Search

Alterations in gastrointestinal (GI) function and the gut-brain axis are associated with progression and pathology of Alzheimer's Disease (AD). Studies in AD animal models show that changes in the gut microbiome and inflammatory markers can contribute to AD development in the central nervous system (CNS). Amyloid-beta (Aβ) accumulation is a major AD pathology causing synaptic dysfunction and neuronal death. Current knowledge of the pathophysiology of AD in enteric neurons is limited, and whether Aβ accumulation directly disrupts enteric neuron function is unknown.In 6-month-old 5xFAD (transgenic AD) and wildtype (WT) male and female mice, GI function was assessed by colonic transit in vivo; propulsive motility and GI smooth muscle contractions ex vivo; electrochemical detection of enteric nitric oxide release in vitro, and changes in myenteric neuromuscular transmission using smooth muscle intracellular recordings. Expression of Aβ in the brain and colonic myenteric plexus in these mice was determined by immunohistochemistry staining and ELISA assay.At 6 months, 5xFAD mice did not show significant changes in GI motility or synaptic neurotransmission in the small intestine or colon. 5xFAD mice, but not WT mice, showed abundant Aβ accumulation in the brain. Aβ accumulation was undetectable in the colonic myenteric plexus of 5xFAD mice.5xFAD AD mice are not a robust model to study amyloidosis in the gut as these mice do not mimic myenteric neuronal dysfunction in AD patients with GI dysmotility. An AD animal model with enteric amyloidosis is required for further study.

Background Neuronal alpha-synuclein (α-Syn) aggregation in the brain is believed to be a central component of the pathogenesis of Parkinson's disease (PD). α-Syn aggregates in the gastrointestinal tract have been suggested as a potential biomarker of PD that may even signal an early event of the Parkinsonian molecular pathology. However, studies further investigating this hypothesis have produced mixed results. Objective To determine whether the prevalence of α-Syn- and serine 129-phosphorylated α-Syn (Ser129p-α-Syn) depositions detected in intestine from PD patients differed from that of non-Parkinsonian controls. Methods In this retrospective study, we examined post-mortem small and large intestine samples of 25 PD patients and 20 age- and sex-matched controls without PD. Specimens were taken from archived paraffin-embedded tissue blocks. Immunohistochemical techniques were applied to detect α-Syn and Ser129p-α-Syn aggregates in situ. Immunoreactivity was quantified by a new approach that employed the detailed assessment of α-Syn- and Ser129p-α-Syn-positive morphological structures of the enteric nervous system (i.e., nerve fibers, myenteric and submucous plexus as well as ganglion cells). Results α-Syn immunoreactivity was a common finding in intestinal tissues from PD patients and controls. Importantly, α-Syn and Ser129p-α-Syn immunoreactivity were significantly reduced in PD patients compared to controls in each of the morphological structures examined. Conclusions Immunohistochemical detection of intestinal α-Syn and Ser129p-α-Syn seems to be a frequent and potentially normal finding. Neither α-Syn nor Ser129p-α-Syn immunoreactivity may, therefore, be regarded as a molecular intestinal biomarker of PD pathology. Reduced intestinal α-Syn and Ser129p-α-Syn immunoreactivity in PD patients rather reflect PD-related neuronal degeneration.

Morphological, morphometric, and ultrastructural analysis of the nerve fibers in the colon mucosa was performed in C57BL/6 mice at various terms of development of acute colitis induced by dextran sodium sulphate. The nerve fibers were labeled with antibodies to pan-neuronal marker βIII-tubulin. The progression of inflammatory and ulcerative processes in the mucosa on days 3-5 was associated with hyperplasia and hypertrophy of nerve fibers that peaked on day 7 after colitis induction. Ultrastructural analysis at all terms of colitis development showed moderate degeneration of axons. Thus, hypertrophy and hyperplasia of the nervous fibers in colon mucosa in experimental acute colitis correlated with aggravation of the ulcerative process in the mucosa. These changes are determined by alteration of histoarchitectonics and regenerative processes in the mucosa.

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.

Purpose/Aim of the study: Auerbach/Meissner network of lower abdominopelvic organs managed by parasympathetic nerve fibres of lumbosacral roots arising from Onuf’s nucleus located in conus ...

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.

The colon is partly controlled by myenteric and submucosal plexuses, which respond to stress and lead to some gastrointestinal disorders. These plexuses play roles in irritable bowel syndrome. Patients suffering from this syndrome can be treated with some antidepressants, including sertraline and nortriptyline. The primary aim of study was to compare the effect of a sertraline and a nortriptyline on the structural changes of the enteric neurons after stress exposure in both sexes. The secondary objectives were to evaluate the effects of stress on the submucosal and myenteric plexuses. Male and female Sprague-Dawley rats were assigned to four subgroups. The first subgroup received no stress. The other three subgroups received chronic variable stress (CVS) and were given phosphate buffer, sertraline (10 mg/kg/day), or nortriptyline (10 mg/kg/day). After 45 days, the neuron number in their colon plexuses was estimated using the stereologic method. The number of neurons increased by 40–51% in the submucosal plexus and by 57–69% in the myenteric plexus in the CVS group compared with the control group (p

Background Obesity is associated with the development of insulin resistance (IR) and type‐2 diabetes mellitus (T2DM); however, not all patients with T2DM are obese. The Goto–Kakizaki (GK) rat is an experimental model of spontaneous and non‐obese T2DM. There is evidence that the intestine contributes to IR development in GK animals. This information prompted us to investigate small intestine remodeling in this animal model. Methods Four‐month‐old male Wistar (control) and GK rats were utilized for the present study. After removing the small intestine, the duodenum, proximal jejunum, and distal ileum were separated. We then measured villi and muscular and mucosa layer histomorphometry, goblet cells abundance, total myenteric and submucosal neuron populations, and inflammatory marker expression in the small intestinal segments and intestinal transit of both groups of animals. Key Results We found that the GK rats exhibited decreased intestinal area (p, Goto–Kakizaki (GK) rat is an experimental model of spontaneous and non‐obese type‐2 diabetes mellitus—T2DM).GK rats exhibit decreased small intestinal area, increased crypt depth, villi height and thickness, and muscular layer thickness, increased content of IL‐1β and NF‐κB p65, decreased density of submucosal neurons, myenteric and submucosal neuronal and ganglionic hypertrophy, and decreased intestinal transit.The development of IR and T2DM in GK rats is associated with small intestine remodeling with marked changes in morphology, enteric nervous system, and transit.

