The small and large intestine contain related mesenchymal subsets that derive from embryonic Gli1+ precursors.

The intestinal lamina propria contains a diverse network of fibroblasts that provide key support functions to cells within their local environment. Despite this, our understanding of the diversity, location and ontogeny of fibroblasts within and along the length of the intestine remains incomplete. Here we show that the small and large intestinal lamina propria contain similar fibroblast subsets that locate in specific anatomical niches. Nevertheless, we find that the transcriptional profile of similar fibroblast subsets differs markedly between the small intestine and colon suggesting region specific functions. We perform in vivo transplantation and lineage-tracing experiments to demonstrate that adult intestinal fibroblast subsets, smooth muscle cells and pericytes derive from Gli1-expressing precursors present in embryonic day 12.5 intestine. Trajectory analysis of single cell RNA-seq datasets of E12.5 and adult mesenchymal cells suggest that adult smooth muscle cells and fibroblasts derive from distinct embryonic intermediates and that adult fibroblast subsets develop in a linear trajectory from CD81+ fibroblasts. Finally, we provide evidence that colonic subepithelial PDGFRαhi fibroblasts comprise several functionally distinct populations that originate from an Fgfr2-expressing fibroblast intermediate. Our results provide insights into intestinal stromal cell diversity, location, function, and ontogeny, with implications for intestinal development and homeostasis.

Protective and restorative effects of sophorolipid on intestinal dystrophy in dextran sulfate sodium-induced colitis mouse model.

The public has gradually begun to regard inflammatory bowel disease (IBD) as a crucial health issue; however, its mode of action has not been fully elucidated. Sophorolipid (SPL), a glycolipid-type biosurfactant, could be used as a potential treatment in physical intestinal dystrophy. We conducted a 2 × 2 factorial experiment to investigate the protective effect of SPL in a dextran sulfate sodium (DSS)-induced colitis mouse model (first factor, presence of SPL in feed; second factor, presence of DSS in water). Forty C57BL/6 mice (8-week-old) were used, and they were allocated to treatments according to their initial body weight. After a 7 d adjustment period, the DSS treatment was initiated in specific groups. At day 14, DSS was withdrawn from mice, and half of the mice were randomly selected and euthanized to collect colon and colon content samples. Three days after the end of DSS treatment, the rest of the mice were euthanized to investigate the therapeutic effect of SPL. Dietary SPL improved the growth performance in 3 d after DSS treatment, and the histopathological score was lower in the DSS-treated SPL group than in the DSS-treated control group. Mucosal thickness and goblet cell numbers significantly increased in the SPL-supplemented groups compared to in the control group. Similarly, SPL supplementation upregulated the gene expression levels of mucin-2, interleukin-10, and transforming growth factor-ß, and increased the concentration of short chain fatty acid compared to the control groups. In conclusion, dietary supplementation with SPL attenuated the pathological response against acute and chronic inflammation by the maintenance of the mucosal barrier and wound healing capacity.

Distinct Age-Specific miRegulome Profiling of Isolated Small and Large Intestinal Epithelial Cells in Mice.

The intestinal epithelium serves as a dynamic barrier to protect the host tissue from exposure to a myriad of inflammatory stimuli in the luminal environment. Intestinal epithelial cells (IECs) encompass differentiated and specialized cell types that are equipped with regulatory genes, which allow for sensing of the luminal environment. Potential inflammatory cues can instruct IECs to undergo a diverse set of phenotypic alterations. Aging is a primary risk factor for a variety of diseases; it is now well-documented that aging itself reduces the barrier function and turnover of the intestinal epithelium, resulting in pathogen translocation and immune priming with increased systemic inflammation. In this study, we aimed to provide an effective epigenetic and regulatory outlook that examines age-associated alterations in the intestines through the profiling of microRNAs (miRNAs) on isolated mouse IECs. Our microarray analysis revealed that with aging, there is dysregulation of distinct clusters of miRNAs that was present to a greater degree in small IECs (22 miRNAs) compared to large IECs (three miRNAs). Further, miRNA-mRNA interaction network and pathway analyses indicated that aging differentially regulates key pathways between small IECs (e.g., toll-like receptor-related cascades) and large IECs (e.g., cell cycle, Notch signaling and small ubiquitin-related modifier pathway). Taken together, current findings suggest novel gene regulation pathways by epithelial miRNAs in aging within the gastrointestinal tissues.

