Mouse Gastrointestinal Tract Research Articles

Candida albicans and Bacterial Microbiota Interactions in the Cecum during Recolonization following Broad-Spectrum Antibiotic Therapy

Candida albicans is a normal member of the gastrointestinal (GI) tract microbiota of healthy humans, but during host immunosuppression or alterations in the bacterial microbiota, C. albicans can disseminate and cause life-threatening illness. The bacterial microbiome of the GI tract, including lactic acid bacteria (LAB), plays a vital role in preventing fungal invasion. However, little is known about the role of C. albicans in shaping the bacterial microbiota during antibiotic recovery. We investigated the fungal burdens in the GI tracts of germfree mice and mice with a disturbed microbiome to demonstrate the role of the microbiota in preventing C. albicans colonization. Histological analysis demonstrated that colonization with C. albicans during antibiotic treatment does not trigger overt inflammation in the murine cecum. Bacterial diversity is reduced long term following cefoperazone treatment, but the presence of C. albicans during antibiotic recovery promoted the recovery of bacterial diversity. Cefoperazone diminishes Bacteroidetes populations long term in the ceca of mice, but the presence of C. albicans during cefoperazone recovery promoted Bacteroidetes population recovery. However, the presence of C. albicans resulted in a long-term reduction in Lactobacillus spp. and promoted Enterococcus faecalis populations. Previous studies have focused on the ability of bacteria to alter C. albicans; this study addresses the ability of C. albicans to alter the bacterial microbiota during nonpathogenic colonization.

Effects of lactobacillus plantarumZJ316 on pig growth and pork quality

BackgroundLactobacillus plantarum is a plant-associated bacterial species but it has also been found in human, mouse and porcine gastrointestinal tracts. It can ferment a broad spectrum of plant carbohydrates; it is tolerant of bile salts and low pH, and it has antagonistic potential against intestinal pathogens. However, experiments reporting the use of L. plantarum as a probiotic are limited. In this study, the effects of L. plantarum ZJ316 isolated from infant fecal samples on pig growth and pork quality were investigated.ResultsOne hundred and fifty newly weaned pigs were selected randomly and divided into five groups. Group 1 was fed a diet supplemented with the antibiotic mequindox; Groups 2, 3 and 4 were fed a diet supplemented with L. plantarum and no antibiotic; and Group 5 was fed a mixture of mequindox and L. plantarum. After a 60 days initial treatment, samples were collected for evaluation. The results showed that, the L. plantarum ZJ316 has probiotic effects on pig growth and that these effects are dose dependent. The effects of a dose of 1 × 109 CFU/d were more pronounced than those of a dose of 5 × 109 CFU/d or 1 × 1010 CFU/d. In Group 2 (1 × 109 CFU/d), the diarrhea (p = 0.000) and mortality rates (p = 0.448) were lower than in antibiotic-treated pigs (Group 1), and the daily weight gain (p = 0.001) and food conversion ratios were better (p = 0.005). Improved pork quality was associated with Lactobacillus treatment. pH (45 min, p = 0.020), hardness (p = 0.000), stickiness (p = 0.044), chewiness (p = 0.000), gumminess (p = 0.000) and restoring force (p = 0.004) were all significantly improved in Lactobacillus-treated pigs (Group 2). Although we found that L. plantarum exerted probiotic effects on pig growth and pork quality, the mechanisms underlying its action require further study. Polymerase chain reaction-denaturing gradient gel electrophoresis results showed that the gut bacterial communities in Lactobacillus- and antibiotic-treated pigs were very similar and the quantity of L. plantarum ZJ316 was below the detection limits of DGGE-band sequencing. The concentration of short-chain fatty acids in Lactobacillus- and antibiotic-treated fecal samples were not significantly different (p = 0.086). However, the villus height of ilea (p = 0.003), jejuna (p = 0.000) and duodena (p = 0.036) were found to be significantly improved by Lactobacillus treatment.ConclusionL. plantarum ZJ316 was found to have probiotic effects, improving pig growth and pork quality. The probiotic mechanism might not involve L. plantarum colonization and alteration of the gut bacterial community. Rather, it might be related to the inhibition of the growth of opportunistic pathogens and promotion of increased villus height.

