Effect of myenteric denervation on intestinal epithelium proliferation and migration of suckling and weanling rats

The effects of myenteric denervation on the cell kinetics of the intestinal epithelium of suckling and weanling rats were investigated. The myenteric plexus of an ileal segment was partially ablated by serosal application of benzalkonium chloride (BAC) in three groups of rats: those that underwent surgery at 13 days and were killed 15 (13/28-day-old) or 23 (13/36-day-old) days after treatment, and those that were operated at 21 days (21/36-day-old) and were killed 15 days after treatment. The extent of denervation was assessed in whole-mount preparations. The cell bodies of myenteric neurones were stained by NADH-diaphorase histochemical technique. Cell proliferation was estimated by the mitotic index (MI) and morphometric analysis of villus and crypt lengths using an image analysis system. Thickness of the muscle layers was also assessed by morphometry. Cell migration on the villi was estimated by the position of the leading labelled cell 24 h after tritiated thymidine injection. The number of neurones was reduced by around 80% in rats operated at 13 days, and reduced by 98% in those operated at 21 days. The thickness of the muscle layers was increased in all groups of treated animals. MI was significantly higher 15 days after BAC-treatment in the 13/28 group. Morphological changes in the intestinal mucosa were observed 15 days after BAC-treatment, when there was an increase in villus height (13/28 group) and crypt depth (13/28 and 21/36 groups). Cell migration rate was accelerated in the 21/36 group. No differences where found in the 13/36 group. These results show the strong effect of myenteric ablation on cell proliferation and migration in the ileal epithelium in the first 15 days of treatment in suckling and in weanling rats, and the subsequent recovery of intestinal mucosa homeostasis later on.     

Ileal VIP submucous neurons: confocal study of the area enlargement induced by myenteric denervation in weanling rats

Vasoactive Intestinal Peptide (VIP) neurons are maturing during suckling and weaning periods and the neuropeptide VIP is thought to be neurotrophic during ontogenesis. We have previously demonstrated that suckling rats with myenteric ablation have significantly higher mitotic index and an increase on villus height and crypt depth 15 days after treatment. In the current study, we measured the area of VIP neurons of submucous plexus in the ileum of weanling rats, in which myenteric neurons were ablated by serosal application of benzalkonium chloride (BAC). The area of VIP immunoreactive cell bodies, reconstructed under confocal microscope, was significantly increased in response to denervation. This result suggests that the myenteric plexus may have an inhibitory role over submucous plexus in the normal intestine. The enhanced production of VIP may be correlated with the increased epithelial proliferation induced by denervation in a critical period of life, from suckling to weaning time.     

Consequences of Citrobacter rodentium infection on enteroendocrine cells and the enteric nervous system in the mouse colon

We tested the hypothesis that Citrobacter rodentium infection leads to changes in the mucosal enteroendocrine signalling and the enteric nervous system and that the host's immune response contributes to these changes. Enteroendocrine cells, serotonin (5-HT) reuptake transporter (SERT), 5-HT release, and inducible nitric oxide synthase (iNOS) expression were assessed in the colon of infected wild-type or severe combined immunodeficient (SCID) mice. Immunoreactivity for iNOS and neuropeptides were examined in the submucosal and myenteric plexuses. Mice were orogastrically infected with C. rodentium and experiments were conducted during the injury phase (10 days) and the recovery phase (30 days). 5-HT and somatostatin enteroendocrine cells and SERT were significantly reduced 10 days after infection, with numbers returning to control values at 30 days. 5-HT release was increased at 10 days. Changes to the mucosal serotonin signalling system were not observed in SCID mice. iNOS immunoreactivity was increased in the submucosa and mucosa at 10 days and returned to baseline levels by 30 days. No differences were observed in neuropeptide or iNOS immunoreactivity in the enteric plexuses following infection. The host's immune response underlies changes to enteroendocrine cells, SERT expression and 5-HT release in C. rodentium infection. These changes could contribute to disturbances in gut function arising from enteric infection.     

