Individual distribution and colocalization of nitric oxide synthase with vasoactive intestinal polypeptide and neuropeptide Y in the developing human fetal small intestine

The appearance, the individual distribution and the pattern of colocalization of nitric oxide synthase (NOS-immunoreactivity, NOSi), vasoactive intestinal peptide (VIP-immunoreactivity, VIPi) and neuropeptide Y (NPY-immunoreactivity, NPYi) immunoreactivity were examined in the developing human fetal small intestine at weeks 12 and 18 of gestation. Neurons expressing VIPi, NPYi and NOSi were observed in the small intestine of the 12-week-old human fetuses and from this age on a gradual increase in the immunoreactivities appeared until week 18 of gestation when a dense network of immunopositive fibres and cell bodies were observed both in the submucous (SmP) and in the myenteric plexuses (MP). The double-labelling immunocytochemistry showed different pattern of the overlapping immunoreactive structures within the myenteric and submucous plexuses. The cellular colocalization of VIPi and NOSi in submucous ganglia were revealed around week 12 of gestation while in the myenteric ganglia cells with overlapping immunoreactivity appeared around week 18. A limited cellular colocalization of NPYi and NOSi were noticed before week 18, and NOSi neurons in the MP of the 12-week-old fetuses were preferentially innervated by NPYi varicosities. These results suggest that VIP, NPY and NO may exert a cooperative action in human fetal gastrointestinal motility

Differences in distribution of the 210 kDa neurofilament subunit and the S-100 protein in the small intestine of a human fetus with trisomy 21 and in that of a normal fetus of the same age

The enteric nervous system of the small intestine of a 22-week-old male fetus with trisomy 21 was examined and compared with gut specimens from a fetus with normal karyotype at the same developmental stage. After the therapeutic termination of the pregnancies, paraffin sections and whole-mounts were prepared, which were processed for histology and immunohistochemistry, with the use of antibodies against the 210 kDA neurofilament subunit and the glial marker S-100 protein. The reduced length of the small intestine, the histologically observed fewer and shorter villi and the frequently appearing pseudostratified epithelium indicate an overall delay in the intestinal development in the trisomic fetus. Both the S-100 protein-immunopositive glial cells and the neurofilament protein-immunopositive nerve cells were distributed differentially in the gut specimens of the trisomic fetus and in the fetus with normal karyotype. While the immunohistochemical expression of the S-100 protein differed only in the circular axis of the gut wall, the distribution of the neurofilament protein-immunoreactive nerve cells also differed along the longitudinal axis of the gastrointestinal tract. Not only the distribution, but also the morphology of the neurofilament protein-immunoreactive myenteric ganglion cells differed in the trisomic fetus as compared with the normal one. The neurofilament protein-immunopositive ganglion cells of the normal fetus possessed lamellar dendrites and one long axon, while the ganglion cells of the trisomic fetus did not exhibit special morphological characteristics. These observations suggest that the enteric nervous system of the fetus with trisomy 21 is involved in the overall delay of the gut development

Gut region-dependent alterations of nitrergic myenteric neurons after chronic alcohol consumption.

Chronic alcohol abuse damages nearly every organ in the body. The harmful effects of ethanol on the brain, the liver and the pancreas are well documented. Although chronic alcohol consumption causes serious impairments also in the gastrointestinal tract like altered motility, mucosal damage, impaired absorption of nutrients and inflammation, the effects of chronically consumed ethanol on the enteric nervous system are less detailed. While the nitrergic myenteric neurons play an essential role in the regulation of gastrointestinal peristalsis, it was hypothesised, that these neurons are the first targets of consumed ethanol or its metabolites generated in the different gastrointestinal segments. To reinforce this hypothesis the effects of ethanol on the gastrointestinal tract was investigated in different rodent models with quantitative immunohistochemistry, in vivo and in vitro motility measurements, western blot analysis, evaluation of nitric oxide synthase enzyme activity and bio-imaging of nitric oxide synthesis. These results suggest that chronic alcohol consumption did not result significant neural loss, but primarily impaired the nitrergic pathways in gut region-dependent way leading to disturbed gastrointestinal motility. The gut segment-specific differences in the effects of chronic alcohol consumption highlight the significance the ethanol-induced neuronal microenvironment involving oxidative stress and intestinal microbiota