Intestinal organoids in farm animals.

In livestock species, the monolayer of epithelial cells covering the digestive mucosa plays an essential role for nutrition and gut barrier function. However, research on farm animal intestinal epithelium has been hampered by the lack of appropriate in vitro models. Over the past decade, methods to culture livestock intestinal organoids have been developed in pig, bovine, rabbit, horse, sheep and chicken. Gut organoids from farm animals are obtained by seeding tissue-derived intestinal epithelial stem cells in a 3-dimensional culture environment reproducing in vitro the stem cell niche. These organoids can be generated rapidly within days and are formed by a monolayer of polarized epithelial cells containing the diverse differentiated epithelial progeny, recapitulating the original structure and function of the native epithelium. The phenotype of intestinal organoids is stable in long-term culture and reflects characteristics of the digestive segment of origin. Farm animal intestinal organoids can be amplified in vitro, cryopreserved and used for multiple experiments, allowing an efficient reduction of the use of live animals for experimentation. Most of the studies using livestock intestinal organoids were used to investigate host-microbe interactions at the epithelial surface, mainly focused on enteric infections with viruses, bacteria or parasites. Numerous other applications of farm animal intestinal organoids include studies on nutrient absorption, genome editing and bioactive compounds screening relevant for agricultural, veterinary and biomedical sciences. Further improvements of the methods used to culture intestinal organoids from farm animals are required to replicate more closely the intestinal tissue complexity, including the presence of non-epithelial cell types and of the gut microbiota. Harmonization of the methods used to culture livestock intestinal organoids will also be required to increase the reproducibility of the results obtained in these models. In this review, we summarize the methods used to generate and cryopreserve intestinal organoids in farm animals, present their phenotypes and discuss current and future applications of this innovative culture system of the digestive epithelium.

Gut commensals suppress interleukin-2 production through microRNA-200/BCL11B and microRNA-200/ETS-1 axes in lamina propria leukocytes of murine large intestine.

The role of microRNAs (miRNAs) in how microbiota influence the host intestinal immune system is not fully understood. We compared the expression profiles of miRNAs and mRNAs in lamina propria leukocytes (LPL) in the large intestines of germ-free (GF) and specific pathogen-free (SPF) mice. Microarray analysis revealed different expression profiles of miRNAs and mRNAs between GF and SPF mice. Quantitative real time-PCR (qRT-PCR) showed that the level of miR-200 family members was significantly higher in SPF mice than in GF mice. In silico prediction followed by qRT-PCR suggested that Bcl11b, Ets1, Gbp7, Stat5b, and Zeb1 genes were downregulated by the miR-200 family. Western blotting revealed that the expression of BCL11B and ETS-1, but not ZEB1, in large intestinal LPL was significantly lower in SPF mice than in GF mice. Interleukin (IL)-2 production in cultured LPL upon stimulation with phorbol 12-myristate 13-acetate and ionomycin for 24 h was significantly lower in SPF mice than in GF mice. Conventionalization of GF mice substantially recapitulated SPF mice in terms of the expression of miR-200 family members and their target genes and IL-2 production in large intestinal LPL. Considering that BCL11B and ETS-1 reportedly function as transcription factors to activate the Il2 gene, we propose that the presence of gut commensals suppresses IL-2 production in large intestinal LPL, at least in part through post-transcriptional downregulation of Bcl11b and Ets1 genes by miR-200 family members.

Non-invasive evaluation of the equine gastrointestinal mucosal transcriptome.

