Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno-associated virus capsid.

BACKGROUND: Intravenous administration of adeno-associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV-PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV-PHP.S with a tunable two-component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). METHODS: A vector system including a tetracycline inducer with a tet-responsive element driving three separate fluorophores was packaged in the AAV-PHP.S capsid. The vectors were injected retro-orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. KEY RESULTS: The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon-axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. CONCLUSIONS & INFERENCES: The multicolor AAV-PHP.S-packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro-circuitry.

Role of PD-L1 Expression during the Progression of Submucosal Gastric Cancer.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) expression is a prognostic marker for gastric cancer that correlates with tumor diameter and depth of penetration. But the role of PD-L1 and mechanism(s) employed in the initial phase of invasion in early gastric cancer is yet to be understood. OBJECTIVE: This study aims to elucidate the role of PD-L1 during the progression of gastric cancer, specifically invading the submucosa beyond the lamina muscularis mucosa. METHODS: Using 107 patients with pathological submucosal gastric cancer, we determined the expression of PD-L1 based on the staining of the cell membrane or cytoplasm of tumor cells in the central and invasive front of the tumor. Samples were categorized into 3 groups based on the intensity of PD-L1 expression. CD8+ lymphocytes expressing PD-1 and CD163+ macrophages were used to determine the number of cell nuclei at the invasive front, similar to PD-L1. CMTM6 levels were determined and used to stratify samples into 3 groups. RESULTS: PD-L1 expression was higher in the invasive front (26.2%) than in the central portion of the tumors (7.4%; p < 0.001). Moreover, lymphatic and vascular invasion were more frequently observed in samples with high levels of PD-L1 (lymphatic invasion: 60.7 vs. 35.4%, p = 0.0026, and vascular invasion: 39.3 vs. 16.5%, p = 0.0018). There was no correlation between PD-L1 expression and the levels of PD-1, CD8, CD163, and CMTM6. CONCLUSIONS: PD-L1-expressing cancer cells at the invasive front of gastric cancer influence the initial stages of tumor invasion and lymphovascular permeation in early-stage gastric cancers. Immune checkpoint signaling may be the driving force in the invasive front during the invasion of the submucosa beyond the lamina muscularis mucosa.

The Endocrine Disruptor Bisphenol A (BPA) Affects the Enteric Neurons Immunoreactive to Neuregulin 1 (NRG1) in the Enteric Nervous System of the Porcine Large Intestine.

The enteric nervous system (ENS), located in the wall of the gastrointestinal (GI) tract, is characterized by complex organization and a high degree of neurochemical diversity of neurons. One of the less known active neuronal substances found in the enteric neurons is neuregulin 1 (NRG1), a factor known to be involved in the assurance of normal development of the nervous system. During the study, made up using the double immunofluorescence technique, the presence of NRG1 in the ENS of the selected segment of porcine large intestine (caecum, ascending and descending colon) was observed in physiological conditions, as well as under the impact of low and high doses of bisphenol A (BPA) which is commonly used in the production of plastics. In control animals in all types of the enteric plexuses, the percentage of NRG1-positive neurons oscillated around 20% of all neurons. The administration of BPA caused an increase in the number of NRG1-positive neurons in all types of the enteric plexuses and in all segments of the large intestine studied. The most visible changes were noted in the inner submucous plexus of the ascending colon, where in animals treated with high doses of BPA, the percentage of NRG1-positive neurons amounted to above 45% of all neuronal cells. The mechanisms of observed changes are not entirely clear, but probably result from neurotoxic, neurodegenerative and/or proinflammatory activity of BPA and are protective and adaptive in nature.

Effects and sites of action of a M1 receptor positive allosteric modulator on colonic motility in rats and dogs compared with 5-HT4 agonism and cholinesterase inhibition.

