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2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), a persistent organic pollutant and a potent aryl hydrocarbon receptor (AHR) ligand, causes delayed intestinal motility and affects the survival of enteric neurons. In this study, we investigated the specific signaling pathways and molecular targets involved in TCDD-induced enteric neurotoxicity. Immortalized fetal enteric neuronal (IM-FEN) cells treated with 10 nM TCDD exhibited cytotoxicity and caspase 3/7 activation, indicating apoptosis. Increased cleaved caspase-3 expression with TCDD treatment, as assessed by immunostaining in enteric neuronal cells isolated from WT mice but not in neural crest cell-specific Ahr deletion mutant mice ( Wnt1Cre +/- /Ahr b(fl/fl ) ), emphasized the pivotal role of AHR in this process. Importantly, the apoptosis in IM-FEN cells treated with TCDD was mediated through a ceramide-dependent pathway, independent of endoplasmic reticulum stress, as evidenced by increased ceramide synthesis and the reversal of cytotoxic effects with myriocin, a potent inhibitor of ceramide biosynthesis. We identified Sptlc2 and Smpd2 as potential gene targets of AHR in ceramide regulation by a chromatin immunoprecipitation (ChIP) assay in IM-FEN cells. Additionally, TCDD downregulated phosphorylated Akt and phosphorylated Ser9-GSK-3β levels, implicating the PI3 kinase/AKT pathway in TCDD-induced neurotoxicity. Overall, this study provides important insights into the mechanisms underlying TCDD-induced enteric neurotoxicity and identifies potential targets for the development of therapeutic interventions.

Bisphenols are dangerous endocrine disruptors that pollute the environment. Due to their chemical properties, they are globally used to produce plastics. Structural similarities to oestrogen allow bisphenols to bind to oestrogen receptors and affect internal body systems. Most commonly used in the plastic industry is bisphenol A (BPA), which also has negative effects on the nervous, immune, endocrine, and cardiovascular systems. A popular analogue of BPA-bisphenol S (BPS) also seems to have harmful effects similar to BPA on living organisms. Therefore, with the use of double immunofluorescence labelling, this study aimed to compare the effect of BPA and BPS on the enteric nervous system (ENS) in mouse jejunum. The study showed that both studied toxins impact the number of nerve cells immunoreactive to substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), the neuronal isoform of nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VAChT). The observed changes were similar in the case of both tested bisphenols. However, the influence of BPA showed stronger changes in neurochemical coding. The results also showed that long-term exposure to BPS significantly affects the ENS.

Side effects associated with nonsteroidal anti-inflammatory drugs (NSAIDs) treatment are a serious limitation of their use in anti-inflammatory therapy. The negative effects of taking NSAIDs include abdominal pain, indigestion nausea as well as serious complications such as bleeding and perforation. The enteric nervous system is involved in regulation of gastrointestinal functions through the release of neurotransmitters. The present study was designed to determine, for the first time, the changes in pituitary adenylate cyclase-activating polypeptide (PACAP), substance P (SP) and galanin (GAL) expression in porcine jejunum after long-term treatment with aspirin, indomethacin and naproxen. The study was performed on 16 immature pigs. The animals were randomly divided into four experimental groups: control, aspirin, indomethacin and naproxen. Control animals were given empty gelatin capsules, while animals in the test groups received selected NSAIDs for 28 days. Next, animals from each group were euthanized. Frozen sections were prepared from collected jejunum and subjected to double immunofluorescence staining. NSAIDs supplementation caused a significant increase in the population of PACAP-, SP- and GAL-containing enteric neurons in the porcine jejunum. Our results suggest the participation of the selected neurotransmitters in regulatory processes of the gastrointestinal function and may indicate the direct toxic effect of NSAIDs on the ENS neurons.

Bisphenol A (BPA) is a substance used in the manufacture of plastics which shows multidirectional adverse effects on living organisms. Since the main path of intoxication with BPA is via the gastrointestinal (GI) tract, the stomach and intestine are especially vulnerable to the impact of this substance. One of the main factors participating in the regulation of intestinal functions is the enteric nervous system (ENS), which is characterized by high neurochemical diversity. Neuregulin 1 (NRG1) is one of the lesser-known active substances in the ENS. During the present study (performed using the double immunofluorescence method), the co-localization of NRG1 with other neuronal substances in the ENS of the caecum and the ascending and descending colon has been investigated under physiological conditions and after the administration of BPA. The obtained results indicate that NRG1-positive neurons also contain substance P, vasoactive intestinal polypeptide, a neuronal isoform of nitric oxide synthase and galanin and the degree of each co-localization depend on the type of enteric plexus and the particular fragment of the intestine. Moreover, it has been shown that BPA generally increases the degree of co-localization of NRG1 with other substances.

