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The enteric nervous system (ENS) orchestrates a broad range of important gastrointestinal functions such as intestinal motility and gastric secretion. The ENS can be affected by environmental factors, diet and disease. Changes due to these alterations are often hard to evaluate in detail when whole gut samples are used. Analyses based on pure ENS tissue can more effectively reflect the ongoing changes during pathological processes. Here, we present an optimized approach for the isolation of pure myenteric plexus (MP) from adult mouse and human. To do so, muscle tissue was individually digested with a purified collagenase. After incubation and a gentle mechanical disruption step, MP networks could be collected with anatomical integrity. These tissues could be stored and used either for immediate genomic, proteomic or in vitro approaches and enteric neurospheres could be generated and differentiated. In a pilot experiment, the influence of bacterial lipopolysaccharide on human MP was analyzed using 2-dimensional gel electrophoresis. The method also allows investigation of factors that are secreted by myenteric tissue in vitro. The isolation of pure MP in large amounts allows new analytical approaches that can provide a new perspective in evaluating changes of the ENS in experimental models, human disease and aging.

Motility disorders of the human intestine are so variable that they cannot be diagnosed by just one technique. Their aetiology is obviously so varied that they have to be approached with a broad range of technical methods. These reach from the simple haematoxylin-stained section to the isolation of stem or precursor cells. In this study, various methods to investigate the enteric nervous system and its surrounding tissue are demonstrated. While sections from paraffin-embedded material or cryostat sections provide only a two-dimensional perspective of the ENS, the whole-mount method yields three-dimensional perspectives of large areas of the gut wall. The three-dimensional impression can even be enhanced by electron microscopy of the isolated ENS. Dynamical aspects of ENS development can be tackled by in vitro studies. The myenteric plexus can be isolated and cultivated under the influence of the microenvironment (protein extracts). Although the postnatal myenteric plexus is not fully developed, the choice of embryological neuronal cells seems to be more effective for certain approaches. They can be isolated from the embryonic mouse gut and cultivated under the influence of various factors. This method seems to us a valuable tool for the investigation of the aetiology of motility disorders, although only a "complete" approach which considers all available methods will yield at the end a clear understanding which might lead to new therapeutical concepts.

Enteric neurons and blood vessels form intricate networks throughout the gastrointestinal tract. To support the hypothesis of a possible interaction of both networks, we investigated whether primary mesenteric vascular cells (MVCs) and enteric nervous system (ENS)-derived cells (ENSc) depend on each other using two- and three-dimensional in vitro assays. In a confrontation assay, both cell types migrated in a target-oriented manner towards each other. The migration of MVCs was significantly increased when cultured in ENSc-conditioned medium. Co-cultures of ENSc with MVCs resulted in an improved ENSc proliferation and differentiation. Moreover, we analysed the formation of the vascular and nervous system in developing mice guts. It was found that the patterning of newly formed microvessels and neural stem cells, as confirmed by nestin and SOX2 stainings, is highly correlated in all parts of the developing gut. In particular in the distal colon, nestin/SOX2-positive cells were found in the tissues adjacent to the capillaries and in the capillaries themselves. Finally, in order to provide evidences for a mutual interaction between endothelial and neural cells, the vascular patterns of a RET((-/-)) knockout mouse model as well as human Hirschsprung's cases were analysed. In the distal colon of postnatal RET((-/-)) knockout mice, the vascular and neural networks were similarly disrupted. In aganglionic zones of Hirschsprung's patients, the microvascular density was significantly increased compared with the ganglionic zone within the submucosa. Taken together, these findings indicate a strong interaction between the enteric nervous and vascular system.

