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ALIMENTARY |
1 Nerve-Gut Research Laboratory, Department of Gastroenterology, Hepatology and General Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia
2 Discipline of Physiology
3 Department of Medicine
4 Discipline of Paediatrics, University of Adelaide, Adelaide, South Australia 5000, Australia
5 Gastroenterology Department, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia
6 School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, Adelaide, Australia
7 School of Biological Sciences, Flinders University, South Australia, Australia
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Abstract |
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(Received 19 October 2006;
accepted after revision 27 November 2006;
first published online 30 November 2006)
Corresponding author L. A. Blackshaw: Nerve-Gut Research Laboratory, Level 1, Hanson Institute, Frome Road, Adelaide, South Australia 5000, Australia. Email: ashley.blackshaw{at}adelaide.edu.au
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Introduction |
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It is well documented that inflammation can have a sensitizing effect on sensory nerves elsewhere in the body leading to increased sensitivity to chemical stimuli, but increased mechanical responsiveness remains controversial (Kocher et al. 1987; Schaible & Schmidt, 1988; Habler et al. 1990; Andrew & Greenspan, 1999; Koltzenburg et al. 1999). Regardless of the cause, increased afferent signals to the central nervous system are likely to result in false perception of the environment and pain. Our aim in the present study was to determine whether increased chemosensitivity or mechanosensitivity of colonic afferent fibres occurs during inflammation, and therefore to gain insight into the role of 5-HT in visceral hypersensitivity. We examined changes in afferent 5-HT sensitivity in a rat model of colonic inflammation. Colitis was induced by ingestion of dextran sulphate sodium (DSS) and this model was chosen for its reproducibility, its morphological similarities with human ulcerative colitis and its proven effectiveness in rat studies (Howarth et al. 1998). We determined the effects of 5-HT and the mast cell degranulator compound 48/80 on colonic afferents in normal rats and those with colitis using an isolated preparation in which the activity of lumbar splanchnic afferent fibres can be recorded during delivery of controlled stimuli to the colon (Lynn & Blackshaw, 1999). We also localized 5-HT-immunoreactive mast cells to the colonic serosa and mesentery and determined their relation to calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres; CGRP is an established marker for colonic afferents (Christianson et al. 2006).
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Methods |
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A total of 98 adult Sprague-Dawley rats (180–200 g) were provided by the Institute of Medical and Veterinary Sciences (IMVS) (Adelaide, South Australia). Animals were housed in groups under standard laboratory conditions (22°C, 12 h light–dark cycle) until the study period for convenience of care and stress minimization. They were then transferred to individual metabolism cages, fed a powdered diet suitable for metabolism cage use, and allowed free access to water (Howarth et al. 1998). All experiments were carried out with the approval of, and according to the guidelines of, the animal ethics committees of the Institute of Medical and Veterinary Science and University of Adelaide.
Induction of colonic inflammation
50 animals were designated for induction of colonic inflammation, with 37 animals used for inflamed studies and 20 used for studies after recovery from inflammation. Animals were housed individually in Tecniplast ® metabolism cages and acclimatized for a period of 3 days. Colonic inflammation was induced by addition of 2%w/v dextran sulphate sodium (DSS) (MW 40000, ICN Biochemicals, Cleveland, Ohio) to the animal's drinking water for a period of 7 days. This produced mild symptoms as described in Table 1, but with no observable behavioural disturbances. 36 animals were designated for controls and were housed in identical conditions but without DSS treatment for the same duration. Recovery animals were allowed a 21-day recovery period post DSS treatment before subjection to the electrophysiological protocol. Afferent recordings were obtained from the majority of preparations, with numbers of fibres given for each protocol in Results.
