Myenteric Research Articles

Morphology and Chemical Coding of Rat Duodenal Enteric Neurons following Prenatal Exposure to Fumonisins.

Simple SummaryFood contamination with toxins produced by various species of fungi (hereafter: mycotoxins) occurs frequently, and, for this reason, their activity in the bodies of animals is the subject of wide-ranging research. Although many aspects of the activity of fumonisins (mycotoxins produced by Fusarium) have been described, an open question is whether prenatal exposure to fumonisins may result in morphological changes, including in the enteric nervous system (ENS). The present study revealed that fumonisin administered via the gastrointestinal tract to pregnant rats did not substantially change the structure of the intestine/ENS of the offspring, but altered the neurochemical profile of its enteric neurons.Fumonisins (FBs), including fumonisin B1 and B2 produced by the fungus Fusarium verticillioides, are widespread mycotoxins contaminating crop plants as well as processed food. The aim of the experiment was to determine whether the exposure of 5-week-old pregnant rats to FBs at 60 mg/kg b.w. (group FB60) or 90 mg/kg b.w. (group FB90) results in morphological changes in the duodenum of weaned offspring, particularly the enteric nervous system (ENS). In addition, the levels of expression of galanin and vasoactive intestinal polypeptide (VIP) in the ENS were analysed by immunofluorescence in the control and experimental groups of animals. No significant morphological changes in the thickness of the muscle layer or submucosa of the duodenum were noted in group FB60 or FB90. In group FB90 (but not FB60), there was a significant increase in the width of the villi and in the density of the intestinal crypts. Immunofluorescence analysis using neuronal marker Hu C/D showed no significant changes in group FB60 or FB90 in the morphology of the duodenal ENS, i.e., the myenteric plexus (MP) and submucosal plexus (SP), in terms of the density of enteric ganglia in the MP and SP, surface area of MP and SP ganglia, length and width of MP and SP ganglia, surface area of myenteric and submucosal neurons, diameter of myenteric and submucosal neurons, density of myenteric and submucosal neurons, and number of myenteric and submucosal neurons per ganglion. In both groups, there was an increase (relative to the control) in the percentage of Hu C/D-IR/VIP-IR (IR-immunoreactive) and Hu C/D-IR/galanin-IR myenteric and submucosal neurons in the ganglia of both the MP and SP of the duodenum. In addition, in groups FB60 and FB90, there was an increase in the number of nerve fibres showing expression of VIP and galanin in the mucosa, submucosa and circular muscle layer of the duodenum. The results indicate that prenatal exposure to FBs does not significantly alter the histological structure of the duodenum (including the ENS) in the weaned offspring. The changes observed in the chemical code of the myenteric and submucosal neurons in both experimental groups suggest harmful activity of FBs, which may translate into activation of repair mechanisms via overexpression of neuroprotective neuropeptides (VIP and galanin).

Evaluation of myenteric neurons in the colon of rats exposed to 2,4 dichlorophenoxyacetic acid herbicide

The assessment of the enteric nervous system provides a better understanding of the effects that contaminants can have on the health and well-being of organisms. It has been reported that 2,4-dichlorophenoxyacetic acid (2,4-D) is a highly persistent herbicide in the environment that is responsible for neurotoxic changes in different myenteric neuronal subpopulations. The current study aimed to evaluate the effects of 2,4-D on myenteric neurons in the colon of Rattus norvegicus for the first time. A dose of 2,4-D (5 mg/kg/day) was administered to the experimental group (2,4-D) for 15 days. Then, the proximal colon was collected and submitted to Giemsa and NADPH-d histochemical techniques for the disclosure of total and nitrergic neurons. The 2,4-D group presented a higher density of total neurons (p = 0.05, t-test), which together with the maintenance of nitrergic neuronal density, may be related to the increase in the expression of the neurotransmitter acetylcholine by colocalization, responsible for stimulating the intestinal smooth muscle and increasing the chances of the expulsion of the harmful content present in the lumen. Over 15 days, the neurotoxic effects of 2,4-D in the myenteric plexus influenced an increase in the general population of myenteric neurons in the colon.

