Expression of the Rai (Shc C) adaptor protein in the human enteric nervous system.

The adaptor protein Rai (ShcC/N-Shc) is almost exclusively present in the nervous system, although little is documented about its expression in the gut and the enteric nervous system (ENS). As Rai is a physiological substrate of Ret, an important factor for the development of ENS, we have evaluated the expression of Rai in the ENS in various segments of the human gastrointestinal tract. The expression of Rai was assessed by immunohistochemistry in disease-free human gut samples (oesophagus, stomach, small bowel and colon) obtained from subjects undergoing surgical procedures. Rai was not expressed in the epithelia or lymphoid tissue, whereas a moderate level of expression was observed in the endothelial cells of blood vessels and on the outer membrane of smooth muscle cells in both the muscularis mucosae and the muscularis propria. In the ENS, strong positivity was observed only in enteric glial cells, overlapping with GFAP and S100. In conclusion, Rai is expressed in the human gut, especially in the enteric glial cells. We conclude that Rai may provide an additional marker for this cell type.     

Intercellular calcium waves in cultured enteric glia from neonatal guinea pig

Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura-2 AM microfluorimetry. Single-cell stimulation was performed with a micromanipulator-driven glass pipette. Data were expressed as mean +/- SEM and analyzed by Student's t-test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% +/- 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 microM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 microM) significantly decreased the percentage of cells responding to mechanical stimulation (6% +/- 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% +/- 13% vs. 60% +/- 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles.     

Chromosomal study of enteric glial cells and neurons by fluorescence in situ hybridization in slow transit constipation

The pathogenesis of slow transit constipation is still elusive. However, a genetic basis may be present. We investigated possible chromosomal abnormalities in enteric neurons and glial cells in patients with slow transit constipation. Colonic specimens from 22 patients with slow transit constipation undergoing surgery for intractable symptoms were obtained, and investigated by fluorescence in situ hybridization (FISH) for chromosomal abnormalities (chromosomes 1, 8, 17 and XY). These specimens were compared with of those obtained in 12 control subjects. Data analysis showed that 45.5% of patients displayed significant (>10%) aneusomy of chromosome 1 in enteric neurons. Aneusomy <10% for the same chromosome, but less than the cutoff suggested (10%), was found in enteric glial cells in 45.4% of the same patients. One patient had <10% aneusomy in enteric neurons for chromosome 8. No other abnormalities were found for the remaining probes, and no abnormalities were found in controls. We concluded that in a subgroup of patients with slow transit constipation a genetic basis may be present.     

Role of enteric glial cells in inflammatory bowel disease

Enteric glial cells (EGCs) represent an extensive but relatively poorly described cell population within the gastrointestinal tract. Accumulating data suggest that EGCs represent the morphological and functional equivalent of CNS astrocytes within the enteric nervous system (ENS). The EGC network has trophic and protective functions toward enteric neurons and is fully implicated in the integration and the modulation of neuronal activities. Moreover, EGCs seem to be active elements of the ENS during intestinal inflammatory and immune responses, sharing with astrocytes the ability to act as antigen-presenting cells and interacting with the mucosal immune system via the expression of cytokines and cytokine receptors. Transgenic mouse systems have demonstrated that specific ablation of EGC by chemical ablation or autoimmune T-cell targeting induces an intestinal pathology that shows similarities to the early intestinal immunopathology of Crohn's disease. EGCs may also share with astrocytes the ability to regulate tissue integrity, thereby postulating that similar interactions to those observed for the blood-brain barrier may also be partly responsible for regulating mucosal and vascular permeability in the gastrointestinal tract. Disruption of the EGC network in Crohn's disease patients may represent one possible cause for the enhanced mucosal permeability state and vascular dysfunction that are thought to favor mucosal inflammation.     

