In vivo imaging of vagal-induced myenteric plexus responses in gastrointestinal tract with an optical window.

The vagus nerve (VN) extensively innervates the gastric enteric nervous system (ENS), but its influence on gastric ENS functionality and motility in vivo remains unclear due to technical challenges. Here we describe a method for stable, long-term observation of gastric ENS activity and muscle dynamics at cellular resolution, which can also be extended to intestinal applications. This method involves ENS-specific labeling and the implantation of an abdominal wall window for optical recording in male mice. In vivo calcium imaging reveals a linear relationship between vagal stimulation frequency and myenteric neuron activation in gastric antrum. Furthermore, the motility of gastric antrum is significantly enhanced and shows a positive correlation with the intensity and number of activated myenteric neurons. While vagal stimulation also activates proximal colonic myenteric neurons, this activation is not frequency-dependent and does not induce proximal colonic motility. The method and results provide important insights into VN-ENS interactions in vivo, advancing our understanding of gastrointestinal motility regulation.

Microbiota-gut-brain axis in health and neurological disease: Interactions between gut microbiota and the nervous system.

Along with mounting evidence that gut microbiota and their metabolites migrate endogenously to distal organs, the 'gut-lung axis,' 'gut-brain axis,' 'gut-liver axis' and 'gut-renal axis' have been established. Multiple animal recent studies have demonstrated gut microbiota may also be a key susceptibility factor for neurological disorders such as Alzheimer's disease, Parkinson's disease and autism. The gastrointestinal tract is innervated by the extrinsic sympathetic and vagal nerves and the intrinsic enteric nervous system, and the gut microbiota interacts with the nervous system to maintain homeostatic balance in the host gut. A total of 1507 publications on the interactions between the gut microbiota, the gut-brain axis and neurological disorders are retrieved from the Web of Science to investigate the interactions between the gut microbiota and the nervous system and the underlying mechanisms involved in normal and disease states. We provide a comprehensive overview of the effects of the gut microbiota and its metabolites on nervous system function and neurotransmitter secretion, as well as alterations in the gut microbiota in neurological disorders, to provide a basis for the possibility of targeting the gut microbiota as a therapeutic agent for neurological disorders.

Olfactory deficit and gastrointestinal dysfunction precede motor abnormalities in alpha-Synuclein G51D knock-in mice.

Parkinson's disease (PD) is typically a sporadic late-onset disorder, which has made it difficult to model in mice. Several transgenic mouse models bearing mutations in SNCA, which encodes alpha-Synuclein (α-Syn), have been made, but these lines do not express SNCA in a physiologically accurate spatiotemporal pattern, which limits the ability of the mice to recapitulate the features of human PD. Here, we generated knock-in mice bearing the G51D SNCA mutation. After establishing that their motor symptoms begin at 9 mo of age, we then sought earlier pathologies. We assessed the phosphorylation at Serine 129 of α-Syn in different tissues and detected phospho-α-Syn in the olfactory bulb and enteric nervous system at 3 mo of age. Olfactory deficit and impaired gut transit followed at 6 mo, preceding motor symptoms. The SncaG51D mice thus parallel the progression of human PD and will enable us to study PD pathogenesis and test future therapies.

Effects of exogenous hydrogen sulfide and honokiol intervention on the proliferation, apoptosis, and calcium signaling pathway of rat enteric glial cells.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that influences digestive and nervous system functions. Enteric glial cells (EGCs) are integral to the enteric nervous system and play a role in regulating gastrointestinal motility. This study explored the dual effects of exogenous H2S on EGCs and the influence of apoptosis-related pathways and ion channels in EGCs. We also administered honokiol for further interventional studies. The results revealed that low-concentration H2S increased the mitochondrial membrane potential (MMP) of EGCs, decreased the whole-cell membrane potential, downregulated BAX and caspase-3, upregulated Bcl2 expression, reduced apoptosis, and promoted cell proliferation. The Ca2+ concentration, Cx43 mRNA, and protein expression were also increased. A high concentration of H2S had the opposite effect. In addition, GFAP mRNA expression was upregulated in the test-low group, downregulated in the test-high group, and upregulated in the test-high + Hon group. Honokiol treatment increased MMP, reduced whole-cell membrane potential, inhibited BAX and caspase-3 expression, increased Bcl2 expression, decreased cell apoptosis, and increased cell proliferation. The Ca2+ concentration, Cx43 mRNA, and protein expression were also upregulated. In conclusion, our study showed that exogenous H2S can bidirectionally regulate EGC proliferation and apoptosis by affecting MMP and cell membrane potential via the Bcl2/BAX/caspase-3 pathway and modulate Cx43-mediated Ca2+ responses in EGCs to regulate colonic motility bidirectionally. Honokiol can ameliorate the damage to EGCs induced by high H2S concentrations through the Bcl2/BAX/caspase-3 pathway and improve colon motility by increasing Cx43 expression and Ca2+ concentration.

