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Abstract
Background: Neonatal maternal deprivation induces colonic alterations in adult rats, such as hypersensitivity to distension or an increase in paracellular permeability, characteristics of irritable bowel syndrome (IBS) patients. Recent studies described neuroimmune alterations in the colonic mucosa of IBS patients.
Methods: Male Wistar rats were submitted to maternal deprivation for 3 h daily during postnatal days 2–14, and were sacrificed at 4 or 12 weeks of age. Control pups were left undisturbed with their dam.
Results: Colonic mast cell hyperplasia was observed at 4 and 12 weeks in maternally deprived rats, and was associated with an increase in protease content. Mucosal nerve fibre density assessed by protein gene product (PGP) 9.5 immunoreactivity was increased at 12 weeks but not at 4 weeks, while calcitonin gene-related protein (CGRP)-immunoreactive fibres remain constant. Synaptogenesis assessed by synaptophysin immunostaining was increased at 4 weeks but not at 12 weeks. The number of mast cells in close proximity to PGP 9.5- or CGRP-immunoreactive fibres was greater at both 4 and 12 weeks. Expression of neurokinin NK1 receptors in the spinal cord was enhanced at 12 weeks. No significant change in total mast cell number, PGP 9.5 immunoreactivity and mast cells associated with PGP 9.5-immunoreactive fibres was observed in the jejunum. Treatment of pups with anti-nerve growth factor (NGF) antibodies abolished the increases in synaptogenesis and in the number of mast cells in close proximity to nerve fibres observed 4 weeks after maternal deprivation.
Conclusions: Neonatal maternal deprivation induces closer association of colonic mast cells with nerves, which is similar to that seen in IBS patients. NGF is a possible mediator of this effect.
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Neonatal maternal deprivation is known to induce alterations of colonic functions in adult rats. Among these are a hypersensitivity to colorectal distension,1–3 an increase in paracellular epithelial permeability,4 alterations of the cytokine profile,4 an infiltration of polymorphonuclear neutrophils,4 an increase in mucosal mast cell density4 and a decrease in epithelial ionic absorption.5 Moreover, neonatal maternal deprivation enhances some effects of acute stress applied in adults, such as hypersensitivity to distension,1 ,2 ,6 increase in faecal output6 or uptake of macromolecule by colonocytes.5 Some of these features have been described in patients presenting an irritable bowel syndrome (IBS): hypersensitivity to rectal or colonic distension,7 increase in paracellular permeability,8 ,9 infiltration of immune cells8 and an increase in mast cell number.10 ,11 However, no correlation has been found between the severity of symptoms and one of these alterations.
Mast cells are located in close proximity to nerve fibres in the intestinal mucosa.12 They can be activated by neuropeptides and, in turn, secrete mediators able to stimulate sensory neurons.13 Moreover, afferent nerves exert a trophic effect on intestinal mast cells.14 The number of mast cells in close apposition (within 5 μm) to nerves has been found to be greatly increased in biopsies of colonic mucosa of IBS patients, in comparison with controls.11 ,15 Interestingly, the severity and the frequency of abdominal pain were significantly correlated with the number of mast cells closely apposed to colonic nerves.11 This enhancement of nerve–mast cell proximity, which can be considered as essential in the genesis of pain in IBS, has never been described in rats submitted to neonatal maternal deprivation. Moreover, the increased mast cell density in IBS colonic mucosa has been found to be associated with an increase in mucosal content of tryptase and a greater ability of mast cells to release tryptase and histamine spontaneously, as compared with controls.11 ,16
Alterations characterising IBS are not limited to the colon, but also involve the small intestine. Full-thickness biopsies revealed a low grade jejunal inflammation and neuronal degeneration.17 They also revealed a marked increase in jejunal mast cell number in diarrhoea-prone IBS patients, associated with an increase in tryptase concentration in jejunal fluid.18 Maternal deprivation has been found to induce in adult rats a moderate, but significant, jejunal inflammation and an increase in epithelial paracellular permeability.19
On the other hand, activation of spinal neurokinin 1 (NK1) receptors contributes to somatic hyperalgesia20 and to stress-induced visceral hyperalgesia.21 ,22 It has been recently shown that repeated stress in rats induces an upregulation of NK1 receptors in the dorsal horn, and an intrathecal administration of an NK1 receptor antagonist reduces the abdominal response to colorectal distension enhanced by repeated stress.22 Moreover, an alteration of the spinal modulation of nociceptive processing has been described in IBS patients.23
Consequently, the aim of the present study was to identify colonic alterations that can be involved in the visceral hyperalgesia induced by neonatal maternal deprivation: nerve fibre density, mast cell hyperplasia, morphology and ability of mast cells to release tryptase, nerve–mast cell proximity and expression of NK1 receptors in the spinal cord. Since alterations in the jejunal wall have been observed in IBS patients and in maternally deprived rats, changes in nerve fibre and mast cell density have been sought in the jejunum of maternally deprived rats. Moreover, most of the alterations already described have been found in adult rats, but it is not known whether they appear early in young individuals. For example, it has been found that overexpression of nerve growth factor (NGF) in the colonic wall appears at the end of the period of maternal deprivation (postnatal days 2–14) and persists until the age of 12 weeks.3 Alterations were thus sought at both 4 and 12 weeks of age.