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Three animals from each of the groups were used for validation of the inflammatory protocol. Inflammation was validated by disease activity index score, histopathological assessment and myeloperoxidase (MPO) assay. The disease activity was scored on a severity scale of 0–4 based on physical observations of weight loss, stool consistency and colonic bleeding. Histopathological assessment using haematoxylin and eosin staining was used to show morphological changes associated with DSS treatment over the 7 day period and subsequent recovery after a further 21 days. Animals from control, inflamed and recovery groups were anaesthetized with sodium pentobarbitone (Nembutal, 60 mg kg–1 I.P.) and the abdomen opened by midline incision. A 2 cm length of distal colon was removed and divided into 1 cm segments of which one was snap frozen in liquid nitrogen for MPO assay and the other fixed in 10% formaldehyde and processed for histopathological assessment (Department of Tissue Pathology, IMVS). Animals were then killed by exsanguination and excision of thoracic organs while under deep anaesthesia. Haematoxylin and eosin-stained sections were assessed for colonic damage, oedema and inflammatory cell infiltration. Neutrophil infiltration as a measure of inflammation was also assessed by MPO assay according to the protocol described by Morris et al. (1989).
Electrophysiological protocol
Dissection was carried out according to a previously described protocol (Lynn & Blackshaw, 1999). Rats from all groups were anaesthetized with sodium pentobarbitone (as above) and the abdomen opened by midline incision. Next, 4–5 cm of distal colon was removed with attached lumbar colonic nerves (LCN) and a bundle containing the inferior mesenteric artery, abdominal aorta, inferior mesenteric ganglion, intermesenteric nerve and lumbar splanchnic nerve (LSN) according to the classification of Baron et al. (1988), and placed in cold modified Krebs solution (bicarbonate buffer; 4°C). Rats were killed as in the histology protocols. The distal colon was opened longitudinally and pinned flat, mucosal side down, in an isolated perfused organ bath (Danz Instruments, Blackwood, South Australia) with the LCN insertions orientated to lie along the edge of the opened preparation. Connective tissue was dissected away from the neurovascular bundle which was then drawn through into a separate paraffin-filled recording chamber. The attached LSN was finely dissected from the aorta and de-sheathed from the transected nerve stump to expose the nerve fibres; these were divided into fine bundles to be placed onto fine platinum recording electrodes for single afferent fibre recording. Extraluminal receptive fields on the serosal surface and the mesentery were located by responses to blunt probing with a glass rod and discriminated as high-threshold serosal/mesenteric afferents by their lack of response to fine stroking with a calibrated von-Frey hair (10 mg) or circular stretch (8 mm) as previously described (Lynn & Blackshaw, 1999; Berthoud et al. 2001; Hicks et al. 2002). Signals from the recording electrode were differentially amplified, filtered and the analog signal sampled at a rate of 20 kHz via a 1401 data interface (CED, Cambridge, UK). This was recorded to a Pentium III notebook computer and analysed using Spike II software (CED). 5-HT (10–10–10–3 M) was applied to the receptive fields via a 1 cm diameter stainless steel ring placed directly around the corresponding receptive field, after residual Krebs solution had been aspirated. Each concentration of 5-HT was applied for a period of 2–3 min followed by a 10 min washout. 5-HT (10–4 M) was used to determine whether or not a fibre was responsive to 5-HT as this was shown previously to be a maximal concentration (Hicks et al. 2002). Responses to drugs were counted as having occurred if a reproducible > 25% increase in discharge occurred with the maximum concentration. For quantification of responses, activity induced by 5-HT was measured over the second minute of response. This period was selected to accurately reflect the peak of response and compensate for latency of response onset especially at lower concentrations. Concentration–response curves for 5-HT (10–10–10–3 M) were constructed and analysed for changes in response between control and inflamed colons and colons after recovery from inflammation. No differences in 5-HT responsiveness or histological scores were seen when inflammation was induced instead by 5% DSS in male rats housed in wire-bottomed cages. Responses to probing were measured prior to testing of chemosensitivity as total spikes evoked by a 1 s probe application, and as mean peak instantaneous frequency. These responses were averaged over eight trials. No changes in probing responses were observed before and after treatment with 5-HT. Spontaneous discharge was measured over 1 min prior to testing of chemosensitivity. Mechanical thresholds were determined with 10, 50, 200 and 1000 mg von Frey hairs applied perpendicularly to the receptive field. In studies with antagonists, colons were incubated for 2 min with alosetron (2 x 10–7 M) alone, followed by 2 min at the same concentration with 5-HT (10–4 M). In three of these studies, the adenylate cyclase inhibitor MDL 12330 (30 µM) was added to alosetron and the response to 5-HT tested in the presence of both antagonists.
