Su2051 The Non-Diabetic BB-Rat: A Spontaneous Model for Impaired Gastric Accommodation
Transcript of Su2051 The Non-Diabetic BB-Rat: A Spontaneous Model for Impaired Gastric Accommodation
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sSu2050
Involvement of Ghrelin Signaling Dysfunction in Acute Restraint Stress-Induced Delayed Gastric EmptyingShunsuke Ohnishi, Shuichi Muto, Koji Nakagawa, Chiharu Sadakane, Yayoi Saegusa,Miwa Nahata, Chihiro Yamada, Tomohisa Hattori, Masahiro Asaka, Naoya Sakamoto,Hiroshi Takeda
Background/Aim: Stress affects gastrointestinal function such as gastric emptying and gastro-intestinal motility. Ghrelin, an appetite-stimulating hormone, has been shown to stimulategastric emptying and gastrointestinal motility. The aim of this study was to elucidate therole of ghrelin on gastrointestinal function during acute stress and examine the effect ofrikkunshito, a traditional Japanese medicine known as a ghrelin signal potentiator (TranslPsychiatry. 2011;1:e23), on delayed gastric emptying in the restraint-stressed mice. Methods:Fasted ICR mice were fed with standard feed for 20 min and were immediately placed in50 mL tubes in order to subject them to restraint. Their blood, stomach, and hypothalamuswere harvested 15-60 min after restraint stress was induced. Ghrelin levels were measuredby ELISA, and the expression of the genes related to ghrelin signaling was measured by RT-PCR. In addition, fasted mice that received feed for 20 min were subsequently administeredwith acyl ghrelin (3 nmol/mouse, i.p.) or rikkunshito (250mg/kg, p.o.), and were immediatelysubjected to restraint stress. The stomach was excised to measure solid gastric emptying 60min after restraint stress was induced. In addition, a ghrelin receptor antagonist (D-[Lys3]-GHRP-6, 0.2 μmol/mouse, i.p.) was administered simultaneously with rikkunshito, and theeffects on gastric emptying were examined 60 min after restraint stress was induced. Results:Gastric emptying was significantly decreased 60 min after restraint stress loading comparedto control mice. Restraint stress did not alter the plasma acyl ghrelin levels, but it significantlyincreased the plasma desacyl ghrelin levels 15, 30 and 60 min after restraint stress wasinduced (Figs. 1 and 2). In the stomach, restraint stress did not alter the acyl and desacylghrelin contents, preproghrelin mRNA expression, ghrelin O-acyl transferase contents ormRNA expression. In the hypothalamus, the desacyl ghrelin contents was significantlyincreased 30 min after restraint stress loading compared to control mice. Moreover, adminis-tration of acyl ghrelin and rikkunshito improved the restraint stress-induced delayed gastricemptying. This effect of rikkunshito was abolished by the simultaneous administration ofD-[Lys3]-GHRP-6. Conclusions: Our findings revealed that restraint stress in mice enhancesacyl ghrelin metabolism or desacyl ghrelin secretion, and that supplementation of exogenousghrelin or stimulation of endogenous ghrelin signaling causes an improvement in stress-induced delayed gastric emptying. These results suggest that under acute stress, the effectsof acyl ghrelin may be masked by an increase in desacyl ghrelin.
Fig. 1 Effects of restraint stress loading on plasma acyl ghrelin levels. # p , 0.05 vs. ‘−20min' of control group. N = 8.
Fig. 2 Effects of restraint stress loading on plasma desacyl ghrelin levels. *, **p , 0.05,0.01 vs. control at each time point. N = 8.
