Enteric glia.

The structure of the enteric nervous system (ENS) is different from that of extraenteric peripheral nerve. Collagen is excluded from the enteric plexuses and support for neuronal elements is provided by astrocyte-like enteric glial cells. Enteric glia differ from Schwann cells in that they do not form basal laminae and they ensheath axons, not individually, but in groups. Although enteric glia are rich in the S-100 and glial fibrillary acidic proteins, it has been difficult to find a single chemical marker that distinguishes enteric glia from non-myelinating Schwann cells. Nevertheless, two monoclonal antibodies have been obtained that recognize antigens that are expressed on Schwann cells (Ran-1 in rats and SMP in avians) but not enteric glia. Functional differences between enteric glia and non-myelinating Schwann cells, including responses to gliotoxins and in vitro proliferative rates, have also been observed. Developmentally, enteric glia, like Schwann cells, are derived from the neural crest. In both mammals and birds the precursors of the ENS appear to migrate to the bowel from sacral as well as vagal levels of the crest. These crest-derived emigrés give rise to both enteric glia and neurons; however, analyses of the ontogeny of the enteric innervation in a mutant mouse (the ls/ls), in which the original colonizing waves of crest-derived precursor cells are unable to invade the terminal colon, suggest that enteric glia can also arise from Schwann cells that enter the gut with the extrinsic innervation. When induced to leave back-transplanted segments of avian bowel, enteric crest-derived cells migrate into peripheral nerves and form Schwann cells. Enteric glia and Schwann cells thus appear to be different cell types, but ones that derive from lineages that diverge relatively late in ontogeny.     

Inability of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice: requirement for a permissive microenvironment

The enteric system is formed by cells that migrate to the bowel from the neural crest. In order to gain insight into intraenteric factors that influence this migration, the colonization of the bowel of the ls/ls mouse was investigated. The terminal 2 mm of ls/ls intestine fails to become colonized by crest cells and thus remains aganglionic. The entire bowel of control mice and ls/ls mice was explanted before the appearance in situ of recognizable neurons and grown in organotypic tissue culture. Neurons, detected by the histochemical demonstration of acetylcholinesterase activity, developed throughout the length of the control gut, but, even in vitro, were excluded from the terminal segment of the ls/ls intestine. Co-culture experiments were done, in which primary and secondary sources of crest cells were combined with recipient segments of bowel, to test the ability of the recipient tissue to become colonized by neural precursors. The primary source was murine crest cells migrating away from an explant of the neuraxis. Secondary sources included avian and murine foregut (control and ls/ls) containing migratory crest cells as well as the quail ganglion of Remak. Recipient segments of bowel included control avian and murine hindgut, explanted before the tissue had become colonized by crest cells in situ, as well as the presumptive aganglionic bowel of ls/ls mice. Both primary and secondary sources of crest cells proved to be able to contribute neurons to the control segments of recipient hindgut. Species differences were no barrier to the colonization of the bowel in vitro. Moreover, the ls/ls foregut was as good a source of neural precursors for a normal recipient bowel, as was control avian or murine foregut. In contrast, none of the sources of crest cells that were utilized contributed neurons to the presumptive aganglionic gut of ls/ls mice. Both cells and processes of enteric neurons developing in vitro (detected by demonstrating neurofilament immunoreactivity) tended to be excluded from the presumptive aganglionic tissue. On the other hand, neurites, but not cell bodies, of dorsal root ganglia co-cultured with presumptive aganglionic ls/ls bowel did enter the abnormal zone. These data are consistent with the hypothesis that nonneuronal elements of the wall of the presumptive aganglionic region of the ls/ls gut are abnormal and prevent the colonization of this segment of the gut with viable neural precursors from the neural crest.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glial fibrillary acidic polypeptides in peripheral glia. Molecular weight, heterogeneity and distribution

