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Journal of Biosciences 2023Enteric glial cells (EGCs) were shown to maintain the barrier integrity and immune homeostasis of the bowel. Postnatally, EGCs develop from progenitor cells located in...
Enteric glial cells (EGCs) were shown to maintain the barrier integrity and immune homeostasis of the bowel. Postnatally, EGCs develop from progenitor cells located in the myenteric plexus and are continuously replenished through adulthood. Both, murine EGC development and replenishment were shown to depend on the microbiome. The underlying mechanisms are still unknown, and we hypothesized that the myeloid differentiation primary response protein 88 (Myd88) or toll-like receptor signaling pathways may be involved. Adult and neonatal C57BL/6 wild-type (wt) and Myd88 mice were housed under specific pathogen-free (SPF) or germ-free (GF) conditions. GF mice were further conventionalized by gavaging stools from, and cohousing with, SPF mice having intact microbiomes. The small bowels were harvested at various time points, and immunohistochemistry and qPCR analysis of EGC markers in the muscularis externa and mucosa were performed. In wt mice, after conventionalization, the glial cell-specific markers, glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein β (S100β), were upregulated in the mucosa and muscularis externa. In Myd88 mice, this upregulation did not occur. Importantly, GFAP (only in the mucosa) and S100β (in both the mucosa and muscularis externa) were significantly reduced in conventionalized Myd88 mice compared with the conventionalized wt mice. In neonatal mice, the gene expressions of and increased between the day of birth (P0) and postnatal day 15 (P15) in the mucosa and muscularis externa of both wt and Myd88 mice. Notably, in the mucosa but not the muscularis externa, at P15, the gene expressions of and were significantly reduced in Myd88. Our data demonstrated that postnatal development and replenishment of EGCs require intestinal microbiota and depend on Myd88. The specific upstream mechanisms may involve toll-like-receptor recognition of the microbiota and will be the subject of further research.
Topics: Mice; Animals; Myeloid Differentiation Factor 88; Mice, Inbred C57BL; Neuroglia; Microbiota; Cell Differentiation
PubMed: 36785487
DOI: No ID Found -
Frontiers in Zoology Feb 2023Gastrointestinal (GI) functions are controlled by the enteric nervous system (ENS) in vertebrates, but data on snakes are scarce, as most studies were done in mammals....
BACKGROUND
Gastrointestinal (GI) functions are controlled by the enteric nervous system (ENS) in vertebrates, but data on snakes are scarce, as most studies were done in mammals. However, the feeding of many snakes, including Crotalus atrox, is in strong contrast with mammals, as it consumes an immense, intact prey that is forwarded, stored, and processed by the GI tract. We performed immunohistochemistry in different regions of the GI tract to assess the neuronal density and to quantify cholinergic, nitrergic, and VIPergic enteric neurons. We recorded motility patterns and determined the role of different neurotransmitters in the control of motility. Neuroimaging experiments complemented motility findings.
RESULTS
A well-developed ganglionated myenteric plexus (MP) was found in the oesophagus, stomach, and small and large intestines. In the submucous plexus (SMP) most neurons were scattered individually without forming ganglia. The lowest number of neurons was present in the SMP of the proximal colon, while the highest was in the MP of the oesophagus. The total number of neurons in the ENS was estimated to be approx. 1.5 million. In all regions of the SMP except for the oesophagus more nitric oxide synthase+ than choline-acetyltransferase (ChAT)+ neurons were counted, while in the MP ChAT+ neurons dominated. In the SMP most nerve cells were VIP+, contrary to the MP, where numerous VIP+ nerve fibers but hardly any VIP+ neuronal cell bodies were seen. Regular contractions were observed in muscle strips from the distal stomach, but not from the proximal stomach or the colon. We identified acetylcholine as the main excitatory and nitric oxide as the main inhibitory neurotransmitter. Furthermore, 5-HT and dopamine stimulated, while VIP and the ß-receptor-agonist isoproterenol inhibited motility. ATP had only a minor inhibitory effect. Nerve-evoked contractile responses were sodium-dependent, insensitive to tetrodotoxin (TTX), but sensitive to lidocaine, supported by neuroimaging experiments.
CONCLUSIONS
The structure of the ENS, and patterns of gastric and colonic contractile activity of Crotalus atrox are strikingly different from mammalian models. However, the main excitatory and inhibitory pathways appear to be conserved. Future studies have to explore how the observed differences are an adaptation to the particular feeding strategy of the snake.
