Key elements determining the intestinal region-specific environment of enteric neurons in type 1 diabetes.

Diabetes, as a metabolic disorder, is accompanied with several gastrointestinal (GI) symptoms, like abdominal pain, gastroparesis, diarrhoea or constipation. Serious and complex enteric nervous system damage is confirmed in the background of these diabetic motility complaints. The anatomical length of the GI tract, as well as genetic, developmental, structural and functional differences between its segments contribute to the distinct, intestinal region-specific effects of hyperglycemia. These observations support and highlight the importance of a regional approach in diabetes-related enteric neuropathy. Intestinal large and microvessels are essential for the blood supply of enteric ganglia. Bidirectional morpho-functional linkage exists between enteric neurons and enteroglia, however, there is also a reciprocal communication between enteric neurons and immune cells on which intestinal microbial composition has crucial influence. From this point of view, it is more appropriate to say that enteric neurons partake in multidirectional communication and interact with these key players of the intestinal wall. These interplays may differ from segment to segment, thus, the microenvironment of enteric neurons could be considered strictly regional. The goal of this review is to summarize the main tissue components and molecular factors, such as enteric glia cells, interstitial cells of Cajal, gut vasculature, intestinal epithelium, gut microbiota, immune cells, enteroendocrine cells, pro-oxidants, antioxidant molecules and extracellular matrix, which create and determine a gut region-dependent neuronal environment in diabetes.

Orai1 calcium channel inhibition prevents progression of chronic pancreatitis.

Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.

Gut Region-Specific Interleukin 1ß Induction in Different Myenteric Neuronal Subpopulations of Type 1 Diabetic Rats.

Interleukin 1ß (IL1ß) is a pro-inflammatory cytokine that may play a crucial role in enteric neuroinflammation in type 1 diabetes. Therefore, our goal is to evaluate the effects of chronic hyperglycemia and insulin treatment on IL1ß immunoreactivity in myenteric neurons and their different subpopulations along the duodenum-ileum-colon axis. Fluorescent immunohistochemistry was used to count IL1ß expressing neurons as well as the neuronal nitric oxide synthase (nNOS)- and calcitonin gene-related peptide (CGRP)-immunoreactive myenteric neurons within this group. Tissue IL1ß level was measured by ELISA in muscle/myenteric plexus-containing homogenates. IL1ß mRNA was detected by RNAscope in different intestinal layers. The proportion of IL1ß-immunoreactive myenteric neurons was significantly higher in the colon than in the small intestine of controls. In diabetics, this proportion significantly increased in all gut segments, which was prevented by insulin treatment. The proportion of IL1ß-nNOS-immunoreactive neurons only increased in the diabetic colon, while the proportion of IL1ß-CGRP-immunoreactive neurons only increased in the diabetic ileum. Elevated IL1ß levels were also confirmed in tissue homogenates. IL1ß mRNA induction was detected in the myenteric ganglia, smooth muscle and intestinal mucosa of diabetics. These findings support that diabetes-related IL1ß induction is specific for the different myenteric neuronal subpopulations, which may contribute to diabetic motility disturbances.

Gut region-specific TNFR expression: TNFR2 is more affected than TNFR1 in duodenal myenteric ganglia of diabetic rats.