BACKGROUND: Muscarinic receptor 1 positive allosteric modulators (M1PAMs) enhance colonic propulsive contractions and defecation through the facilitation of M1 receptor (M1R)-mediated signaling. We examined M1R expression in the colons of 5 species and compared colonic propulsion and defecation caused by the M1PAM, T440, the 5-HT4 agonist, prucalopride, and the cholinesterase inhibitor, neostigmine, in rats and dogs. METHODS: M1R expression was profiled by immunostaining and in situ hybridization. In vivo studies utilized male SD rats and beagle dogs. Colonic propulsive contractions were recorded by manometry in anesthetized rats. Gut contractions in dogs were assessed using implanted force transducers in the ileum, proximal, mid, and distal colons. KEY RESULTS: M1R was localized to neurons of myenteric and submucosal plexuses and the epithelium of the human colon. A similar receptor localization was observed in rat, dog, mouse, and pig. T440 enhanced normal defecation in rats in a dose-dependent manner. Prucalopride also enhanced defecation in rats, but the maximum effect was half that of T440. Neostigmine and T440 were similarly effective in enhancing defecation, but the effective dose of neostigmine was close to its lethal dose. In rats, all 3 compounds induced colonic contractions, but the associated propulsion was strongest with T440. In dogs, intestinal contractions elicited by T440 propagated from ileum to distal colon. Prucalopride and neostigmine also induced intestinal contractions, but these were less well coordinated. No loss of effectiveness of T440 on defecation occurred after 5 days of repeated dosing. CONCLUSION AND INFERENCES: These results suggest that M1PAMs produce highly coordinated propagating contraction by actions on the enteric nervous system of the colon. The localization of M1R to enteric neurons in both animals and humans suggests that the M1PAM effects would be translatable to human. M1PAMs provide a potential novel therapeutic option for constipation disorders.

Increased expression of CART, nNOS, VIP, PACAP, SP and GAL in enteric neurons of the porcine stomach prepyloric region following hydrochloric acid infusion.

INTRODUCTION: Stomach hyperacidity leads to damage of the mucus/bicarbonate barrier, ulcerations and the development of stomach cancer. Key regulators of the mucosal barrier/luminal acid balance are neurotransmitters secreted by intramural neurons. The aim of the current study was to determine the expression of gastric neuropeptides and nNOS in the porcine stomach following hydrochloric acid instillation. We report on increased expression of enteric neurotransmitters involved in adaptive reaction to an experimentally-induced hyperacidity state. MATERIAL AND METHODS: The investigation was conducted on eight 12-18 kg pigs. The influence of intragastric infusion of hydrochloric acid on the expression of cocaine- and amphetamine-regulated transcript peptide (CART), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), substance P (SP) and galanin (GAL) in the submucous and myenteric gastric neurons of the pig has been studied with double immunofluorescence. RESULTS: A mimicked hyperacidity state significantly increased the proportion of enteric neurons immunoreactive to CART, nNOS, VIP, PACAP, SP and GAL in the submucous gastric neurons. In the myenteric plexus, a significant increase of the number of VIP-, CART- and GAL-immunoreactive (IR) neurons was found. Similarly, the percentage of myenteric nNOS-IR and PACAP-IR neurons tended to increase, while the fraction of SP-IR cells did not change. CONCLUSIONS: Stomach hyperacidity modifies the expression of the studied neurotransmitters in a specific way depending on the location of the neurons in particular plexuses of the stomach. Increased numbers of neurons expressing CART, nNOS, VIP, PACAP, SP and GAL clearly indicate their regulatory engagement in the restoration of the physiological gastric balance following hyperacidity.

Influence of Acrylamide Administration on the Neurochemical Characteristics of Enteric Nervous System (ENS) Neurons in the Porcine Duodenum.

The digestive tract, especially the small intestine, is one of the main routes of acrylamide absorption and is therefore highly exposed to the toxic effect of acrylamide contained in food. The aim of this experiment was to elucidate the effect of low (tolerable daily intake-TDI) and high (ten times higher than TDI) doses of acrylamide on the neurochemical phenotype of duodenal enteric nervous system (ENS) neurons using the pig as an animal model. The experiment was performed on 15 immature gilts of the Danish Landrace assigned to three experimental groups: control (C) group-pigs administered empty gelatine capsules, low dose (LD) group-pigs administered capsules with acrylamide at the TDI dose (0.5 µg/kg body weight (b.w.)/day), and the high dose (HD) group-pigs administered capsules with acrylamide at a ten times higher dose than the TDI (5 µg/kg b.w./day) with a morning feeding for 4 weeks. Administration of acrylamide, even in a low (TDI) dose, led to an increase in the percentage of enteric neurons immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), neuronal nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VACHT) in the porcine duodenum. The severity of the changes clearly depended on the dose of acrylamide and the examined plexus. The obtained results suggest the participation of these neuroactive substances in acrylamide-inducted plasticity and the protection of ENS neurons, which may be an important line of defence from the harmful action of acrylamide.

