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.

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.

Neurochemical profile of the myenteric plexus in the rat colorectal region.

The enteric nervous system, a major subdivision of the autonomic nervous system, is known for its neurochemical heterogeneity and complexity. The myenteric plexus, one of its two principal components, primarily controls peristalsis and its dysfunction may lead to a number of gastrointestinal motility disorders. The myenteric neurons have been described to use a wide variety of neurotransmitters although no evidence has been reported for the existence of adrenergic neurons in the hindgut. This study aims at elucidating the chemical coding of neurons in the myenteric plexus of the rat colon and anorectal region with particular emphasis on cholinergic and the so-called nonadrenergic, noncholinergic (NANC) transmitter systems. The immunostaining for choline acetyltransferase revealed an intense staining of the myenteric ganglia with clear delineation of their neuronal cell bodies and without local distributional differences in the colonic region. The myenteric ATPergic structures were mostly limited to fiber bundles surrounding unstained myenteric neurons and penetrating the two muscle layers. We also observed an abundance of intensely stained varicose substance P-immunopositive fibers, ensheathing the immunonegative myenteric neuronal cell bodies in a basket-like manner. Applying NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry, we were able to demonstrate numerous nitrergic somata of myenteric neurons with Dogiel Type I morphology. Apart from the observed nitrergic distributional patterns, no distinct variations were found in the staining intensity or distribution of myenteric structures in the colon and anorectal area. Our results suggest that myenteric neurons in the distal intestinal portion utilize a broad spectrum of enteric transmitters, including classical and NANC transmitters.

Postnatal development of the enteric neurons expressing neuronal nitric oxide synthase.

Neurons, expressing neuronal nitric oxide synthase (nNOS) in the enteric ganglia are inhibitory motor neurons or interneurons. The aim of the study was to identify the percentage, cross-sectional area of nNOS-immunoreactive (IR) neurons and their colocalization with choline acetyltransferase (ChAT), vasoactive intestinal polypeptide (VIP), and neuropeptide Y in the intramural ganglia of the myenteric (MP) and submucous plexus (SP) of the small intestine (SI) and large intestine (LI) of rats of different age groups using immunohistochemical methods. In the intramural ganglia of the MP, the largest percentage of nNOS-IR neurons was detected in newborn rats in the LI (81 ± 0.9%) and SI (48 ± 4.1%). Subsequently, it decreased in ontogenesis up to 60 days of life (26 ± 0.9% LI, 29 ± 3.2% SI), and did not change until senescence. In the SP, abundant nNOS-IR neurons were also detected in newborns (82 ± 7.0% SI, 85 ± 3.2% LI), while their percentage decreased significantly in the next 20 days. Furthermore, a very small number of nNOS-IR neurons was detected in 30-day- and 2-month-old animals, but they again appeared in large numbers in aged rats. In the MP, the highest percentage of nNOS+/ChAT+ neurons was in 1-day-old, 10-day-old, and 2-year-old rats. In the SP, the largest number of nNOS-IR neurons colocalized ChAT regardless of age. In the MP of all rats, many nNOS-IR neurons colocalized VIP, and the maximal percentage of nNOS+/VIP+ neurons was found in 2-year-old rats, minimal-in newborns. In conclusion, nNOS expression in neurons of the gut is decreased in early postnatal ontogenesis and subsequently increased in aged rats.

Galanin Receptors (GALR1, GALR2, and GALR3) Immunoexpression in Enteric Plexuses of Colorectal Cancer Patients: Correlation with the Clinico-Pathological Parameters.