Evaluating the health and function of the gastrointestinal tract can be challenging in all species, but is especially difficult in horses due to their size and length of the gastrointestinal (GI) tract. Isolation of mRNA of cells exfoliated from the GI mucosa into feces (i.e., the exfoliome) offers a novel means of non-invasively examining the gene expression profile of the GI mucosa. This approach has been utilized in people with colorectal cancer. Moreover, we have utilized this approach in a murine model of GI inflammation and demonstrated that the exfoliome reflects the tissue transcriptome. The ability of the equine exfoliome to provide non-invasive information regarding the health and function of the GI tract is not known. The objective of this study was to characterize the gene expression profile found in exfoliated intestinal epithelial cells from normal horses and compare the exfoliome data with the tissue mucosal transcriptome. Mucosal samples were collected from standardized locations along the GI tract (i.e. ileum, cecum, right dorsal colon, and rectum) from four healthy horses immediately following euthanasia. Voided feces were also collected. RNA isolation, library preparation, and RNA sequencing was performed on fecal and intestinal mucosal samples. Comparison of gene expression profiles from the tissue and exfoliome revealed correlation of gene expression. Moreover, the exfoliome contained reads representing the diverse array of cell types found in the GI mucosa suggesting the equine exfoliome serves as a non-invasive means of examining the global gene expression pattern of the equine GI tract.

Single cell transcriptomic profiling of large intestinal enteroendocrine cells in mice - Identification of selective stimuli for insulin-like peptide-5 and glucagon-like peptide-1 co-expressing cells.

OBJECTIVE: Enteroendocrine cells (EECs) of the large intestine, found scattered in the epithelial layer, are known to express different hormones, with at least partial co-expression of different hormones in the same cell. Here we aimed to categorize colonic EECs and to identify possible targets for selective recruitment of hormones. METHODS: Single cell RNA-sequencing of sorted enteroendocrine cells, using NeuroD1-Cre x Rosa26-EYFP mice, was used to cluster EECs from the colon and rectum according to their transcriptome. G-protein coupled receptors differentially expressed across clusters were identified, and, as a proof of principle, agonists of Agtr1a and Avpr1b were tested as candidate EEC secretagogues in vitro and in vivo. RESULTS: EECs from the large intestine separated into 7 clear clusters, 4 expressing higher levels of Tph1 (enzyme required for serotonin (5-HT) synthesis; enterochromaffin cells), 2 enriched for Gcg (encoding glucagon-like peptide-1, GLP-1, L-cells), and the 7th expressing somatostatin (D-cells). Restricted analysis of L-cells identified 4 L-cell sub-clusters, exhibiting differential expression of Gcg, Pyy (Peptide YY), Nts (neurotensin), Insl5 (insulin-like peptide 5), Cck (cholecystokinin), and Sct (secretin). Expression profiles of L- and enterochromaffin cells revealed the clustering to represent gradients along the crypt-surface (cell maturation) and proximal-distal gut axes. Distal colonic/rectal L-cells differentially expressed Agtr1a and the ligand angiotensin II was shown to selectively increase GLP-1 and PYY release in vitro and GLP-1 in vivo. CONCLUSION: EECs in the large intestine exhibit differential expression gradients along the crypt-surface and proximal-distal axes. Distal L-cells can be differentially stimulated by targeting receptors such as Agtr1a.

Wall thickness and mucous cell distribution in the rabbit large intestine.

To achieve a better understanding of rabbit large intestinal functions, such as production of hard and soft feces and cecal fermentation, knowledge of the intestinal wall structure is essential. However, such knowledge is far from complete. Therefore, the aims of this study were to measure the thickness of the wall and its constituent layers and describe distribution of mucous cells in each segment of the large intestine in New Zealand White rabbits. Results showed that the cecum had the thinnest entire wall throughout the large intestine, and the fusus coli and rectum had a thicker entire wall in comparison to the cecum, the first segment of the proximal colon, the second segment of the proximal colon, and the distal colon. Moreover, the thickness of the mucosa in the fusus coli and that of the inner and outer layers of the tunica muscularis in the rectum were greater than that of the other segments. Mucous cells in the mucosa were the fewest in the cecum and most numerous in the fusus coli. This study provides detailed knowledge of the wall thickness and distribution of mucous cells in the large intestine of the rabbit. These findings are important for improving our understanding of rabbit intestinal physiology and pathology.