BACKGROUND Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce. METHODS We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut. KEY RESULTS Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7 ± 2.4% of all submucosal neurons (n = 6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α1 , α2 or α3 -subunit). Hexamethonium (200 µM) reduced nerve-evoked chloride secretion by 34.3 ± 18.6% (n = 14 patients), whereas D-APV had no effect. CONCLUSION & INFERENCE Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered.

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 enteric nervous system (ENS) controls gastrointestinal functions. In large mammals’ intestine, it comprises an inner (ISP) and outer (OSP) submucous plexus and a myenteric plexus (MP). This study quantifies enteric neurons in the ISP, OSP, and MP of the pig ascending (AC) and descending colon (DC) using the HuC/D, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) neuronal markers in whole mount preparations with multiple labeling immunofluorescence. We established that the ISP contains the highest number of HuC/D neurons/mm2, which were more abundant in AC vs. DC, followed by OSP and MP with similar density in AC and DC. In the ISP, the density of ChAT immunoreactive (IR) neurons was very similar in AC and DC (31% and 35%), nNOS-IR neurons were less abundant in AC than DC (15% vs. 42%, P < 0.001), and ChAT/nNOS-IR neurons were 5% and 10%, respectively. In the OSP, 39–44% of neurons were ChAT-IR in AC and DC, while 45% and 38% were nNOS-IR and 10–12% were ChAT/nNOS-IR (AC vs. DC P < 0.05). In the MP, ChAT-IR neurons were 44% in AC and 54% in DC (P < 0.05), nNOS-IR neurons were 50% in both, and ChAT/nNOS-IR neurons were 12 and 18%, respectively. The ENS architecture with multilayered submucosal plexuses and the distribution of functionally distinct groups of neurons in the pig colon are similar to humans, supporting the suitability of the pig as a model and providing the platform for investigating the mechanisms underlying human colonic diseases.

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-HTM1R 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.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.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.

AIM To investigate the intestinal segment-specific effects of diabetes and insulin replacement on the density of different subpopulations of submucous neurons. METHODS Ten weeks after the onset of type 1 diabetes samples were taken from the duodenum, ileum and colon of streptozotocin-induce diabetic, insulin-treated diabetic and sex- and age-matched control rats. Whole-mount preparations of submucous plexus were prepared from the different gut segments for quantitative fluorescent immunohistochemistry. The following double-immunostainings were performed: neuronal nitric oxide synthase (nNOS) and HuC/D, heme oxygenase (HO) 1 and peripherin, as well as HO2 and peripherin. The density of nNOS-, HO1- and HO2-immunoreactive (IR) neurons was determined as a percentage of the total number of submucous neurons. RESULTS The total number of submucous neurons and the proportion of nNOS-, HO1- and HO2-IR subpopulations were not affected in the duodenal ganglia of control, diabetic and insulin-treated rats. While the total neuronal number did not change in either the ileum or the colon, the density of nitrergic neurons exhibited a 2- and 3-fold increase in the diabetic ileum and colon, respectively, which was further enhanced after insulin replacement. The presence of HO1- and HO2-IR submucous neurons was robust in the colon of controls (38.4%-50.8%), whereas it was significantly lower in the small intestinal segments (0.0%-4.2%, P < 0.0001). Under pathophysiological conditions the only alteration detected was an increase in the ileum and a decrease in the colon of the proportion of HO-IR neurons in insulin-treated diabetic animals. CONCLUSION Diabetes and immediate insulin replacement induce the most pronounced region-specific alterations of nNOS-, HO1- and HO2-IR submucous neuronal density in the distal parts of the gut.

© 2017 International Pediatric Research Foundation, Inc. Background:Enteric neural stem/progenitor cells (ENSCs) offer an innovative approach to treating Hirschsprung disease (HSCR) and other enteric neuropathies. However, postnatal-derived human ENSCs have not been thoroughly characterized and their behavior in the embryonic and postnatal intestinal environment is unknown.Methods:ENSCs were isolated from the intestines of 25 patients undergoing bowel resection, including 7 children with HSCR. Neuronal differentiation and proliferation of ENSCs from submucosal and myenteric plexuses from patients with and without HSCR were characterized. ENSC migration and differentiation were studied following transplantation into embryonic chick neural crest, embryonic chick hindgut, and postnatal mouse aganglionic colon.Results:The proliferative and neurogenic potential of ENSCs from HSCR intestine is equivalent to that of non-HSCR controls. Similarly, no difference was observed between myenteric- A nd submucosal-derived ENSCs. Postnatal ENSCs transplanted to embryonic neural crest pathways and to aneural hindgut migrate normally and differentiate into appropriate neural crest-derived cell types. ENSCs in postnatal mouse aganglionic colon differentiate into neurons and glia both ex vivo and in vivo.Conclusions:ENSCs isolated from the postnatal intestine of patients with and without HSCR can behave like embryonic neural crest-derived cells. These results support the feasibility of cell-based therapy for future treatment of neurointestinal disease.