Distribution and ultrastructural characteristics of enteric glial cell in the chicken cecum.

Enteric glial cell (EGC) is involved in neuroimmune regulation within the enteric nervous system (ENS); however, limited information exists on the distribution and ultrastructure of EGC in the poultry gut. We aim to investigate the morphological features and distribution of EGC in the chicken cecum. Here, we investigated the distribution and ultrastructural features of chicken cecum EGC using immunohistochemistry (IHC) and transmission electron microscopy (TEM). IHC showed that EGC was widely distributed throughout the chicken cecum. In the mucosal layer, EGC was morphologically irregular, with occasionally interconnecting protrusions that outlined signal-negative neurons. The morphology of EGC in the submucosal layer was also irregular. In the inner circular muscle layer and between the inner circular and outer longitudinal muscle layers, EGC aligned parallel to the circular muscle cells. A small number of EGC with an irregular morphology were found in the outer longitudinal muscle layer. In addition, in the submucosal and myenteric plexus, EGC were aggregated, and the protrusions of the immunoreactive cells interconnected to outline the bodies of nonreactive neurons. TEM-guided ultrastructural characterization confirmed the IHC findings that EGC were morphologically irregular and revealed they developed either a star, bipolar, or fibrous shape. The nucleus was also irregular, with electron-dense heterochromatin distributed in the center of the nucleus or on the nuclear membrane. The cytoplasm contained many glial filaments and vesicle-containing protrusions from neuronal cells; organelles were rare. EGC was in close contact with other cells in their vicinity. These findings suggest that EGC is well-situated to exert influence on intestinal motility and immune functions through mechanical contraction and chemical secretion.

Diet-microbiome interactions promote enteric nervous system resilience following spinal cord injury.

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.

Modulation of Ceramide-Induced Apoptosis in Enteric Neurons by Aryl Hydrocarbon Receptor Signaling: Unveiling a New Pathway beyond ER Stress.

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-3ß 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.

ELP1, the Gene Mutated in Familial Dysautonomia, Is Required for Normal Enteric Nervous System Development and Maintenance and for Gut Epithelium Homeostasis.

Familial dysautonomia (FD) is a rare sensory and autonomic neuropathy that results from a mutation in the ELP1 gene. Virtually all patients report gastrointestinal (GI) dysfunction and we have recently shown that FD patients have a dysbiotic gut microbiome and altered metabolome. These findings were recapitulated in an FD mouse model and moreover, the FD mice had reduced intestinal motility, as did patients. To understand the cellular basis for impaired GI function in FD, the enteric nervous system (ENS; both female and male mice) from FD mouse models was analyzed during embryonic development and adulthood. We show here that not only is Elp1 required for the normal formation of the ENS, but it is also required in adulthood for the regulation of both neuronal and non-neuronal cells and for target innervation in both the mucosa and in intestinal smooth muscle. In particular, CGRP innervation was significantly reduced as was the number of dopaminergic neurons. Examination of an FD patient's gastric biopsy also revealed reduced and disoriented axons in the mucosa. Finally, using an FD mouse model in which Elp1 was deleted exclusively from neurons, we found significant changes to the colon epithelium including reduced E-cadherin expression, perturbed mucus layer organization, and infiltration of bacteria into the mucosa. The fact that deletion of Elp1 exclusively in neurons is sufficient to alter the intestinal epithelium and perturb the intestinal epithelial barrier highlights a critical role for neurons in regulating GI epithelium homeostasis.

