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ObjectiveThe enteric nervous system (ENS) plays a key role in controlling the gut-brain axis under normal and pathological conditions, such as type 2 diabetes. The discovery of intestinal actors, such as enterosynes, able to modulate the ENS-induced duodenal contraction is considered an innovative approach. Among all the intestinal factors, the understanding of the role of gut microbes in controlling glycaemia is still developed. We studied whether the modulation of gut microbiota by prebiotics could permit the identification of novel enterosynes.DesignWe measured the effects of prebiotics on the production of bioactive lipids in the intestine and tested the identified lipid on ENS-induced contraction and glucose metabolism. Then, we studied the signalling pathways involved and compared the results obtained in mice to human.ResultsWe found that modulating the gut microbiota with prebiotics modifies the actions of enteric neurons, thereby controlling duodenal contraction and subsequently attenuating hyperglycaemia in diabetic mice. We discovered that the signalling pathway involved in these effects depends on the synthesis of a bioactive lipid 12-hydroxyeicosatetraenoic acid (12-HETE) and the presence of mu-opioid receptors (MOR) on enteric neurons. Using pharmacological approaches, we demonstrated the key role of the MOR receptors and proliferator-activated receptor γ for the effects of 12-HETE. These findings are supported by human data showing a decreased expression of the proenkephalin and MOR messanger RNAs in the duodenum of patients with diabetic.ConclusionsUsing a prebiotic approach, we identified enkephalin and 12-HETE as new enterosynes with potential real beneficial and safety impact in diabetic human.

This study investigates the therapeutic mechanisms of electroacupuncture (EA) in regulating the vagal nerve for functional dyspepsia (FD) using an integrated multi-omics approach. A rat model of FD was established via iodoacetamide gavage combined with tail-clamp stress. Rats were randomly assigned to five groups (n=6 per group): control (CON), model (MOD), electroacupuncture (EA), subdiaphragmatic vagotomy and electroacupuncture (SDV+EA), and subdiaphragmatic vagotomy (SDV). EA was administered at ST36 (Zusanli) and ST37 (Shangjuxu) for 20 minutes per session, once daily for 14 days. EA treatment restored vagal tone, improved sympathovagal balance, and enhanced gastrointestinal motility in FD model rats. 16S rDNA sequencing revealed that EA modulated vagus nerve-dependent changes in the relative abundance of 12 microbial taxa, including and . Crucially, the vagotomy procedure significantly attenuated EA's restorative effects on these microbial populations. Metabolomics identified 24 differential metabolites regulated by EA through the vagus nerve, including Cholesta-3,5-dien-7-one, Licofelone, Digoxigenin, 7-Hydroxymethotrexate, Hydroxymethylbilane, among others. Similarly, subdiaphragmatic vagotomy largely reversed the normalizing effects of EA on these metabolite levels. Transcriptomics, on the other hand, identified 23 differential genes, including Prss22, Lypd3, and Tnfrsf12a. KEGG analysis of differential metabolites and differential genes suggested that arachidonic acid metabolism may represent a potential therapeutic target for EA in the treatment of FD through vagus nerve modulation. Mechanistic analyses of the key differentially expressed gene and the arachidonic acid metabolic pathway demonstrated that EA attenuated inflammatory responses by suppressing TWEAK/Fn14/NF-κB pathway activation and arachidonic acid metabolism, leading to decreased levels of TNF-α, IL-1β, IL-6, and PGE . Importantly, the anti-inflammatory effects of EA were significantly attenuated in the SDV+EA group, confirming that vagal integrity is essential for EA to fully exert its suppressive action on these key inflammatory pathways and mediators. EA ameliorates FD by modulating vagal nerve activity, concurrently suppressing TWEAK/Fn14/NF-κB pathway activation and arachidonic acid metabolism, thus attenuating duodenal low-grade inflammation in FD model rats. These findings demonstrate the potential of EA as an effective therapeutic intervention for FD.