Glial fibrillary acidic (GFA) polypeptides are present in major categories of rat peripheral glia including non-myelin-forming Schwann cells, enteric glia and some satellite cells. They can be detected both immunochemically and immunohistochemically. The immunoreactivity is associated with a polypeptide which has an MW of 49 000, indistinguishable from that of glial fibrillary acidic protein (GFAP) from rat brain. In spite of this, the GFA polypeptides found in the peripheral nervous system and central nervous system are not identical since they can be distinguished both immunohistochemically and immunochemically by a monoclonal GFAP antibody which recognizes GFAP in astrocytes and some enteric glia, but not GFAP in non-myelin-forming Schwann cells, satellite cells and many enteric glia. GFA-related molecules can also be detected in human Schwann cells by immunofluorescence. The results suggest, however, that the glial filament polypeptides of peripheral glia and astrocytes are less closely related in the human than in the rat. The glial distribution of GFAP is closely paralleled by 2 cell surface proteins, Ran-2 and A5E3 antigen. Although GFAP, Ran-2 and A5E3 are individually expressed by diverse cell types, the phenotype GFAP+, Ran-2+, A5E3+ defines a narrow group including only non-myelin-forming Schwann cells, enteric glia and astrocytes. These observations suggest that the non-myelin-forming cells of the central and peripheral nervous system may share some common functions.     

Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia

The similarities between the enteric nervous system of the gut and the central nervous system (CNS), both of which function as complex integrative nervous networks, include striking ultrastructural similarities between the glia of the enteric nervous system and the astrocytic glia of the CNS. In this paper we have determined whether this anatomical resemblance also extends to the molecular level by examining the enteric glial cells to see whether they express several surface and intracellular molecules which are highly restricted to glia and to astrocytes in particular. Indirect immunofluorescence was used to visualize the antigens in frozen sections of gut wall and in whole mount, tissue culture, and freshly dissected preparations of myenteric and submucous plexuses from rats of various ages. It was found that enteric glial cells expressed the intracellular proteins glial fibrillary acidic protein, glutamine synthetase, and vimentin both in situ and in culture. The surface antigen Ran-2 was expressed in situ but not in culture, and the surface antigen Ran-1 was expressed in culture but not in situ. Cultured enteric glial cells did not express fibronectin in significant quantity, nor did they make galactocerebroside. From these results we conclude that the adult phenotype of enteric glia in situ closely resembles that of astrocytes, while in culture some of their cell surface features change, reverting to those seen during development. Because these cells possess distinctive molecular features and numerically form one of the major populations of peripheral glia, it is appropriate to classify them as a third distinctive category of peripheral glial cells, in addition to satellite and Schwann cells. The molecular similarities between these cells and astrocytes, in addition to their anatomical resemblance, suggest that a further study of enteric glia will provide new insights into the role of glia in integrative nervous tissues.     

An immunohistochemical study of glial fibrillary acidic (GFA) protein and S-100 protein in the colon affected by Hirschsprung's disease

Summary The supportive cells of the enteric nervous system were examined in gut tissues from 15 patients with Hirschsprung's disease by means of immunohistochemistry, utilizing antisera to glial fibrillary acidic (GFA) protein and S-100 protein. In the normoganglionic segment, GFA protein immunoreactivity was predominantly found in association with the myenteric plexus and to a lesser extent in the submucous plexus. On the other hand, the extrinsic, hypertrophic nerve fasciculi were selectively immunostained with GFA protein antiserum throughout the entire length of the aganglionic intestinal walls from all children studied. The large fasciculi were numerous in the distal aganglionic segment and commonly appeared in the intermuscular zone and submucosal connective tissue. Both small-and mediumsized nerve fasciculi with GFA protein immunoreactivity were also encountered within the circular muscle layer of the proximal aganglionic segment. A subpopulation of supportive cells within the hypertrophic nerve fasciculi showed immunoreactivity for GFA protein, while all supportive elements of these fasciculi were stained for S-100 protein. The intrinsic nerve fibers within the circular muscle layer of normoganglionic segments were stained for S-100 protein, but not for GFA protein. The present study supports our previous findings that two types of supportive cells can be differentiated by immunohistochemistry in the enteric nervous system, utilizing antisera to GFA protein and S-100 protein. It is also concluded that the demonstration of GFA protein by immunohistochemical methods favors the diagnosis of aganglionic colons with Hirschsprung's disease, since GFA protein immunoreactivity is confined to the extrinsic, hypertrophic nerve fasciculi characteristic of aganglionic bowels.     

