increased responsiveness of rat colonic splanchnic afferents to 5-ht after inflammation and recovery

J Physiol 579.1 (2007) pp 203–213 203

Increased responsiveness of rat colonic splanchnicafferents to 5-HT after inflammation and recovery

Jonathan R. Coldwell1,2, Benjamin D. Phillis1, Kate Sutherland1,2, Gordon S. Howarth2,4,5,6,7

and L. Ashley Blackshaw1,2,3

1Nerve-Gut Research Laboratory, Department of Gastroenterology, Hepatology and General Medicine, Royal Adelaide Hospital, Adelaide,South Australia, Australia2Discipline of Physiology, 3Department of Medicine and 4Discipline of Paediatrics, University of Adelaide, Adelaide, South Australia 5000, Australia5Gastroenterology Department, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia6 School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, Adelaide, Australia7 School of Biological Sciences, Flinders University, South Australia, Australia

5-Hydroxytryptamine (5-HT) activates colonic splanchnic afferents, a mechanism by which ithas been implicated in generating symptoms in postinfectious and postinflammatory states inhumans. Here we compared mechanisms of colonic afferent activation by 5-HT and mechanicalstimuli in normal and inflamed rat colon, and after recovery from inflammation. Colonicinflammation was induced in rats by dextran sulphate sodium. Single-fibre recordings of coloniclumbar splanchnic afferents revealed that 58% of endings responded to 5-HT (10−4 M) incontrols, 88% in acute inflammation (P < 0.05) and 75% after 21 days recovery (P < 0.05 versuscontrol). Maximal responses to 5-HT were also larger, and the estimated EC50 was reduced from3.2 × 10−6 to 8 × 10−7 M in acute inflammation and recovered to 2 × 10−6 M after recovery.Responsiveness to mechanical stimulation was unaffected. 5-HT3 receptor antagonism withalosetron reduced responses to 5-HT in controls but not during inflammation. Responses to themast cell degranulator 48/80 mimicked those to 5-HT in inflamed tissue but not in controls, andmore 5-HT-containing mast cells were seen close to calcitonin gene-related peptide-containingfibres in inflamed serosa. We conclude that colonic serosal and mesenteric endings exhibitincreased sensitivity to 5-HT in inflammation, with both an increase in proportion of respondersand an increase in sensitivity, which is maintained after healing of inflammation. This isassociated with alterations in the roles of 5-HT3 receptors and mast cells.

(Resubmitted 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: [email protected]

A large proportion of the 5-HT in the body is foundin the gastrointestinal tract, primarily contained withinenterochromaffin cells and mast cells (Yu et al. 1999). Itis released by food, toxins and chemotherapeutic agents(Andrews et al. 1990; Bearcroft et al. 1998). 5-HT releaseis well known to activate vagal afferent endings in theupper gastrointestinal tract (Blackshaw & Grundy, 1993;Hillsley et al. 1998; Zhu et al. 2001). 5-HT also activatescutaneous nociceptive primary afferents contributing to arole in inflammatory pain (Zeitz et al. 2002). It was shownrecently that high-threshold extrinsic primary afferentsfrom the colon also respond to 5-HT (Hicks et al. 2002).5-HT is thus implicated in mediating symptoms fromthe colon. Correspondingly, patients suffering from post-infectious irritable bowel syndrome (IBS) have increasedsources of 5-HT in the form of increased numbers ofenterochromaffin cells (Spiller et al. 2000) and increased

mast cell populations (O’Sullivan et al. 2000; Barbara et al.2004). They show increased postprandial 5-HT release(Bearcroft et al. 1998; Houghton et al. 2003) and a decreasein symptoms of IBS with 5-HT3 receptor antagonists(Camilleri et al. 2000; Camilleri, 2001). Metabolism of5-HT may also be disrupted in both IBS and inflammatorybowel disease (IBD) (Coates et al. 2004). Patients withIBD in the recovery period between episodes of activeinflammation may also have symptoms from the colon(Isgar et al. 1983).