A Crypt-Specific Core Microbiota Resides in the Mouse Colon

ABSTRACTIn an attempt to explore the microbial content of functionally critical niches of the mouse gastrointestinal tract, we targeted molecular microbial diagnostics of the crypts that contain the intestinal stem cells, which account for epithelial regeneration. As current evidence indicates, the gut microbiota affects epithelial regeneration; bacteria that are likely to primarily participate in this essential step of the gut, microbiota cross talk, have been identified. We show in this article that only the cecal and colonic crypts harbor resident microbiota in the mouse and that regardless of the line and breeding origin of these mice, this bacterial population is unexpectedly dominated by aerobic genera. Interestingly, this microbiota resembles the restricted microbiota found in the midgut of invertebrates; thus, the presence of our so-called “crypt-specific core microbiota” (CSCM) in the mouse colon potentially reflects a coevolutionary process under selective conditions that can now be addressed. We suggest that CSCM could play both a protective and a homeostatic role within the colon. This article is setting the bases for such studies, particularly by providing a bona fide—and essentially cultivable—crypt microbiota of reference.

Gsdma3 is a new factor needed for TNF-α-mediated apoptosis signal pathway in mouse skin keratinocytes

Gsdma3, a newly found gene, is expressed restrictedly in mouse skin keratinocytes and gastrointestinal tract. But until now, there is little information on the regulation and the function of Gsdma3 in skin keratinocytes. In our previous study, we found that Gsdma3 mutation resulted in a decrease in catagen-associated apoptosis of hair follicle keratinocytes. Apoptosis of skin keratinocytes is strictly regulated by a series of signal pathways, among of which, tumor necrosis factor (TNF)-α-induced signal pathway has been extensively studied. To further investigate the role and the pathway of Gsdma3 involved in skin keratinocyte apoptosis, using immunofluorescence, RT-PCR, western blot and TUNEL analysis, we showed here that accompanying TNF-α-induced apoptosis and Caspase-3 expression in mouse skin keratinocytes in vivo and in vitro, Gsdma3 expression was significantly upregulated. After Gsdma3 gene mutation, TNF-α-induced apoptosis and Caspase-3 expression in skin keratinocytes were reduced. The injection of Gsdma3 expression plasmid could directly enhance the apoptosis and Caspase-3 expression in skin keratinocytes. These results, taken together, indicated that in mouse skin keratinocytes, Gsdma3 expression could be regulated by TNF-α. Gsdma3 was not only involved in but also necessary for the TNF-α-induced apoptosis pathway by directly enhancing the Caspase3 expression as well as the apoptosis induction.

Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways

4',5,7-Trihydroxy-3',5'-dimethoxyflavone (Tricin), a naturally occurring flavone, has anti-inflammatory potential and exhibits diverse biological activities including antigrowth activity in several human cancer cell lines and cancer chemopreventive effects in the gastrointestinal tract of mice. The present study aimed to investigate the biological actions of tricin on hepatic stellate cells (HSCs) in vitro, exploring its potential as a treatment of liver fibrosis, since HSC proliferation is closely related to the progression of hepatic fibrogenesis in chronic liver diseases leading to irreversible liver cirrhosis and hepatocellular carcinoma. Tricin inhibited platelet-derived growth factor (PDGF)-BB-induced cell proliferation by blocking cell cycle progression and cell migration in the human HSC line LI90 and culture-activated HSCs. It also reduced the phosphorylation of PDGF receptor β and the downstream signaling molecules ERK1/2 and Akt, which might be due to its tyrosine kinase inhibitor properties rather than inhibition of the direct binding between PDGF-BB and its receptor. Our findings suggest that tricin might be beneficial in HSC-targeting therapeutic or chemopreventive applications for hepatic fibrosis.

