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

Single Haemoglobin Nanocapsules as Test Materials for Artificial Blood

Single protein nanocapsules (SPNs) means that each individual protein molecules are coated with a very thin polymer layer. The polymer chains which are porous enough to allow enzymatic functions are bound covalently to the protein molecule. According to our previous results the polymer layer can essentially stabilize different types of enzymes, e.g. its stability became to 50-70 times longer than that of the native ones. The heat stability (at 80 °C SPNs has activity after 24 hours) and the pH-stability (from pH = 1.5 to pH = 12.0) of the covered enzyme can essentially be improved comparing to the native enzymes. Our results show, that SPNs have a good features as drug carriers: acrylamide-bisacrylamide copolymer layer can carries bovine serum albumin molecules across the blood brain barrier in rat brain. We synthesized single haemoglobin nanocapsules with acrylamide-bisacrylamide copolymer on the surface of the molecules (PAAHgB) and their size, homogeneity, aggregation status, zeta potential were investigated compared with other nanomaterials

Novel features of the rat model of inflammatory bowel disease based on 2,4,6-trinitrobenzenesulfonic acidinduced acute colitis

The 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute inflammatory bowel disease (IBD) model in the rat is discussed, focusing on the details of the TNBS instillation and highlighting the advantages and limitations of this model. For determination of the time-dependent action of 50% ethanol and different doses of TNBS, male Wistar rats were treated with 50% ethanol or 10 mg or 30 mg of TNBS dissolved in 50% ethanol. The TNBS-induced inflammation peaked 48-72 h after installation and the colitis caused by 30 mg of TNBS was more severe than that caused by 10 mg of TNBS. To test the effectiveness of sulfasalazine (SASP), male rats were treated with 10 mg of TNBS or with 10 mg of TNBS and SASP, and 72 h later the extent of mucosal damage was determined. Orally administered 50 mg/kg/day SASP proved to reduce the TNBS-induced colonic inflammation in rats significantly. The TNBS-induced colitis model facilitates a better understanding of the immunopathological mechanisms of IBD. Optimization of the dose of TNBS and oral SASP as positive control in TNBS-induced colitis in rats furnishes an appropriate test system for new anti-IBD drugs

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

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