BACKGROUND: Cytokines are essential in autoimmune inflammatory processes that accompany type 1 diabetes. Tumor necrosis factor alpha plays a key role among others in modulating enteric neuroinflammation, however, it has a dual role in cell degeneration or survival depending on different TNFRs. In general, TNFR1 is believed to trigger apoptosis, while TNFR2 promotes cell regeneration. The importance of the neuronal microenvironment has been recently highlighted in gut region-specific diabetic enteric neuropathy, however, the expression and alterations of different TNFRs in the gastrointestinal tract has not been reported. AIM: To investigate the TNFR1 and TNFR2 expression in myenteric ganglia and their environment in different intestinal segments of diabetic rats. METHODS: Ten weeks after the onset of hyperglycemia, gut segments were taken from the duodenum, ileum and colon of streptozotocin-induced (60 mg/body weight kg i.p.) diabetic (n = 17), insulin-treated diabetic (n = 15) and sex- and age-matched control (n = 15) rats. Myenteric plexus whole-mount preparations were prepared from different gut regions for TNFR1/HuCD or TNFR2/HuCD double-labeling fluorescent immunohistochemistry. TNFR1 and TNFR2 expression was evaluated by post-embedding immunogold electron microscopy on ultrathin sections of myenteric ganglia. TNFRs levels were measured by enzyme-linked immun-osorbent assay in muscle/myenteric plexus-containing (MUSCLE-MP) tissue homogenates from different gut segments and experimental conditions. RESULTS: A distinct region-dependent TNFRs expression was detected in controls. The density of TNFR1-labeling gold particles was lowest, while TNFR2 density was highest in duodenal ganglia and a decreased TNFRs expression from proximal to distal segments was observed in MUSCLE-MP homogenates. In diabetics, the TNFR2 density was only significantly altered in the duodenum with decrease in the ganglia (0.32 ± 0.02 vs 0.45 ± 0.04, P < 0.05), while no significant changes in TNFR1 density was observed. In diabetic MUSCLE-MP homogenates, both TNFRs levels significantly decreased in the duodenum (TNFR1: 4.06 ± 0.65 vs 20.32 ± 3.1, P < 0.001; TNFR2: 11.72 ± 0.39 vs 15.91 ± 1.04, P < 0.01), which markedly influenced the TNFR2/TNFR1 proportion in both the ganglia and their muscular environment. Insulin treatment had controversial effects on TNFR expression. CONCLUSION: Our findings show diabetes-related region-dependent changes in TNFR expression and suggest that TNFR2 is more affected than TNFR1 in myenteric ganglia in the duodenum of type 1 diabetic rats.

Thiopurines impair the apical plasma membrane expression of CFTR in pancreatic ductal cells via RAC1 inhibition.

BACKGROUND AND AIMS: Thiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear. METHODS: To model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl- and HCO3- secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization. RESULTS: We demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3- and Cl- secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR. CONCLUSIONS: AZA impaired the ductal HCO3- and Cl- secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future.

Intestinal Region-Dependent Alterations of Toll-Like Receptor 4 Expression in Myenteric Neurons of Type 1 Diabetic Rats.

Toll-like receptor 4 (TLR4) can activate pro-inflammatory cascades in the gastrointestinal tract. Our aim was to determine TLR4 expression in myenteric neurons of different gut regions using a type 1 diabetic model. Ten weeks after the onset of hyperglycemia, myenteric whole-mount preparations from the duodenum, ileum and colon of streptozotocin-induced diabetic, insulin-treated diabetic and control rats were prepared for TLR4/peripherin double-labelling fluorescent immunohistochemistry. Immunogold electron microscopy was applied to evaluate TLR4 expression in the myenteric perikaryon and neuropil. Tissue TLR4 levels were measured by enzyme-linked immunosorbent assay. In controls, the number and proportion of the TLR4-immunoreactive myenteric neurons showed an increasing tendency to aboral direction. These values were significantly higher in diabetics compared to controls in the duodenum and ileum, but were significantly lower in the colon. In diabetics, the distribution of TLR4-labelling gold particles between the perikaryon and neuropil of myenteric neurons varied in a different way by intestinal segment. TLR4 tissue concentration changed only in the diabetic duodenum, and it decreased in muscle/myenteric plexus-containing homogenates, while it increased in mucosa/submucosa/submucous plexus-containing samples relative to controls. Insulin had beneficial effects on TLR4 expression. These findings support that chronic hyperglycemia has segment-specific effects on TLR4 expression, contributing to gastrointestinal disorders in diabetic patients.

The ontogenies of endometrial and myometrial leptin and adiponectin receptors in pregnant rats: Their putative impact on uterine contractility.

