Marianne W. Furnes

Relevance of TNBS-Colitis in Rats: A Methodological Study with Endoscopic, Histologic and Transcriptomic Characterization and Correlation to IBD

Background Rectal instillation of trinitrobenzene sulphonic acid (TNBS) in ethanol is an established model for inflammatory bowel disease (IBD). We aimed to 1) set up a TNBS-colitis protocol resulting in an endoscopic and histologic picture resembling IBD, 2) study the correlation between endoscopic, histologic and gene expression alterations at different time points after colitis induction, and 3) compare rat and human IBD mucosal transcriptomic data to evaluate whether TNBS-colitis is an appropriate model of IBD. Methodology/Principal Findings Five female Sprague Daley rats received TNBS diluted in 50% ethanol (18 mg/0.6 ml) rectally. The rats underwent colonoscopy with biopsy at different time points. RNA was extracted from rat biopsies and microarray was performed. PCR and in situ hybridization (ISH) were done for validation of microarray results. Rat microarray profiles were compared to human IBD expression profiles (25 ulcerative colitis Endoscopic score demonstrated mild to moderate colitis after three and seven days, but declined after twelve days. Histologic changes corresponded with the endoscopic appearance. Over-represented Gene Ontology Biological Processes included: Cell Adhesion, Immune Response, Lipid Metabolic Process, and Tissue Regeneration. IL-1α, IL-1β, TLR2, TLR4, PRNP were all significantly up-regulated, while PPARγ was significantly down-regulated. Among genes with highest fold change (FC) were SPINK4, LBP, ADA, RETNLB and IL-1α. The highest concordance in differential expression between TNBS and IBD transcriptomes was three days after colitis induction. ISH and PCR results corresponded with the microarray data. The most concordantly expressed biologically relevant pathways included TNF signaling, Cell junction organization, and Interleukin-1 processing. Conclusions/Significance Endoscopy with biopsies in TNBS-colitis is useful to follow temporal changes of inflammation visually and histologically, and to acquire tissue for gene expression analyses. TNBS-colitis is an appropriate model to study specific biological processes in IBD.

Mechanism of gastric bypass-induced body weight loss: one-year follow-up after micro-gastric bypass in rats.

Bariatric surgery (Roux-en-Y or mini-gastric bypass) is designed to limit food intake by creating a small gastric pouch and to reduce nutrient absorption by bypassing the long limb of the intestine. We report 1-year follow-up results after micro-gastric bypass in rats. Micro-gastric bypass was performed by anastomosis of the esophagus and the proximal jejunum. Body weight, body composition, bone mineral density, food intake, and serum levels of ghrelin and obestatin were measured. Growing rats had a 40% weight reduction 2 months after micro-gastric bypass surgery compared to 20% after gastrectomy and 30% after stomach bypass (anastomosis of the esophagus and duodenal bulb). Six months after micro-gastric bypass surgery, the rats stopped growing compared to controls that gained continuously due to expansion of the fat compartment. Adult rats (600 g) lost 30% of their body weight 5 months after the micro-gastric bypass, while food intake was not reduced. Serum levels of obestatin (but not ghrelin) were reduced in rats with micro-gastric bypass. The results suggest that micro-gastric bypass efficiently reduced body weight, particularly fat mass; loss of the weight after micro-gastric bypass was not due to reduced food intake; and lean tissue and bone development were impaired in growing subjects after gastric bypass.

Feeding Behavior in Rats Subjected to Gastrectomy or Gastric Bypass Surgery

Background/Aim: Gastric bypass (GB) is usually designed to restrict food intake and to induce malabsorption. Gastric hormones have been thought to play a role in the regulation of food intake and body weight. The aim of the present study was to analyze feeding behavior after total gastrectomy (Gx) or GB in rats. Methods: Animals were subjected to Gx, GB, or sham operations. Eating and drinking behaviors after surgeries were assessed by a comprehensive laboratory animal monitoring system. Gastric hormones were measured by radioimmunoassay and energy density in feces by adiabatic bomb calorimeter. Results: Compared with sham operation, both Gx and GB reduced the body weight as measured during 3–8 weeks postoperatively, which was associated with increased energy expenditure per 100 g body weight. Daily accumulated food intake and meal size (during nighttime) were reduced following Gx, but not GB. The water intake (during daytime) was increased after Gx and GB. The energy density in feces was unchanged. Serum concentrations of ghrelin, obestatin, leptin, gastrin, and pancreastatin were greatly reduced after Gx. Conclusions: Control of food intake and meal size was independent of the food reservoir function of the stomach. Surgical depletion of gastric hormones is associated with reduced meal size, but increased water intake.

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease

Abstract Objective. Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn’s disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. Material and methods. Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). Results. GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3′,5′-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. Conclusions. GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

Cellular localization of guanylin and uroguanylin mRNAs in human and rat duodenal and colonic mucosa.

