Tu1989

Real Time In Vitro Monitoring of Inflammation Induced Motility Changes andProtective Effects of STW 5 in a Newly Designed ModelAndrei Sibaev, Birol Yuoce, Olaf Kelber, Dieter Weiser, Heba Abdel-Aziz, Burkhard Göke,Martin Storr

Background and Aims: The question was raised, whether the herbal medicine STW 5 actson contractions elicited after electrical field stimulation (EFS) and on intestinal slow waveactivity in the small intestine of mice, in a new type of an In Vitro model of trinitrobenzenesulfonic acid (TNBS) triggered inflammation. Methods: In the organ bath, segments ofdistal ileum of Balb/C mice were used for registration of spontaneous and EFS stimulatedcontractions. In the electrophysiological studies, segments of distal ileum (with minimum50% increase in EFS induced contraction after 90 min of intraluminal TNBS application)were used for intracellular recordings, after removal of mucosa and submucosa. STW 5 wastested in a dilution of 1:100 in an organ bath and compared to vehicle (31% ethanol solutiondiluted 1:100), following intraluminal application of TNBS (0,01M solution, 3 cm H2Opressure) or Krebs solution for 90 min. Results: TNBS induced a significant time dependentenhancement of contractility (90 min: 155.1 ± 6.5% vs. control, n=15). STW 5 applied intothe organ bath reduced basal tone (-22.1±2.1% TNBS vs. -17.7±1.5% no TNBS n=15), EFSinduced contractility (-61.3±3.5% TNBS vs. -67.6±3.7% no TNBS n=10) and significantlyprevented TNBS induced increase of EFS induced contractions (97.4 ± 4.1% STW5+TNBS vs.TNBS/no STW 5 155.1 ± 6.5% n=10). Electrophysiological parameters were not significantlyinfluenced in TNBS pretreated or control preparations. No significant effects on intracellularrecordings of resting membrane potential, slow wave amplitude and frequency were observedmaking neuronal and muscular effects unlikely. Summary and Conclusions: Stimulationwith TNBS results in inflammation induced changes of motility, which are reversed orprevented by STW 5. The underlying mechanisms may be of relevance in the treatment offunctional gastrointestinal and subclinical inflammatory gastrointestinal diseases.

Tu1990

Expression of Mucosal Defense Mediators and Cytokines are Dependent onthe Number of Exposures to C. Jejuni in a Rat Model of Post-Infectious IBSZachary Marsh, Walter Morales, Joel Alpern, Emily Rooks, Gene Kim, Venkata B.Pokkunuri, Jaekyu Sung, Stacy Weitsman, Christopher Chang, Mark Pimentel

Elevated gut expression of specific mediators of innate immunity such as, TLR-4 and β-Defensin-2, and cytokines such as IL-6, and IL-8 have been foundin individuals with irritablebowel syndrome (IBS) and post-infectious IBS. In a recently validated animal model of PI-IBS, rats develop a change in bowel form, small intestinal bacterial overgrowth (SIBO) andincreased rectal intraepithelial lymphocytes—mimicking IBS and PI-IBS in humans. In thisstudy, we utilize this animal model to examine the role of mucosal defense mediators andcytokines in relation to the development of IBS features, and further examine the effect ofone versus two exposures to Campylobacter jejuni. Methods: A total of 120 rats wereanalyzed. 50 rats were gavaged with 108 CFU of C.jejuni 81-176 both at age 21 days andagain as adults (2.5 months old). Another 50 rats were infected only once with 108 CFUof C. jejuni as adults. The remaining 20 rats served as uninfected controls. Clearance of C.jejuni infection was based on 2 consecutive days of negative stool cultures on Campylobacter-selective plates. 3 months after clearance of adult exposure, animals were euthanized andileal expression of β-Defensin-2, TLR-4, IL-8, β-Defensin-6, TNF-α, IL-6, and IL1β weredetermined through quantitative RT-PCR (BioRad iQSYBR Kit, BioRad iCycler). Expressionof these genes was normalized to β-Actin expression in each rat. Results: Rats exposed toC. jejuni twice were more likely to have SIBO compared to rats singly exposed as adults(47% vs. 26%, P=0.02). Relative expression of β-Defensin-2, TLR-4, IL-8, and β-Defensin-6 were all significantly elevated (p<0.05) in infected rats compared to controls. Expressionof these genes was stepwise significantly greater with second exposure compared to singleexposure to C. jejuni (p<0.05 for these outcome biomarkers). In contrast, TNF-α expressionwas only significantly increased insingly infected rats compared to controls (p<0.05). ForIL-6 expression, singly infected rats had decreased levels compared to the control group(p<0.01). Finally, expression of IL1β, while exhibiting a decreasing trend between singleinfected and double infected versus controls, did not reach significance. Significantly, thesecytokine and mucosal defense alterations did not depend on the development of SIBO orchronic bowel alterations. Conclusions: Months after clearance of C. jejuni, rats have apersistent change in expression of ileal mucosal immunity and defense mediators. The extentof these changes is not dependent on the development of a SIBO phenotype but rather onthe number of exposures to C. jejuni. These data in animal models suggest that alterationsin cytokine levels found in IBS may be a reflection of past gastroenteritis and not of thedevelopment of IBS specifically, although this hypothesis remains to be tested in humans.

