9 research outputs found
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
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
Intestinal Region-Specific and Layer-Dependent Induction of TNF alpha in Rats with Streptozotocin-Induced Diabetes and after Insulin Replacement
Tumour necrosis factor alpha (TNF alpha) is essential in neuroinflammatory modulation. Therefore, the goal of this study is to reveal the effects of chronic hyperglycaemia and insulin treatment on TNF alpha expression in different gut segments and intestinal wall layers. TNF alpha expression was mapped by fluorescent immunohistochemistry and quantitative immunogold electron microscopy in myenteric ganglia of duodenum, ileum and colon. Tissue TNF alpha levels were measured by enzyme-linked immunosorbent assays in muscle/myenteric plexus-containing (MUSCLE-MP) and mucosa/submucosa/submucous plexus-containing (MUC-SUBMUC-SP) homogenates. Increasing density of TNF alpha-labelling gold particles is observed in myenteric ganglia from proximal to distal segments and TNF alpha tissue levels are much more elevated in MUSCLE-MP homogenates than in MUC-SUBMUC-SP samples in healthy controls. In the diabetics, the number of TNF alpha gold labels is significantly increased in the duodenum, decreased in the colon and remained unchanged in the ileal ganglia, while insulin does not prevent these diabetes-related TNF alpha changes. TNF alpha tissue concentration is also increased in MUSCLE-MP homogenates of diabetic duodenum, while decreased in MUC-SUBMUC-SP samples of diabetic ileum and colon. These findings support that type 1 diabetes has region-specific and intestinal layer-dependent effects on TNF alpha expression, contributing to the regional damage of myenteric neurons and their intestinal milieu