The enteric nervous system (ENS) consists of an extensive network of neurons and glial cells embedded within the wall of the gastrointestinal (GI) tract. Alterations in neuronal distribution and function are strongly associated with GI dysfunction. Current methods for assessing neuronal distribution suffer from undersampling, partly due to challenges associated with imaging and analyzing large tissue areas, and operator bias due to manual analysis. We present the Gut Analysis Toolbox (GAT), an image analysis tool designed for characterization of enteric neurons and their neurochemical coding using 2D images of GI wholemount preparations. It is developed in Fiji, has a user-friendly interface and offers rapid and accurate segmentation via custom deep learning (DL) based cell segmentation models developed using StarDist, and a ganglion segmentation model in deepImageJ. We use proximal neighbor-based spatial analysis to reveal differences in cellular distribution across gut regions using a public dataset. In summary, GAT provides an easy-to-use toolbox to streamline routine image analysis tasks in ENS research. GAT enhances throughput allowing unbiased analysis of larger tissue areas, multiple neuronal markers and numerous samples rapidly.

Spinal cord injury (SCI) results in numerous systemic dysfunctions, including intestinal dysmotility and enteric nervous system (ENS) atrophy. The ENS has capacity to recover following perturbation, yet intestinal pathologies persist. With emerging evidence demonstrating SCI-induced alterations to gut microbiome composition, we hypothesized that microbiome modulation contributes to post-injury enteric recovery. Here, we show that intervention with the dietary fiber, inulin, prevents SCI-induced ENS atrophy and dysmotility in mice. While SCI-associated microbiomes and specific injury-sensitive gut microbes are not sufficient to modulate intestinal dysmotility after injury, intervention with microbially-derived short-chain fatty acid (SCFA) metabolites prevents ENS dysfunctions in injured mice. Notably, inulin-mediated resilience is dependent on IL-10 signaling, highlighting a critical diet-microbiome-immune axis that promotes ENS resilience post-injury. Overall, we demonstrate that diet and microbially-derived signals distinctly impact ENS survival after traumatic spinal injury and represent a foundation to uncover etiological mechanisms and future therapeutics for SCI-induced neurogenic bowel.

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+/-/Ahrb(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.

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Somatostatin (SST) is a peptide expressed in the peripheral and central nervous systems, as well as in endocrine and immune cells. The aim of the current study is to determine the percentage of SST immunoreactive (IR) neurons and their colocalization with choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and glial fibrillary acidic protein (GFAP) in the myenteric plexus (MP) and submucous plexus (SP) of the small intestine (SI) and large intestine (LI) of rats across different age groups from newborn to senescence using immunohistochemistry. In the MP of the SI and LI, the percentage of SST-IR neurons significantly increased during early postnatal development from 12 ± 2.4 (SI) and 13 ± 3.0 (LI) in newborn rats to 23 ± 1.5 (SI) and 18 ± 1.6 (LI) in 20-day-old animals, remaining stable until 60 days of age. The proportion of SST-IR cells then decreased in aged 2-year-old animals to 14 ± 2.0 (SI) and 10 ± 2.6 (LI). In the SP, the percentage of SST-IR neurons significantly rose from 22 ± 3.2 (SI) and 23 ± 1.7 (LI) in newborn rats to 42 ± 4.0 in 20-day-old animals (SI) and 32 ± 4.9 in 30-day-old animals (LI), before declining in aged 2-year-old animals to 21 ± 2.6 (SI) and 28 ± 7.4 (LI). Between birth and 60 days of age, 97-98% of SST-IR neurons in the MP and SP colocalized with ChAT in both plexuses of the SI and LI. The percentage of SST/ChAT neurons decreased in old rats to 85 ± 5.0 (SI) and 90 ± 3.8 (LI) in the MP and 89 ± 3.2 (SI) and 89 ± 1.6 (LI) in the SP. Conversely, in young rats, only a few SST-IR neurons colocalized with nNOS, but this percentage significantly increased in 2-year-old rats. The percentage of SST/NPY-IR neurons exhibited considerable variation throughout postnatal development, with no significant differences across different age groups in both the MP and SP of both intestines. No colocalization of SST with GFAP was observed in any of the studied animals. In conclusion, the expression of SST in enteric neurons increases in young rats and decreases in senescence, accompanied by changes in SST colocalization with ChAT and nNOS.