PubMed: 36759847
DOI: 10.1186/s12983-023-00484-1 -
The Journal of Physiology Apr 2023The enteric nervous system (ENS) regulates the motor, secretory and defensive functions of the gastrointestinal tract. Enteric neurons integrate mechanical and chemical...
The enteric nervous system (ENS) regulates the motor, secretory and defensive functions of the gastrointestinal tract. Enteric neurons integrate mechanical and chemical inputs from the gut lumen to generate complex motor outputs. How intact enteric neural circuits respond to changes in the gut lumen is not well understood. We recorded intracellular calcium in live-cell confocal recordings in neurons from intact segments of mouse intestine in order to investigate neuronal response to luminal mechanical and chemical stimuli. Wnt1-, ChAT- and Calb1-GCaMP6 mice were used to record neurons from the jejunum and colon. We measured neuronal calcium response to KCl (75 mM), veratridine (10 μM), 1,1-dimethyl-4-phenylpiperazinium (DMPP; 100 μM) or luminal nutrients (Ensure®), in the presence or absence of intraluminal distension. In the jejunum and colon, distension generated by the presence of luminal content (chyme and faecal pellets, respectively) renders the underlying enteric circuit unresponsive to depolarizing stimuli. In the distal colon, high levels of distension inhibit neuronal response to KCl, while intermediate levels of distension reorganize Ca response in circumferentially propagating slow waves. Mechanosensitive channel inhibition suppresses distension-induced Ca elevations, and calcium-activated potassium channel inhibition restores neuronal response to KCl, but not DMPP in the distended colon. In the jejunum, distension prevents a previously unknown tetrodotoxin-resistant neuronal response to luminal nutrient stimulation. Our results demonstrate that intestinal distension regulates the excitability of ENS circuits via mechanosensitive channels. Physiological levels of distension locally silence or synchronize neurons, dynamically regulating the excitability of enteric neural circuits based on the content of the intestinal lumen. KEY POINTS: How the enteric nervous system of the gastrointestinal tract responds to luminal distension remains to be fully elucidated. Here it is shown that intestinal distension modifies intracellular calcium levels in the underlying enteric neuronal network, locally and reversibly silencing neurons in the distended regions. In the distal colon, luminal distension is integrated by specific mechanosensitive channels and coordinates the dynamics of neuronal activation within the enteric network. In the jejunum, distension suppresses the neuronal calcium responses induced by luminal nutrients. Physiological levels of distension dynamically regulate the excitability of enteric neuronal circuits.
Topics: Mice; Animals; Calcium; Enteric Nervous System; Neurons; Intestine, Small; Jejunum; Colon; Myenteric Plexus
PubMed: 36752210
DOI: 10.1113/JP284171 -
Frontiers in Neurology 2022The enteric nervous system (ENS) plays a central role in developing Parkinson's disease (PD) constipation, and the regulation of the ENS may be a key component in...
BACKGROUND
The enteric nervous system (ENS) plays a central role in developing Parkinson's disease (PD) constipation, and the regulation of the ENS may be a key component in treating PD constipation. Electroacupuncture (EA) can effectively treat constipation symptoms in PD, but research on its specific mechanisms, especially in terms of ENS, is relatively lacking. Therefore, we investigated whether EA at ST25 promotes the restoration of ENS structure and colonic motor function in the rotenone-induced PD constipation rat model.
METHODS
In this study, we evaluated constipation symptoms by stool characteristics, excretion and water volume, and whole gut transit time and observed colonic motility regulation through colonic motion detection and pathological changes in the colonic myenteric nervous plexus by transmission electron microscopy and immunofluorescence staining.
RESULTS
EA significantly improved the constipation symptoms and positively adjusted the colonic motility in rotenone-induced PD constipation rats. At the same time, EA reversed the rotenone-induced colonic myenteric nervous plexus injury and regulated the ratio of inhibitory and excitatory neurotransmitters.
CONCLUSION
Our results indicate that EA treatment of PD constipation may be mediated through the adjustment of ENS.
PubMed: 36733445
DOI: 10.3389/fneur.2022.1092127 -
BioRxiv : the Preprint Server For... Jan 2023Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to...
BACKGROUND
Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI tract function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression.
METHODS
We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation as well as a rod-mounted peeling technique. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis.
KEY RESULTS AND CONCLUSIONS
We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin, neuronal nitric oxide synthase (nNOS), or somatostatin (SST) in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased marker labeling for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in ileum and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling, demonstrating a critical variability in wholemount muscularis dissection methods.