Background Obesity is associated with the development of insulin resistance (IR) and type‐2 diabetes mellitus (T2DM); however, not all patients with T2DM are obese. The Goto–Kakizaki (GK) rat is an experimental model of spontaneous and non‐obese T2DM. There is evidence that the intestine contributes to IR development in GK animals. This information prompted us to investigate small intestine remodeling in this animal model. Methods Four‐month‐old male Wistar (control) and GK rats were utilized for the present study. After removing the small intestine, the duodenum, proximal jejunum, and distal ileum were separated. We then measured villi and muscular and mucosa layer histomorphometry, goblet cells abundance, total myenteric and submucosal neuron populations, and inflammatory marker expression in the small intestinal segments and intestinal transit of both groups of animals. Key Results We found that the GK rats exhibited decreased intestinal area (p < 0.0001), decreased crypt depth in the duodenum (p = 0.01) and ileum (p < 0.0001), increased crypt depth in the jejunum (p < 0.0001), longer villi in the jejunum and ileum (p < 0.0001), thicker villi in the duodenum (p < 0.01) and ileum (p < 0.0001), thicker muscular layers in the duodenum, jejunum, and ileum (p < 0.0001), increased IL‐1β concentrations in the duodenum and jejunum (p < 0.05), and increased concentrations of NF‐κB p65 in the duodenum (p < 0.01), jejunum and ileum (p < 0.05). We observed high IL‐1β reactivity in the muscle layer, myenteric neurons, and glial cells of the experimental group. GK rats also exhibited a significant reduction in submucosal neuron density in the jejunum and ileum, ganglionic hypertrophy in all intestinal segments studied (p < 0.0001), and a slower intestinal transit (about 25%) compared to controls. Conclusions The development of IR and T2DM in GK rats is associated with small intestine remodeling that includes marked alterations in small intestine morphology, local inflammation, and reduced intestinal transit. Goto–Kakizaki (GK) rat is an experimental model of spontaneous and non‐obese type‐2 diabetes mellitus—T2DM).GK rats exhibit decreased small intestinal area, increased crypt depth, villi height and thickness, and muscular layer thickness, increased content of IL‐1β and NF‐κB p65, decreased density of submucosal neurons, myenteric and submucosal neuronal and ganglionic hypertrophy, and decreased intestinal transit.The development of IR and T2DM in GK rats is associated with small intestine remodeling with marked changes in morphology, enteric nervous system, and transit.

Background The prevalence of obesity has increased at alarming rates, particularly because of the increased consumption of high-fat diets (HFDs). The influence of HFDs on intrinsic innervation and the intestinal wall has not been fully characterized. The aim of this study was to investigate the morpho-quantitative aspects of myenteric neurons and the wall of the small intestine in mice fed a HFD. Methods Swiss mice were fed a HFD (59% kcal from fat) or standard chow (9% Kcal from fat) for 8 weeks. Segments of the duodenum, jejunum, and ileum were subjected to histological processing for morpho-quantitative examination of the intestinal wall and mucosal cells, and immunohistochemistry was performed to evaluate myenteric neurons. The data for each segment were compared between the groups using an unpaired Student’s t -test or an equivalent nonparametric test. Results The HFD increased body weight and visceral fat and decreased the length of the small intestine and the circumference of the ileum. In the duodenum, the HFD increased the density of the nitrergic subpopulation and decreased the area of nitrergic neurons and vasoactive intestinal peptide (VIP) varicosities. In the jejunum, the density of the nitrergic subpopulation was increased and the neuronal areas of the general population, nitrergic subpopulation and (VIP) varicosities were reduced. In the ileum, the density of the general population and nitrergic subpopulation were increased and the neuronal areas of the general population, nitrergic subpopulation and (VIP) varicosities were reduced. The morphometric parameters of the villi, crypts, muscular layer and total wall generally increased in the duodenum and jejunum and decreased in the ileum. In the duodenum and jejunum, the HFD promoted a decreased in the proportion of intraepithelial lymphocytes. In the ileum, the proportion of intraepithelial lymphocytes and goblet cells reduced, and the enteroendocrine cells increased. Conclusions The high-fat diet induces changes in the myenteric innervation of the small intestine, intestinal wall and mucosal cells responsible for the secretion of hormones and maintenance of the protective intestinal barrier. The morpho-quantitative data provide a basis for further studies to clarify the influence of HFD in the motility, digestive and absorptive capacity, and intestinal barrier.