Non-sulfated cholecystokinin-8 increases enteric and hindbrain Fos-like immunoreactivity in male Sprague Dawley rats.

Recently, we reported that non-sulfated cholecystokinin-8 (NS CCK-8) reduces food intake by cholecystokinin-B receptors (CCK-BR). To examine a possible site of action for this peptide, and based on the fact that both NS CCK-8 and CCK-BR are found centrally and peripherally, in the current study we hypothesized that NS CCK-8 increases Fos-like immunoreactivity (Fos-LI, a neuronal activation marker) in the dorsal vagal complex (DVC) of the hindbrain and the myenteric and submucosal plexuses of the small intestine. We found that intraperitoneal NS CCK-8 (0.5 nmol/kg) increases Fos-LI in the DVC, the myenteric and the submucosal plexuses of the duodenum and the myenteric plexus of the jejunum. The findings suggest, but does not prove, a potential role for the DVC and the enteric neurons in the feeding responses evoked by NS CCK-8.

Characterisation of tau in the human and rodent enteric nervous system under physiological conditions and in tauopathy.

Tau is normally a highly soluble phosphoprotein found predominantly in neurons. Six different isoforms of tau are expressed in the adult human CNS. Under pathological conditions, phosphorylated tau aggregates are a defining feature of neurodegenerative disorders called tauopathies. Recent findings have suggested a potential role of the gut-brain axis in CNS homeostasis, and therefore we set out to examine the isoform profile and phosphorylation state of tau in the enteric nervous system (ENS) under physiological conditions and in tauopathies. Surgical specimens of human colon from controls, Parkinson's disease (PD) and progressive supranuclear palsy (PSP) patients were analyzed by Western Blot and immunohistochemistry using a panel of anti-tau antibodies. We found that adult human ENS primarily expresses two tau isoforms, localized in the cell bodies and neuronal processes. We did not observe any difference in the enteric tau isoform profile and phosphorylation state between PSP, PD and control subjects. The htau mouse model of tauopathy also expressed two main isoforms of human tau in the ENS, and there were no apparent differences in ENS tau localization or phosphorylation between wild-type and htau mice. Tau in both human and mouse ENS was found to be phosphorylated but poorly susceptible to dephosphorylation with lambda phosphatase. To investigate ENS tau phosphorylation further, primary cultures from rat enteric neurons, which express four isoforms of tau, were pharmacologically manipulated to show that ENS tau phosphorylation state can be regulated, at least in vitro. Our study is the first to characterize tau in the rodent and human ENS. As a whole, our findings provide a basis to unravel the functions of tau in the ENS and to further investigate the possibility of pathological changes in enteric neuropathies and tauopathies.

Cocaine- and amphetamine-regulated transcript (CART) peptide in the enteric nervous system of the porcine esophagus.

Cocaine- and amphetamine-regulated transcript peptide (CART) is widely distributed within the central and peripheral nervous system. In the brain, CART is considered as the main anorectic peptide involved in the regulation of food intake. Contrary to the central nervous system, a lot of aspects connected with the distribution and functions of CART within the enteric nervous system (ENS) still remain unknown. The aim of the present study was to investigate, for the first time, the population of CART-like immunoreactive (CART-LI) neurons within the porcine esophagus and the denotation of their neurochemical coding. During this experiment, the distribution of CART-LI neurons and the colocalization of CART with other neuronal active substances were examined using standard double- and triple-immunofluorescence techniques in enteric plexuses of cervical, thoracic, and abdominal esophagus fragments. The obtained results showed that CART is present in a relatively high percentage of esophageal neurons (values fluctuated from 45.2±0.9% in the submucous plexus of the thoracic esophagus to 58.1±5.0% in the myenteric plexus of the same fragment of the esophagus). Moreover, CART colocalized with a wide range of other active neuronal substances, mainly with the vesicular acetylcholine transporter (VAChT, a marker of cholinergic neurons), neuronal isoform of nitric oxide synthase (nNOS, a marker of nitrergic neurons), vasoactive intestinal polypeptide (VIP) and galanin (GAL). The number of CART-positive neuronal cells and their neurochemical coding clearly depended on the fragment of esophagus studied and the type of enteric plexus. The obtained results suggest that CART may play important and multidirectional roles in the neuronal regulation of esophageal functions.