Impaired nitrergic relaxation in pyloric sphincter of the 6-OHDA Parkinson's disease rat.

Patients with Parkinson's disease (PD) often suffer from delayed gastric emptying, but the underlying mechanism remains unclear. We have shown previously that a PD rat model comprising bilateral substantia nigra destruction by 6-hydroxydopamine (6-OHDA rats) exhibits gastroparesis with alteration of neural nitric oxide synthase (nNOS) and acetylcholine in gastric corpus. However, changes in pyloric motility in the 6-OHDA rats have not been characterized. Solid gastric emptying test, immunofluorescence, Western blot, and in vitro pyloric motility recordings were used to assess pyloric motor function in the 6-OHDA rats. The 6-OHDA-treated rats displayed delayed solid gastric emptying and a lower basal pyloric motility index. In the 6-OHDA rats, high K+-induced transient contractions were weaker in pyloric sphincters. Electric field stimulation (EFS)-induced pyloric sphincter relaxation was lower in the 6-OHDA rats. NG-nitro-l-arginine methyl ester (l-NAME), a nonselective inhibitor of NOS, markedly inhibited the EFS-induced relaxation in both control and 6-OHDA rats. Pretreatment of tetrodotoxin abolished the effect of EFS on the pyloric motility. In addition, nNOS-positive neurons were extensively distributed in the pyloric myenteric plexus, whereas the number of nNOS-immunoreactive neurons and the protein expression of nNOS were significantly decreased in the pyloric muscularis of 6-OHDA rats. However, sodium nitroprusside-induced pyloric relaxations were similar between the control and 6-OHDA rats. These results indicate that the pyloric sphincters of 6-OHDA rats exhibit both weakened contraction and relaxation. The latter may be due to reduced nNOS in the pyloric myenteric plexus. The dysfunction of the pyloric sphincter might be involved in the delayed gastric emptying.NEW & NOTEWORTHY Reduced nitrergic neurons in pyloric myenteric plexus potently contributed to the attenuated relaxation in 6-hydroxydopamine (6-OHDA) rats, subsequently affecting gastric emptying. SNP could well improve the relaxation of pylori in 6-OHDA rats. The present study provides new insight into the diagnosis and treatment of delayed gastric emptying in patients with PD.

Multisystem screening reveals SARS-CoV-2 in neurons of the myenteric plexus and in megakaryocytes.

SARS‐CoV‐2, the causative agent of COVID‐19, typically manifests as a respiratory illness, although extrapulmonary involvement, such as in the gastrointestinal tract and nervous system, as well as frequent thrombotic events, are increasingly recognised. How this maps onto SARS‐CoV‐2 organ tropism at the histological level, however, remains unclear. Here, we perform a comprehensive validation of a monoclonal antibody against the SARS‐CoV‐2 nucleocapsid protein (NP) followed by systematic multisystem organ immunohistochemistry analysis of the viral cellular tropism in tissue from 36 patients, 16 postmortem cases and 16 biopsies with polymerase chain reaction (PCR)‐confirmed SARS‐CoV‐2 status from the peaks of the pandemic in 2020 and four pre‐COVID postmortem controls. SARS‐CoV‐2 anti‐NP staining in the postmortem cases revealed broad multiorgan involvement of the respiratory, digestive, haematopoietic, genitourinary and nervous systems, with a typical pattern of staining characterised by punctate paranuclear and apical cytoplasmic labelling. The average time from symptom onset to time of death was shorter in positively versus negatively stained postmortem cases (mean = 10.3 days versus mean = 20.3 days, p = 0.0416, with no cases showing definitive staining if the interval exceeded 15 days). One striking finding was the widespread presence of SARS‐CoV‐2 NP in neurons of the myenteric plexus, a site of high ACE2 expression, the entry receptor for SARS‐CoV‐2, and one of the earliest affected cells in Parkinson's disease. In the bone marrow, we observed viral SARS‐CoV‐2 NP within megakaryocytes, key cells in platelet production and thrombus formation. In 15 tracheal biopsies performed in patients requiring ventilation, there was a near complete concordance between immunohistochemistry and PCR swab results. Going forward, our findings have relevance to correlating clinical symptoms with the organ tropism of SARS‐CoV‐2 in contemporary cases as well as providing insights into potential long‐term complications of COVID‐19. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Three-dimensional imaging of intramural perineural invasion in colorectal cancer: Three-dimensional reconstruction approach with multiple immunohistochemically stained sections.

