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

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

Oxidative-Stress-Related Alterations in Metabolic Panel, Red Blood Cell Indices, and Erythrocyte Morphology in a Type 1 Diabetic Rat Model

Diabetes mellitus is often associated with vascular complications in which hyperglycemia-induced oxidative stress may be the cause of the impaired vessels and circulating blood cells. The aim of this study was to follow the hyperglycemia-related metabolic and morphological changes in blood and urine samples of Wistar rats. Animals were divided into streptozotocin-induced diabetic (acute and chronic), insulin-treated diabetic, reversed diabetic, and control groups. In chronic diabetic rats, decreases in albumin, total protein, and antioxidant glutation concentration were measured, while glutamic-pyruvic transaminase, alkaline phosphatase, red blood cell (RBC) count, hematocrit, and hemoglobin levels were increased. Moreover, an increased level of the phenotypic variants was detected in the RBC population of the diabetic animals. In conclusion, we verified the sensitivity of RBCs to long-lasting hyperglycemia, and to insulin deficiency, which were both accompanied with an increased level of RBC-derived parameters and the presence of eccentrocytes, hemolyzed RBCs, and codocytes. Moreover, our results show that the response of the RBC glutation system to oxidative stress depends on the duration of hyperglycemia, and that the short-term activation of this defense system is exhausted in a long-lasting oxidative environment. Insulin therapy was effective in the case of most parameters, which clearly emphasizes the importance of maintaining blood glucose at physiological level

Perturbation of the mucosa-associated anaerobic gut microbiota in streptozotocin-induced diabetic rats

Our aim was to map the gut region-specific differences of the mucosa-associated microbiome distribution in a streptozotocin-induced diabetic rat model. Tissue samples from the duodenum, ileum and colon were collected 10 weeks after the onset of hyperglycaemia to analyse the mucosa-associated microbiota using next-generation DNA sequencing. Striking differences were observed in the mucosa-associated microbiota of the duodenum between diabetic and control rats. A significant invasion of the aerobic genus Mycoplasma was apparent in diabetes, and the abundance of the anaerobic phylum Firmicutes decreased massively. It is noteworthy that insulin treatment eliminated the Mycoplasma invasion in the duodenum and apparently restored the anaerobic environment in the mucosa. In the ileum the abundance of the phylum Firmicutes increased in the diabetic samples. Although the proportion of the phylum Proteobacteria decreased moderately, its composition changed significantly, and insulin treatment induced only minor alterations. In the diabetic samples of colon, the abundance of the phylum Firmicutes decreased slightly, the relative number of the bacteria in the phylum Bacteroidetes increased strongly as compared to the control values, and after insulin treatment this increase was more significant. Chronic hyperglycaemia has the most prominent effect on the mucosa-associated microbiota in the duodenum

Structural and molecular features of intestinal strictures in rats with Crohn's-like disease

AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn’s disease (CD). METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations. The frequency and macroscopic extent of the strictures were measured on digital photographs. The structural features of strictured gut wall were studied by light- and electron microscopy. Inflammation related alterations in TGF-beta 2 and 3, matrix metalloproteinases 9 (MMP9) and TIMP1 mRNA and protein expression were determined by quantitative real-time PCR and western blot analysis. The quantitative distribution of caspase 9 was determined by post-embedding immunohistochemistry. RESULTS: Intestinal strictures first appeared 60 d after TNBS treatments and the frequency of them increased up to day 120. From day 90 an intact lamina epithelialis, reversible thickening of lamina muscularis mucosae and irreversible thickening of the muscularis externa were demonstrated in the strictured colonic segments. Nevertheless the morphological signs of apoptosis were frequently seen and excess extracellular matrix deposition was recorded between smooth muscle cells (SMCs). Enhanced caspase 9 expression on day 90 in the SMCs and on day 120 also in myenteric neurons indicated the induction of apoptosis. The mRNA expression profile of TGF-betas after repeated TNBS doses was characteristic to CD, TGF-beta 2, but not TGF-beta 3 was up-regulated. Overexpression of MMP9 and down-regulation of TIMP1 were demonstrated. The progressive increase in the amount of MMP9 protein in the strictures was also obvious between days 90 and 120 but TIMP1 protein was practically undetectable at this time. CONCLUSION: These findings indicate that aligned structural and molecular changes in the gut wall rather than neuronal cell death play the primary role in stricture formation