AIMS: Limited data are available about the functions and expressions of leptin and adiponectin receptors (LEPR, AdipoRs) in the uterus. Our aim was to investigate the effects of leptin and adiponectin on the contractions of intact and denuded nonpregnant and pregnant uteri, as well as the changes in mRNA and protein expressions of LEPR and AdipoRs during the gestational period. MAIN METHODS: Contractions of nonpregnant and 5-, 15-, 18-, 20- or 22-day pregnant uterine rings were measured in an isolated organ bath system. The tissue contractions were stimulated with KCl and modified by cumulative concentrations of leptin or adiponectin. The mRNAs, protein expressions and localizations of LEPR and AdipoRs were determined by RT-PCR, Western blot and immunohistochemistry, respectively. KEY FINDINGS: Both adipokines relaxed the nonpregnant intact uterus more effectively than the denuded myometrium. Leptin inhibited the contractions of endometrium-denuded uteri throughout pregnancy, while its action was weakened on intact uteri towards term. The changes in LEPR receptor densities were independent of the relaxing effect. Adiponectin inhibited contractions, but this effect ceased on pregnancy day 22, while a gradual decrease was detected towards term on denuded myometria. These modifications were in harmony with changes in the expressions of AdipoRs. SIGNIFICANCE: Both leptin and adiponectin play a role in the relaxation of the pregnant uterus, but their efficacy significantly decreases towards the end of gestation. Their endometrial receptors may have a fine-tuning role in uterine contractions, predicting the importance of these adipokines in uterine contractions under altered adipokine level conditions.

Intestinal Region-Specific and Layer-Dependent Induction of TNFα in Rats with Streptozotocin-Induced Diabetes and after Insulin Replacement.

Tumour necrosis factor alpha (TNFα) is essential in neuroinflammatory modulation. Therefore, the goal of this study is to reveal the effects of chronic hyperglycaemia and insulin treatment on TNFα expression in different gut segments and intestinal wall layers. TNFα expression was mapped by fluorescent immunohistochemistry and quantitative immunogold electron microscopy in myenteric ganglia of duodenum, ileum and colon. Tissue TNFα 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 TNFα-labelling gold particles is observed in myenteric ganglia from proximal to distal segments and TNFα 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 TNFα 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 TNFα changes. TNFα 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 TNFα expression, contributing to the regional damage of myenteric neurons and their intestinal milieu.

Diabetes-related intestinal region-specific thickening of ganglionic basement membrane and regionally decreased matrix metalloproteinase 9 expression in myenteric ganglia.

BACKGROUND: The importance of the neuronal microenvironment has been recently highlighted in gut region-specific diabetic enteric neuropathy. Regionally distinct thickening of endothelial basement membrane (BM) of intestinal capillaries supplying the myenteric ganglia coincide with neuronal damage in different intestinal segments. Accelerated synthesis of matrix molecules and reduced degradation of matrix components may also contribute to the imbalance of extracellular matrix dynamics resulting in BM thickening. Among the matrix degrading proteinases, matrix metalloproteinase 9 (MMP9) and its tissue inhibitor (TIMP1) are essential in regulating extracellular matrix remodelling. AIM: To evaluate the intestinal segment-specific effects of diabetes and insulin replacement on ganglionic BM thickness, MMP9 and TIMP1 expression. METHODS: Ten weeks after the onset of hyperglycaemia gut segments were taken from the duodenum and ileum of streptozotocin-induced diabetic, insulin-treated diabetic and sex- and age-matched control rats. The thickness of BM surrounding myenteric ganglia was measured by electron microscopic morphometry. Whole-mount preparations of myenteric plexus were prepared from the different gut regions for MMP9/TIMP1 double-labelling fluorescent immunohistochemistry. Post-embedding immunogold electron microscopy was applied on ultrathin sections to evaluate the MMP9 and TIMP1 expression in myenteric ganglia and their microenvironment from different gut segments and conditions. The MMP9 and TIMP1 messenger ribonucleic acid (mRNA) level was measured by quantitative polymerase chain reaction. RESULTS: Ten weeks after the onset of hyperglycaemia, the ganglionic BM was significantly thickened in the diabetic ileum, while it remained intact in the duodenum. The immediate insulin treatment prevented the diabetes-related thickening of the BM surrounding the ileal myenteric ganglia. Quantification of particle density showed an increasing tendency for MMP9 and a decreasing tendency for TIMP1 from the proximal to the distal small intestine under control conditions. In the diabetic ileum, the number of MMP9-indicating gold particles decreased in myenteric ganglia, endothelial cells of capillaries and intestinal smooth muscle cells, however, it remained unchanged in all duodenal compartments. The MMP9/TIMP1 ratio was also decreased in ileal ganglia only. However, a marked segment-specific induction was revealed in MMP9 and TIMP1 at the mRNA levels. CONCLUSION: These findings support that the regional decrease in MMP9 expression in myenteric ganglia and their microenvironment may contribute to extracellular matrix accumulation, resulting in a region-specific thickening of ganglionic BM.