Perineural invasion (PNI) at Auerbach's plexus in colorectal cancer (CRC), known as intramural PNI, is associated with adverse prognostic outcomes. This study aimed to characterize the three-dimensional (3D) architecture of CRC with intramural PNI and to evaluate the morphological features of tumor invasion around nerve tissue. Serial tissue sections from two cases of CRC were stained with cytokeratin AE1/AE3 and an anti-S-100 protein antibody. 3D models were reconstructed by scanning the virtual slides. In one case, intramural PNI was observed at the horizontal invasive front. The 3D reconstruction model showed tumor cells that appeared to infiltrate along the nervous meshwork, the structure of which was preserved. In the other case, intramural PNI was observed both at and behind the horizontal invasive front, and the 3D reconstruction model showed that the tumor cells appeared to be involved with nerve cells at the focal part of the horizontal invasive front. However, the nervous meshwork structure was not well identified in cancer-involved areas. This is the first study to characterize the 3D structure of tumor invasion around nerve tissue in CRC, demonstrating the morphological features of intramural PNI in CRC.

Sympathetic Pathways Target Cholinergic Neurons in the Human Colonic Myenteric Plexus.

BackgroundThe sympathetic nervous system inhibits human colonic motility largely by effects on enteric neurons. Noradrenergic axons, which branch extensively in the myenteric plexus, are integral to this modulatory role, but whether they contact specific types of enteric neurons is unknown. The purpose of this study was to determine the association of noradrenergic varicosities with types of enteric neurons.MethodsHuman colonic tissue from seven patients was fixed and dissected prior to multi-layer immunohistochemistry for human RNA binding proteins C and D (HuC/D) (pan-neuronal cell body labelling), tyrosine hydroxylase (TH, catecholaminergic labelling), Enkephalin (ENK), choline acetyltransferase (ChAT, cholinergic labelling) and/or nitric oxide synthase (NOS, nitrergic labelling) and imaged using confocal microscopy. TH-immunoreactive varicose nerve endings and myenteric cell bodies were reconstructed as three dimensional digital images. Data was exported to a purpose-built software package which quantified the density of varicosities close to the surface of each myenteric cell body.ResultsTH-immunoreactive varicosities had a greater mean density within 1 μm of the surface of ChAT +/NOS− nerve cell bodies compared with ChAT−/NOS + cell bodies. Similarly, ENK-immunoreactive varicosities also had a greater mean density close to ChAT +/NOS− cell bodies compared with ChAT−/NOS + cells.ConclusionA method for quantifying close associations between varicosities and nerve cell bodies was developed. Sympathetic axons in the myenteric plexus preferentially target cholinergic excitatory cells compared to nitrergic neurons (which are largely inhibitory). This connectivity is likely to be involved in inhibitory modulation of human colonic motility by the sympathetic nervous system.

Identification of MrgprD expression in mouse enteric neurons.