The enteric nervous system (ENS) has to respond to continuously changing microenvironmental challenges within the gut and is therefore dependent on a neural stem cell niche to keep the ENS functional throughout life. In this study, we hypothesize that this stem cell niche is also affected during inflammation and therefore investigated lipopolysaccharides (LPS) effects on enteric neural stem/progenitor cells (NSPCs). NSPCs were derived from the ENS and cultured under the influence of different LPS concentrations. LPS effects upon proliferation and differentiation of enteric NSPC cultures were assessed using immunochemistry, flow cytometry, western blot, Multiplex ELISA and real-time PCR. LPS enhances the proliferation of enteric NSPCs in a dose-dependent manner. It delays and modifies the differentiation of these cells. The expression of the LPS receptor toll-like receptor 4 on NSPCs could be demonstrated. Moreover, LPS induces the secretion of several cytokines. Flow cytometry data gives evidence for individual subgroups within the NSPC population. ENS-derived NSPCs respond to LPS in maintaining at least partially their stem cell character. In the case of inflammatory disease or trauma where the liberation and exposure to LPS will be increased, the expansion of NSPCs could be a first step towards regeneration of the ENS. The reduced and altered differentiation, as well as the induction of cytokine signalling, demonstrates that the stem cell niche may take part in the LPS-transmitted inflammatory processes in a direct and defined way.

Pharmaceutical compounds enter the body via several major natural gateways; i.e. the lung, the skin and the gastrointestinal tract. Drug application during surgical operations can lead to severe impairment of gastrointestinal motility, which can contribute to a paralytic ileus. Here we investigated an ex vivo perfused small intestine model that allows us to ascertain the influence of pharmaceuticals upon the gut. Corresponding segments from the proximal jejunum of adult rats were used. Their mesenteric arteries and veins were cannulated and the jejunal segment excised. The individual segments were placed in a custom designed perfusion chamber and perfusion performed through the intestinal lumen as well as the mesenteric superior artery. Three test drugs, which are commonly used in anesthesiology; i.e. pentobarbital, propofol and ketamine were administered via the blood vessels. Their effects upon gastrointestinal motility patterns were evaluated by optical measurements. Longitudinal and pendular movements were distinguishable and separately analyzed. Pharmacological effects of the individual substances could be investigated. Propofol (50-200 μg/ml) was found to decrease intestinal motility, especially longitudinal movements in a dose dependent manner. Pentobarbital decreased intestinal motility only at high concentrations, above 2.5 mg/ml. A dose of 2.5 mg/ml lead to an increase in longitudinal- and pendular movements in comparison to control, while ketamine (2.5-10 mg/ml) did not alter intestinal motility at all. Histological examination of the perfused segments revealed only minor changes in tissue morphology after perfusion. The perfusion approach shown here allows for the identification of compounds which interfere with gut motility in a highly sophisticated way. It is suitable for characterization of drug and dose specific changes in motility patterns and can be used in drug development and preclinical studies.

The enteric nervous system (ENS) controls and modulates gut motility and responds to food intake and to internal and external stimuli such as toxins or inflammation. Its plasticity is maintained throughout life by neural progenitor cells within the enteric stem cell niche. Granulocyte-colony stimulating factor (G-CSF) is known to act not only on cells of the immune system but also on neurons and neural progenitors in the central nervous system (CNS). Here, we demonstrate, for the first time, that G-CSF receptor is present on enteric neurons and progenitors and that G-CSF plays a role in the expansion and differentiation of enteric neural progenitor cells. Cultured mouse ENS-neurospheres show increased expansion with increased G-CSF concentrations, in contrast to CNS-derived spheres. In cultures from differentiated ENS- and CNS-neurospheres, neurite outgrowth density is enhanced depending on the amount of G-CSF in the culture. G-CSF might be an important factor in the regeneration and differentiation of the ENS and might be a useful tool for the investigation and treatment of ENS disorders.

In Alzheimer's disease (AD), fatal neuronal cell loss occurs long before relevant evidence can lead to a reliable diagnosis. If characteristic pathological alterations take place in the enteric nervous system (ENS), it could be one of the most promising targets for an early diagnosis, using submucosal biopsies from the gut. We therefore investigated time- and spatial-dependent changes in an amyloid-beta protein precursor (A beta PP) overexpressing transgenic mouse model to examine early changes within the ENS. Wholemount preparations and paraffin sections were analyzed for the expression of neuronal, glial, and innate immunity markers. Isolated myenteric networks were screened for differences in overall protein expression, and a motility analysis delivered functional data. The level of A beta PP in the gut was significantly higher in the AD mouse model than in wild-type mice and also higher in the gut than in the brain at all ages investigated. The transcriptional level of Nestin, GFAP, and TLR4 increased with age with a peak at 3 months. At the protein level, human amyloid-beta was located in myenteric neurons. Myenteric networks showed a reduction of the neuronal density in A beta PP compared to wild-type mice, which was functionally relevant as revealed by motility analysis. The ENS undergoes significant changes during the early onset of A beta PP expression in AD mouse models that appear before those seen in the brain as demonstrated in this study. Thus, there is a chance of determining similar alterations in the human gut of AD patients, which could be used to develop early diagnostic approaches.