Lipopolysaccharides (LPS), also known as lipoglycans or endotoxins, form part of the outer membrane of Gram-negative bacteria. Previous studies have described the various harmful impacts of LPS on humans and animals. Nevertheless, many aspects of these effects are still not fully explained. One of them is the influence of endotoxins on the neurochemical characterization of neurons within the enteric nervous system (ENS), which is found in the intestinal wall and plays important adaptive roles during pathological processes and exposures. In this study, the impact of a low single dose of Salmonella Enteritidis LPS on the duodenal enteric neurons immunoreactive to substance P (SP), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating peptide (PACAP-27), and cocaine- and amphetamine-regulated transcript (CART) was studied using a double immunofluorescence technique. During the study, it was shown that even a low dose of LPS affects the number of enteric neurons containing the neuropeptides studied, and these changes were dependent on the type of the enteric plexus. The most visible changes concerned the SP-like immunoreactive (LI) neurons in the outer submucous plexus (LPS caused an increase in the percentage of these neurons from15.74 ± 0.61 to 21.72 ± 0.79%). Furthermore, the VIP-LI neurons in the inner submucous plexus were seen to decrease from 12.64 ± 0.83 to 5.96 ± 0.58%. The mechanisms behind these noted fluctuations are not clear, but it may be connected with the pro-inflammatory and neurotoxic activity of LPS.

The digestive tract, especially the small intestine, is one of the main routes of acrylamide absorption and is therefore highly exposed to the toxic effect of acrylamide contained in food. The aim of this experiment was to elucidate the effect of low (tolerable daily intake—TDI) and high (ten times higher than TDI) doses of acrylamide on the neurochemical phenotype of duodenal enteric nervous system (ENS) neurons using the pig as an animal model. The experiment was performed on 15 immature gilts of the Danish Landrace assigned to three experimental groups: control (C) group—pigs administered empty gelatine capsules, low dose (LD) group—pigs administered capsules with acrylamide at the TDI dose (0.5 μg/kg body weight (b.w.)/day), and the high dose (HD) group—pigs administered capsules with acrylamide at a ten times higher dose than the TDI (5 μg/kg b.w./day) with a morning feeding for 4 weeks. Administration of acrylamide, even in a low (TDI) dose, led to an increase in the percentage of enteric neurons immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), neuronal nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VACHT) in the porcine duodenum. The severity of the changes clearly depended on the dose of acrylamide and the examined plexus. The obtained results suggest the participation of these neuroactive substances in acrylamide-inducted plasticity and the protection of ENS neurons, which may be an important line of defence from the harmful action of acrylamide.

Obesity and type 2 diabetes are increasing in prevalence at an alarming rate in developed and developing nations and over 50 % of patients with prolonged stages of disease experience forms of autonomic neuropathy. These patients have symptoms indicating disrupted enteric nervous system function including gastric discomfort, gastroparesis and intestinal dysmotility. Previous assessments have examined enteric neuronal injury within either type 1 diabetic or transgenic type 2 diabetic context. This study aims to assess damage to myenteric neurons within the duodenum of high-fat diet ingesting mice experiencing symptoms of type 2 diabetes, as this disease context is most parallel to the human condition and disrupted duodenal motility underlies negative gastrointestinal symptoms. Mice fed a high-fat diet developed symptoms of obesity and diabetes by 4 weeks. After 8 weeks, the total number of duodenal myenteric neurons and the synaptophysin density index were reduced and transmission electron microscopy showed axonal swelling and loss of neurofilaments and microtubules, suggesting compromised neuronal health. High-fat diet ingestion correlated with a loss of neurons expressing VIP and nNOS but did not affect the expression of ChAT, substance P, calbindin and CGRP. These results correlate high-fat diet ingestion, obesity and type 2 diabetes symptoms with a loss of duodenal neurons, biasing towards those with inhibitory nature. This pathology may underlie dysmotility and other negative GI symptoms experienced by human type 2 diabetic and obese patients.