Detection of nutritional and noxious food components in the gut is a crucial component of gastrointestinal function. Contents in the gut lumen interact with enteroendocrine cells dispersed throughout the gut epithelium. Enteroendocrine cells release many different hormones, neuropeptides, and neurotransmitters that communicate either directly or indirectly with the central nervous system and the enteric nervous system, a network of neurons and glia located within the gut wall. Several populations of enteric neurons extend processes that innervate the gastrointestinal lamina propria; however, how these processes develop and begin to transmit information from the mucosa is not fully understood. In this study, we found that Tuj1-immunoreactive neurites begin to project out of the myenteric plexus at embryonic day (E)13.5 in the mouse small intestine, even before the formation of villi. Using live calcium imaging, we discovered that neurites were capable of transmitting electrical information from stimulated villi to the plexus by E15.5. In unpeeled gut preparations where all layers were left intact, we also mimicked the basolateral release of 5-HT from enteroendocrine cells, which triggered responses in myenteric cell bodies at postnatal day (P)0. Altogether, our results show that enteric neurons extend neurites out of the myenteric plexus early during mouse enteric nervous system development, innervating the gastrointestinal mucosa, even before villus formation in mice of either sex. Neurites are already able to conduct electrical information at E15.5, and responses to 5-HT develop postnatally.NEW & NOTEWORTHY How enteric neurons project into the gut mucosa and begin to communicate with the epithelium during development is not known. Our study shows that enteric neurites project into the lamina propria as early as E13.5 in the mouse, before development of the submucous plexus and before formation of intestinal villi. These neurites are capable of transmitting electrical signals back to their cell bodies by E15.5 and respond to serotonin applied to neurite terminals by birth. ispartof: AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY vol:318 issue:1 pages:G53-G65 ispartof: location:United States status: published

Gastrointestinal (GI) symptoms can originate from severe dysmotility due to enteric neuropathies. Current methods used to demonstrate enteric neuropathies are based mainly on classic qualitative histopathological/immunohistochemical evaluation. This study was designed to identify an objective morphometric method for paraffin-embedded tissue samples to quantify the interganglionic distance between neighboring myenteric ganglia immunoreactive for neuron-specific enolase, as well as the number of myenteric and submucosal neuronal cell bodies/ganglion in jejunal specimens of patients with severe GI dysmotility. Jejunal full-thickness biopsies were collected from 32 patients (22 females; 16–77 yr) with well-characterized severe dysmotility and 8 controls (4 females; 47–73 yr). A symptom questionnaire was filled before surgery. Mann-Whitney U test, Kruskal-Wallis coupled with Dunn’s posttest and nonparametric linear regression tests were used for analyzing morphometric data and clinical correlations, respectively. Compared with controls, patients with severe dysmotility exhibited a significant increase in myenteric interganglionic distance ( P = 0.0005) along with a decrease in the number of myenteric ( P < 0.00001) and submucosal ( P < 0.0004) neurons. A 50% reduction in the number of submucosal and myenteric neurons correlated with an increased interganglionic distance and severity of dysmotility. Our study proposes a relatively simple tool that can be applied for quantitative evaluation of paraffin sections from patients with severe dysmotility. The finding of an increased interganglionic distance may aid diagnosis and limit the direct quantitative analysis of neurons per ganglion in patients with an interganglionic distance within the control range. NEW & NOTEWORTHY Enteric neuropathies are challenging conditions characterized by a severe impairment of gut physiology, including motility. An accurate, unambiguous assessment of enteric neurons provided by quantitative analysis of routine paraffin sections may help to define neuropathy-related gut dysmotility. We showed that patients with severe gut dysmotility exhibited an increased interganglionic distance associated with a decreased number of myenteric and submucosal neurons, which correlated with symptoms and clinical manifestations of deranged intestinal motility.

Aim: Ileocolonoscopy allows early detection of recurrence after surgical resection for Crohn’s disease [CD]. Plexitis, defined as presence of inflammatory cells in or around enteric ganglia or nerve bundles, in the proximal surgical margin has been associated with an increased overall recurrence risk. We investigated prospectively whether plexitis can predict endoscopic recurrence [ER] in a consecutive cohort of CD patients undergoing ileocolonic resection. Methods: All CD patients undergoing ileocolonic resection in our institution between October 2009 and December 2012 were eligible for this study. Clinical data were obtained prospectively from the patients’ files, and biopsies from the proximal surgical margins were analysed immunohistochemically for inflammation at the myenteric and submucosal plexus [lymphocytes, mast cells, eosinophils]. The degree of plexitis was correlated with the presence of ER at 6 months, defined as a modified Rutgeerts’ score of ≥ i2b. Multivariate models were developed and tested to predict posterior probability of ER. Results: A total of 74 patients were included. Six months after ileocolonic resection, 50% showed ER. Known risk factors such as penetrating disease, previous resections, and active smoking, showed no relation with ER. On the other hand, submucosal lymphocytic plexitis was associated with ER [ p = 0.020]. The predictive value of lymphocytic cell count increased with more extensive biopsy sampling and with application of immunohistochemistry. Conclusions: Submucosal lymphocytic plexitis in the proximal surgical margin was significantly related with a higher risk for ER after ileocolonic resection. These data support development of a postoperative prevention trial with vedolizumab, which may block lymphocytic trafficking in the postoperative bowel.

Oxaliplatin, currently used for treatment of colorectal and other cancers, causes severe gastrointestinal side effects, including nausea, vomiting, diarrhea, and constipation that are attributed to mucosal damage. However, delayed onset and long-term persistence of these side effects suggest that damage to the enteric nervous system (ENS) regulating physiological function of the gastrointestinal tract may also occur. The ENS comprises myenteric and submucosal neurons and enteric glial cells (EGCs). This study aimed to investigate the effects of oxaliplatin treatment on enteric neurons and EGCs within the mouse ileum. BALB/c mice received repeated intraperitoneal injections of oxaliplatin (3 mg/kg, 3 injections/week). Tissues were collected 3, 7, 14, and 21 days from the commencement of treatment. Decreases in glial fibrillary acidic protein–immunoreactive (IR) EGCs and protein gene product 9.5/β-Tubulin III-IR neurons as well as increase in s100β-IR EGCs after chronic oxaliplatin administration were observed in both the myenteric and submucosal plexi. Changes in EGCs were further observed in cross-sections of the ileum at day 14 and confirmed by Western blotting. Alterations in EGCs correlated with loss of myenteric and submucosal neurons in the ileum from oxaliplatin-treated mice. These changes to the ENS may contribute to the mechanisms underlying gastrointestinal side effects associated with oxaliplatin treatment.