It is well documented that inflammation can havea sensitizing effect on sensory nerves elsewhere inthe body leading to increased sensitivity to chemicalstimuli, but increased mechanical responsiveness remainscontroversial (Kocher et al. 1987; Schaible & Schmidt,1988; Habler et al. 1990; Andrew & Greenspan, 1999;Koltzenburg et al. 1999). Regardless of the cause, increased

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/jphysiol.2006.123158

204 J. R. Coldwell and others J Physiol 579.1

Table 1. Observations of disease activity in 25 inflamed and 20 recovered preparations, and histopathology in three of each duringtreatment with 2% dextran sulphate sodium and subsequent recovery after 21 days

Disease activity Histology

Days Index Stool Occult or Mucosal Basal cell CryptDSS score consistency gross bleeding ulceration hyperplasia Oedema destruction

1 0 Normal Negative − − − −2 0 Normal Negative − − − −3 1 Loose Negative − − − −4 1 Loose Negative + + + +5 2 Loose Positive ++ ++ + +6 3 Diarrhoea Positive ++ ++ ++ ++7 4 Diarrhoea Positive ++ ++ +++ +++Recovery 0 Normal/Loose Negative − − − −Severity of histological findings is indicated as: −, +, mild; ++, moderate; +++, severe; according to standard diagnostic criteria.Disease activity index is calibrated as described in the Methods.

afferent signals to the central nervous system are likelyto result in false perception of the environment andpain. Our aim in the present study was to determinewhether increased chemosensitivity or mechanosensitivityof colonic afferent fibres occurs during inflammation, andtherefore to gain insight into the role of 5-HT in visceralhypersensitivity. We examined changes in afferent 5-HTsensitivity in a rat model of colonic inflammation. Colitiswas induced by ingestion of dextran sulphate sodium(DSS) and this model was chosen for its reproducibility,its morphological similarities with human ulcerative colitisand its proven effectiveness in rat studies (Howarth et al.1998). We determined the effects of 5-HT and the mastcell degranulator compound 48/80 on colonic afferentsin normal rats and those with colitis using an isolatedpreparation in which the activity of lumbar splanchnicafferent fibres can be recorded during delivery of controlledstimuli to the colon (Lynn & Blackshaw, 1999). We alsolocalized 5-HT-immunoreactive mast cells to the colonicserosa and mesentery and determined their relation tocalcitonin gene-related peptide (CGRP)-immunoreactivenerve fibres; CGRP is an established marker for colonicafferents (Christianson et al. 2006).

Methods

Animals

A total of 98 adult Sprague-Dawley rats (180–200 g)were provided by the Institute of Medical and VeterinarySciences (IMVS) (Adelaide, South Australia). Animalswere housed in groups under standard laboratoryconditions (22◦C, 12 h light–dark cycle) until the studyperiod for convenience of care and stress minimization.They were then transferred to individual metabolismcages, fed a powdered diet suitable for metabolism cageuse, and allowed free access to water (Howarth et al.1998). All experiments were carried out with the approvalof, and according to the guidelines of, the animal ethicscommittees of the Institute of Medical and VeterinaryScience and University of Adelaide.

Induction of colonic inflammation

50 animals were designated for induction of colonicinflammation, with 37 animals used for inflamed studiesand 20 used for studies after recovery from inflammation.Animals were housed individually in Tecniplast ®

metabolism cages and acclimatized for a period of3 days. Colonic inflammation was induced by addition of2%w/v dextran sulphate sodium (DSS) (MW 40000, ICNBiochemicals, Cleveland, Ohio) to the animal’s drinkingwater for a period of 7 days. This produced mild symptomsas described in Table 1, but with no observable behaviouraldisturbances. 36 animals were designated for controls andwere housed in identical conditions but without DSStreatment for the same duration. Recovery animals wereallowed a 21-day recovery period post DSS treatmentbefore subjection to the electrophysiological protocol.Afferent recordings were obtained from the majorityof preparations, with numbers of fibres given for eachprotocol in Results.

