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ABSTRACT Background Aim Methods Key Results Conclusions and Inferences Intestinal ischemia affects the functioning of the Enteric Nervous System (ENS). Pannexin‐1 channel participates in cell communication and extracellular signaling. Probenecid (PB) is a pannexin‐1 channel inhibitor, which can be a potential treatment for intestinal ischemia.Study the effects of ileal ischemia and reperfusion (I/R) and PB treatment on myenteric neurons and in rats.Male Wistar rats were used for I/R induction, the ileal vessels were occluded for 45 min and reperfusion was performed after this time. The Sham groups underwent all surgical procedures without obstruction of the ileal vessels. Animals were euthanized 24 h or 14d post‐I/R. The PB group received an injection of PB post‐I/R. Ileal segments were collected for immunofluorescence analyses to identify neurons calretinin immunoreactive (−ir) and pannexin‐1‐ir. Neuronal density (cells/field), area (μm2), intestinal motility, and ultrastructural analyses were performed.The pannexin‐1 channel was double‐labeled with calretinin‐ir neurons. Neuronal density reduced by 21% reduction in calretinin‐ir neurons in the I/R 24 h group and recovered 26% in the PB 24 h group. In the 14d group, there was a 23% reduction in calretinin‐ir neurons in the I/R 14d group and a recovery of 26% in the PB 14d group. The analysis of the contraction after electrical simulation was lower in the I/R 14 d group and recovered in the PB 14d.Intestinal I/R affects myenteric neurons and causes morphological and functional changes. PB was able to attenuate the effects of I/R and could constitute a therapeutic tool for intestinal I/R. [ABSTRACT FROM AUTHOR]

Background/Objectives: Type 1 diabetes affects cytokines as potential inducers of NFκB signalling involved in inflammation and neuronal survival. Our goal was to assess the expression of NFκB p65 and its negative regulator, Nrf2, in myenteric neurons and adjacent smooth muscle of different gut segments after chronic hyperglycaemia and immediate insulin treatment. Methods: After ten weeks of hyperglycaemia, intestinal samples of control, streptozotocin-induced diabetic and insulin-treated diabetic rats were prepared for fluorescent immunohistochemistry, immunogold electron microscopy, ELISA and qPCR. Results: In the diabetic rats, the proportion of NFκB p65-immunoreactive myenteric neurons decreased significantly in the duodenum and increased in the ileum. The density of NFκB p65-labelling gold particles increased in the ileal but remained unchanged in the duodenal ganglia. Meanwhile, both total and nuclear Nrf2 density increased in the myenteric neurons of the diabetic duodenum. In smooth muscle, NFκB p65 and Nrf2 density increased in the small intestine of diabetic rats. While on the mRNA level, NFκB p65 and Nrf2 were induced, on the protein level, NFκB p65 increased and Nrf2 decreased in muscle/myenteric plexus homogenates. Insulin treatment had protective effects. Conclusions: Our findings reveal a segment-specific NFκB and Nrf expression in myenteric neurons and ganglionic muscular environments, which may contribute to regional neuronal survival and motility disturbances in diabetes. [ABSTRACT FROM AUTHOR]

Constipation is a common symptom in patients with Parkinson's disease (PD) and is often associated with depression. Enteric glial cells (EGCs) are crucial for regulating intestinal inflammation and colon motility, and their activation can lead to the death of intestinal neurons. Glial cell line-derived neurotrophic factor (GDNF) has been recognized for its neuroprotective properties in various neurological disorders, including PD. This study explores the potential of GDNF in alleviating intestinal reactive gliosis and inflammation, thereby improving constipation and depressive behavior in a rat model of PD. A PD model was established via unilateral stereotaxic injection of 6-hydroxydopamine (6-OHDA). Five weeks post-injury, AAV5-GDNF (2 ~ 5 × 10^11) was intraperitoneally injected into experimental and control rats. Fecal moisture percentage (FMP) and colonic propulsion rate (CPPR) were used to evaluate colon motility. Colon-related inflammation and colonic epithelial morphology were assessed, and depressive behavior was analyzed one week before sampling. PD rats exhibited reduced colonic motility and GDNF expression, along with increased EGC reactivity and elevated levels of pro-inflammatory cytokines IL-1, IL-6, and TNF-α. Additionally, there was an up-regulation of CX43 and a decrease in PGP 9.5 expression. The intraperitoneal injection of AAV-GDNF significantly protected colonic neurons by inhibiting EGC activation and down-regulating CX43. This treatment also led to a notable reduction in depressive-like symptoms in PD rats with constipation. GDNF effectively reduces markers of reactive gliosis and inflammation, and promotes the survival of colonic neurons, and improves colonic motility in PD rats by regulating CX43 activity. Furthermore, GDNF treatment alleviates depressive behavior, suggesting that GDNF or its agonists could be promising therapeutic agents for managing gastrointestinal and neuropsychiatric symptoms associated with PD. [ABSTRACT FROM AUTHOR]

Background and Aims: The enteric nervous system independently controls gastrointestinal function including motility, which is primarily mediated by the myenteric plexus, therefore also playing a crucial role in functional intestinal disorders. Live recordings from human myenteric neurons proved to be challenging due to technical difficulties. Using the neuroimaging technique, we are able to record human colonic myenteric neuronal activity and investigate their functional properties in a large cohort of patients. Methods: Activity from myenteric neurons in wholemount preparations of different sampling sites of fresh, human colonic tissue was recorded using neuroimaging with the voltage sensitive dye 1-(3-sulfanatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine. Neuronal responses were analyzed following stimulation with nicotine and serotonin (5-HT) for differences based on the donor’s age, the disorder indicative for surgery and the colonic region. Immunohistochemistry was performed to calculate the total neuronal numbers. Results: Stimulation with nicotine and 5-HT elicited reproducible action potential discharge in a proportion of human myenteric neurons. The responses to 5-HT were significantly greater in tissues from older patients and from those with inflammatory disorders, while neuronal activity to nicotinergic stimulation was comparable in all patients. Neuronal numbers declined with rising patient’s age and was highest in the sigmoid colon. Conclusion: Neuroimaging with 1-(3-sulfanatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine was successfully adapted to record reproducible responses from human colonic myenteric neurons upon pharmacological stimulation. Evidence exists for an impact of age and inflammation on the serotonergic neuronal signaling and for differences in neuronal numbers in the distinct colonic regions as well as a neuronal decrease with age.

Introduction: We previously showed enteric nerve activation after application of colonic mucosal biopsy supernatants from patients with irritable bowel syndrome (IBS). The question remains whether this is a region-specific or a generalized sensitization. We tested the nerve-activating properties of supernatants from large and small intestinal regions of IBS patients with diarrhea (IBS-D) in comparison to those from mastocytosis patients with diarrhea (MC-D) or non-IBS/non-MC patients with GI-complaints. MC-D patients were included to test samples from patients with an established, severe mast cell disorder, because mast cells are suggested to play a role in IBS. Methods: Voltage-sensitive dye imaging was used to record the effects of mucosal biopsy supernatants from IBS-D, MC-D, and non-IBS/non-MC on guinea pig submucous neurons. Mast cell density and histamine concentrations were measured in all samples. Results: The median neuroindex (spike frequency × % responding neurons in Hz × %) was significantly (all p < 0.001) increased for IBS-D (duodenum and colon, proximal and distal each, 49.3; 50.5; 63.7; 71.9, respectively) compared to non-IBS/non-MC (duodenum and colon, proximal and distal each, 8.7; 4.9; 6.9; 5.4, respectively) or MC-D supernatants (duodenum and colon, proximal and distal each, 9.4; 11.9; 0.0; 7.9, respectively). Nerve activation by MC-D and non-IBS/non-MC supernatants was comparable (p>0.05). Mast cell density or histamine concentrations were not different between IBS-D, MC-D, and non-IBS/non-MC samples. Discussion: Nerve activation by biopsy supernatants is an IBS hallmark that occurs throughout the gut, unrelated to mast cell density or histamine concentration. At least as important is our finding that GI complaints per se were not associated with biopsy supernatant-induced nerve activation, which further stresses the relevance of altered nerve behavior in IBS. [ABSTRACT FROM AUTHOR]