Localization of 5-HT- and CGRP-like immunoreactivity in normal and inflamed tissue
Distal colon was removed from rats after transcardial perfusion with 4% paraformaldehyde under deep anaesthesia with sodium pentobarbitone (60 mg kg–1 I.P.). The region corresponding to that used for electrophysiological study, including the mesenteric attachment, was taken and fixed for 2 h at room temperature (RT, 20°C) in 4% paraformaldehyde, after which the tissue was washed three times for 20 min with phosphate-buffered saline (PBS) at pH 7.4 then cryoprotected with 30% sucrose in PBS for 24 h at 4°C. The colon was then frozen and 20 µm transverse sections cut. Sections were air dried at RT for 10 min, after which the tissue was washed with PBS (pH 7.4) including 0.1% Triton X-100 (PBST). Tissue was then blocked with 2% goat serum, 1% bovine serum albumin, 0.005% Tween 20 and 0.1% gelatin in PBST for 60 min at RT and subsequently incubated with monoclonal anti-5-HT (1 : 100; Dako, Sydney, Australia) and rabbit anti-CGRP (1 : 200; Calbiochem) in blocking solution in PBST overnight at 4°C. Tissue was then washed three times with PBST, then incubated a mixture of secondary antibodies: goat anti-rabbit Alexa Fluor 350 and donkey anti-mouse Alexa Fluor 555 (5 µg ml–1; Molecular Probes, USA) for 45 min at RT. The tissue was then washed three times with PBST and mounted with ProLong Antifade (Molecular Probes, USA). Slides were allowed to dry overnight before viewing with an Olympus BX51 epifluorescence microscope. Images were taken with a Photometrics CoolSnap fx monochrome camera then re-coloured. Negative controls were prepared as above with the primary antibody omitted. Both antibodies have been shown to be specific for their respective epitope in previous studies (Ito et al. 1986; Christianson et al. 2006).
Data analysis
Electrophysiological data are presented as means ± S.E.M., and are compared by one-way ANOVA with Dunnet's post hoc test or by two-way ANOVA for concentration–response curves. Proportions of fibres responding were compared with a Fisher's exact test. Statistical analyses, EC50 values and curve fits were all determined using Graphpad Prism Software (v 3.02; San Diego, CA, USA) according to standard pharmacological methods. Associations between CGRP-containing fibres and 5-HT-containing mast cells were counted from 15 randomly selected sections from four rats.
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Results |
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The DSS model of colitis has been used extensively in rats. We re-validated this model in our laboratory in both active and recovery phases of inflammation. Disease activity by morphological examination showed a progressive deterioration of the colonic mucosa over the 7 day DSS treatment programme and subsequent recovery 21 days after DSS (Table 1). Ulceration of the colonic mucosa and infiltration of inflammatory cells were first detected by light microscopy at day 4 of DSS treatment and increased in severity until day 7. This was accompanied by submucosal oedema and destruction of colonic crypts. After recovery, 21 days after DSS treatment, morphological changes had reversed; morphology was indistinguishable from controls (Fig. 1). Blood was detected in the stool from day 4 and gross colonic bleeding was apparent after 7 days. MPO assay for neutrophil infiltration (Fig. 2) showed a greater than 500% increase in inflamed animals at day 7 compared with controls (n = 3). MPO levels after recovery from inflammation had returned to basal levels.
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As we have reported previously (Lynn & Blackshaw, 1999; Berthoud et al. 2001; Hicks et al. 2002), serosal/mesenteric afferents were located throughout the length of the preparation and were clustered towards the mesenteric border with receptive fields located on or near blood vessels. Many colonic serosal/mesenteric afferents had multiple receptive fields in both the mesentery and serosal surface. Responses to 5-HT were maintained throughout the period of superfusion, showing little if any adaptation during exposure, and normally continued for approximately 2 min after washout before discharge returned to basal levels (Fig. 3), as we have found previously (Hicks et al. 2002).