IL-1β and IL-10 have dual effects on enteric glial cell proliferation

Inflammatory bowel disease is typically accompanied by functional and structural changes of the enteric nervous system. In pathological studies, cellular loss and axonal degeneration have been described in the myenteric plexus. However, more recent studies suggest that the proliferation rate of myenteric glial cells is enhanced in animal models of intestinal inflammation. Therefore, we have investigated the effect of different cytokines on the proliferative response of enteric glial cells (EGCs), comparing transformed enteric glial cell lines, primary astrocyte cultures and transformed oligodendrocytes. Cells were incubated in serum-free chemically defined medium in the presence or absence of either interleukin (IL)-1beta or IL-10 at concentrations ranging between 0.1 and 100 ng mL(-1) for 48 h. Subsequently, [3H]thymidine was added to each culture dish for an additional 6 h, and the amount of incorporated [3H] was assessed. IL-1beta significantly and dose-dependently suppressed [3H]-uptake by EGCs. In contrast, IL-10 induced a biphasic response; IL-10 at low concentrations (0.1 ng mL(-1)) caused a significant suppression of [3H]-uptake, whereas high concentrations (5-100 ng mL(-1)) significantly enhanced [3H] uptake. These results indicate that EGC proliferation can be modulated by cytokines. The differential effects of IL-1beta and IL-10 suggest that during intestinal inflammation there may be a regulatory interplay between different classes of cytokines modulating EGC proliferation.     

Colonization of the bowel by the precursors of enteric glia: studies of normal and congenitally aganglionic mutant mice

The terminal portion of the ls/ls mouse is congenitally aganglionic because the precursors of enteric neurons fail to enter this region. This animal was studied in order to gain insight into the origin of enteric glia and into the process by which the precursors of these cells colonize the gut. In control (CD-1) mice, immunoreactivity of the glial marker, glial fibrillary acidic protein, appeared for the first time in the fetal bowel at day E16 and, in adults, was much more intense within intraenteric neural elements than in nerves outside the bowel. Glial fibrillary acidic protein developed in tissue cultures of fetal intestine explanted before the protein appeared in situ, and before the bowel became innervated by extrinsic nerves; thus, the precursors of cells able to elaborate glial fibrillary acidic protein must have been present, but unrecognizable, in the original explants. This explant assay demonstrated that these glial precursors were present in all regions of the bowel of control mice, but not in the presumptive aganglionic bowel of ls/ls mice. The nerves (of extrinsic origin) in the aganglionic tissue of ls/ls mice showed a high level of immunoreactive glial fibrillary acidic protein; nevertheless, their ultrastructure was typical of peripheral nerve, not enteric plexus, and they contained Schwann cells, not enteric glia. These observations support the view that enteric glia are derived from the single wave of neural crest colonists that populates the enteric nervous system before the gut receives its extrinsic innervation. These glial precursors, like neuronal precursors, tend to be excluded from the presumptive aganglionic ls/ls bowel. In contrast, Schwann cells grow into the abnormal ls/ls gut with the extrinsic innervation. The enteric microenvironment appears to promote the expression of glial fibrillary acidic protein in both enteric glia and Schwann cells; however, even within the bowel, Schwann cells retain their characteristic morphology. It is thus probable that the normal enteric nervous system contains supporting cells of separate lineages, enteric glia and Schwann cells.     

Effect of old age on the subpopulations of enteric glial cells in human descending colon

Old age is associated with a higher incidence of lower bowel conditions such as constipation. Recent evidence suggest that colonic motility may be influenced by enteric glial cells (EGCs). Little is known about the effect of aging on the subpopulation of EGCs in the human colon. We assessed and compared the pattern of distribution of EGCs in adult and elderly human colon. Human descending colon were obtained from 23 cancer patients comprising of adults (23–63 years; 6 male, 7 female) and elderly (66–81 year; 6 male, 4 female). Specimens were serially-sectioned and immunolabeled with anti-Sox-10, anti-S100 and anti-GFAP for morphometric analysis. Standardized procedures were utilized to ensure unbiased counting and densitometric evaluation of EGCs. The number of Sox-10 immunoreactive (IR) EGCs were unaltered with age in both the myenteric plexus (MP) (respectively, in adult and elderly patients, 1939 ± 82 and 1760 ± 44/mm length; p > .05) and submucosal plexus; there were no apparent differences between adult males and females. The density of S100-IR EGCs declined among the elderly in the circular muscle and within the MP per ganglionic area. In the adult colon, there were more S100-IR EGCs distributed in the circular muscle per unit area than the Taenia coli. There was little or no GFAP-IR EGCs in both adult and elderly colon. We concluded that aging of the human descending colon does not result in a loss of Sox-10-IR EGCs in the MP and SMP but reduces S100-IR EGCs density within the musculature. This alteration in myenteric EGCs density with age may contribute to colonic dysfunction.     