Background: Neurons of the enteric nervous system are characterised by high neuronal plasticity, with their number likely to change in response to various endogenous and exogenous substances. Materials and methods: Fifteen sexually immature gilts divided into 3 groups were used: control — animals receiving empty gelatin capsules; G1 — animals receiving a low dose of glyphosate — 0.05mg/kg bw/day; G2 — animals receiving a higher dose of glyphosate — 0.5 mg/kg/day in gelatin capsules orally for 28days. Frozen sections were then subjected to the procedure of double immunofluorescent staining. Results: With low-dose supplementation, no effect on the SP- and CART-positive neuron population was observed. However, a reduction in the number of VAChT-positive neurons in the internal submucosal plexus was described, while the number of CGRP-positive neurons increased in all enteric plexuses. In response to a high glyphosate dose, the quantitative variability of the neurons was significantly more pronounced than that for a low dose. There was an increase in the number of SP- and CGRP-positive neurons and a decrease in the number of VAChT-positive neurons in both the myenteric plexus and the submucosal plexuses. The response of CART-positive neurons was the weakest, as a high dose of glyphosate led to an increase in the number of neurons only in the myenteric plexus. Conclusions: The above data show that glyphosate is an exogenous substance that affects neuronal populations of the enteric nervous system, in this case, the descending colon. [ABSTRACT FROM AUTHOR]

Phosphatase and tensin homolog (Pten) is a key regulator of cell proliferation and a potential target to stimulate postnatal enteric neuro- and/or gliogenesis. To investigate this, we generated two tamoxifen-inducible Cre recombinase murine models in which Pten was conditionally ablated, (1) in glia (Plp1-expressing cells) and (2) in neurons (Calb2-expressing cells). Tamoxifen-treated adult (7–12 weeks of age; n = 4–15) mice were given DSS to induce colitis, EdU to monitor cell proliferation, and were evaluated at two timepoints: (1) early (3–4 days post-DSS) and (2) late (3–4 weeks post-DSS). We investigated gut motility and evaluated the enteric nervous system. Pten inhibition in Plp1-expressing cells elicited gliogenesis at baseline and post-DSS (early and late) in the colon, and neurogenesis post-DSS late in the proximal colon. They also exhibited an increased frequency of colonic migrating motor complexes (CMMC) and slower whole gut transit times. Pten inhibition in Calb2-expressing cells did not induce enteric neuro- or gliogenesis, and no alterations were detected in CMMC or whole gut transit times when compared to the control at baseline or post-DSS (early and late). Our results merit further research into Pten modulation where increased glia and/or slower intestinal transit times are desired (e.g., short-bowel syndrome and rapid-transit disorders). [ABSTRACT FROM AUTHOR]

Summary: Systemic inflammation is a key contributor to the onset and progression of several obesity‐associated diseases and is thought to predominantly arise from the hyperplasia and hypertrophy of white adipose tissue. However, a growing body of works suggests that early changes in the gastrointestinal (GI) barrier may contribute to both local, within the GI lining, and systemic inflammation in obesity. Intestinal barrier dysfunction is well‐characterized in inflammatory GI disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) and is known to contribute to systemic inflammation. Thus, drawing parallels between GI disorders, where intestinal permeability and systemic inflammation are prominent features, and obesity‐induced GI manifestations may provide insights into the potential role of the intestinal barrier in systemic inflammation in obesity. This review summarizes the current literature surrounding intestinal barrier dysfunction in obesity and explores the potential role of intestinal hyperpermeability and intestinal barrier dysfunction in the development of systemic inflammation and GI dysfunction in obesity. [ABSTRACT FROM AUTHOR]

IntroductionWe previously showed enteric nerve activation after application of colonic mucosal biopsy supernatants from patients with irritable bowel syndrome (IBS). The question remains whether this is a region-specific or a generalized sensitization. We tested the nerve-activating properties of supernatants from large and small intestinal regions of IBS patients with diarrhea (IBS-D) in comparison to those from mastocytosis patients with diarrhea (MC-D) or non-IBS/non-MC patients with GI-complaints. MC-D patients were included to test samples from patients with an established, severe mast cell disorder, because mast cells are suggested to play a role in IBS.MethodsVoltage-sensitive dye imaging was used to record the effects of mucosal biopsy supernatants from IBS-D, MC-D, and non-IBS/non-MC on guinea pig submucous neurons. Mast cell density and histamine concentrations were measured in all samples.ResultsThe median neuroindex (spike frequency × % responding neurons in Hz × %) was significantly (all p < 0.001) increased for IBS-D (duodenum and colon, proximal and distal each, 49.3; 50.5; 63.7; 71.9, respectively) compared to non-IBS/non-MC (duodenum and colon, proximal and distal each, 8.7; 4.9; 6.9; 5.4, respectively) or MC-D supernatants (duodenum and colon, proximal and distal each, 9.4; 11.9; 0.0; 7.9, respectively). Nerve activation by MC-D and non-IBS/non-MC supernatants was comparable (p>0.05). Mast cell density or histamine concentrations were not different between IBS-D, MC-D, and non-IBS/non-MC samples.DiscussionNerve activation by biopsy supernatants is an IBS hallmark that occurs throughout the gut, unrelated to mast cell density or histamine concentration. At least as important is our finding that GI complaints per se were not associated with biopsy supernatant-induced nerve activation, which further stresses the relevance of altered nerve behavior in IBS.

Background/Objectives: Type 1 diabetes affects cytokines as potential inducers of NFκB signalling involved in inflammation and neuronal survival. Our goal was to assess the expression of NFκB p65 and its negative regulator, Nrf2, in myenteric neurons and adjacent smooth muscle of different gut segments after chronic hyperglycaemia and immediate insulin treatment. Methods: After ten weeks of hyperglycaemia, intestinal samples of control, streptozotocin-induced diabetic and insulin-treated diabetic rats were prepared for fluorescent immunohistochemistry, immunogold electron microscopy, ELISA and qPCR. Results: In the diabetic rats, the proportion of NFκB p65-immunoreactive myenteric neurons decreased significantly in the duodenum and increased in the ileum. The density of NFκB p65-labelling gold particles increased in the ileal but remained unchanged in the duodenal ganglia. Meanwhile, both total and nuclear Nrf2 density increased in the myenteric neurons of the diabetic duodenum. In smooth muscle, NFκB p65 and Nrf2 density increased in the small intestine of diabetic rats. While on the mRNA level, NFκB p65 and Nrf2 were induced, on the protein level, NFκB p65 increased and Nrf2 decreased in muscle/myenteric plexus homogenates. Insulin treatment had protective effects. Conclusions: Our findings reveal a segment-specific NFκB and Nrf expression in myenteric neurons and ganglionic muscular environments, which may contribute to regional neuronal survival and motility disturbances in diabetes.