Recently, the involvement of the nervous system in the pathology of allergic diseases has attracted increasing interest. However, the precise pathophysiological role of enteric neurons in food allergies has not been elucidated. We report the presence of functional high-affinity IgE receptors (FcεRIs) in enteric neurons. FcεRI immunoreactivities were observed in approximately 70% of cholinergic myenteric neurons from choline acetyltransferase-eGFP mice. Furthermore, stimulation by IgE-antigen elevated intracellular Ca 2+ concentration in isolated myenteric neurons from normal mice, suggesting that FcεRIs are capable of activating myenteric neurons. Additionally, the morphological investigation revealed that the majority of mucosal mast cells were in close proximity to enteric nerve fibers in the colonic mucosa of food allergy mice. Next, using a newly developed coculture system of isolated myenteric neurons and mucosal-type bone-marrow-derived mast cells (mBMMCs) with a calcium imaging system, we demonstrated that the stimulation of isolated myenteric neurons by veratridine caused the activation of mBMMCs, which was suppressed by the adenosine A3 receptor antagonist MRE 3008F20. Moreover, the expression of the adenosine A3 receptor gene was detected in mBMMCs. Therefore, in conclusion, it is suggested that, through interaction with mucosal mast cells, IgE-antigen-activated myenteric neurons play a pathological role in further exacerbating the pathology of food allergy.

Until recently, glia were considered to be a structural support for neurons, however further investigations showed that glial cells are equally as important as neurons. Among many different types of glia, enteric glial cells (EGCs) found in the gastrointestinal tract, have been significantly underestimated, but proved to play an essential role in neuroprotection, immune system modulation and many other functions. They are also said to be remarkably altered in different physiopathological conditions. A nutraceutical is defined as any food substance or part of a food that provides medical or health benefits, including prevention and treatment of the disease. Following the description of these interesting peripheral glial cells and highlighting their role in physiological and pathological changes, this article reviews all the studies on the effects of nutraceuticals as modulators of their functions. Currently there are only a few studies available concerning the effects of nutraceuticals on EGCs. Most of them evaluated molecules with antioxidant properties in systemic conditions, whereas only a few studies have been performed using models of gastrointestinal disorders. Despite the scarcity of studies on the topic, all agree that nutraceuticals have the potential to be an interesting alternative in the prevention and/or treatment of enteric gliopathies (of systemic or local etiology) and their associated gastrointestinal conditions.

Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAP cre x Ai14 floxed mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor.

Four drugs are currently approved for the treatment of Alzheimer's disease (AD) by the FDA. Three of these drugs-donepezil, rivastigmine, and galantamine-belong to the class of acetylcholine esterase inhibitors. Memantine, a NMDA receptor antagonist, represents the fourth and a combination of donepezil and memantine the fifth treatment option. Recently, the gut and its habitants, its microbiome, came into focus of AD research and added another important factor to therapeutic considerations. While the first data provide evidence that AD patients might carry an altered microbiome, the influence of administered drugs on gut properties and commensals have been largely ignored so far. However, the occurrence of digestive side effects with these drugs and the knowledge that cholinergic transmission is crucial for several gut functions enforces the question if, and how, this medication influences the gastrointestinal system and its microbial stocking. Here, we investigated aspects such as microbial viability, colonic propulsion, and properties of enteric neurons, affected by assumed intestinal concentration of the four drugs using the mouse as a model organism. All ex vivo administered drugs revealed no direct effect on fecal bacteria viability and only a high dosage of memantine resulted in reduced biofilm formation of E. coli . Memantine was additionally the only compound that elevated calcium influx in enteric neurons, while all acetylcholine esterase inhibitors significantly reduced esterase activity in colonic tissue specimen and prolonged propulsion time. Both, acetylcholine esterase inhibitors and memantine, had no effect on general viability and neurite outgrowth of enteric neurons. In sum, our findings indicate that all AD symptomatic drugs have the potential to affect distinct intestinal functions and with this-directly or indirectly-microbial commensals.

Acrylamide is one of the harmful substances present in food. The present study aimed to establish the effect of acrylamide supplementation in tolerable daily intake (TDI) dose (0.5 µg/kg b.w./day) and a dose ten times higher than TDI (5 µg/kg b.w./day) on the population of vasoactive intestinal peptide-like immunoreactive (VIP-LI) neurons in the porcine small intestine and the degree of the co-localization of VIP with other neuroactive substances (neuronal nitric oxide synthase (nNOS), substance P (SP), and cocaine- and amphetamine-regulated transcript peptide (CART)). In our work, 15 Danish landrace gilts (5 in each experimental group) received capsules (empty or with low or high doses of acrylamide) for a period of 28 days with their morning feeding. Using double immunofluorescence staining, we established that acrylamide supplementation increased the number of neurons showing immunoreactivity towards VIP in all types of enteric nervous system (ENS) plexuses and fragments of the small intestine studied. Moreover, both doses of acrylamide led to changes in the degree of co-localization of VIP with nNOS, SP, and CART in intramural neurons. The observed changes may be the adaptation of neurons to local inflammation, oxidative stress, or the direct toxic effects of acrylamide on intestinal neurons, also referred to as neuronal plasticity.