The Adenomatous Polyposis Coli (APC) mutation spectra in different anatomical regions of the large intestine in colorectal cancer.

The enhanced incidence of colorectal cancer (CRC) in the U.S.A. has been linked to promutagens, such as heterocyclic aromatic amines, in the western diet that are produced by high temperature cooking of meat. However, a prior analysis of driver nonsense mutations in the Adenomatous Polyposis Coli (APC) gene, which is mutated in 75% of human CRC, indicated that the C·G → A·T transversions produced by this class of mutagens were not enriched but actually lower than what would be statistically anticipated. Moreover, the APC mutation patterns in the U.S.A. vs. China were indistinguishable despite differences in diet. In the present study, we have dissected the APC mutation pattern in tumors that arise in the different anatomical regions of the large intestine. The results show that the nonsense mutation pattern in APC differ in the different regions and that there is a statistically significant increase in C·G → A·T transversions in the rectum vs. the other regions, albeit, the percent of C·G → A·T mutations still remains lower than predicted based on random mutagenesis.

Effect of deoxynivalenol on the levels of toll-like receptors 2 and 9 and their mRNA expression in enterocytes in the porcine large intestine: a preliminary study.

Deoxynivalenol (DON), one of the most prevalent mycotoxins in the world, and is capable of inducing immune disorders in humans and animals. The aim of this study was to determine the effect of feed contaminated with DON on the number of TLR2- and TLR9-positive cells and their mRNA expression in the porcine large intestine. The experiment was conducted on two equal groups of pigs (n=4). The experimental group (E) was administered feed contaminated with DON (1008 µg/kg of feed) for 6 weeks, and the control group (C) was administered non-contaminated feed over the same period of time. A decrease in the expression of TLR2 mRNA was noted in the cecum. The percentage of TLR9-positive enterocytes increased in the ascending colon and decreased in the cecum. The results of this study indicate that DON can modify the local immune response by changing the expression of TLRs.

Maintenance of Distal Intestinal Structure in the Face of Prolonged Fasting: A Comparative Examination of Species From Five Vertebrate Classes.

It was recently shown that fasting alters the composition of microbial communities residing in the distal intestinal tract of animals representing five classes of vertebrates [i.e., fishes (tilapia), amphibians (toads), reptiles (leopard geckos), birds (quail), and mammals (mice)]. In this study, we tested the hypothesis that the extent of tissue reorganization in the fasted distal intestine was correlated with the observed changes in enteric microbial diversity. Segments of intestine adjacent to those used for the microbiota study were examined histologically to quantify cross-sectional and mucosal surface areas and thicknesses of mucosa, submucosa, and tunica muscularis. We found no fasting-induced differences in the morphology of distal intestines of the mice (3 days), quail (7 days), or geckos (28 days). The toads, which exhibited a general increase in phylogenetic diversity of their enteric microbiota with fasting, also exhibited reduced mucosal circumference at 14 and 21 days of fasting. Tilapia showed increased phylogenetic diversity of their enteric microbiota, and showed a thickened tunica muscularis at 21 days of fasting; but this morphological change was not related to microbial diversity or absorptive surface area, and thus, is unlikely to functionally match the changes in their microbiome. Given that fasting caused significant increases and reductions in the enteric microbial diversity of mice and quail, respectively, but no detectable changes in distal intestine morphology, we conclude that reorganization is not the primary factor shaping changes in microbial diversity within the fasted colon, and the observed modest structural changes are more related to the fasted state. Anat Rec, 300:2208-2219, 2017. © 2017 Wiley Periodicals, Inc.

Modeling and analysis of the Delta-Notch dependent boundary formation in the Drosophila large intestine.