Environmental contamination and the resulting food contamination represent a serious problem and pose a major threat to animal and human health. The gastrointestinal tract is directly exposed to a variety of substances. One is glyphosate, whose presence in the soil is commonly observed. This study demonstrates the effects of low and high glyphosate doses on the populations of intramural neurons of the porcine descending colon. An analysis was performed on neurons ex-pressing the vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, a neuronal isoform of nitrogen oxide synthase, and galanin. Even a low dose of glyphosate increased the number of neurons immunoreactive against the studied substances. However, the changes depended on both the plexus analysed and the substance tested. Meanwhile, a high glyphosate dose resulted in quantitative changes (an increase in the number) within neurons immunoreactive against all the studied neuropeptides/enzymes in the myenteric plexus and both submucosal plexuses. The response of the enteric nervous system in the form of an increase in the number of neurons immunoreactive against neuroprotective substances may suggest that glyphosate has a toxic effect on enteric neurons which attempt to increase their survivability through the released neuroprotective substances. [ABSTRACT FROM AUTHOR]

Short bowel syndrome (SBS) is a severe, life-threatening condition and one of the leading causes of intestinal failure in children. Here we were interested in changes in muscle layers and especially in the myenteric plexus of the enteric nervous system (ENS) of the small bowel in the context of intestinal adaptation. Twelve rats underwent a massive resection of the small intestine to induce SBS. Sham laparotomy without small bowel transection was performed in 10 rats. Two weeks after surgery, the remaining jejunum and ileum were harvested and studied. Samples of human small bowel were obtained from patients who underwent resection of small bowel segments due to a medical indication. Morphological changes in the muscle layers and the expression of nestin, a marker for neuronal plasticity, were studied. Following SBS, muscle tissue increases significantly in both parts of the small bowel, i.e., jejunum and ileum. The leading pathophysiological mechanism of these changes is hypertrophy. Additionally, we observed an increased nestin expression in the myenteric plexus in the remaining bowel with SBS. Our human data also showed that in patients with SBS, the proportion of stem cells in the myenteric plexus had risen by more than twofold. Our findings suggest that the ENS is tightly connected to changes in intestinal muscle layers and is critically involved in the process of intestinal adaptation to SBS. [ABSTRACT FROM AUTHOR]

Introduction: The Enteric Nervous System (ENS) contributes to the innervation of the gut and is considered the second brain in the gut. It is a part of the peripheral nervous system and the ganglionated plexus (GP) of enteric neurons, present in the layers of the digestive tract, is more complex than any other structure of the peripheral nervous system. The establishment of a valid standard method for quantitative analysis of the myenteric plexus has been a topic of debate among enteric neuropathologists and is essential for better characterisation of patients with gut dysmotility. Aim: To determine the histomorphometric analysis of the interganglion distance of the myenteric plexus in the entire large intestine of aborted human foetuses. Materials and Methods: A cross-sectional descriptive study was carried out in the Department of Anatomy at Pondicherry Institute of Medical Sciences, Pondicherry, India. The study duration was eight years and seven months, from October 2014 to May 2022. A total of 50 aborted human foetus samples were collected from Rajiv Gandhi Government Women and Children’s Hospital, Puducherry, India, from the Department of Obstetrics and Gynaecology. The age of the aborted human foetuses was determined based on Biparietal Diameter (BPD) and Crown Rump Length (CRL) and correlated with clinical history. The aborted human foetuses were assigned to two groups: group A (n=29), ≤20 Weeks of Gestation (WG), and group B (n=21), ≥20 WG. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) diaphorase histochemistry, an enzyme histochemistry technique, was used to study the tissue preparations. Statistical significance was determined using Statistical Package for Social Sciences (SPSS) version 20.0, International Business Machines (IBM) corporation. The independent sample t-test was conducted for parametric analysis. Results: The interganglion distance was measured from the caecum (A1 segment) to the anal canal (A7 segment) in aborted foetuses of less than 20 WG. The mean values ranged from 78.86±38.09 μm to 126.94±23.87 µm in foetuses of less than 20 WG. The mean values ranged from 66.41±8.05 μm to 107.67±30.17 µm in aborted human foetuses of more than 20 WG. Conclusion: The anal canal showed a long interganglion distance. The average reference values were found to be helpful as a diagnostic tool for identifying disorders related to gut innervation and motility.

Introduction: Bisphenol A (BPA) is used in large quantities for the production of plastics and is present in various everyday objects. It penetrates living organisms and shows multidirectional adverse influence on many internal organs. For this reason, BPA is often replaced in plastic production by other substances. One of them is bisphenol S (BPS), whose effects on the enteric nervous system (ENS) have not been explained. Methods: Therefore, the present study compares the influence of BPA and BPS on the number of enteric neurons immunoreactive to cocaine-and amphetamine-regulated transcript (CART) peptide located in the ENS of the stomach, jejunum and colon with the use of double immunofluorescence method. Results: The obtained results have shown that both bisphenols studied induced an increase in the number of CART-positive enteric neurons, and the severity of changes depended on the type of enteric ganglion, the dose of bisphenols and the segment of the digestive tract. The most visible changes were noted in the myenteric ganglia in the colon. Moreover, in the colon, the changes submitted by BPS are more noticeable than those observed after BPA administration. In the stomach and jejunum, bisphenol-induced changes were less visible, and changes caused by BPS were similar or less pronounced than those noted under the impact of BPA, depending on the segment of the gastrointestinal tract and ganglion type studied. Discussion: The results show that BPS affects the enteric neurons containing CART in a similar way to BPA, and the BPS impact is even stronger in the colon. Therefore, BPS is not neutral for the gastrointestinal tract and ENS. [ABSTRACT FROM AUTHOR]

Recent accessibility to specialized high-throughput “omics” technologies including single cell RNA sequencing allows researchers to capture cell type- and subtype-specific expression signatures. These omics methods are used in the enteric nervous system (ENS) to identify potential subtypes of enteric neurons and glia. ENS omics data support the known gene and/or protein expression of functional neuronal and glial cell subtypes and suggest expression patterns of novel subtypes. Gene and protein expression patterns can be further used to infer cellular function and implications in human disease. In this review we discuss how high-throughput “omics” data add additional depth to the understanding of established functional subtypes of ENS cells and raise new questions by suggesting novel ENS cell subtypes with unique gene and protein expression patterns. Then we investigate the changes in these expression patterns during pathology observed by omics research. Although current ENS omics studies provide a plethora of novel data and therefore answers, they equally create new questions and routes for future study.

Objectives: Sacral nerve stimulation (SNS) has been employed for treating constipation. However, its mechanisms involving enteric nervous system (ENS) and motility are largely unknown. In this study, we investigated the possible ENS involvement of SNS in treating Loperamide-induced constipation in rats. Methods: Experiment-1 was designed to study the effects of acute SNS on whole colon transit time (CTT). In experiment-2, we induced constipation by Loperamide and then applied daily SNS or sham-SNS for 1 week. Choline acetyltransferase (ChAT), nitric oxide synthase (nNOS), and PGP9.5 in colon tissue were examined at the end of the study. Moreover, survival factors such as phosphorylated AKT (p-AKT) and Glial cell-derived neurotrophic factor (GDNF) were measures by immunohistochemistry (IHC) and western blot (WB). Key results: (1) SNS with one set of parameters shortened CTT starting at 90 min after phenol red administration (p < 0.05). (2) While Loperamide induced slow transit constipation with a significant reduction in fecal pellet number and feces wet weight, daily SNS for a week resolved constipation. (3) Moreover, SNS was able to shorten whole gut transit time comparing to sham-SNS (p = 0.01). (4) Loperamide reduced the number of PGP9.5 and ChAT positive cells, and downregulated ChAT protein expression and upregulated nNOS protein expression, whereas these detrimental effects were significantly reversed by SNS. (5) Furthermore, SNS increased expressions of both GDNF and p-AKT in colon tissue. (6) Vagal activity was reduced following Loperamide (p < 0.01); yet SNS normalized vagal activity. Conclusion: SNS with appropriate parameters improves opioid-induced constipation and reversed the detrimental effects of Loperamide on enteric neurons possibly via the GDNF-PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]

IntroductionBisphenol A (BPA) is used in large quantities for the production of plastics and is present in various everyday objects. It penetrates living organisms and shows multidirectional adverse influence on many internal organs. For this reason, BPA is often replaced in plastic production by other substances. One of them is bisphenol S (BPS), whose effects on the enteric nervous system (ENS) have not been explained.MethodsTherefore, the present study compares the influence of BPA and BPS on the number of enteric neurons immunoreactive to cocaine-and amphetamine-regulated transcript (CART) peptide located in the ENS of the stomach, jejunum and colon with the use of double immunofluorescence method.ResultsThe obtained results have shown that both bisphenols studied induced an increase in the number of CART-positive enteric neurons, and the severity of changes depended on the type of enteric ganglion, the dose of bisphenols and the segment of the digestive tract. The most visible changes were noted in the myenteric ganglia in the colon. Moreover, in the colon, the changes submitted by BPS are more noticeable than those observed after BPA administration. In the stomach and jejunum, bisphenol-induced changes were less visible, and changes caused by BPS were similar or less pronounced than those noted under the impact of BPA, depending on the segment of the gastrointestinal tract and ganglion type studied.DiscussionThe results show that BPS affects the enteric neurons containing CART in a similar way to BPA, and the BPS impact is even stronger in the colon. Therefore, BPS is not neutral for the gastrointestinal tract and ENS.