Intestinal epithelial secretion is coordinated by the submucosal plexus (SMP). Chemical mediators from SMP regulate the immunobiological response and direct actions against infectious agents. Toxoplasma gondii is a worldwide parasite that causes toxoplasmosis. This study aimed to determine the effects of chronic infection with T. gondii on the morphometry of the mucosa and the submucosal enteric neurons in the proximal colon of rats. Male adult rats were distributed into a control group (n = 10) and an infected group (n = 10). Infected rats received orally 500 oocysts of T. gondii (ME-49). After 36 days, the rats were euthanized and samples of the proximal colon were processed for histology to evaluate mucosal thickness in sections. Whole mounts were stained with methylene blue and subjected to immunohistochemistry to detect vasoactive intestinal polypeptide. The total number of submucosal neurons decreased by 16.20%. Vasoactive intestinal polypeptide-immunoreactive neurons increased by 26.95%. Intraepithelial lymphocytes increased by 62.86% and sulfomucin-producing goblet cells decreased by 22.87%. Crypt depth was greater by 43.02%. It was concluded that chronic infection with T. gondii induced death and hypertrophy in the remaining submucosal enteric neurons and damage to the colonic mucosa of rats.

The present study analysed changes in the distribution pattern of cocaine- and amphetamine-regulated transcript (CART) in the enteric nervous system (ENS) of the human colon challenged by adenocarcinoma invasion, using the double-labelling immunofluorescence technique. In control specimens, CART immunoreactivity was found in neurons of all studied plexuses, representing 30.1 ± 4.1%, 12.9 ± 5.2%, and 4.1 ± 1.3% of all neurons forming the myenteric plexus (MP), outer submucous plexus (OSP), and inner submucous plexus (ISP), respectively. Tumour growth into the colon wall caused an increase in the relative frequency of CART-like immunoreactive (CART-LI) neurons in enteric plexuses located in the vicinity of the infiltrating neoplasm (to 36.1 ± 6.7%, 32.7 ± 7.3% and 12.1 ± 3.8% of all neurons in MP, OSP and ISP, respectively). The density of CART-LI nerves within particular layers of the intestinal wall did not differ between control and adenocarcinoma-affected areas of the human colon. This is the first detailed description of the CART distribution pattern within the ENS during the adenocarcinoma invasion of the human colon wall. The obtained results suggest that CART probably acts as a neuroprotective factor and may be involved in neuronal plasticity evoked by the progression of a neoplastic process.

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.

Phase-amplitude coupling of two pacemaker activities of the small intestine, the omnipresent slow wave activity generated by interstitial cells of Cajal of the myenteric plexus (ICC-MP) and the stimulus-dependent rhythmic transient depolarizations generated by ICC of the deep muscular plexus (ICC-DMP), was recently hypothesized to underlie the orchestration of the segmentation motor pattern. The aim of the present study was to increase our understanding of phase-amplitude coupling through modeling. In particular the importance of propagation velocity of the ICC-DMP component was investigated. The outcome of the modeling was compared with motor patterns recorded from the rat or mouse intestine from which propagation velocities within the different patterns were measured. The results show that the classical segmentation motor pattern occurs when the ICC-DMP component has a low propagation velocity (

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.

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.

Although sigmoid volvulus (SV) causes acute obstruction, its pathogenesis and mechanism of torsion are unknown, and few reports have described its pathological findings. Here, we evaluated the clinicopathological characteristics of volvulus and factors contributing to volvulus of the sigmoid colon.We compared 14 patients with SV (10 men and 4 women; median age, 78.5 years) with 14 age- and sex-matched control patients for differences in clinical characteristics, focusing on dysmotility (enteric visceral myopathy, neuropathy, and mesenchymopathy).Of the 14 SV patients, 7 had recurrent volvulus, 11 had an associated condition, and 5 required emergency surgery. Atrophy and fibrosis of the inner muscle were more prevalent in the SV than control patients (p = 0.041). Median extent (per centimeter of muscularis propria) of the myenteric plexus (12.5 versus 17.5, p 0.001) and submucous plexus (15.0 versus 25.5, p 0.001) was lower in the SV patients, as were the median numbers of myenteric (9.7 versus 30.4, p 0.001) and submucous ganglion cells (10.0 versus 23.2, p 0.001). Inflammatory neuropathy was more prevalent in the SV than control patients (p = 0.046); whereas, the prevalence of mesenchymopathy did not differ (p = 0.481).A decrease in the extent of enteric plexus and ganglion cells precedes the clinical manifestation of SV. Although further elucidation is needed, this decrease may play an important role in the diagnosis of SV and in identifying the mechanism leading to torsion in SV.

Context.— Colonic inertia is a debilitating form of primary chronic constipation with unknown etiology and diagnostic criteria, often requiring pancolectomy. We have occasionally observed massively enlarged submucosal ganglia containing at least 20 perikarya, in addition to previously described giant ganglia with greater than 8 perikarya, in cases of colonic inertia. These massively enlarged ganglia have yet to be formally recognized. Objective.— To determine whether such “massive submucosal ganglia,” defined as ganglia harboring at least 20 perikarya, characterize colonic inertia. Design.— We retrospectively reviewed specimens from colectomies of patients with colonic inertia and compared the prevalence of massive submucosal ganglia occurring in this setting to the prevalence of massive submucosal ganglia occurring in a set of control specimens from patients lacking chronic constipation. Results.— Seven of 8 specimens affected by colonic inertia harbored 1 to 4 massive ganglia, for a total of 11 massive ganglia. One specimen lacked massive ganglia but had limited sampling and nearly massive ganglia. Massive ganglia occupied both superficial and deep submucosal plexus. The patient with 4 massive ganglia also had 1 mitotically active giant ganglion. Only 1 massive ganglion occupied the entire set of 10 specimens from patients lacking chronic constipation. Conclusions.— We performed the first, albeit distinctly small, study of massive submucosal ganglia and showed that massive ganglia may be linked to colonic inertia. Further, larger studies are necessary to determine whether massive ganglia are pathogenetic or secondary phenomena, and whether massive ganglia or mitotically active ganglia distinguish colonic inertia from other types of 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