Ca(2+) permeability of human heteromeric nAChRs expressed by transfection in human cells

The distribution of the calcium binding protein neurocalcin a has been examined in the enteric nervous system of young adult (3 months) and aged (24+ months) male rats by immunofluorescence. Neurocalcin-immunoreactive (NC-ir) neurons were observed in the submucous and myenteric plexuses throughout the gastrointestinal tract from the oesophagus to the distal large intestine. NC-ir nerve terminals were also seen on NC-ir and NC-negative neurons. Semiquantitative estimates revealed fewer NC-ir neurons in the submucous plexus than in the myenteric plexus. The greatest occurrence of NC-ir neurons was in the small and large intestine. NC-ir axons were seen in the mucosa and also in between the ganglia of the myenteric plexus. In the aged rats, there were no discernible changes in the numbers of NC-ir neurons in th e oesophagus and stomach, with an increase in the pylorus and slight decreases in the small and large intestines. No decrease in NC-ir was observed in the distal large intestine. NC-ir neurons never contained lipofuscin age pigment and many enteric neuro ns devoid of NC-ir contained age pigment. Like other previously investigated calcium-binding proteins in enteric neurons, the distribution of NC shows much variability from one part of the intestine to another. The observed slight decreases in the number of NC-ir enteric neurons in aged rats may compromise the regulation of calcium in these neurons.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.     

Development of Calbindin- and Calretinin-Immunopositive Neurons in the Enteric Ganglia of Rats

Calbindin D28 K (CB) and calretinin (CR) are the members of the EF-hand family of calcium-binding proteins that are expressed in neurons and nerve fibers of the enteric nervous system. CB and CR are expressed differentially in neuronal subpopulations throughout the central and peripheral nervous systems and their expression has been used to selectively target specific cell types and isolate neuronal networks. The present study presents an immunohistochemical analysis of CB and CR in the enteric ganglia of small intestine in rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 60-day-old, 1-year-old, and 2-year-old). The data obtained suggest a number of age-dependent changes in CB and CR expression in the myenteric and submucous plexuses. In the myenteric plexus, the lowest percentage of CB-immunoreactive (IR) and CR-IR neurons was observed at birth, after which the number of IR cells increased in the first 10 days of life. In the submucous plexus, CB-IR and CR-IR neurons were observed from 10-day-old onwards. The percentage of CR-IR and CB-IR neurons increased in the first 2 months and in the first 20 days, respectively. In all animals, the majority of the IR neurons colocalized CR and CB. From the moment of birth, the mean of the cross-sectional area of the CB-IR and CR-IR neuronal profiles was larger than that of CB- and CR-negative cells.     

The chronological relationship between the thickening of smooth muscle, epithelial cell proliferation and myenteric neural denervation in the rat jejunum

The jejunum of rats was treated by serosal application of a 0.2% solution of benzalkonium chloride (BAC) for 30 min. Control animals were treated with saline (0.9% NaC1). The animals were allocated to eight groups of 10 rats each and sacrificed 15, 30, 45, 60 days after BAC treatment. Segments were removed from the jejunum for neuronal counting, measurement of the smooth muscle area and morphokinetic study of the epithelium. There was a significant reduction in neuron number in the myenteric plexus 30 days after BAC treatment, thickening of smooth muscle 15-60 days after BAC treatment, but no change in epithelial cell proliferation in the jejunum at either time.     

