A␦ fibers, up-regulation of the central connectivity of nociceptive afferences, or involvement of skin mast cells.14,20,24 However, NGF depletion by autoimmunization produces thermal hypoalgesia, associated with high serum titer of anti-NGF immunoglobulin G, and an absence of anti-NGF immunoglobulin G in the cerebrospinal fluid.47 This strongly supports that antibodies directed against NGF do not cross the blood-brain barrier and that the thermal hypoalgesia observed is the consequence of peripheral NGF neutralization. Accord-

Figure 6. Effects of (A) anti-NGF antibodies and (B) doxantrazole on rectal sensitivity of 12-week-old rats previously submitted to neonatal MD. Values are means ⫾ SEM (n ⫽ 8). *P ⬍ 0.05, significantly different from nondeprived rats; †P ⬍ 0.05, significantly different from deprived rats.

expression was increased.21 Moreover, IP administration of a nonpeptidic NGF antagonist (ALE-0540) has an antiallodynia effect in a model of neuropathic pain.43 Coupled with previous findings showing that NGF can provoke and exacerbate hyperalgesic states, this result further supports the hypothesis that NGF is involved in the signaling pathway associated with persistent pain states. Indeed, NGF can directly or indirectly induce a rapid sensitization of most pelvic afferences innervating the bladder, supporting the hypothesis that NGF may be an endogenous mediator in some persistent pain states.44 In agreement with this study, we observed that NGF expression is increased in deprived rats and that antiNGF antibodies restore a normal response to RD in

Figure 7. Effects of (A) anti-NGF antibodies and (B) doxantrazole on gut paracellular permeability of 12-week-old rats previously submitted to neonatal MD. Values are means ⫾ SEM (n ⫽ 8). *P ⬍ 0.05, significantly different from deprived rats treated with inactivated antiNGF antibodies or doxantrazole vehicle.

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ingly, we can also hypothesize that visceral hyperalgesia induced by neonatal MD is mainly induced by a peripheral mechanism. In agreement with this latter hypothesis, we observed an enhanced colonic mRNA and protein expression of NGF in deprived rats that can be related to visceral hyperalgesia. This enhanced NGF expression induced by neonatal MD could be explained by possible physiologic relationships between adrenocortical activity and NGF.48 Indeed, some studies have reported that corticosterone and adrenocorticotropic hormone may play an important role in the regulation of NGF expression. Glucocorticoids49 and adrenocorticotropic hormone50 enhance brain NGF expression, which modulates the activation of the hypothalamic-pituitary-adrenal axis during the stress response. Furthermore, neonatal MD has been reported to strongly enhance hypothalamicpituitary-adrenal axis activity,51 which can increase NGF expression. Enhanced hypothalamic-pituitary-adrenal axis activity may indeed explain the high expression of colonic NGF mRNA and protein in maternally deprived rats. Growing evidence recently emerged showing that a close link exists between impaired gut paracellular permeability and visceral hypersensitivity. Increased paracellular permeability favors the passage of pathogens, bacteria, and toxins, activating the submucosal immune system, which in turn sensitize terminal endings of nociceptive fibers. The existence of this link is supported by the recent observation of elevated gut paracellular permeability in patients with postdysenteric irritable bowel syndrome,52 characterized by a hypersensitivity to colonic distention.53 Moreover, experimental activation of colonic PAR-2 receptors has been found to be associated with increased permeability and subsequent rectal hyperalgesia.54 The increase in colonic paracellular permeability that we observed in deprived rats and rats neonatally treated with NGF are consistent with a mechanism that may involve colonic mucosal mast cells. Indeed, repeated stress increases the number of colonic mucosal mast cells, and no increase in gut paracellular permeability was observed after stress in mast cell– deficient Ws/Ws rats.55 In deprived rats and in rats neonatally treated with NGF, an increase of colonic mucosal mast cell numbers was observed. Similar massive mast cell hyperplasia has already been described in neonatal rats treated with NGF over the first 14 days of life, with this massive mast cell hyperplasia dependent on the ability of NGF to degranulate mast cells.26 We have also shown herein that mast cell degranulation participates to maintain a hypersensitivity to distention and an increased gut permeability in adult maternally deprived rats, in agreement with an