Galanin (GAL) is an important neurotransmitter released by the enteric nervous system (ENS) neurons located in the muscularis externa and submucosa enteric plexuses that acts by binding to GAL receptors 1, 2 and 3 (GALR1, 2 and 3). In our previous studies, the GAL immunoexpression was compared in colorectal cancer (CRC) tissue and the adjacent parts of the large intestine wall including myenteric and submucosal plexuses. Recently we have also found that expression levels of GALR1 and GALR3 proteins are elevated in CRC tissue as compared with their expression in epithelial cells of unchanged mucosa. Moreover, higher GALR3 immunoreactivity in CRC cells correlated with better prognosis of CRC patients. To understand the distribution of GALRs in enteric plexuses distal and close to CRC invasion, in the present study we decided to evaluate GALRs expression within the myenteric and submucosal plexuses located proximally and distally to the cancer invasion and correlated the GALRs expression levels with the clinico-pathological data of CRC patients. The immunohistochemical and immunofluorescent methods showed only slightly decreased immunoexpression of GALR1 and GALR3 in myenteric plexuses close to cancer but did not reveal any correlation in the immunoexpression of all three GAL receptors in myenteric plexuses and tumour progression. No significant changes were found between the expression levels of GALRs in submucosal plexuses distal and close to the tumour. However, elevated GALR1 expression in submucosal plexuses in vicinity of CRC correlated with poor prognosis, higher tumour grading and shorter overall survival. When myenteric plexuses undergo morphological and functional alterations characteristic for atrophy, GALRs maintain or only slightly decrease their expression status. In contrast, the correlation between high expression of GALR1 in the submucosal plexuses and overall survival of CRC patients suggest that GAL and GALRs can act as a components of local neuro-paracrine pro-proliferative pathways accelerating the invasion and metastasis of cancer cell. The obtained results suggest an important role of GALR1 in submucosal plexuses function during the progression of CRC and imply that GALR1 expression in submucosal plexuses of ENS could be an important predictive factor for CRC progression.

Nitrergic and Purinergic Nerves in the Small Intestinal Myenteric Plexus and Circular Muscle of Mice and Guinea Pigs.

ATP is an excitatory and inhibitory neurotransmitter, while nitric oxide (NO) is an inhibitory neurotransmitter in the enteric nervous system (ENS). We used a vesicular nucleotide transporter (SLC17A9, VNUT) antibody and a nitric oxide synthase (NOS) antibody to identify purinergic and nitrergic nerves in mouse and guinea ileum. Mouse: VNUT-immunoreactivity (ir) was detected in nerve fibers in myenteric ganglia and circular muscle. VNUT-ir fibers surrounded choline acetyltransferase (ChAT), nitric oxide synthase (nNOS), and calretinin-ir neurons. VNUT-ir nerve cell bodies were not detected. Tyrosine hydroxylase (TH)-ir nerves were detected in myenteric ganglia and the tertiary plexus. Guinea pig: VNUT-ir was detected in neurons and nerves fibers and did not overlap with NOS-ir nerve fibers. VNUT-ir was detected in nerve fibers in ganglia but not nerve cell bodies. VNUT-ir nerve fibers surrounded NOS-ir and NOS- neurons. NOS-ir and VNUT-ir nerve fibers did not overlap in myenteric ganglia or circular muscle. VNUT-ir nerves surrounded some ChAT-ir neurons. VNUT-ir and ChAT-ir were detected in separate nerves in the CM. VNUT-ir nerve fibers surrounded calretinin-ir neurons.Conclusions: VNUT-ir neurons likely mediate purinergic signaling in small intestinal myenteric ganglia and circular muscle. ATP and NO are likely released from different inhibitory motorneurons. VNUT-ir and ChAT-ir interneurons mediate cholinergic and purinergic synaptic transmission in the myenteric plexus.

Identifying Types of Neurons in the Human Colonic Enteric Nervous System.

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon.

PTEN Immunohistochemistry.