Effect of Chemical Ablation of Myenteric Neurones On Intestinal Cell Proliferation

Abstract. The duodenum or descending colon of male Wistar rats (average weight 60 g) was treated by a serosal application of a 0.2% solution of benzalkonium chloride (BAC) for 30 min. Control animals were treated with 0.9% (physiological) saline. the rats were allocated to four groups: Group DC ( N = 8) in which the duodenum was treated with physiological saline; Group DB ( N = 8) in which the duodenum was treated with BAC; Group CC ( N = 7) in which the descending colon was treated with physiological saline and Group CB ( N = 7) in which the descending colon was treated with BAC. After treatment, the animals were followed up for 5 months. At the end of the experiment, the animals were injected intraperitoneally with vincristine sulphate before sacrifice. Three segments were removed from the duodenum and descending colon for neuronal counting, catecholamine and serotonin measurements and morphokinetic studies of the epithelium. the following results were obtained: (1) there was a significant reduction in neurone number in the myenteric plexus of segments treated with BAC; (2) in the denervated intestinal segments, catecholamine levels were unchanged whereas serotonin levels were increased; (3) epithelial hyperplasia was observed in the denervated duodenum and descending colon; and (4) crypt cell production rate in the duodenum was similar in groups DC and DB but was significantly increased in the descending colon in group CB as compared with controls (CC). the present findings indicate that selective myenteric neuronal denervation caused by benzalkonium chloride plays a causative role in the hyperplasia and crypt cell production rate of the intestinal epithelium (duodenum and descending colon). These changes are probably induced by functional imbalance by the surviving neuronal elements in the gut, implicating neurotransmitters such as acetylcholine, noradrenaline, serotonin, somatostatin and vasoactive intestinal peptide.     

Effect of chemical ablation of myenteric neurones on intestinal cell proliferation

The duodenum or descending colon of male Wistar rats (average weight 60 g) was treated by a serosal application of a 0.2% solution of benzalkonium chloride (BAC) for 30 min. Control animals were treated with 0.9% (physiological) saline. The rats were allocated to four groups: Group DC (N = 8) in which the duodenum was treated with physiological saline; Group DB (N = 8) in which the duodenum was treated with BAC; Group CC (N = 7) in which the descending colon was treated with physiological saline and Group CB (N = 7) in which the descending colon was treated with BAC. After treatment, the animals were followed up for 5 months. At the end of the experiment, the animals were injected intraperitoneally with vincristine sulphate before sacrifice. Three segments were removed from the duodenum and descending colon for neuronal counting, catecholamine and serotonin measurements and morphokinetic studies of the epithelium. The following results were obtained: (1) there was a significant reduction in neurone number in the myenteric plexus of segments treated with BAC; (2) in the denervated intestinal segments, catecholamine levels were unchanged whereas serotonin levels were increased; (3) epithelial hyperplasia was observed in the denervated duodenum and descending colon; and (4) crypt cell production rate in the duodenum was similar in groups DC and DB but was significantly increased in the descending colon in group CB as compared with controls (CC). The present findings indicate that selective myenteric neuronal denervation caused by benzalkonium chloride plays a causative role in the hyperplasia and crypt cell production rate of the intestinal epithelium (duodenum and descending colon). These changes are probably induced by functional imbalance by the surviving neuronal elements in the gut, implicating neurotransmitters such as acetylcholine, noradrenaline, serotonin, somatostatin and vasoactive intestinal peptide.     

Neuroplasticity in the smooth muscle of the myenterically and extrinsically denervated rat jejunum