Tu1996 Glycogen Metabolism Defects Produce Gastrointestinal Myopathy With Polyglucosan Bodies

INTRODUCTION The intestine is a significant site of gluconeogenesis, with high expression of glucose 6 phosphatase. Abnormal forms of glycogen, including polyglucosan bodies, occur in the muscularis propria in a subset of patients with gastrointestinal motility disorders. Little is known, however, of the distribution of glycogen metabolic enzymes and related proteins in normal gastrointestinal tract, or effects of deficiencies of these proteins on the bowel. METHODS Key enzymes in glycogen metabolism, including glycogen synthase (GS), glycogen branching enzyme (GBE), malin and laforin were examined in stomach, ileum and colon of two mouse strains and in normal human bowel using immunohistochemistry with semiquantitiative analysis of epithelium, muscle, submucosal and myenteric plexus, validated by western blotting and positive and negative controls. The pathological effects of GBE, laforin and malin gene knockout on mouse gastrointestinal tract were also studied. RESULTS GS was present at high levels in the smooth muscle of muscularis propria, GBE in the majority of large ganglion cells of the submucosal and myenteric plexuses, and laforin and malin in submucosal ganglia. Analysis of human bowel showed no significant regional differences in the distribution of GBE, laforin and malin, GS was more prominent in the muscularis propria of the small intestine, compared with stomach and colon. GBE knockout produced a progressive severe myopathy with numerous polyglucosan bodies and vacuolation. Polyglucosan bodies were also present in both laforin and malin knockout models, but with a less severe phenotype than that seen in the GBE knockout, and were not seen in controls. CONCLUSION The distribution of glycogen metabolic proteins in the bowel wall of mouse and man shows a specific neuromuscular distribution for each pathway component. Defects in glycogen metabolism in mouse knockout models reproduce histological features of polyglucosan body myopathy in human digestive motility disorders.

Tu1997 A Novel In Vivo Method to Measure Gastrointestinal Transit in Mice

INTRODUCTION The intestine is a significant site of gluconeogenesis, with high expression of glucose 6 phosphatase. Abnormal forms of glycogen, including polyglucosan bodies, occur in the muscularis propria in a subset of patients with gastrointestinal motility disorders. Little is known, however, of the distribution of glycogen metabolic enzymes and related proteins in normal gastrointestinal tract, or effects of deficiencies of these proteins on the bowel. METHODS Key enzymes in glycogen metabolism, including glycogen synthase (GS), glycogen branching enzyme (GBE), malin and laforin were examined in stomach, ileum and colon of two mouse strains and in normal human bowel using immunohistochemistry with semiquantitiative analysis of epithelium, muscle, submucosal and myenteric plexus, validated by western blotting and positive and negative controls. The pathological effects of GBE, laforin and malin gene knockout on mouse gastrointestinal tract were also studied. RESULTS GS was present at high levels in the smooth muscle of muscularis propria, GBE in the majority of large ganglion cells of the submucosal and myenteric plexuses, and laforin and malin in submucosal ganglia. Analysis of human bowel showed no significant regional differences in the distribution of GBE, laforin and malin, GS was more prominent in the muscularis propria of the small intestine, compared with stomach and colon. GBE knockout produced a progressive severe myopathy with numerous polyglucosan bodies and vacuolation. Polyglucosan bodies were also present in both laforin and malin knockout models, but with a less severe phenotype than that seen in the GBE knockout, and were not seen in controls. CONCLUSION The distribution of glycogen metabolic proteins in the bowel wall of mouse and man shows a specific neuromuscular distribution for each pathway component. Defects in glycogen metabolism in mouse knockout models reproduce histological features of polyglucosan body myopathy in human digestive motility disorders.

Abstract 4048: Multiphoton tomographic imaging is an effective optical biopsy tool for detecting gastrointestinal inflammation and neoplasia