Mas-related G protein-coupled receptor D (MrgprD) was first identified in small-diameter sensory neurons of mouse dorsal root ganglion (DRG). The role of MrgprD has been studied in somatosensation, especially in pain and itch response. We recently showed that MrgprD also participated in the modulation of murine intestinal motility. The treatment of MrgprD receptor agonist suppressed the spontaneous contractions in the isolated intestinal rings of mice, indicating the intrinsic expression of MrgprD in the murine gastrointestinal (GI) tract. Although the expression of Mrgprd in GI tract has been previously detected by the way of quantitative real-time PCR, the cell-type-specific expression of MrgprD in GI tract is no yet determined. Herein, we employed Mrgprd-tdTomato reporter mouse line and the whole-mount immunohistochemistry to observe the localization of MrgprD in the smooth muscle layers of ileum and colon. We show that tdTomato-positive cells colocalized with NeuN-immunostaining in the myenteric plexus in the whole-mount preparations of the ileum and the colon. Further immunohistochemistry using the commercially available MrgprD antibody revealed the expression of MrgprD in NeuN-labeled enteric neurons in the myenteric plexus. Our results demonstrate the expression of MrgprD in the enteric neurons in the murine GI tract, highlighting the implications of MrgprD in the physiology and pathophysiology of the GI tract.

A Case of Leporine Dysautonomia from Croatia

Abstract Leporine dysautonomia (or dysautonomia of hares) is an idiopathic disorder associated with degeneration of neurons of the peripheral nervous system with loss of sympathetic and parasympathetic nerve function. The disease has so far been described in rabbits and hares from Great Britain, and a similar disorder has also been described in horses (grass sickness), dogs and cats (Key-Gaskell syndrome) throughout the world. We describe a case of leporine dysautonomia in a rabbit from Croatia, characterized by gross findings of cachexia, dehydration, dilated intestines and urinary bladder. Histologic findings included severe vacuolation of neurons of the submucous and myenteric plexus in the intestinal tract. This disease presents a rare disorder of rabbits, and based on current report, it should be considered as a possible differential diagnosis is rabbits with neurologic clinical signs and mild gross findings including cachexia and distension of intestine and urinary bladder.

Impact of Dietary Fiber on West Nile Virus Infection.

Dietary fiber supports healthy gut bacteria and their production of short-chain fatty acids (SCFA), which promote anti-inflammatory cell development, in particular, regulatory T cells. It is thus beneficial in many diseases, including influenza infection. While disruption of the gut microbiota by antibiotic treatment aggravates West Nile Virus (WNV) disease, whether dietary fiber is beneficial is unknown. WNV is a widely-distributed neurotropic flavivirus that recruits inflammatory monocytes into the brain, causing life-threatening encephalitis. To investigate the impact of dietary fiber on WNV encephalitis, mice were fed on diets deficient or enriched with dietary fiber for two weeks prior to inoculation with WNV. To induce encephalitis, mice were inoculated intranasally with WNV and maintained on these diets. Despite increased fecal SCFA acetate and changes in gut microbiota composition, dietary fiber did not affect clinical scores, leukocyte infiltration into the brain, or survival. After the brain, highest virus loads were measured in the colon in neurons of the submucosal and myenteric plexuses. Associated with this, there was disrupted gut homeostasis, with shorter colon length and higher local inflammatory cytokine levels, which were not affected by dietary fiber. Thus, fiber supplementation is not effective in WNV encephalitis.

Transplanted neural crest cells migrate toward Auerbach's plexus layer instead of the colon surface in recipient colon pretreated with collagenase and fibronectin

The enteric nervous system (ENS) regulates gastrointestinal motility, secretion, and absorption. Developmental ENS dysplasia causes intestinal ganglion dysfunction, including Hirschsprung's disease. Given their potential ability to replenish insufficient neurons, transplantation of enteric neural cells provides the prospect of a cure. In this study, we used an ex vivo mouse colon transplant model to demonstrate that treatment with collagenase and fibronectin altered the migration of transplanted cells from the direction of the colon surface toward the lumen. Collagenase-treated colons exhibited enhanced expression of type III and VI collagens, which inhibited fibronectin-induced enteric neural crest cell (ENCC) migration. Invasion of neurospheres into colon was dependent on preoperative treatment of recipient colon with collagenase and fibronectin, which enhanced neurosphere motility towards the direction of colon lumen. Infiltration of transplanted ENCCs into the colon increased proportionally to the degree of dedifferentiation of surrounding smooth muscle cells, which was induced in a neurosphere-dependent manner in collagenase-treated colon. Furthermore, induction of GDNF expression, a Ret ligand that promotes enteric neural cell migration, was observed in treated colons. Our results suggest that the environment provided by the extracellular matrix of the colon surface affects the direction of transplanted ENCC migration. Moreover, these findings demonstrating that ENCCs can be accepted by the recipient colon will help to refine current strategies for cell therapy.