Sustained Maternal Smoking Triggers Endothelial-Mediated Oxidative Stress in the Umbilical Cord Vessels, Resulting in Vascular Dysfunction.

Nitric oxide (NO) bioavailability is fundamental in the regulation of redox balance and functionality of the endothelium, especially in the case of the umbilical cord (UC), which has no innervation. The analysis of UC vessel-related complications could serve as a useful tool in the understanding of the pathophysiological mechanisms leading to neonatal cardiovascular disorders. Therefore, the aim of this study was to characterize the mechanisms that rule the severity of prenatal endothelial dysfunction, induced by the long-term effect of maternal smoking. Our analysis describes the initiation and the consequences of endothelial nitric oxide synthase (NOS3) deactivation, along with the up-regulation of possible compensatory pathways, using structural, molecular and biochemical approaches. This study was carried out on both the UC arteries and veins originated from neonates born to non-smoking and heavy-smoking mothers. The alterations stimulated by maternal smoking are vessel-specific and proportional to the level of exposure to harmful materials passed through the placenta. Typically, in the primarily exposed veins, an increased formation of reactive oxygen species and an up-regulation of the highly-efficient NOS2-NO producing pathway were detected. Despite all the extensive structural and functional damages, the ex vivo heat and cadmium ion-treated UC vein pieces still support the potential for stress response.

The ontogeny of kisspeptin receptor in the uterine contractions in rats: Its possible role in the quiescence of non-pregnant and pregnant uteri.

The objectives of this study were to investigate the effects of KISS1 94-121 fragment on the contractility of non-pregnant and pregnant rat uteri, and to determine the uterine and myometrial expressions of Kiss1r. Uterine muscle strips were obtained from non-pregnant Sprague-Dawley rats in oestrous phase and from pregnant rats on gestational days 5, 15, 18, 20 or 22. The in vitro contractility measurements were carried out in an isolated organ bath in the presence of KISS1 94-121. Experiments with 5-day pregnant tissues were also performed in the presence of kisspeptin-234 trifluoroacetate. The mRNA and protein expressions of Kiss1r were measured by RT-PCR and Western blot analysis, while localizations of receptors were defined by fluorescent immunohistochemistry. KISS1 94-121 induced a dose-dependent relaxation both in non-pregnant and pregnant intact and endometrium-denuded uteri. A gradual decrease was found in the uterine expressions of Kiss1r mRNA and protein towards the end of the gestational period, and it was further confirmed by the immunohistochemical results. The significant majority of Kiss1r is found in the myometrium, however the few endometrial Kiss1r also influences the uterine contractions. The relaxing effect of kisspeptin is continuously reduced towards the end of gestational period in parallel with the reduction of Kiss1r expression. Our results suggest a putative role of kisspeptin in the maintenance of uterine quiescence that may have significance in miscarriage or preterm contractions.

Regulation of (Pro)Renin Receptor in Renin-Positive Smooth Muscle Cells of Kidney Arterioles in Rats with STZ-Induced Diabetes.