Intestinal helminth infection triggers a type 2 immune response that promotes a \'weep-and sweep\' response characterised by increased mucus secretion and intestinal hypermotility, which function to dislodge the worm from its intestinal habitat. Recent studies have discovered that several other pathogens cause intestinal dysmotility through major alterations to the immune and enteric nervous systems (ENS), and their interactions, within the gastrointestinal tract. However, the involvement of these systems has not been investigated for helminth infections. Eosinophils represent a key cell type recruited by the type 2 immune response and alter intestinal motility under steady-state conditions. Our study aimed to investigate whether altered intestinal motility driven by the murine hookworm, Nippostrongylus brasiliensis, infection involves eosinophils and how the ENS and smooth muscles of the gut are impacted. Eosinophil deficiency did not influence helminth-induced intestinal hypermotility and hypermotility did not involve gross structural or functional changes to the ENS. Hypermotility was instead associated with a dramatic increase in smooth muscle thickness and contractility, an observation that extended to another rodent nematode, Heligmosomoides polygyrus. In summary our data indicate that, in contrast to other pathogens, helminth-induced intestinal hypermotility is driven by largely by myogenic, rather than neurogenic, alterations with such changes occurring independently of eosinophils. (<300 words).

This study aimed to investigate the antioxidant and anti-inflammatory properties of quercetin on the cellular components of the Enteric Nervous System in the ileum of rats with arthritis. Rats were distributed into five groups: control (C), arthritic (AIA), arthritic treated with ibuprofen (AI), arthritic treated with quercetin (AQ) and arthritic treated with both ibuprofen and quercetin (AIQ). The ileum was processed for immunohistochemical techniques for HuC/D, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. Measurements in histological sections, chemiluminescence assays, and total antioxidant capacity were also performed. Rheumatoid arthritis resulted in a decrease in neuronal density, yet neuroplasticity mechanisms were evident through observed changes in varicosities size and neuronal area compared to the control group. Reduced paw edema and neuroprotective effects were predominantly noted in both plexuses, as evidenced by the increased density preservation of HuC/D-IR neurons in the AIQ group. The increase of lipoperoxidation levels and paw edema volume in the AQ group was observed compared to the arthritic, whereas the AIQ group mainly showed similar results to those observed in the control. The enteropathy associated with arthritis proved to be significant in the field of gastroenterology, and the combination of quercetin and ibuprofen demonstrated promising anti-inflammatory and neuroprotective effects.

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UNASSIGNED: Enteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed.
UNASSIGNED: We examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3\' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice.
UNASSIGNED: We identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1β treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation.
UNASSIGNED: Together, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.

BACKGROUND: Spontaneous neuronal network activity is essential to the functional maturation of central and peripheral circuits, yet whether this is a feature of enteric nervous system development has yet to be established. Although enteric neurons are known exhibit electrophysiological properties early in embryonic development, no connection has been drawn between this neuronal activity and the development of gastrointestinal (GI) motility patterns.
METHODS: We use ex vivo GI motility assays with newly developed unbiased computational analyses to identify GI motility patterns across mouse embryonic development.
RESULTS: We find a previously unknown pattern of neurogenic contractions termed \"clustered ripples\" that arises spontaneously at embryonic day 16.5, an age earlier than any identified mature GI motility patterns. We further show that these contractions are driven by nicotinic cholinergic signaling.
CONCLUSIONS: Clustered ripples are neurogenic contractile activity that arise from spontaneous ENS activity and precede all known forms of neurogenic GI motility. This earliest motility pattern requires nicotinic cholinergic signaling, which may inform pharmacology for enhancing GI motility in preterm infants.