PubMed: 36711933
DOI: 10.1101/2023.01.17.524014 -
Revista Espanola de Enfermedades... Jan 202354-year-old woman with acromegaly due to pituitary macroadenoma that consulted for dysphagia for solids. In gastroscopy, it is observed difficulty in passing in the...
CASE REPORT
54-year-old woman with acromegaly due to pituitary macroadenoma that consulted for dysphagia for solids. In gastroscopy, it is observed difficulty in passing in the esophagogastric junction (EGJ). Under the suspect of Achalasia, a high-resolution esophageal manometry (HRM) was performed, observing a complete absence of motility of the esophageal body, panesophageal pressurization in > 20% of swallows and IRP of 21mmHg, confirming the diagnosis of type II Achalasia. Peroral endoscopic myotomy (POEM) is performed. After the intervention, the patient presented clinical improvement. In the control HRM carried out one year after the POEM, in addition to a decrease in IRP, is remarkable a partial recovery of motility in the upper and middle third of the esophagus.
DISCUSSION
Achalasia is an esophageal motor disorder characterized by incomplete relaxation of the lower esophageal sphincter and a complete absence of peristalsis in the esophageal body. The cause of the absence of motility is the loss of the inhibitory neurons of the myenteric plexus, but the accurate etiopathogenesis is still unknown. Therefore, there is no curative treatment and all therapeutic options are symptomatic, aimed to relieve the obstruction of the EGJ. POEM is the newest method. Absence of motility of the esophageal body in patients with achalasia was believed to be irreversible. Nevertheless, more and more studies describe a partial recovery of motility observed in manometry after POEM, especially in type II Achalasia. The exact pathophysiological mechanism of this recovery is still unknown.
IMAGES DESCRIPTION
Figure 1 (Before POEM): High-resolution manometry with a diagnosis of type II Achalasia: Absence of motility, panesophageal pressurization and IRP 21mmHg Figure 2 (After POEM): High resolution manometry showing ineffective esophageal motility (partial recovery of motility) after POEM with IRP of 4mmHg.
PubMed: 36688437
DOI: 10.17235/reed.2023.9420/2022 -
World Journal of Diabetes Jan 2023Cytokines are essential in autoimmune inflammatory processes that accompany type 1 diabetes. Tumor necrosis factor alpha plays a key role among others in modulating...
BACKGROUND
Cytokines are essential in autoimmune inflammatory processes that accompany type 1 diabetes. Tumor necrosis factor alpha plays a key role among others in modulating enteric neuroinflammation, however, it has a dual role in cell degeneration or survival depending on different TNFRs. In general, TNFR1 is believed to trigger apoptosis, while TNFR2 promotes cell regeneration. The importance of the neuronal microenvironment has been recently highlighted in gut region-specific diabetic enteric neuropathy, however, the expression and alterations of different TNFRs in the gastrointestinal tract has not been reported.
AIM
To investigate the TNFR1 and TNFR2 expression in myenteric ganglia and their environment in different intestinal segments of diabetic rats.
METHODS
Ten weeks after the onset of hyperglycemia, gut segments were taken from the duodenum, ileum and colon of streptozotocin-induced (60 mg/body weight kg i.p.) diabetic ( = 17), insulin-treated diabetic ( = 15) and sex- and age-matched control ( = 15) rats. Myenteric plexus whole-mount preparations were prepared from different gut regions for TNFR1/HuCD or TNFR2/HuCD double-labeling fluorescent immunohistochemistry. TNFR1 and TNFR2 expression was evaluated by post-embedding immunogold electron microscopy on ultrathin sections of myenteric ganglia. TNFRs levels were measured by enzyme-linked immun-osorbent assay in muscle/myenteric plexus-containing (MUSCLE-MP) tissue homogenates from different gut segments and experimental conditions.
RESULTS
A distinct region-dependent TNFRs expression was detected in controls. The density of TNFR1-labeling gold particles was lowest, while TNFR2 density was highest in duodenal ganglia and a decreased TNFRs expression from proximal to distal segments was observed in MUSCLE-MP homogenates. In diabetics, the TNFR2 density was only significantly altered in the duodenum with decrease in the ganglia (0.32 ± 0.02 0.45 ± 0.04, < 0.05), while no significant changes in TNFR1 density was observed. In diabetic MUSCLE-MP homogenates, both TNFRs levels significantly decreased in the duodenum (TNFR1: 4.06 ± 0.65 20.32 ± 3.1, < 0.001; TNFR2: 11.72 ± 0.39 15.91 ± 1.04, < 0.01), which markedly influenced the TNFR2/TNFR1 proportion in both the ganglia and their muscular environment. Insulin treatment had controversial effects on TNFR expression.
