BACKGROUND AND PURPOSEGut microbiota is essential for the development of the gastrointestinal system, including the enteric nervous system (ENS). Perturbations of gut microbiota in early life have the potential to alter neurodevelopment leading to functional bowel disorders later in life. We examined the hypothesis that gut dysbiosis impairs the structural and functional integrity of the ENS, leading to gut dysmotility in juvenile mice. EXPERIMENTAL APPROACHTo induce gut dysbiosis, broad-spectrum antibiotics were administered by gavage to juvenile (3weeks old) male C57Bl/6 mice for 14 days. Bile acid composition in the intestinal lumen was analysed by liquid chromatography-mass spectrometry. Changes in intestinal motility were evaluated by stool frequency, transit of a fluorescent-labelled marker and isometric muscle responses of ileal full-thickness preparations to receptor and non-receptor-mediated stimuli. Alterations in ENS integrity were assessed by immunohistochemistry and Western blot analysis. KEY RESULTSAntibiotic treatment altered gastrointestinal transit, luminal bile acid metabolism and bowel architecture. Gut dysbiosis resulted in distorted glial network, loss of myenteric plexus neurons, altered cholinergic, tachykininergic and nitrergic neurotransmission associated with reduced number of nNOS neurons and different ileal distribution of the toll-like receptor TLR2. Functional defects were partly reversed by activation of TLR2 signalling. CONCLUSIONS AND IMPLICATIONSGut dysbiosis caused complex morpho-functional neuromuscular rearrangements, characterized by structural defects of the ENS and increased tachykininergic neurotransmission. Altogether, our findings support the beneficial role of enteric microbiota for ENS homeostasis instrumental in ensuring proper gut neuromuscular function during critical stages of development. Abbreviations

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.

Objective: Toll-like receptors (TLRs) play a pivotal role in the homeostatic microflora-host crosstalk. TLR4-mediated modulation of both motility and enteric neuronal survival has been reported mainly for colon with limited information on the role of TLR4 in tuning structural and functional integrity of enteric nervous system (ENS) and in controlling small bowel motility.Methods: Male TLR4 knockout (TLR4-/-, 9 ± 1 weeks old) and sex- and age-matched wild-type (WT) C57BL/6J mice were used for the experiments. Alterations in ENS morphology and neurochemical code were assessed by immunohistochemistry whereas neuromuscular function was evaluated by isometric mechanical activity of ileal preparations following receptor and non-receptor-mediated stimuli and by gastrointestinal transit.Results: The absence of TLR4 induced gliosis and reduced the total number of neurons, mainly nNOS+ neurons, in ileal myenteric plexus. Furthermore, a lower cholinergic excitatory response with an increased inhibitory neurotransmission was found together with a delayed gastrointestinal transit. These changes were dependent on increased ileal non-adrenergic non-cholinergic (NANC) relaxations mediated by a complex neuronal-glia signaling constituted by P2X7 and P2Y1 receptors, and NO produced by nNOS and iNOS.Conclusion: We provide novel evidence that TLR4 signaling is involved in the fine-tuning of P2 receptors controlling ileal contractility, ENS cell distribution, and inhibitory NANC neurotransmission via the combined action of NO and adenosine-5′-triphosphate (ATP). For the first time, this study implicates TLR4 at regulating the crosstalk between glia and neurons in small intestine and helps to define its role in gastrointestinal motor abnormalities during dysbiosis.

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.

Inflammatory bowel diseases (IBD) are chronic relapsing disorders that have a negative impact on quality of life. They can be highly disabling and have been associated with sleep disturbance. The aim of our study was to evaluate the sleep quality of a large cohort of IBD patients to identify possible associated cofactors. We prospectively recruited consecutive patients attending the IBD Unit of “Azienda Ospedaliera” of Padua from November 2018 to May 2019 and collected demographics and clinical characteristics. The patients completed the Pittsburgh Sleep Quality Index (PSQI), the IBD questionnaire (IBDQ), the IBD-Disability Index (IBD-DI) questionnaire, and the Hospital Anxiety and Depression Scale (9-HADS). A multivariate regression model was applied to assess independent risk factors of sleep disturbance among IBD-related variables, disability, quality of life, anxiety, and depression. We investigated the sleep quality of 166 patients with IBD, finding 67.5% of them suffering from sleep disturbance. In particular, low quality of life, presence of disability and extraintestinal manifestations were identified as independent risk factors of sleep disturbance. We discovered that all depressed patients were also affected by sleep disturbance, while we found no difference in sleep disturbance between patients with or without anxiety state. However, a positive correlation was reported between both anxiety and depression scores and PSQI score (Spearman correlation: r = 0.31 and r = 0.38 respectively). Our study showed that sleep quality is not directly associated with an active or inactive IBD state or with the ongoing treatment, but it is mostly correlated with the patients’ mood state, disability, and quality of life. Gastroenterologists and psychologists should join forces during clinical outpatients’ visits to evaluate emotional states for a better IBD management.

Edible seaweeds have been consumed by Asian coastal communities since ancient times. Fucus vesiculosus and Ascophyllum nodosum extracts have been traditionally used for the treatment of obesity and several gastrointestinal diseases. We evaluated the ability of extracts obtained from these algae to inhibit the digestive enzymes α-amylase and α-glucosidase in vitro, and control postprandial plasma glucose levels in a mouse model of non-alcoholic steatohepatitis (NASH); a liver disease often preceding the development of Type 2 diabetes (T2DM). This model was obtained by the administration of a high-fat diet. Our results demonstrate that these algae only delayed and reduced the peak of blood glucose (p < 0.05) in mice fed with normal diet, without changing the area under the blood glucose curve (AUC). In the model of NASH, the phytocomplex was able to reduce both the postprandial glycaemic peak, and the AUC. The administration of the extract in a diet particularly rich in fat is associated with a delay in carbohydrate digestion, but also with a decrease in its assimilation. In conclusion, our results indicate that this algal extract may be useful in the control of carbohydrate digestion and absorption. This effect may be therapeutically exploited to prevent the transition of NASH to T2DM.

Adenosine A receptors (AR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of AR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic AR localization was examined by immunofluorescence. The role of AR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, AR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the AR antagonist MRS1754 enhanced electrically induced NK-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both AR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of AR expression. AR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.

The identification of AMPK subunits responsible for specific anti-inflammatory actions and the understanding of the underlying molecular mechanisms will promote the generation of novel AMPK activators, endowed with improved pharmacodynamic and pharmacokinetic profiles. These new tools will aid us to utilize AMPK pathway activation in the management of acute and chronic inflammatory diseases, while minimizing potential adverse reactions related to the effects of AMPK on metabolic energy.