Total colonic aganglionosis with ileum extension Hirschsprung's disease in an adult: A case report.

IntroductionHirschsprung's disease is a gastrointestinal anomaly affecting neuronal development and function. The primary abnormality is the absent ganglionic cells in the submucosal and myenteric neural plexuses. Disease presentation can vary, and patients may present with delayed meconium passage or late in adulthood. Total colonic aganglionosis is considered a rare subtype and even rarer to extend proximally into the small bowel. Therefore, adult Hirschsprung disease is frequently misdiagnosed as chronic constipation until comorbid events such as volvulus or perforation occur.Case presentationA 34 years South-Sudanese male presented with chronic constipation since early childhood, abdominal discomfort, pain, and distension. His bowel habits were infrequent, requiring occasional enemas. A strong family history of Hirschsprung disease was identified. He underwent total abdominal colectomy and end ileostomy. Postoperative diagnosis of TCA and small bowel extension of more than 50 cm was confirmed. The patient was discharged home after ileostomy education and possible future reconstruction.Clinical discussionHirschsprung's ileal extension resembles a risk of morbidities and mortality. It increases as the segment extends proximally. No international consensus on surgical management. It should be tailored to patient condition, diseased segment length, and the possible definitive primary reconstruction. Our patient's surgical diversion considered the patient's long-standing constipation history and dilated fecal-loaded ileum.ConclusionTCA requires a high index suspension during the workup of chronic constipation patients. There is no reported superior surgical approach, and it depends on the institution's experience and surgeon's expertise. Primary reconstruction can be achieved, but we recommend an ileostomy for such cases.

A6 MECHANISMS UNDERLYING GUT DYSFUNCTION FOLLOWING C. DIFFICILE INFECTION AND IMPLICATIONS FOR TREATMENT

BackgroundRecent evidence suggests an increasing prevalence of gut dysfunction following C. difficile infection (CDI). The accompanying prolonged antibiotic (AB) exposure likely contributes to chronic gut dysfunction and our ability to induce gut dysfunction in germ free (GF) mice colonized with microbiota from a patient with severe slow transit post CDI (PCDI) supports this notion (10.1093/jcag/gwz047.117). Furthermore, we were able to restore gut function following fecal microbial transfer from healthy murine donors. Our studies have implicated a role for macrophages in the destruction of the Interstitial Cell of Cajal (ICC) network underlying slow colonic transit in the humanized mouse model. These findings prompted us to evaluate microbiota-directed therapy in normalising gut function in this model.Aims 1)To investigate whether dietary psyllium rescues the development of slow colonic transit (SCT) through modulating host function via microbiota mediated immune mechanisms; and 2)To evaluate the mechanisms underlying the beneficial effects of psylliumMethodsGF mice were colonized with either microbiota from the PCDI patient or healthy control (HC) for 3 weeks following which PCDI mice were treated with either a control diet or a 15% psyllium diet (PSY). Colonic motility was assessed before and after the diet intervention using the bead expulsion test. Stool samples were collected for microbial profiling, and short and branched-chain fatty acids (SCFA/BCFA) analysis. Colonic muscle layers encompassing myenteric plexus (MP) were collected for gene expression analysis and to evaluate activated macrophages and ICC degeneration using immunohistochemistry.ResultsMicrobiota from a PCDI patient induced a SCT phenotype in GF mice (n=13) as compared to mice colonized with HC microbiota (p=0.0002). Psyllium rescued this SCT phenotype in mice (PCDI(n=7) vs.PSY(n=6):p=0.0014). The psyllium-induced rescue was accompanied by normalization of the ICC network and morphological alterations in infiltrating macrophages. This was supported by changes in immune-related gene expression in the MP including CD11b, NOS, Myd88, Mapk1 and NF-κB. Additionally, bacterial composition was different between PCDI and PSY group (p=0.003). SCFAs like acetic and propionic acid were increased, while BCFA like isobutyric and isovaleric acid were decreased following PSY treatment. These alterations in SCFA/BCFA were supported by fluctuations in specific bacteria like Butyricimonas, Phascolarctobacterium and Allistipes.ConclusionsOur results provide evidence that chronic gut dysfunction following CDI and AB exposure is microbiota-driven. Furthermore, microbiota-directed therapy using psyllium could serve as a novel therapeutic strategy to normalize gut function via microbiota-mediated restoration of immune homeostasis in these patients.Funding AgenciesW. Garfield Weston Foundation