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±1°C) and maintained on a 12 h light/dark cycle (lights on at 07:00). 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. All experimental protocols described in this study were approved by the Local Institutional Animal Care and Use Committee.
Maternal deprivation
Maternal deprivation was performed according to a previously described method.2–4 After delivery (day 1), litters were culled to 10 pups. Maternal deprivation was performed daily for three consecutive hours (from 9:00 to 12:00), during which pups were removed from their home cage and kept singly in temperature-controlled cages at 28±1°C, where bedding was changed every day. This procedure was applied between postnatal days 2 and 14. Control—that is, non-deprived, pups were left undisturbed with their dam. From days 15 to 22, all control and maternally deprived pups were left with their dam. Weaning was performed on day 22, siblings were sex-matched and males were selected.
Experimental protocol
Experiments were performed in six groups of eight rats. Group 1 (control) and group 2 (deprived) were sacrificed at 4 weeks. Samples of distal colon and spinal cord were taken for immunohistological studies. Similarly, group 3 (control) and group 4 (deprived) were sacrificed at 12 weeks, and a piece of proximal colon was taken to investigate mast cell degranulation, and a segment of mid-jejunum for identification of nerve fibres and mast cells. Group 5 was treated intraperitoneally (10 μl) with inactivated anti-NGF antibodies 10 min before each session of separation, and sacrificed at 4 weeks. Samples of distal colon were taken for immunohistological studies. Group 6 received anti-NGF antibodies in the same conditions. Anti-NGF antibodies (fractionated antiserum against 2.5S-NGF purified from mouse submaxillary glands, Sigma Chemical Co, St Louis, Missouri, USA) were diluted according to a previously described protocol.3 For inactivation, anti-NGF antibodies were boiled at 100°C for 30 mins.
Immunohistochemistry
Rat mast cell protease II, protein gene product 9.5 and calcitonin gene-related protein in colonic or jejunal tissue
Specimens were fixed in 4% buffered formalin and immersed for 24 h in 30% sucrose at 4°C. Samples were embedded in Tissue Tek medium (Euromedex, Souffelweyersheim, France) and frozen in isopentane at −45°C. Cryostat sections (10 μm) were post-fixed with acetone (10 min, −20°C) and hydrated in phosphate-buffered saline (PBS)–milk. After incubation in blocking solution (PBS with 0.25% Triton X-100 containing 0.1% bovine serum albumin (BSA)), sections were incubated overnight at 4°C with sheep anti-rat mast cell protease (RMCP) II antibodies (1/500) (Moredun, Midlothian, UK), and/or rabbit anti-protein gene product (PGP) 9.5 (1/4000) (AbCys, Paris, France) and/or rabbit anti-calcitonin gene-related protein (CGRP) (1/5000) (Sigma, Saint Quentin Fallavier, France). Sections were then washed in PBS–milk and incubated for 1.5 h at room temperature with Alexa fluor 594-conjugated immunoglobulin G (IgG) donkey anti-sheep (1/2000) and Alexa fluor 488-conjugated IgG donkey anti-rabbit (1/2000) antibodies (Invitrogen, Cergy Pontoise, France). Sections were mounted in Vectashield Hard set mounting medium (Clinisciences, Montrouge, France), and examined under a Nikon 90i fluorescence microscope (Nikon, Champigny-sur-Marne, France).