The objective of this study was to examine the effects of two different denervation procedures on the distribution of nerve fibers and neurotransmitter levels in the rat jejunum. Extrinsic nerves were eliminated by crushing the mesenteric pedicle to a segment of jejunum. The myenteric plexus and extrinsic nerves were eliminated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). The effects of these two denervation procedures were evaluated at 15 and 45 days. The level of norepinephrine in whole segments of jejunum was initially reduced by more than 76% after both denervation procedures, but by 45 days the level of norepinephrine was the same as in control tissue. Tyrosine hydroxylase (nor-adrenergic nerve marker) immunostaining was absent at 15 days, but returned by 45 days. However, the pattern of noradrenergic innervating axons was altered in the segment deprived of myenteric neurons. Immunohistochemical studies showed protein gene product 9.5 (PGP 9.5)-immunoreactive fibers in whole-mount preparations of the circular smooth muscle in the absence of the myenteric plexus and extrinsic nerves. At 45 days, the number of nerve fibers in the circular smooth muscle increased. Vasoactive intestinal polypeptide (VIP)-immunoreactive fibers, a subset of the PGP 9.5 nerve fibers, were present in the circular smooth muscle at both time points examined. Choline acetyltransferase (CAT) activity and VIP and leucine enkephalin levels were measured in separated smooth muscle and submucosa-musosal layers of the denervated jejunum. VIP and leucine-enkephalin levels were no different from control in tissue that was extrinsically denervated alone. However, the levels of these peptides were elevated two-fold in the smooth muscle 15 and 45 days after myenteric and extrinsic denervation. In the submucosa-mucosa, VIP and leucine enkephalin levels also were elevated two-fold at 15 days, but comparable to control at 45 days. CAT activity was equal to control in the smooth muscle but elevated two-fold in the submucosa-mucosa at both times. These results provide evidence for innervation of the circular smooth muscle by the submucosal plexus. Moreover, these nerve fibers originating from the submucosal plexus proliferate in the absence of the myenteric plexus. Furthermore, the myenteric neurons appear to be essential for normal innervation of the smooth muscle by the sympathetic nerve fibers. It is speculated that the sprouting of the submucosal plexus induced by myenteric plexus ablation is mediated by increased production of trophic factors in the hyperplastic smooth muscle.     

A Time-Limited and Partially Reversible Model of Hypoganglionosis Induced by Benzalkonium Chloride Treatment

Serosal application of benzalkonium chloride (BAC) has been previously applied to produce a model of aganglionosis; however, confusion remains regarding the extent of chemical ablation of enteric myenteric plexus after BAC treatment. The time sequence of BAC-induced effects on the myenteric plexus of the rat colon was determined and followed the morphologic changes. After sacrifice of animals 7, 14, 28, 56, 84 or 168 days postintervention, colonic tissue samples were removed, fixed in formalin, and cut into 5-μm longitudinal sections for histological analysis. The neural analysis was used to re-evaluate BAC treatments for the appropriate model. Compared with rats in sham groups, rats in 0.1 %-30-min BAC group maintained only 15.27 ± 4.80 % of ganglia per section in a 1-cm/5-μm slice and 11.76 ± 2.30 % of ganglionic cells after 28 days, the lower and stable number of ganglionic cells between Day 7 and 84 (from 11.67 ± 2.10 to 19.05 ± 5.10 %). Although an increase, ganglionic cell numbers did not recover at Day168 when compared with the numbers in sham groups. The results showed that characteristics of rats in the 0.1 %-30-min BAC group between Day 7 and 84 most closely kept in stable state, suggesting that these treatment parameters are ideal for producing a hypoganglia model of hypoganglionosis.     

急性力竭运动对大鼠胃肠传输速率及回肠肌间神经丛氮能神经的影响

目的：研究力竭运动对大鼠胃肠动力的影响及其肠神经机制。方法：24只大鼠随机分成对照组和急性力竭运动组,建立力竭运动大鼠模型,测定胃肠传输速率,用酶组织化学方法和计算机图像分析技术对两组大鼠回肠肌间神经丛内氮能神经元的数目和一氧化氮合酶（NOS）的表达进行测定。结果：急性力竭运动组大鼠胃肠传输速率明显延迟,回肠肌间神经丛内氮能神经元的数目明显增多和NOS的表达显著增强（P〈0．05和P〈0．01）。结论：大鼠力竭运动后小肠肌问神经丛内氮能神经元的数目增多和NOS的表达增强可能是导致胃肠传输速率延迟的重要原因之一。     