CONCLUSION
Our findings show diabetes-related region-dependent changes in TNFR expression and suggest that TNFR2 is more affected than TNFR1 in myenteric ganglia in the duodenum of type 1 diabetic rats.
PubMed: 36684383
DOI: 10.4239/wjd.v14.i1.48 -
The Journal of Biological Chemistry Mar 2023Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important....
Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP) and its precursor inositol hexakisphosphate (IP). Although their absolute levels in the GIT are diet dependent, elevated IP metabolism still exists under dietary regimens devoid of exogenous IP. Of the major GIT cells, enteric neurons selectively express the IP-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP-a highly active IP6K2-IP pathway is conducive to the enteric nervous system.
Topics: Animals; Mice; Diphosphates; Enteric Nervous System; Inositol Phosphates; Mice, Knockout; Neurons; Phosphotransferases (Phosphate Group Acceptor); Phytic Acid; Gastrointestinal Tract; Transcriptome
PubMed: 36681123
DOI: 10.1016/j.jbc.2023.102928 -
Biomedicines Jan 2023Toll-like receptor 4 (TLR4) can activate pro-inflammatory cascades in the gastrointestinal tract. Our aim was to determine TLR4 expression in myenteric neurons of...
Toll-like receptor 4 (TLR4) can activate pro-inflammatory cascades in the gastrointestinal tract. Our aim was to determine TLR4 expression in myenteric neurons of different gut regions using a type 1 diabetic model. Ten weeks after the onset of hyperglycemia, myenteric whole-mount preparations from the duodenum, ileum and colon of streptozotocin-induced diabetic, insulin-treated diabetic and control rats were prepared for TLR4/peripherin double-labelling fluorescent immunohistochemistry. Immunogold electron microscopy was applied to evaluate TLR4 expression in the myenteric perikaryon and neuropil. Tissue TLR4 levels were measured by enzyme-linked immunosorbent assay. In controls, the number and proportion of the TLR4-immunoreactive myenteric neurons showed an increasing tendency to aboral direction. These values were significantly higher in diabetics compared to controls in the duodenum and ileum, but were significantly lower in the colon. In diabetics, the distribution of TLR4-labelling gold particles between the perikaryon and neuropil of myenteric neurons varied in a different way by intestinal segment. TLR4 tissue concentration changed only in the diabetic duodenum, and it decreased in muscle/myenteric plexus-containing homogenates, while it increased in mucosa/submucosa/submucous plexus-containing samples relative to controls. Insulin had beneficial effects on TLR4 expression. These findings support that chronic hyperglycemia has segment-specific effects on TLR4 expression, contributing to gastrointestinal disorders in diabetic patients.
PubMed: 36672637
DOI: 10.3390/biomedicines11010129 -
Biomolecules Jan 2023Glutamate is the major excitatory neurotransmitter in the central nervous system, and there is evidence that Group-I metabotropic glutamate receptors (mGlu1 and mGlu5)...
Glutamate is the major excitatory neurotransmitter in the central nervous system, and there is evidence that Group-I metabotropic glutamate receptors (mGlu1 and mGlu5) have established roles in excitatory neurotransmission and synaptic plasticity. While glutamate is abundantly present in the gut, it plays a smaller role in neurotransmission in the enteric nervous system. In this study, we examined the roles of Group-I mGlu receptors in gastrointestinal function. We investigated the expression of (mGlu1) and (mGlu5) in the mouse myenteric plexus using RNAscope in situ hybridization. Live calcium imaging and motility analysis were performed on ex vivo preparations of the mouse colon. mGlu5 was found to play a role in excitatory enteric neurotransmission, as electrically-evoked calcium transients were sensitive to the mGlu5 antagonist MPEP. However, inhibition of mGlu5 activity did not affect colonic motor complexes (CMCs). Instead, inhibition of mGlu1 using BAY 36-7620 reduced CMC frequency but did not affect enteric neurotransmission. These data highlight complex roles for Group-I mGlu receptors in myenteric neuron activity and colonic function.
Topics: Mice; Animals; Calcium; Central Nervous System; Neurons; Glutamic Acid; Synaptic Transmission
PubMed: 36671524
DOI: 10.3390/biom13010139