The number of mast cells per mm2 of mucosa, the number of mast cells with RMCP II granulation greater than 80 μm2 per mm2 of mucosa, and PGP 9.5-immunoreactive (IR) areas per mm2 of mucosa were quantified using the Lucia G software (version 4.8, Nikon) under a 20× objective. The number of mast cells in the close vicinity of PGP 9.5-IR or CGRP-IR nerve fibres was determined using the same software. Mast cells were considered tightly associated with a nerve fibre when the two stainings (RMCP II and PGP 9.5 or CGRP) merged. According to microscope characteristics, merging of staining corresponds to a distance less than 10 μm. Analyses were done on five fields of four control and four maternally deprived rats.
Synaptophysin in colonic tissues
Specimens were fixed and cryostat sections were made as above described. Then, after inhibition of endogenous peroxidases with 0.6% H2O2 in PBS for 30 min and incubation in blocking solution (PBS with 0.25% Triton X-100 containing 0.1% BSA), sections were incubated overnight at 4°C with mouse monoclonal anti-synaptophysin antibodies (AbCys). Sections were then washed in PBS–milk and incubated for 1.5 h at room temperature with a biotinylated goat anti-mouse IgG immune serum and subsequently with ABC complexes coupled to peroxidase (Vectastain Elite ABC kit, Clinisciences). Antigen–antibody complexes were revealed using 3,3′-diaminobenzidine contrasted with nickel (Clinisciences). As negative controls, sections were treated with the same procedure, but with omission of primary antibody.
Immunohistochemical analysis was performed in a blinded fashion using a Nikon 90i microscope. Mucosal areas occupied by synaptophysin immunostaining were measured using the software Nis-Elements Ar (Nikon). Results were expressed in μm2 per mm2 of mucosa. Analyses were done on five fields of four control and four maternally deprived rats.
NK1 receptors in spinal cord
Rats were anaesthetised with urethane and immediately perfused intracardially with PBS, and subsequently with 4% paraformaldehyde at 4°C. L1–L2 segments of spinal cord were removed, postfixed overnight in the same fixative, and cryoprotected by incubating with 30% sucrose in phosphate buffer (4°C for 24 h). Samples were embedded in Tissue Tek medium (Euromedex) and frozen in isopentane at −45°C. Cryostat sections (25 μm) hydrated in PBS were incubated in blocking solution (PBS with 0.3% Triton X-100 containing 1% donkey serum) and then overnight at 4°C with rabbit anti-NK1 receptor antibodies (1/3000) (AbCys) diluted in PBS–0.3% Triton–5% donkey serum. Sections were washed in PBS and incubated for 2 h at room temperature with Alexa fluor 488-conjugated IgG donkey anti-rabbit (1/4000) diluted in PBS–0.3% Triton. Sections were rinsed in PBS and mounted in Vectashield Hard set mounting medium (Clinisciences).
Preparations were examined with a Nikon 90i fluorescence microscope. A 40× objective (Nikon CFI Planfluor) was used to quantify NK1 receptor (NK1R)-IR neurons. Counting was done according to established protocols.19 The total number of NK1R-IR neurons was counted in 10 sections from the L1–L2 segment of the spinal cord of each rat (n = 4). Only neurons in lamina I of the dorsal horn were considered.
After 20 min incubation in Ringer’s solution, colonic strips from 12-week-old maternally deprived rats released more RMCP II than controls, under basal conditions and in response to substance P or A-23187 (table 1). However, the ratio of the concentration of RMCP II and the number of mast cells per mm2 did not differ between maternally deprived and control rats.
Mast cell degranulation
In each 12-week-old rat, three pieces of proximal colon, 0.5×0.5 cm, were taken to assess the release of RMCP II in basal conditions or after stimulation. Each piece was incubated for 20 min at 37°C in an oxygenated Ringer’s buffer solution, supplemented or not with substance P (30 μM), which induces a receptor-mediated degranulation,24 or A-23187 (1 μM), a calcium ionophore inducing a non-receptor-mediated exocytosis in mast cells.25 RMCP II concentration was measured in supernatants by ELISA. A monoclonal antibody against RMCP II raised in mouse was diluted to a concentration of 2 μg/ml in a 0.2 M carbonate buffer at pH 9.6. Coated plates were incubated with the antibody at 4°C for 24 h before use. A 30 min incubation with 4% (w/v) BSA was done before loading standard and unknown samples to avoid an unspecific reaction. Sample incubation was for 1.5 h at 37°C. A sheep anti-RMCP II and horseradish peroxidase conjugate was added afterward and incubated for 1 h. Plates were developed using o-phenylenediamine as substrate and read at 492 nm after the reaction was stopped. RMCP II concentration was expressed in nmol/g of tissue.