Background The principal components of the enteric nervous system (ENS) are two neuronal networks, the myenteric and submucosal plexus (SMP), which are primarily involved in the regulation of gastrointestinal (GI) motility and secretion, respectively. These two plexus are made up of intrinsic neurons receiving input from the extrinsic sympathetic and parasympathetic innervation of the gut. Both the intrinsic and extrinsic innervations of the gut are affected by Lewy pathology in Parkinson's disease (PD). A recent autopsy survey indicated that there was no global or dopaminergic loss in the myenteric plexus in PD but the SMP was not examined. Objective The aim of the present work was to compare the relative abundance of dopaminergic and noradrenergic neurons in colonic biopsies between PD patients and control individuals. Methods Colonic biopsies were taken during the course of a colonoscopy in 35 PD patients and 10 control subjects. Density of dopaminergic neurons and expression of the dopaminergic and noradrenergic markers were analyzed by tyrosine hydroxylase (TH) immunofluorescence and Western blot using anti-dopamine transporter (DAT) and anti-dopamine beta-hydroxylase (DBH), respectively. Results No significant differences were observed in the density of dopaminergic neurons and in the expression levels of dopaminergic and noradrenergic markers in colonic biopsies from PD patients as compared to controls. Conclusion Our results indicate that there is no evidence of dopaminergic and noradrenergic neuronal loss in the SMP in PD, thereby suggesting that neuropathology in submucosal neurons is unlikely to be a causative factor for GI dysfunction in PD.

This study aimed to determine whether enteric neurons are involved in multiple system atrophy (MSA). Four-μm-thick slices of small intestine were prepared from 10%-formalin-fixed and paraffin-embedded materials obtained from autopsied cases. Enteric neurons were stained using an anti-peripherin antibody. Immunostaining of phosphorylated α-synuclein was also performed. Areas of the cytoplasm and nucleus that showed nucleoli were measured using computer software. Both areas of myenteric neurons were significantly smaller in MSA cases (n = 3) than in control subjects (n = 3) (P 0.0001); however, no deposits of phosphorylated α-synuclein were observed. These findings suggest that myenteric neurons in MSA are affected independent of α-synuclein accumulation.

The cocaine and amphetamine regulated transcript (CART) belongs to the group of peptides with anorexigenic properties and is present in many areas of the central and peripheral nervous systems of numerous mammalian species. Research has suggested an effect on the feeling of appetite and satiety; however, there are no clear clues as to the role of CART in specific organs, including the stomach. Considering the specificity of cattle feeding and digestion, CART may play a highly significant role possibly associated with the option of administering greater amounts of high-volume feeds. Based on the results of immunohistochemical staining of abomasum samples prepared from hybrid bulls, the presence of CART-positive structures and CART distribution were determined in the mucosa, submucosa and muscularis layers of the stomach. Abundant sites of CART were found in the myenteric plexus, nerve fibers innervating the myocytes of the myenteron, neuroendocrine cells of the diffuse neuroendocrine system and the submucous plexus. The preliminary stage of abomasal CART detection suggests that CART is an agent that strongly affects the regulation of motor activity involved in stomach emptying and in secretory functions of the stomach. However, further research is necessary to explain the relationship.

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.

In the 1970s, by using classic histological methods, close topographical relationships between special areas of enteric ganglia and capillaries were shown in the pig. In this study, by application of double and triple immunohistochemistry, we confirmed this neurovascular interface and demonstrated that these zones are mainly confined to nitrergic neurons in the myenteric and the external submucosal plexus. In the upper small intestine of the pig, the respective neurons display type III morphology, i.e. they have long, slender and branched dendrites and a single axon. In another set of experiments, we prepared specimens for electron-microscopical analysis of these zones. Both ganglia and capillaries display continuous basement membranes, the smallest distances between them being 1,000 nm at the myenteric and 300 nm at the external submucosal level. The capillary endothelium was mostly continuous but, at the external submucosal level, scattered fenestrations were observed. This particular neurovascular relationship suggests that nitrergic neurons may require a greater amount of oxygen and/or nutrients. In guinea pig and mouse, previous ischemia/reperfusion experiments showed that nitrergic neurons are selectively damaged. Thus, a preferential blood supply of enteric nitrergic neurons may indicate that these neurons are more vulnerable in ischemia.

In diabetes mellitus (DM), hyperglycemia promotes changes in biochemical mechanisms that induce oxidative stress. Oxidative stress has been closely linked to adverse consequences that affect the function of the gastrointestinal tract caused by injuries to the enteric nervous system (ENS) that in turn cause neurodegeneration and enteric glial loss. Therapeutic approaches have shown that diet supplementation with antioxidants, such as quercetin, reduce oxidative stress. This work sought to evaluate neurons and enteric glial cells in the myenteric and submucosal plexuses of the duodenum in diabetic rats supplemented with quercetin. The duodenum of 24 rats, including a control group (C), control quercetin supplementation group (CQ), diabetic group (D), and diabetic quercetin supplementation group (DQ), were used to investigate whole mounts of muscular and submucosal layers subjected to immunohistochemistry to detect vasoactive intestinal peptide in the myenteric layer and double-staining for HuC-D/neuronal nitric oxide synthase (nNOS) and HuC-D/S100. A reduction of the general neuronal population (HuC/D) was found in the myenteric and submucosal plexuses (p

Background Most direct understanding of enteric nerve (patho)physiology has been obtained by electrode and imaging techniques in animal models and human surgical samples. Until now, neuronal activity recordings from a more accessible human tissue source have remained a true challenge. Objectives To record nerve activity in human intestinal biopsies using imaging techniques. Design Submucous plexus was isolated from duodenal biopsies. Enteric nerves were functionally and morphologically examined using calcium (Ca 2+ ) imaging and immunohistochemistry. Exogenous application of high-K + solution, the nicotinic cholinergic receptor agonist (1,1-dimethyl-4-phenylpiperazinium; DMPP) or serotonin (5-HT), and electrical stimulation of interganglionic fibre tracts were used to activate the neurons, and intracellular Ca 2+ concentrations ([Ca 2+ ] i ) were monitored. Enteric ganglia were stained with neuronal and glial markers. Results Using high-K + solution, 146 neurons were identified in 70 ganglia (44 biopsies from 29 subjects). The exogenous application of DMPP or 5-HT caused a transient [Ca 2+ ] i increase, respectively, in 68% and 63% of the neurons identified by high-K + . Electrical stimulation evoked responses in 57% of the neurons; these responses were totally or partly suppressed by tetrodotoxin or zero-Ca 2+ solution, respectively. Immunohistochemical analysis showed both isolated neurons and ganglia interconnected by typical interganglionic fibre bundles. The average number of ganglia was 7.7±6.0 per biopsy and each ganglion contained on average 4.5±1.2 neurons. Conclusion In this study, for the first time, live recordings were performed of nerve activity in intestinal biopsies. This novel approach is of key importance to study living neurons in both health and disease and to test newly developed compounds in an in-vitro human tissue model.

