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Archives of Pathology & Laboratory... May 2022Intestinal neuronal dysplasia type B (IND B) is a complex entity involving the enteric nervous system, clinically manifested with constipation in infancy. Diagnosis has...
CONTEXT.—
Intestinal neuronal dysplasia type B (IND B) is a complex entity involving the enteric nervous system, clinically manifested with constipation in infancy. Diagnosis has been established by histopathologic analysis of rectal biopsies. However, the criteria for the diagnosis have been questioned and modified, hindering diagnostic practice.
OBJECTIVE.—
To analyze the applicability of PTEN immunohistochemistry in the diagnosis of IND B and to compare with control cases and cases of Hirschsprung disease (HD).
DESIGN.—
PTEN immunohistochemical expression was analyzed in colorectal samples from 29 cases of IND B and compared with 4 control cases and 6 cases of HD. The pattern of PTEN immunoexpression was analyzed in glial cells of the submucosal and myenteric nerve plexuses and in neural fibrils of the muscularis propria using a scoring system.
RESULTS.—
Marked reduction or absence of PTEN expression was observed in glial cells of the submucosal nerve plexuses in all cases of the IND B group and in the myenteric nerve plexuses in 28 of 29 cases (96.5%). Lack of PTEN expression was detected in neural fibrils within the muscularis propria in 21 of 29 cases (72%) of the IND B group. PTEN expression was positive in the same neural structures of the control and HD groups.
CONCLUSIONS.—
PTEN immunohistochemistry may be a valuable tool in the diagnostic evaluation of IND B. Lack of or reduction of PTEN expression in neural fibrils within the muscularis propria suggests that involvement of the neuromuscular junction may be a key event in the pathogenesis of the motility disturbance occurring in IND B.
Topics: Humans; Immunohistochemistry; Enteric Nervous System; Myenteric Plexus; Hirschsprung Disease; Constipation; PTEN Phosphohydrolase
PubMed: 35943858
DOI: 10.5858/arpa.2021-0424-OA -
Nature Neuroscience Jan 2021Autonomous regulation of the intestine requires the combined activity of functionally distinct neurons of the enteric nervous system (ENS). However, the variety of...
Autonomous regulation of the intestine requires the combined activity of functionally distinct neurons of the enteric nervous system (ENS). However, the variety of enteric neuron types and how they emerge during development remain largely unknown. Here, we define a molecular taxonomy of 12 enteric neuron classes within the myenteric plexus of the mouse small intestine using single-cell RNA sequencing. We present cell-cell communication features and histochemical markers for motor neurons, sensory neurons and interneurons, together with transgenic tools for class-specific targeting. Transcriptome analysis of the embryonic ENS uncovers a novel principle of neuronal diversification, where two neuron classes arise through a binary neurogenic branching and all other identities emerge through subsequent postmitotic differentiation. We identify generic and class-specific transcriptional regulators and functionally connect Pbx3 to a postmitotic fate transition. Our results offer a conceptual and molecular resource for dissecting ENS circuits and predicting key regulators for directed differentiation of distinct enteric neuron classes.
Topics: Animals; Cell Communication; Enteric Nervous System; Homeodomain Proteins; Interneurons; Mice; Mice, Inbred C57BL; Motor Neurons; Myenteric Plexus; Neurons; Proto-Oncogene Proteins; RNA; Sensory Receptor Cells; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome
PubMed: 33288908
DOI: 10.1038/s41593-020-00736-x -
Journal of Crohn's & Colitis Jan 2024Pain is a cardinal symptom in inflammatory bowel disease [IBD]. An important structure in the transduction of pain signalling is the myenteric plexus [MP]. Nevertheless,...
BACKGROUND AND AIMS
Pain is a cardinal symptom in inflammatory bowel disease [IBD]. An important structure in the transduction of pain signalling is the myenteric plexus [MP]. Nevertheless, IBD-associated infiltration of the MP by immune cells lacks in-depth characterisation. Herein, we decipher intra- and periganglionic immune cell infiltrations in Crohn´s disease [CD] and ulcerative colitis [UC] and provide a comparison with murine models of colitis.
METHODS
Full wall specimens of surgical colon resections served to examine immune cell populations by either conventional immuno-histochemistry or immunofluorescence followed by either bright field or confocal microscopy. Results were compared with equivalent examinations in various murine models of intestinal inflammation.
RESULTS
Whereas the MP morphology was not significantly altered in IBD, we identified intraganglionic IBD-specific B cell- and monocyte-dominant cell infiltrations in CD. In contrast, UC-MPs were infiltrated by CD8+ T cells and revealed a higher extent of ganglionic cell apoptosis. With regard to the murine models of intestinal inflammation, the chronic dextran sulphate sodium [DSS]-induced colitis model reflected CD [and to a lesser extent UC] best, as it also showed increased monocytic infiltration as well as a modest B cell and CD8+ T cell infiltration.
CONCLUSIONS
In CD, MPs were infiltrated by B cells and monocytes. In UC, mostly CD8+ cytotoxic T cells were found. The chronic DSS-induced colitis in the mouse model reflected best the MP-immune cell infiltrations representative for IBD.