Validation of inflammation

Three animals from each of the groups wereused for validation of the inflammatory protocol.Inflammation was validated by disease activity indexscore, histopathological assessment and myeloperoxidase(MPO) assay. The disease activity was scored on a severityscale of 0–4 based on physical observations of weight loss,stool consistency and colonic bleeding. Histopathologicalassessment using haematoxylin and eosin staining wasused to show morphological changes associated with DSStreatment over the 7 day period and subsequent recoveryafter a further 21 days. Animals from control, inflamedand recovery groups were anaesthetized with sodiumpentobarbitone (Nembutal, 60 mg kg−1 i.p.) and theabdomen opened by midline incision. A 2 cm length ofdistal colon was removed and divided into 1 cm segmentsof which one was snap frozen in liquid nitrogen forMPO assay and the other fixed in 10% formaldehyde andprocessed for histopathological assessment (Department

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society

J Physiol 579.1 Afferent 5-HT sensitivity in inflammation and recovery 205

of Tissue Pathology, IMVS). Animals were then killedby exsanguination and excision of thoracic organs whileunder deep anaesthesia. Haematoxylin and eosin-stainedsections were assessed for colonic damage, oedema andinflammatory cell infiltration. Neutrophil infiltration asa measure of inflammation was also assessed by MPOassay according to the protocol described by Morris et al.(1989).

Electrophysiological protocol

Dissection was carried out according to a previouslydescribed protocol (Lynn & Blackshaw, 1999). Rats fromall 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 attachedlumbar colonic nerves (LCN) and a bundle containingthe inferior mesenteric artery, abdominal aorta, inferiormesenteric ganglion, intermesenteric nerve and lumbarsplanchnic nerve (LSN) according to the classification ofBaron et al. (1988), and placed in cold modified Krebssolution (bicarbonate buffer; 4◦C). Rats were killed asin the histology protocols. The distal colon was openedlongitudinally and pinned flat, mucosal side down, inan isolated perfused organ bath (Danz Instruments,Blackwood, South Australia) with the LCN insertionsorientated to lie along the edge of the opened preparation.Connective tissue was dissected away from the neuro-vascular bundle which was then drawn through into aseparate paraffin-filled recording chamber. The attachedLSN was finely dissected from the aorta and de-sheathedfrom the transected nerve stump to expose the nervefibres; these were divided into fine bundles to be placedonto fine platinum recording electrodes for single afferentfibre recording. Extraluminal receptive fields on the serosalsurface and the mesentery were located by responsesto blunt probing with a glass rod and discriminated ashigh-threshold serosal/mesenteric afferents by their lackof response to fine stroking with a calibrated von-Freyhair (10 mg) or circular stretch (8 mm) as previouslydescribed (Lynn & Blackshaw, 1999; Berthoud et al. 2001;Hicks et al. 2002). Signals from the recording electrodewere differentially amplified, filtered and the analog signalsampled at a rate of 20 kHz via a 1401 data interface(CED, Cambridge, UK). This was recorded to a Pentium IIInotebook computer and analysed using Spike II software(CED). 5-HT (10−10–10−3 m) was applied to the receptivefields via a 1 cm diameter stainless steel ring placed directlyaround the corresponding receptive field, after residualKrebs solution had been aspirated. Each concentration of5-HT was applied for a period of 2–3 min followed by a10 min washout. 5-HT (10−4 m) was used to determinewhether or not a fibre was responsive to 5-HT as thiswas shown previously to be a maximal concentration(Hicks et al. 2002). Responses to drugs were counted

as having occurred if a reproducible > 25% increase indischarge occurred with the maximum concentration. Forquantification of responses, activity induced by 5-HT wasmeasured over the second minute of response. This periodwas selected to accurately reflect the peak of responseand compensate for latency of response onset especiallyat lower concentrations. Concentration–response curvesfor 5-HT (10−10–10−3 m) were constructed and analysedfor changes in response between control and inflamedcolons and colons after recovery from inflammation. Nodifferences in 5-HT responsiveness or histological scoreswere seen when inflammation was induced instead by5% DSS in male rats housed in wire-bottomed cages.Responses to probing were measured prior to testing ofchemosensitivity as total spikes evoked by a 1 s probeapplication, and as mean peak instantaneous frequency.These responses were averaged over eight trials. Nochanges in probing responses were observed before andafter treatment with 5-HT. Spontaneous discharge wasmeasured over 1 min prior to testing of chemosensitivity.Mechanical thresholds were determined with 10, 50, 200and 1000 mg von Frey hairs applied perpendicularly to thereceptive field. In studies with antagonists, colons wereincubated for 2 min with alosetron (2 × 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 cyclaseinhibitor MDL 12330 (30 µm) was added to alosetronand the response to 5-HT tested in the presence of bothantagonists.