Abstract Background: Improved early detection methods are needed to reduce the risk of gastrointestinal (GI) cancers for both average and high risk individuals. Standard colonoscopes and low magnification adjuncts (e.g., narrow band imaging) are unable to provide cellular resolution. Thus, any suspicious lesions must be biopsied. Individuals with longstanding inflammatory bowel disease (IBD) are at increased risk for colorectal cancer (CRC), requiring frequent colonoscopic evaluations including multiple random biopsies to exclude dysplasia. Technologies that facilitate directed biopsies should decrease both procedure-related morbidity and costs. Here, we explore the use of multiphoton microscopy (MPM) as a novel optical biopsy tool with several potential advantages: (1) no need for exogenous contrast; (2) greater depth of imaging; and (3) access to multiple signals from different tissue sources using a single illumination. Study design and results: Phase 1: Generating an MPM atlas of normal mouse GI tract. Tissues along the mouse GI tract from esophagus to anus were imaged using a single excitation wavelength; three distinct signals were collected, color-coded and overlaid to obtain cellular-resolution 3D images: (1) Second Harmonic Generation - a higher order scattering signal from collagen; (2) Short wavelength autofluorescence - from cytoplasm of epithelial and certain immune cells, and elastin; (3) Long wavelength autofluorescence - from macrophages, presumably originating from lipofuscin. After MPM imaging, each specimen was fixed, H&E stained, and images from both MPM and H&E-stained slides were archived and annotated by a pathologist. MPM provided sufficient histological details to identify all relevant tissue substructures. Phase 2: Imaging disease progression in mouse models of dextran sodium sulfate (DSS) induced colitis and azoxymethane (AOM) induced CRC. We identified both acute and resolving stages of colitis in the DSS model, which correlated with pathological findings. In the AOM model, we followed the progression from aberrant crypts to aberrant crypt foci to micro-adenoma to adenoma. Three independent pathologists identified all lesion types from the MPM images with 100% concordance. Phase 3: Translating mouse model findings to human disease. Patients with both IBD and celiac sprue are at increased risk of inflammation-related GI malignancy. Hence, both IBD and celiac sprue were evaluated. Additionally, adenomatous polyps and CRC were imaged. Normal tissue was imaged in each case as control, and all MPM images were compared with H&E slides prepared from the same specimens. MPM images demonstrated striking similarity to those generated by H&E staining. Conclusion: MPM is a promising optical biopsy technique that identifies relevant tissue substructures without exogenous contrast, and might have distinct advantages over competing technologies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4048. doi:1538-7445.AM2012-4048

Red chlorophyll: the new barium?

Imaging of gastrointestinal (GI) motility remotely through the abdominal wall has always been a tradeoff between resolution and invasiveness. Skin reflects and/or absorbs wavelengths of radiation in the ultraviolet and visible ranges, but is largely transparent to both high-energy radiation (Gamma to X-rays; <0.1-10 nm) and low-energy radiation (infrared to radio waves; 700 nm-10 m). Imaging using short wavelength radiation such as X-ray cinematography has excellent spatial and temporal resolution, but ionization can produce acute and long-term deleterious effects to the patient or animal. Other 'slice-based' imaging techniques such as ultrasound/MRI/CT minimize tissue damage, but are limited in the planar area that can be imaged in a timely fashion. This viewpoint article will summarize and explore the implications of recent advances in infrared imaging of the GI tract, in particular, an article published in this issue of NGM entitled 'In vivo dynamic imaging of intestinal motions using diet-related autofluorescence' in which the authors have used infrared imaging in combination with that most elusive ingredient, standard mouse chow, to capture the motions of the mouse GI tract.

Histochemical demonstration of mucosubstances in the mouse gastrointestinal tract treated with Origanum hypericifolium O. Schwartz and P.H. Davis extract

Glycoconjugate-containing mucous in the gastrointestinal tract has lubricant and cytoprotective effects. This study was performed to demonstrate the intensity of mucous in the stomach, small and large intestine tissues of female BALB/c mice injected intraperitoneally with 0.2 ml of the Origanum hypericifolium extract for six weeks. The staining procedures employed were Alcian Blue (AB) at pH 1 and 2.5, periodic acid-Schiff (PAS) and lectins including Galanthus nivalis agglutinin (GNA),Arachis hypogaea (PNA, Peanut Agglutinin) and Maackia amurensis leucoagglutinin (MAL). The mucous glands in the large intestine of the experimental groups were found to show stronger positive reaction for AB at pH 2.5 and PAS when compared with the controls. In lectin histochemistry, the mucous glands which reacted with lectins exhibited less density for PNA in the stomach and for MAL and PNA in the small intestine in the experimental groups than the controls. In the large intestine of the experimental groups, less staining pattern of GNA was observed on the surface epithelium of villi when compared with the controls. The results suggest that O. hypericifolium extract increases acidic and neutral mucosubstances in the large intestine and also changes some glycan structures in the gastrointestinal tract. Key words: Mucosubstances, Gastrointestinal tract, Glycoconjugates, Origanum hypericifolium.