At the end of the embryonic period of human development, interstitial cells of Cajal (ICC) are present in the esophagus, stomach, and proximal duodenum, around the inception of the myenteric plexus (MP) ganglia. In the small and large bowel, ICC appear later. The object of the present study was to determine the timing of appearance and pattern of distribution of ICC in the human embryonic and fetal distal colon. Human distal colon specimens were obtained from 8 embryos and 14 fetuses without gastrointestinal disorders. The specimens were 7–16 weeks of gestational age. The specimens were exposed to anti-c-kit antibodies to investigate ICC differentiation. Enteric plexuses were immunohistochemically examined using anti-neuron-specific enolase, and the differentiation of smooth muscle cells was studied with anti-desmin antibodies. In the distal colon, ICC emerged at weeks 10–11 of the fetal period in the form of two parallel belts of densely packed cells extending at the submucous plexus (SMP) and the MP level. These cells correspond to ICC of the SMP (ICC-SMP) and ICC of the MP (ICC-MP). The simultaneous appearance of ICC at the SMP and MP level in the distal colon can be explained by the fact that there are differences in the migration of neural crest cells in particular portions of the digestive tube. In conclusion, in humans, there was a difference in the patterns of development of ICC in the distal colon compared to the rest of the gut.

The mechanisms underlying distension-evoked peristalsis in the colon are incompletely understood. It is well known that, following colonic distension, 5-hydroxytryptamine (5-HT) is released from enterochromaffin (EC) cells in the intestinal mucosa. It is also known that exogenous 5-HT can stimulate peristalsis. These observations have led some investigators to propose that endogenous 5-HT release from EC cells might be involved in the initiation of colonic peristalsis, following distension. However, because no direct evidence exists to support this hypothesis, the aim of this study was to determine directly whether release of 5-HT from EC cells was required for distension-evoked colonic peristalsis. Real-time amperometric recordings of 5-HT release and video imaging of colonic wall movements were performed on isolated segments of guinea pig distal colon, during distension-evoked peristalsis. Amperometric recordings revealed basal and transient release of 5-HT from EC cells before and during the initiation of peristalsis, respectively. However, removal of mucosa (and submucosal plexus) abolished 5-HT release but did not inhibit the initiation of peristalsis nor prevent the propagation of fecal pellets or intraluminal fluid. Maintained colonic distension by fecal pellets induced repetitive peristaltic waves, whose intrinsic frequency was also unaffected by removal of the submucosal plexus and mucosa, although their propagation velocities were slower. In conclusion, the mechanoreceptors and sensory neurons activated by radial distension to initiate peristalsis lie in the myenteric plexus and/or muscularis externa, and their activation does not require the submucosal plexus, release of 5-HT from EC cells, nor the presence of the mucosa. The propagation of peristalsis and propulsion of liquid or solid content along the colon is entrained by activity within the myenteric plexus and/or muscularis externa and does not require sensory feedback from the mucosa, nor neural inputs arising from submucosal ganglia.

We and others have shown that gastrin-releasing peptide (GRP) reduces food intake. In this study, we determined the activation of the gastrointestinal and dorsal vagal complex (DVC) neurons by various forms of GRP to determine the pathway involved in this reduction. We found the following: (1) GRP-10, -27 and -29 (2.1 nmol/kg, i.p.) increased the Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of the stomach and the area postrema (AP) of the DVC; (2) GRP-27 and GRP-29 increased the Fos-LI in the myenteric plexus of the duodenum; and (3) only GRP-29 increased the Fos-LI in the submucosal plexus of the duodenum. In conclusion, GRP may reduce food intake by activating the area postrema. The enteric neurons may have a potential role in this reduction through the direct activation of the AP or exerting local gut actions, such as the stimulation of gut motility or secretions.

The aim of the study was to compare 2 different forceps designed to perform biopsies of the rectal mucosa, those of Noblett and Scheye, the latter having a similar design and differing by the disposable cutting system.This historical study compares biopsies obtained with the Noblett forceps in 13 girls and 20 boys (mean ± SD age, 13 ± 30 months) and biopsies obtained with the Scheye forceps in 19 girls and 21 boys (mean ± SD age, 8.5 ± 19 months).The thickness of the material obtained with the Scheye forceps was significantly greater for the specimens obtained with the Scheye forceps (total biopsy: 1.74 ± 0.46 mm vs 0.67 ± 0.2 mm, P.0001; submucosa: 1.12 ± 0.4 mm vs 0.14 ± 0.17 mm, P.001). The Scheye forceps considerably increased the yield of neuronal structures, both for submucosal plexus (P.003) and ganglia (P.0001). No complication occurred in either group.The Scheye disposable rectal biopsy system provides larger mucosal biopsy samples than the Noblett with increased recovery of neuronal structures.