Data analysis
Statistical analysis of differences between control and maternally deprived rats was performed using the Student t test for unpaired data. Data are expressed as the mean (SEM).
RESULTS
Colonic mast cells
As previously shown,4 the density of colonic mucosal mast cells was strongly increased in maternally deprived 12-week-old rats, in comparison with controls. A similar increase in mast cell number was observed in 4-week-old rats, but there was no significant difference between 4- and 12-week old rats (p>0.05) (fig 1). Moreover, in 12-week-old rats, the number of mast cell with an RMCP II-IR granulation surface greater than 80 μm2/mm2 was doubled in maternally deprived rats, in comparison with controls (fig 2).
Colonic nerve density and mast cell–nerve interactions
In 4-week-old rats, there was no difference in the density of colonic nerve fibres identified by PGP 9.5 immunostaining between control and maternally deprived rats, fibre density being assessed by the surface (μm2) occupied by PGP 9.5 immunoreactivity per mm2 of mucosa. In both control and maternally deprived rats there was a significant decrease in PGP 9.5 immunoreactivity between 4 and 12 weeks. However, in 12-week-old rats, the mucosal surface of PGP 9.5 immunoreactivity was significantly higher (p<0.05) in maternally deprived than in control rats (fig 3). In contrast, the mucosal surface of CGRP immunoreactivity remained unchanged between 4 and 12 weeks in control and maternally deprived rats, and there was no significant difference between control and maternally deprived rats at either 4 or 12 weeks (fig 4).
The evolution of synaptophysin immunoreactivity did not parallel that of PGP 9.5 or CGRP. At 4 weeks, the mucosal surface of synaptophysin immunostaining was significantly higher (p<0.05) in maternally deprived than in control rats, while the immunostained areas were similar in 12-week-old rats. Moreover, only in control rats was the synaptophysin-IR surface significantly increased (p<0.05) between 4 and 12 weeks of age (fig 5).
About 20 mast cells per mm2 were found in close proximity (<10 μm) to PGP 9.5-IR nerve fibres in 4- and 12-week-old control rats. This number was nearly doubled in maternally deprived rats, at both 4 and 12 weeks, compared with their respective controls. In both control and maternally deprived rats, the number of mast cell–nerve interactions remained unchanged between 4 and 12 weeks (fig 6). The number of mast cells in close proximity to CGRP fibres was also significantly greater (p<0.05) in maternally deprived rats than in controls, at both 4 and 12 weeks. However, the number of mast cell–CGRP-IR fibre interactions was significantly increased (p<0.05) from 4 to 12 weeks in both control and maternally deprived rats (fig 7).
Expression of NK1 receptors in the spinal cord
In maternally deprived 4-week-old rats, there was a tendency towards an increase in the number of NK1R-IR cells in lamina I of the dorsal horn in comparison with controls, but a statistically significant difference was not reached (p>0.05). In 12-week-old rats, the number of NK1Rs was significantly higher (p<0.05) in deprived rats in comparison with controls. Moreover, in both control and maternally deprived rats, there was a significant decrease (p<0.05) in the number of NK1R-IR cells (fig 8).
Alterations of the jejunal mucosa
In the jejunal mucosa of 12-week-old rats, the surface of PGP 9.5-IR nerve fibres was similar in control and maternally deprived rats (fig 9). Despite a strong tendency to an increase in maternally deprived rats, there was also no significant statistical difference (p>0.05) in the density of mast cells (p = 0.11) or in the number of mast cells closely apposed to PGP 9.5-IR fibres (p = 0.19).
Effects of anti-NGF antibody treatment
In the colonic mucosa of 4-week-old rats, anti-NGF antibody treatment during maternal separation did not modify the increase in mast cell density induced by maternal separation. On the contrary, the increases in synaptophysin-IR surface and in the number of mast cells in close proximity to PGP 9.5-IR or CGRP-IR nerve fibres were abolished (fig 10). The density of PGP 9.5-IR and CGRP-IR nerve fibres, which was unaffected by maternal deprivation, remained unchanged after anti-NGF antibodies treatment.