Tu1991

Vagal Efferent Neuronal Integrity and Response to TRH in an Animal Model ofSpinal Cord Injury-Induced GastroparesisEmily M. Swartz, Gregory M. Holmes

Our previous data utilizing a rat model of experimental spinal cord injury (SCI) has demon-strated that SCI elicits immediate and profound gastric dysmotility as well as a prolongedreduction in mesenteric blood flow to the digestive tract. The stomach is modulated bygastric sensory signals transmitted via the vagus nerve to the nucleus tractus solitarius (NTS).The NTS integrates sensory information with signals from throughout the CNS and projectsto nuclei which include preganglionic efferent neurons located in the dorsal motor nucleusof the vagus (DMV). This vago-vagal reflex remains anatomically intact after SCI and ourprevious reports begin to suggest that SCI diminishes vagal afferent sensitivity. However,neurons comprising the DMV in the rat are known to respond more severely to axonalinsult than other peripherally projecting neurons and may be adversely affected by chronicmesenteric hypoperfusion. Therefore, derangements in efferent signals to the stomach mayalso account for reduced motility after SCI. Thyrotropin releasing hormone (TRH) profoundly

S-895 AGA Abstracts

modulates gastricmotility by a direct depolarization of DMVneurons and offers a pharmacolo-gical tool to test DMV efferent integrity. We first tested the hypothesis that experimentalSCI induces functional impairment of rat DMV motoneurons by microinjecting TRH intothe DMV after SCI or control surgery. Male Wistar rats (n=40) were chosen at random forSCI or surgical control. The animals were anaesthetized and spinal T3 was exposed vialaminectomy. SCI rats were subjected to a 300 kDyne midline contusion injury. Three dayslater, the rats were re-anaesthetized, gastric motility and tone were recorded with straingauges applied to the corpus surface and TRH (0, 3, 10, 30 or 100 pmoles/60nl) wasmicroinjected in the left DMV adjacent to area postrema. Our data reveal a rightward dose-response shift to unilateral DMVmicroinjection of TRH. Specifically, gastric motility responseto 3pmoles TRH was reduced in SCI animals vs. surgical controls. Secondly, we began totest the hypothesis that experimental SCI induces anatomical changes in DMV neurons. Sixdays prior to spinal surgery, rats (n=8) were anaesthetized, the stomach isolated, and Choleratoxin B (CTB; 0.5%, 5ul total) was injected into the anterior gastric corpus. Rats weretranscardially perfused and brainstem sections were prepared for CTB immunohistochemistry9 days after spinal surgery. There were no differences between SCI and controls regardingthe number of CTB immuno-positive, DMV neurons in the region corresponding to ourTRH microinjection site. Combined with our previous reports, these data begin to suggestthat SCI may also impair vagal preganglionic motoneurons. The mechanism leading to thisimpairment remains to be determined but does not include DMV neuron loss. Funding:NINDS 49117