Protein profiles of developing neural circuits undergo manifold changes. The aim of this proteomic analysis was to quantify postnatal changes in two auditory brainstem areas in a comparative approach. Protein samples from the inferior colliculus (IC) and the superior olivary complex (SOC) were obtained from neonatal (P4) and young adult (P60) rats. The cytosolic fractions of both areas were examined by 2-D DIGE, and the plasma membrane-enriched fraction of the IC was analyzed via iTRAQ. iTRAQ showed a regulation in 34% of the quantified proteins. DIGE revealed 12% regulated spots in both the SOC and IC and, thus, numeric congruency. Although regulation in KEGG pathways displayed a similar pattern in both areas, only 13 of 71 regulated DIGE proteins were regulated in common, implying major area-specific differences. 89% of regulated glycolysis/gluconeogenesis and citrate cycle proteins were up-regulated in the SOC or IC, suggesting a higher energy demand in adulthood. Seventeen cytoskeleton proteins were regulated, consistent with complex morphological reorganization between P4 and P60. Fourteen were uniquely regulated in the SOC, providing further evidence for area-specific differences. Altogether, we provide the first elaborate catalog of proteins involved in auditory brainstem development, several of them possibly of particular developmental relevance.

Ultrasound is widely used in clinical practice, mostly in diagnostic studies, but increasingly in therapeutic applications as well. This may be the case in acceleration of wound healing or treatment of cancer. Still, little is known about the direct effect of frequency or energy density of the ultrasound upon the cells themselves. We therefore investigated the impact of three different protocols using high, medium and low energy densities at three different frequencies on normal endothelial and epithelial as well as carcinoma cell lines (neuroblastoma and adenocarcinoma cell lines). Proliferation of endothelial and epithelial cell lines was significantly increased depending on the frequency and energy density applied. No influence on actin cytoskeleton formation was seen in these cells after treatment, while a significant decrease in the density of microvilli and the length of filopodia in the epithelial cell line could be noted. The proliferation rate of the carcinoma cell lines was reduced and cells destroyed. Apoptosis was induced in the adenocarcinoma cells after ultrasound exposure. Additionally, the expression of neurofilament was increased in neuroblastoma cells as evidence of beginning differentiation. So, different settings of frequency and energy density in an ultrasonic treatment protocol lead to different impacts on proliferation, morphology and differentiation and might be used to stimulate or inhibit the growth of individual cell types.

Breast-feeding plays an important role for the development of the newborn. Non-breast fed premature born infants show a significantly higher risk of developing diseases like infantile diarrhoea and necrotizing enterocolitis. In this study, the content of neurotrophic factors and cytokines, which might influence the postnatal development of the enteric nervous system (ENS), was determined in human breast milk. Glial cell-line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) as well as a panel of cytokines were analyzed using single factor or multiplex ELISA. In order to link their presence in milk with possible effects on the development of the ENS, rat myenteric neurons were cultured in protein extracts from breast milk. Neurite outgrowth, neuron survival and nestin expression in glial cells were measured. Growth factors and cytokines were found in all breast milk samples at varying concentrations. It could be demonstrated that protein extracts of breast milk increased the amount of surviving enteric neurones as well as neurite outgrowth. Additionally it was shown, that the number of nestin and S100-expressing glial cells increased significantly after incubating in breast milk protein extracts. The data suggest that milk-born proteins support the development of the enteric nervous system.