Peripheral N-methyl-D-aspartate receptor localization and role in gastric acid secretion regulation: immunofluorescence and pharmacological studies.

The enteric nervous system (ENS) and a glutamate receptor (GluR), N-methyl-D-aspartate receptor (NMDAR), participate in gastric acid secretion (GAS) regulation. NMDARs are localized in different stomach cells; however, knowledge of NMDAR expression and function in the ENS is limited. In the present study, we clarified the types of stomach cells that express the NMDARs that are involved in GAS regulation. The pharmacological method of isolated stomach perfusion by Ghosh and Shild combined with direct mapping of NMDARs by fluorescence microscopy in the rat stomach was employed. By immunofluorescence labeling with an anti-NMDA-NR1 antibody, NMDARs were found to be highly expressed in nerve cells of the submucosal and myenteric plexuses in the stomach. The exact localization of the NMDARs relevant to GAS and its mechanism of action were determined by stimulating different receptors of neuronal and stomach cells using specific secretagogues for NMDA and by selectively blocking those receptors. NMDARs relevant to GAS stimulation are mainly localized in cholinergic interneurons; however, all of the nerve cells of the submucosal ganglia are involved in the stimulating process. In addition, the NMDARs in parietal cells are involved in gastric acid inhibition via influencing H2-histamine receptors.

Calbindin D28k-Immunoreactivity in Human Enteric Neurons.

Calbindin (CALB) is well established as immunohistochemical marker for intrinsic primary afferent neurons in the guinea pig gut. Its expression by numerous human enteric neurons has been demonstrated but little is known about particular types of neurons immunoreactive for CALB. Here we investigated small and large intestinal wholemount sets of 26 tumor patients in order to evaluate (1) the proportion of CALB⁺ neurons in the total neuron population, (2) the colocalization of CALB with calretinin (CALR), somatostatin (SOM) and vasoactive intestinal peptide (VIP) and (3) the morphology of CALB+ neurons. CALB+ neurons represented a minority of myenteric neurons (small intestine: 31%; large intestine: 25%) and the majority of submucosal neurons (between 72 and 95%). In the submucosa, most CALB⁺ neurons co-stained for CALR and VIP (between 69 and 80%) or for SOM (between 20 and 3%). In the myenteric plexus, 85% of CALB+ neurons did not co-stain with the other markers investigated. An unequivocal correlation between CALB reactivity and neuronal morphology was found for myenteric type III neurons in the small intestine: uniaxonal neurons with long, slender and branched dendrites were generally positive for CALB. Since also other neurons displayed occasional CALB reactivity, this protein is not suited as an exclusive marker for type III neurons.

Downregulation of neuronal vasoactive intestinal polypeptide in Parkinson's disease and chronic constipation.

BACKGROUND: Chronic constipation (CC) is a common and severe gastrointestinal complaint in Parkinson's disease (PD), but its pathogenesis remains poorly understood. This study evaluated functionally distinct submucosal neurons in relation to colonic motility and anorectal function in PD patients with constipation (PD/CC) vs both CC and controls. METHODS: Twenty-nine PD/CC and 10 Rome III-defined CC patients were enrolled. Twenty asymptomatic age-sex matched subjects served as controls. Colonic transit time measurement and conventional anorectal manometry were evaluated in PD/CC and CC patients. Colonoscopy was performed in all three groups. Colonic submucosal whole mounts from PD/CC, CC, and controls were processed for immunohistochemistry with antibodies for vasoactive intestinal polypeptide (VIP) and peripheral choline acetyltransferase, markers for functionally distinct submucosal neurons. The mRNA expression of VIP and its receptors were also assessed. KEY RESULTS: Four subgroups of PD/CC patients were identified: delayed colonic transit plus altered anorectal manometry (65%); delayed colonic transit (13%); altered manometric pattern (13%); and no transit and manometric impairment (9%). There were no differences in the number of neurons/ganglion between PD/CC vs CC or vs controls. A reduced number of submucosal neurons containing VIP immunoreactivity was found in PD/CC vs controls (P<.05). VIP, VIPR1, and VIPR2 mRNA expression was significantly reduced in PD/CC vs CC and controls (P<.05). CONCLUSIONS AND INFERENCES: Colonic motor and rectal sensory functions are impaired in most PD/CC patients. These abnormalities are associated with a decreased VIP expression in submucosal neurons. Both sensory-motor abnormalities and neurally mediated motor and secretory mechanisms are likely to contribute to PD/CC pathophysiology.