Phosphatase and tensin homolog (Pten) is a key regulator of cell proliferation and a potential target to stimulate postnatal enteric neuro- and/or gliogenesis. To investigate this, we generated two tamoxifen-inducible Cre recombinase murine models in which Pten was conditionally ablated, (1) in glia (Plp1-expressing cells) and (2) in neurons (Calb2-expressing cells). Tamoxifen-treated adult (7–12 weeks of age; n = 4–15) mice were given DSS to induce colitis, EdU to monitor cell proliferation, and were evaluated at two timepoints: (1) early (3–4 days post-DSS) and (2) late (3–4 weeks post-DSS). We investigated gut motility and evaluated the enteric nervous system. Pten inhibition in Plp1-expressing cells elicited gliogenesis at baseline and post-DSS (early and late) in the colon, and neurogenesis post-DSS late in the proximal colon. They also exhibited an increased frequency of colonic migrating motor complexes (CMMC) and slower whole gut transit times. Pten inhibition in Calb2-expressing cells did not induce enteric neuro- or gliogenesis, and no alterations were detected in CMMC or whole gut transit times when compared to the control at baseline or post-DSS (early and late). Our results merit further research into Pten modulation where increased glia and/or slower intestinal transit times are desired (e.g., short-bowel syndrome and rapid-transit disorders).

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence‐activated cell sorting (FACS). We find that CD56/CD90/CD24 co‐expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co‐expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single‐cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease. Synopsis: This study describes flow‐cytometry based enrichment of human enteric neurons and glia using a combination of cell surface markers (CD56/CD90/CD24), which enables studies of the human ENS. Based on CD56/CD90/CD24 co‐expression, enteric nervous system cells can be enriched by flow cytometry with higher specificity and resolution than previous methods.Enteric neurons representing a minority of the ENS cluster can be resolved from enteric glial cells based on CD24 levels.CD56/CD90/CD24‐positive cells can be further processed for downstream analysis, such as scRNAseq, enabling interrogation of the human ENS transcriptome. [ABSTRACT FROM AUTHOR]

Nausea and vomiting are common gastrointestinal side effects of oxaliplatin chemotherapy used for the treatment of colorectal cancer. However, the mechanism underlying oxaliplatin-induced nausea and vomiting is unknown. The stomach is involved in the emetic reflex but no study investigated the effects of oxaliplatin treatment on the stomach. In this study, the in vivo effects of oxaliplatin treatment on eating behaviour, stomach content, intrinsic gastric neuronal population, extrinsic innervation to the stomach, levels of mucosal serotonin (5-hydroxytryptamine, 5-HT), and parasympathetic vagal efferent nerve activity were analysed. Chronic systemic oxaliplatin treatment in mice resulted in pica, indicated by increased kaolin consumption and a reduction in body weight. Oxaliplatin treatment significantly increased the stomach weight and content. The total number of myenteric and nitric oxide synthase-immunoreactive neurons as well as the density of sympathetic, parasympathetic, and sensory fibres in the stomach were decreased significantly with oxaliplatin treatment. Oxaliplatin treatment significantly increased the levels in mucosal 5-HT and the number of enterochromaffin-like cells. Chronic oxaliplatin treatment also caused a significant increase in the vagal efferent nerve activity. The findings of this study indicate that oxaliplatin exposure has adverse effects on multiple components of gastric innervation, which could be responsible for pica and gastric dysmotility. [ABSTRACT FROM AUTHOR]

Background: Diarrhea is present in up to 30–50% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte–enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of damage associated molecular patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. Methods: Primary mouse enteric neurons (EN) were exposed to a conditioned medium from ACE2-expressing Caco-2 colonic epithelial cells infected with SARS-CoV-2 or treated with tunicamycin (ER stress inducer). Vasoactive intestinal peptides (VIP) expression and secretion by EN were assessed by RT-PCR and ELISA, respectively. Membrane expression of NHE3 was determined by surface biotinylation. Results: SARS-CoV-2 infection led to increased expression of BiP/GRP78, a marker and key regulator for ER stress in Caco-2 cells. Infected cells secreted the DAMP protein, heat shock protein 70 (HSP70), into the culture media, as revealed by proteomic and Western analyses. The expression of VIP mRNA in EN was up-regulated after treatment with a conditioned medium of SARS-CoV-2-infected Caco-2 cells. CD91, a receptor for HSP70, is abundantly expressed in the cultured mouse EN. Tunicamycin, an inducer of ER stress, also induced the release of HSP70 and Xbp1s, mimicking SARS-CoV-2 infection. Co-treatment of Caco-2 with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in membrane expression of Na+/H+ exchanger (NHE3), an important transporter that mediates intestinal Na+/fluid absorption. Conclusions: Our findings demonstrate that SARS-CoV-2 enterocyte infection leads to ER stress and the release of DAMPs that up-regulates the expression and release of VIP by EN. VIP in turn inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in enterocytes. These data highlight the role of epithelial-enteric neuronal crosstalk in COVID-19-related diarrhea. [ABSTRACT FROM AUTHOR]

Dietary advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed when reducing sugars are heated with proteins, amino acids, or lipids at high temperatures for a prolonged period. The presence and accumulation of AGEs in numerous cell types and tissues are known to be prevalent in the pathology of many diseases. Modern diets, which contain a high proportion of processed foods and therefore a high level of AGE, cause deleterious effects leading to a multitude of unregulated intracellular and extracellular signalling and inflammatory pathways. Currently, many studies focus on investigating the chemical and structural aspects of AGEs and how they affect the metabolism and the cardiovascular and renal systems. Studies have also shown that AGEs affect the digestive system. However, there is no complete picture of the implication of AGEs in this area. The gastrointestinal tract is not only the first and principal site for the digestion and absorption of dietary AGEs but also one of the most susceptible organs to AGEs, which may exert many local and systemic effects. In this review, we summarise the current evidence of the association between a high-AGE diet and poor health outcomes, with a special focus on the relationship between dietary AGEs and alterations in the gastrointestinal structure, modifications in enteric neurons, and microbiota reshaping. [ABSTRACT FROM AUTHOR]

Background: Respiratory inflammation is the body's response to lung infection, trauma or hypersensitivity and is often accompanied by comorbidities, including gastrointestinal (GI) symptoms. Why respiratory inflammation is accompanied by GI dysfunction remains unclear. Here, we investigate the effect of lipopolysaccharide (LPS)-induced lung inflammation on intestinal barrier integrity, tight-junctions, enteric neurons and inflammatory marker expression. Methods: Female C57bl/6 mice (6–8 weeks) were intratracheally administered LPS (5 µg) or sterile saline, and assessed after either 24 or 72 h. Total and differential cell counts in bronchoalveolar lavage fluid (BALF) were used to evaluate lung inflammation. Intestinal barrier integrity was assessed via cross sectional immunohistochemistry of tight junction markers claudin-1, claudin-4 and EpCAM. Changes in the enteric nervous system (ENS) and inflammation in the intestine were quantified immunohistochemically using neuronal markers Hu + and nNOS, glial markers GFAP and S100β and pan leukocyte marker CD45. Results: Intratracheal LPS significantly increased the number of neutrophils in BALF at 24 and 72 h. These changes were associated with an increase in CD45 + cells in the ileal mucosa at 24 and 72 h, increased goblet cell expression at 24 h, and increased expression of EpCAM at 72 h. LPS had no effect on the expression of GFAP, S100β, nor the number of Hu + neurons or proportion of nNOS neurons in the myenteric plexus. Conclusions: Intratracheal LPS administration induces inflammation in the ileum that is associated with enhanced expression of EpCAM, decreased claudin-4 expression and increased goblet cell density, these changes may contribute to systemic inflammation that is known to accompany many inflammatory diseases of the lung. [ABSTRACT FROM AUTHOR]

