Lonicerin alleviates intestinal myenteric neuron injury induced by hypoxia/reoxygenation treated macrophages by downregulating EZH2.

Intestinal ischemia-reperfusion (IR) injury is a common gastrointestinal disease that induces severe intestinal dysfunction. Intestinal myenteric neurons participate in maintaining the intestinal function, which will be severely injured by IR. Macrophages are widely reported to be involved in the pathogenesis of organ IR injury, including intestine, which is activated by NLRP3 signaling. Lonicerin (LCR) is a natural extracted monomer with inhibitory efficacy against the NLRP3 pathway in macrophages. The present study aims to explore the potential protective function of LCR in intestinal IR injury. Myenteric neurons were extracted from mice. RAW 264.7 cells were stimulated by H/R with or without 10 µM and 30 µM LCR. Remarkable increased release of IL-6, MCP-1, and TNF-α were observed in H/R treated RAW 264.7 cells, along with an upregulation of NLRP3, cleaved-caspase-1, IL-1ß, and EZH2, which were sharply repressed by LCR. Myenteric neurons were cultured with the supernatant collected from each group. Markedly decreased neuron number and shortened length of neuron axon were observed in the H/R group, which were signally reversed by LCR. RAW 264.7 cells were stimulated by H/R, followed by incubated with 30 µM LCR with or without pcDNA3.1-EZH2. The inhibition of LCR on NLRP3 signaling in H/R treated RAW 264.7 cells was abolished by EZH2 overexpression. Furthermore, the impact of LCR on neuron number and neuron axon length in myenteric neurons in the H/R group was abated by EZH2 overexpression. Collectively, LCR alleviated intestinal myenteric neuron injury induced by H/R treated macrophages via downregulating EZH2.

The recruitment of mechanosensitive enteric neurons in the guinea pig gastric fundus is dependent on ganglionic stretch level.

BACKGROUND: Serving as a reservoir, the gastric fundus can expand significantly, with an initial receptive and a following adaptive relaxation, controlled by extrinsic and intrinsic reflex circuits, respectively. We hypothesize that mechanosensitive enteric neurons (MEN) are involved in the adaptive relaxation, which is initiated when a particular gastric volume and a certain stretch of the stomach wall is reached. To investigate whether the responsiveness of MEN in the gastric fundus is dependent on tissue stretch, we performed mechanical stimulations in stretched versus ganglia "at rest". METHODS: Responses of myenteric neurons in the guinea pig gastric fundus were recorded with membrane potential imaging using Di-8-ANEPPS. MEN were identified by small-volume intraganglionic injection in ganglia stretched to different degrees using a self-constructed stretching tool. Immunohistochemical staining identified the neurochemical phenotype of MEN. Hexamethonium and capsaicin were added to test their effect on recruited MEN. KEY RESULTS: In stretched compared to "at rest" ganglia, significantly more MEN were activated. The change in the ganglionic area correlated significantly with the number of additional recruited MEN. The additional recruitment of MEN was independent from nicotinic transmission and the ratio of active MEN in stretched ganglia shifted towards a nitrergic phenotype. CONCLUSION AND INFERENCES: The higher number of active MEN with increasing stretch of the ganglia and their greater share of nitrergic phenotype might indicate their contribution to the adaptive relaxation. Further experiments are necessary to address the receptors involved in mechanotransduction.

Accelerated Electron Ionization-Induced Changes in the Myenteric Plexus of the Rat Stomach.

The influence of accelerated electrons on neuronal structures is scarcely explored compared to gamma and X-rays. This study aims to investigate the effects of accelerated electron radiation on some pivotal neurotransmitter circuits (cholinergic and serotonergic) of rats' myenteric plexus. Male Wistar rats were irradiated with an electron beam (9 MeV, 5 Gy) generated by a multimodality linear accelerator. The contractile activity of isolated smooth muscle samples from the gastric corpus was measured. Furthermore, an electrical stimulation (200 µs, 20 Hz, 50 s, 60 V) was performed on the samples and an assessment of the cholinergic and serotonergic circuits was made. Five days after irradiation, the recorded mechanical responses were biphasic-contraction/relaxation in controls and contraction/contraction in irradiated samples. The nature of the contractile phase of control samples was cholinergic with serotonin involvement. The relaxation phase involved ACh-induced nitric oxide release from gastric neurons. There was a significant increase in serotonergic involvement during the first and second contractile phases of the irradiated samples, along with a diminished role of acetylcholine in the first phase. This study demonstrates an increased involvement of serotonergic neurotransmitter circuits in the gastric myenteric plexus caused by radiation with accelerated electrons.