Topics: Animals; Mice; Colitis, Ulcerative; Crohn Disease; Myenteric Plexus; Inflammatory Bowel Diseases; Colitis; Neurotransmitter Agents; Pain; Inflammation
PubMed: 37565754
DOI: 10.1093/ecco-jcc/jjad122 -
Cellular and Molecular Gastroenterology... 2021Neuroinflammation in the gut is associated with many gastrointestinal (GI) diseases, including inflammatory bowel disease. In the brain, neuroinflammatory conditions are...
BACKGROUND & AIMS
Neuroinflammation in the gut is associated with many gastrointestinal (GI) diseases, including inflammatory bowel disease. In the brain, neuroinflammatory conditions are associated with blood-brain barrier (BBB) disruption and subsequent neuronal injury. We sought to determine whether the enteric nervous system is similarly protected by a physical barrier and whether that barrier is disrupted in colitis.
METHODS
Confocal and electron microscopy were used to characterize myenteric plexus structure, and FITC-dextran assays were used to assess for presence of a barrier. Colitis was induced with dextran sulfate sodium, with co-administration of liposome-encapsulated clodronate to deplete macrophages.
RESULTS
We identified a blood-myenteric barrier (BMB) consisting of extracellular matrix proteins (agrin and collagen-4) and glial end-feet, reminiscent of the BBB, surrounded by a collagen-rich periganglionic space. The BMB is impermeable to the passive movement of 4 kDa FITC-dextran particles. A population of macrophages is present within enteric ganglia (intraganglionic macrophages [IGMs]) and exhibits a distinct morphology from muscularis macrophages, with extensive cytoplasmic vacuolization and mitochondrial swelling but without signs of apoptosis. IGMs can penetrate the BMB in physiological conditions and establish direct contact with neurons and glia. Dextran sulfate sodium-induced colitis leads to BMB disruption, loss of its barrier integrity, and increased numbers of IGMs in a macrophage-dependent process.
CONCLUSIONS
In intestinal inflammation, macrophage-mediated degradation of the BMB disrupts its physiological barrier function, eliminates the separation of the intra- and extra-ganglionic compartments, and allows inflammatory stimuli to access the myenteric plexus. This suggests a potential mechanism for the onset of neuroinflammation in colitis and other GI pathologies with acquired enteric neuronal dysfunction.
Topics: Animals; Biomarkers; Colitis; Disease Models, Animal; Disease Susceptibility; Enteric Nervous System; Extracellular Matrix; Fluorescent Antibody Technique; Immunohistochemistry; Immunophenotyping; Macrophages; Mice; Myenteric Plexus; Neuroglia; Neuroinflammatory Diseases; Neutrophil Infiltration
PubMed: 34246810
DOI: 10.1016/j.jcmgh.2021.07.003 -
STAR Protocols Mar 2022The myenteric plexus is located between the longitudinal and circular layers of muscularis externa in the gastrointestinal tract. It contains a large network of enteric...
The myenteric plexus is located between the longitudinal and circular layers of muscularis externa in the gastrointestinal tract. It contains a large network of enteric neurons that form the enteric nervous system (ENS) and control intestinal functions, such as motility and nutrient sensing. This protocol describes the method for physical separation (peeling) of muscularis and submucosal layers of the mouse intestine. Subsequently, the intestinal layers are then processed for flow cytometry and/or immunofluorescence analysis. For complete details on the use and execution of this profile, please refer to Ahrends et al. (2021).
Topics: Animals; Flow Cytometry; Fluorescent Antibody Technique; Gastrointestinal Tract; Mice; Mice, Inbred C57BL; Myenteric Plexus; Submucous Plexus
PubMed: 35146454
DOI: 10.1016/j.xpro.2022.101157 -
International Journal of Molecular... Jan 2018Calbindin (CALB) is well established as immunohistochemical marker for intrinsic primary afferent neurons in the guinea pig gut. Its expression by numerous human enteric...
Calbindin (CALB) is well established as immunohistochemical marker for intrinsic primary afferent neurons in the guinea pig gut. Its expression by numerous human enteric neurons has been demonstrated but little is known about particular types of neurons immunoreactive for CALB. Here we investigated small and large intestinal wholemount sets of 26 tumor patients in order to evaluate (1) the proportion of CALB⁺ neurons in the total neuron population, (2) the colocalization of CALB with calretinin (CALR), somatostatin (SOM) and vasoactive intestinal peptide (VIP) and (3) the morphology of CALB neurons. CALB neurons represented a minority of myenteric neurons (small intestine: 31%; large intestine: 25%) and the majority of submucosal neurons (between 72 and 95%). In the submucosa, most CALB⁺ neurons co-stained for CALR and VIP (between 69 and 80%) or for SOM (between 20 and 3%). In the myenteric plexus, 85% of CALB neurons did not co-stain with the other markers investigated. An unequivocal correlation between CALB reactivity and neuronal morphology was found for myenteric type III neurons in the small intestine: uniaxonal neurons with long, slender and branched dendrites were generally positive for CALB. Since also other neurons displayed occasional CALB reactivity, this protein is not suited as an exclusive marker for type III neurons.