BACKGROUND: The boundary formation in the Drosophila large intestine is widely studied as an important biological problem. It has been shown that the Delta-Notch signaling pathway plays an essential role in the formation of boundary cells. RESULTS: In this paper, we propose a mathematical model for the Delta-Notch dependent boundary formation in the Drosophila large intestine in order to better interpret related experimental findings of this biological phenomenon. To achieve this, we not only perform stability analysis on the model from a theoretical point of view, but also perform numerical simulations to analyze the model with and without noises, the phenotype change with the change of Delta or Notch expression, and the perturbation influences of binding and inhibition parameters on the boundary formation. CONCLUSIONS: By doing all these work, we can assure that our model can better interpret the biological findings related to the boundary formation in the Drosophila large intestine.

Commensal microbiota-induced microRNA modulates intestinal epithelial permeability through the small GTPase ARF4.

The intestinal tract contains many commensal bacteria that modulate various physiological host functions. Dysbiosis of commensal bacteria triggers dysfunction of the intestinal epithelial barrier, leading to the induction or aggravation of intestinal inflammation. To elucidate whether microRNA plays a role in commensal microbiome-dependent intestinal epithelial barrier regulation, we compared transcripts in intestinal epithelial cells (IECs) from conventional and germ-free mice and found that commensal bacteria induced the expression of miR-21-5p in IECs. miR-21-5p increased intestinal epithelial permeability and up-regulated ADP ribosylation factor 4 (ARF4), a small GTPase, in the IEC line Caco-2. We also found that ARF4 expression was up-regulated upon suppression of phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4), which are known miR-21-5p targets, by RNAi. Furthermore, ARF4 expression in epithelial cells of the large intestine was higher in conventional mice than in germ-free mice. ARF4 suppression in the IEC line increased the expression of tight junction proteins and decreased intestinal epithelial permeability. These results indicate that commensal microbiome-dependent miR-21-5p expression in IECs regulates intestinal epithelial permeability via ARF4, which may therefore represent a target for preventing or managing dysfunction of the intestinal epithelial barrier.

Distribution and characterisation of CCK containing enteroendocrine cells of the mouse small and large intestine.

There is general consensus that enteroendocrine cells, EEC, containing the enteric hormone cholecystokinin (CCK) are confined to the small intestine and predominate in the duodenum and jejunum. Contrary to this, EEC that express the gene for CCK have been isolated from the large intestine of the mouse and there is evidence for EEC that contain CCK-like immunoreactivity in the mouse colon. However, the human and rat colons do not contain CCK cells. In the current study, we use immunohistochemistry to investigate CCK peptide presence in endocrine cells, PCR to identify cck transcripts and chromatography to identify CCK peptide forms in the mouse small and large intestine. The colocalisation of CCK and 5-HT, hormones that have been hypothesised to derive from cells of different lineages, was also investigated. CCK immunoreactivity was found in EEC throughout the mouse small and large intestine but positive cells were rare in the rectum. Immunoreactive EEC were as common in the caecum and proximal colon as they were in the duodenum and jejunum. CCK gene transcripts were found in the mucosa throughout the intestine but mRNA for gastrin, a hormone that can bind some anti-CCK antibodies, was only found in the stomach and duodenum. Characterisation of CCK peptides of the colon by extraction, chromatographic separation and radioimmunoassay revealed bioactive amidated and sulphated forms, including CCK-8 and CCK-33. Moreover, CCK-containing EEC in the large intestine bound antibodies that target the biologically active sulfated form. Colocalisation of CCK and 5-HT occurred in a proportion of EEC throughout the small intestine and in the caecum but these hormones were not colocalised in the colon, where there was CCK and PYY colocalisation. It is concluded that authentic, biologically active, CCK occurs in EEC of the mouse large intestine.

Morphometric profile of large intestinal neuronal plexuses in normal perinatal autopsies and Hirschsprung disease.