CONTEXT.­: Intestinal neuronal dysplasia type B (IND B) is a complex entity involving the enteric nervous system, clinically manifested with constipation in infancy. Diagnosis has been established by histopathologic analysis of rectal biopsies. However, the criteria for the diagnosis have been questioned and modified, hindering diagnostic practice. OBJECTIVE.­: To analyze the applicability of PTEN immunohistochemistry in the diagnosis of IND B and to compare with control cases and cases of Hirschsprung disease (HD). DESIGN.­: PTEN immunohistochemical expression was analyzed in colorectal samples from 29 cases of IND B and compared with 4 control cases and 6 cases of HD. The pattern of PTEN immunoexpression was analyzed in glial cells of the submucosal and myenteric nerve plexuses and in neural fibrils of the muscularis propria using a scoring system. RESULTS.­: Marked reduction or absence of PTEN expression was observed in glial cells of the submucosal nerve plexuses in all cases of the IND B group and in the myenteric nerve plexuses in 28 of 29 cases (96.5%). Lack of PTEN expression was detected in neural fibrils within the muscularis propria in 21 of 29 cases (72%) of the IND B group. PTEN expression was positive in the same neural structures of the control and HD groups. CONCLUSIONS.­: PTEN immunohistochemistry may be a valuable tool in the diagnostic evaluation of IND B. Lack of or reduction of PTEN expression in neural fibrils within the muscularis propria suggests that involvement of the neuromuscular junction may be a key event in the pathogenesis of the motility disturbance occurring in IND B.

Intestinal Region-Specific and Layer-Dependent Induction of TNFα in Rats with Streptozotocin-Induced Diabetes and after Insulin Replacement.

Tumour necrosis factor alpha (TNFα) is essential in neuroinflammatory modulation. Therefore, the goal of this study is to reveal the effects of chronic hyperglycaemia and insulin treatment on TNFα expression in different gut segments and intestinal wall layers. TNFα expression was mapped by fluorescent immunohistochemistry and quantitative immunogold electron microscopy in myenteric ganglia of duodenum, ileum and colon. Tissue TNFα levels were measured by enzyme-linked immunosorbent assays in muscle/myenteric plexus-containing (MUSCLE-MP) and mucosa/submucosa/submucous plexus-containing (MUC-SUBMUC-SP) homogenates. Increasing density of TNFα-labelling gold particles is observed in myenteric ganglia from proximal to distal segments and TNFα tissue levels are much more elevated in MUSCLE-MP homogenates than in MUC-SUBMUC-SP samples in healthy controls. In the diabetics, the number of TNFα gold labels is significantly increased in the duodenum, decreased in the colon and remained unchanged in the ileal ganglia, while insulin does not prevent these diabetes-related TNFα changes. TNFα tissue concentration is also increased in MUSCLE-MP homogenates of diabetic duodenum, while decreased in MUC-SUBMUC-SP samples of diabetic ileum and colon. These findings support that type 1 diabetes has region-specific and intestinal layer-dependent effects on TNFα expression, contributing to the regional damage of myenteric neurons and their intestinal milieu.

Optogenetic activation of the distal colon epithelium engages enteric nervous system circuits to initiate motility patterns.

Digestive functions of the colon depend on sensory-motor reflexes in the enteric nervous system (ENS), initiated by intrinsic primary afferent neurons (IPANs). IPAN terminals project to the mucosal layer of the colon, allowing communication with epithelial cells comprising the colon lining. The chemical nature and functional significance of this epithelial-neural communication in regard to secretion and colon motility are of high interest. Colon epithelial cells can produce and release neuroactive substances such as ATP and 5-hydroxytryptamine (5-HT), which can activate receptors on adjacent nerve fibers, including IPAN subtypes. In this study, we examined if stimulation of epithelial cells alone is sufficient to activate neural circuits that control colon motility. Optogenetics and calcium imaging were used in ex vivo preparations of the mouse colon to selectively stimulate the colon epithelium, measure changes in motility, and record activity of neurons within the myenteric plexus. Light-mediated activation of epithelial cells lining the distal, but not proximal, colon caused local contractions and increased the rate of colonic migrating motor complexes. Epithelial-evoked local contractions in the distal colon were reduced by both ATP and 5-HT receptor antagonists. Our findings indicate that colon epithelial cells likely use purinergic and serotonergic signaling to initiate activity in myenteric neurons, produce local contractions, and facilitate large-scale coordination of ENS activity responsible for whole colon motility patterns.NEW & NOTEWORTHY Using an all-optical approach to measure real-time cell-to-cell communication responsible for colon functions, we show that selective optogenetic stimulation of distal colon epithelium produced activity in myenteric neurons, as measured with red genetically encoded calcium indicators. The epithelial-induced neural response led to local contractions, mediated by both purinergic and serotonergic signaling, and facilitated colonic motor complexes that propagate from proximal to distal colon.