Quercetin and ibuprofen combination displayed anti-inflammatory effects and also extenuates the enteric neurons damage of arthritic rats.

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.

Extracellular matrix substrates differentially influence enteric glial cell homeostasis and immune reactivity.

Introduction: 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. Methods: 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. Results: 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-1ß 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. Discussion: 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.

Nobiletin restores HFD-induced enteric nerve injury by regulating enteric glial activation and the GDNF/AKT/FOXO3a/P21 pathway.

BACKGROUND: To explore whether nobiletin has a protective effect on high-fat diet (HFD)-induced enteric nerve injury and its underlying mechanism. METHODS: An obesity model was induced by a HFD. Nobiletin (100 mg/kg and 200 mg/kg) and vehicle were administered by gastric gavage for 4 weeks. Lee's index, body weight, OGTT and intestinal propulsion assays were performed before sacrifice. After sampling, lipids were detected using Bodipy 493/503; lipid peroxidation was detected using MDA and SOD kits and the expression of PGP 9.5, Trem2, GFAP, ß-tubulin 3, Bax, Bcl2, Nestin, P75 NTR, SOX10 and EDU was detected using immunofluorescence. The GDNF, p-AKT, AKT, p-FOXO3a, FOXO3a and P21 proteins were detected using western blotting. The relative mRNA expression levels of NOS2 were detected via qPCR. Primary enteric neural stem cells (ENSCs) were cultured. After ENSCs were treated with palmitic acid (PA) and nobiletin, CCK-8 and caspase-3/7 activity assays were performed to evaluate proliferation and apoptosis. RESULTS: HFD consumption caused colon lipid accumulation and peroxidation, induced enteric nerve damage and caused intestinal motor dysfunction. However, nobiletin reduced lipid accumulation and peroxidation in the colon; promoted Trem2, ß-tubulin 3, Nestin, P75NTR, SOX10 and Bcl2 expression; inhibited Bax and GFAP expression; reduced NOS2 mRNA transcription; and regulated the GDNF/AKT/FOXO3a/P21 pathway. Nobiletin also promoted PA-induced impairment of ENSCs. CONCLUSIONS: Nobiletin restored HFD-induced enteric nerve injury, which may be associated with inhibiting enteric nerve apoptosis, promoting enteric nerve survival and regulating the GDNF/AKT/FOXO3a/P21 pathway.

Helminth infection driven gastrointestinal hypermotility is independent of eosinophils and mediated by alterations in smooth muscle instead of enteric neurons.

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).

Tetrodotoxin-resistant mechanosensitivity and L-type calcium channel-mediated spontaneous calcium activity in enteric neurons.

Gut motility undergoes a switch from myogenic to neurogenic control in late embryonic development. Here, we report on the electrical events that underlie this transition in the enteric nervous system, using the GCaMP6f reporter in neural crest cell derivatives. We found that spontaneous calcium activity is tetrodotoxin (TTX) resistant at stage E11.5, but not at E18.5. Motility at E18.5 was characterized by periodic, alternating high- and low-frequency contractions of the circular smooth muscle; this frequency modulation was inhibited by TTX. Calcium imaging at the neurogenic-motility stages E18.5-P3 showed that CaV1.2-positive neurons exhibited spontaneous calcium activity, which was inhibited by nicardipine and 2-aminoethoxydiphenyl borate (2-APB). Our protocol locally prevented muscle tone relaxation, arguing for a direct effect of nicardipine on enteric neurons, rather than indirectly by its relaxing effect on muscle. We demonstrated that the ENS was mechanosensitive from early stages on (E14.5) and that this behaviour was TTX and 2-APB resistant. We extended our results on L-type channel-dependent spontaneous activity and TTX-resistant mechanosensitivity to the adult colon. Our results shed light on the critical transition from myogenic to neurogenic motility in the developing gut, as well as on the intriguing pathways mediating electro-mechanical sensitivity in the enteric nervous system. HIGHLIGHTS: What is the central question of this study? What are the first neural electric events underlying the transition from myogenic to neurogenic motility in the developing gut, what channels do they depend on, and does the enteric nervous system already exhibit mechanosensitivity? What is the main finding and its importance? ENS calcium activity is sensitive to tetrodotoxin at stage E18.5 but not E11.5. Spontaneous electric activity at fetal and adult stages is crucially dependent on L-type calcium channels and IP3R receptors, and the enteric nervous system exhibits a tetrodotoxin-resistant mechanosensitive response. Abstract figure legend Tetrodotoxin-resistant Ca2+ rise induced by mechanical stimulation in the E18.5 mouse duodenum.