Decreased expression of NEDL2 in Hirschsprung's disease.

PURPOSE: NEDD4-like ubiquitin protein ligase 2 (NEDL2) plays an important role in many physiological and pathological processes. NEDL2 is a positive regulator of GDNF/Ret signaling during enteric neurogenesis. Mice lacking NEDL2 exhibit decreased numbers of enteric neurons, progressive bowel dysmotility and intestinal hypoganglionosis. We designed this study to investigate the expression of NEDL2 in the normal human colon and in HSCR. METHODS: HSCR tissue specimens (n=10) were collected at the time of pull-through surgery and divided into aganglionic and ganglionic segments. Colonic control samples (n=10) were obtained from patients with imperforate anus at the time of colostomy closure. Immunolabeling of NEDL2 was visualized using confocal microscopy to assess protein distribution, while Western blot analysis was undertaken to quantify NEDL2 protein expression. RESULTS: Confocal microscopy revealed that NEDL2-immunoreactivity colocalized with ICCs and neurons within the submucosa, myenteric plexus and smooth muscle in controls and ganglionic specimens, with markedly reduced NEDL2-immunoreactivity in aganglionic specimens. Western blotting revealed high levels of the NEDL2 protein in normal controls and the ganglionic region of HSCR, while there was a marked decrease in NEDL2 protein expression in the aganglionic region of HSCR. CONCLUSION: We report, for the first time, the expression of NEDL2 in the human colon. The decreased expression of NEDL2 in the aganglionic colon suggests that NEDL2 may play a role in the pathophysiology of HSCR.

Myenteric plexuses atrophy in the vicinity of colorectal cancer tissue is not caused by apoptosis or necrosis.

INTRODUCTION: The previously performed studies showed that the presence of colorectal cancer (CRC) tumor is associated with the atrophy of myenteric plexuses in the vicinity of cancer invasion; however, the possible mechanisms of this phenomenon are not known. The aim of the present study was to determine whether the atrophic changes of the enteric nervous system (ENS) within an intestine wall of the CRC patients were caused by apoptosis or necrosis and whether they were associated with changes in the number of galanin-immunore-active (GAL-Ir) neurons. MATERIAL AND METHODS: Samples of the large intestine wall located close to the CRC invasion and control, distally-located part of the colon, were collected from 9 CRC patients. The size of ENS plexuses and the number of neurons were compared. Triple immunofluorescent staining was used to visualize the co-expression of caspase 3 (CASP3) or caspase 8 (CASP8) with GAL and protein gene-product 9.5 (PGP 9.5, panneuronal marker) in the submucosal and myenteric ENS plexuses. The cells expressing myeloperoxidase (MPO, marker of neutrophils) and CD68 (marker of macrophages) were detected by immunohistochemistry around/in myenteric plexuses (MPs) and in the muscularis externa of the colon wall in the vicinity of tumor invasion. RESULTS: Myenteric plexuses in the vicinity of the CRC tissue were significantly smaller and had lower number of neurons per plexus than distantly located plexuses. The number of CASP8- and CASP3-Ir neurons in the ENS plexuses was similar in the colon wall both close to and distally from tumor invasion. The number of CASP8-Ir neurons within MPs located close to CRC invasion was higher than of CASP3-Ir neurons. The percentage of neurons co-expressing CASP8 and GAL in myenteric plexuses close and distantly from tumor was three-fold lower than of those co-expressing CASP3 and GAL. The mean number of neutrophils and macrophages inside and around myenteric plexuses located close to tumor invasion was higher or similar, respectively, as compared with adjacent muscularis externa. CONCLUSIONS: The atrophy of myenteric plexuses in the vicinity of CRC invasion is not caused by apoptosis or necrosis. The differences in the proportions of neurons expressing galanin and the studied caspases suggest as yet unknown role of this neuropeptide in the mechanisms of neuron's atrophy in MPs located close to CRC tumor.