Irritable bowel syndrome (IBS) is characterized by episodic bouts of abdominal pain, bloating, and altered bowel habit. Accumulating evidence has linked immune activation with IBS, including reports of increases in circulating levels of the proinflammatory cytokine interleukin (IL)-6. However, it is unknown whether IL-6 contributes directly to disease manifestation. As enteric nervous activity mediates motility and secretory function, the aims of this study were to determine the effects of IL-6 on submucosal neurons and related gastrointestinal (GI) function. In these studies, we examined the colons of maternally separated (MS) rats, which exhibit elevated circulating levels of IL-6 in addition to GI dysfunction. To our knowledge, these studies are the first to provide evidence of the sensitivity of submucosal neurons to colonic secretions from MS rats ( n = 50, P < 0.05), thus recapitulating clinical biopsy data. Moreover, we demonstrated that the excitatory action is IL-6 dependent. Thereafter, the impact of IL-6 on neuronal and glial activation and absorpto/secretory function was pharmacologically characterized. Other proinflammatory cytokines including IL-8 ( n = 30, P > 0.05), IL-1β ( n = 56, P > 0.05), and TNF-α ( n = 56, P > 0.05) excited fewer neurons. Both muscarinic and nicotinic cholinergic receptors participate in the effect and cause downstream activation of ERK, JAK-STAT, and NF-κB signaling cascades. Functionally, IL-6 increases transepithelial resistance and enhances neurally and cholinergically mediated ion transport. These data provide a role for IL-6 in colonic secretory functions and relate these effects to GI dysfunction in an animal model of IBS, thereby elucidating a potential relationship between circulating levels of IL-6 and aberrant GI function.

The aim of the study was to describe and depict the spatial arrangement of the colon microcirculatory bed as a whole. Various parts of the large intestine and terminal ileum were harvested from either cadaver or section material or gained peroperatively. Samples were then injected with India ink or methylmetacrylate Mercox resin for microdissection and corrosion casting for scanning electron microscopy. The results showed that extramural vasa recta ramified to form the subserous plexus, some of them passing underneath the colon taeniae. Branches of both short and long vasa recta merged in the colon wall, pierced the muscular layer and spread out as the submucous plexus, which extended throughout the whole intestine without any interruption. The muscular layer received blood via both the centrifugal branches of the submucous plexus and the minor branches sent off by the subserous plexus. The mucosa was supplied by the mucous plexus, which sent capillaries into the walls of intestinal glands. The hexagonal arrangement of the intestinal glands reflected their vascular bed. All three presumptive critical points are only gross anatomical points of no physiological relevance in healthy individuals. Neither microscopic weak points nor regional differences were proven within the wall of the whole large intestine. The corrosion casts showed a huge density of capillaries under the mucosa of the large intestine. A regular hexagonal pattern of the vascular bed on the inner surface was revealed. No microvascular critical point proofs were confirmed and a correlation model to various pathological states was created.

Vasoactive intestinal peptide (VIP) immunoreactive neurons are important secretomotor neurons in the submucous plexus. They are the only submucosal neurons to receive inhibitory inputs and exhibit both noradrenergic and nonadrenergic inhibitory synaptic potentials (IPSPs). The former are mediated by α2-adrenoceptors, but the receptors mediating the latter have not been identified. We used standard intracellular recording, RT-PCR, and confocal microscopy to test whether 5-HT1A, SST1, and/or SST2receptors mediate nonadrenergic IPSPs in VIP submucosal neurons in guinea pig ileum in vitro. The specific 5-HT1Areceptor antagonist WAY 100135 (1 μM) reduced the amplitude of IPSPs, an effect that persisted in the presence of the α2-adrenoceptor antagonist idazoxan (2 μM), suggesting that 5-HT might mediate a component of the IPSPs. Confocal microscopy revealed that there were many 5-HT-immunoreactive varicosities in close contact with VIP neurons. The specific SSTR2antagonist CYN 154806 (100 nM) and a specific SSTR1antagonist SRA 880 (3 μM) each reduced the amplitude of nonadrenergic IPSPs and hyperpolarizations evoked by somatostatin. In contrast with the other antagonists, CYN 154806 also reduced the durations of nonadrenergic IPSPs. Effects of WAY 100135 and CYN 154806 were additive. RT-PCR revealed gene transcripts for 5-HT1A, SST1, and SST2receptors in stripped submucous plexus preparations consistent with the pharmacological data. Although the involvement of other neurotransmitters or receptors cannot be excluded, we conclude that 5-HT1A, SST1, and SST2receptors mediate nonadrenergic IPSPs in the noncholinergic (VIP) secretomotor neurons. This study thus provides the tools to identify functions of enteric neural pathways that inhibit secretomotor reflexes.

Evidence from patients with inflammatory bowel disease (IBD) and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro video microscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sodium sulfate [DSS, 5% (wt/vol)] colitis. Using a luciferase-based ATP assay, we examined the degradation of ATP and utilized real-time PCR, Western blotting, and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 microM) but did constrict in the presence of its nonhydrolyzable analog alpha,beta-methylene ATP (1 microM). alpha,beta-Methylene ADP (100 microM), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from mice with DSS-induced colitis. CD39 protein and mRNA expression was markedly increased during colitis. Immunohistochemical analysis demonstrated that, in addition to vascular CD39, F4/80-immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining during colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis.

We report an autopsied 20-year-old man case of intestinal necrosis associated with megacolon from hypoganglionosis, a pseudo-Hirschsprung's disease. The patient had suffered from severe constipation since two years of age, and presented abdominal distention from age ten. Autopsy revealed marked dilatation and necrosis of the entire large intestine. Although ganglion cells in the intestinal plexus were found throughout the large intestine, their number was reduced to 12-20% of that in the normal control. In pseudo-Hirschsprung's disease, there are occasional cases where an acute abdomen first presents itself in adulthood after running its course as chronic constipation.

Diabetes affects many aspects of gastrointestinal motility, in part due to changes in interstitial cells of Cajal (ICC). The effect of diabetes on the colon, however, is not well characterized, and the aim of the present study was to investigate possible relationships between altered colonic motility as a consequence of streptozotocin-induced diabetes and injury to ICC. Physiological, immunohistochemical, and ultrastructural techniques were employed. The motor pattern of the rat colon was dominated by rhythmic high-amplitude, low-frequency contractions that were primarily myogenic in origin. These rhythmic contractions were induced by stretch associated with increased tension; the amplitude of the superimposed rhythmic contractions increased with increasing applied tension. In diabetic rats, the stretch-induced rhythmic contractile activity remained robust and of similar frequency but was significantly higher in amplitude compared with that in control rats. At 700 mg of applied tension, the force of contraction in circular colonic muscle strips of the diabetic rats was 370% of control values. This robust presence of low-frequency contractions is consistent with the unaffected pacemaker, the ICC associated with Auerbach's plexus, and the increased amplitude correlates with loss of and injury to ICC of the submuscular plexus and intramuscular ICC. Loss of inhibitory nitrergic nerves does not appear to be a factor based on unaltered nNOS immunoreactivity.