Biphenotypic Cells and α-Synuclein Accumulation in Enteric Neurons of Leucine-Rich Repeat Kinase 2 Knockout Mice.

BACKGROUND: Leucine-rich repeat kinase 2 is a molecule that is responsible for familial Parkinson's disease. Our previous findings revealed that leucine-rich repeat kinase 2 is expressed in the enteric nervous system. However, which cells in the enteric nervous system express leucine-rich repeat kinase 2 and whether leucine-rich repeat kinase 2 is associated with the structure of the enteric nervous system remain unclear. The enteric nervous system is remarkable because some patients with Parkinson's disease experience gastrointestinal symptoms before developing motor symptoms. AIMS: We established a leucine-rich repeat kinase 2 reporter mouse model and performed immunostaining in leucine-rich repeat kinase 2 knockout mice. METHODS: Longitudinal muscle containing the myenteric plexus prepared from leucine-rich repeat kinase 2 reporter mice was analyzed by immunostaining using anti-green fluorescent protein (GFP) antibody. Immunostaining using several combinations of antibodies characterizing enteric neurons and glial cells was performed on intestinal preparations from leucine-rich repeat kinase 2 knockout mice. RESULTS: GFP expression in the reporter mice was predominantly in enteric glial cells rather than in enteric neurons. Immunostaining revealed that differences in the structure and proportion of major immunophenotypic cells were not apparent in the knockout mice. Interestingly, the number of biphenotypic cells expressing the neuronal and glial cell markers increased in the leucine-rich repeat kinase 2 knockout mice. Moreover, there was accumulation of α-synuclein in the knockout mice. CONCLUSIONS: Our present findings suggest that leucine-rich repeat kinase 2 is a newly recognized molecule that potentially regulates the integrity of enteric nervous system and enteric α-synuclein accumulation.

Isolation of Myenteric and Submucosal Plexus from Mouse Gastrointestinal Tract and Subsequent Co-Culture with Small Intestinal Organoids.

Intestinal homeostasis results from the proper interplay among epithelial cells, the enteric nervous system (ENS), interstitial cells of Cajal (ICCs), smooth muscle cells, the immune system, and the microbiota. The disruption of this balance underpins the onset of gastrointestinal-related diseases. The scarcity of models replicating the intricate interplay between the ENS and the intestinal epithelium highlights the imperative for developing novel methods. We have pioneered a sophisticated tridimensional in vitro technique, coculturing small intestinal organoids with myenteric and submucosal neurons. Notably, we have made significant advances in (1) refining the isolation technique for culturing the myenteric plexus, (2) enhancing the isolation of the submucosal plexus-both yielding mixed cultures of enteric neurons and glial cells from both plexuses, and (3) subsequently co-culturing myenteric and submucosal neurons with small intestinal organoids. This co-culture system establishes neural innervations with intestinal organoids, allowing for the investigation of regulatory interactions in the context of gastrointestinal diseases. Furthermore, we have developed a method for microinjecting the luminal space of small intestinal organoids with fluorescently labeled compounds. This technique possesses broad applicability such as the assessment of intestinal permeability, transcytosis, and immunocytochemical and immunofluorescence applications. This microinjection method could be extended to alternative experimental setups, incorporating bacterial species, or applying treatments to study ENS-small intestinal epithelium interactions. Therefore, this technique serves as a valuable tool for evaluating the intricate interplay between neuronal and intestinal epithelial cells (IECs) and shows great potential for drug screening, gene editing, the development of novel therapies, the modeling of infectious diseases, and significant advances in regenerative medicine. The co-culture establishment process spans twelve days, making it a powerful asset for comprehensive research in this critical field.

A Novel Objective Pathologic Criterion for Isolated Hypoganglionosis.

