GDNF induces synaptic vesicle markers in enteric neurons.

Regulation of intestinal motility depends on an intact synaptic vesicle apparatus. Thus, we investigated the expression of the synaptic vesicle markers synaptophysin and synaptobrevin in the human enteric nervous system (ENS) and their regulation by glial cell line-derived neurotrophic factor (GDNF) in cultured enteric neurons. Full-thickness specimens of the human colon were assessed for expression of synaptophysin and synaptobrevin and neuronal localization was assessed by dual-label immunocytochemistry with PGP 9.5. Effects of GDNF on both synaptic markers were monitored in enteric nerve cell cultures and the presence of varicosities was determined by applying electron microscopy to the cultures. Human colonic specimens showed immunoreactivity for synaptophysin and synaptobrevin in both myenteric and submucosal ganglia as well as in nerve fibers. Both synaptic vesicle markers co-localized with the neuronal marker PGP 9.5 and exhibited granular accumulation patterns in the human and rat ENS. In cultured rat myenteric neurons GDNF treatment promoted expression of both synaptic vesicle markers and the formation of neuronal varicosities. The regulation of synaptophysin and synaptobrevin in enteric neurons by GDNF argues for the induction of functional neuronal networks in culture characterized by an increase of synaptogenesis.

Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon.

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.

Expression and function of the Transforming Growth Factor-b system in the human and rat enteric nervous system.

BACKGROUND: Transforming growth factor-betas (TGF-bs) are pleiotropic growth factors exerting neurotrophic functions upon various neuronal populations of the central nervous system. In contrast, the role of TGF-b isoforms in the enteric nervous system (ENS) is largely unknown. We therefore analyzed the gene expression pattern of the TGF-b system in the human colon and in rat myenteric plexus, and smooth muscle cell cultures and determined the effect of TGF-b isoforms on neuronal differentiation. METHODS: Human colonic samples as well as cultured rat myenteric plexus, and smooth muscle cells were assessed for mRNA expression levels of the TGF-b system (TGF-b1-3, TbR-1-3) by qPCR. The colonic wall was separated into mucosa and tunica muscularis and enteric ganglia were isolated by laser microdissection (LMD) to allow site-specific gene expression analysis. Effects of TGF-b isoforms on neurite outgrowth and branching pattern of cultured myenteric neurons were monitored. KEY RESULTS: mRNA expression of the TGF-b system was detected in all compartments of the human colonic wall as well as in LMD-isolated myenteric ganglia. Cultured myenteric neurons and smooth muscle cells of rat intestine also showed mRNA expression of all ligands and receptors. Transforming growth factor-b2 treatment increased neurite length and branching pattern in cultured myenteric neurons. CONCLUSIONS & INFERENCES: The TGF-b system is abundantly expressed in the human and rat ENS arguing for an auto-/paracrine function of this system on enteric neurons. Transforming growth factor-b2 promotes neuronal differentiation and plasticity characterizing this molecule as a relevant neurotrophic factor for the ENS.

Scanning electron microscopy analysis of two methods of root-end preparation.

AIM: This study evaluated changes in dental tissues of the apical third after root-end preparation. METHODS: Sixty permanent single-rooted human teeth were used after apicectomy at 90 degrees to the long axis of the tooth. Crown removal was performed with a double-faced diamond disk, on a straight handpiece, and specimen standard length was set at 8 mm. Root-end cavities were prepared with an ultrasound system in 30 teeth (G1); in the other teeth, the cavities were prepared with a bur using a contra-angle and micro-handpiece (G2). The width of the root-end cavity was the diameter of the tip or bur, and its depth was 3 mm. Each group was divided into two subgroups with 15 teeth each; 37% phosphoric acid was applied to specimens in subgroups G1B and G2B. All specimens were photographed under scanning electron microscopy. Images were evaluated descriptively and data were compared for fractures, smear layer, uniform inner surface, regular edges, and whether root-end preparation including the whole foramen. A chi-square test and the kappa index were used to analyze results statistically. RESULTS: Only two variables, uniform inner surface and regular edge, varied according to the method used (bur or ultrasound). The presence of smear layer was associated with the use of phosphoric acid. CONCLUSION: Both methods seemed to be adequate for use in endodontic surgeries.