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern released by dying cells to stimulate the immune response. During cell death, HMGB1 is translocated from the nucleus to the cytoplasm and passively released. High levels of secreted HMGB1 are observed in the faeces of inflammatory bowel disease (IBD) patients, indicating its role in IBD pathophysiology and potential as a non-invasive IBD biomarker. HMGB1 is important in regulating neuronal damage in the central nervous system; its pathological activity is intertwined with oxidative stress and inflammation. In this study, HMGB1 expression in the enteric nervous system and its relevance to intestinal neuroinflammation is explored in organotypic cultures of the myenteric plexus exposed to oxidative stimuli and in Winnie mice with spontaneous chronic colitis. Oxidative stimuli induced cytoplasmic translocation of HMGB1 in myenteric neurons in organotypic preparations. HMGB1 translocation correlated with enteric neuronal loss and oxidative stress in the myenteric ganglia of Winnie mice. Inhibition of HMGB1 by glycyrrhizic acid ameliorated HMGB1 translocation and myenteric neuronal loss in Winnie mice. These data highlight modulation of HMGB1 signalling as a therapeutic strategy to reduce the consequences of enteric neuroinflammation in colitis, warranting the exploration of therapeutics acting on the HMGB1 pathway as an adjunct treatment with current anti-inflammatory agents. [ABSTRACT FROM AUTHOR]

The enteric nervous system (ENS) regulates several functional and immunological processes in the gastrointestinal tract. However, some diseases can disrupt the ENS functionality, impacting the behavior of enteric neurons and enteric glial cells by increasing the accumulation of reactive oxygen species. Oxidative stress is considered to be a trigger for alterations in these cells' morphology, density, and neurochemical patterns. In light of this, nutritional strategies are a growing field of investigation regarding their potential to modulate enteric neurons and enteric glial cells through reduced reactive oxygen species production. Moreover, several lines of evidence show that nutrients are related to counteracting oxidative stress. Some studies have evaluated the potential of nutrients with antioxidant roles (such as amino acids, polyphenols, prebiotics, vitamins, and specific extracts obtained from foods) to modulate the ENS. Thus, this review discusses how bioactive compounds and nutrients can impact the ENS by alleviating oxidative stress. [ABSTRACT FROM AUTHOR]

Introduction: The enteric nervous system (ENS) in the wall of the gastrointestinal tract is complex and comprises many neurons, which are differentiated in terms of structure, function and neurochemistry. Neuregulin 1 (NRG 1) is one of the neuronal factors synthesised in the ENS about the distribution and functions of which relatively little is known. The present study is the first description of the distribution of NRG 1 in the ENS in various segments of the porcine small intestine., Material and Methods: Fragments were excised from the duodenum, jejunum and ileum of five euthanised Piétrain × Duroc sows, 18-20 kg in weight and eight weeks of age. Paraformaldehyde-fixed and dehydrated tissue was sectioned and double-labelling immunofluorescence was performed using Alexa Fluor-conjugated secondary antibodies to visualise neuregulin 1 and its colocalisation with vasoactive intestinal polypeptide (VIP), galanin (GAL), and the neuronal isoform of nitric oxide synthase (nNOS) in the myenteric and inner and outer submucosal plexuses, with PGP 9.5 serving as a pan-neuronal marker., Results: Neuregulin 1 was observed in all enteric plexuses in each segment of the small intestine. The percentage of NRG 1-positive neurons ranged from 8.38 ± 0.55% of all neurons in the jejunal inner submucous plexus to 21.52 ± 0.98% in the duodenal myenteric plexus. Cells which were NRG 1-positive also contained VIP, GAL and nNOS in all segments of the small intestine to a degree which varied by small intestine segment and enteric plexus type., Conclusion: The results indicate that NRG 1-positive neurons are present in the ENS of the porcine small intestine and differ significantly neurochemically, which may suggest a multifaceted role for NRG-1 in the controlling of the small intestine activity., Competing Interests: Conflicts of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2024 Łukasz Puchała et al., published by Sciendo.)

The gut contains the largest macrophage pool in the body, with populations of macrophages residing in the mucosa and muscularis propria of the gastrointestinal (GI) tract. Muscularis macrophages (MMs), which are located within the muscularis propria, interact with cells essential for GI function, such as interstitial cells of Cajal, enteric neurons, smooth muscle cells, enteric glia, and fibroblast-like cells, suggesting that these immune cells contribute to several aspects of GI function. This review focuses on the latest insights on the factors contributing to MM heterogeneity and the functional interaction of MMs with other cell types essential for GI function. This review integrates the latest findings on macrophages in other organs with increasing knowledge of MMs to better understand their role in a healthy and diseased gut. We describe the factors that contribute to (muscularis macrophage) MM heterogeneity, and the nature of MM interactions with cells regulating GI function. Finally, we also describe the increasing evidence suggesting a critical role of another immune cell type, the mast cell, in normal and diseased GI physiology.

The enteric nervous system (ENS) regulates numerous functional and immunological attributes of the gastrointestinal tract. Alterations in ENS cell function have been linked to intestinal outcomes in various metabolic, intestinal, and neurological disorders. Chronic kidney disease (CKD) is associated with a challenging intestinal environment due to gut dysbiosis, which further affects patient quality of life. Although the gut-related repercussions of CKD have been thoroughly investigated, the involvement of the ENS in this puzzle remains unclear. ENS cell dysfunction, such as glial reactivity and alterations in cholinergic signaling in the small intestine and colon, in CKD are associated with a wide range of intestinal pathways and responses in affected patients. This review discusses how the ENS is affected in CKD and how it is involved in gut-related outcomes, including intestinal permeability, inflammation, oxidative stress, and dysmotility. [Display omitted] [ABSTRACT FROM AUTHOR]

The gastrointestinal epithelium is critical for maintaining a symbiotic relationship with commensal microbiota. Chronic morphine exposure can compromise the gut epithelial barrier in mice and lead to dysbiosis. Recently, studies have implicated morphine-induced dysbiosis in the mechanism of antinociceptive tolerance and reward, suggesting the presence of a gut-brain axis in the pharmacological effects of morphine. However, the mechanism(s) underlying morphine-induced changes in the gut microbiome remains unclear. The pro-inflammatory cytokine, Interleukin-18 (IL-18), released by enteric neurons can modulate gut barrier function. Therefore, in the present study we investigated the effect of morphine on IL-18 expression in the mouse ileum. We observed that chronic morphine exposure in vivo induces IL-18 expression in the ileum myenteric plexus that is attenuated by naloxone. Given that mu-opioid receptors (MORs) are mainly expressed in enteric neurons, we also characterized morphine effects on the excitability of cholinergic (excitatory) and vasoactive intestinal peptide (VIP)-expressing (inhibitory) myenteric neurons. We found fundamental differences in the electrical properties of cholinergic and VIP neurons such that VIP neurons are more excitable than cholinergic neurons. Furthermore, MORs were primarily expressed in cholinergic neurons, although a subset of VIP neurons also expressed MORs and responded to morphine in electrophysiology experiments. In conclusion, these data show that morphine increases IL-18 in ileum myenteric plexus neurons via activation of MORs in a subset of cholinergic and VIP neurons. Thus, understanding the neurochemistry and electrophysiology of MOR-expressing enteric neurons can help to delineate mechanisms by which morphine perturbs the gut barrier. [ABSTRACT FROM AUTHOR]

