Telocytes and Lymphatics of the Human Colon

Background: Telocytes (TCs) are a peculiar morphological type of stromal cells. They project long and moniliform telopodes, visible on various bidimensional sections. Originally regarded as “interstitial Cajal-like cells”, gastrointestinal TCs were CD34+. Further double-labelling studies found that colon TCs are negative for the expressions of the PDGFR-α and α-SMA. However, the TCs in colon were not distinguished specifically from endothelial cells (ECs), vascular or lymphatic. A combinational approach is important for accurate TC identification. Hence, we designed an immunohistochemical study of human colon to check whether ECs and CD34+ TCs express different markers. Methods: Immunohistochemistry was performed on archived paraffin-embedded samples of human colon (nine cases) for the following markers: CD31, CD34, CD117/c-kit and D2-40 (podoplanin). Results: A distinctive population of CD34+ TCs was found coating the myenteric ganglia. However, also perivascular cells and vascular ECs were CD34+. c-kit expression was equally found in interstitial Cajal cells (ICCs) and perivascular cells. The CD34 TCs did not express c-kit. As they were equally CD31- and D2-40- they were assessed as different from ECs. Conclusions: Testing specific markers of ECs, vascular and lymphatic, in the same tissues in which CD34+ TCs are found, is much more relevant than to identify TCs by transmission electron microscopy alone.

Intestinal Region-Specific and Layer-Dependent Induction of TNFα in Rats with Streptozotocin-Induced Diabetes and after Insulin Replacement

Tumour necrosis factor alpha (TNFα) is essential in neuroinflammatory modulation. Therefore, the goal of this study is to reveal the effects of chronic hyperglycaemia and insulin treatment on TNFα expression in different gut segments and intestinal wall layers. TNFα expression was mapped by fluorescent immunohistochemistry and quantitative immunogold electron microscopy in myenteric ganglia of duodenum, ileum and colon. Tissue TNFα levels were measured by enzyme-linked immunosorbent assays in muscle/myenteric plexus-containing (MUSCLE-MP) and mucosa/submucosa/submucous plexus-containing (MUC-SUBMUC-SP) homogenates. Increasing density of TNFα-labelling gold particles is observed in myenteric ganglia from proximal to distal segments and TNFα tissue levels are much more elevated in MUSCLE-MP homogenates than in MUC-SUBMUC-SP samples in healthy controls. In the diabetics, the number of TNFα gold labels is significantly increased in the duodenum, decreased in the colon and remained unchanged in the ileal ganglia, while insulin does not prevent these diabetes-related TNFα changes. TNFα tissue concentration is also increased in MUSCLE-MP homogenates of diabetic duodenum, while decreased in MUC-SUBMUC-SP samples of diabetic ileum and colon. These findings support that type 1 diabetes has region-specific and intestinal layer-dependent effects on TNFα expression, contributing to the regional damage of myenteric neurons and their intestinal milieu.

P061 Immune cell infiltrations of myenteric plexus in IBD – characterization and implications