Cisplatin is a chemotherapeutic agent widely used for the treatment of solid cancers. Its administration is commonly associated with acute and chronic gastrointestinal dysfunctions, likely related to mucosal and enteric nervous system (ENS) injuries, respectively. Glucagon-like peptide-2 (GLP-2) is a pleiotropic hormone exerting trophic/reparative activities on the intestine, via antiapoptotic and pro-proliferating pathways, to guarantee mucosal integrity, energy absorption and motility. Further, it possesses anti-inflammatory properties. Presently, cisplatin acute and chronic damages and GLP-2 protective effects were investigated in the mouse distal colon using histological, immunohistochemical and biochemical techniques. The mice received cisplatin and the degradation-resistant GLP-2 analog ([Gly2]GLP-2) for 4 weeks. Cisplatin-treated mice showed mucosal damage, inflammation, IL-1β and IL-10 increase; decreased number of total neurons, ChAT- and nNOS-immunoreactive (IR) neurons; loss of SOX-10-IR cells and reduced expression of GFAP- and S100β-glial markers in the myenteric plexus. [Gly2]GLP-2 co-treatment partially prevented mucosal damage and counteracted the increase in cytokines and the loss of nNOS-IR and SOX-10-IR cells but not that of ChAT-IR neurons. Our data demonstrate that cisplatin causes mucosal injuries, neuropathy and gliopathy and that [Gly2]GLP-2 prevents these injuries, partially reducing mucosal inflammation and inducing ENS remodeling. Hence, this analog could represent an effective strategy to overcome colonic injures induced by cisplatin.

The enteric nervous system (ENS), located in the wall of the gastrointestinal (GI) tract, is characterized by complex organization and a high degree of neurochemical diversity of neurons. One of the less known active neuronal substances found in the enteric neurons is neuregulin 1 (NRG1), a factor known to be involved in the assurance of normal development of the nervous system. During the study, made up using the double immunofluorescence technique, the presence of NRG1 in the ENS of the selected segment of porcine large intestine (caecum, ascending and descending colon) was observed in physiological conditions, as well as under the impact of low and high doses of bisphenol A (BPA) which is commonly used in the production of plastics. In control animals in all types of the enteric plexuses, the percentage of NRG1-positive neurons oscillated around 20% of all neurons. The administration of BPA caused an increase in the number of NRG1-positive neurons in all types of the enteric plexuses and in all segments of the large intestine studied. The most visible changes were noted in the inner submucous plexus of the ascending colon, where in animals treated with high doses of BPA, the percentage of NRG1-positive neurons amounted to above 45% of all neuronal cells. The mechanisms of observed changes are not entirely clear, but probably result from neurotoxic, neurodegenerative and/or proinflammatory activity of BPA and are protective and adaptive in nature.

Mycotoxins are secondary metabolites produced by various fungal species. They are commonly found in a wide range of agricultural products. Mycotoxins contained in food enter living organisms and may have harmful effects on many internal organs and systems. The gastrointestinal tract, which first comes into contact with mycotoxins present in food, is particularly vulnerable to the harmful effects of these toxins. One of the lesser-known aspects of the impact of mycotoxins on the gastrointestinal tract is the influence of these substances on gastrointestinal innervation. Therefore, the present study is the first review of current knowledge concerning the influence of mycotoxins on the enteric nervous system, which plays an important role, not only in almost all regulatory processes within the gastrointestinal tract, but also in adaptive and protective reactions in response to pathological and toxic factors in food.

Enteric glial cells (EGCs) influence nitric oxide (NO) - and adenosine diphosphate (ADP) - mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4 -/- mice. Ileal segments from male TLR4 -/- and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4 -/- ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO - and ADP - mediated relaxation in the TLR4 -/- mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4 -/- myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4 -/- mice.

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., Competing Interests: The authors declare no conflicts of interest.