Distribution of hyaluronic acid and chondroitin sulfate proteoglycans in the presumptive aganglionic terminal bowel of ls/ls fetal mice: an ultrastructural analysis

The terminal colon of the ls/ls mouse is aganglionic because an intrinsic defect prevents its colonization by cells migrating from the neural crest. Previous studies showed that laminin, type IV collagen, and glycosaminoglycans accumulate in the region of the presumptive aganglionic ls/ls bowel through which crest-derived cells would be expected to migrate. It was suggested that crest-derived cells might fail to enter the abnormal bowel because they receive inappropriate signals from a defective extracellular matrix. This hypothesis was evaluated by analyzing the ultrastructure of the extracellular matrix in mutant and control gut. Tissue was fixed in the presence of ruthenium red before or after selective enzymatic digestion. Heparan sulfate proteoglycan (diameter approximately equal to 15 nm) and chondroitin sulfate proteoglycan (diameter approximately equal to 20-50 nm) granules were found in both control and presumptive aganglionic gut. The heparan sulfate proteoglycan granules were primarily located within formed basal laminae, while chondroitin sulfate proteoglycan granules decorated plasma membranes and 5 nm hyaluronic acid microfibrils that formed a network in the extracellular matrix. At day E11.5, the mutant gut differed from the control in the following: 1) Hyaluronic acid microfibrils were longer and more numerous. 2) There were larger numbers of chondroitin sulfate proteoglycan granules associated with cell membranes and with hyaluronic acid microfibrils. By day E13 the spaces between mesenchymal cells of the outer wall of the control bowel contained a regular lattice of hyaluronic acid microfibrils studded with chondroitin sulfate proteoglycan granules. Instead of this lattice, tangles of excessively long hyaluronic acid microfibrils, coated more heavily than in the control with chondroitin sulfate proteoglycan granules, were found in the presumptive aganglionic gut. These results confirm that the extracellular matrix is abnormal in the presumptive aganglionic bowel of the ls/ls mouse; moreover, they also indicate that the defect involves not one, but several components of the extracellular matrix, as well as their distribution. The defective extracellular matrix is apparent at a time when crest-derived cells would be expected to be migrating in the terminal bowel and is located in their path. The observations thus support the idea that a localized abnormality of the extracellular matrix interferes with the colonization of the terminal bowel by crest-derived cells in the ls/ls mouse.     

The olfactory nerve contains two populations of glia, identified both in vivo and in vitro.

The peripheral olfactory nervous system exhibits, uniquely, neuronal cell body replacement and reestablishment of central connections in adult mammals. The role of the olfactory nerve glia in these phenomena is unknown, but information might be provided by in vitro systems. This paper reports on the characterization of olfactory nerve glia in dissociated cell cultures of newborn rat nasal mucosal tissues. The predominant type of glial cell resembled Schwann cells and immunostained for the S-100 protein, found in all glial cell types; glial fibrillary acidic protein (GFAP), found in astrocytes and nonmyelinating Schwann cells; and showed binding of 217C, a monoclonal Schwann-cell marker that binds to the low-affinity NGF receptor in glioma cells. They were negative for A2B5. The Schwann-cell-like olfactory glia changed morphology upon culturing in serum-free medium, with further shape changes after plating on laminin. Plating on laminin increased cell numbers. A second population, found only after GFAP-immunostaining, was astrocyte-like in morphology and represented approximately 10 percent of all glial cells. These were S-100-, A2B5-, and 217C-negative, a unique glial cell immunological profile. At low dilutions of anti-GFAP (1/10,000), or with weak fluorescent secondary antibodies, astrocyte-like glia were immunostained but Schwann-cell-like glia were not detectable. Astrocyte-like glia were not an artifact of the dissection, since they were detectable in tissue sections of newborn-rat olfactory nerves immunostained with a low dilution of anti-GFAP. The presence of two types of glial cells in culture suggests similarities between olfactory glia and enteric glia.     

Novel insights into the glia limitans of the olfactory nervous system

Olfactory ensheathing cells (OECs) are often described as being present in both the peripheral and the central nervous systems (PNS and CNS). Furthermore, the olfactory nervous system glia limitans (the glial layer defining the PNS–CNS border) is considered unique as it consists of intermingling OECs and astrocytes. In contrast, the glia limitans of the rest of the nervous system consists solely of astrocytes which create a distinct barrier to Schwann cells (peripheral glia). The ability of OECs to interact with astrocytes is one reason why OECs are believed to be superior to Schwann cells for transplantation therapies to treat CNS injuries. We have used transgenic reporter mice in which glial cells express DsRed fluorescent protein to study the cellular constituents of the glia limitans. We found that the glia limitans layer of the olfactory nervous system is morphologically similar to elsewhere in the nervous system, with a similar low degree of intermingling between peripheral glia and astrocytes. We found that the astrocytic layer of the olfactory bulb is a distinct barrier to bacterial infection, suggesting that this layer constitutes the PNS–CNS immunological barrier. We also found that OECs interact with astrocytes in a similar fashion as Schwann cells in vitro. When cultured in three dimensions, however, there were subtle differences between OECs and Schwann cells in their interactions with astrocytes. We therefore suggest that glial fibrillary acidic protein–reactive astrocyte layer of the olfactory bulb constitutes the glia limitans of the olfactory nervous system and that OECs are primarily “PNS glia.”     