Enteric neural stem cell transplant restores gut motility in mice with Hirschsprung disease.

The goal of this study was to determine if transplantation of enteric neural stem cells (ENSCs) can rescue the enteric nervous system, restore gut motility, reduce colonic inflammation, and improve survival in the Ednrb-KO mouse model of Hirschsprung disease (HSCR). ENSCs were isolated from mouse intestine, expanded to form neurospheres, and microinjected into the colons of recipient Ednrb-KO mice. Transplanted ENSCs were identified in recipient colons as cell clusters in "neo-ganglia." Immunohistochemical evaluation demonstrated extensive cell migration away from the sites of cell delivery and across the muscle layers. Electrical field stimulation and optogenetics showed significantly enhanced contractile activity of aganglionic colonic smooth muscle following ENSC transplantation and confirmed functional neuromuscular integration of the transplanted ENSC-derived neurons. ENSC injection also partially restored the colonic migrating motor complex. Histological examination revealed a significant reduction in inflammation in ENSC-transplanted aganglionic recipient colon compared with that of sham-operated mice. Interestingly, mice that received cell transplant also had prolonged survival compared with controls. This study demonstrates that ENSC transplantation can improve outcomes in HSCR by restoring gut motility and reducing the severity of Hirschsprung-associated enterocolitis, the leading cause of death in human HSCR.

Schwann cells and enteric glial cells: Emerging stars in colorectal cancer.

Cancer neuroscience, a promising field dedicated to exploring interactions between cancer and the nervous system, has attracted growing attention. The gastrointestinal tracts exhibit extensive innervation, notably characterized by intrinsic innervation. The gut harbors a substantial population of glial cells, including Schwann cells wrapping axons of neurons in the peripheral nervous system and enteric glial cells intricately associated with intrinsic innervation. Glial cells play a crucial role in maintaining the physiological functions of the intestine, encompassing nutrient absorption, barrier integrity, and immune modulation. Nevertheless, it has only been in recent times that the significance of glial cells within colorectal cancer (CRC) has begun to receive considerable attention. Emerging data suggests that glial cells in the gut contribute to the progression and metastasis of CRC, by interacting with cancer cells, influencing inflammation, and modulating the tumor microenvironment. Here, we summarize the significant roles of glial cells in the development and progression of CRC and discuss the latest technologies that can be integrated into this field for in-depth exploration, as well as potential specific targeted strategies for future exploration to benefit patients.

Autonomic and Enteric Profiling May Help Predict Response to Diverse Obesity Therapies.

PURPOSE: Changes in autonomic (ANS) and enteric nervous systems (ENS) may be involved in pathogenesis of obesity. We hypothesized that baseline autonomic and enteric parameters may predict outcomes of diverse obesity therapies. MATERIAL AND METHODS: We studied ANS and ENS physiology in 37 patients (8 male, 29 female, age 45 years, weight 129.7 kg) at 4 centers in patients undergoing medical (9: low-calorie diet) versus invasive (22: 16 sleeve, 6 bypass) and semi-invasive (6: 2 band, 2 high energy stimulation, 2 aspiration) weight loss therapies. Weight loss was reported as percent weight loss from baseline to latest values at 1 year and in some up to 5 years; classified as < or > /= 20% for each group. ANS testing included sympathetic adrenergic function by measuring reflex vasoconstriction and postural adjustment ratio. ENS was measured non-invasively using cutaneous low-resolution electrogastrogram. RESULTS: Percent weight loss was greater with the invasive (28.5%) than semi-invasive (9.1%) or non-invasive low-calorie diet (4.4%) (p < .001). Percent weight loss at 1 year (and up to 5 years) corresponded to the adrenergic measure of postural adjustment ratio (r = .42, p = .012), total pulse amplitude at rest (r = .56, p < .001), and electrogastrogram standing-to-rest difference (r = .33, p = .056). CONCLUSION: Baseline autonomic and enteric function measures correspond to percentage with loss in this pilot study using diverse weight loss methods. Autonomic and enteric profiling has potential clinical use for evaluation and treatment of obesity but needed larger controlled trials.