Objective. The nephron (pro)renin receptor may play a pathophysiological role in renal disorders in hypertension or diabetes. The aim of this study was to determine the relationship of (pro)renin receptors and transdifferentiation between the renin-negative and renin-positive SMCs in the afferent arteriole by estimating the distribution of (pro)renin receptors in renin-positive and renin-negative SMCs of the afferent arteriole of kidneys in normal and streptozotocin- (STZ-) induced diabetic rats. Therefore in diabetes the renin granulation of afferent arterioles is different as in normal, the diabetes model for finding the differences to normal in distribution of (pro)renin receptors of afferent arterioles was used. Method. To estimate the number of (pro)renin receptors in arteriolar SMCs a special protocol of immunohistochemistry to stereology was followed. Results. Our results showed that on the surface of renin-positive SMCs the number of (pro)renin receptors was upregulated, while in the cytoplasm of SMCs there was downregulation in comparison to renin-negative SMCs. There is a significant difference between the number of (pro)renin receptors on the surface and in the cytoplasm of renin-positive SMCs in normal rats. These differences in the number of (pro)renin receptors were not present in rats with STZ-induced diabetes. Any other differences in the number of (pro)renin receptors between the STZ-induced diabetic and normal rats were not detected. The tissue level of angiotensin II did not change in the kidneys of STZ-induced diabetic rats. Conclusion. The distribution of (pro)renin receptors in afferent arteriolar SMCs is related to renin granulation of SMCs, but independent of angiotensin II plasma or tissue levels in the kidney.

The Anti-Inflammatory Role of Mannich Curcuminoids; Special Focus on Colitis.

The incidence of inflammatory bowel disease (IBD) increases gradually in Western countries with high need for novel therapeutic interventions. Mannich curcuminoids, C142 or C150 synthetized in our laboratory, have been tested for anti-inflammatory activity in a rat model of TNBS (2,4,6-trinitrobenzenesulphonic acid) induced colitis. Treatment with C142 or C150 reduced leukocyte infiltration to the submucosa and muscular propria of the inflamed gut. C142 or C150 rescued the loss of body weight and C150 decreased the weight of standard colon preparations proportional with 20% less tissue oedema. Both C142 and C150 curcumin analogues caused 25% decrease in the severity of colonic inflammation and haemorrhagic lesion size. Colonic MPO (myeloperoxidase) enzyme activity as an indicator of intense neutrophil infiltration was 50% decreased either by C142 or C150 Mannich curcuminoids. Lipopolysaccharide (LPS) co-treatment with Mannich curcuminoids inhibited NF-κB (nuclear factor kappa B) activity on a concentration-dependent manner in an NF-κB-driven luciferase expressing reporter cell line. Co-treatment with LPS and curcuminoids, C142 or C150, resulted in NF-κB inhibition with 3.57 µM or 1.6 µM half maximal effective concentration (EC50) values, respectively. C150 exerted a profound inhibition of the expression of inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-4 (IL-4) in human PBMCs (peripheral blood mononuclear cells) upon LPS stimulus. Mannich curcuminoids reported herein possess a powerful anti-inflammatory activity.

Nitrergic Enteric Neurons in Health and Disease-Focus on Animal Models.

Nitrergic enteric neurons are key players of the descending inhibitory reflex of intestinal peristalsis, therefore loss or damage of these neurons can contribute to developing gastrointestinal motility disturbances suffered by patients worldwide. There is accumulating evidence that the vulnerability of nitrergic enteric neurons to neuropathy is strictly region-specific and that the two main enteric plexuses display different nitrergic neuronal damage. Alterations both in the proportion of the nitrergic subpopulation and in the total number of enteric neurons suggest that modification of the neurochemical character or neuronal death occurs in the investigated gut segments. This review aims to summarize the gastrointestinal region and/or plexus-dependent pathological changes in the number of nitric oxide synthase (NOS)-containing neurons, the NO release and the cellular and subcellular expression of different NOS isoforms. Additionally, some of the underlying mechanisms associated with the nitrergic pathway in the background of different diseases, e.g., type 1 diabetes, chronic alcoholism, intestinal inflammation or ischaemia, will be discussed.