Localization of 5-HT- and CGRP-like immunoreactivityin normal and inflamed tissue

Distal colon was removed from rats after trans-cardial perfusion with 4% paraformaldehyde under deepanaesthesia with sodium pentobarbitone (60 mg kg−1

i.p.). The region corresponding to that used for electro-physiological 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 washedthree times for 20 min with phosphate-buffered saline(PBS) at pH 7.4 then cryoprotected with 30% sucrosein PBS for 24 h at 4◦C. The colon was then frozen and20 µm transverse sections cut. Sections were air dried atRT for 10 min, after which the tissue was washed withPBS (pH 7.4) including 0.1% Triton X-100 (PBST). Tissuewas then blocked with 2% goat serum, 1% bovine serumalbumin, 0.005% Tween 20 and 0.1% gelatin in PBST for60 min at RT and subsequently incubated with monoclonalanti-5-HT (1 : 100; Dako, Sydney, Australia) and rabbitanti-CGRP (1 : 200; Calbiochem) in blocking solution inPBST overnight at 4◦C. Tissue was then washed threetimes with PBST, then incubated a mixture of secondaryantibodies: goat anti-rabbit Alexa Fluor 350 and donkeyanti-mouse Alexa Fluor 555 (5 µg ml−1; Molecular Probes,



USA) for 45 min at RT. The tissue was then washedthree times with PBST and mounted with ProLong Anti-fade (Molecular Probes, USA). Slides were allowed todry overnight before viewing with an Olympus BX51epifluorescence microscope. Images were taken with aPhotometrics CoolSnap fx monochrome camera thenre-coloured. Negative controls were prepared as above withthe primary antibody omitted. Both antibodies have beenshown to be specific for their respective epitope in previousstudies (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 withDunnet’s post hoc test or by two-way ANOVA forconcentration–response curves. Proportions of fibresresponding were compared with a Fisher’s exact test.Statistical analyses, EC50 values and curve fits were alldetermined using Graphpad Prism Software (v 3.02; SanDiego, CA, USA) according to standard pharmacologicalmethods. Associations between CGRP-containing fibres

Figure 1. Examples of disease progression and regression in haematoxylin and eosin-stained colonicsections (20 µm)A, control with normal colonic morphology. B, inflamed colon of a dextran sulphate sodium (DSS)-treated (7 days)animal. The field of view shows examples of crypt destruction, ulceration, oedema and polymorph infiltration inthe mucosa and submucosa (arrows), whereas outer muscle and serosa appear unchanged. C, animal 21 daysafter DSS treatment exhibiting recovery to morphology as in controls.

and 5-HT-containing mast cells were counted from 15randomly selected sections from four rats.

Results

Inflammatory response to DSS

The DSS model of colitis has been used extensivelyin rats. We re-validated this model in our laboratoryin both active and recovery phases of inflammation.Disease activity by morphological examination showed aprogressive deterioration of the colonic mucosa over the7 day DSS treatment programme and subsequent recovery21 days after DSS (Table 1). Ulceration of the colonicmucosa and infiltration of inflammatory cells were firstdetected by light microscopy at day 4 of DSS treatmentand increased in severity until day 7. This was accompaniedby submucosal oedema and destruction of colonic crypts.After recovery, 21 days after DSS treatment, morphologicalchanges had reversed; morphology was indistinguishablefrom controls (Fig. 1). Blood was detected in the stool fromday 4 and gross colonic bleeding was apparent after 7 days.



MPO assay for neutrophil infiltration (Fig. 2) showed agreater than 500% increase in inflamed animals at day 7compared with controls (n = 3). MPO levels after recoveryfrom inflammation had returned to basal levels.

Afferent fibre responses to 5-HT and mechanicalstimulation

As we have reported previously (Lynn & Blackshaw, 1999;Berthoud et al. 2001; Hicks et al. 2002), serosal/mesentericafferents were located throughout the length of thepreparation and were clustered towards the mesentericborder with receptive fields located on or nearblood vessels. Many colonic serosal/mesenteric afferentshad multiple receptive fields in both the mesentery andserosal surface. Responses to 5-HT were maintainedthroughout the period of superfusion, showing little ifany adaptation during exposure, and normally continuedfor approximately 2 min after washout before dischargereturned to basal levels (Fig. 3), as we have foundpreviously (Hicks et al. 2002).