Background IBD frequently causes chronic abdominal pain and visceral hypersensitivity. To understand development of pain in IBD in more detail, we analyzed the inflammatory infiltration of the enteric nervous system (ENS). A crucial signaling point for pain transmission is the myenteric plexus situated in the smooth muscle layer of the colon wall. We investigated (i) the immune cell infiltration within the myenteric plexus, (ii) neuronal cell survival and (iii) expression of neurotransmitters by immunostaining in surgical colonic samples from patients with Crohn′s disease (CD) or ulcerative colitis (UC). Methods FFPE material from surgeries (ileocecal resections and colectomies) was collected from 12 UC, nine CD and 11 patients that received surgeries for non-inflammatory reasons (controls). Immune cell composition, neurotransmitter expression and cell survival were analyzed by quantifying immune cell infiltration of the myenteric ganglia in immunohistochemistry sections. Immune cells within the myenteric plexus were defined as intraganglionar cells. The neurotransmitters CGRP and substance P as well as annexin V as a cell death marker were quantified based on their expression within the myenteric ganglia. Results CD3+CD4+ intraganglionar T-cells (216 ± 44 cells/mm²) were found to be increased for CD myenteric plexus compared to controls (79 ± 35 cells/mm²) (p=0.04) (see Fig. 1). For CD and UC, the cell counts of CD3+CD8+ lymphocytes infiltrating the myenteric plexus were significantly increased (controls: 39 ± 18 cells/mm², CD: 281 ± 105 cells/mm² (p=0.0004), UC: 177 ± 59 cells/mm² (p=0.04), Intraganglionar Foxp3+ T-cells were not significantly changed. Intraganglionar CD163+ and CD68+ monocytes were increased in CD (CD68+ monocytes: control: 377 ± 50 cells/mm², CD: 1278 ± 264 cells/mm², p=0.002 and CD163+ monocytes: control: 501 ± 97 cells/mm², CD: 963 ± 236 cells/mm², p=0.04). Expression levels of the neurotransmitter CGRP were found to be increased for UC myenteric plexus (control: 1.8 ± 0.2 units of intensity, UC: 2.8 ± 0.2 units of intensity, p=0.005). Substance P was found to be reduced in the myenteric plexus of CD patients if compared to controls (control: 2.1 ± 0.1 units of intensity, CD: 1.5 ± 0.2 units of intensity, p=0.007). UC myenteric plexus showed more annexin V-positive cells than control patients. Conclusion The intraganglionar immune cell composition of myenteric plexus in IBD comprises CD3+CD4+, CD3+CD8 T-cells in UC and CD3+CD4+, CD3+CD8+ and CD3+Foxp3+ T-cells as well as CD68+ and CD163+ monocytes in CD. In UC myenteric ganglia levels of the neurotransmitter CGRP are increased whereas substance P expression is reduced in CD. UC-affected myenteric plexus show increased levels of apoptosis in comparison to controls.

Microstructure of the nerve plexus of the muscular membrane of the gut of domestic ducks (Anas platyrhynchos domesticus) of different ages

As evidenced by the publications of recent years, contrary to the existing dogma about the immutability of the state of the enteric nervous system during the postnatal period of ontogenesis, the population of intestinal neurons is a dynamic formation, decreasing with age and changing due to the action of environmental factors. The current article presents the results of study of the microscopic structure of the nerve plexus of the muscular membrane of the enteric nervous system of domestic ducks (Anas platyrhynchos domesticus) of the black white–breasted breed, of nine age groups of 1–365 days of age. The topography, number, area of nerve nodes, as well as the density of neurons in them were determined on transverse sections of the duodenum, jejunum, ileum, caecum and rectum. For the purpose of a generalized assessment of the morphofunctional state of the nerve plexuses, two parameters were determined: the average age indicator of the gut and intestines. The average age indicator of a certain structure of each intestine was determined as the arithmetical average of its nine age indicators. The average age indicator of a certain gut structure was determined as the arithmetic average of the average age indicator of the structure of all five intestines. It has been established that the nerve plexus of the muscular membrane (myenteric, plexus Auerbachi) of the gut of domestic ducks, in contrast to mammals, is not located between the layers of the muscular membrane, but in its outer layer. On a transverse section of the gut wall, the myenteric ganglia and cords that connect have a predominantly elliptical shape. Despite a significant increase with age in the diameter and thickness of the gut wall, the total number of myenteric ganglia changed little, increasing or decreasing with varying degrees of reliability relative to the previous age. In the gut of ducks, during the first year of the postnatal period of ontogenesis, the smallest number of myenteric ganglia was found in the cecum, and the largest – in the ileum. The general pattern of the dynamics of the size of the myenteric ganglia of the gut of ducks was an increase in their area with age. Moreover, this indicator reached the greatest value at different ages of ducks: at 30 days of age in the ileum and cecum, at 180 days of age – in the rectum and at 365 days of age – in the duodenum. The smallest area of the myenteric ganglia was found in the jejunum, and the largest – in the duodenum and ileum. The smallest number of neurons in the ganglion was found in the cecum, and the largest – in the rectum, the lowest density of neurons in the ganglion was found in the cecum, and the largest – in the jejunum. The general quantitative pattern of neurons in the ganglion was the decrease in their density with age. Changes in the morphometric parameters of the ganglia of the nerve plexus of the muscular membrane of the ducks’ gut indicate the plasticity of the enteric nervous system, its ability to dynamically respond to the action of factors of the internal and external environment. It is promising to study the state of the submucous nerve plexus, as well as the cellular composition of the population of neurons of the enteric nervous system of domestic and wild poultry.