The identification and characterization of fungal commensals of the human gut (the mycobiota) is ongoing, and the effects of their various secondary metabolites on the health and disease of the host is a matter of current research. While the neurons of the central nervous system might be affected indirectly by compounds from gut microorganisms, the largest peripheral neuronal network (the enteric nervous system) is located within the gut and is exposed directly to such metabolites. We analyzed 320 fungal extracts and their effect on the viability of a human neuronal cell line (SH-SY5Y), as well as their effects on the viability and functionality of the most effective compound on primary enteric neurons of murine origin. An extract from P. coprobium was identified to decrease viability with an EC 50 of 0.23 ng/µL in SH-SY5Y cells and an EC 50 of 1 ng/µL in enteric neurons. Further spectral analysis revealed that the effective compound was patulin, and that this polyketide lactone is not only capable of evoking ROS production in SH-SY5Y cells, but also diverse functional disabilities in primary enteric neurons such as altered calcium signaling. As patulin can be found as a common contaminant on fruit and vegetables and causes intestinal injury, deciphering its specific impact on enteric neurons might help in the elaboration of preventive strategies., Competing Interests: The authors declare no conflict of interest.

Epidemiological studies have shown that coffee consumption decreases the risk of Parkinson's disease (PD). Caffeic acid (CA) and chlorogenic acid (CGA) are coffee components that have antioxidative properties. Rotenone, a mitochondrial complex I inhibitor, has been used to develop parkinsonian models, because the toxin induces PD-like pathology. Here, we examined the neuroprotective effects of CA and CGA against the rotenone-induced degeneration of central dopaminergic and peripheral enteric neurons. Male C57BL/6J mice were chronically administered rotenone (2.5 mg/kg/day), subcutaneously for four weeks. The animals were orally administered CA or CGA daily for 1 week before rotenone exposure and during the four weeks of rotenone treatment. Administrations of CA or CGA prevented rotenone-induced neurodegeneration of both nigral dopaminergic and intestinal enteric neurons. CA and CGA upregulated the antioxidative molecules, metallothionein (MT)-1,2, in striatal astrocytes of rotenone-injected mice. Primary cultured mesencephalic or enteric cells were pretreated with CA or CGA for 24 h, and then further co-treated with a low dose of rotenone (1⁻5 nM) for 48 h. The neuroprotective effects and MT upregulation induced by CA and CGA in vivo were reproduced in cultured cells. Our data indicated that intake of coffee components, CA and CGA, enhanced the antioxidative properties of glial cells and prevents rotenone-induced neurodegeneration in both the brain and myenteric plexus.

Researches on spicatoside A (SpiA)-containing natural products suggest the possibility of SpiA as a potential laxative to alleviate chronic constipation. However, no studies have been conducted with single compound administration of SpiA. To verify the laxative effects and mechanism of action of SpiA on chronic constipation, we investigated alterations in the excretion parameters, histological structure, and cholinergic regulation of the enteric nerve in the colons of Institute of Cancer Research (ICR) mice with loperamide (Lop)-induced constipation after exposure to 20 mg/kg of SpiA. Decrease in the number, weight and water contents of stools in the Lop+Vehicle treated group significantly recovered after SpiA treatment, and alterations in the histological structure and transmission electron microscopy (TEM) images were improved in the Lop+SpiA treated group. Similar recovery effects were observed in the ability for mucin secretion and expression of the membrane water channel gene (aquaporin 8, AQP8). Furthermore, significant improvements were observed in the acetylcholinesterase (AChE) activity and acetylcholine receptors' (AChRs) downstream signaling pathway after treatment of SpiA. The levels of gastrointestinal (GI) hormones including cholecystokinin (CCK) and gastrin were also remarkably enhanced in the Lop+SpiA treated group as compared to the Lop+Vehicle treated group. The expression of receptor tyrosine kinase (C-kit) and protein gene product 9.5 (PGP9.5) in Cajal and neural cells, as well as the phosphorylation of myosin light chain (MLC) in smooth muscle cells, were recovered after SpiA exposure. Taken together, the results of the present study provide the first strong evidence that SpiA improves chronic constipation through muscarinic cholinergic regulation of the enteric nerve in a Lop-induced constipation ICR mice model.

Bisphenol A, used in the production of plastic, is able to leach from containers into food and cause multidirectional adverse effects in living organisms, including neurodegeneration and metabolic disorders. Knowledge of the impact of BPA on enteric neurons is practically non-existent. The destination of this study was to investigate the influence of BPA at a specific dose (0.05 mg/kg body weight/day) and at a dose ten times higher (0.5 mg/kg body weight/day), given for 28 days, on the porcine ileum. The influence of BPA on enteric neuron immunoreactive to selected neuronal active substances, including substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL), vesicular acetylcholine transporter (VAChT-used here as a marker of cholinergic neurons), and cocaine- and amphetamine-regulated transcript peptide (CART), was studied by the double immunofluorescence method. Both doses of BPA affected the neurochemical characterization of the enteric neurons. The observed changes depended on the type of enteric plexus but were generally characterized by an increase in the number of cells immunoreactive to the particular substances. More visible fluctuations were observed after treatment with higher doses of BPA. The results confirm that even low doses of BPA may influence the neurochemical characterization of the enteric neurons and are not neutral for living organisms., Competing Interests: The authors declare that they have no conflict of interest.