Mas-related G protein-coupled receptor D (MrgprD) was first identified in small-diameter sensory neurons of mouse dorsal root ganglion (DRG). The role of MrgprD has been studied in somatosensation, especially in pain and itch response. We recently showed that MrgprD also participated in the modulation of murine intestinal motility. The treatment of MrgprD receptor agonist suppressed the spontaneous contractions in the isolated intestinal rings of mice, indicating the intrinsic expression of MrgprD in the murine gastrointestinal (GI) tract. Although the expression of Mrgprd in GI tract has been previously detected by the way of quantitative real-time PCR, the cell-type-specific expression of MrgprD in GI tract is no yet determined. Herein, we employed Mrgprd-tdTomato reporter mouse line and the whole-mount immunohistochemistry to observe the localization of MrgprD in the smooth muscle layers of ileum and colon. We show that tdTomato-positive cells colocalized with NeuN-immunostaining in the myenteric plexus in the whole-mount preparations of the ileum and the colon. Further immunohistochemistry using the commercially available MrgprD antibody revealed the expression of MrgprD in NeuN-labeled enteric neurons in the myenteric plexus. Our results demonstrate the expression of MrgprD in the enteric neurons in the murine GI tract, highlighting the implications of MrgprD in the physiology and pathophysiology of the GI tract. [ABSTRACT FROM AUTHOR]

Hirschsprung disease is most often characterized by aganglionosis limited to the distal colon and rectum, and mice lacking the Endothelin receptor type B (Ednrb) faithfully recapitulate this phenotype. However, despite the presence of enteric ganglia in the small intestine, both human patients and Ednrb−/− mice suffer from dysmotility and altered gastrointestinal function, thus raising the possibility of enteric nervous system (ENS) abnormalities proximal to the aganglionic region. We undertook the present study to determine whether abnormalities with the ENS in ganglionated regions may account for abnormal gastrointestinal function. We performed single-cell RNA sequencing on ENS cells from the small intestine of Ednrb−/− mice and compared the results to a published single-cell dataset. Our results identified a missing population of neurons marked by the enzyme Gad2, which catalyzes the production of γ-Aminobutyric acid (GABA), in the small intestine of Ednrb−/− animals. This result was confirmed by immunostaining enteric ganglia from Ednrb−/− mice and their wild-type littermates. These data show for the first time that ganglionated regions of the Hirschsprung gut lack a neuronal subpopulation, which may explain the persistent gastrointestinal dysfunction after surgical correction of Hirschsprung disease.

Dietary fiber supports healthy gut bacteria and their production of short-chain fatty acids (SCFA), which promote anti-inflammatory cell development, in particular, regulatory T cells. It is thus beneficial in many diseases, including influenza infection. While disruption of the gut microbiota by antibiotic treatment aggravates West Nile Virus (WNV) disease, whether dietary fiber is beneficial is unknown. WNV is a widely-distributed neurotropic flavivirus that recruits inflammatory monocytes into the brain, causing life-threatening encephalitis. To investigate the impact of dietary fiber on WNV encephalitis, mice were fed on diets deficient or enriched with dietary fiber for two weeks prior to inoculation with WNV. To induce encephalitis, mice were inoculated intranasally with WNV and maintained on these diets. Despite increased fecal SCFA acetate and changes in gut microbiota composition, dietary fiber did not affect clinical scores, leukocyte infiltration into the brain, or survival. After the brain, highest virus loads were measured in the colon in neurons of the submucosal and myenteric plexuses. Associated with this, there was disrupted gut homeostasis, with shorter colon length and higher local inflammatory cytokine levels, which were not affected by dietary fiber. Thus, fiber supplementation is not effective in WNV encephalitis. [ABSTRACT FROM AUTHOR]

Background: The enteric nervous system (ENS) is an extensive neural network embedded in the wall of the gastrointestinal tract that regulates digestive function and gastrointestinal homeostasis. The ENS consists of two main cell types; enteric neurons and enteric glial cells. In vitro techniques allow simplified investigation of ENS function, and different culture methods have been developed over the years helping to understand the role of ENS cells in health and disease. Purpose: This review focuses on summarizing and comparing available culture protocols for the generation of primary ENS cells from adult mice, including dissection of intestinal segments, enzymatic digestions, surface coatings, and culture media. In addition, the potential of human ENS cultures is also discussed. [ABSTRACT FROM AUTHOR]

The physiopathology of digestive disorders in patients with spinal cord injury (SCI) remains largely unknown, particularly the involvement of the enteric nervous system (ENS). We aimed in a rat model of chronic thoracic SCI to characterize (1) changes in the neurochemical coding of enteric neurons and their putative consequences upon neuromuscular response, and (2) the inflammatory response of the colon. Ex vivo motility of proximal and distal colon segments of SCI and control (CT) rats were studied in an organ chamber in response to electrical field stimulation (EFS) and bethanechol. Immunohistochemical analysis of proximal and distal segments was performed using antibodies again Hu, neuronal nitric oxide synthase, (nNOS), and choline acetyltransferase. Colonic content of acetylcholine and acetylcholinesterase was measured; messenger RNA (mRNA) expression of inflammatory cytokines was measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) approaches. Compared with the CT rats, the contractile response to bethanechol was significantly decreased in the proximal colon of SCI rats but not in the distal colon. The proportion of nNOS immunoreactive (IR) neurons was significantly reduced in the proximal but not distal colon of SCI rats. No change in proportion of choline acetyltransferase (ChAT)-IR was reported; the tissue concentration of acetylcholine was significantly decreased in the proximal colon of SCI rats. The expression of tumor necrosis factor alpha (TNF-?) and intercellular adhesion molecule-1 (ICAM-1) was significantly reduced in the proximal and distal colon of SCI rats. This study demonstrates that functional motor and enteric neuroplastic changes affect preferentially the proximal colon compared with the distal colon. The underlying mechanisms and factors responsible for these changes remain to be discovered.

Dietary fiber supports healthy gut bacteria and their production of short-chain fatty acids (SCFA), which promote anti-inflammatory cell development, in particular, regulatory T cells. It is thus beneficial in many diseases, including influenza infection. While disruption of the gut microbiota by antibiotic treatment aggravates West Nile Virus (WNV) disease, whether dietary fiber is beneficial is unknown. WNV is a widely-distributed neurotropic flavivirus that recruits inflammatory monocytes into the brain, causing life-threatening encephalitis. To investigate the impact of dietary fiber on WNV encephalitis, mice were fed on diets deficient or enriched with dietary fiber for two weeks prior to inoculation with WNV. To induce encephalitis, mice were inoculated intranasally with WNV and maintained on these diets. Despite increased fecal SCFA acetate and changes in gut microbiota composition, dietary fiber did not affect clinical scores, leukocyte infiltration into the brain, or survival. After the brain, highest virus loads were measured in the colon in neurons of the submucosal and myenteric plexuses. Associated with this, there was disrupted gut homeostasis, with shorter colon length and higher local inflammatory cytokine levels, which were not affected by dietary fiber. Thus, fiber supplementation is not effective in WNV encephalitis.

Nausea and vomiting are common gastrointestinal side effects of oxaliplatin chemotherapy used for the treatment of colorectal cancer. However, the mechanism underlying oxaliplatin-induced nausea and vomiting is unknown. The stomach is involved in the emetic reflex but no study investigated the effects of oxaliplatin treatment on the stomach. In this study, the in vivo effects of oxaliplatin treatment on eating behaviour, stomach content, intrinsic gastric neuronal population, extrinsic innervation to the stomach, levels of mucosal serotonin (5-hydroxytryptamine, 5-HT), and parasympathetic vagal efferent nerve activity were analysed. Chronic systemic oxaliplatin treatment in mice resulted in pica, indicated by increased kaolin consumption and a reduction in body weight. Oxaliplatin treatment significantly increased the stomach weight and content. The total number of myenteric and nitric oxide synthase-immunoreactive neurons as well as the density of sympathetic, parasympathetic, and sensory fibres in the stomach were decreased significantly with oxaliplatin treatment. Oxaliplatin treatment significantly increased the levels in mucosal 5-HT and the number of enterochromaffin-like cells. Chronic oxaliplatin treatment also caused a significant increase in the vagal efferent nerve activity. The findings of this study indicate that oxaliplatin exposure has adverse effects on multiple components of gastric innervation, which could be responsible for pica and gastric dysmotility.