Development of the enteric nervous system: bringing together cells, signals and genes

Abstract  The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract that controls essential functions such as motility, secretion and blood flow, comprises a vast number of neurons and glial cells that are organized into complex networks of interconnected ganglia distributed throughout the entire length of the gut wall. Enteric neurons and glia are derived from neural crest cells that undergo extensive migration, proliferation, differentiation and survival in order to form a functional ENS. Investigations of the developmental processes that underlie ENS formation in animal models, and of the common human congenital ENS abnormality Hirschsprung's disease, have been intimately related and recently led to major advances in the field. This review touches on some of these advances and introduces two topics that are elaborated upon in this journal issue: (i) genome wide approaches for profiling gene expression in wild type and mutant ENS that have been used to identify novel molecules with important roles in enteric neurogenesis, and (ii) the use of multilineage ENS progenitors isolated from embryonic or postnatal gut as novel cell replacement therapies for Hirschsprung's disease. Such studies will not only unravel the mechanisms underlying ENS development, but will also shed light on the pathogenesis of ENS developmental disorders and help to establish novel therapeutic strategies for restoring or repairing malfunctioning enteric neural circuits prevalent in numerous gastrointestinal diseases.     

Differential distribution of inositol 1,4,5-triphosphate receptors in the rat olfactory bulb

The inositol 1,4,5-triphosphate receptor (IP₃R) regulates the release of calcium from intracellular stores. In the present study, the distribution of IP₃R in the rat main olfactory bulb was determined by immunohistochemistry. Immunofluorescence was used to double label for IP₃R and for γ-aminobutyric acid (GABA) or for projection neurons, which were retrogradely labeled following dextran injection into the lateral olfactory tract (LOT). The expression profile of IP₃R changes dramatically during development. In the glomerular layer of adults, many juxtaglomerular neurons are IP₃R immunoreactive [IP₃R(+)]; the majority of these cells are also GABA immunoreactive [GABA(+)]. Scattered sparsely throughout the external plexiform layer are small numbers of IP₃R(+) neurons, a small number of which are LOT-projecting tufted cells. Significant numbers of IP₃R(+) neurons are in the granule cell layer; however, most of these cells are GABA(−). The vast majority of mitral cells contain little or no IP₃R immunoreactivity. These findings indicate that, in the olfactory bulb of adult rats, IP₃R is preferentially localized in specific classes of intrinsic neurons and that it is rarely expressed in projection neurons. In contrast, during the first postnatal week, the receptor is detected almost exclusively in mitral cells. Expression of IP₃R in subclasses of intrinsic neurons begins during the second and third weeks, concomitant with a decrease in immunostaining of mitral cells. Adult patterns of IP₃R immunostaining are apparent by the fourth postnatal week. These observations raise the possibility that the expression of IP₃R in specific classes of neurons during development is activity dependent. J. Comp. Neurol. 389:224–234, 1997. © 1997 Wiley-Liss, Inc.     