Pathophysiological Roles of Neuro-Immune Interactions between Enteric Neurons and Mucosal Mast Cells in the Gut of Food Allergy Mice.

Recently, the involvement of the nervous system in the pathology of allergic diseases has attracted increasing interest. However, the precise pathophysiological role of enteric neurons in food allergies has not been elucidated. We report the presence of functional high-affinity IgE receptors (FcεRIs) in enteric neurons. FcεRI immunoreactivities were observed in approximately 70% of cholinergic myenteric neurons from choline acetyltransferase-eGFP mice. Furthermore, stimulation by IgE-antigen elevated intracellular Ca2+ concentration in isolated myenteric neurons from normal mice, suggesting that FcεRIs are capable of activating myenteric neurons. Additionally, the morphological investigation revealed that the majority of mucosal mast cells were in close proximity to enteric nerve fibers in the colonic mucosa of food allergy mice. Next, using a newly developed coculture system of isolated myenteric neurons and mucosal-type bone-marrow-derived mast cells (mBMMCs) with a calcium imaging system, we demonstrated that the stimulation of isolated myenteric neurons by veratridine caused the activation of mBMMCs, which was suppressed by the adenosine A3 receptor antagonist MRE 3008F20. Moreover, the expression of the adenosine A3 receptor gene was detected in mBMMCs. Therefore, in conclusion, it is suggested that, through interaction with mucosal mast cells, IgE-antigen-activated myenteric neurons play a pathological role in further exacerbating the pathology of food allergy.

Evidence of a Myenteric Plexus Barrier and Its Macrophage-Dependent Degradation During Murine Colitis: Implications in Enteric Neuroinflammation.

BACKGROUND & AIMS: Neuroinflammation in the gut is associated with many gastrointestinal (GI) diseases, including inflammatory bowel disease. In the brain, neuroinflammatory conditions are associated with blood-brain barrier (BBB) disruption and subsequent neuronal injury. We sought to determine whether the enteric nervous system is similarly protected by a physical barrier and whether that barrier is disrupted in colitis. METHODS: Confocal and electron microscopy were used to characterize myenteric plexus structure, and FITC-dextran assays were used to assess for presence of a barrier. Colitis was induced with dextran sulfate sodium, with co-administration of liposome-encapsulated clodronate to deplete macrophages. RESULTS: We identified a blood-myenteric barrier (BMB) consisting of extracellular matrix proteins (agrin and collagen-4) and glial end-feet, reminiscent of the BBB, surrounded by a collagen-rich periganglionic space. The BMB is impermeable to the passive movement of 4 kDa FITC-dextran particles. A population of macrophages is present within enteric ganglia (intraganglionic macrophages [IGMs]) and exhibits a distinct morphology from muscularis macrophages, with extensive cytoplasmic vacuolization and mitochondrial swelling but without signs of apoptosis. IGMs can penetrate the BMB in physiological conditions and establish direct contact with neurons and glia. Dextran sulfate sodium-induced colitis leads to BMB disruption, loss of its barrier integrity, and increased numbers of IGMs in a macrophage-dependent process. CONCLUSIONS: In intestinal inflammation, macrophage-mediated degradation of the BMB disrupts its physiological barrier function, eliminates the separation of the intra- and extra-ganglionic compartments, and allows inflammatory stimuli to access the myenteric plexus. This suggests a potential mechanism for the onset of neuroinflammation in colitis and other GI pathologies with acquired enteric neuronal dysfunction.

The α isoform of cGMP-dependent protein kinase 1 (PKG1α) is expressed and functionally important in intrinsic primary afferent neurons of the guinea pig enteric nervous system.