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In separate experiments to those described above (n = 6 controls and n = 7 inflamed preparations), the afferent response to the mast cell degranulator, compound 48/80 (1–30 µg ml–1), was tested. No significant increases in afferent discharge were recorded in afferents from control animals after application of the maximum concentration. In inflamed tissue, four out of seven endings responded to compound 48/80 with an increase of discharge greater than 25% (P < 0.01, Fisher's exact test, Fig. 7). Proportions of responsive endings and magnitude of response are therefore similar to that seen on application of 5-HT.
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Tissue from five control and five inflamed preparations revealed different immunolabelling for 5-HT and for the sensory neural marker CGRP, which labels a large proportion of rat colonic splanchnic afferents (Christianson et al. 2006). DSS-treated colon showed both a higher level of background 5-HT-like immunoreactivity and an increased number of 5-HT-containing cells in the mucosa and submucosa (data not shown), which were of comparable size and distribution to those previously shown to be mast cells in inflamed colon (Oshima et al. 1999; O'Hara et al. 2004). 5-HT-immunoreactive cells in thick sections of serosa and mesentery were observed in both inflamed and normal tissue (Fig. 8), which corresponded in morphology to mast cells when examined under Nomarsky optics (Yu et al. 1999; Nagata et al. 2001). In this location, no other 5-HT-immunoreactive structures were seen. Mast cells were found close to blood vessels before entering the gut wall, often in pairs or threes, and were particularly closely associated with CGRP-immunoreactive fibres, which normally followed a circuitous path around the blood vessel and mast cell(s). The distance between mast cells and CGRP fibres was measured and the number of mast cells that were within 25 µm of CGRP fibres was counted, although previous data indicate that these associations may be much closer (Stead et al. 1987). In inflamed specimens, the number of mast cells per section close to fibres was significantly greater (34.5 ± 6.3%) than in non-inflamed specimens (17.0 ± 4.9%, data from 15 random sections each, P < 0.05, t test).
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Discussion |
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In contrast to the changes in sensitivity to 5-HT we observed in lumbar splanchnic serosal and mesenteric afferent endings, their responsiveness to mechanical stimuli or capsaicin was not affected by inflammation. This is contrary to the findings of a recent report of sacral pelvic distension-sensitive fibres in rat colon after trinitrobenzene sulphonic acid (TNBS) colitis (Wynn et al. 2004), which found increases in mechanosensitivity that were associated with increased purinoceptor expression. Together, these findings suggest that endings closer to the site of inflammation may undergo changes in mechanosensitivity, whereas those more distant (or in a different pathway) may not. Our findings also indicate that increases in responsiveness are relatively confined to serotonergic responses, and are not a consequence of general increases in excitability, although this is evident in other disease models (Beyak et al. 2004). In a previous study, we showed that mechanical sensitivity of oesophageal mucosal afferents was in fact slightly reduced during oesophageal inflammation in ferrets, but it was indirectly increased during chemical activation (Page et al. 2000). In active colonic inflammation in humans, mechanical sensitivity may also be decreased (Chang et al. 2000), but may be increased paradoxically during periods of remission (Isgar et al. 1983).
