Short communication: Tryptic ß-casein hydrolysate modulates enteric nervous system development in primary culture.

The intestinal tract of the newborn is particularly sensitive to gastrointestinal disorders, such as infantile diarrhea or necrotizing colitis. Perinatal development of the gut also encompasses the maturation of the enteric nervous system (ENS), a main regulator of intestinal motility and barrier functions. It was recently shown that ENS maturation can be enhanced by nutritional factors to improve intestinal maturation. Bioactivity of milk proteins is often latent, requiring the release of bioactive peptides from inactive native proteins. Several casein-derived hydrolysates presenting immunomodulatory properties have been described recently. Furthermore, accumulating data indicate that milk-derived hydrolysate can enhance gut maturation and enrichment of milk formula with such hydrolysates has recently been proposed. However, the capability of milk-derived bioactive hydrolysate to target ENS maturation has not been analyzed so far. We, therefore, investigated the potential of a recently described tryptic ß-casein hydrolysate to modulate ENS growth parameters in an in vitro model of rat primary culture of ENS. Rat primary cultures of ENS were incubated with a bioactive tryptic ß-casein hydrolysate and compared with untreated controls or to cultures treated with native ß-casein or a Prolyve ß-casein hydrolysate (Lyven, Colombelles, France). Differentiation of enteric neurons and enteric glial cells, and establishment of enteric neural network were analyzed using immunohistochemistry and quantitative PCR. Effect of tryptic ß-casein hydrolysate on bone morphogenetic proteins (BMP)/Smad pathway, an essential regulator of ENS development, was further assessed using quantitative PCR and immunochemistry. Tryptic ß-casein hydrolysate stimulated neurite outgrowth and simultaneously modulated the formation of enteric ganglia-like structures, whereas native ß-casein or Prolyve ß-casein hydrolysate did not. Additionally, treatment with tryptic bioactive ß-casein hydrolysate increased the expression of the glial marker glial fibrillary acidic protein and induced profound modifications of enteric glial cells morphology. Finally, expression of BMP2 and BMP4 and activation of Smad1/5 was altered after treatment with tryptic bioactive ß-casein hydrolysate. Our data suggests that this milk-derived bioactive hydrolysate modulates ENS maturation through the regulation of BMP/Smad-signaling pathway. This study supports the need for further investigation on the influence of milk-derived bioactive peptides on ENS and intestinal maturation in vivo.

SNAP-25 is abundantly expressed in enteric neuronal networks and upregulated by the neurotrophic factor GDNF.

Control of intestinal motility requires an intact enteric neurotransmission. Synaptosomal-associated protein 25 (SNAP-25) is an essential component of the synaptic vesicle fusion machinery. The aim of the study was to investigate the localization and expression of SNAP-25 in the human intestine and cultured enteric neurons and to assess its regulation by the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF). SNAP-25 expression and distribution were analyzed in GDNF-stimulated enteric nerve cell cultures, and synaptic vesicles were evaluated by scanning and transmission electron microscopy. Human colonic specimens were processed for site-specific SNAP-25 gene expression analysis and SNAP-25 immunohistochemistry including dual-labeling with the pan-neuronal marker PGP 9.5. Additionally, gene expression levels and distributional patterns of SNAP-25 were analyzed in colonic specimens of patients with diverticular disease (DD). GDNF-treated enteric nerve cell cultures showed abundant expression of SNAP-25 and exhibited granular staining corresponding to synaptic vesicles. SNAP-25 gene expression was detected in all colonic layers and isolated myenteric ganglia. SNAP-25 co-localized with PGP 9.5 in submucosal and myenteric ganglia and intramuscular nerve fibers. In patients with DD, both SNAP-25 mRNA expression and immunoreactive profiles were decreased compared to controls. GDNF-induced growth and differentiation of cultured enteric neurons is paralleled by increased expression of SNAP-25 and formation of synaptic vesicles reflecting enhanced synaptogenesis. The expression of SNAP-25 within the human enteric nervous system and its downregulation in DD suggest an essential role in enteric neurotransmission and render SNAP-25 as a marker for impaired synaptic plasticity in enteric neuropathies underlying intestinal motility disorders.

Characterization of human, mouse, and rat cultures of enteric glial cells and their effect on intestinal epithelial cells.

BACKGROUND: Enteric glial cells (EGC) are major regulators of neuronal and intestinal epithelial cell (IEC) functions. Simple isolation methods of EGC, especially human tissues, remain scarce and limit their study. We present herein a method to isolate EGC and we characterize EGC phenotype and their functional impact on IEC. METHODS: Longitudinal muscle and myenteric plexus preparations of rat, mouse, or human intestine were obtained by microdissection. After mechanical and enzymatic dissociation, individual ganglionic or interganglionic structures were seeded into plates, maintained in culture several weeks and passaged up to 4 times. Purity of cultures was assessed by immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), S100ß and Sox10 or smooth muscle actin. Effects of adenosine triphosphate (ATP) on intracellular Ca²âº signaling in EGC were studied. Co-cultures of EGC with IEC line, Caco-2, were performed for 2-6 days to analyze their impact on monolayer resistance, cell proliferation, and cell spreading. KEY RESULTS: More than 80% of DAPI-positive cells were GFAP, S100ß, and Sox10-immunoreactive. EGC expressed these glial markers over 4 consecutive passages, and the majority of them responded to ATP by an increase in intracellular Ca²âº concentration. In addition, rat, mouse, and human EGC increased intestinal barrier resistance, IEC size, and reduced IEC number. CONCLUSIONS & INFERENCES: We have developed a simple method to isolate and culture human, rat, or mouse EGC. EGC exhibit similar functional properties on the intestinal barrier independently of the species. This study sets the basis for exploring glial biology and functions in human health and diseases.