Fructose consumption impairs serotonergic signaling in the murine enteric nervous system.

The intake of free fructose has increased substantially since the development of high-fructose corn syrup. This has not only been associated with metabolic disorders but recent evidence also indicates that chronic fructose consumption can affect neuronal and cognitive function. In this study we investigated the effects of fructose consumption on serotonergic signaling and neuronal activity in the mouse submucous plexus. Male mice were put on a control or fructose (23% solution) diet for 6 weeks or were assigned to a recovery group that received normal water (2 weeks) after 4 weeks of fructose. At the end of the diet, gene expressions and enteric neuronal activity, after depolarization with high K(+) and 5-HT, were measured using Ca(2+) imaging and RT-qPCR, respectively. Even in the lack of gain weight and the absence of changes in duodenal permeability, the total number of 5-HT-responding neurons and the depolarization and 5-HT-evoked Ca(2+) amplitudes were significantly lower after fructose consumption. Expression of synaptobrevin CaV 2.1 and CaV 2.2 mRNA did not differ after fructose intake; however, CaV 2.1 mRNA levels were significantly higher in the recovery animals. SERT mRNA concentration, isolated from submucosal plexus containing mucosal epithelium, was significantly decreased after fructose consumption. Chronic fructose consumption impairs serotonergic signaling in the mouse submucous plexus, prior to weight gain and detectable intestinal permeability problems.

Post-translational modifications of α-synuclein contribute to neurodegeneration in the colon of elderly individuals.

Synucleinopathies and abnormalities in the nerves of the enteric nervous system are hypothesized to be involved in age-associated motility disorders. The aim of the present study was to investigate the expression of various antigens, including α­synuclein (Syn) and its post­translational modified forms, in the human colon at various ages. In addition, the study aimed to correlate the expression of Syn with neurodegeneration. Immunohistochemistry was used to detect the expression of neurofilament (NF), Syn, as well as its nitrated (N) form in the healthy colonic tissue of 12 young (34.08±5.12 years), 10 middle­aged (51.80±3.52 years), and 11 elderly (75.82±7.70 years) individuals. To the best of our knowledge, the current study is the first to demonstrate the presence of N­Syn in the colonic tissue. N­Syn was identified in the upper layer of the mucosa and submucosa layer. Furthermore, Syn (wild­type) was present in the mucosa and submucosa. The number of NF­positive neurons in the submucosal layer declined significantly with age (P<0.01). In addition, Syn and N­Syn significantly increased during aging (P<0.01). Furthermore, a negative correlation was identified between neuron number and synucleinopathies, indicating the abnormal accumulation of both wild-type Syn and N­Syn in the mucosa, submucosa, muscle layer and myenteric plexus. The present study demonstrates that the Syn pathology may be linked to colic neuronal degeneration during normal aging, and this link may cause functional deficits.

Activation of the prostaglandin D2 metabolic pathway in Crohn's disease: involvement of the enteric nervous system.

BACKGROUND: Recent works provide evidence of the importance of the prostaglandin D2 (PGD2) metabolic pathway in inflammatory bowel diseases. We investigated the expression of PGD2 metabolic pathway actors in Crohn's disease (CD) and the ability of the enteric nervous system (ENS) to produce PGD2 in inflammatory conditions. METHODS: Expression of key actors involved in the PGD2 metabolic pathway and its receptors was analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in colonic mucosal biopsies of patients from three groups: controls, quiescent and active CD patients. To determine the ability of the ENS to secrete PGD2 in proinflammatory conditions, Lipocalin-type prostaglandin D synthase (L-PGDS) expression by neurons and glial cells was analyzed by immunostaining. PGD2 levels were determined in a medium of primary culture of ENS and neuro-glial coculture model treated by lipopolysaccharide (LPS). RESULTS: In patients with active CD, inflamed colonic mucosa showed significantly higher COX2 and L-PGDS mRNA expression, and significantly higher PGD2 levels than healthy colonic mucosa. On the contrary, peroxysome proliferator-activated receptor Gamma (PPARG) expression was reduced in inflamed colonic mucosa of CD patients with active disease. Immunostaining showed that L-PGDS was expressed in the neurons of human myenteric and submucosal plexi. A rat ENS primary culture model confirmed this expression. PGD2 levels were significantly increased on primary culture of ENS treated with LPS. This production was abolished by AT-56, a specific competitive L-PGDS inhibitor. The neuro-glial coculture model revealed that each component of the ENS, ECG and neurons, could contribute to PGD2 production. CONCLUSIONS: Our results highlight the activation of the PGD2 metabolic pathway in Crohn's disease. This study supports the hypothesis that in Crohn's disease, enteric neurons and glial cells form a functional unit reacting to inflammation by producing PGD2.