OBJECTIVES: To derive objective values for the diagnosis of Hirschsprung disease (HSCR) from a comparison of the morphometric profile of large intestinal neuronal plexuses in normal perinatal autopsies and surgical specimens of HSCR. METHODS: A cross-sectional comparative study with 40 subjects each in (i) non-HSCR perinatal group encompassing neonates and stillborn babies beyond 30 weeks of gestation on whom autopsies were conducted and (ii) HSCR group comprising all patients clinicoradiologically diagnosed as HSCR. The morphometric assessment was done on hematoxylin-and-eosin-stained sections. KEY RESULTS: The morphometric profile in terms of average number of ganglia/linear mm of colon, interganglion distance, number of ganglion cells/ganglion, average ganglion cell length, ganglion cell nuclear area, ganglion cell nuclear diameter, nerve trunk thickness, and density has been outlined. On comparison with the neuroanatomically normal zone of HSCR, the cut-offs to identify hypertrophic nerve trunks (nerve trunk thickness of >37.85 µm) and reduced number of ganglia (number of ganglia/linear mm of colon <2.05 and interganglion distance of >229 µm) were derived. CONCLUSIONS & INFERENCES: The determined objective values, after testing on diagnostic rectal biopsies, may serve to formulate a diagnostic algorithm along with immunostaining for diagnosis of HSCR in colorectal specimens.

Optimizing neurogenic potential of enteric neurospheres for treatment of neurointestinal diseases.

BACKGROUND: Enteric neurospheres derived from postnatal intestine represent a promising avenue for cell replacement therapy to treat Hirschsprung disease and other neurointestinal diseases. We describe a simple method to improve the neuronal yield of spontaneously formed gut-derived neurospheres. MATERIALS AND METHODS: Enteric neurospheres were formed from the small and large intestines of mouse and human subjects. Neurosphere size, neural crest cell content, cell migration, neuronal differentiation, and neuronal proliferation in culture were analyzed. The effect of supplemental neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF) and endothelin-3, was also assessed. RESULTS: Mouse small intestine-derived neurospheres contained significantly more P75-expressing neural crest-derived cells (49.9 ± 15.3% versus 21.6 ± 11.9%, P < 0.05) and gave rise to significantly more Tuj1-expressing neurons than colon-derived neurospheres (69.9 ± 8.6% versus 46.2 ± 15.6%, P < 0.05). A similar pattern was seen in neurospheres isolated from human small and large intestine (32.6 ± 17.5% versus 10.2 ± 8.2% neural crest cells, P < 0.05; 29.7 ± 16.4% versus 16.0 ± 13.5% enteric neurons, P < 0.05). The addition of GDNF to the culture media further improved the neurogenic potential of small intestinal neurospheres (75.9 ± 4.0% versus 67.8 ± 5.8%, P < 0.05) whereas endothelin-3 had no effect. CONCLUSIONS: Enteric neurospheres formed from small intestine and supplemented with GDNF yield an enriched population of neural crest-derived progenitor cells and give rise to a high density of enteric neurons.

Doublecortin-like kinase 1-positive enterocyte - a new cell type in human intestine.

Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated kinase. In murine intestine, DCLK1 marks tuft cells with characteristic microvilli, features of neuroendocrine cells and also quiescent stem cell-like properties. The occurrence and pathological role of DCLK1-positive cells in human intestinal mucosa is unknown. We analysed DCLK1 expression in healthy duodenal, jejunal and colorectal mucosa samples (n = 35), and in duodenal specimens from patients with coeliac disease (n = 20). The samples were immunohistochemically double-stained with DCLK1, and synaptophysin, chromogranin A and Ki-67. Ultrastructure of DCLK1-expressing duodenal cells was assessed using correlative light and electron microscopy. DCLK1 expression was seen in about 1% of epithelial cells diffusely scattered through the intestinal epithelium. Electron microscopy showed that the duodenal DCLK1-positive cells had short apical microvilli similar to neighbouring enterocytes and cytoplasmic granules on the basal side. DCLK1-positive cells were stained with synaptophysin. The number of DCLK1-positive cells was decreased in villus atrophy in coeliac disease. Our findings indicate that in human intestinal epithelium, DLCK1-positive cells form a subpopulation of non-proliferating neuroendocrine cells with apical brush border similar to that in enterocytes, and their number is decreased in untreated coeliac disease.