Salicylic acid-induced ROS production by mitochondrial electron transport chain depends on the activity of mitochondrial hexokinases in tomato (Solanum lycopersicum L.).

The growth regulator, salicylic acid (SA) plays an important role in the induction of cell death in plants. Production of reactive oxygen species (ROS) by mitochondrial electron transport chain (mtETC), cytochrome c (cyt c) release from mitochondria and loss of mitochondrial integrity can be observed during cell death execution in plant tissues. The aim of this work was to study the putative role of hexokinases (HXKs) in the initiation of cell death using tomato (Solanum lycopersicum L.) leaves and mitochondria isolated from plants exposed to a sublethal, 0.1 mM and a cell death-inducing, 1 mM concentrations of SA. Both treatments enhanced ROS and nitric oxide (NO) production in the leaves, which contributed to a concentration-dependent loss of membrane integrity. Images prepared by transmission electron microscopy showed swelling and disorganisation of mitochondrial cristae and vacuolization of mitochondria after SA exposure. Using post-embedding immunohistochemistry, cyt c release from mitochondria was also detected after 1 mM SA treatment. Both SA treatments decreased the activity and transcript levels of HXKs in the leaves and the total mtHXK activity in the mitochondrial fraction. The role of mitochondrial hexokinases (mtHXKs) in ROS and NO production of isolated mitochondria was investigated by the addition of HXK substrate, glucose (Glc) and a specific HXK inhibitor, N-acetylglucosamine (NAG) to the mitochondrial suspension. Both SA treatments enhanced ROS production by mtETC in the presence of succinate and ADP, which was slightly inhibited by Glc and increased significantly by NAG in control and in 0.1 mM SA-treated mitochondria. These changes were not significant at 1 mM SA, which caused disorganisation of mitochondrial membranes. Thus the inhibition of mtHXK activity can contribute to the mitochondrial ROS production, but it is not involved in NO generation in SA-treated leaf mitochondria suggesting that SA can promote cell death by suppressing mtHXK transcription and activity.

Reduction of nitrosative stress by methane: Neuroprotection through xanthine oxidoreductase inhibition in a rat model of mesenteric ischemia-reperfusion.

Our aim was to characterize the main components of the nitrosative response with quantitative changes of the nitrergic myenteric neurons in adjacent intestinal segments after transient superior mesenteric artery occlusion. We also tested the hypothesis that exogenous methane may modulate the evolution of nitroxidation by influencing xanthine oxidoreductase (XOR) activity. The microcirculatory consequences of a 50 min ischemia or ischemia-reperfusion were investigated in anesthetized rats (n = 124) inhaling normoxic air with or without 2.2% methane. XOR activities, nitrogen monoxide (NO), nitrite/nitrate (NOx), and nitrotyrosine levels were measured, together with relative nitrergic neuron ratios from duodenum, ileum and colon samples. The effects of methane on XOR were also examined in vitro. The intramural flow stopped only in the ileum during ischemia. The highest baseline XOR activity was found in the duodenum, which increased further during ischemia. NO and nitrotyrosine levels rose, and the nNOS-immunopositive neuron ratio and NOx level both dropped. Reperfusion uniformly elevated XOR activity and nitrotyrosine formation, with the highest level attained in the duodenum, where the nitrergic neuron ratio remained depressed. These alterations were eliminated in methane-treated animals, XOR activity and nitrotyrosine formation decreased in all sites, and the duodenal nitrergic neuron ratio was re-established. The inhibitory effect of methane on XOR-linked nitrate reductase activity was also demonstrated in vitro. With segment-specific microcirculatory alterations, the risk for nitrosative stress is highest in transiently hypoxic tissues with high endogenous XOR activities. The XOR-inhibitory effect of methane can reduce nitroxidation and protects the nitrergic neuron population in such conditions.

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

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. CONCLUSION: Diabetes and immediate insulin replacement induce the most pronounced region-specific alterations of nNOS-, HO1- and HO2-IR submucous neuronal density in the distal parts of the gut.

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.

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.

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.