Isolated hypoganglionosis (IHG) is histologically characterized by small numbers of myenteric ganglion cells and small myenteric ganglia; however, no numerical diagnostic criteria for IHG have been established. Therefore, this study aimed to develop quantitative pathologic criteria for IHG. We evaluated 160 resected intestinal tissue specimens from 29 pediatric autopsies and 10 IHG cases. These specimens were obtained from the jejunum, ileum, ascending colon, transverse colon, and rectum. Morphologic features of the myenteric ganglion cells and myenteric ganglia were quantified and analyzed in digitized HuC/HuD-immunostained and CD56-immunostained sections, respectively. Quantitative criteria were developed with a scoring system that used parameters with the area under the receiver operating characteristic curve (AUC) values >0.7 and sensitivity and specificity exceeding 70%. The selected parameters were the number of myenteric ganglion cells per cm and the number of myenteric ganglia with an area >2500 µm 2 per cm. The score for each parameter ranged from -1 to 2, and the total score of the scoring system ranged from -2 to 4. With a cutoff value of ≥2 (AUC, 0.98; 95% CI: 0.96-1.00), the scoring system had a sensitivity of 96% (95% CI: 0.82-1.00) and a specificity of 99% (95% CI: 0.95-1.00). We devised a novel pathologic criterion based on the quantification of the number of myenteric ganglion cells and ganglia. Furthermore, this criterion showed high diagnostic accuracy and could lead to a definitive diagnosis of IHG in clinical practice.

Muscularis macrophages controlled by NLRP3 maintain the homeostasis of excitatory neurons.

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites ß-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.

Protocol to culture enteric glial cells from the submucosal and myenteric plexi of neonatal and juvenile pig colons.

Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al.1.

A novel method for culturing enteric neurons generates neurospheres containing functional myenteric neuronal subtypes.

BACKGROUND: The enteric nervous system (ENS) is comprised of neurons, glia, and neural progenitor cells that regulate essential gastrointestinal functions. Advances in high-efficiency enteric neuron culture would facilitate discoveries surrounding ENS regulatory processes, pathophysiology, and therapeutics. NEW METHOD: Development of a simple, robust, one-step method to culture murine enteric neurospheres in a 3D matrix that supports neural growth and differentiation. RESULTS: Myenteric plexus cells isolated from the entire length of adult murine small intestine formed ≥3000 neurospheres within 7 days. Matrigel-embedded neurospheres exhibited abundant neural stem and progenitor cells expressing Sox2, Sox10 and Msi1 by day 4. By day 5, neural progenitor cell marker Nestin appeared in the periphery of neurospheres prior to differentiation. Neurospheres produced extensive neurons and neurites, confirmed by Tubulin beta III, PGP9.5, HuD/C, and NeuN immunofluorescence, including neural subtypes Calretinin, ChAT, and nNOS following 8 days of differentiation. Individual neurons within and external to neurospheres generated depolarization induced action potentials which were inhibited in the presence of sodium channel blocker, Tetrodotoxin. Differentiated neurospheres also contained a limited number of glia and endothelial cells. COMPARISON WITH EXISTING METHODS: This novel one-step neurosphere growth and differentiation culture system, in 3D format (in the presence of GDNF, EGF, and FGF2), allows for ∼2-fold increase in neurosphere count in the derivation of enteric neurons with measurable action potentials. CONCLUSION: Our method describes a novel, robust 3D culture of electrophysiologically active enteric neurons from adult myenteric neural stem and progenitor cells.

Role of ICAM-1 in the Adhesion of T Cells to Enteric Glia: Perspectives in the Formation of Plexitis in Crohn's Disease.

BACKGROUND & AIMS: The presence of myenteric plexitis in the proximal resection margins is a predictive factor of early postoperative recurrence in Crohn's disease. To decipher the mechanisms leading to their formation, T-cell interactions with enteric neural cells were studied in vitro and in vivo. METHODS: T cells close to myenteric neural cells were retrospectively quantified in ileocolonic resections from 9 control subjects with cancer and 20 patients with Crohn's disease. The mechanisms involved in T-cell adhesion were then investigated in co-cultures of T lymphocytes with enteric glial cells (glia). Finally, the implication of adhesion molecules in the development of plexitis and colitis was studied in vitro but also in vivo in Winnie mice. RESULTS: The mean number of T cells close to glia, but not neurons, was significantly higher in the myenteric ganglia of relapsing patients with Crohn's disease (2.42 ± 0.5) as compared with controls (0.36 ± 0.08, P = .0007). Co-culture experiments showed that exposure to proinflammatory cytokines enhanced T-cell adhesion to glia and increased intercellular adhesion molecule-1 (ICAM-1) expression in glia. We next demonstrated that T-cell adhesion to glia was inhibited by an anti-ICAM-1 antibody. Finally, using the Winnie mouse model of colitis, we showed that the blockage of ICAM-1/lymphocyte function-associated antigen-1 (LFA-1) with lifitegrast reduced colitis severity and decreased T-cell infiltration in the myenteric plexus. CONCLUSIONS: Our present work argues for a role of glia-T-cell interaction in the development of myenteric plexitis through the adhesion molecules ICAM-1/LFA-1 and suggests that deciphering the functional consequences of glia-T-cell interaction is important to understand the mechanisms implicated in the development and recurrence of Crohn's disease.

Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

Clinicopathologic evaluation of congenital idiopathic megaesophagus in a Gordon Setter puppy: a case report and development and application of peripherin immunohistochemistry for detection of ganglion cells.

We examined a case of congenital idiopathic megaesophagus (CIM) in a 5-wk-old female Gordon Setter puppy by means of contrast radiography, autopsy, histopathology, and immunohistochemistry. Clinical and radiologic findings included weight stagnation and marked generalized esophageal dilation with ventral displacement of the heart and lungs. These findings were confirmed at autopsy, and segments of the thoracic esophagus were sampled for histopathology. On histopathology, diffuse esophageal muscular atrophy, mucosal erosions, mononuclear inflammation, and a marked reduction in the number of myenteric plexus structures and number of ganglion cells were present (aganglionosis). The latter was determined immunohistochemically using an anti-peripherin antibody as the primary reagent, which provides a strong tool for the histologic confirmation of CIM. The histologic findings share some similarities to lesions associated with megaesophagus in Friesian foals, as well as esophageal achalasia and Hirschsprung disease in humans.

Differences in enteric neuronal density in the NSE-Noggin mouse model across institutes.

The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.

Effect of the Flavonoid Rutin on the Modulation of the Myenteric Plexuses in an Experimental Model of Parkinson's Disease.

Recent discoveries have shown that enteric glial cells play an important role in different neurodegenerative disorders, such as Parkinson's disease (PD), which is characterized by motor dysfunctions caused by the progressive loss of dopaminergic neurons in the substance nigra pars compacta and non-motor symptoms including gastrointestinal dysfunction. In this study, we investigated the modulatory effects of the flavonoid rutin on the behavior and myenteric plexuses in a PD animal model and the response of enteric glia. Adult male Wistar rats were submitted to stereotaxic injection with 6-hydroxydopamine or saline, and they were untreated or treated with rutin (10 mg/kg) for 14 days. The ileum was collected to analyze tissue reactivity and immunohistochemistry for neurons (HuC/HuD) and enteric glial cells (S100ß) in the myenteric plexuses. Behavioral tests demonstrated that treatment with rutin improved the motor capacity of parkinsonian animals and improved intestinal transit without interfering with the cell population; rutin treatment modulated the reactivity of the ileal musculature through muscarinic activation, reducing relaxation through the signaling pathway of nitric oxide donors, and increased the longitudinal contractility of the colon musculature in parkinsonian animals. Rutin revealed modulatory activities on the myenteric plexus, bringing relevant answers regarding the effect of the flavonoid in this system and the potential application of PD adjuvant treatment.

The P2Y1 receptor in the colonic myenteric plexus of rats and its correlation with opioid-induced constipation.

This study was designed to explore the expression changes of P2Y1 receptors in the distal colonic myenteric layer of rats. An opioid induced constipation(OIC) rat model was generated by intraperitoneal (i.p) injection of loperamide. At 7 days post-treatment, the model rats were assessed by calculating the fecal water content and the gastrointestinal transit ratio. The immunofluorescence (IF)-based histochemical study was used to observe the distribution of P2Y1 receptors in the distal colonic myenteric plexus. Western blotting (WB) was performed to evaluate the expression changes of P2Y1 proteins in the myenteric layer, and the electrophysiological approaches were carried out to determine the regulatory roles of P2Y1 receptors on distal colonic motor function. IF showed that P2Y1 receptors are co-expressed MOR in the enteric nerve cells of the distal colonic myenteric plexus. Moreover, the WB revealed that the protein levels of P2Y1 were significantly decreased in the distal colonic myenteric layer of OIC rats. In vitro tension experiments exhibited that the P2Y1 receptor antagonist MRS2500 enhanced the spontaneous contraction amplitude, adding EM2 and ß-FNA did not have any effect on MRS2500. Therefore, P2Y1 receptor expression could be associated with the occurrence of OIC in this rat model and the regulation of colonic motility by MOR may be related to the release of purine neurotransmitters such as ATP in the colonic nervous system.