Two modes of microtubule-associated protein 1B phosphorylation are differentially regulated during peripheral nerve regeneration

Two major modes of MAP1B phosphorylation (I and II), respectively recognized by monoclonal antibodies 150 and 125, have been related to remodeling and formation of processes in the mature nervous system. To gain insight into the cytoskeletal modifications underlying peripheral nerve regeneration, the pattern of expression of both MAP1B phosphorylated modes was studied during this process. Sciatic nerves from adult Wistar rats were crushed and animals allowed to survive for 5, 7, 10 or 14 days. After those survival periods, damaged and undamaged sciatic nerves, dorsal root ganglia (DRG), and spinal cords, were subjected to immunohistochemistry and Western blot, using antibodies 150 and 125. At all survival periods analysed, MAP1B phosphorylated at mode I was concentrated at the distal region of regenerating nerves whereas mode II phosphorylation underwent an overall decrease in regenerating axons that was less evident in more proximal nerve regions. Very high levels of MAP1B phosphorylated at mode II were detected in the bodies of DRG neurons and in bodies and dendrites of spinal motor neurons. This phosphorylation mode was also encountered in some Schwann cells and oligodendroglia associated with more proximal regions of regenerating axons. In this study we conclude that MAP1B was differentially phosphorylated depending on the cell type, subcellular compartment and stage of the regenerative process and discuss the possible functional implications that differential expression of each MAP1B phosphorylation mode might have during nerve regeneration.     

Generalized neuromuscular hypoplasia,reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging

Background Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging‐associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make‐up of the small intestinal and colonic neuromuscular apparatus. Methods Klotho expression was studied by RT‐PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron‐specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy, and three‐dimensional reconstruction. HuC/D⁺ myenteric neurons were enumerated by fluorescent microscopy. Key Results Klotho protein was detected in neurons, smooth muscle cells, and some ICC classes. Small intestinal transit was slower but whole‐gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines. Conclusions & Inferences Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted postweaning growth. As reduced fecal output in these mice occurs in the presence of accelerated colonic and whole‐gut transit, it likely reflects reduced food intake rather than intestinal dysmotility.     

Morphological changes in interstitial cells of Cajal in the deep muscular plexus and enteric motor neurons of the intestine in rats with multiple organ dysfunction syndrome

BACKGROUND:Gastrointestinal motility dysfunction in multiple organ dysfunction syndrome (MODS) has been reported to be related to damage to interstitial cells of Cajal (ICC). In the entedc nervous system, ICC and smooth muscle cells are connected in a network to form a special functional unit. Many gastrointestinal motility dysfunction diseases are associated with damage to this network.OBJECTIVE:To investigate the morphological changes of intestinal ICC, and to explore the mechanisms underlying gastrointestinal motility dysfunction in rats with MODS.DESIGN, TIME AND SE'I-FING:The randomized, controlled, experiment was performed at the Central Laboratory of the First Affiliated Hospital of Dalian Medical University of China between June 2007 and March 2009.MATERIALS:Escherichia coli (E. colistrain O127 H6) and bovine serum albumin were purchased from Sigma, USA.METHODS:A total of 40 Wistar rats were equally and randomly divided into MODS group and control group. Suspension of E. coil strain O127 H6 containing BaSO4 and saline were sterilely injected into the abdominal cavity of rats in the MODS and control groups, respectively.MAIN OUTCOME MEASURES:Immunohistochemical double-staining and confocal laser scanning microscopy were used to observe the morphological changes in intestinal cholinergic nerves and ICC in the deep muscular plexus network. Electron microscopy was employed to evaluate the ultrastructural features of ICC in the deep muscular plexus of rats with MODS.RESULTS:Compared with the control group, the distributions and densities of cholinergic/nitrergic newes and ICC in the deep muscular plexus were significantly decreased in the MODS group (P < 0.01). The enteric nerve-ICC network were disrupted.CONCLUSION:There is ultrastructural injury in the ICC in the deep muscular plexus and enteric nerves of the intestine in rats with MODS, which may be associated with the dysmotility of the gastrointestinal tract in MODS.     

Neuroprotective effects of willed-movement therapy in a rat model of cerebral ischemia/reperfusion

The present study established rat models of middle cerebral artery ischemia/reperfusion using the thread method.Rats performed willed-movement(climbing a ladder or wall in a box) when induced by food and water.Results revealed that Longa scores of neurological deficits significantly de-creased in the willed-movement group at 15 days after reperfusion,while expression of glial fibrillary acidic protein,neurotrophic factor-3,and growth-associated protein-43 significantly increased at 7 and 15 days after reper...