Plasticity of interstitial cells of Cajal: a study of mouse colon

Ablation of the myenteric plexus in mouse colon with the detergent benzalkonium chloride (BAC) is followed by considerable recovery of the nerves, indicating that this plexus is capable of regeneration and has plasticity. Interstitial cells of Cajal (ICC) are closely associated with enteric nerves, and the acquisition and maintenance of their adult phenotype are nerve-dependent. Little is known about the regenerative processes of ICC or about the possible dependence of these processes on neurons. To address these questions, we ablated the myenteric plexus in the mouse colon with BAC and followed changes in the adjacent ICC (ICC-MP) from day 2 to day 70 after treatment, by using c-kit-immunohistochemistry and electron microscopy. In the untreated area, c-kit-positive cells and ICC-MP with normal ultrastructural features were always present. The region partially affected by BAC contained some c-kit-positive cells, and either normal or vacuolated ICC-MP were observed by electron microscopy. Moreover, at days 60–70, ICC-MP with particularly extended rough endoplasmic reticulum were present in this area. In the treated area, either denervated or reinnervated, c-kit-positive cells were always absent. By day 14 after BAC treatment, nerve fibers had started to grow back into the treated region and, in the reinnervated area, cells with fibroblast-like features appeared and were seen to contact both nerve endings and smooth muscle cells and to acquire some typical ICC features. Thus, ICC are vulnerable to external insult but appear to have some ability to regenerate.This work was supported by the US-Israel Binational Science Foundation (BSF, 98-00185; to M.H.) and University funds “quota di ateneo ex 60%” (M.-S. F.-P.).     

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Growth of enteric neurones from isolated myenteric ganglia in dissociated cell culture

Summary Ganglia of the myenteric plexus from the newborn guinea-pig, isolated by microdissection, were dissociated by a combination of enzymatic and mechanical methods. The neurones and glial cells in the resulting cell suspension were cultured for up to 21 days in vitro. The growth of the enteric ganglion cells in serum-free, hormone-supplemented (N1) medium and in serum-supplemented medium containing a mitotic inhibitor was compared over a period of 14 days in vitro. Enteric neurones were outnumbered by glia in both culture media, although glial cell proliferation was inhibited in both media compared with that in serum-supplemented medium without mitotic inhibitors. Glial cell numbers appeared to decline in serum-free medium after the first week in vitro. Neurites tended to be more varicose in the serum-free medium, and the morphology of the enteric glial cells also differed markedly in the two media. This is the first report of the dissociation and subsequent culture of myenteric ganglia that had previously been completely isolated from the remainder of the gut wall.     

Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons

Summary Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.     

Morphological and Functional Alterations of the Myenteric Plexus in Rats with TNBS-Induced Colitis

The 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced model of experimental colitis was used to investigate the time-course of alterations in enteric neurotransmission and/or smooth muscle function that occur in chronic inflammation. Myenteric plexus morphology (immunocytochemical markers), functional integrity of cholinergic neurons (³H-choline uptake, acetylcholine release and contractile response to electrical field stimulation) and smooth muscle integrity (contractile response to exogenous acetylcholine) were determined 2, 7, 15, and 30 days after TNBS treatment. In TNBS-treated rats extensive ulcerations of the mucosa and/or the submucosa and increase in colonic weights were accompanied by significant reduction in ³H-choline uptake, acetylcholine release and contractile response to stimulation of enteric nerves. These changes were maximal 7 and 15 days after TNBS treatment. Immunocytochemical marker (PGP 9.5, SNAP 25, synaptophysin and S100 protein) expression was absent in necrotic areas of colons removed 7 days post-injury and partially reduced in colons removed 15 days after TNBS treatment. By contrast, the contractile response to exogenous acetylcholine was significantly increased after 7 days in both inflamed and uninflamed regions and returned to control values by day 30. Likewise, an almost complete recovery of neural cholinergic function and of myenteric plexus morphology was observed 30 days after TNBS treatment. These data suggest that TNBS-induced colitis is associated with progressive and selective alterations in myenteric plexus structure and function, with consequent reduction of cholinergic neurotransmission and abnormality in colonic contractility. The reversibility of myenteric plexus disruption is a clear indication of neuronal plasticity within enteric nervous system as an adaptative mechanism against inflammatory challenges.     

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.