Mature enteric neurons have the capacity to reinnervate the intestine with glial cells as their guide.

Here, we establish that plasticity exists within the postnatal enteric nervous system by demonstrating the reinnervation potential of post-mitotic enteric neurons (ENs). Employing BAF53b-Cre mice for selective neuronal tracing, the reinnervation capabilities of mature postnatal ENs are shown across multiple model systems. Isolated ENs regenerate neurites in vitro, with neurite complexity and direction influenced by contact with enteric glial cells (EGCs). Nerve fibers from transplanted ENs exclusively interface and travel along EGCs within the muscularis propria. Resident EGCs persist after Cre-dependent ablation of ENs and govern the architecture of the myenteric plexus for reinnervating ENs, as shown by nerve fiber projection tracing. Transplantation and optogenetic experiments in vivo highlight the rapid reinnervation potential of post-mitotic neurons, leading to restored gut muscle contractile activity within 2 weeks. These studies illustrate the structural and functional reinnervation capacity of post-mitotic ENs and the critical role of EGCs in guiding and patterning their trajectories.

An iPSC-derived small intestine-on-chip with self-organizing epithelial, mesenchymal, and neural cells.

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids are valuable tools for researching developmental biology and personalized therapies, but their closed topology and relative immature state limit applications. Here, we use organ-on-chip technology to develop a hiPSC-derived intestinal barrier with apical and basolateral access in a more physiological in vitro microenvironment. To replicate growth factor gradients along the crypt-villus axis, we locally expose the cells to expansion and differentiation media. In these conditions, intestinal epithelial cells self-organize into villus-like folds with physiological barrier integrity, and myofibroblasts and neurons emerge and form a subepithelial tissue in the bottom channel. The growth factor gradients efficiently balance dividing and mature cell types and induce an intestinal epithelial composition, including absorptive and secretory lineages, resembling the composition of the human small intestine. This well-characterized hiPSC-derived intestine-on-chip system can facilitate personalized studies on physiological processes and therapy development in the human small intestine.

Electroacupuncture alleviates intestinal inflammation via a distinct neuro-immune signal pathway in the treatment of postoperative ileus.

BACKGROUND: The induction of intestinal inflammation as a result of abdominal surgery is an essential factor in postoperative ileus (POI) development. Electroacupuncture (EA) at ST36 has been demonstrated to relieve intestinal inflammation and restore gastrointestinal dysmotility in POI. This study aims to elucidate the neuroimmune pathway involved in the anti-inflammatory properties of EA in POI. METHODS: After intestinal manipulation (IM) was performed to induce POI, intestinal inflammation and motility were assessed 24 h post-IM, by evaluating gastrointestinal transit (GIT), cytokines expression, and leukocyte infiltration. Experimental surgery, pharmacological intervention, and genetic knockout mice were used to elucidate the neuroimmune mechanisms of EA. RESULTS: EA at ST36 significantly improved GIT and reduced the expression of pro-inflammatory cytokines and leukocyte infiltration in the intestinal muscularis following IM in mice. The anti-inflammatory effectiveness of EA treatment was abolished by sub-diaphragmatic vagotomy, whereas splenectomy did not hinder the anti-inflammatory benefits of EA treatment. The hexamethonium chloride (HEX) administration contributes to a notable reduction in the EA capacity to suppress inflammation and enhance motility dysfunction, and EA is ineffective in α7 nicotinic acetylcholine receptor (α7nAChR) knockout mice. CONCLUSIONS: EA at ST36 prevents intestinal inflammation and dysmotility through a neural circuit that requires vagal innervation but is independent of the spleen. Further findings revealed that the process involves enteric neurons mediating the vagal signal and requires the presence of α7nAChR. These findings suggest that utilizing EA at ST36 may represent a possible therapeutic approach for POI and other immune-related gastrointestinal diseases.

Developmental role of macrophages modeled in human pluripotent stem cell-derived intestinal tissue.