Tu1992

The Neuroligin 3 Arg451cys (Nl-3) Mouse Model of Autism Shows AlteredColonic Function In VitroMelina Ellis, Ali M. Taher, Sonja McKeown, Elisa L. Hill, Joel C. Bornstein

Purpose: Gastrointestinal (GI) symptoms and disorders affect up to 80% of autism spectrumdisorder (ASD) patients, but the underlying mechanisms are unknown. Mutations in ASDpatients that alter synaptic function in the brain may also alter function of the enteric nervoussystem (ENS) to produce bowel disorders. This study aimed to determine if synaptic proteinsknown to be mutated in autism are expressed in the ENS and if isolated colon from theneuroligin 3 Arg451Cys knockin (NL-3) mouse model of ASD shows altered function.Methods: Qualitative RT-PCR using designed primers for neuroligins 1-3 and neurexins 1-2, cell adhesion molecules implicated in ASD, was performed using RNA isolated fromduodenum and colon of control mice. Video recordings of isolated segments of NL-3 andwild type (WT) mouse colon were processed to create spatiotemporal maps of motor patternsusing in-house edge detection software. Colonic migrating motor complexes (CMMCs),motor patterns observed in empty colon, were assessed in control and in the presence ofbath applied antagonists acting on GABA (GABAA: bicuculline 10 μM, gabazine 10 μM;GABAB: CGP 54626 10 μM), and serotonin (tropisetron, 5-HT3,4, 10 μM) receptors. Statisticalcomparisons were made using two-way analysis of variance with p values < 0.05 indicatingsignificance.Results: mRNA for neuroligins 1-3 and neurexin 1 and 2 was expressed inmyenteric plexus smooth muscle preparations from colon and duodenum, but not in mucosa.There were no observed strain differences in CMMCs in control solutions (WT: 4.3 ± 0.4,NL-3: 4.1 ± 0.4; both n = 50; data are expressed as number of CMMCs every 15 min).Bicuculline reversibly depressed CMMCs in NL-3 colon, but had no effect in WT (WT: 4.5± 0.8, NL-3: 2.8 ± 0.6; both n = 11, p = 0.001). Other properties of CMMCs like propagationspeed did not differ between NL-3 and WT in bicuculline, indicating that GABAA blockadedepressed initiation of the CMMCs and not their propagation. Gabazine slightly depressedCMMC frequency in WT, but was significantly more effective in NL-3 (WT: 2.7 ± 0.6, NL-3: 1.7 ± 0.4; both n = 8 p < 0.05) confirming that the effect was on GABAA receptors. CGP54626 (n = 5) had no effect. The 5-HT3/5-HT4 antagonist tropisetron decreased CMMCsin WT, but the depression was significantly more pronounced in NL-3 (WT: 3.6 ± 0.8, NL-3: 1.6 ± 0.8; n ≥ 8, p < 0.01). Conclusion: This study reveals the first evidence that amutation associated with autism in humans produces gastrointestinal dysfunction specificto the ENS and suggests that GI problems experienced by autistic patients can originate inthe ENS, rather than being secondary to behaviour or a disorder of the gut-brain axis. Thisshould have major consequences for treatment of GI symptoms in autistic patients byfocusing attention on peripheral, rather than central, dysfunction.

Tu1993

Mucosal Mast Cells Maintain Normal Contractility of the Colon ThroughNGF-Dependent Mechanisms in a Rat Model of Post-Infectious IBSFerran Jardi, Joan Antoni Fernandez-Blanco, Vicente Martinez, Patri Vergara