Glial fibrillary acidic protein (GFAP) immunoreactivity in enteric ganglia of the chick embryo

We examined by immunohistochemistry the expression of glial fibrillary acidic protein (GFAP) in enteric ganglia of the chick embryo, using a polyclonal antibody. The morphology of enteric ganglion cells was examined by electron microscopy. Faint GFAP immunoreactivity was detected in ganglion cells and cell processes from around day 7 in ovo. Later in development the intensity of the immunofluorescence increased and it became more evident that immunoreactive small ganglion cells (interpreted as primitive glial cells), and their processes, surrounded larger negative cell profiles (interpreted as primitive neuronal cells); GFAP immunofluorescence was also evident in intramuscular and mucosal nerve trunks. In colocalization experiments, GFAP immunoreactivity was detected in a proportion of HNK-1/N-CAM immunoreactive ganglion cells, in both the myenteric and submucosal plexus. In addition, we observed GFAP immunoreactive nerves in wholemount preparations of chick gut from as early as day 4.5 in ovo. In the ganglionated nerve of Remak, GFAP immunoreactive satellite and Schwann cells were in evidence from day 5 of incubation. Neuronal markers, such as neurofilament, have been detected very early in development in neural crest cell populations in chick enteric ganglia. In contrast, the expression of markers of the glial phenotype has previously been observed only in the late stages of embryonic development. From our experiments, we conclude that neuronal and glial phenotypes are immunohistochemically distinct from as early as day 4.5 of incubation, even if by ultrastructural criteria glial cells are clearly distinguishable from neurons only after day 16 in ovo.     

Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells

Background The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly. Methods We studied the effect of acetylcholine (ACh), serotonin (5‐HT), and adenosine triphosphate (ATP) on intracellular Ca²⁺ signaling using aequorin‐expressing and Fluo‐4 AM‐loaded CRL‐2690 rat and human enteric glial cell cultures devoid of neurons. The influence of these neurotransmitters on the proliferation of glia was measured and their effect on the expression of c‐Fos as well as glial fibrillary acidic protein (GFAP), Sox10, and S100 was examined by immunohistochemistry and quantitative RT‐PCR. Key Results Apart from ATP, also ACh and 5‐HT induced a dose‐dependent increase in intracellular Ca²⁺ concentration in CRL‐2690 cells. Similarly, these neurotransmitters also evoked Ca²⁺ transients in human primary enteric glial cells obtained from mucosal biopsies. In contrast with ATP, stimulation with ACh and 5‐HT induced early gene expression in CRL‐2690 cells. The proliferation of enteric glia and their expression of GFAP, Sox10, and S100 were not affected following stimulation with these neurotransmitters. Conclusions & Inferences We provide evidence that enteric glial cells respond to fast excitatory neurotransmitters by changes in intracellular Ca²⁺. On the basis of our experimental in vitro setting, we show that enteric glia are not only directly responsive to purinergic but also to serotonergic and cholinergic signaling mechanisms.     

An immunohistochemical study of somatostatin-containing nerves in the aganglionic colon of human and rat

Summary The distribution of somatostatin-like immunoreactive (SOM-LI) nerves was elucidated immunohistochemically in the gut tissues from patients with Hirschsprung's disease and congenital aganglionosis rats. In the normoganglionic human colon, SOM-LI nerve cell bodies were found to a greater extent in the submucous plexus and to a lesser extent in the myenteric plexus. However, they were rarely observed in both the plexuses of the oligoganglionic segment. SOM-LI nerve fibres were widely distributed in the aganglionic bowel. The circular muscle layer of the distal aganglionic segment was densely innervated by SOM-LI nerve fibres which are probably derived from the extrinsic, hypertrophic nerve bundles. A decreased number of the intramuscular nerves fibres were seen in the proximal aganglionic segment. In the colon and rectum from adult and 21-day-old rats, SOM-LI cell bodies were numerous in both plexuses. On the other hand, enteric neurons were completely lacking from the colon and rectum of congenital aganglionosis rats of 21 days old. No neuronal elements staining for SOM were disclosed in these aganglionic segments of mutant rats. A possible origin and pathophysiological role of the extrinsic nerve fibres containing SOM in the diseased bowel are discussed. It is concluded that SOM-LI nerves in the human distal colon comprise both intrinsic and extrinsic elements, while SOM nerves in the rat colon and rectum are of only intrinsic origin.     

Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system

Enteric glial cells are vital for the autonomic control of gastrointestinal homeostasis by the enteric nervous system. Several different functions have been assigned to enteric glial cells but whether these are performed by specialized subtypes with a distinctive phenotype and function remains elusive. We used Mosaic Analysis with Double Markers and inducible lineage tracing to characterize the morphology and dynamic molecular marker expression of enteric GLIA in the myenteric plexus. Functional analysis in individually identified enteric glia was performed by Ca²⁺ imaging. Our experiments have identified four morphologically distinct subpopulations of enteric glia in the gastrointestinal tract of adult mice. Marker expression analysis showed that the majority of glia in the myenteric plexus co‐express glial fibrillary acidic protein (GFAP), S100β, and Sox10. However, a considerable fraction (up to 80%) of glia outside the myenteric ganglia, did not label for these markers. Lineage tracing experiments suggest that these alternative combinations of markers reflect dynamic gene regulation rather than lineage restrictions. At the functional level, the three myenteric glia subtypes can be distinguished by their differential response to adenosine triphosphate. Together, our studies reveal extensive heterogeneity and phenotypic plasticity of enteric glial cells and set a framework for further investigations aimed at deciphering their role in digestive function and disease. GLIA 2015;63:229–241     

Expression of glial antigens C1 and M1 in the peripheral nervous system during development and regeneration

The expression of C1 and M1 antigens was studied by indirect immunofluorescence methods in histological sections of peripheral nerves and ganglia of C57BL/6J mice during development and regeneration. In sciatic nerves of adult mice, C1 but not M1 antigen is found in vimentin- and glial fibrillary acidic protein (GFAP)-positive Schwann cells. A similar distribution is also seen in trigeminal nerve, dorsal root and superior cervical ganglia, and olfactory nerve. In all cases vimentin-positive structures outnumber GFAP- or C1 antigen-positive ones. At birth, C1 antigen and vimentin are expressed in sciatic nerves, but GFAP is not yet detectable. M1 antigen cannot be detected in Schwann cells. In monolayer cultures of neonatal mouse dorsal root ganglia, C1 antigen is expressed in a fibrillary staining pattern in some, but not all morphologically identified Schwann cells. In vitro, M1 antigen is not detectable in Schwann cells. After lesioning sciatic nerves of adult mice by cut or crush, detectable levels of C1 antigen rise after 4-6 days: The number of immunofluorescently labeled structures and their relative intensities are drastically augmented, first distally more so than proximally, over control values from non-lesioned, i.e. contralateral nerves. A similar augmentation is also observed for vimentin and GFAP. M1 antigen expression does not reach detectable levels in Schwann cells under these conditions. The increased detectability of C1 antigen persists up to 150 days after lesioning, the longest time period tested.     

Monoclonal antibody to glial fibrillary acidic protein reveals a parcellation of individual barrels in the early postnatal mouse somatosensory cortex

The relative dispositions of cells in immature and mature barrel field cortices that bind antibody to glial fibrillary acidic protein (GFAP) were examined and photographed under the light microscope. Light micrographs demonstrate that radially oriented glial cells are present in the barrel field of postnatal day 6 cortices and that they are located predominantly within the presumptive barrel sides and/or septae, thus sharply delineating individual barrels from each other. The relative dispositions of radial glial fibers observed at this time implicate glia in development of topographic order during early postnatal development of the somatosensory cortex. In contrast, no such delineation could be detected in the cortices of more mature mice, because GFAP-positive astrocytes are present throughout the barrel field and are not confined to barrel sides. This ephemeral nature of the GFAP-delineated barrel field is of interest with respect to the recently reported ephemeral lectin-delineated barrel field.     