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increased density of mast cells. However, it remains to be determined whether NGF promotes the differentiation of precursors of colonic mast cells in situ or whether it enhances the differentiation of cells already committed in specific sources such as the thymus, spleen, and bone marrow and their subsequent mobilization toward colonic tissue. Another possible explanation for this elevated gut paracellular permeability is linked to the ability of NGF to enhance synthesis and release of cytokines by mast cells.56 Neonatal MD increases expression of both proinflammatory and anti-inflammatory cytokines,28 and NGF is known to enhance peritoneal mast cell expression of anti-inflammatory (interleukin 4 and interleukin 10) and proinflammatory (tumor necrosis factor ␣) cytokines.56,57 All of these cytokines are reported to be involved in controlling gut paracellular permeability58 by acting on membrane receptors of epithelial cells.59 We can thus hypothesize that long-term elevation of colonic NGF level may result in increased concentrations of colonic cytokines that in turn enhance gut paracellular permeability. However, because mast cells are the major structures releasing NGF and because TrkA and P75NTR receptors are present on epithelial cells, we can speculate that mast cell degranulation is responsible for the increase in permeability through NGF release. In summary, this study shows that MD (1) increases colonic NGF protein and mRNA expression level in neonate rats and later in adults and that this synthesis and release are responsible for long-term alterations in epithelial barrier and rectal sensitivity as evidenced by the similarities of the effects of MD and neonatal NGF treatment and (2) favors a high level of NGF expression and mucosal mast cell hyperplasia in adult deprived rats responsible for long-term visceral hypersensitivity to RD and elevated gut paracellular permeability. Our study confirms that stressful events early in life play a pivotal role in the regulation of the colonic mucosal barrier later in life and could have implications in the development of intestinal disorders such as irritable bowel syndrome and/or inflammatory bowel disease. Furthermore, these data highlight a potentially important role for NGF in the maintenance of visceral pain in adults.

References 1. Ringel Y, Drossman DA. Psychosocial aspects of Crohn’s disease. Surg Clin North Am 2001;81:231–252. 2. Hislop IG. Childhood deprivation: an antecedent of the irritable bowel syndrome. Med J Aust 1979;1:372–374. 3. Lowman BC, Drossman DA, Cramer EM, McKee DC. Recollection of childhood events in adults with irritable bowel syndrome. J Clin Gastroenterol 1987;9:324 –330. 4. Ackerman SH, Hofer MA, Weiner H. Predisposition to gastric erosions in the rat: behavioral and nutritional effects of early maternal separation. Gastroenterology 1978;75:649 – 654.

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5. Soderholm JD, Yates DA, Gareau MG, Yang PC, MacQueen G, Perdue MH. Neonatal maternal separation predisposes adult rats to colonic barrier dysfunction in response to mild stress. Am J Physiol 2002;283:G1257–G1263. 6. Coutinho SV, Plotsky PM, Sablad M, Miller JC, Zhou H, Bayati AI, McRoberts JA, Mayer EA. Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in rat. Am J Physiol 2002;282:G307–G316. 7. Rosztoczy A, Fioramonti J, Jarmay K, Barreau F, Wittmann T, Bueno L. Influence of sex and experimental protocol on the effect of maternal deprivation on rectal sensitivity to distension in the adult rat. Neurogastroenterol Motil 2003;15:679 – 686. 8. Whitehead WE, Holtkotter B, Enck P, Hoelzl R, Holmes KD, Anthony J, Shabsin HS, Schuster MM. Tolerance for rectosigmoid distention in irritable bowel syndrome. Gastroenterology 1990; 98:1187–1192. 9. Cirulli F, Micera A, Alleva E, Aloe L. Early maternal separation increases NGF expression in the developing rat hippocampus. Pharmacol Biochem Behav 1998;59:853– 858. 10. Cirulli F, Alleva E, Antonelli A, Aloe L. NGF expression in the developing rat brain: effects of maternal separation. Brain Res Dev 2000;123:129 –134. 11. Cirulli F. Role of environmental factors on brain development and nerve growth factor expression. Physiol Behav 2001;73:321– 330. 12. Lewin GR, Barde YA. Physiology of the neurotrophins. Annu Rev Neurosci 1996;19:289 –317. 13. Mobley WC, Rutkowski JL, Tennekoon GI, Buchanan K, Johnston MV. Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor. Science 1985;229:284 –287. 14. Lewin GR, Rueff A, Mendell LM. Peripheral and central mechanisms of NGF-induced hyperalgesia. Eur J Neurosci 1994;6:1903–1912. 15. Theodosiou M, Rush RA, Zhou XF, Hu D, Walker JS, Tracey DJ. Hyperalgesia due to nerve damage: role of nerve growth factor. Pain 1999;81:245–255. 16. Kobayashi H, Yamataka A, Fujimoto T, Lane GJ, Miyano T. Mast cells and gut nerve development: implications for Hirschsprung’s disease and intestinal neuronal dysplasia. J Pediatr Surg 1999; 34:543–548. 17. Leon A, Buriani A, Dal Toso R, Fabris M, Romanello S, Aloe L, Levi-Montalcini R. Mast cells synthesize, store, and release nerve growth factor. Proc Natl Acad Sci U S A 1994;91:3739 – 3743. 18. Otten U, Scully JL, Ehrhard PB, Gadient RA. Neurotrophins: signals between the nervous and immune systems. Prog Brain Res 1994;103:293–305. 19. Chao MV, Hempstead BL. p75 and Trk: a two-receptor system. Trends Neurosci 1995;18:321–326. 20. Woolf CJ. Phenotypic modification of primary sensory neurons: the role of nerve growth factor in the production of persistent pain. Philos Trans R Soc Lond B Biol Sci 1996;351:441– 448. 21. di Mola FF, Friess H, Zhu ZW, Koliopanos A, Bley T, Di Sebastiano P, Innocenti P, Zimmermann A, Buchler MW. Nerve growth factor and Trk high affinity receptor (TrkA) gene expression in inflammatory bowel disease. Gut 2000;46:670 – 679. 22. McMahon SB. NGF as a mediator of inflammatory pain. Philos Trans R Soc Lond B Biol Sci 1996;351:431– 440. 23. Lamb K, Kang YM, Gebhart GF, Bielefeldt K. Nerve growth factor and gastric hyperalgesia in the rat. Neurogastroenterol Motil 2003;15:355–361. 24. Lewin GR, Ritter AM, Mendell LM. Nerve growth factor-induced hyperalgesia in the neonatal and adult rat. J Neurosci 1993;13: 2136 –2148. 25. Pearce FL, Thompson HL. Some characteristics of histamine secretion from rat peritoneal mast cells stimulated with nerve growth factor. J Physiol 1986;372:379 –393. 26. Marshall JS, Stead RH, McSharry C, Nielsen L, Bienenstock J. The role of mast cell degranulation products in mast cell hyper-