Luminal nutrients activate distinct patterns in submucosal and myenteric neurons in the mouse small intestine

Nutrient signals sensed by enteroendocrine cells are conveyed to the enteric nervous system (ENS) to initiate intestinal reflexes. We addressed whether there are specific enteric pathways dedicated to detecting different luminal nutrients. Calcium imaging was performed on intact jejunal preparations from Wnt1-cre;R26R-GCaMP3 and Villin-cre;R26R-GCaMP3 mice which express a fluorescent calcium indicator in their ENS or intestinal epithelium, respectively. Glucose, acetate, and L-phenylalanine were perfused onto the mucosa whilst imaging underlying enteric neurons. Nutrient transport or diffusion across the mucosa was mimicked by applying nutrients onto sensory nerve endings in a villus, or onto myenteric ganglia. The nutrients perfused onto the mucosa each elicited Ca2+ transients in submucosal neurons and in distinct patterns of myenteric neurons. Notably, the neurochemical subtypes of myenteric neurons that responded differed between the nutrients, while submucosal responders were predominantly cholinergic. Nutrients applied into villi or onto ganglia did not elicit specific neuronal responses but did stimulate Ca2+ signaling in the mucosal epithelium. These data suggest that nutrients are first detected at the level of the epithelium and that the ENS is capable of discriminating between different compositions of luminal content. Furthermore, our data show that responses to mucosal stimulation are primarily in the myenteric plexus and submucosal neurons respond secondarily.

Bisphenol A (BPA) Affects the Enteric Nervous System in the Porcine Stomach

Bisphenol A (BPA) is widely utilized in plastic production process all over the world. Previous studies have shown that BPA, with its similarity to estrogen, may negatively affect living organisms. It is acknowledged that BPA distorts the activity of multiple internal systems, including the nervous, reproductive, urinary, and endocrine systems. BPA also affects the gastrointestinal tract and enteric nervous system (ENS), which is placed throughout the wall from the esophagus to the rectum. Contrary to the intestine, the influence of BPA on the ENS in the stomach is still little known. This study, performed using the double immunofluorescence method, has revealed that BPA affects the number of nervous structures in the porcine gastric wall immunoreactive to vesicular acetylcholine transporter (VAChT, a marker of cholinergic neurons), substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL) and cocaine- and amphetamine-regulated transcript peptide (CART). The character and severity of noted alterations depended on the part of the ENS, the BPA dose, and the type of neuronal substance. Administration of BPA resulted in an increase in the number of nervous structures containing SP, GAL, and/or CART, and a decrease in the number of cholinergic neurons in all parts of the gastric wall. The number of VIP-positive nervous structures increased in the enteric myenteric ganglia, along with the muscular and mucosal layers, whilst it decreased in the submucous ganglia. The exact mechanism of noted changes was not absolutely obvious, but they were probably related to the neuroprotective and adaptive processes constituting the response to the impact of BPA.

COUNTEN – an AI-driven tool for rapid, and objective structural analyses of the Enteric Nervous System

Healthy gastrointestinal functions require a healthy Enteric Nervous System (ENS). ENS health is often defined by the presence of normal ENS structure. However, we currently lack a comprehensive understanding of normal ENS structure as current methodologies of manual enumeration of neurons within tissue and ganglia can only parse limited tissue regions; and are prone to error, subjective bias, and peer-to-peer discordance. Thus, there is a need to craft objective methods and robust tools to capture and quantify enteric neurons over a large area of tissue and within multiple ganglia. Here, we report on the development of an AI-driven tool COUNTEN which parses HuC/D-immunolabeled adult murine myenteric ileal plexus tissues to enumerate and classify enteric neurons into ganglia in a rapid, robust, and objective manner. COUNTEN matches trained humans in identifying, enumerating and clustering myenteric neurons into ganglia but takes a fraction of the time, thus allowing for accurate and rapid analyses of a large tissue region. Using COUNTEN, we parsed thousands of myenteric neurons and clustered them in hundreds of myenteric ganglia to compute metrics that help define the normal structure of the adult murine ileal myenteric plexus. We have made COUNTEN freely and openly available to all researchers, to facilitate reproducible, robust, and objective measures of ENS structure across mouse models, experiments, and institutions.

Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ

BACKGROUND & AIMSThe enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (EN). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions.METHODSHuman myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA-Seq. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA-Seq (snRNA-Seq), totaling over four billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse snRNA-Seq data. Comparative informatics between human and mouse datasets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction (HCR).RESULTSSeveral EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of one or more marker genes.CONCLUSIONSOur combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human GI studies.Graphical Abstract