A132 ACHALASIA FOLLOWING A SARS-COV-2 INFECTION: A CASE REPORT

BackgroundSARS-CoV-2 is a novel virus currently causing a major pandemic. Recent studies have shown that SARS-CoV-2 has an affinity to ACE2 receptors which are present in the olfactory epithelium as well as the pulmonary and gastrointestinal (GI) systems. It is stipulated that these receptors are responsible for the entry of the virus into cells explaining the respiratory and GI symptoms of this infection. The anosmia related to COVID-19 is likely due to an aberrant immune response to the virus rather than direct damage to the olfactory epithelium. Similarly, achalasia is also believed to result from an aberrant immune response through which there is degeneration of inhibitory neurons in the myenteric plexus. In this case report, we present the first case of achalasia thought to be linked to a SARS-CoV-2 infection in contemporary literature.AimsWe hypothesize that SARS-CoV-2 caused an aberrant immune response in our patient, leading him to develop achalasia. We hope to expose clinicians to this potential complication of COVID-19, allowing them to consider it sooner in their differential.MethodsThe patient’s file was reviewed to extract the relevant information. A second gastroenterologist’s opinion was obtained on the manometry to confirm the diagnosis of type III achalasia.ResultsA 61-year-old man with a medical history of myasthenia gravis, chronic obstructive pulmonary disease, hypothyroidism, and chronic renal failure was treated in our establishment for a SARS-CoV-2 pneumonia. He experienced new severe dysphagic symptoms, regurgitations, and lost 10 pounds during his hospitalization. He also developed anosmia and diarrhea. A CT-scan of his neck and thorax showed no extrinsic compression of his esophagus. No neoplasm was found on his gastroscopy. A barium study was then conducted showing a stagnation of contrast in the distal third of his esophagus. No relaxation of the inferior gastroesophageal sphincter was noted. A manometry was then performed and confirmed a type III achalasia. The mean lower esophageal sphincter residual pressure was elevated at 43.7 mmHg. A calculated 69% of wet swallows were premature contractions with a distal latency value less than 4.5 seconds. The mean distal contractile integral was 13613 mmHg. This corroborates the spastic nature of type III achalasia.ConclusionsConsidering that our patient developed anosmia and knowing its pathogenesis in the context of COVID-19, an inappropriate immune response to SARS-CoV-2 could have equally taken place in the esophagus. This inflammatory response might have caused the degeneration of the inhibitory neurons in the myenteric plexus responsible for the achalasia. Furthermore, given the fact that our patient had diarrhea, this suggests that the virus has a GI tropism supporting our hypothesis. We therefore believe that achalasia may be a potential complication of COVID-19. High resolution esophageal motility study showing a type III achalasia pattern.Funding AgenciesNone

Morphologies, dimensions and targets of gastric nitric oxide synthase neurons

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

Chronic Morphine Induces IL-18 in Ileum Myenteric Plexus Neurons Through Mu-opioid Receptor Activation in Cholinergic and VIPergic Neurons.