A stromal cell population in the large intestine identified by tissue factor expression that is lost during colorectal cancer progression.

Colorectal cancer (CRC) is a major cause of morbidity and mortality, and the composition of the tumour stroma is a strong predictor of survival in this cancer type. Tissue factor (TF) functions as the trigger of haemostasis together with its ligand coagulation factor VII/VIIa, and TF expression has been found in tumour cells of colorectal tumours. However, TF expression in the CRC tumour stroma or its relationship to patient outcome has not yet been studied. To address this question we developed and validated a specific anti-TF antibody using standardised methods within the Human Protein Atlas project. We used this antibody to investigate TF expression in normal colorectal tissue and CRC using immunofluorescence and immunohistochemistry in two patient cohorts. TF was strongly expressed in a cell population immediately adjacent to the colorectal epithelium. These TF-positive cells were ACTA2-negative but weakly vimentin-positive, defining a specific population of pericryptal sheath cells. In colorectal tumours, TF-positive sheath cells were progressively lost after the adenoma-to-carcinoma transition, demonstrating downregulation of this source of TF in CRC. Furthermore, loss of sheath cell TF was significantly associated with poor overall and disease-specific survival in rectal but not colon cancers. In conclusion, we demonstrate that TF is a marker of a specific cell population in the large intestine, which is lost during CRC progression. Our results highlight the role of the tumour stroma in this cancer type and suggest TF to be a potential prognostic biomarker in rectal cancers through the identification of pericryptal sheath cells.

Macrophage Isolation from the Mouse Small and Large Intestine.

Macrophages play important roles in maintaining intestinal homeostasis via their ability to orchestrate responses to the normal microbiota as well as pathogens. One of the most important steps in beginning to understand the functions of these cells is the ability to effectively isolate them from the complex intestinal environment. Here, we detail methodology for the isolation and phenotypic characterization of macrophages from the mouse small and large intestine.

Identification of different functional types of spinal afferent neurons innervating the mouse large intestine using a novel CGRPα transgenic reporter mouse.

Spinal afferent neurons detect noxious and physiological stimuli in visceral organs. Five functional classes of afferent terminals have been extensively characterized in the colorectum, primarily from axonal recordings. Little is known about the corresponding somata of these classes of afferents, including their morphology, neurochemistry, and electrophysiology. To address this, we made intracellular recordings from somata in L6/S1 dorsal root ganglia and applied intraluminal colonic distensions. A transgenic calcitonin gene-related peptide-α (CGRPα)-mCherry reporter mouse, which enabled rapid identification of soma neurochemistry and morphology following electrophysiological recordings, was developed. Three distinct classes of low-threshold distension-sensitive colorectal afferent neurons were characterized; an additional group was distension-insensitive. Two of three low-threshold classes expressed CGRPα. One class expressing CGRPα discharged phasically, with inflections on the rising phase of their action potentials, at low frequencies, to both physiological (<30 mmHg) and noxious (>30 mmHg) distensions. The second class expressed CGRPα and discharged tonically, with smooth, briefer action potentials and significantly greater distension sensitivity than phasically firing neurons. A third class that lacked CGRPα generated the highest-frequency firing to distension and had smaller somata. Thus, CGRPα expression in colorectal afferents was associated with lower distension sensitivity and firing rates and larger somata, while colorectal afferents that generated the highest firing frequencies to distension had the smallest somata and lacked CGRPα. These data fill significant gaps in our understanding of the different classes of colorectal afferent somata that give rise to distinct functional classes of colorectal afferents. In healthy mice, the majority of sensory neurons that respond to colorectal distension are low-threshold, wide-dynamic-range afferents, encoding both physiological and noxious ranges.