Macrophages populate the embryo early in gestation, but their role in development is not well defined. In particular, specification and function of macrophages in intestinal development remain little explored. To study this event in the human developmental context, we derived and combined human intestinal organoid and macrophages from pluripotent stem cells. Macrophages migrate into the organoid, proliferate, and occupy the emerging microanatomical niches of epithelial crypts and ganglia. They also acquire a transcriptomic profile similar to that of fetal intestinal macrophages and display tissue macrophage behaviors, such as recruitment to tissue injury. Using this model, we show that macrophages reduce glycolysis in mesenchymal cells and limit tissue growth without affecting tissue architecture, in contrast to the pro-growth effect of enteric neurons. In short, we engineered an intestinal tissue model populated with macrophages, and we suggest that resident macrophages contribute to the regulation of metabolism and growth of the developing intestine.

The Oxidative Stress and Nervous Distress Connection in Gastrointestinal Disorders.

Oxidative stress is increasingly recognized as a central player in a range of gastrointestinal (GI) disorders, as well as complications stemming from therapeutic interventions. This article presents an overview of the mechanisms of oxidative stress in GI conditions and highlights a link between oxidative insult and disruption to the enteric nervous system (ENS), which controls GI functions. The dysfunction of the ENS is characteristic of a spectrum of disorders, including neurointestinal diseases and conditions such as inflammatory bowel disease (IBD), diabetic gastroparesis, and chemotherapy-induced GI side effects. Neurons in the ENS, while essential for normal gut function, appear particularly vulnerable to oxidative damage. Mechanistically, oxidative stress in enteric neurons can result from intrinsic nitrosative injury, mitochondrial dysfunction, or inflammation-related pathways. Although antioxidant-based therapies have shown limited efficacy, recognizing the multifaceted role of oxidative stress in GI diseases offers a promising avenue for future interventions. This comprehensive review summarizes the literature to date implicating oxidative stress as a critical player in the pathophysiology of GI disorders, with a focus on its role in ENS injury and dysfunction, and highlights opportunities for the development of targeted therapeutics for these diseases.

Anti-Hyperalgesic Efficacy of Acetyl L-Carnitine (ALCAR) Against Visceral Pain Induced by Colitis: Involvement of Glia in the Enteric and Central Nervous System.

The management of abdominal pain in patients affected by inflammatory bowel diseases (IBDs) still represents a problem because of the lack of effective treatments. Acetyl L-carnitine (ALCAR) has proved useful in the treatment of different types of chronic pain with excellent tolerability. The present work aimed at evaluating the anti-hyperalgesic efficacy of ALCAR in a model of persistent visceral pain associated with colitis induced by 2,4-dinitrobenzene sulfonic acid (DNBS) injection. Two different protocols were applied. In the preventive protocol, ALCAR was administered daily starting 14 days to 24 h before the delivery of DNBS. In the interventive protocol, ALCAR was daily administered starting the same day of DNBS injection, and the treatment was continued for 14 days. In both cases, ALCAR significantly reduced the establishment of visceral hyperalgesia in DNBS-treated animals, though the interventive protocol showed a greater efficacy than the preventive one. The interventive protocol partially reduced colon damage in rats, counteracting enteric glia and spinal astrocyte activation resulting from colitis, as analyzed by immunofluorescence. On the other hand, the preventive protocol effectively protected enteric neurons from the inflammatory insult. These findings suggest the putative usefulness of ALCAR as a food supplement for patients suffering from IBDs.

GABAergic signaling between enteric neurons and intestinal smooth muscle promotes innate immunity and gut defense in Caenorhabditis elegans.

The nervous system is critical for intestinal homeostasis and function, but questions remain regarding its impact on gut immune defense. By screening the major neurotransmitters of C. elegans, we found that γ-aminobutyric acid (GABA) deficiency enhanced susceptibility to pathogenic Pseudomonas aeruginosa PA14 infection. GABAergic signaling between enteric neurons and intestinal smooth muscle promoted gut defense in a PMK-1/p38-dependent, but IIS/DAF-16- and DBL-1/TGF-ß-independent, pathway. Transcriptomic profiling revealed that the neuropeptide, FLP-6, acted downstream of enteric GABAergic signaling. Further data determined that FLP-6 was expressed and secreted by intestinal smooth muscle cells and functioned as a paracrine molecule on the intestinal epithelium. FLP-6 suppressed the transcription factors ZIP-10 and KLF-1 that worked in parallel and converged to the PMK-1/p38 pathway in the intestinal epithelia for innate immunity and gut defense. Collectively, these findings uncover an enteric neuron-muscle-epithelium axis that may be evolutionarily conserved in higher organisms.