Background and Aims: The enteric nervous system independently controls gastrointestinal function including motility, which is primarily mediated by the myenteric plexus, therefore also playing a crucial role in functional intestinal disorders. Live recordings from human myenteric neurons proved to be challenging due to technical difficulties. Using the neuroimaging technique, we are able to record human colonic myenteric neuronal activity and investigate their functional properties in a large cohort of patients., Methods: Activity from myenteric neurons in wholemount preparations of different sampling sites of fresh, human colonic tissue was recorded using neuroimaging with the voltage sensitive dye 1-(3-sulfanatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine. Neuronal responses were analyzed following stimulation with nicotine and serotonin (5-HT) for differences based on the donor's age, the disorder indicative for surgery and the colonic region. Immunohistochemistry was performed to calculate the total neuronal numbers., Results: Stimulation with nicotine and 5-HT elicited reproducible action potential discharge in a proportion of human myenteric neurons. The responses to 5-HT were significantly greater in tissues from older patients and from those with inflammatory disorders, while neuronal activity to nicotinergic stimulation was comparable in all patients. Neuronal numbers declined with rising patient's age and was highest in the sigmoid colon., Conclusion: Neuroimaging with 1-(3-sulfanatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine was successfully adapted to record reproducible responses from human colonic myenteric neurons upon pharmacological stimulation. Evidence exists for an impact of age and inflammation on the serotonergic neuronal signaling and for differences in neuronal numbers in the distinct colonic regions as well as a neuronal decrease with age., (© 2024 The Authors.)

Irritable bowel syndrome (IBS) is a prevalent functional gastrointestinal disorder characterized by visceral pain and gut dysmotility. However, the specific mechanisms by which Lactobacillus strains relieve IBS remain unclear. Here, we screened Lactobacillus strains from traditional Chinese fermented foods with potential IBS-alleviating properties through in vitro and in vivo experiments. We demonstrated that Lactiplantibacillus plantarum D266 ( Lp D266) administration effectively modulates intestinal peristalsis, enteric neurons, visceral hypersensitivity, colonic inflammation, gut barrier function, and mast cell activation. Additionally, Lp D266 shapes gut microbiota and enhances tryptophan (Trp) metabolism, thus activating the aryl hydrocarbon receptor (AhR) and subsequently enhancing IL-22 production to maintain gut homeostasis. Mechanistically, Lp D266 potentially modulates colonic physiology and enteric neurons by microbial tryptophan metabolites. Further, our study indicates that combining Lp D266 with Trp synergistically ameliorates IBS symptoms. Together, our experiments identify the therapeutic efficacy of tryptophan-catabolizing Lp D266 in regulating gut physiology and enteric neurons, providing new insights into the development of probiotic-mediated nutritional intervention for IBS management.

Background: Gastrointestinal (GI) function is critically dependent on the control of the enteric nervous system (ENS), which is situated within the gut wall and organized into two ganglionated nerve plexuses: the submucosal and myenteric plexus. The ENS is optimally positioned and together with the intestinal epithelium, is well‐equipped to monitor the luminal contents such as microbial metabolites and to coordinate appropriate responses accordingly. Despite the heightened interest in the gut microbiota and its influence on intestinal physiology and pathophysiology, how they interact with the host ENS remains unclear. Methods: Using full‐thickness proximal colon preparations from transgenic Villin‐CreERT2;R26R‐GCaMP3 and Wnt1‐Cre;R26R‐GCaMP3 mice, which express a fluorescent Ca2+ indicator in their intestinal epithelium or in their ENS, respectively, we examined the effects of key luminal microbial metabolites (SCFAs and 5‐HT) on the mucosa and underlying enteric neurons. Key Results: We show that the SCFAs acetate, propionate, and butyrate, as well as 5‐HT can, to varying extents, acutely elicit epithelial and neuronal Ca2+ responses. Furthermore, SCFAs exert differential effects on submucosal and myenteric neurons. Additionally, we found that submucosal ganglia are predominantly aligned along the striations of the transverse mucosal folds in the proximal colon. Conclusions & Inferences: Taken together, our study demonstrates that different microbial metabolites, including SCFAs and 5‐HT, can acutely stimulate Ca2+ signaling in the mucosal epithelium and in enteric neurons. [ABSTRACT FROM AUTHOR]

Background: Diarrhea is present in up to 30–50% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte–enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of damage associated molecular patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. Methods: Primary mouse enteric neurons (EN) were exposed to a conditioned medium from ACE2-expressing Caco-2 colonic epithelial cells infected with SARS-CoV-2 or treated with tunicamycin (ER stress inducer). Vasoactive intestinal peptides (VIP) expression and secretion by EN were assessed by RT-PCR and ELISA, respectively. Membrane expression of NHE3 was determined by surface biotinylation. Results: SARS-CoV-2 infection led to increased expression of BiP/GRP78, a marker and key regulator for ER stress in Caco-2 cells. Infected cells secreted the DAMP protein, heat shock protein 70 (HSP70), into the culture media, as revealed by proteomic and Western analyses. The expression of VIP mRNA in EN was up-regulated after treatment with a conditioned medium of SARS-CoV-2-infected Caco-2 cells. CD91, a receptor for HSP70, is abundantly expressed in the cultured mouse EN. Tunicamycin, an inducer of ER stress, also induced the release of HSP70 and Xbp1s, mimicking SARS-CoV-2 infection. Co-treatment of Caco-2 with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in membrane expression of Na+/H+ exchanger (NHE3), an important transporter that mediates intestinal Na+/fluid absorption. Conclusions: Our findings demonstrate that SARS-CoV-2 enterocyte infection leads to ER stress and the release of DAMPs that up-regulates the expression and release of VIP by EN. VIP in turn inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in enterocytes. These data highlight the role of epithelial-enteric neuronal crosstalk in COVID-19-related diarrhea.

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern released by dying cells to stimulate the immune response. During cell death, HMGB1 is translocated from the nucleus to the cytoplasm and passively released. High levels of secreted HMGB1 are observed in the faeces of inflammatory bowel disease (IBD) patients, indicating its role in IBD pathophysiology and potential as a non-invasive IBD biomarker. HMGB1 is important in regulating neuronal damage in the central nervous system; its pathological activity is intertwined with oxidative stress and inflammation. In this study, HMGB1 expression in the enteric nervous system and its relevance to intestinal neuroinflammation is explored in organotypic cultures of the myenteric plexus exposed to oxidative stimuli and in Winnie mice with spontaneous chronic colitis. Oxidative stimuli induced cytoplasmic translocation of HMGB1 in myenteric neurons in organotypic preparations. HMGB1 translocation correlated with enteric neuronal loss and oxidative stress in the myenteric ganglia of Winnie mice. Inhibition of HMGB1 by glycyrrhizic acid ameliorated HMGB1 translocation and myenteric neuronal loss in Winnie mice. These data highlight modulation of HMGB1 signalling as a therapeutic strategy to reduce the consequences of enteric neuroinflammation in colitis, warranting the exploration of therapeutics acting on the HMGB1 pathway as an adjunct treatment with current anti-inflammatory agents.