Morphological, molecular, and functional characterization of mouse glutamatergic myenteric neurons.

The enteric nervous system (ENS) functions largely independently of the central nervous system (CNS). Glutamate, the dominant neurotransmitter in the CNS and sensory afferents, is not a primary neurotransmitter in the ENS. Only a fraction (∼2%) of myenteric neurons in the mouse distal colon and rectum (colorectum) are positive for vesicular glutamate transporter type 2 (VGLUT2), the structure and function of which remain undetermined. Here, we systematically characterized VGLUT2-positive enteric neurons (VGLUT2-ENs) through sparse labeling with adeno-associated virus, single-cell mRNA sequencing (scRNA-seq), and GCaMP6f calcium imaging. Our results reveal that the majority of VGLUT2-ENs (29 of 31, 93.5%) exhibited Dogiel type I morphology with a single aborally projecting axon; most axons (26 of 29, 89.7%) are between 4 and 10 mm long, each traversing 19 to 34 myenteric ganglia. These anatomical features exclude the VGLUT2-ENs from being intrinsic primary afferent or motor neurons. The scRNA-seq conducted on 52 VGLUT2-ENs suggests different expression profiles from conventional descending interneurons. Ex vivo GCaMP6f recordings from flattened colorectum indicate that almost all VGLUT2-EN (181 of 215, 84.2%) are indirectly activated by colorectal stretch via nicotinic cholinergic neural transmission. In conclusion, VGLUT2-ENs are a functionally unique group of enteric neurons with single aborally projecting long axons that traverse multiple myenteric ganglia and are activated indirectly by colorectal mechanical stretch. This knowledge will provide a solid foundation for subsequent studies on the potential interactions of VGLUT2-EN with extrinsic colorectal afferents via glutamatergic neurotransmission.NEW & NOTEWORTHY We reveal that VGLUT2-positive enteric neurons (EN), although constituting a small fraction of total EN, are homogeneously expressed in the myenteric ganglia, with a slight concentration at the intermediate region between the colon and rectum. Through anatomic, molecular, and functional analyses, we demonstrated that VGLUT2-ENs are activated indirectly by noxious circumferential colorectal stretch via nicotinic cholinergic transmission, suggesting their participation in mechanical visceral nociception.

Improving morphological and functional properties of enteric neuronal networks in vitro using a novel upside-down culture approach.

The enteric nervous system (ENS) comprises millions of neurons and glia embedded in the wall of the gastrointestinal tract. It not only controls important functions of the gut but also interacts with the immune system, gut microbiota, and the gut-brain axis, thereby playing a key role in the health and disease of the whole organism. Any disturbance of this intricate system is mirrored in an alteration of electrical functionality, making electrophysiological methods important tools for investigating ENS-related disorders. Microelectrode arrays (MEAs) provide an appropriate noninvasive approach to recording signals from multiple neurons or whole networks simultaneously. However, studying isolated cells of the ENS can be challenging, considering the limited time that these cells can be kept vital in vitro. Therefore, we developed an alternative approach cultivating cells on glass samples with spacers (fabricated by photolithography methods). The spacers allow the cells to grow upside down in a spatially confined environment while enabling acute consecutive recordings of multiple ENS cultures on the same MEA. Upside-down culture also shows beneficial effects on the growth and behavior of enteric neural cultures. The number of dead cells was significantly decreased, and neural networks showed a higher resemblance to the myenteric plexus ex vivo while producing more stable signals than cultures grown in the conventional way. Overall, our results indicate that the upside-down approach not only allows to investigate the impact of neurological diseases in vitro but could also offer insights into the growth and development of the ENS under conditions much closer to the in vivo environment.NEW & NOTEWORTHY In this study, we devised a novel approach for culturing and electrophysiological recording of the enteric nervous system using custom-made glass substrates with spacers. This allows to turn cultures of isolated myenteric plexus upside down, enhancing the use of the microelectrode array technique by allowing recording of multiple cultures consecutively using only one chip. In addition, upside-down culture led to significant improvements in the culture conditions, resulting in a more in vivo-like growth.