Mini-review: Intercellular communication between enteric glia and neurons.

The enteric nervous system is a dense network of enteric neurons and glia housed in the gastrointestinal tract. This system is responsible for performing several functions that enable digestion as well as maintaining gut homeostasis through diverse signaling processes including those that arise from interactions with the immune system. Bidirectional communication between enteric neurons and enteric glia has gained increased attention for playing essential roles in enteric nervous system function. Neuronal mediators such as neurotransmitters stimulate enteric glia and subsequent gliotransmission processes refine neuronal signaling during intestinal motor control. In this mini-review, we present and discuss the basis of intercellular signaling between neurons and glia in the enteric nervous system and the relevance of these interactions to gut function.

Who's talking to whom: microbiome-enteric nervous system interactions in early life.

The enteric nervous system (ENS) is the intrinsic nervous system of the gastrointestinal tract (GI) and regulates important GI functions, including motility, nutrient uptake, and immune response. The development of the ENS begins during early organogenesis and continues to develop once feeding begins, with ongoing plasticity into adulthood. There has been increasing recognition that the intestinal microbiota and ENS interact during critical periods, with implications for normal development and potential disease pathogenesis. In this review, we focus on insights from mouse and zebrafish model systems to compare and contrast how each model can serve in elucidating the bidirectional communication between the ENS and the microbiome. At the end of this review, we further outline implications for human disease and highlight research innovations that can lead the field forward.

LRRK2 expression in normal and pathologic human gut and in rodent enteric neural cell lines.

Leucine-rich repeat kinase 2 (LRRK2) gene, which is the gene most commonly associated with Parkinson's disease (PD), is also a susceptibility gene for Crohn's disease, thereby suggesting that LRRK2 may sit at the crossroads of gastrointestinal inflammation, Parkinson's, and Crohn's disease. LRRK2 protein has been studied intensely in both CNS neurons and in immune cells, but there are only few studies on LRRK2 in the enteric nervous system (ENS). LRRK2 is present in ENS ganglia and the existing studies on LRRK2 expression in colonic biopsies from PD subjects have yielded conflicting results. Herein, we propose to extend these findings by studying in more details LRRK2 expression in the ENS. LRRK2 expression was evaluated in full thickness segments of colon of 16 Lewy body, 12 non-Lewy body disorders cases, and 3 non-neurodegenerative controls and in various enteric neural cell lines. We showed that, in addition to enteric neurons, LRRK2 is constitutively expressed in enteric glial cells in both fetal and adult tissues. LRRK2 immunofluorescence intensity in the myenteric ganglia was not different between Lewy body and non-Lewy body disorders. Additionally, we identified the cAMP pathway as a key signaling pathway involved in the regulation of LRRK2 expression and phosphorylation in the enteric glial cells. Our study is the first detailed characterization of LRRK2 in the ENS and the first to show that enteric glial cells express LRRK2. Our findings provide a basis to unravel the functions of LRRK2 in the ENS and to further investigate the pathological changes in enteric synucleinopathies.

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system.