We used 12 animals from each group to determine theproportion of colonic afferent fibres responding to 5-HT(Fig. 4A). Seven out of 12 fibres tested (58.3%) respondedto 10−4 m 5-HT in controls compared with 14 out of 16(87.5%) in inflammation and nine out of 12 (75%) inrecovery (both P < 0.05, Fisher’s exact test). It was possibleto determine concentration–response relationships overa range of 5-HT concentrations (10−10–10−4 m) in most5-HT-responsive fibres from each group (Fig. 4B). Thisshowed a significant increase in sensitivity to 5-HT inboth the inflamed and recovered state (P < 0.01, two-wayANOVA). The response at maximum concentration,indicative of efficacy, evoked on application of 5-HTwas significantly larger during inflammation and thissignificance was sustained in animals after recovery at10−5 and 10−4 m 5-HT (P < 0.05). EC50 was estimated

Figure 2. Myeloperoxidase (MPO) assayResults showed a significant increase from 2.34 ± 0.57 × 10−4 incontrols to 1.93 ± 0.15 × 10−3 U mg−1 in inflamed colons. MPOlevels in animals after recovery had returned to basal levels of2.56 ± 0.63 × 10−4 U mg−1 (P < 0.05, n = 3, one-way ANOVA withDunnet’s post hoc test).

from non-linear regression of the concentration–responsecurves. Because a plateau was not reached, an accuratequantification of EC50 was not possible. Estimated EC50

for 5-HT was reduced from 3.2 × 10−6 in controlsto 8.2 × 10−7 during inflammation and increased to2.0 × 10−6 m in animals after recovery. In order toinvestigate whether other aspects of chemosensitivity orgeneral excitability were affected during inflammation,the response to a maximal concentration of capsaicin(3 × 10−6 m), which is a very potent activator of thesefibres (Berthoud et al. 2001), was compared betweenthree controls and five inflamed preparations, and therewas a comparable 5-fold mean increase in discharge in

Figure 3. Examples of serosal/mesenteric afferent responses onapplication of 5-HT (10 µm) for 3 min in spontaneously activefibresControl (A), a dextran sulphate sodium (DSS)-treated animal (B) andanimal following recovery (C). A–C, upper traces show rate of nervefiring (bin size, 2 s). The lower traces show a raw record of nerveactivity, with one active fibre evident. 5-HT was introduced over theperiod indicated by first arrows, and was washed out at the pointshown by second arrows. Note increase in size of response in B and Ccompared with control (A).



responsive fibres from both groups, indicating that theincrease in chemosensitivity of afferents was relativelyspecific to 5-HT.

The effects of 5-HT3 receptor antagonism on afferentresponses to 5-HT was tested in five control and inflamedpreparations (Fig. 5). As we showed in our previous study(Hicks et al. 2002), a maximally effective concentrationof alosetron significantly reduced responses to 5-HT incontrols. Administration of the adenylate cyclase inhibitorMDL 12330 (30 µm) in addition to alosetron completelyabolished the remaining response to 5-HT in threepreparations. In inflamed preparations, alosetron had nosignificant effect on the response to 5-HT (Fig. 5).

In nine preparations from each group, mechanicalsensitivity to a 1 s standardized blunt probe of thereceptive field was assessed systematically (Fig. 6A), andshowed no significant difference between control andinflamed preparations in terms of total spikes evokedper stimulus (Fig. 6B), or in terms of mean peak

Figure 4. Proportion of responders and size of response to 5-HTA, percentage of high threshold serosal/mesenteric afferent fibresresponding to 5-HT was 58.3% in controls and significantly increasedto 87.5% in inflamed and 75% in recovered preparations (P < 0.05,n = 12 each, Fisher’s exact test). B, concentration–responserelationships to 5-HT were performed in serosal/mesenteric afferentsshowed a significant increase of activity in inflamed and recoveredpreparations (P < 0.01, n = 7–9, two-way ANOVA). Activity wasaveraged over the first 60 s of response. From theconcentration–response curves an estimate of curve fit was performedusing Prism software, from which EC50 for 5-HT in both groups wasestimated. EC50 was 3.2 × 10−6 M 5-HT in controls, 8.2 × 10−7 M

5-HT in inflamed tissue and 2.0 × 10−6 M after recovery.

instantaneous frequency (Fig. 6C). Spontaneous dischargeof serosal/mesenteric afferent endings was not significantlyaltered in inflamed conditions, or following recovery(Fig. 6D). During the investigation of mechanically evokedresponses, no changes were observed either in the sizeor the distribution of receptive fields. The von Freythreshold of serosal/mesenteric afferents in control colonswas reproducibly 200 mg when tested with 10, 50, 200and 1000 mg stimuli and this was unchanged in bothinflammation and recovery.