Myenteric Plexus Immune Cell Infiltrations and Neurotransmitter Expression in Crohn's Disease and Ulcerative Colitis.

BACKGROUND AND AIMS: Pain is a cardinal symptom in inflammatory bowel disease [IBD]. An important structure in the transduction of pain signalling is the myenteric plexus [MP]. Nevertheless, IBD-associated infiltration of the MP by immune cells lacks in-depth characterisation. Herein, we decipher intra- and periganglionic immune cell infiltrations in Crohn´s disease [CD] and ulcerative colitis [UC] and provide a comparison with murine models of colitis. METHODS: Full wall specimens of surgical colon resections served to examine immune cell populations by either conventional immuno-histochemistry or immunofluorescence followed by either bright field or confocal microscopy. Results were compared with equivalent examinations in various murine models of intestinal inflammation. RESULTS: Whereas the MP morphology was not significantly altered in IBD, we identified intraganglionic IBD-specific B cell- and monocyte-dominant cell infiltrations in CD. In contrast, UC-MPs were infiltrated by CD8+ T cells and revealed a higher extent of ganglionic cell apoptosis. With regard to the murine models of intestinal inflammation, the chronic dextran sulphate sodium [DSS]-induced colitis model reflected CD [and to a lesser extent UC] best, as it also showed increased monocytic infiltration as well as a modest B cell and CD8+ T cell infiltration. CONCLUSIONS: In CD, MPs were infiltrated by B cells and monocytes. In UC, mostly CD8+ cytotoxic T cells were found. The chronic DSS-induced colitis in the mouse model reflected best the MP-immune cell infiltrations representative for IBD.

Comparison of wholemount dissection methods for neuronal subtype marker expression in the mouse myenteric plexus.

BACKGROUND: Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study, we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression. METHODS: We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation and a third rod-mounted peeling technique. We also tested a reversed orientation variation of flat-sheet peeling, two step-by-step variations of the rod peeling technique, and whole-gut fixation as a tube. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis. KEY RESULTS AND CONCLUSIONS: We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin or neuronal nitric oxide synthase (nNOS) as a proportion of total neurons in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased proportion of neurons labeled for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in distal colon and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling. The step-by-step variations of this method point to mechanical manipulation of the tissue as the likely cause of decreased labeling. Our study thereby demonstrates a critical variability in wholemount muscularis dissection methods.

Effects of weaning on intestinal longitudinal muscle-myenteric plexus function in piglets.

Weaning piglets usually suffer from severe diarrhea (commonly known as postweaning diarrhea, PWD) along with intestinal motility disorder. Intestinal peristalsis is mainly regulated by the longitudinal muscle-myenteric plexus (LM-MP). To understand the relationship between intestinal LM-MP function and the development of PWD, we compared the intestinal electrical activity, and the transcriptional profile of the LM-MP between 21-day-old piglets (just weaned, n=7) and 24-day-old piglets (suffered the most severe weaning stress, n=7). The results showed that 24-day-old piglets exhibited different degrees of diarrhea. A significant increase in the slow-wave frequency in the ileum and colon was observed in 24-day-old piglets, while c-kit expression in the intestinal LM-MPs was significantly decreased, indicating that PWD caused by elevated slow-wave frequency may be associated with loss of c-kit. The real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) showed that intestinal LM-MPs in 24-day-old piglets may undergo inflammation and oxidative stress. Significant increases in 8-hydroxy-2'-deoxyguanosine and decreases in thioredoxin suggest that weaning may lead to DNA damage in the LM-MP of 24-day-old piglets. In addition, activating transcription factor 3 was significantly upregulated, indicating nerve damage in the LM-MP of 24-day-old piglets. The transcriptomic results showed that most of the differentially expressed genes in the ileal LM-MP after weaning were downregulated and closely related to the cell cycle process. Subsequent RT-qPCR analysis showed that the relative expression of p21 was upregulated, while the expression of cyclin A2, cyclin B1, and proliferating cell nuclear antigen was downregulated in the ileal and colonic LM-MP of 24-day-old piglets, suggesting that weaning may inhibit cell proliferation and cause G1/S cell cycle arrest in ileal and colonic LM-MP. In conclusion, weaning may lead to cell cycle arrest by causing DNA damage in the LM-MP, impairing intestinal motility regulation, and ultimately leading to diarrhea in piglets.