Background: Regional inflammation of the gut leads to structural and functional disturbancesat various levels of the gastrointestinal tract, including remote locations not directly affectedby the inflammatory insult. In this line, during experimental T. Spiralis (TS) infection inrodents a primary jejunitis is observed; however, mast cell (MC) infiltration can be observedalso in the colon. We have shown that ovalbumin-induced colonic dysmotility in rats mightbe associated to alterations in MC- and Nerve Growth Factor (NGF)-dependent mechanisms.Aim: To characterize colonic motor alterations and the potential implication of MCs andNGF in TS-induced post-infectious gut dysfunction model, which courses with a primaryjejunitis, in rats. Methods: Male SD rats received TS (7500 larvae/rat, PO) or vehicle (1 mL,PO). At the post-infective phase (day 30±2 post-infection), colonic samples were obtained.Density of Connective Tissue (CTMC) and Mucosal MCs (MMC) was determined by IHQ(RMCPVI and RMCPII, respectively). In a separate experiment, animals were treated withthe MC stabilizer ketotifen (0.1 mg/mL in drinking water) during the same time period.From these animals, colonic strips were obtained and spontaneous contractility and contract-ile responses to NGF (100 mg/ml), carbachol (CCh; 0.1-10 μM) and the NO inhibitor L-NNA (1 mM) were assessed In Vitro (organ bath). Results: As expected, TS infection inducedjejunitis with significant MC hyperplasia and structural changes persisting up to day 30post-infection. In the same animals, increased MMC density (cells/x40 field; vehicle: 6±1.2;TS: 35.6±2.3, n=4-6; P<0.01) was observed in the colon, in the absence of histopathologicalsigns of inflammation. CTMC counts were unaffected by TS infection (cells/section; vehicle:9.4±1.7; TS: 10.4±2.8, n=6-7). Colonic spontaneous activity and contractile responses to

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sCCh were not affected by TS infection (Table). However, in TS-infected rats receivingketotifen contractile responses to CCh were increased by 60%; an effect completely preventedby pre-incubation with NGF, while the peptide did not affect contractility by itself (Table).Overall, ketotiken had also a tendency to increase cholinergic responses in non-infectedanimals (Table). L-NNA responses were unaltered by TS infection or ketotifen. Conclusions:During TS infection a significant MMC infiltrate appears in the colon without other structuralor functional (as contractility relates) alterations. Altered colonic contractile responses duringketotifen treatment and prevention by NGF suggests that MCs play a protective role normaliz-ing altered motility, likely through a NGF-related mechanism. Overall, these observationssuggest that the MMC infiltrate that characterize functional gastrointestinal disorders mightrepresent a protective mechanism aiming the normalization of colonic motility.Contractility (g)

Mean±SEM (N=5-6 per group) *:P<0.05 vs. vehicle, †:P<0.01 vs. respective contractileresponse to CCh alone (ANOVA)

Tu1994

Nitrergic Neurotransmission is Impaired in Non-Diabetic BB-Rats GastricFundusChristophe Vanormelingen, Tim Vanuytsel, Tatsuhiro Masaoka, Shadea Salim Rasoel,Pieter Vanden Berghe, Inge Depoortere, Ricard Farré, Jan F. Tack

Nitric oxide (NO) is believed to play an important role mediating gastric accommodationto a meal. Intestinal inflammation leads to loss of nitrergic myenteric neurons and disturbedmotor function, but spontaneous animal models to study the relationship between thesechanges are missing at the moment. The Biobreeding (BB) rat consists of a diabetes-resistant(BBDR), control strain, and a diabetes-prone (BBDP) strain. Disturbed gastric emptying isreported in hyperglycemic rats. We reported ganglionic inflammation, loss of nNOS expres-sion and nitrergic motor control in the small intestine of this model, independent of thedevelopment of hyperglycemia. The aim of this study was to evaluate in the BB rat modelthe neuromuscular neurotransmission and the presence of inflammation at the gastric fundus.Methods: Gastric fundus muscle strips of 220 days old rats (BBDR, non-diabetic (BBDP)and diabetic (BBDP-H) rats;all n=5) were suspended along their circular axis. Responses toelectrical field stimulation (EFS; 8V, 35ms and 1-16Hz) under NANC conditions wereevaluated, as well as the impact of NO synthase inhibitor L-NAME (3x10-4) and the P2Y1receptor antagonist MRS2179 (10-5), separately or in combination. Relaxation during thestimulation period (on-response) was evaluated as area under the curve (AUC) and amplitude,values were corrected for cross-sectional area. Nitrergic and P2Y1 mediated componentwere evaluated by considering the relaxation under L-NAME and MRS2179, corrected forrelaxation under NANC. Myeloperoxidase (MPO)-activity was determined for the fundicneuromuscular layer. Results: In all animals, muscle relaxation was inhibited by L-NAMEand MRS2179 (decrease in amplitude and AUC, ANOVA p<0.001). Relaxation under NANCconditions, expressed as AUC, was reduced in a similar magnitude in BBDP (ANOVAp<0.0001) and BBDP-H rats (ANOVAp<0.0001) in comparison to BBDR rats at all frequenciestested (at 1Hz, 37 ± 3.5 and 30 ± 3 vs. 59 ± 1.8 g/mm2/s; p<0.0001), while amplitude didnot differ. The nitrergic component was significantly smaller in BBDP and BBDP-H ratscompared to BBDR rats (ANOVA p<0.001). No significant differences in relaxation werefound when evaluating the P2Y1 component. NG induced a similar relaxatory response inall animal groups. Increased MPO activity was found in the fundic neuromuscular layer ofBBDP (0.61±0.20 IU/mg tissue) and BBDP-H (0.49±0.32 IU/mg tissue) rats compared toBBDR rats (0.005±0.002 IU/mg tissue, p<0.05). Conclusion: The BBDP rats showed alteredfundic muscle function, which is partially related to loss of nitrergic function in the myentericplexus, and may be related to local inflammation. These fundic changes seem to developat least partially independent from diabetes. The non-diabetic BBDP rat may provide aspontaneous model for post-inflammatory impaired gastric accommodation.