Biodistribution and stability of CdSe core quantum dots in mouse digestive tract following per os administration: Advantages of double polymer/silica coated nanocrystals

CdSe-core, ZnS-capped semiconductor quantum dots (QDs) are of great potential for biomedical applications. However, applications in the gastrointestinal tract for in vivo imaging and therapeutic purposes are hampered by their sensitivity to acidic environments and potential toxicity. Here we report the use of coatings with a combination of polythiol ligands and silica shell (QDs PolyT–APS) to stabilize QDs fluorescence under acidic conditions. We demonstrated the stability of water-soluble QDs PolyT–APS both in vitro, in strong acidic solutions, and in vivo. The biodistribution, stability and photoluminescence properties of QDs in the gastrointestinal tract of mice after per os administration were assessed. We demonstrated that QDs coated with current traditional materials – mercapto compounds (QDs MPA) and pendant thiol group (QDs PolyT) – are not capable of protecting QDs from chemically induced degradation and surface modification. Polythiol ligands and silica shell quantum dots (QDs PolyT–APS) are suitable for biological and biomedical applications in the gastrointestinal tract.

Transient expression of the calcitonin receptor by enteric neurons of the embryonic and early post-natal mouse

Calcitonin receptor-immunoreactivity (CTR-ir) was found in enteric neurons of the mouse gastrointestinal tract from embryonic day 13.5 (E13.5) to post-natal day 28 (P28). CTR-ir occurred in cell bodies in ganglia of the myenteric plexus extending from the esophagus to the colon and in nerve cells of the submucosal ganglia of the small and large intestines. CTR-ir was also found in vagal nerve trunks and mesenteric nerves. Counts in the ileal myenteric plexus revealed CTR-ir in 80% of neurons. CTR-ir was clearly evident in the cell bodies of enteric neurons by E15.5. The immunoreactivity reached maximum intensity between P1.5 and P12 but was weaker at P18 and barely detectable at P28. The receptor was detected in nerve processes in the intestine for only a brief period around E17.5, when it was present in one to two axonal processes per villus in the small intestine. In late gestation and soon after birth, CTR-ir was also evident in the mucosal epithelium. The perinatal expression of CTR within the ENS suggests that the calcitonin/CTR system may have a role in the maturation of enteric neurons. Signals may reach enteric neurons in milk, which contains high levels of calcitonin.

Runx1 is a tumor suppressor gene in the mouse gastrointestinal tract

The Runx1 transcription factor plays an important role in tissue homeostasis through its effects on stem/progenitor cell populations and differentiation. The effect of Runx1 on epithelial differentiation of the secretory cell lineage of the colon was recently demonstrated. This study aimed to examine the role of Runx1 in tumor development in epithelial cells of the gastrointestinal tract. Conditional knockout mice that lacked Runx1 expression in epithelial cells of the GI tract were generated. These mice were crossed onto the Apc(Min) background, killed and their intestinal tumor phenotypes were compared with Apc(Min) Runx1 wild-type control mice. Apc-wild-type Runx1-mutant mice were also examined for tumor development. Colons from Runx1 knockout and wild-type mice were used for genome-wide mRNA expression analyses followed by gene-specific quantitative RT-PCR of whole colon and colon epithelium to identify Runx1 target genes. Runx1 deficiency in intestinal epithelial cells significantly enhanced tumorigenesis in Apc(Min) mice. Notably, epithelial Runx1 deficiency in Apc-wild-type mice was sufficient to cause tumor development. Absence of Runx1 was associated with global changes in the expression of genes involved in inflammation and intestinal metabolism, and with gene sets indicative of a metastatic phenotype and poor prognosis. Gene-specific analysis of Runx1-deficient colon epithelium revealed increased expression of genes linked to an expansion of the stem/progenitor cell population. These results identify Runx1 as a novel tumor suppressor gene for gastrointestinal tumors and support a role for Runx1 in maintaining the balance between the intestinal stem/progenitor cell population and epithelial differentiation of the GI tract.