Afferent fibre responses to mast cell degranulation

In separate experiments to those described above (n = 6controls and n = 7 inflamed preparations), the afferentresponse to the mast cell degranulator, compound 48/80(1–30 µg ml−1), was tested. No significant increases inafferent discharge were recorded in afferents from controlanimals after application of the maximum concentration.In inflamed tissue, four out of seven endings respondedto compound 48/80 with an increase of discharge greaterthan 25% (P < 0.01, Fisher’s exact test, Fig. 7). Proportionsof responsive endings and magnitude of response aretherefore similar to that seen on application of 5-HT.

Localization of 5-HT-like immunoreactivity in normaland inflamed tissue

Tissue from five control and five inflamed preparationsrevealed different immunolabelling for 5-HT and forthe sensory neural marker CGRP, which labels alarge proportion of rat colonic splanchnic afferents(Christianson et al. 2006). DSS-treated colon showed botha higher level of background 5-HT-like immunoreactivityand an increased number of 5-HT-containing cells in themucosa and submucosa (data not shown), which were

Figure 5. Effects of 5-HT3 receptor antagonism onserosal/mesenteric afferent responses to 5-HT (100 µm) incontrol and inflamed tissueAlosetron (200 nM) significantly reduced responses to 5-HT in controls(n = 5), whereas it had no significant effect in the inflamed group(n = 5).



of comparable size and distribution to those previouslyshown to be mast cells in inflamed colon (Oshima et al.1999; O’Hara et al. 2004). 5-HT-immunoreactive cellsin thick sections of serosa and mesentery were observedin both inflamed and normal tissue (Fig. 8), whichcorresponded in morphology to mast cells when examinedunder Nomarsky optics (Yu et al. 1999; Nagata et al.2001). In this location, no other 5-HT-immunoreactivestructures were seen. Mast cells were found close toblood vessels before entering the gut wall, often in pairsor threes, and were particularly closely associated withCGRP-immunoreactive fibres, which normally followeda circuitous path around the blood vessel and mastcell(s). The distance between mast cells and CGRP fibreswas measured and the number of mast cells that werewithin 25 µm of CGRP fibres was counted, althoughprevious data indicate that these associations may bemuch closer (Stead et al. 1987). In inflamed specimens,the number of mast cells per section close to fibres wassignificantly greater (34.5 ± 6.3%) than in non-inflamedspecimens (17.0 ± 4.9%, data from 15 random sectionseach, P < 0.05, t test).

Discussion

The results of the present study show that excitation ofcolonic afferents by 5-HT is greater after a period ofinflammation in three ways: in terms of the proportionof fibres responding; in the size of the response; and in thereduced threshold for response. Furthermore, most of thechanges in 5-HT sensitivity are maintained after healingof the mucosa as assessed histologically and biochemically– a situation closely resembling postinfectious or post-inflammatory hypersensitivity in humans (Spiller, 2003).A particularly striking feature of the changes we observedis the fact that the afferent endings that were affected arelocated in the serosa and mesentery away from the siteof inflammation. This means that mediators released atthe site of inflammation may evoke changes in afferentendings after they have diffused over some distance orreached the endings via the venous outflow. We oftennoticed that the endings we studied were located at thejunctions of mesenteric blood vessels, which would placethem in an optimal location to be influenced by mediatorsreleased into the venous outflow from the colon or arrivingvia its arterial supply. There are a number of candidates formediators causing changes in afferent function, probablythe best characterized of which is nerve growth factor,which has been shown to be responsible for increases inchemo- and thermosensitivity in skin afferents (Bevan &Winter, 1995; Koltzenburg et al. 1999).

In contrast to the changes in sensitivity to 5-HT weobserved in lumbar splanchnic serosal and mesentericafferent endings, their responsiveness to mechanicalstimuli or capsaicin was not affected by inflammation.