Immune landscape of the enteric nervous system differentiates Parkinson's disease patients from controls: The PADUA-CESNE cohort.

BACKGROUND: Gastrointestinal dysfunction has emerged as a prominent early feature of Parkinson's Disease, shedding new light on the pivotal role of the enteric nervous system in its pathophysiology. However, the role of immune-cell clusters and inflammatory and glial markers in the gut pathogenetic process needs further elucidation. OBJECTIVES: We aimed to study duodenum tissue samples to characterize PD's enteric nervous system pathology further. Twenty patients with advanced PD, six with early PD, and 18 matched controls were included in the PADUA-CESNE cohort. METHODS: Duodenal biopsies from 26 patients with early to advanced stage PD and 18 age-matched HCs were evaluated for the presence of surface markers (CD3+, CD4+, CD8+, CD20+, CD68+, HLA-DR), presence of misfolded alpha-synuclein and enteric glial alteration (GFAP). Correlation of immulogic pattern and clinical characteristic were analyzed. RESULTS: The findings validate that in patients with Parkinson's Disease, the activation and reactive gliosis are linked to the neurodegeneration triggered by the presence of misfolded alpha-synuclein in the enteric nervous system. This process intensifies from the initial to the advanced stages of the disease. The clusters of T- and B-lymphocytes in the enteric system, along with the overall expression of HLA-DR in antigen-presenting cells, exceeded those in the control group. Conversely, no differences in terms of macrophage populations were found. CONCLUSIONS: These findings broaden our understanding of the mechanisms underlying the enteric nervous system's involvement in PD and point to the gastrointestinal system as a potential therapeutic target, especially in the early stages of the disease. Moreover, our results propose a role of T- and B-lymphocytes in maintaining inflammation and ultimately influencing alpha-synuclein misfolding and aggregation.

Moniezia benedeni drives the SNAP-25 expression of the enteric nerves in sheep's small intestine.

BACKGROUND: The neuroimmune network plays a crucial role in regulating mucosal immune homeostasis within the digestive tract. Synaptosome-associated protein 25 (SNAP-25) is a presynaptic membrane-binding protein that activates ILC2s, initiating the host's anti-parasitic immune response. METHODS: To investigate the effect of Moniezia benedeni (M. benedeni) infection on the distribution of SNAP-25 in the sheep's small intestine, the recombinant plasmid pET-28a-SNAP-25 was constructed and expressed in BL21, yielding the recombinant protein. Then, the rabbit anti-sheep SNAP-25 polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of SNAP-25 in the intestines of normal and M. benedeni-infected sheep were detected by ELISA. RESULTS: The results showed that the SNAP-25 recombinant protein was 29.3 KDa, the titer of the prepared immune serum reached 1:128,000. It was demonstrated that the rabbit anti-sheep SNAP-25 polyclonal antibody could bind to the natural protein of sheep SNAP-25 specifically. The expression levels of SNAP-25 in the sheep's small intestine revealed its primary presence in the muscular layer and lamina propria, particularly around nerve fibers surrounding the intestinal glands. Average expression levels in the duodenum, jejunum, and ileum were 130.32 pg/mg, 185.71 pg/mg, and 172.68 pg/mg, respectively. Under conditions of M. benedeni infection, the spatial distribution of SNAP-25-expressing nerve fibers remained consistent, but its expression level in each intestine segment was increased significantly (P < 0.05), up to 262.02 pg/mg, 276.84 pg/mg, and 326.65 pg/mg in the duodenum, jejunum, and ileum, and it was increased by 101.06%, 49.07%, and 89.16% respectively. CONCLUSIONS: These findings suggest that M. benedeni could induce the SNAP-25 expression levels in sheep's intestinal nerves significantly. The results lay a foundation for further exploration of the molecular mechanism by which the gastrointestinal nerve-mucosal immune network perceives parasites in sheep.