Topics: Adult; Aged; Aged, 80 and over; Calbindin 1; Female; Humans; Male; Middle Aged; Myenteric Plexus; Neurons; Somatostatin; Submucous Plexus; Vasoactive Intestinal Peptide
PubMed: 29316719
DOI: 10.3390/ijms19010194 -
American Journal of Physiology.... Mar 2023Vagal preganglionic neurons innervate myenteric ganglia. These autonomic efferents are distributed so densely within the ganglia that it has been impractical to track...
Vagal preganglionic neurons innervate myenteric ganglia. These autonomic efferents are distributed so densely within the ganglia that it has been impractical to track individual vagal axons through the myenteric plexus with tracer labeling. To evaluate whether vagal efferent axons evidence selectivity, particularly for nitrergic or non-nitrergic myenteric neurons within the plexus, we limited the numbers and volumes of brainstem dextran biotin tracer injections per animal. Reduced labeling and the use of immunohistochemistry generated cases in which some individual axons could be distinguished and traced in three dimensions (Neurolucida) within and among successive (up to 46) myenteric ganglia. In the myenteric plexus of all stomach regions, the majority (∼86%) of vagal efferents were organized into two distinct subtypes. One subtype (∼24% of dextran-labeled efferents, designated "primarily nitrergic") selectively contacted and linked-both within and between ganglia-nitric oxide synthase positive (nNOS+) neurons into presumptive motor modules. A second subtype (∼62% of efferents, designated "primarily non-nitrergic") appeared to selectively contact and link-both within and between ganglia-non-nitrergic enteric neurons into a second type of effector ensemble. A third candidate type (∼14% of labeled preganglionics), appeared to lack "nitrergic selectivity" and to contact both nNOS+ and nNOS- enteric neurons. In addition to the quantitative assessment of the efferent axons in stomach, qualitative observations of the proximal duodenum indicated similar selective vagal efferent projections, in proportions comparable with those evaluated in the stomach. Limited injections of tracer, three-dimensional (3-D) tracing of individual axons, and histochemistry of myenteric neurons might distinguish additional efferent phenotypes. The present study highlights the following: ) one type of vagal efferent axon selectively innervates nitrergic upper gastrointestinal myenteric neurons; ) a second type of vagal efferent selectively innervates non-nitrergic gastrointestinal myenteric neurons; and ) the two types of vagal efferents might modulate peristalsis reciprocally and cooperatively.
Topics: Animals; Myenteric Plexus; Dextrans; Vagus Nerve; Axons; Neurons
PubMed: 36622086
DOI: 10.1152/ajpregu.00260.2022 -
The Journal of Histochemistry and... Oct 2018Oxaliplatin (platinum-based chemotherapeutic agent) is a first-line treatment of colorectal malignancies; its use associates with peripheral neuropathies and...
Oxaliplatin (platinum-based chemotherapeutic agent) is a first-line treatment of colorectal malignancies; its use associates with peripheral neuropathies and gastrointestinal side effects. These gastrointestinal dysfunctions might be due to toxic effects of oxaliplatin on the intestinal innervation and glia. Male Balb/c mice received intraperitoneal injections of sterile water or oxaliplatin (3 mg/kg/d) triweekly for 2 weeks. Colon tissues were collected for immunohistochemical assessment at day 14. The density of sensory, adrenergic, and cholinergic nerve fibers labeled with calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), and vesicular acetylcholine transporter (VAChT), respectively, was assessed within the myenteric plexus of the distal colon. The number and proportion of excitatory neurons immunoreactive (IR) against choline acetyltransferase (ChAT) were counted, and the density of glial subpopulations was determined by using antibodies specific for glial fibrillary acidic protein (GFAP) and s100β protein. Oxaliplatin treatment induced significant reduction of sensory and adrenergic innervations, as well as the total number and proportion of ChAT-IR neurons, and GFAP-IR glia, but increased s100β expression within the myenteric plexus of the distal colon. Treatment with oxaliplatin significantly alters nerve fibers and glial cells in the colonic myenteric plexus, which could contribute to long-term gastrointestinal side effects following chemotherapeutic treatment.
Topics: Animals; Antineoplastic Agents; Colon; Colorectal Neoplasms; Male; Mice, Inbred BALB C; Motor Neurons; Myenteric Plexus; Neuroglia; Oxaliplatin
PubMed: 29741434
DOI: 10.1369/0022155418774755 -
Gut Mar 1970
Review
Topics: Cathartics; Chagas Disease; Colonic Diseases; Duodenum; Esophageal Achalasia; Gastrointestinal Motility; Humans; Intestinal Obstruction; Malabsorption Syndromes; Megacolon; Myenteric Plexus; Nervous System Diseases; Neurons; Pyloric Stenosis
PubMed: 4987386
DOI: 10.1136/gut.11.3.271 -
Cell and Tissue Research Apr 2022We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide...
We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.
Topics: Animals; Myenteric Plexus; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Rats; Stomach; Submucous Plexus
PubMed: 35146560
DOI: 10.1007/s00441-022-03594-0