BACKGROUND: Intrinsic primary afferent neurons (IPANs) enable the gut to manifest reflexes in the absence of CNS input. PKG1α is selectively expressed in a subset of neurons in dorsal root ganglia (DRG) and has been linked to nociception and long-term hyperexcitability. METHODS: We used immunoblotting, immunocytochemistry, and in vitro assays of IPAN-dependent enteric functions to test hypotheses that subsets of primary neurons of the ENS and DRG share a reliance on PKG1α expression. KEY RESULTS: PKG1α immunoreactivity was demonstrated in immunoblots from isolated myenteric ganglia. PKG1α, but not PKG1ß, immunoreactivity, was coincident with that of neuronal markers (HuC/D; ß3-tubulin) in both enteric plexuses. PKG1α immunoreactivity also co-localized with the immunoreactivities of the IPAN markers, calbindin (100%; myenteric plexus) and cytoplasmic NeuN (98 ± 1% submucosal plexus). CGRP-immunoreactive DRG neurons, identified as visceral afferents by retrograde transport, were PKG1α-immunoreactive. We used intraluminal cholera toxin to determine whether PKG1α was necessary to enable stimulation of the mucosa to activate Fos in enteric neurons. Tetrodotoxin (1.0 µM), low Ca2+ /high Mg2+ media, and the PKG inhibitor, N46 (100 µM), all inhibited Fos activation in myenteric neurons. N46 also concentration dependently inhibited peristaltic reflexes in isolated preparations of distal colon (IC50  = 83.3 ± 1.3 µM). CONCLUSIONS & INFERENCES: These data suggest that PKG1α is present and functionally important in IPANs and visceral afferent nociceptive neurons.

Vincristine leads to colonic myenteric neurons injury via pro-inflammatory macrophages activation.

Vincristine is widely used in treatment of various malignant tumors. The clinical application of vincristine is accompanied by peripheral neurotoxicity which might not be strictly related to the mechanism of anti-tumor action. There are several possible mechanisms but the effect of vincristine on enteric neurons and the underlying mechanism are still unclear. C57BL6/J mice were systematically treated with vincristine for 10 days, and macrophages were depleted using clodronate liposomes. The colonic myenteric plexus neurons were extracted and cultured in vitro. Macrophages from different parts were extracted in an improved way. In the current study, we demonstrated that system treatment of vincristine resulted in colonic myenteric neurons injury, pro-inflammatory macrophages activation and total gastrointestinal transport time increase. Vincristine promoted the pro-inflammatory macrophages activation individually or in coordination with LPS and increased the expression of pro-inflammatory factors IL-1ß, IL-6, TNF-α via increasing the phosphorylation of ERK1/2 and p38. In addition, pro-inflammatory macrophages led to colonic myenteric neurons apoptosis targeting on SGK1-FOXO3 pathway. These effects were attenuated by inhibitors of the ERK1/2 and p38-MAPK pathways. Importantly, macrophages depletion alleviated colonic myenteric neurons injury and the delay of gastrointestinal motility caused by system treatment of vincristine. Taken together, system treatment of vincristine led to colonic myenteric neurons injury via pro-inflammatory macrophages activation which was alleviated by depletion of macrophages.

Correlation between intestinal BMP2, IFNγ, and neural death in experimental infection with Trypanosoma cruzi.