Guanylin (GUCA2A/Guca2a/GN) and uroguanylin (GUCA2B/Guca2b/UGN) are expressed in the gastrointestinal tract and have been implicated in ion and fluid homeostasis, satiety, abdominal pain, growth and intestinal barrier integrity. Their cellular sources are debated and include goblet cells, entero-/colonocytes, enteroendocrine (EE) cells and tuft cells. We therefore investigated the cellular sources of GN and UGN mRNAs in human and rat duodenal and colonic epithelium with in situ hybridization (ISH) to determine co-expression with Chromogranin A (CHGA/Chga/CgA; enterochromaffin [EC] cells), defensin alpha 6 (DEFA6/Defa6; Paneth cells), mucin 2 (MUC2/Muc2; goblet cells) and selected tuft cell markers. GUCA2A/Guca2a expression was localized to goblet cells and colonocytes in human and rat colon. In human duodenum, GUCA2A was expressed in Paneth cells and was scarce in villous epithelial cells. In rat duodenum, Guca2a was only localized to goblet cells. Guca2b was focally expressed in rat colon. In human and rat duodenum and in human colon, GUCA2B/Guca2b was expressed in dispersed solitary epithelial cells, some with a tuft cell-like appearance. Neither GUCA2A nor GUCA2B were co-expressed with CHGA in human duodenal cells. Consequently, EC cells are probably not the major source of human GN or UGN but other EE cells as a source of GN or UGN are not entirely excluded. No convincing overlap with tuft cell markers was found. For the first time, we demonstrate the cellular expression of GUCA2B in human duodenum. The specific cellular distribution of both GN and UGN differs between duodenum and colon and between human and rat intestines.

Mechanistic Comparison between Gastric Bypass vs. Duodenal Switch with Sleeve Gastrectomy in Rat Models

Background Both gastric bypass (GB) and duodenal switch with sleeve gastrectomy (DS) have been widely used as bariatric surgeries, and DS appears to be superior to GB. The aim of this study was to better understand the mechanisms leading to body weight loss by comparing these two procedures in experimental models of rats. Methods Animals were subjected to GB, DS or laparotomy (controls), and monitored by an open-circuit indirect calorimeter composed of comprehensive laboratory animal monitoring system and adiabatic bomb calorimeter. Results Body weight loss was greater after DS than GB. Food intake was reduced after DS but not GB. Energy expenditure was increased after either GB or DS. Fecal energy content was increased after DS but not GB. Conclusion GB induced body weight loss by increasing energy expenditure, whereas DS induced greater body weight loss by reducing food intake, increasing energy expenditure and causing malabsorption in rat models.

Su2070 Comparison of Different Surgical Procedures in Animal Models for a Better Consideration of Personalized Bariatric Surgery

S A T A b st ra ct s increase in serum amylase, AST, ALT was observed in the Aged group compared to the Young group (p<0.05). Two hours after AP a transient liver mitochondrial dysfunction occurred in young animals, mainly due to uncoupling of oxidative phosphorylation, and that was partially recovered. Liver mitochondrial dysfunction did not occur in the Sham group of young animals. However, in aged animals two hours after AP there was a liver mitochondrial dysfunction that was also noted in sham aged animals, suggesting a previous degenerative process similar to that found in cellular ischemia. Likewise, it was observed an increase of MDA content in young animals two hours after AP in comparison to the sham group. The aged animals showed an increase of MDA content both in the sham group and in the AP group. Conclusion: This study demonstrates that liver mitochondrial function is transiently compromised in young animals submitted to AP. However, in aged animals unexpectedly the pre-existing severe mitochondrial dysfunction remained unchanged after induction of AP associated with a sustained oxidative stress. These findings may have significant therapeutic implications in the clinical setting.

Tu2069 Gastric Bypass and Duodenal Switch Cause Body Weight Loss Through Different Mechanisms in Rats

Background/Aim: It is still a challenge how to select the most suitable surgical procedure for each individual obese patient. Both gastric bypass (GB) and duodenal switch associated with sleeve gastrectomy (DS) have been widely used as bariatric surgery, and DS appears to be superior to GB, particularly for morbid obesity. The aim of the present study was to compare these procedures with respect to the mechanisms leading to body weight loss in rats. Methods: Male Sprague-Dawley rats were subjected to GB, DS, or laparotomy (as controls) and followed for 2-14 weeks by an open-circuit indirect calorimeter composed in comprehensive laboratory animalmonitoring system and adiabatic bomb calorimeter. Results: Body weight loss was greater after DS than GB. Calorie intake in terms of kcal/day/rat, kcal/ day/100 g body weight, and kcal/meal was reduced after DS but not GB. The fecal energy content (expressed as J/g) was increased after DS but not after GB. Energy expenditure (kcal/ hr/100 g body weight) was increased during nighttime at 3 weeks and then during daytime at 14 weeks after GB. The energy expenditure was increased both at 2 weeks (during daytime) and 8 weeks (during both daytime and nighttime) after DS. Respiratory exchange ratio, i.e., VCO2/VO2, was unchanged after GB, but reduced after DS. Serum ghrelin levels were reduced at 3 weeks after GB but no longer afterwards. Serum CCK levels were greatly increased at least at 8 weeks after DS. Conclusion: GB induced body weight loss by increasing energy expenditure, whereas DS induced body weight loss by reducing food intake (probably due to hyperCCK-emia), causing malabsorption, and increasing both fat metabolism and energy expenditure. Acknowledgements: The research leading to these results has received funding from the Central Norway Regional FUGE programme, Central Norway Regional Health Authority, and the European Union Seventh Framework Programme (FP7/20072013) under grant agreement n°266408.