NGF AND COLONIC MUCOSAL INTEGRITY

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44. 45.

46.

533

plasia. I. Mechanism of action of nerve growth factor. J Immunol 1990;144:1886 –1892. Barreau F, Ferrier L, Fioramonti J, Bueno L. Neonatal maternal deprivation triggers long term alterations in colonic epithelial barrier and mucosal immunity in rats. Gut 2004;53:501–506. Ruckebusch M, Fioramonti J. Electrical spiking activity and propulsion in small intestine in fed and fasted rats. Gastroenterology 1975;68:1500 –1508. White JR, Pearce FL. Effect of anti-allergic and cyclic AMP-active drugs on histamine secretion from rat mast cells treated with the novel calcium ionophore chlortetracycline. Int Arch Allergy Appl Immunol 1983;71:352–356. Morteau O, Hachet T, Caussette M, Bueno L. Experimental colitis alters visceromotor response to colorectal distension in awake rats. Dig Dis Sci 1994;39:1239 –1248. Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206 – 209. Coelho AM, Fioramonti J, Bueno L. Systemic lipopolysaccharide influences rectal sensitivity in rats: role of mast cells, cytokines, and vagus nerve. Am J Physiol 2000;279:G781–G790. Anton PM, Theodorou V, Fioramonti J, Bueno L. Chronic low-level administration of diquat increases the nociceptive response to gastric distension in rats: role of mast cells and tachykinin receptor activation. Pain 2001;92:219 –227. Lafreniere GF, Persinger MA, Lafreniere RF. Effects of permanent residence with foster mothers and new siblings upon numbers of mast cells within the thalamus of preweaned rats. Psychol Rep 2001;88:625– 626. Mulholland MW, Romanchuk G, Lally K, Simeone DM. Nerve growth factor promotes neurite outgrowth in guinea pig myenteric plexus ganglia. Am J Physiol 1994;267:G716 –G722. Yasuda T, Sobue G, Ito T, Mitsuma T, Takahashi A. Nerve growth factor enhances neurite arborization of adult sensory neurons; a study in single-cell culture. Brain Res 1990;524:54 – 63. Malcangio M, Ramer MS, Boucher TJ, McMahon SB. Intrathecally injected neurotrophins and the release of substance P from the rat isolated spinal cord. Eur J Neurosci 2000;12:139 –144. Skoff AM, Resta C, Swamydas M, Adler JE. Nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) regulate substance P release in adult spinal sensory neurons. Neurochem Res 2003;28:847– 854. Vedder H, Affolter HU, Otten U. Nerve growth factor (NGF) regulates tachykinin gene expression and biosynthesis in rat sensory neurons during early postnatal development. Neuropeptides 1993;24:351–357. Schuligoi R, Amann R. Differential effects of treatment with nerve growth factor on thermal nociception and on calcitonin generelated peptide content of primary afferent neurons in the rat. Neurosci Lett 1998;252:147–149. Plourde V, St-Pierre S, Quirion R. Calcitonin gene-related peptide in viscerosensitive response to colorectal distension in rats. Am J Physiol 1997;273:G191–G196. Kamp EH, Beck DR, Gebhart GF. Combinations of neurokinin receptor antagonists reduce visceral hyperalgesia. J Pharmacol Exp Ther 2001;299:105–113. Owolabi JB, Rizkalla G, Tehim A, Ross GM, Riopelle RJ, Kamboj R, Ossipov M, Bian D, Wegert S, Porreca F, Lee DK. Characterization of antiallodynic actions of ALE-0540, a novel nerve growth factor receptor antagonist, in the rat. J Pharmacol Exp Ther 1999;289: 1271–1276. Dmitrieva N, McMahon SB. Sensitisation of visceral afferents by nerve growth factor in the adult rat. Pain 1996;66:87–97. Delafoy L, Raymond F, Doherty AM, Eschalier A, Diop L. Role of nerve growth factor in the trinitrobenzene sulfonic acid-induced colonic hypersensitivity. Pain 2003;105:489 – 497. Farquhar-Smith WP, Jaggar SI, Rice AS. Attenuation of nerve