The gastrointestinal epithelium is critical for maintaining a symbiotic relationship with commensal microbiota. Chronic morphine exposure can compromise the gut epithelial barrier in mice and lead to dysbiosis. Recently, studies have implicated morphine-induced dysbiosis in the mechanism of antinociceptive tolerance and reward, suggesting the presence of a gut-brain axis in the pharmacological effects of morphine. However, the mechanism(s) underlying morphine-induced changes in the gut microbiome remains unclear. The pro-inflammatory cytokine, Interleukin-18 (IL-18), released by enteric neurons can modulate gut barrier function. Therefore, in the present study we investigated the effect of morphine on IL-18 expression in the mouse ileum. We observed that chronic morphine exposure in vivo induces IL-18 expression in the ileum myenteric plexus that is attenuated by naloxone. Given that mu-opioid receptors (MORs) are mainly expressed in enteric neurons, we also characterized morphine effects on the excitability of cholinergic (excitatory) and vasoactive intestinal peptide (VIP)-expressing (inhibitory) myenteric neurons. We found fundamental differences in the electrical properties of cholinergic and VIP neurons such that VIP neurons are more excitable than cholinergic neurons. Furthermore, MORs were primarily expressed in cholinergic neurons, although a subset of VIP neurons also expressed MORs and responded to morphine in electrophysiology experiments. In conclusion, these data show that morphine increases IL-18 in ileum myenteric plexus neurons via activation of MORs in a subset of cholinergic and VIP neurons. Thus, understanding the neurochemistry and electrophysiology of MOR-expressing enteric neurons can help to delineate mechanisms by which morphine perturbs the gut barrier.

Gastric GIST or Gastric Schwannoma – A Diagnostic Conundrum

Background: Gastric schwannoma (GS) accounts for 0,2% of all gastric tumors and derives from Schwann cells in the Auerbach's plexus. It frequently presents as a solitary mass, commonly located in the body of the stomach, with predilection for adult females in the fifth to eighth decade. The symptoms can range from abdominal discomfort to hematemesis and due to its slow growth and homogeneous appearance, it is easily mistaken for a gastrointestinal stromal tumor (GIST) on upper endoscopy.

A rare association of ileal atresia and total colonic Hirschsprung's disease

Total colonic aganglionosis is the most rare form of Hirschsprung's disease and is defined as the absence of ganglion cells in both myenteric and submucosal plexus extending from the anus to at least the ileocecal valve. Intrauterine intussusceptions, on the second hand, is also extremely rare. It may occur in late stage of pregnancy and cause impairment of blood supply to a segment of intestine leading to its resorption and atresia. The coexistence of total colonic Hirschsprung's disease, intrauterine intussusception and ileal atresia has yet to be described in the literature. We present this rare case of intrauterine intussusception causing an association of ileal atresia and total colonic Hirschsprung's disease. The diagnosis was performed per-operatively.

CNS-Sparing Histamine H3 Receptor Antagonist as a Candidate to Prevent the Diabetes-Associated Gastrointestinal Symptoms.

Among the histamine receptors, growing evidence points to the histamine H3 receptor as a pharmacological candidate to counteract the autonomic neuropathy associated with diabetes. The study aimed to evaluate the effect of PF00868087 (also known as ZPL-868), a CNS-sparing histamine H3 receptor antagonist, on the autonomic neuropathy of the intestinal tract associated with diabetes. Diabetes was induced in male BALB/c mice by a single high dose of streptozotocin (150 mg/kg). Colorectal specimens from control and diabetic mice, randomized to vehicle or PF0086087 (10, 30, 100 mg/kg/day by oral gavage for 14 days), were processed for morphological and immunohistochemical analysis. A significant overproduction of mucus in the intestinal mucosa of diabetic mice compared to the controls was observed. PF0086087 at the highest dose prevented mucin overproduction. The immunohistochemistry analysis demonstrated that diabetes causes a decrease in the inhibitory component of enteric motility, measured as the percentage of neuronal nitric oxide synthase-positive neurons (p < 0.05) and a parallel increase in the excitatory component evaluated as substance P-positive fibres (p < 0.01). PF0086087 dose-dependently prevented these pathophysiological events. In conclusion, PF0086087 may be an essential tool in preventing nitrergic dysfunction in the myenteric plexus of the distal colon and diabetes-induced gastrointestinal complications.