Symptoms of diabetic gastrointestinal dysmotility indicate neuropathy of the enteric nervous system. Long-standing diabetic enteric neuropathy has not been fully characterized, however. We used prolonged high fat diet ingestion (20 weeks) in a mouse model to mimic human obese and type 2 diabetic conditions, and analyzed changes seen in neurons of the duodenal myenteric plexus. Ganglionic and neuronal size, number of neurons per ganglionic area, density indices of neuronal phenotypes (immunoreactive nerve cell bodies and varicosities per ganglion or tissue area) and nerve injury were measured. Findings were compared with results previously seen in mice fed the same diet for 8 weeks. Compared to mice fed standard chow, those on a prolonged high fat diet had smaller ganglionic and cell soma areas. Myenteric VIP- and ChAT-immunoreactive density indices were also reduced. Myenteric nerve fibers were markedly swollen and cytoskeletal protein networks were disrupted. The number of nNOS nerve cell bodies per ganglia was increased, contrary to the reduction previously seen after 8 weeks, but the density index of nNOS varicosities was reduced. Mice fed high fat and standard chow diets experienced an age-related reduction in total neurons, with bias towards neurons of sensory phenotype. Meanwhile, ageing was associated with an increase in excitatory neuronal markers. Collectively, these results support a notion that nerve damage underlies diabetic symptoms of dysmotility, and reveals adaptive ENS responses to the prolonged ingestion of a high fat diet. This highlights a need to mechanistically study long-term diet-induced nerve damage and age-related impacts on the ENS.

T2 toxin synthetized by Fusarium spp. negatively affects various internal organs and systems, including the digestive tract and the immune, endocrine, and nervous systems. However, knowledge about the effects of T2 on the enteric nervous system (ENS) is still incomplete. Therefore, during the present experiment, the influence of T2 toxin with a dose of 12 µg/kg body weight (b.w.)/per day on the number of enteric nervous structures immunoreactive to neuronal isoform nitric oxide synthase (nNOS—used here as a marker of nitrergic neurons) in the porcine duodenum was studied using the double immunofluorescence method. Under physiological conditions, nNOS-positive neurons amounted to 38.28 ± 1.147%, 38.39 ± 1.244%, and 35.34 ± 1.151 of all enteric neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively. After administration of T2 toxin, an increase in the number of these neurons was observed in all types of the enteric plexuses and nNOS-positive cells reached 46.20 ± 1.453% in the MP, 45.39 ± 0.488% in the OSP, and 44.07 ± 0.308% in the ISP. However, in the present study, the influence of T2 toxin on the intramucosal and intramuscular nNOS-positive nerves was not observed. The results obtained in the present study indicate that even low doses of T2 toxin are not neutral for living organisms because they may change the neurochemical characterization of the enteric neurons.

Background Most direct understanding of enteric nerve (patho)physiology has been obtained by electrode and imaging techniques in animal models and human surgical samples. Until now, neuronal activity recordings from a more accessible human tissue source have remained a true challenge. Objectives To record nerve activity in human intestinal biopsies using imaging techniques. Design Submucous plexus was isolated from duodenal biopsies. Enteric nerves were functionally and morphologically examined using calcium (Ca2+) imaging and immunohistochemistry. Exogenous application of high-K+ solution, the nicotinic cholinergic receptor agonist (1,1-dimethyl-4-phenylpiperazinium; DMPP) or serotonin (5-HT), and electrical stimulation of interganglionic fibre tracts were used to activate the neurons, and intracellular Ca2+ concentrations ([Ca2+]i) were monitored. Enteric ganglia were stained with neuronal and glial markers. Results Using high-K+ solution, 146 neurons were identified in 70 ganglia (44 biopsies from 29 subjects). The exogenous application of DMPP or 5-HT caused a transient [Ca2+]i increase, respectively, in 68% and 63% of the neurons identified by high-K+. Electrical stimulation evoked responses in 57% of the neurons; these responses were totally or partly suppressed by tetrodotoxin or zero-Ca2+ solution, respectively. Immunohistochemical analysis showed both isolated neurons and ganglia interconnected by typical interganglionic fibre bundles. The average number of ganglia was 7.7±6.0 per biopsy and each ganglion contained on average 4.5±1.2 neurons. Conclusion In this study, for the first time, live recordings were performed of nerve activity in intestinal biopsies. This novel approach is of key importance to study living neurons in both health and disease and to test newly developed compounds in an in-vitro human tissue model.