Megacolon is one of the main late complications of Chagas disease, affecting approximately 10% of symptomatic patients. However, studies are needed to understand the mechanisms involved in the progression of this condition. During infection by Trypanosoma cruzi (T. cruzi), an inflammatory profile sets in that is involved in neural death, and this destruction is known to be essential for megacolon progression. One of the proteins related to the maintenance of intestinal neurons is the type 2 bone morphogenetic protein (BMP2). Intestinal BMP2 homeostasis is directly involved in the maintenance of organ function. Thus, the aim of this study was to correlate the production of intestinal BMP2 with immunopathological changes in C57Bl/6 mice infected with the T. cruzi Y strain in the acute and chronic phases. The mice were infected with 1000 blood trypomastigote forms. After euthanasia, the colon was collected, divided into two fragments, and a half was used for histological analysis and the other half for BMP2, IFNγ, TNF-α, and IL-10 quantification. The infection induced increased intestinal IFNγ and BMP2 production during the acute phase as well as an increase in the inflammatory infiltrate. In contrast, a decreased number of neurons in the myenteric plexus were observed during this phase. Collagen deposition increased gradually throughout the infection, as demonstrated in the chronic phase. Additionally, a BMP2 increase during the acute phase was positively correlated with intestinal IFNγ. In the same analyzed period, BMP2 and IFNγ showed negative correlations with the number of neurons in the myenteric plexus. As the first report of BMP2 alteration after infection by T. cruzi, we suggest that this imbalance is not only related to neuronal damage but may also represent a new route for maintaining the intestinal proinflammatory profile during the acute phase.

T cells show preferential adhesion to enteric neural cells in culture and are close to neural cells in the myenteric ganglia of Crohn's patients.

Plexitis in the proximal margin of intestinal resections are associated with post-operative recurrence of Crohn's disease. To understand their formation, in vitro analyzes were performed. T cells adhered preferentially to neuron and glial cells in mixed primary cultures of enteric nervous system and T cell activation increased their adhesion capacity. Higher number of T lymphocytes in close proximity to enteric glial cells was also observed in the myenteric ganglia of Crohn's patients as compared to control. These data show that close proximity between lymphocytes and enteric neural cells exists and may contribute to the formation of plexitis.

Neurochemical Plasticity of nNOS-, VIP- and CART-Immunoreactive Neurons Following Prolonged Acetylsalicylic Acid Supplementation in the Porcine Jejunum.

Aspirin, also known as acetylsalicylic acid (ASA), is a commonly used anti-inflammatory drug that has analgesic and antipyretic properties. The side effects are well known, however, knowledge concerning its influence on gastric and intestinal innervation is limited. The enteric nervous system (ENS) innervates the whole gastrointestinal tract (GIT) and is comprised of more than one hundred million neurons. The capacity of neurons to adapt to microenvironmental influences, termed as an enteric neuronal plasticity, is an essential adaptive response to various pathological stimuli. Therefore, the goal of the present study was to determine the influence of prolonged ASA supplementation on the immunolocalization of neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP) and cocaine- and amphetamine- regulated transcript peptide (CART) in the porcine jejunum. The experiment was performed on 8 Pietrain × Duroc immature gilts. Using routine double-labelling immunofluorescence, we revealed that the ENS nerve cells underwent adaptive changes in response to the induced inflammation, which was manifested by upregulated or downregulated expression of the studied neurotransmitters. Our results suggest the participation of nNOS, VIP and CART in the development of inflammation and may form the basis for further neuro-gastroenterological research.

Anti- and pro-oxidant effects of quercetin stabilized by microencapsulation on interstitial cells of Cajal, nitrergic neurons and M2-like macrophages in the jejunum of diabetic rats.

Given the well-known antioxidant and neuroprotective properties of quercetin, the aim of this work was to evaluate the effects of quercetin stabilized by microencapsulation at two doses (10 mg kg-1 and 100 mg kg-1) on the oxidative/antioxidant status, number and morphological features of ICC, nitrergic neurons and M2-like macrophages in jejunum of diabetic rats. The rats were randomly distributed into six groups: normoglycemic control (N), diabetic control (D) and either normoglycemic or diabetic groups treated with quercetin-loaded microcapsules at a dose of 10 mg kg-1 (NQ10 and DQ10, respectively) or 100 mg kg-1 (NQ100 and DQ100, respectively). After 60 days, the jejunum was collected. Whole mounts were immunostained for Ano1, nNOS and CD206, and oxidative stress levels and total antioxidant capacity of the jejunum were measured. Diabetes led to a loss of ICC and nitrergic neurons, but increased numbers of M2-like macrophages and elevated levels of oxidative stress were seen in diabetic animals. High-dose administration of quercetin (100 mg kg-1) further aggravated the diabetic condition (DQ100) but this treatment resulted in harmful effects on healthy rats (NQ100), pointing to a pro-oxidant activity. However, low-dose administration of quercetin (10 mg kg-1) gave rise to antioxidant and protective effects on ICC, nNOS, macrophages and oxidative/antioxidant status in DQ100, but NQ100 displayed infrequent negative outcomes in normoglycemic animals. Microencapsulation of the quercetin may become promising alternatives to reduce diabetes-induced oxidative stress but antioxidant therapies should be careful used under healthy status to avoid toxic effects.