P044 Enteric dopaminergic pathways in mouse and human intestinal inflammation

Abstract Background Changes in dopamine beta-hydroxylase (DBH) and anomalies in dopaminergic machinery have been shown in inflammatory bowel disease (IBD) patients and related animal models. Thus, we aimed to evaluate the dopaminergic pathways in IBD patients as well as in a mouse model of dextran sodium sulphate (DSS)-induced ileitis. Methods Colon biopsies (CB) obtained from healthy volunteers (N=3) and matched-IBD patients (N=3), were used to evaluate DBH immunoreactivity by confocal microscopy. Male C57/Bl6 (8±2 weeks old; N=16 mice) received 1.5% DSS in drinking water for 5 days, then switched to regular drinking water for 3 days. Inflammatory cytokines (IL-1β, TNFα, IL-6) were measured to assess ileitis severity. Changes in ileal muscle tension were isometrically recorded following 30 μM dopamine or 30 μM SKF38393 (a dopamine receptor 1 (D1R) agonist) or 30 μM bromocriptine (a D2R agonist). Immunofluorescence distribution of Iba1 (a macrophage specific marker), D1R, DBH and dopamine transporter (DAT) were determined in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs) by confocal microscopy. D1R and D2R mRNA transcripts were evaluated by qRT-PCR. Results CB from IBD patients and LMMPs from DSS mice showed a significant increase of DBH immunoreactivity compared to healthy patients and sham mice (+25% [p&amp;lt;0.01], +20% [p&amp;lt;0.01], respectively). DSS treatment caused a significant increase of DAT and D1R immunoreactivity as well as D1R mRNA levels (+27% [p&amp;lt;0.05], +24% [p&amp;lt;0.05], +6-fold [p&amp;lt;0.05], respectively), accompanied by a significant reduction of dopamine-mediated relaxation (-27% [p&amp;lt;0.01]). SKF38393 determined a marked inhibitory response in ileal preparations from DSS mice compared to sham mice (+73% [p&amp;lt;0.01]), suggesting that dopamine responses are mainly mediated through D1R. A 2-fold increase of resident Iba1+ macrophages was observed in the myenteric plexus of DSS mice associated with a 2.9- and 1.5-fold enhancement of IL-6 and IL-1β mRNA levels, respectively. Conclusion Human colitis and mouse ileitis affect dopamine machinery in the enteric nervous system. Experimentally induced ileitis impairs dopaminergic neurotransmission altering D1R-mediated responses. These findings provide novel information on the involvement of dopaminergic pathways in IBD.

The Colombian Strain of Trypanosoma cruzi Induces a Proinflammatory Profile, Neuronal Death, and Collagen Deposition in the Intestine of C57BL/6 Mice Both during the Acute and Early Chronic Phase.

The objective of this study was to evaluate the histopathological changes caused by infection with the Colombian strain of Trypanosoma cruzi (T. cruzi) in the acute and chronic experimental phases. C57Bl/6 mice were infected with 1000 trypomastigote forms of the Colombian strain of T. cruzi. After 30 days (acute phase) and 90 days (early chronic phase) of infection, the animals were euthanized, and the colon was collected and divided into two parts: proximal and distal. The distal portion was used for histopathological analysis, whereas the proximal portion was used for quantification of pro- and anti-inflammatory cytokines. In addition, the weight of the animals and parasitemia were assessed. The infection induced gradual weight loss in the animals. In addition, the infection induced an increase in interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α) in the intestine in the acute phase, in which this increase continued until the early chronic phase. The same was observed in relation to the presence of intestinal inflammatory infiltrates. In relation to interleukin (IL)-10, there was an increase only in the early chronic phase. The Colombian strain infection was also able to induce neuronal loss in the myenteric plexus and deposition of the collagen fibers during the acute phase. The Colombian strain of T. cruzi is capable of causing histopathological changes in the intestine of infected mice, especially in inducing neuronal destructions. Thus, this strain can also be used to study the intestinal form of Chagas disease in experimental models.