The chemical code of porcine enteric neurons and the number of enteric glial cells are altered by dietary probiotics

Background The enteric nervous system (ENS) contains chemically coded populations of neurons that serve specific functions for the control of the gastrointestinal tract. The ability of neurons to modify their chemical code in response to luminal changes has recently been discovered. It is possible that enteric neuronal plasticity may sustain the adaptability of the gut to changes in intestinal activity or injury, and that gut neurons may respond to an altered intestinal environment by changing their neuropeptide expression. Methods We used immunohistochemical methods to investigate the presence and localization of several neuronal populations and enteric glia in both the small (ileum) and large (cecum) intestine of piglets. We assessed their abundance in submucosal and myenteric plexus from animals treated with the probiotic Pediococcus acidilactici compared with untreated controls. Key Results The treated piglets had a larger number of galanin‐ and calcitonin gene‐related peptide (CGRP)‐immunoreactive neurons than controls, but this was limited to the submucosal plexus ganglia of the ileum. Moreover, immunohistochemistry revealed that glial fibrillary acidic protein‐positive enteric glial cells were significantly higher in the inner and outer submucosal plexuses of treated animals. Conclusions & Inferences The neuronal and glial changes described here illustrate plasticity of the ENS in response to an altered luminal environment in the gastrointestinal tract.     

Central nervous system stem/progenitor cells form neurons and peripheral glia after transplantation to the dorsal root ganglion

We asked whether neural stem/progenitor cells from the cerebral cortex of E14.5 enhanced green fluorescent protein transgenic mice are able to survive grafting and differentiate in the adult rat dorsal root ganglion. Neurospheres were placed in lumbar dorsal root ganglion cavities after removal of the dorsal root ganglia. Alternatively, dissociated neurospheres were injected into intact dorsal root ganglia. Enhanced green fluorescent protein-positive cells in the dorsal root ganglion cavity were located in clusters and expressed beta-III-tubulin or glial fibrillary acidic protein after 1 month, whereas after 3 months, surviving grafted cells expressed only glial fibrillary acidic protein. In the intact adult DRG, transplanted neural stem/progenitor cells surrounded dorsal root ganglion cells and fibers, and expressed glial but not neuronal markers. These findings show that central nervous system stem/progenitor cells can survive and differentiate into neurons and peripheral glia after xenotransplantation to the adult dorsal root ganglion.     

The relationship between glial distortion and neuronal changes following intestinal ischemia and reperfusion

Background Damage to mucosal epithelial cells, muscle cells and enteric neurons has been extensively studied following intestinal ischemia and reperfusion (I/R). Interestingly, the effects of intestinal I/R on enteric glia remains unexplored, despite knowledge that glia contribute to neuronal maintenance. Here, we describe structural damage to enteric glia and associated changes in distribution and immunoreactivity of the neuronal protein Hu. Methods The mouse small intestine was made ischemic for 3 h and reperfused from 1 to 12 h. Immunohistochemical localisation of glial fibrillary acidic protein (GFAP), Hu and TUNEL were used to evaluate changes. Key Results At all time points glial cells became distorted, which was evident by their altered GFAP immunoreactivity, including an unusual appearance of bright perinuclear GFAP staining and the presence of GFAP globules. The numbers of neurons per ganglion area were significantly fewer in ganglia that contained distorted glia when compared with ganglia that contained glia of normal appearance. The distribution of Hu immunoreactivity was altered at all reperfusion time points. The presence of vacuoles and Hu granules in neurons was evident and an increase in nuclear Hu, relative to cytoplasmic Hu, was observed in ganglia that contained both normal and distorted glial cells. A number of neurons appeared to lose their Hu immunoreactivity, most noticeably in ganglia that contained distorted glial cells. TUNEL reaction occurred in a minority of glial cells and neurons. Conclusions & Inferences Structural damage to gliofilaments occurs following I/R and may be associated with damage to neighboring neurons.     

Advances in ontogeny of the enteric nervous system

The neurons and glia that comprise the enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, are derived from vagal and sacral regions of the neural crest. In order to form the ENS, neural crest-derived precursors undergo a number of processes including survival, migration and proliferation, prior to differentiation into neuronal subtypes, some of which form functional connections with the gut smooth muscle. Investigation of the developmental processes that underlie ENS formation has progressed dramatically in recent years, in no small part due to the attention of scientists from a range of disciplines on the genesis of Hirschsprung's disease (aganglionic megacolon), the major congenital abnormality of the ENS. This review summarizes recent advances in the field of early ENS ontogeny and focuses on: (i) the spatiotemporal migratory pathways followed by vagal and sacral neural crest-derived ENS precursors, including recent in vivo imaging of migrating crest cells within the gut, (ii) the roles of the RET and EDNRB signalling pathways and how these pathways interact to control ENS development, and (iii) how perpendicular migrations of neural crest cells within the gut lead to the formation of the myenteric and submucosal plexi located between the smooth muscle layers of the gut wall.