The vertebrate enteric nervous system (ENS) is a crucial network of enteric neurons and glia resident within the entire gastrointestinal tract (GI). Overseeing essential GI functions such as gut motility and water balance, the ENS serves as a pivotal bidirectional link in the gut-brain axis. During early development, the ENS is primarily derived from enteric neural crest cells (ENCCs). Disruptions to ENCC development, as seen in conditions like Hirschsprung disease (HSCR), lead to absence of ENS in the GI, particularly in the colon. In this study, using zebrafish, we devised an in vivo F0 CRISPR-based screen employing a robust, rapid pipeline integrating single-cell RNA sequencing, CRISPR reverse genetics, and high-content imaging. Our findings unveil various genes, including those encoding for opioid receptors, as possible regulators of ENS establishment. In addition, we present evidence that suggests opioid receptor involvement in neurochemical coding of the larval ENS. In summary, our work presents a novel, efficient CRISPR screen targeting ENS development, facilitating the discovery of previously unknown genes, and increasing knowledge of nervous system construction.

A New Transgenic Tool to Study the Ret Signaling Pathway in the Enteric Nervous System.

The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g., Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent, allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in females and males. In homozygous mutants of ret and sox10-another important ENS developmental regulator gene-GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood.

Neurons, Macrophages, and Glia: The Role of Intercellular Communication in the Enteric Nervous System.

Neurons of the enteric nervous system (ENS) are the primary controllers of gastrointestinal functions. Although the ENS has been the central focus of research areas such as motility, this has now expanded to include the modulatory roles that non-neuronal cells have on neuronal function. This review discusses how enteric glia (EGC) and resident muscularis macrophages (mMacs) influence ENS communication. It highlights how the understanding of neuroglia interactions has extended beyond EGCs responding to exogenously applied neurotransmitters. Proposed mechanisms for neuron-EGC and glio-glia communication are discussed. The significance of these interactions is evidenced by gut functions that rely on these processes. mMacs are commonly known for their roles as immune cells which sample and respond to changes in the tissue environment. However, a more recent theory suggests that mMacs and enteric neurons are mutually dependent for their maintenance and function. This review summarizes the supportive and contradictory evidence for this theory, including potential mechanisms for mMac-neuron interaction. The need for a more thorough classification scheme to define how the "state" of mMacs relates to neuron loss or impaired function in disease is discussed. Despite the growing literature suggesting EGCs and mMacs have supportive or modulatory roles in ENS communication and gut function, conflicting evidence from different groups suggests more investigation is required. A broader understanding of why enteric neurons may need assistance from EGCs and mMacs in neurotransmission is still missing.

Central and Enteric Neuroprotective Effects by Eucommia ulmoides Extracts on Neurodegeneration in Rotenone-induced Parkinsonian Mouse.

Parkinson's disease (PD) is a progressive neurodegenerative disease of both the central and peripheral / enteric nervous systems. Oxidative stress and neuroinflammation are associated with the pathogenesis of PD, suggesting that anti-oxidative and anti-inflammatory compounds could be neuroprotective agents for PD. Eucommia ulmoides (EU) is a traditional herbal medicine which exerts neuroprotective effects by anti-inflammatory and anti-oxidative properties. Our previous study showed that treatment with chlorogenic acid, a component of EU, protected against neurodegeneration in the central and enteric nervous systems in a PD model. In this study, we examined the effects of EU extract (EUE) administration on dopaminergic neurodegeneration, glial response and α-synuclein expression in the substantia nigra pars compacta (SNpc), and intestinal enteric neurodegeneration in low-dose rotenone-induced PD model mice. Daily oral administration of EUE ameliorated dopaminergic neurodegeneration and α-synuclein accumulation in the SNpc. EUE treatment inhibited rotenone-induced decreases in the number of total astrocytes and in those expressing the antioxidant molecule metallothionein. EUE also prevented rotenone-induced microglial activation. Furthermore, EUE treatment exerted protective effects against intestinal neuronal loss in the PD model. These results suggest that EU exerts neuroprotective effects in the central and enteric nervous systems of rotenone-induced parkinsonism mice, in part by glial modification.

P2X7 receptor blockade decreases inflammation, apoptosis, and enteric neuron loss during Clostridioides difficile toxin A-induced ileitis in mice.