Neuroprotection and immunomodulation of progesterone in the gut of a mouse model of Parkinson's disease.

Gastrointestinal symptoms appear in Parkinson's disease patients many years before motor symptoms, suggesting the implication of dopaminergic neurones of the gut myenteric plexus. Inflammation is also known to be increased in PD. We previously reported neuroprotection with progesterone in the brain of mice lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and hypothesised that it also has neuroprotective and immunomodulatory activities in the gut. To test this hypothesis, we investigated progesterone administered to adult male C57BL/6 mice for 10 days and treated with MPTP on day 5. In an additional experiment, progesterone was administered for 5 days following MPTP treatment. Ilea were collected on day 10 of treatment and microdissected to isolate the myenteric plexus. Dopaminergic neurones were reduced by approximately 60% and pro-inflammatory macrophages were increased by approximately 50% in MPTP mice compared to intact controls. These changes were completely prevented by progesterone administered before and after MPTP treatment and were normalised by 8 mg kg-1 progesterone administered after MPTP. In the brain of MPTP mice, brain-derived neurotrophic peptide (BDNF) and glial fibrillary acidic protein (GFAP) were associated with progesterone neuroprotection. In the myenteric plexus, increased BDNF levels compared to controls were measured in MPTP mice treated with 8 mg kg-1 progesterone started post MPTP, whereas GFAP levels remained unchanged. In conclusion, the results obtained in the present study show neuroprotective and anti-inflammatory effects of progesterone in the myenteric plexus of MPTP mice that are similar to our previous findings in the brain. Progesterone is non-feminising and could be used for both men and women in the pre-symptomatic stages of the disease.

Increased Fibronectin Impairs the Function of Excitatory/Inhibitory Synapses in Hirschsprung Disease.

Although approximately 50% of cases have a known genetic defect, the precise pathogenesis of Hirschsprung disease (HSCR) is still unclear. We recently reported that expression of fibronectin (FN), which is involved in the migration, colonization, and differentiation of enteric neural crest cells (ENCCs), is increased in aganglionic colonic segments obtained from patients. We hypothesized that abnormally high levels of FN might play a role in the etiology of HSCR. Here, to test this hypothesis, we investigated aganglionic, transitional, and ganglionic colon segments from 63 children with HSCR and distal colon from thirty healthy Wistar rats at embryonic day 20, in addition to in vitro studies with PC12 Adh neural crest cells. We measured the protein and mRNA expression levels of FN, together with a panel of excitatory (VGLUT1, GluA1, GluN1, PSD-95, and NL-1) and inhibitory (GAD67, GABA AR-α1, NL-2, and SLC32) synaptic markers. Expression of all these synaptic markers was significantly decreased in aganglionic colon, compared to ganglionic colon, whereas expression of FN was significantly increased. In a neural crest cell line, PC12 Adh, knockdown of FN with small-interfering RNA increased the expression of synaptic markers. Co-culture of colons from embryonic day 20 rats with RGD recombinant protein, which contains the RGD motif of FN, reduced the expression of excitatory and inhibitory synaptic markers. These results are consistent with the idea that the etiology of HSCR involves aberrant overexpression of FN, which may impair synaptic function and enteric nervous system development, leading to motor dysfunction of intestinal muscles.