Oit1/Fam3D, a gut-secreted protein displaying nutritional status-dependent regulation

Oncoprotein-induced transcript 1 (Oit1) was previously identified as a dietary fat-induced gene in the small intestine of C57Bl/6J mice. In this study, we further characterized Oit1 and its human ortholog family with sequence similarity 3, member D (Fam3D), on the messenger RNA as well as the protein level. Oit1 and Fam3D were found to be predominantly expressed in the gastrointestinal tract of mice and humans, respectively. Dietary fat induced a clear and acute up-regulation of Oit1, especially in the jejunum, whereas fasting led to a reduced gene expression in the small intestine. Regarding protein expression, we found a remarkable pattern of Oit1 along the longitudinal axis of the intestine, a predominant villus-restricted expression in the proximal small intestine and a more pronounced crypt expression in the distal parts of the intestine. Using transfection experiments, we confirmed secretion of the Oit1 protein, as was predicted by a signal peptide sequence. Detection of Oit1 and Fam3D in plasma samples indicated that both proteins are secreted to the basolateral site of enterocytes. Moreover, in human plasma samples, we also found an effect of nutritional status on Fam3D levels, with a postprandial elevation and a reduction after fasting. In conclusion, Oit1 and Fam3D are gut-derived proteins that are expressed and secreted in a nutritional status-dependent manner.

Biodistribution and endocytosis of ICAM-1-targeting antibodies versus nanocarriers in the gastrointestinal tract in mice

Drug delivery to the gastrointestinal (GI) tract is key for improving treatment of GI maladies, developing oral vaccines, and facilitating drug transport into circulation. However, delivery of formulations to the GI tract is hindered by pH changes, degradative enzymes, mucus, and peristalsis, leading to poor GI retention. Targeting may prolong residence of therapeutics in the GI tract and enhance their interaction with this tissue, improving such aspects. We evaluated nanocarrier (NC) and ligand-mediated targeting in the GI tract following gastric gavage in mice. We compared GI biodistribution, degradation, and endocytosis between control antibodies and antibodies targeting the cell surface determinant intercellular adhesion molecule 1 (ICAM-1), expressed on GI epithelium and other cell types. These antibodies were administered either as free entities or coated onto polymer NCs. Fluorescence and radioisotope tracing showed proximal accumulation, with preferential retention in the stomach, jejunum, and ileum; and minimal presence in the duodenum, cecum, and colon by 1 hour after administration. Upstream (gastric) retention was enhanced in NC formulations, with decreased downstream (jejunal) accumulation. Of the total dose delivered to the GI tract, ∼60% was susceptible to enzymatic (but not pH-mediated) degradation, verified both in vitro and in vivo. Attenuation of peristalsis by sedation increased upstream retention (stomach, duodenum, and jejunum). Conversely, alkaline NaHCO3, which enhances GI transit by decreasing mucosal viscosity, favored downstream (ileal) passage. This suggests passive transit through the GI tract, governed by mucoadhesion and peristalsis. In contrast, both free anti-ICAM and anti-ICAM NCs demonstrated significantly enhanced upstream (stomach and duodenum) retention when compared to control IgG counterparts, suggesting GI targeting. This was validated by transmission electron microscopy and energy dispersive X-ray spectroscopy, which revealed anti-ICAM NCs in vesicular compartments within duodenal epithelial cells. These results will guide future work aimed at improving intraoral delivery of targeted therapeutics for the treatment of GI pathologies.

Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis.

The gastrointestinal (GI) tract is lined by a single layer of epithelial cells which function in secretion, absorption, and digestion. In addition, most GI tract tumors develop from epithelial cells (carcinomas). This protocol describes isolation of the surface epithelium from the underlying stroma, muscular layer and submucosa in the GI tract. In this protocol, epithelial cell adhesions are weekend by chelating Ca +2 ions followed by mechanical separation of the cells by vortexing. Analysis of protein levels and gene expression patterns in isolated epithelial cells versus whole GI tissue minimizes the potential for confounding contributions from contaminating stromal cells.

Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer

BackgroundColorectal-cancer (CRC) research has greatly benefited from the availability of small animal tumor models. Spontaneous and chemically-induced CRC models are widely used yet limited in their resemblance to human disease and are often prolonged, not accurately repetitive, and associated with inflammatory side effects. In-situ murine or human tumor implantation in the gastrointestinal tract of mice is extremely challenging, and limited by inter-animal variability and procedure-related complications and mortality. As a result, in frequent studies CRC is implanted in distal sites, most commonly the subcutaneous region, an approach that is greatly limited by the absence of normal gastrointestinal tumor milieu and has substantial effects on tumor development.AimsIn this study we aimed to develop a well-tolerated repetitive tool to study CRC in small animals by adapting the murine colonoscopy system to serve as a platform for colonic sub-mucosal orthotopic implantation of human and murine CRC tumor cells.ResultsWe report the establishment of a novel small-animal CRC model that is minimally invasive, rapid, well-tolerated, highly reproducible, and confers precise control of tumor number, location and growth rate. Moreover, we show that this model uniquely allows the side-by-side induction of distinct genetically manipulated tumors, enabling the mechanistic study of tumor interaction and cross-talk within the native intestinal microenvironment.ConclusionsEmployment of this new approach may represent a major technical advance for the in-vivo study of CRC.

Suppression of Tumorigenicity-14, encoding matriptase, is a critical suppressor of colitis and colitis-associated colon carcinogenesis

Colitis-associated colorectal cancers are an etiologically distinct subgroup of colon cancers that occur in individuals suffering from inflammatory bowel disease and arise as a consequence of persistent exposure of hyperproliferative epithelial stem cells to an inflammatory microenvironment. An intrinsic defect in the intestinal epithelial barrier has been proposed to be one of several factors that contribute to the inappropriate immune response to the commensal microbiota that underlies inflammatory bowel disease. Matriptase is a membrane-anchored serine protease encoded by Suppression of Tumorigenicity-14 (ST14) that strengthens the intestinal epithelial barrier by promoting tight junction formation. Here we show that intestinal epithelial-specific ablation of St14 in mice causes formation of colon adenocarcinoma with very early onset and high penetrance. Neoplastic progression is preceded by a chronic inflammation of the colon that resembles human inflammatory bowel disease and is promoted by the commensal microbiota. This study demonstrates that inflammation-associated colon carcinogenesis can be initiated and promoted solely by an intrinsic intestinal permeability barrier perturbation, establishes St14 as a critical tumor suppressor gene in the mouse gastrointestinal tract, and adds matriptase to the expanding list of pericellular proteases with tumor suppressive functions.

The Role of β-Arrestin2 in the Mechanism of Morphine Tolerance in the Mouse and Guinea Pig Gastrointestinal Tract

β-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of β-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 μM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC(50) values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from β-arrestin2 knockout mice. β-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of β-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 μM) exposure. Naloxone prevented the decrease in β-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in β-arrestin2 occurred. We conclude that a decrease in β-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

Pitfalls using tyramide signal amplification (TSA) in the mouse gastrointestinal tract: Endogenous streptavidin-binding sites lead to false positive staining

Highly sensitive immunohistochemical detection systems such as tyramide signal amplification (TSA) are widely used, since they allow using two primary antibodies raised in the same species. Most of them are based on the streptavidin–biotin–peroxidase system and include streptavidin-coupled secondary antibodies. Using TSA in cryostat-sectioned tissues of mouse esophagus, we were puzzled by negative controls with unexpected staining mostly in the ganglionic areas. This prompted us to search for the causing agent and to include also other parts of the mouse gastrointestinal tract for comparison. Streptavidin-coupled antibodies bound to endogenous binding sites yet to be characterized, which are present throughout the mouse intestines. Staining was mainly localized around neuronal cell bodies of enteric ganglia. Thus, caution is warranted when applying streptavidin-coupled antibodies in the mouse gastrointestinal tract. The use of endogenous biotin-blocking kits combined with a prolonged post-fixation time could significantly reduce unintentional staining.