Many reports describing inflammation-induced changes in afferent sensitivity have emerged from studies of cutaneous afferents. In the skin, mechanical hyperalgesia in inflammation is obvious, yet changes in mechanical responsiveness of primary afferents is controversial (Reeh et al. 1986; Kocher et al. 1987; Andrew & Greenspan, 1999; Koltzenburg et al. 1999; Schlegel et al. 2004), and reports of increased mechanosensitivity show that this is restricted to a small subpopulation of high-threshold afferents (Reeh et al. 1986; Kocher et al. 1987). However, there are much broader changes in chemical sensitivity, and these may prime afferent endings before a mechanical stimulus, and thereby contribute indirectly to the phenomenon of mechanical hyperalgesia (Blackshaw & Grundy, 1993; Koltzenburg et al. 1999; Banik et al. 2001). All this evidence point to the importance of increased chemosensitivity as a cause of increased apparent mechanosensitivity. Once afferent signals converge in the central nervous system, the potentiating effect of chemosensory pathways on mechanosensory inputs can be observed on second order neurons or their subsequent connections. We found, during activation of chemosensory pathways with bile (Lynn & Blackshaw, 1999), that an increase in spinal dorsal horn responses to colonic distension could be detected in vivo (Andrew & Blackshaw, 2001). Because serosal and mesenteric afferents are unlikely to be activated by physiological levels of distension, and are activated only by intense or rapid stretch (Lynn & Blackshaw, 1999; Brierley et al. 2004), the chemosensory role we have found for them may therefore be of importance in colouring the overall sensory input from the colon in the whole animal.
The present study was conducted in vitro without vascular perfusion, which makes it unlikely that appreciable levels of endogenous 5-HT release from the mucosa would reach endings. In vivo, on the other hand, circulating concentrations of 5-HT would exceed the threshold for activation of these endings, in particular, levels in the plasma seen after meals in symptomatic IBS patients (mean 6 x 10–8 M 3 h after a meal (Houghton et al. 2003) up to 1.4 x 10–6 M (Bearcroft et al. 1998)). The relevance of this is highlighted by the lowered threshold concentration of 5-HT we observed in inflammation. The lack of ongoing endogenous 5-HT action in vitro may also explain why we saw no significant change in spontaneous afferent fibre activity in inflammation. Increases in spontaneous discharge in vagal mucosal afferents are readily observed after mucosal deterioration or injury in vivo, and may be blocked by a 5-HT3 receptor antagonist (Blackshaw & Grundy, 1993).
We saw robust responses to mast cell degranulation only in inflamed preparations, which may be attributable to 5-HT release, although there are several other candidate mediators. In any case these data indicate greater likelihood of functional associations between sensory nerves and mast cells in the diseased state, as has already been shown anatomically in IBS (Barbara et al. 2004). Because the response to mast cell degranulation is not reproducible (presumably as a result of depletion of mast cell contents), we did not have the opportunity to investigate its pharmacology. However the response to 48/80 closely mimicked the response to 5-HT, and there was an increased association of 5-HT-containing cells with CGRP-immunoreactive fibres in the region of colon where we found receptive fields, as previously shown in a study of rat DSS colitis (Oshima et al. 1999). This would suggest that 5-HT is involved in the responses to 48/80, but not exclusively.
Our previous data on afferent sensitivity to 5-HT in non-inflamed colon show that the 5-HT-induced excitation was significantly reduced by the 5-HT3 receptor antagonist alosetron (Hicks et al. 2002), at a concentration that abolished excitation by a 5-HT3 receptor agonist. We confirmed the effect of alosetron on 5-HT responses in controls, and went on to determine the relative contribution of 5-HT3 receptors to the increased responses in inflammation. We found that reduction in the response to 5-HT was no longer observable after application of alosetron. This indicates that other excitatory 5-HT receptors are likely to play a larger role either because of downregulation of 5-HT3 receptors, or upregulation of others, or both. Alternatively, changes in relative expression of the two 5-HT3 receptor subunits in colonic afferents, or their phosphorylation state, may account for this result (e.g. Zeitz et al. 2002). Our findings with the adenylate cyclase inhibitor MDL 12330, which blocked alosetron-resistant responses, would indicate that the other subtypes involved signal via a pathway involving the G-protein GS and are therefore most likely to be 5-HT4, 6 or 7 subtypes (Gershon et al. 1990; Hoyer et al. 2002).
In conclusion, we have described a marked sensitization to 5-HT of lumbar splanchnic afferents from the colon in acute inflammation that is maintained after healing of the mucosa. This sensitization is sufficient to potentially underlie persistent sensory disturbances in humans after a bout of colonic infection or inflammation.
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References |
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