This is contrary to the findings of a recent reportof sacral pelvic distension-sensitive fibres in ratcolon after trinitrobenzene sulphonic acid (TNBS)colitis (Wynn et al. 2004), which found increases inmechanosensitivity that were associated with increased

Figure 6. Mechanically-evoked and spontaneous activity ofserosal/mesenteric afferent fibresExamples of mechanical stimulation with a blunt probe (A) in colonicafferent fibres from control (a), inflamed (b) and recovered (c)preparations. Impulses evoked by a 1 s calibrated blunt probe (B) andmean peak instantaneous frequency of the probe response (C) showedno significant changes between colonic afferent fibres from control,inflamed or recovered preparations (P > 0.05, n = 9, one-way ANOVAwith Dunnet’s post hoc test). Spontaneous discharge in responsivefibres was not significantly altered in inflammation and recovery.



purinoceptor expression. Together, these findings suggestthat endings closer to the site of inflammation mayundergo changes in mechanosensitivity, whereas thosemore distant (or in a different pathway) may not. Ourfindings also indicate that increases in responsiveness arerelatively confined to serotonergic responses, and are not aconsequence of general increases in excitability, althoughthis is evident in other disease models (Beyak et al. 2004).In a previous study, we showed that mechanical sensitivity

Figure 7. Colonic afferent responsiveness to mast cell degranulation in control and inflamed tissueA, original record of response to compound 48/80 (30 µg ml−1) added to the ring around the receptive fieldfor approximately 3 min from a splanchnic afferent fibre innervating the inflamed colon. B, occurrence ofresponders and non-responders to 48/80 in control (n = 6) and inflamed (n = 7) tissue, showing significantly moreresponders (n = 4) in inflamed than control tissue (n = 0; P < 0.01). C, group data showing concentration–responserelationship for compound 48/80 in all fibres tested in control and inflamed tissue. At the maximum dose(30 µg kg−1), there was a significant increase in response in inflamed tissue (P < 0.01, one-way ANOVA withDunnet’s post hoc test), which was not seen in controls.

of oesophageal mucosal afferents was in fact slightlyreduced during oesophageal inflammation in ferrets, but itwas indirectly increased during chemical activation (Pageet al. 2000). In active colonic inflammation in humans,mechanical sensitivity may also be decreased (Chang et al.2000), but may be increased paradoxically during periodsof remission (Isgar et al. 1983).

Many reports describing inflammation-inducedchanges in afferent sensitivity have emerged from



studies of cutaneous afferents. In the skin, mechanicalhyperalgesia in inflammation is obvious, yet changesin mechanical responsiveness of primary afferents iscontroversial (Reeh et al. 1986; Kocher et al. 1987; Andrew& Greenspan, 1999; Koltzenburg et al. 1999; Schlegelet al. 2004), and reports of increased mechanosensitivityshow that this is restricted to a small subpopulationof high-threshold afferents (Reeh et al. 1986; Kocheret al. 1987). However, there are much broader changesin chemical sensitivity, and these may prime afferentendings before a mechanical stimulus, and therebycontribute indirectly to the phenomenon of mechanicalhyperalgesia (Blackshaw & Grundy, 1993; Koltzenburget al. 1999; Banik et al. 2001). All this evidence pointto the importance of increased chemosensitivity as acause of increased apparent mechanosensitivity. Onceafferent signals converge in the central nervous system,the potentiating effect of chemosensory pathways onmechanosensory inputs can be observed on second orderneurons or their subsequent connections. We found,during activation of chemosensory pathways with bile(Lynn & Blackshaw, 1999), that an increase in spinal dorsalhorn responses to colonic distension could be detectedin vivo (Andrew & Blackshaw, 2001). Because serosaland mesenteric afferents are unlikely to be activated byphysiological levels of distension, and are activated onlyby intense or rapid stretch (Lynn & Blackshaw, 1999;Brierley et al. 2004), the chemosensory role we have foundfor them may therefore be of importance in colouring theoverall sensory input from the colon in the whole animal.