Inflammatory bowel disease is a recurrent intestinal inflammatory disorder that in adults has been associated with changes in enteric nervous system neuropeptide expression. The aim of the present study was to determine whether similar changes were observed in paediatric Crohn's disease. The distribution of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) was determined in colonic tissues from children with ileo-colonic ( n = 4) and colonic ( n = 3) Crohn's disease. The submucosal plexus of inflamed regions showed significant increase in density of VIP immunoreactive neurons (margin, 48% vs. inflamed tissue, 82% of HuC/D positive neurons). The density of submucosal plexus nNOS immunoreactive neurons was too low to be reliably quantified. Using the pan-neuronal marker HuC/D, no significant difference in numbers of HuC/D positive submucosal neurons was evident except where neurons were normalized to length of tissue (margins, 3.6 ± 0.7 vs. inflamed tissue, 4.0 ± 0.6 neurons/ganglia, p = 0.33; margins, 2.7 ± 0.4 vs. inflamed tissue, 5.7 ± 1.2, neurons/mm, p = 0.03). In the myenteric plexus, there was a significant increase in the percent of NOS neurons (38% vs. 82% of HuC/D positive neurons) while there was no significant difference in percent of VIP neurons (4% vs. 8%). No difference in number of HuC/D positive myenteric neurons among margin and inflamed tissues was observed (margin, 12.2 ± 3.0 vs. inflamed tissue, 12.5 ± 5.1 neurons/ganglia, p = 0.50; margins 9.1 ± 2.1 vs. inflamed tissue, 13.7 ± 2.3 neurons/mm, p = 0.11). These data demonstrate that inflammation is associated with a differential expression of VIP and nNOS neuronal subpopulations within the two major enteric plexi, likely due to phenotypic switch. Such changes might contribute to the pathogenesis of IBD and ongoing symptoms even in quiescent disease.

The submucosal plexus (SMP) of the large intestine plays important roles in secretion and motility, functions that are compromised in the aged. To determine the effects of aging on intrinsic SMP neurons and their extrinsic inputs, whole mounts from the colon of ad libitum fed virgin male Fischer 344 rats ranging in age from 6 to 27 months were processed to visualize neurons and nerve fibers immunoreactive for either tyrosine hydroxylase (TH-IR) or calcitonin gene-related peptide (CGRP-IR). Significant age-related loss of SMP neurons occurred as early as 12 months of age and progressed in a roughly linear manner with age, dropping, for example, by 38% in the distal colon. In aging rats, the colonic SMP routinely contained markedly swollen TH-IR axons and terminals and exhibited a reduction of ganglionic neuropil, whereas CGRP-IR fibers evidenced only modest axonopathies. These findings point to selective deterioration of the SMP and its extrinsic inputs as one possible mechanism for the age-related decline in large intestinal function evidenced in the elderly.

The Nav1.9 sodium channel is expressed in nociceptive DRG neurons where it contributes to spontaneous pain behavior after peripheral inflammation. Here, we used a newly developed antibody to investigate the distribution of Nav1.9 in rat and mouse trigeminal ganglion (TG) nerve endings and in enteric nervous system (ENS). In TGs, Nav1.9 was expressed in the soma of small- and medium-sized, peripherin-positive neurons. Nav1.9 was present along trigeminal afferent fibers and at terminals in lip skin and dental pulp. In the ENS, Nav1.9 was detected within the soma and proximal axons of sensory, Dogiel type II, myenteric and submucosal neurons. Immunological data were correlated with the detection of persistent TTX-resistant Na(+) currents sharing similar properties in DRG, TG and myenteric neurons. Collectively, our data support a potential role of Nav1.9 in the transmission of trigeminal pain and the regulation of intestinal reflexes. Nav1.9 might therefore constitute a molecular target for therapeutic treatments of orofacial pain and gastrointestinal syndromes.

5-HT released by gastrointestinal mucosa and enteric interneurons has powerful effects on gut behavior. However, the targets of 5-HT-containing neurons within enteric circuits are not well characterized. We used antisera against 5-HT and selected markers of known enteric neuron types to investigate the connections made by 5-HT-containing neurons in the guinea-pig jejunum. Confocal microscopy was used to quantify the number of 5-HT-immunoreactive varicosities apposed to immunohistochemically identified cell bodies. Large numbers of varicosities were identified apposing cholinergic secretomotor neurons, immunoreactive for neuropeptide Y, in both myenteric and submucous plexuses. Subgroups of neurons identified by calretinin (ascending interneurons) and nitric oxide synthase (descending interneurons and inhibitory motor neurons) immunoreactivity were also apposed by many varicosities. Longitudinal muscle motor neurons (calretinin immunoreactive) and AH/Dogiel type II (sensory) neurons (calbindin immunoreactive) were apposed by small numbers of varicosities. Combined retrograde tracing and immunohistochemistry were used to identify excitatory circular muscle motor neurons; these were encircled by 5-HT-immunoreactive varicosities, but the appositions could not be quantified. We suggest that 5-HT-containing interneurons are involved in secretomotor pathways and pathways to subgroups of other interneurons, but not longitudinal muscle motor neurons. There also appear to be connections between 5-HT-containing interneurons and excitatory circular muscle motor neurons. Physiological evidence demonstrates a functional connection between 5-HT-containing interneurons and AH/Dogiel type II neurons, but few 5-HT-immunoreactive varicosities were observed apposing calbindin-immunoreactive cell bodies. Taken together these results suggest that neural 5-HT may have significant roles in excitatory pathways regulating both motility and secretion.