Search

Introduction: The transplantation of neural crest derived stem cells (NCSC) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. Influences of HSCR-smooth-muscle-protein extracts upon isolated myenteric plexus cells, dissociated dorsal root ganglia and NCSC were studied in vitro to investigate the quality of this microenvironment effects., Methods: Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) and transforming-growth-factor-β-1 (TGFβ-1) concentration were measured in the homogenates from the individual segment using ELISA. Myenteric plexus and dissociated dorsal root ganglia (DRG) cultures, as well as NCSCs were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival, and branching was evaluated., Results and Conclusions: The amount of the factors varied considerably between the individual segments and also from patient to patient. Four major expression patterns could be detected. While all extracts tested lead to a significant increase in neurite outgrowth compared to the control, extracts from proximal segments tended to have more prominent effects. In one experiment, extracts from all individual segments of a single patient were tested. Neurite outgrowth, neuronal survival, and branching pattern varied from segment to segment, but all HSCR-muscle-protein extracts increased neuronal survival and network formation. Smooth muscle protein from aganglionic bowel supports the survival and outgrowth of myenteric neurons and NCSCs and is so an appropriate target for neural stem cell treatment.

Background and Aims: The transplantation of neural crest derived stem cells (NCSC's) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. To investigate the quality of this microenvironment, effects of HSCR-smooth-muscle-protein extracts upon NCSC's were studied in vitro., Methods: Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) concentration was measured in the homogenates from the individual segment using ELISA. NCSC's were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival and branching was evaluated., Results: The amount of the factors varied considerably between the proximal and distal segments, and also from patient to patient. While extracts from proximal segments tended to have more prominent effects, all HSCR-muscle-protein extracts increased neuronal survival and network formation., Conclusion: Muscle protein from aganglionic bowel supports the survival and outgrowth of NCSC's and is so an appropriate target for neural stem cell treatment.

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.

In contrast to adult surgery, the neonatal small intestine confronts the surgeon, depending on the age of the patient, with variable diameters of the intestine. Therefore, anastomoses are usually performed by hand with interrupted sutures. In the presented study, a new technique is demonstrated. An anastomosis in the distal ileum of Sprague Dawley rats was performed with a single clamp applicator (Anastoclip). Small bowel anastomoses were performed in 32 rats. The clipped bowel anastomosis was evaluated concerning stenosis, leakage, and adhesions in comparison to the sutured anastomosis. Tension test and X-ray examination were performed to measure the stability. The rats were sacrificed at day 3 or 14 after laparotomy. The clipped anastomosis is feasible, and faster to perform than the conventional hand-sutured anastomosis (Operation time: control group: 18.5 min versus clipped group 4 min; p > 0.05). Furthermore, there were differences in the mechanical stability, with higher tension forces needed for rupturing the clipped anastomosis. There were fewer stenoses (16.5 mm stenotic diameter in the control group versus 20.6 mm in the clipped group) and fewer adhesions in the group of the clipped anastomosis. Histological examinations were performed and did not show significant differences between the two groups. In the animal model presented, the clipped, intestinal, non-perforating anastomosis (CINPA) shows advantages compared to the common hand-sutured anastomosis.

Background: The human enteric nervous system (ENS) descends from migrating neural crest cells (NCC) and is structured into different plexuses embedded in the gastrointestinal tract wall. The development of this entity strongly depends on the supply of an appropriate support with trophic factors during organogenesis. The lack of important factors, such as glial cell line-derived neurotrophic factor, leads to severe disturbances in the ENS and, thus, to motility disorders in children. The isolation of neuronal precursor cells as well as their transplantation after expansion in vitro is therefore a hopeful new approach concerning all forms of dysganglionosis in children., Methods: We therefore established a way to isolate and expand precursor cells from the developing and postnatal human ENS. Bowel samples were obtained from human fetuses and children (from the 9th week of gestation to 5 years postnatal). Myenteric plexus was isolated by enzymatical digestion and cultivated until spheroid aggregates, the so-called neurospheres, developed. These neurospheres could be differentiated and also be transplanted after dissociation into aganglionic bowel in vitro., Results: Enteric neurospheres could be grown from different gestational ages, including postmortem material. Undifferentiated proliferating precursor cells were kept in culture for up to 72 days and could be differentiated in neurons and glial cells in vitro., Conclusion: The first results using isolated enteric neurospheres in aganglionic bowel are quite promising and are a basis to develop an appropriate cell therapy for all kinds of dysganglionosis, especially for cases where a surgical approach is not sufficient or not even possible.

Aim: The aim of this study was to establish a three-dimensional model of the innervated mucosal barrier using a co-culture of an enterocyte cell line and enteric glial and nerve cells. Such a model might form the basis for further studies of interactions between the single compartments of the bowel wall, as well as of extrinsic influences on intestinal development and plasticity., Methods: Isolated and dissociated myenteric plexus was resuspended in either collagen or extracellular matrix (ECM) solutions. After incubation at 37 degrees C the solution gelled and formed stable plugs where neurons and glial cells reaggregated to form secondary neuronal networks. HT-29-enterocytes were seeded on top of the gels either immediately (collagen, ECM), or after adding a thin layer of collagen II (ECM)., Results: While the neuronal tissue formed complex networks within the gel, the enterocytes on top of the gels grew differently depending on the substrate and innervation. So enterocytes on ECM gels did not grow to confluence, while on collagen gels or on ECM plus collagen larger patches and increasing confluence could be observed. In general HT-29 grew better on innervated gels than on gels with no neuronal tissue., Conclusions: With the presented model of different compartments of the bowel wall, various parameters of intercellular dependencies and influences can be observed in vitro. Moreover, the first results are also steps towards developing an innervated gut wall in vitro which might be able to restore functional capacity in infants with short bowel syndrome or other disorders that severely impair bowel function.