DISCUSSION
This study provides evidence that maternal deprivation during the first days of life induces in adult rats colonic mucosal alterations presenting similarities with those observed in biopsies from IBS patients. In line with previous studies by our group,3 ,4 we confirmed that maternal deprivation induces mast cell hyperplasia. In addition, we showed that mast cell hyperplasia was accompanied by mast cell hypertrophy. This increase in mast cell density occurs very early, since it has been observed as soon as day 14 of life just at the end of the period of maternal deprivation,3 and persists at least until 12 weeks of age. It has been shown that NGF plays a key role during maternal deprivation for inducing long-term alterations of colonic mucosa.3 It was speculated that NGF overexpression induced by maternal deprivation at the colonic level is an essential cause of mast cell hyperplasia26 since treatment of pups with anti-NGF antibodies prevented mast cell hyperplasia observed 12 weeks after maternal deprivation.3 The results of the present study indicate that NGF is not a primary factor triggering mast cell hyperplasia since anti-NGF antibody treatment did not prevent the increase in mast cell density observed 4 weeks after maternal deprivation.
Mast cell hyperplasia has been described in IBS patients at the level of the ileum,10 ,15 ,27 caecum,15 ,28 ,31 colon,11 ,27 ,30 rectum27 ,29 and jejunum.18 The key role played by mast cells in neuroimmune interactions, their increased number in biopsies from IBS patients and the association between mast cell number and visceral hypersensitivity observed in animals led to a search for a correlation between mast cell number and pain or hypersensitivity characterising IBS patients. In the first study,11 no correlation was found between the number of mast cells in colonic biopsies and the severity or the frequency of abdominal pain or discomfort. In another study,27 the number of mast cells in the ileum, colon or rectum was greater in IBS patients without hypersensitivity to rectal distension than in hypersensitive patients. A possible explanation for this result, given by the authors, was that chronically increased mast cells could lead to tissue desensitisation to its mediators. However, a recent study emphasises the role of mast cells in IBS hypersensitivity.16 Supernatants of incubated colonic biopsies of IBS patients have been found to be able to enhance firing of mesenteric nerves in rats, and to stimulate Ca2+ mobilisation in dorsal root ganglia neurons in culture. Nerve firing and Ca2+ mobilisation were correlated with the number of mast cells in the lamina propria of biopsies.
We observed that mast cell volume was increased in maternally deprived rats compared with controls; more precisely, the number of mast cells with an RMCP II granulation surface greater than 80 μm2 was doubled. The volume of mast cells has not been assessed in IBS patients, but the area occupied by tryptase immunoreactivity on sections of colonic biopsies of IBS patients have been found to be greater than in controls.11 ,16 ,28 We showed that the amount of RMCP II in the colonic wall of maternally deprived rats is greater than in controls, since the spontaneous release of RMCP II or the release after stimulation by two mast cell degranulators was greater in pieces of colon from maternally deprived rats. This is in agreement with data obtained in humans showing that the spontaneous release of tryptase and histamine by colonic biopsies is greater in IBS patients than in controls.11 ,16 However, we observed that the ratios between RMCP II release and mast cell number, in basal and stimulated conditions, were similar in controls and maternally deprived rats, indicating that mast cell hyperplasia is responsible for the increased RMCP II release.
Changes in mucosal innervation were assessed with PGP 9.5, a general marker of nerve elements already used for observation of the enteric nervous system.30 Besides changes induced by maternal deprivation at 12 weeks, we observed a decrease in PGP 9.5-IR nerve density between 4 and 12 weeks in both control and maternally deprived rats. Modifications of the enteric nervous system have been described throughout life in mice.31 The decrease we observed is in agreement with the dramatic decrease in neuron density described in humans during the first 3–4 years of life, at least at the level of the myenteric plexus.32 The increase in mucosal innervation observed at 12 weeks in maternally deprived rats in comparison with controls has been already described in the mucosa of the terminal ileum and rectosigmoid of IBS patients, using neuron-specific enolase (NSE) another marker of neurofilaments.15 A neuroproliferation in the mucosa has also been described in intestinal inflammatory states such as appendicitis33 or Crohn’s disease.34 Nerve remodelling is known to be a consequence of intestinal inflammation,35 ,36 whatever the origin of the inflammation. Inflammation may indeed be a cause of the neuroproliferation observed in both IBS patients and maternally deprived rats since there are data permitting consideration of IBS as a low-grade inflammatory bowel disease,37 and a moderate but significant inflammation has been described in the colon of maternally deprived rats.4 Besides the increase of PGP 9.5 immunoreactivity, we found no change in CGRP innervation induced by maternal deprivation at both 4 and 12 weeks of age. Interestingly, the number of CGRP-positive nerve fibres in colonic biopsies does not differ between controls and IBS patients.15