BACKGROUND: Clostridioides difficile (C. difficile) is the most common pathogen causing health care-associated infections. C. difficile TcdA and TcdB have been shown to activate enteric neurons; however, what population of these cells is more profoundly influenced and the mechanism underlying these effects remain unknown. AIM: To characterize a specific population of TcdA-affected myenteric neurons and investigate the role of the P2X7 receptor in TcdA-induced ileal inflammation, cell death, and the changes in the enteric nervous system in mice. METHODS: Swiss mice were used to model TcdA-induced ileitis in ileal loops exposed to TcdA (50 µg/Loop) for 4 h. To investigate the role of the P2X7 receptor, Brilliant Blue G (50 mg/kg, i.p.), which is a nonspecific P2X7 receptor antagonist, or A438079 (0.7 µg/mouse, i.p.), which is a competitive P2X7 receptor antagonist, were injected one hour prior to TcdA challenge. Ileal samples were collected to analyze the expression of the P2X7 receptor (by quantitative real-time polymerase chain reaction and immunohistochemistry), the population of myenteric enteric neurons (immunofluorescence), histological damage, intestinal inflammation, cell death (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling), neuronal loss, and S100B synthesis (immunohistochemistry). RESULTS: TcdA upregulated (P < 0.05) the expression of the P2X7 receptor gene in the ileal tissues, increasing the level of this receptor in myenteric neurons compared to that in control mice. Comparison with the control mice indicated that TcdA promoted (P < 0.05) the loss of myenteric calretinin+ (Calr) and choline acetyltransferase+ neurons and increased the number of nitrergic+ and Calr+ neurons expressing the P2X7 receptor. Blockade of the P2X7 receptor decreased TcdA-induced intestinal damage, cytokine release [interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-α], cell death, enteric neuron loss, and S100B synthesis in the mouse ileum. CONCLUSION: Our findings demonstrated that TcdA induced the upregulation of the P2X7 receptor, which promoted enteric neuron loss, S100B synthesis, tissue damage, inflammation, and cell death in the mouse ileum. These findings contribute to the future directions in understanding the mechanism involved in intestinal dysfunction reported in patients after C. difficile infection.

Histomorphology of enteric neurons and enteric ganglia in different layers of human fetal colon.

Objective: Literature shows very few studies explaining morphology of enteric neurons and ganglia in humans. This study was aimed at determining the morpho-histology of enteric neurons and ganglia in human fetal colon. Methods: Histological sections of human fetal colon were stained with hematoxylin and eosin, Bielschowsky's silver and Masson's trichrome stains to study the morpho-histology of enteric neurons and ganglia. Results: Enteric neurons scattered in the early weeks of development and ganglionated as the fetal age progresses. Migration of enteric neurons was less and in scattered form during early weeks and as the age progresses it was more and in ganglionated form. Enteric neurons were round, oval, pyramidal and flat in all layers of colon. Enteric ganglia in serosa were oval in early weeks, oval and elongated in late weeks whereas in between the muscle layers and submucosa they were few and oval, irregular and elongated. Distance between the enteric ganglia increased in serosa but fluctuated in the remaining layers as the gestational age progressed. Number of enteric neurons and ganglia was more in serosa and less in other layers during early weeks and as the fetal age progressed they decreased in serosa but increased in other layers. Conclusion: There are various shapes and numbers of enteric neurons and ganglia and distances between the ganglia in different layers of fetal colon.

A Critical Analysis of Intestinal Enteric Neuron Loss and Constipation in Parkinson's Disease.

Constipation afflicts many patients with Parkinson's disease (PD) and significantly impacts on patient quality of life. PD-related constipation is caused by intestinal dysfunction, but the etiology of this dysfunction in patients is unknown. One possible cause is neuron loss within the enteric nervous system (ENS) of the intestine. This review aims to 1) Critically evaluate the evidence for and against intestinal enteric neuron loss in PD patients, 2) Justify why PD-related constipation must be objectively measured, 3) Explore the potential link between loss of enteric neurons in the intestine and constipation in PD, 4) Provide potential explanations for disparities in the literature, and 5) Outline data and study design considerations to improve future research. Before the connection between intestinal enteric neuron loss and PD-related constipation can be confidently described, future research must use sufficiently large samples representative of the patient population (majority diagnosed with idiopathic PD for at least 5 years), implement a consistent neuronal quantification method and study design, including standardized patient recruitment criteria, objectively quantify intestinal dysfunctions, publish with a high degree of data transparency and account for potential PD heterogeneity. Further investigation into other potential influencers of PD-related constipation is also required, including changes in the function, connectivity, mitochondria and/or α-synuclein proteins of enteric neurons and their extrinsic innervation. The connection between enteric neuron loss and other PD-related gastrointestinal (GI) issues, including gastroparesis and dysphagia, as well as changes in nutrient absorption and the microbiome, should be explored in future research.