Regionally Distinct Alterations in the Composition of the Gut Microbiota in Rats with Streptozotocin-Induced Diabetes

The aim of this study was to map the microbiota distribution along the gut and establish whether colon/faecal samples from diabetic rats adequately reflect the diabetic alterations in the microbiome. Streptozotocin-treated rats were used to model type 1 diabetes mellitus (T1D). Segments of the duodenum, ileum and colon were dissected, and the microbiome of the lumen material was analysed by using next-generation DNA sequencing, from phylum to genus level. The intestinal luminal contents were compared between diabetic, insulin-treated diabetic and healthy control rats. No significant differences in bacterial composition were found in the luminal contents from the duodenum of the experimental animal groups, whereas distinct patterns were seen in the ileum and colon, depending on the history of the luminal samples. Ileal samples from diabetic rats exhibited particularly striking alterations, while the richness and diversity obscured some of the modifications in the colon. Characteristic rearrangements in microbiome composition and diversity were detected after insulin treatment, though the normal gut flora was not restored. The Proteobacteria displayed more pronounced shifts than those of the predominant phyla (Firmicutes and Bacteroidetes) in the rat model of T1D. Diabetes and insulin replacement affect the composition of the gut microbiota in different, gut region-specific manners. The luminal samples from the ileum appear more suitable for diagnostic purposes than the colon/faeces. The Proteobacteria should be at the focus of diagnosis and potential therapy. Klebsiella are recommended as biomarkers of T1D

Prenatal development of the myenteric plexus in the human fetal small intestine

The enteric nervous system is large, complex and independent of the central nervous system. Its neuralcrest-derived precursors migrate along defined pathways to colonize the bowel. It has been established that signalling molecules produced by the developing neurons and the mesenchyma of the gut wall play a critical role in the development of the mammalian enteric nervous system. Recent studies have further characterized the roles of the different cellular and molecular elements that are critical for enteric ganglia formation. The application of modern neuroanatomical techniques revealed that the enteric nervous system contains a considerable number of neuronal subpopulations. Most of our knowledge concerning the functional features of the enteric neurons, e.g. chemical coding, neuronal connectivity and electrophysiological behaviour, was derived from studies of the guinea-pig small intestine. In light of the interspecies differences, comparison of the findings on different species is mandatory. Consequently, the investigation of human fetal material is necessary in order to estab-lish the basic rules of the development of the human enteric nervous system and to find the time relation between the morphological and functional maturation, thereby permitting an understanding of the causes of congenital malformation leading to misfunction of the gastrointestinal system

Region-dependent effects of diabetes and insulin-replacement on neuronal nitric oxide synthase- and heme oxygenase-immunoreactive submucous neurons

Abstract AIM To investigate the intestinal segment-specific effects of diabetes and insulin replacement on the density of different subpopulations of submucous neurons. METHODS Ten weeks after the onset of type 1 diabetes samples were taken from the duodenum, ileum and colon of streptozotocin-induce diabetic, insulin-treated diabetic and sex- and age-matched control rats. Whole-mount preparations of submucous plexus were prepared from the different gut segments for quantitative fluorescent immunohistochemistry. The following double-immunostainings were performed: neuronal nitric oxide synthase (nNOS) and HuC/D, heme oxygenase (HO) 1 and peripherin, as well as HO2 and peripherin. The density of nNOS-, HO1- and HO2-immunoreactive (IR) neurons was determined as a percentage of the total number of submucous neurons. RESULTS The total number of submucous neurons and the proportion of nNOS-, HO1- and HO2-IR subpopulations were not affected in the duodenal ganglia of control, diabetic and insulin-treated rats. While the total neuronal number did not change in either the ileum or the colon, the density of nitrergic neurons exhibited a 2- and 3-fold increase in the diabetic ileum and colon, respectively, which was further enhanced after insulin replacement. The presence of HO1- and HO2-IR submucous neurons was robust in the colon of controls (38.4%-50.8%), whereas it was significantly lower in the small intestinal segments (0.0%-4.2%, P < 0.0001). Under pathophysiological conditions the only alteration detected was an increase in the ileum and a decrease in the colon of the proportion of HO-IR neurons in insulin-treated diabetic animals

Diabetes-Related Induction of the Heme Oxygenase System and Enhanced Colocalization of Heme Oxygenase 1 and 2 with Neuronal Nitric Oxide Synthase in Myenteric Neurons of Different Intestinal Segments