Tu1995

Relative Movement Between the Longitudinal and Circular Muscle LayersDuring Vagus Nerve Stimulated Esophageal ContractionsYanfen Jiang, Jane Dong, Valmik Bhargava, Ravinder K. Mittal

Aim and Background: Circular (CM) & longitudinal muscle (LM) layers of the esophaguscontract together in a perfectly synchronized manner during peristalsis. LM contractioncauses esophageal shortening & CM contraction causes luminal occlusion. Goal of our studywas to determine if there is relative movement (or sliding) between the LM & CM duringesophageal contraction. Methods: Studies were conducted in rats. Vagus nerve was isolatedin the neck and stimulated electrically. Two piezo electric crystals were anchored to the LMlayer of the esophageal wall, crystal 2 and 3, 5 mm apart, vertically ((figure 1). Using asuperficial vertical incision, a small tunnel was created between the CM and submucosaand 2 more crystals, crystal 1 and 4, were anchored to the CM layer (from inside) 3 mmapart vertically. Intraluminal pressure of the esophagus was measured by a 2.5 F solid-statepressure transducer catheter. After physiological recordings, esophagus along with the LESand proximal stomach were harvested, formalin fixed, and immuno-stained for skeletal &smooth muscle to determine physical connection between the CM & LM layers. Results:Vagus nerve stimulation caused simultaneous contraction of both CM & LM layers. Themagnitude of esophageal shortening recorded by crystals was significantly greater (p <. 002)with the crystals located on the LM (0.71 ± 0.06 mm) as compared to the CM layer (0.25± 0.06 mm at 2mA; pulse width 10 ms; pulse frequency 20 Hz; and train duration 5s). The

S-896AGA Abstracts

relative movement between crystals 1 and 2 was significantly great (p <.01) than between3 and 4, (0.36 ± 0.1 vs 0.23 ± 0.09 mm at 2mA; pulse width 10 ms; pulse frequency 20Hz; and train duration 5s). Histological studies show that the longitudinal muscle layer wasinserted into the bundles of circular muscle at the level of the LES. On the other hand,there were no such connections between the 2 muscle layers of the esophagus, i.e., cranialto the LES, figure 2. Conclusion: Our studies prove that during esophageal contraction thelongitudinal and circular muscle layer slide against each other. Sliding of the 2 muscle layersmay activate mechanosenstive neurons located in the myenteric plexus which may have aphysiological role in the peristalsis.

Figure 1: Schematics of crystal installation.

Figure 2: Immunostaining of Esophageal Wall: Smooth Muscle α-Actin (green), SkeletalMuscle Heavy Chain (red) and DAPI for Nucleus (blue) Stains: coronal section including LESand stomach. LM: longitudinal muscle; CM: circular muscle; LES: lower esophageal sphincter.