Introduction: After successful experimental autologic-allotopic ileum mucosa transplantation, we investigated the remodeling of the transplanted submucous and mucous plexus, which is essential for the motility of the created colon coat-ileum mucosa complex., Method: In 8 beagles we transplanted ileum mucosa in a demucosed vascularized transverse colon segment, which was reanastomosed with the small bowel immediately after transplantation. Four weeks later the animals were sacrificed and histology specimens taken from the anastomosis site of the colon coat-ileum mucosa complex, allowed comparison between transplanted and normal mucosa in the same section. After fixation in 4% formaldehyde and PBS the samples were embedded in paraffin and 7 micro m sections were prepared. The distribution of nerve fibers and submucous ganglia were examined in dewaxed sections, using antisera against protein gene product (PGP9.5), a general neuronal marker., Results: The submucosal ganglia were prominent in all samples but they were smaller and the submucous nerve cells within the ganglia were less numerous compared to the controls. The innervation of the transplanted ileum mucosa was reduced as the number of nerve fibers in the mucosal villi was decreased. Besides these neuromorphologic changes the transplanted mucosa showed a slightly higher rate of shortened villi compared to normal ileum mucosa., Conclusions: After ileum mucosa transplantation the submucosal ganglia are smaller and less numerous. Furthermore there is a considerable loss of nerve fibers in the mucosal layer. Additionally a loss of microvilli in the transplanted ileum mucosa was found. Whether these findings represent a state of remodeling or a slow atrophy of the enteric nervous system in the transplanted areas is currently under investigation.

The enteric nervous system (ENS) derives from neural crest cells, which migrate from the neural tube into the developing gut. The neuronal and glial precursor cells migrate mainly from the oral towards the anal end of the gastrointestinal tract. So far, knowledge about the multipotent influences upon the ENS development, especially its neurotrophic support, derives mainly from knock-out models. The in vitro technique of isolating enteric neuronal precursor cells allows to study the effects of various factors upon their appropriate development in more detail. We therefore adapted the method of growing neurospheres, which are agglomerates of neuronal precursor cells and differentiated neurones and glial cells, from the central nervous system (CNS) for the ENS. The gut of NMRI mice at E12 were dissected, mildly dissociated and plated in 25-cm(2) culture flasks. The cultures were maintained in N1 supplemented DMEM/F12 medium with the appropriate neurotrophin cocktails (bFGF, GDNF, Neurturin, CNTF). After several days in culture most of the cells die, while the surviving cells form clusters from which domes, and later spheres arise. The spheres could be harvested and processed for further experiments. First investigations revealed, that the amount of precursor cells was much less in enteric neurospheres as seen in corresponding cultures from the CNS. We found about 43% HNK-1-NCAM+ in enteric and approximately 90% Nestin-+ cells in midbrain neurospheres. Differentiation studies of the enteric neurospheres showed that especially ciliary neurotrophic factor (CNTF) increased the number of enteric neurones (PGP positive), while the amount of HNK-1 precursor cells decreased under the influence of all tested neurotrophins but GDNF. The culture of the freshly dissociated enteric neurospheres in a three-dimensional matrix yielded a secondary network which allows to investigate the pattern formation of the ENS. The generation of enteric neurospheres and the following differentiation and 3D culture in vitro can increase our knowledge of the amount and time point of neurotrophic as well as the ECM-protein influence upon the appropriate development of the ENS.

The cellular processes that lead to changes in shape (morphogenesis) and organ formation (organogenesis) are poorly understood. Local contraction of microfilaments can change cell shape and lead to changes of tissue shape. To clarify the role of contractile microfilaments in the foregut morphogenesis of chick embryos, 2- to 4-day-old embryos were exposed to cytochalasin D (CD), which is known to disrupt microfilaments. Untreated age-matched embryos were used as controls. Sections of treated embryos and controls were stained with phalloidin, which binds to actin, and examined with a fluorescence microscope. Microdissected specimens were examined using a scanning electron microscope (SEM). Immunofluorescent staining showed a bright signal belt toward the apical cell region of the foregut epithelium in controls. This signal was not evident in CD-exposed embryos. SEM micrographs of the controls showed the cranial foregut as a smooth, even, cylindrical structure in all stages studied. The lumen was narrow and perfectly straight, the ventral and dorsal walls were in close apposition. The foregut of CD-exposed specimens, however, showed a wide lumen and the walls were separated from each other. The structure seemed atonic and appeared conical, curved, or tilted. We observed a dense microfilament network toward the apical cell pole of the epithelial foregut cells of controls that was no longer evident after CD exposure. This network seems to play an important role in foregut morphogenesis, since actin-filament disruption by CD causes loss of the normal shape.