Gastrointestinal Changes and Alzheimer's Disease.

BACKGROUND: There is a well-described mechanism of communication between the brain and gastrointestinal system in which both organs influence the function of the other. This bi-directional communication suggests that disease in either organ may affect function in the other. OBJECTIVE: To assess whether the evidence supports gastrointestinal system inflammatory or degenerative pathophysiology as a characteristic of Alzheimer's disease (AD). METHODS: A review of both rodent and human studies implicating gastrointestinal changes in AD was performed. RESULTS: Numerous studies indicate that AD changes are not unique to the brain but also occur at various levels of the gastrointestinal tract involving both immune and neuronal changes. In addition, it appears that numerous conditions and diseases affecting regions of the tract may communicate to the brain to influence disease. CONCLUSION: Gastrointestinal changes represent an overlooked aspect of AD, representing a more system influence of this disease.

A Gut Feeling in Amyotrophic Lateral Sclerosis: Microbiome of Mice and Men.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating disease characterized by progressive degeneration of motor neurons. ALS etiology and pathophysiology are not well understood. It could be the consequences of complex interactions among host factors, microbiome, and the environmental factors. Recent data suggest the novel roles of intestinal dysfunction and microbiota in ALS etiology and progression. Although microbiome may indeed play a critical role in ALS pathogenesis, studies implicating innate immunity and intestinal changes in early disease pathology are limited. The gastrointestinal symptoms in the ALS patients before their diagnosis are largely ignored in the current medical practice. This review aims to explore existing evidence of gastrointestinal symptoms and progress of microbiome in ALS pathogenesis from human and animal studies. We discuss dietary, metabolites, and possible therapeutic approaches by targeting intestinal function and microbiome. Finally, we evaluate existing evidence and identify gaps in the knowledge for future directions in ALS. It is essential to understanding the microbiome and intestinal pathogenesis that determine when, where, and whether microbiome and metabolites critical to ALS progression. These studies will help us to develop more accurate diagnosis and better treatment not only for this challenging disease, but also for other neurodegenerative diseases.

The RET gene encodes RET protein, which triggers intracellular signaling pathways for enteric neurogenesis, and RET mutation results in Hirschsprung's disease.

Enteric neurons and ganglia are derived from vagal and sacral neural crest cells, which undergo migration from the neural tube to the gut wall. In the gut wall, they first undergo rostrocaudal migration followed by migration from the superficial to deep layers. After migration, they proliferate and differentiate into the enteric plexus. Expression of the Rearranged During Transfection (RET) gene and its protein RET plays a crucial role in the formation of enteric neurons. This review describes the molecular mechanism by which the RET gene and the RET protein influence the development of enteric neurons. Vagal neural crest cells give rise to enteric neurons and glia of the foregut and midgut while sacral neural crest cells give rise to neurons of the hindgut. Interaction of RET protein with its ligands (glial cell derived neurotrophic factor (GDNF), neurturin (NRTN), and artemin (ARTN)) and its co-receptors (GDNF receptor alpha proteins (GFRα1-4)) activates the Phosphoinositide-3-kinase-protein kinase B (PI3K-PKB/AKT), RAS mitogen-activated protein kinase (RAS/MAPK) and phospholipase Cγ (PLCγ) signaling pathways, which control the survival, migration, proliferation, differentiation, and maturation of the vagal and sacral neural crest cells into enteric neurons. Abnormalities of the RET gene result in Hirschsprung's disease.