Architecture and Chemical Coding of the Inner and Outer Submucous Plexus in the Colon of Piglets.

In the porcine colon, the submucous plexus is divided into an inner submucous plexus (ISP) on the epithelial side and an outer submucous plexus (OSP) on the circular muscle side. Although both plexuses are probably involved in the regulation of epithelial functions, they might differ in function and neurochemical coding according to their localization. Therefore, we examined expression and co-localization of different neurotransmitters and neuronal markers in both plexuses as well as in neuronal fibres. Immunohistochemical staining was performed on wholemount preparations of ISP and OSP and on cryostat sections. Antibodies against choline acetyltransferase (ChAT), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and the pan-neuronal markers Hu C/D and neuron specific enolase (NSE) were used. The ISP contained 1,380 ± 131 ganglia per cm2 and 122 ± 12 neurons per ganglion. In contrast, the OSP showed a wider meshwork (215 ± 33 ganglia per cm2) and smaller ganglia (57 ± 3 neurons per ganglion). In the ISP, 42% of all neurons expressed ChAT. About 66% of ChAT-positive neurons co-localized SP. A small number of ISP neurons expressed SOM. Chemical coding in the OSP was more complex. Besides the ChAT/±SP subpopulation (32% of all neurons), a nNOS-immunoreactive population (31%) was detected. Most nitrergic neurons were only immunoreactive for nNOS; 10% co-localized with VIP. A small subpopulation of OSP neurons was immunoreactive for ChAT/nNOS/±VIP. All types of neurotransmitters found in the ISP or OSP were also detected in neuronal fibres within the mucosa. We suppose that the cholinergic population in the ISP is involved in the control of epithelial functions. Regarding neurochemical coding, the OSP shares some similarities with the myenteric plexus. Because of its location and neurochemical characteristics, the OSP may be involved in controlling both the mucosa and circular muscle.

Expression of Cocaine and Amphetamine Regulated Transcript (CART) in the Porcine Intramural Neurons of Stomach in the Course of Experimentally Induced Diabetes Mellitus.

In the present study, the effect of streptozotocin-induced diabetes on the cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) enteric nervous structures was investigated within the porcine stomach. To induce diabetes, the pigs were administered intravenously streptozotocin at a dose of 150 mg/kg of body weight. A significant decrease of the number of CART-LI perikarya was observed in the myenteric plexus of the gastric antrum, corpus, and pylorus in the experimental group. In contrast, submucous plexus was devoid of CART-positive neuronal cells both in control and experimental animals. In the control group, the highest densities of CART-LI nerve fibers were observed in the circular muscle layer of antrum and slightly less nerve fibers were present in the muscle layer of corpus and pylorus. In turn, submucous layer of all studied stomach regions revealed relatively smaller number of CART-positive nerve fibers. Diabetes caused statistically significant decrease in the expression of CART-LI nerve fibers only in the antrum circular muscle layer. Also, no changes in the CART-like immunoreactivity in the intraganglionic nerve fibers were observed. The obtained results suggest that acute hyperglycemia produced significant reduction of the CART expression in enteric perikarya throughout entire stomach as well as decrease of density the CART-LI fibers in circular muscle layer of the antrum. Additionally, we suggest that CART might be involved in the regulation of stomach function especially in the gastric motility.

Expression of Pattern Recognition Receptors by Nociceptive Metasympathetic Neurons.

Immunohistochemical assay with double label and confocal laser scanning microscopy showed that innate immunity receptor TLR4 is expressed predominantly in neurons of the intestinal Auerbach (myenteric) plexus, while vanilloid nociceptive receptor TRPV1 is expressed by neurons of Meissner (submucous) plexus. Immunohistochemical analysis with triple labeling revealed coexpression of TLR4 and TRPV1 in enteric neurons of rat colon. The results attest to a possibility of functional interaction between Toll-like and vanilloid receptors in the neuron level.