We observed that synaptogenesis, assessed by the expression of the presynaptic vesicle protein synaptophysin, was significantly increased between 4 and 12 weeks in the colonic mucosa of control rats. The increased synaptogenesis observed in maternally deprived animals, in comparison with controls, at 4 weeks of age has not been looked for in biopsies from IBS patients but has been described in inflammatory states. Synaptophysin has been found to be overexpressed in acute appendicitis,38 ulcerative colitis, regional enteritis or non-specific enteritis.39 NGF is known to induce sprouting and synaptogenesis,40 and overexpression of synaptophysin in intestinal inflammation has been found associated with overexpression of NGF receptors.39 In agreement with these data, our results indicate that anti-NGF antibody treatment abolishes the increased synaptogenesis observed 4 weeks after maternal deprivation. Mast cell hyperplasia may be the cause of increased neuronal density and synaptogenesis since mast cells are able to synthesise and release NGF.41 On the contrary, neuroproliferation may be the cause of an increase in mast cell number, in agreement with the trophic effect on mucosal mast cells of nerve fibres, at least from the vagus.14
The close apposition between mast cells and nerve fibres is an anatomical feature well established at the level of the gastrointestinal tract,12 but also in other epithelia such as skin42 or urinary bladder.43 This close apposition, found to be enhanced in maternally deprived rats, may play a crucial role in hypersensitivity and IBS symptoms. Among several parameters found to be increased in colonic biopsies from IBS patients (number of intact or degranulated mast cells, histamine or tryptase release), only the number of mast cells at less than 5 μm from nerves has been found to be significantly correlated with the severity and frequency of abdominal pain or discomfort.11 Maternally deprived rats are characterised by a hypersensitivity to colonic distension.1–3 Interestingly, contact between mast cells and nerves has been found to be enhanced in the rat jejunum after infection with the nematode Nippostrongylus brasiliensis,44 which also induces a hypersensitivity to distension.45 It is well established that in the intestine,44 but also in the skin42 or the urinary bladder,43 mast cells preferentially contact substance P-IR and/or CGRP-IR fibres, and we found an enhancement of the contact between mast cells and CGRP fibres in 4 and 12 week maternally deprived rats. We also found that in control rats the number of mast cells closely apposed to CGRP fibres was nearly tripled between 4 and 12 weeks of age. It can be speculated that this spontaneous reinforcement of the mast cell–nerve interaction may be involved in the spontaneous increase in sensitivity to colorectal distension observed with age in mice.46 At skin level, acute stress in adult mice or rats also induces an increase in mast cell number47 and nerve–mast cell interactions,48 mast cell hyperplasia being mediated through the release of corticotrophin-releasing factor.47
The pathophysiology of visceral hypersensitivity in IBS patients is proabably multifactorial, involving both peripheral and central mechanisms. Several pieces of experimental data provide direct and objective arguments favouring the idea that visceral hypersensitivity reported in IBS patients is associated with hyperexcitability of spinal nociceptive neurons.23 In adult rats, chronic stress induces an increase of NK1R expression in the dorsal spinal cord similar to that described in the present study in 12 week maternally deprived rats.22 This overexpression of spinal NK1Rs very probably plays a role in the stress-induced hypersensitivity which has been found to be abolished by intrathecal administration of an NK1R antagonist.22
At the jejunal level, we found a tendency to an increase in the number of mast cells in maternally deprived rats. A recent study describes a mast cell hyperplasia in the jejunum of diarrhoea-predominant IBS patients.18 We found no alteration in the jejunal innervation assessed by PGP 9.5 immunoreactivity. Neuronal alterations have been reported in the jejunum of IBS patients, but they corresponded to degeneration instead of proliferation as described in the terminal ileum and the rectosigmoid.15 Moreover, an increase in small intestinal paracellular permeability has been reported in subgroups of IBS patients,49 and we have previously shown that jejunal permeability was also increased in 12-week-old maternally deprived rats.19 The alterations observed in the colon but not in the jejunum suggest a role for the colonic flora, which reinforces the hypothesis of a primary role for inflammation since a very low inflammatory state has been observed, after maternal deprivation, in the jejunum in comparison with the colon.19
Our results indicate strong similarities between alterations induced in the colonic mucosa by maternal deprivation and alterations reported in colonic biopsies from IBS patients. Maternal deprivation can be considered as a useful animal model for the study of the pathophysiology of IBS.
REFERENCES
Footnotes
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Competing interests: None.
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