Bidirectional interactions of the gut epithelium with commensal bacteria are critical for maintaining homeostasis within the gut. Chronic opioid exposure perturbs gut homeostasis through a multitude of neuro-immune-epithelial mechanisms, resulting in the development of analgesic tolerance, a major underpinning of the current opioid crisis. Differences in molecular mechanisms of opioid tolerance between the enteric and central pain pathways pose a significant challenge for managing chronic pain without untoward gastrointestinal effects. [ABSTRACT FROM AUTHOR]

Background: Visceral hypersensitivity (VH) is one of the underlying pathophysiologies of irritable bowel syndrome. Mast cell overactivation has been found to be one of the main causes of VH. We investigated the effects and mechanisms of actions of sacral nerve stimulation (SNS) on visceral pain in a rodent model of VH.Methods: The VH was established by an intrarectal infusion of AA in 10-day-old pups. Rats were chronically implanted with electrodes for SNS and recording electromyogram (EMG) and electrocardiogram. The acute study was performed in 2-randomized sessions with SNS (14 Hz, 330 μs, 40% motor threshold or MT, 30 min) or sham-SNS. Later on, rats were randomized into SNS/sham-SNS groups and a chronic study was performed with 2 h-daily SNS or sham-SNS for 21 days. Visceromotor reflexes were assessed by abdominal EMG and withdrawal reflex (AWR). Colon tissues were collected to study colonic acetylcholine (ACh), the enteric neurons (ChAT, nNOS, and PGP9.5), mast cells activity [Tryptase, prostaglandins E2 (PGE2), and cyclooxygenases-2 (COX2)] and pain markers [nerve growth factor (NGF) and Sub-P].Key Results: Sacral nerve stimulation significantly improved visceromotor reflexes assessed by the EMG and AWR, compared with sham-SNS. SNS normalized the protein expressions of ChAT and nNOS and regulated mast cells activity by downregulating Tryptase, COX2, and PGE2. Neonatal AA administration upregulated NGF and Sub-P; chronic SNS significantly decreased these pain biomarkers. Concurrently, chronic SNS increased ACh in colon tissues and vagal efferent activity.Conclusions: Sacral nerve stimulation reduces VH in rats and this ameliorating effect might be attributed to the suppression of mast cell overactivation in the colon tissue via the modulation of autonomic nervous system functions.

Background: Visceral hypersensitivity (VH) is one of the underlying pathophysiologies of irritable bowel syndrome. Mast cell overactivation has been found to be one of the main causes of VH. We investigated the effects and mechanisms of actions of sacral nerve stimulation (SNS) on visceral pain in a rodent model of VH. Methods: The VH was established by an intrarectal infusion of AA in 10-day-old pups. Rats were chronically implanted with electrodes for SNS and recording electromyogram (EMG) and electrocardiogram. The acute study was performed in 2-randomized sessions with SNS (14 Hz, 330 μs, 40% motor threshold or MT, 30 min) or sham-SNS. Later on, rats were randomized into SNS/sham-SNS groups and a chronic study was performed with 2 h-daily SNS or sham-SNS for 21 days. Visceromotor reflexes were assessed by abdominal EMG and withdrawal reflex (AWR). Colon tissues were collected to study colonic acetylcholine (ACh), the enteric neurons (ChAT, nNOS, and PGP9.5), mast cells activity [Tryptase, prostaglandins E2 (PGE2), and cyclooxygenases-2 (COX2)] and pain markers [nerve growth factor (NGF) and Sub-P]. Key Results: Sacral nerve stimulation significantly improved visceromotor reflexes assessed by the EMG and AWR, compared with sham-SNS. SNS normalized the protein expressions of ChAT and nNOS and regulated mast cells activity by downregulating Tryptase, COX2, and PGE2. Neonatal AA administration upregulated NGF and Sub-P; chronic SNS significantly decreased these pain biomarkers. Concurrently, chronic SNS increased ACh in colon tissues and vagal efferent activity. Conclusions: Sacral nerve stimulation reduces VH in rats and this ameliorating effect might be attributed to the suppression of mast cell overactivation in the colon tissue via the modulation of autonomic nervous system functions. [ABSTRACT FROM AUTHOR]

An intact gut epithelium preserves the immunological exclusion of “non-self” entities in the external environment of the gut lumen. Nonetheless, information flows continuously across this interface, with the host immune, endocrine, and neural systems all involved in monitoring the luminal environment of the gut. Both pathogenic and commensal gastrointestinal (GI) bacteria can modulate centrally-regulated behaviors and brain neurochemistry and, although the vagus nerve has been implicated in the microbiota-gut-brain signaling axis, the cellular and molecular machinery that facilitates this communication is unclear. Studies were carried out in healthy Sprague–Dawley rats to understand cross-barrier communication in the absence of disease. A novel colonic-nerve electrophysiological technique was used to examine gut-to-brain vagal signaling by bacterial products. Calcium imaging and immunofluorescent labeling were used to explore the activation of colonic submucosal neurons by bacterial products. The findings demonstrate that the neuromodulatory molecule, glucagon-like peptide-1 (GLP-1), secreted by colonic enteroendocrine L-cells in response to the bacterial metabolite, indole, stimulated colonic vagal afferent activity. At a local level indole modified the sensitivity of submucosal neurons to GLP-1. These findings elucidate a cellular mechanism by which sensory L-cells act as cross-barrier signal transducers between microbial products in the gut lumen and the host peripheral nervous system.

The stomach acts as a buffer between the ingestion of food and its processing in the small intestine. It signals to the brain to modulate food intake and it in turn regulates the passage of a nutrient-rich fluid, containing partly digested food, into the duodenum. These processes need to be finely controlled, for example to restrict reflux into the esophagus and to transfer digesta to the duodenum at an appropriate rate. Thus, the efferent pathways that control gastric volume, gastric peristalsis and digestive juice production are critically important. We review these pathways with an emphasis on the identities of the final motor neurons and comparisons between species. The major types of motor neurons arising from gastric enteric ganglia are as follows: immunohistochemically distinguishable excitatory and inhibitory muscle motor neurons; four neuron types innervating mucosal effectors (parietal cells, chief cells, gastrin cells and somatostatin cells); and vasodilator neurons. Sympathetic efferent neurons innervate intramural arteries, myenteric ganglia and gastric muscle. Vagal efferent neurons with cell bodies in the brain stem do not directly innervate gastric effector tissues; they are pre-enteric neurons that innervate each type of gastric enteric motor neuron. The principal transmitters and co-transmitters of gastric motor neurons, as well as key immunohistochemical markers, are the same in rat, pig, human and other species. [ABSTRACT FROM AUTHOR]

Background: The mucosal barrier damage is recognized as one of the key factors in the pathogenesis of colitis. While sacral nerve stimulation (SNS) was reported to have therapeutic potential for colitis, its mechanisms of actions on colonic permeability remained largely unknown. Methods: In this study, colitis was induced by intrarectal administration of TNBS in rats. Five days later, they were treated with SNS or sham‐SNS for 10 days. The effects of SNS on colonic permeability were assessed by measuring the expression of tight‐junction proteins involved in regulating permeability and the FITC‐dextran test. The mechanism of actions of SNS was investigated by studying the function of the enteric nervous system (ENS) cells and analyzing the autonomic nervous system. Key Results: SNS decreased the disease activity index, microscopic and macroscopic scores, myeloperoxidase activity, and pro‐inflammatory cytokines (TNF‐α, IL‐6). SNS increased the expression of Zonula Occludens‐1, Occludin, Claudin‐1, and Junctional adhesion molecule‐A in the colon tissue. The FITC‐dextran test showed that the colonic permeability was lower with SCS than sham‐SNS. SNS increased ChAT, pancreatic polypeptide, and GDNF and reduced norepinephrine NGF, sub‐P, and mast cell overactivation in the colon tissue. Concurrently, SNS increased acetylcholine in colon tissues and elevated vagal efferent activity. Conclusions & Inferences: SNS ameliorates colonic inflammation and enhances colonic barrier function with the proposed mechanisms involving the increase in parasympathetic activity and modulation of the activity of the ENS and immune system, including mast cells. [ABSTRACT FROM AUTHOR]