Background Pancreatic neuropathy in chronic pancreatitis (CP) and pancreatic cancer (PCa) is characterized by pancreatic neuropathy, i.e. increased neural density and hypertrophy, which are associated with neuropathic pain. To better understand the mechanism of these neuropathic alterations, we aimed at achieving an in-vitro simulation of the intrapancreatic neuroplasticity. Methods Dissociated myenteric plexus (MP) and dorsal root ganglia (DRG) neurons of newborn rats were treated with normal human pancreas (NP), CP or PCa tissue extracts. Furthermore, MP and DRG neurons were cultured in supernatants from different pancreatic cancer cell lines (PCC) and human pancreatic stellate cells (hPSC) obtained from either CP or PCa tissues. For analysis, the neurite density, outgrowth, neuronal branching capacity and perikaryonal size were quantified. Key Results Myenteric plexus and DRG neurons grown in CP and PCa tissue extracts built denser networks than in NP extracts. Both neuronal types showed a strong neurite outgrowth, more complex branching pattern and a somatic hypertrophy in CP and PCa extracts. Pancreatic cancer cell supernatants induced a prominent neurite outgrowth, increased neurite density and perikaryonal hypertrophy in MP and DRG neurons. Supernatants of CP-derived hPSC strongly stimulated neurite outgrowth. Glial density in MP cultures was strikingly increased by PCa tissue extracts. Conclusions & Inferences Intrapancreatic microenvironment in CP and PCa induces neuroplastic alterations under in-vitro conditions, leading to increased neural density and hypertrophy. Thus, due to its neurotrophic attributes, the intrapancreatic microenviroment in CP and PCa seems to be a key player in the generation of pancreatic neuropathy and neuroplasticity.

The enteric nervous system with its intricate network of neurons and glia shows a high plasticity, which not only changes during pre- and postnatal development, but also with disease or changing dietary habits. FGF as a potent neurotrophic factor in the central nervous system might also play a specific role for the ENS development, FGF-2 knockout and corresponding wild-type mice were histologically and functionally analyzed. FGF-2 knockout mice are viable and thrive normally and do apparently not display any obvious neurological deficit. Morphological differences were studied on whole mount preparations of muscle and submucous layer using either cuprolinic blue or immunohistochemical stainings for the neuronal marker PGP 9.5. Ussing-chamber and isometric muscle contraction experiments were performed on isolated gut wall, respectively muscle preparations. Intravital microscopy with GFP-transfected E. coli bacteria was used to investigate influences upon bacterial translocation. In additional experiments the protein pattern of the isolated myenteric plexus of knockout and wild-type mice were compared using 2D-DIGE technology. The morphometric analysis of the myenteric plexus revealed significant differences between FGF-2 knockout and wild-type animals, resulting in larger neurons in the knock out animals, embedded in less densely packed enteric ganglia. While muscle contractility appeared not to be affected, there was a significant difference in bacterial translocation as well as differences in basal chloride secretion to be seen. The observed morphological differences were reflected in the varying protein patterns, which were revealed by 2D-DIGE. A large number of differentially expressed proteins were found in both colonic and duodenal samples. FGF obviously influences the development of well established gastrointestinal functions by various means, thus leading to minor but significant deficiencies. Whether the revealed deficits in the mucous barrier are indebted to the morphological alterations in the ENS cannot yet be proved, but is very likely.

Paraneoplastic neurological syndromes (PNS) are often associated with antineuronal autoantibodies and many of them could be identified in the recent years. However, there are still new antineuronal binding patterns with yet unidentified autoantigens. We here describe a new autoantibody associated with paraneoplastic sensorimotor and autonomic neuropathy in a patient with small cell lung cancer. In indirect immunofluorescence test, the patients serum colocalised with the synaptic protein synaptophysin in the cerebellum and myenteric plexus of the gut. Immunoblotting showed a 38 kDa reactivity, which is also the molecular weight of synaptophysin. Therefore a Western Blot with recombinant synaptophysin has been used and revealed reactivity of the serum against synaptophysin. In patients with non-paraneoplastic neuropathies or healthy controls, anti-synaptophysin autoantibodies were not detectable. In 20 SCLC patients without neurological syndromes, one patient had low-titer anti-synaptophysin autoantibodies. Interestingly, the PNS patient´s serum and IgG fraction showed marked cytotoxicity to primary cultured myenteric plexus neurons.We conclude that synaptophysin is an autoantigen in paraneoplastic neurological syndromes.