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Su2051
The Non-Diabetic BB-Rat: A Spontaneous Model for Impaired GastricAccommodationChristophe Vanormelingen, Ricard Farré, Tim Vanuytsel, Tatsuhiro Masaoka, ShadeaSalim Rasoel, Joran G. Tóth, Theo Thijs, Hanne Vanheel, Lukas Van Oudenhove, IngeDepoortere, Pieter Vanden Berghe, Jan F. Tack
Nitric oxide (NO) is an important mediator of gastric accommodation to a meal. Intestinalinflammation leads to loss of nitrergic myenteric neurons and disturbed motor function,but spontaneous animal models to study the relationship between these changes are missing.The Biobreeding (BB) rat consists of a diabetes-resistant (BBDR), control strain, and a diabetes-prone (BBDP) strain. In our facility 50% of the BBDP rats develop diabetes after the age of90 days. BBDP rats develop ganglionic inflammation, loss of nNOS expression and nitrergicmotor control in the small intestine, independently of hyperglycemia. Aim of this studywas to evaluate the neuromuscular neurotransmission and the presence of inflammation inthe gastric fundus of different BB rat strains. Methods Gastric fundus muscle strips of rats70 and 220 days old (control, non-diabetic (BBDP) and diabetic (BBDP-H) (N=6)), weresuspended along their circular axis. Responses to electrical field stimulation (EFS; 8V, 35msand 1-16Hz) under NANC conditions were evaluated, as well as the impact of NO synthaseinhibitor L-NAME (3x10-4) and the P2Y1 receptor antagonist MRS2179 (10-5), separatelyor combined. Total relaxation during stimulation was evaluated as area under the curve(AUC), and corrected for cross-sectional area and weight. Nitrergic and P2Y1 mediatedcomponents were evaluated by the relaxation under L-NAME and MRS2179. Statistics forstrip experiments were done using mixed model analysis. Expression of nNOS was quantifiedby Western blot relative to PGP9.5. Myeloperoxidase (MPO)-activity was determined forsegments of mucosa and muscularis propria and inflammation was also evaluated histologi-cally. Results Relaxation under NANC conditions was reduced at all frequencies in BBDPand BBDP-H rats of 220 days when compared to control rats (eg. at 1Hz, 29±2.5 and 30±3vs. 55±1 g/mm2/s; p,0.01). In all animals, muscle relaxation was inhibited by L-NAMEand MRS2179. The nitrergic component was significantly smaller in BBDP (70 and 220days) and BBDP-H (220 days) rats compared to controls. Significant loss of nNOS proteinswas seen in BBDP rats of 220 days but not at 70 days. The P2Y1 component was significantlyimpaired in BBDP-H rats of 220 days. Table 1 summarizes data on inflammation. MPOactivity was increased in the fundic mucosa and muscularis propria of BBDP (70 and 220days) and BBDP-H (220 days) rats compared to controls, and this was confirmed by asignificant increase in polymorphonuclear cells (PMN) on histology. Conclusion BBDP ratsshowed altered fundic muscle function, which is at least partially explained by loss ofnitrergic function in the myenteric plexus, and may be related to local inflammation. Thesefundic changes develop independently from diabetes. The non-diabetic BBDP rat may providea spontaneous model for post-inflammatory impaired gastric accommodation.Table1: Data of inflammation in the BB-rat fundus
* p,0,05 vs control ** p,0,01 vs control *** p,0,001 vs control
Su2052
Duodenal Aeromonas SPP Are Increased in Number in a Rat Model of Post-Infectious IBS: Translation of Data From Deep Sequencing of the Microbiomein Humans With IBSGene Kim, Walter Morales, Vincent Funari, Jordan Brown, Stacy Weitsman, Gillian M.Barlow, Christopher Chang, Mark Pimentel
Recent human small bowel culture and qPCR data suggest that subjects with irritable bowelsyndrome (IBS) have small intestinal bacterial overgrowth (SIBO). In a validated animalmodel of post-infectious IBS generated using the most common cause of acute gastroenteritis(Campylobacter jejuni), rats develop IBS-like phenotypes and SIBO, as determined by qPCR.Recently using novel deep sequencing and genus-specific quantitative PCR (qPCR), ourgroup has identified Aeromonas spp as elevated in human IBS subjects. Since Aeromonas isa mild pathogen, in this translational study, we conduct duodenal qPCR for Aeromonas inour rat model, and test whether cytokines and other defense mediators (elevated in ourmodel), correlated with Aeromonas. Methods: Male Sprague-Dawley rats were gavaged withC. jejuni as previously described. Control rats were gavaged with vehicle. Rats were followedfor 3 months after clearance of infection, euthanized and dissected to resect segments ofbowel. After DNA extraction of luminal contents from duodenum, qPCR for Aeromonas sppwas performed using genus-specific primers in three groups of rats: C. jejuni rats thatdeveloped duodenal SIBO (C+/SIBO+); C. jejuni rats that did not develop SIBO (C+/SIBO-); and uninfected controls. SIBO was determined using a universal bacterial 16S primer aspreviously described. cDNA was generated from homogenates of full thickness small boweland qPCR was performed to evaluate mRNA expression of the mucosal defense mediatorsbeta-defensin 2, beta-defensin 6, Toll-like receptor-4 (TLR-4), interleukin-6 (IL-6), IL-8 andTNF-alpha. These results and the presence or absence of SIBO were compared to Aeromonasspp levels. Results: Interestingly, all rat groups had detectable Aeromonas spp in the duodenum.However, Aeromonas spp were significantly elevated in rats that developed duodenal SIBO(C+/SIBO+) (median=1.42x107) compared to rats that did not develop SIBO (C+/SIBO-)