Increase in hyperglycaemia-induced oxidative stress and decreased effectiveness of endogenous defense mechanisms plays an essential role in the initiation of diabetes-related neuropathy. We demonstrated that nitrergic myenteric neurons display different susceptibilities to diabetic damage in different gut segments. Therefore, we aim to reveal the gut segment-specific differences in the expression of heme oxygenase (HO) isoforms and the colocalization of these antioxidants with neuronal nitric oxide synthase (nNOS) in myenteric neurons. After ten weeks, samples from the duodenum, ileum, and colon of control and streptozotocin-induced diabetic rats were processed for double-labelling fluorescent immunohistochemistry and ELISA. The number of both HO-immunoreactive and nNOS/HO-immunoreactive myenteric neurons was the lowest in the ileal and the highest in the colonic ganglia of controls; it increased the most extensively in the ileum and was also elevated in the colon of diabetics. Although the total number of nitrergic neurons decreased in all segments, the proportion of nNOS-immunoreactive neurons colocalizing with HOs was enhanced robustly in the ileum and colon of diabetics. We presume that those nitrergic neurons which do not colocalize with HOs are the most seriously affected by diabetic damage. Therefore, the regional induction of the HO system is strongly correlated with diabetes-related region-specific nitrergic neuropathy

GABA, glutamát és nitrogén monoxid tartalmú myentericus neuronok térbeli mintázatának ontogenetikus és pathológiás változása = Ontogenetical and pathological changes in the spatial pattern of GABA, glutamate and nitrogen oxide-containing myenteric neurons

Vizsgálataink elsőként bizonyították, hogy a krónikus alkoholkezelés bélszakasz-függő módon befolyásolja a nitrogén monoxid szintáz (NOS) enzim aktivitását, a NOS fehérjetartalmat és a nitrerg myentericus neuronok számát patkány bélidegrendszerében (Alcohol Clin Exp Res. 2006 30(6):967-73). A streptozotocin-indukálta diabeteszes patkányok bélcsatornájának különböző szakaszaiban megvizsgáltuk a diabetes által okozott motilitási problémák és a nitrerg myentericus neuronszám változás összefüggéseit. Bizonyítottuk azt is, hogy az inzulinkezelés megakadályozta a rendellenes bélperisztaltika és a nitrerg neuropathia kialalkulását. Ereményeink mutatták az első bizonyítékát annak, hogy a bélcsatorna különböző szakaszaiban a nitrerg neuronok eltérő mértékben érzékenyek a diabeteses állapotra és az inzulinkezelésre (Diabetes Res Clin Pract. 2008 Jan 31; [Epub ahead of print]). Munkánk során immunhisztokémia módszerekkel vizsgáltuk a VGLUT1-3-immunreaktivitás jelenlétét 14 és 23 hetes humán magzatok myentericus plexusában. Mindhárom vezikuláris glutamát transzporter egyedi térbeli és időbeli megoszlást mutatott. A VGLUT1-immunreaktivitás volt a legkifejezettebb az egész vizsgált periódus alatt (Histol Histopathol 2008 in press). | Our study has demonstrated for the first time that chronic ethanol consumption has a differential effect on nitric oxide synthase (NOS) activity, NOS protein content, and the number of nitrergic neurons in different intestinal segments, suggesting that chronic ethanol administration affects the nitric oxide pathways in the enteric nervous system (Alcohol Clin Exp Res. 2006 30:967-73). The streptozotocin-induced diabetic rat model was used to investigate the relation between the deranged gut motility and the segment-specific quantitative changes in the nitrergic myenteric neurons. Additionally, we studied the effectiveness of early insulin replacement to prevent the diabetes-induced changes. These findings comprise the first evidence that the nitrergic neurons located in different intestinal segments exhibit different susceptibilities to a diabetic state and to insulin treatment (Diabetes Res Clin Pract. 2008 Jan 31; [Epub ahead of print]). Immunocytochemistry was applied to examine the distribution of the immunoreactivity of all three VGLUTs during prenatal development of the myenteric plexus in the human small intestine. We also investigated changes in their localization in the different segments of the small intestine and in the different compartments of the developing myenteric ganglia. Each transporter displayed a characteristic spatiotemporal expression pattern, depending on the fetal age, the gut segment or the ganglionic compartment (Histol Histopathol 2008 in press)