The present study was conducted in vitro withoutvascular perfusion, which makes it unlikely thatappreciable levels of endogenous 5-HT release from themucosa would reach endings. In vivo, on the other hand,circulating concentrations of 5-HT would exceed the

Figure 8. Immunohistochemistry for 5-HT andCGRP in rat colon in the region of the mesentericborderSections were taken from dextran sulphate sodium(DSS)-treated rats. A–D, arrowheads indicate calcitoningene-related peptide-immunoreactive fibres (green) inassociation with 5-HT-immunoreactive cells (red,arrows). Section in A shows CGRP labelling also inmyenteric plexus (mp). Section in D is taken from aregion of mesentery several millimetres from the colonicwall. lm, longitudinal muscle; bv, serosal/mesentericblood vessel. Scale bar, 50 µm.

threshold for activation of these endings, in particular,levels in the plasma seen after meals in symptomatic IBSpatients (mean 6 × 10−8 m 3 h after a meal (Houghtonet al. 2003) up to 1.4 × 10−6 m (Bearcroft et al. 1998)). Therelevance of this is highlighted by the lowered thresholdconcentration of 5-HT we observed in inflammation.The lack of ongoing endogenous 5-HT action in vitromay also explain why we saw no significant changein spontaneous afferent fibre activity in inflammation.Increases in spontaneous discharge in vagal mucosalafferents are readily observed after mucosal deteriorationor injury in vivo, and may be blocked by a 5-HT3 receptorantagonist (Blackshaw & Grundy, 1993).

We saw robust responses to mast cell degranulation onlyin inflamed preparations, which may be attributable to5-HT release, although there are several other candidatemediators. In any case these data indicate greaterlikelihood of functional associations between sensorynerves and mast cells in the diseased state, as has alreadybeen shown anatomically in IBS (Barbara et al. 2004).Because the response to mast cell degranulation is notreproducible (presumably as a result of depletion ofmast cell contents), we did not have the opportunity toinvestigate its pharmacology. However the response to48/80 closely mimicked the response to 5-HT, and therewas an increased association of 5-HT-containing cells withCGRP-immunoreactive fibres in the region of colon wherewe found receptive fields, as previously shown in a studyof rat DSS colitis (Oshima et al. 1999). This would suggestthat 5-HT is involved in the responses to 48/80, but notexclusively.

Our previous data on afferent sensitivity to 5-HTin non-inflamed colon show that the 5-HT-inducedexcitation was significantly reduced by the 5-HT3 receptorantagonist 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 responsesin controls, and went on to determine the relativecontribution of 5-HT3 receptors to the increased responsesin inflammation. We found that reduction in the responseto 5-HT was no longer observable after applicationof alosetron. This indicates that other excitatory 5-HTreceptors are likely to play a larger role either becauseof downregulation of 5-HT3 receptors, or upregulationof others, or both. Alternatively, changes in relativeexpression of the two 5-HT3 receptor subunits in colonicafferents, or their phosphorylation state, may account forthis result (e.g. Zeitz et al. 2002). Our findings with theadenylate cyclase inhibitor MDL 12330, which blockedalosetron-resistant responses, would indicate that theother subtypes involved signal via a pathway involving theG-protein GS and are therefore most likely to be 5-HT4, 6or 7 subtypes (Gershon et al. 1990; Hoyer et al. 2002).

In conclusion, we have described a marked sensitizationto 5-HT of lumbar splanchnic afferents from the colonin acute inflammation that is maintained after healing ofthe mucosa. This sensitization is sufficient to potentiallyunderlie persistent sensory disturbances in humans aftera bout of colonic infection or inflammation.

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Acknowledgements

This work was funded by the National Health and MedicalResearch Council Grant no. 104814 and 298942; J.R.C. andK.S. were supported by University of Adelaide PostgraduateScholarships and Sigma Xi grant in aid of research (J.R.C.);L.A.B. was supported by an National Health and MedicalResearch Council (NHMRC) Senior Research Fellowship. Wethank the staff of the Department of Tissue Pathology ofthe IMVS for their help with scoring of histopathologicalfeatures and processing of specimens. Helen Bougesis of theWomen’s and Children’s hospital assisted with animal-relatedwork and Chandra Kirana of Commonwealth Scientific andIndustrial Research Organisation (CSIRO) with biochemicalassay procedures. Chris Martin of the Nerve-Gut ResearchLaboratory assisted with the antagonist experiments.


increased responsiveness of rat colonic splanchnic afferents to 5-ht after inflammation and recovery

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