534

47.

48.

49.

50.

51.

52.

53.

BARREAU ET AL.

growth factor-induced visceral hyperalgesia via cannabinoid CB(1) and CB(2)-like receptors. Pain 2002;97:11–21. Chudler EH, Anderson LC, Byers MR. Nerve growth factor depletion by autoimmunization produces thermal hypoalgesia in adult rats. Brain Res 1997;765:327–330. Otten U, Baumann JB, Girard J. Stimulation of the pituitaryadrenocortical axis by nerve growth factor. Nature 1979;282: 413– 414. Mocchetti I, Spiga G, Hayes VY, Isackson PJ, Colangelo A. Glucocorticoids differentially increase nerve growth factor and basic fibroblast growth factor expression in the rat brain. J Neurosci 1996;16:2141–2148. Shadrina MI, Dolotov OV, Grivennikov IA, Slominsky PA, Andreeva LA, Inozemtseva LS, Limborska SA, Myasoedov NF. Rapid induction of neurotrophin mRNAs in rat glial cell cultures by Semax, an adrenocorticotropic hormone analog. Neurosci Lett 2001;308: 115–118. Levine S. Regulation of the hypothalamic-pituitary-adrenal axis in the neonatal rat: the role of maternal behavior. Neurotox Res 2002;4:557–564. Spiller RC, Jenkins D, Thornley JP, Hebden JM, Wright T, Skinner M, Neal KR. Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome. Gut 2000;47:804 – 811. Bradette M, Delvaux M, Staumont G, Fioramonti J, Bueno L, Frexinos J. Evaluation of colonic sensory thresholds in IBS patients using a barostat. Definition of optimal conditions and comparison with healthy subjects. Dig Dis Sci 1994;39:449 – 457.

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54. Coelho AM, Vergnolle N, Guiard B, Fioramonti J, Bueno L. Proteinases and proteinase-activated receptor 2: a possible role to promote visceral hyperalgesia in rats. Gastroenterology 2002; 122:1035–1047. 55. Santos J, Yang PC, Soderholm JD, Benjamin M, Perdue MH. Role of mast cells in chronic stress induced colonic epithelial barrier dysfunction in the rat. Gut 2001;48:630 – 636. 56. Bullock ED, Johnson EM Jr. Nerve growth factor induces the expression of certain cytokine genes and bcl-2 in mast cells. Potential role in survival promotion. J Biol Chem 1996;271: 27500 –27508. 57. Marshall JS, Gomi K, Blennerhassett MG, Bienenstock J. Nerve growth factor modifies the expression of inflammatory cytokines by mast cells via a prostanoid-dependent mechanism. J Immunol 1999;162:4271– 4276. 58. McKay DM, Baird AW. Cytokine regulation of epithelial permeability and ion transport. Gut 1999;44:283–289. 59. Perdue MH. Mucosal immunity and inflammation. III. The mucosal antigen barrier: cross talk with mucosal cytokines. Am J Physiol 1999;277:G1–G5. Received November 21, 2003. Accepted April 22, 2004. Address requests for reprints to: Lionel Bueno, Ph.D., Neuro-Gastroenterology and Nutrition Unit, Institut National de la Recherche Agronomique, 180 chemin de Tournefeuille, BP3, 31931 Toulouse Cedex 9, France. e-mail: [email protected]; fax: (33) 561 28 53 97. Supported by the Institut National de la Recherche Agronomique (Paris, France). The authors thank Bernard Joseph for technical assistance.