Maternal vitamin A deficiency impairs cholinergic and nitrergic neurons, leading to gastrointestinal dysfunction in rat offspring via RARß.

AIMS: Many gastrointestinal (GI) disorders are developmental in origin and are caused by abnormal enteric nervous system (ENS) formation. Maternal vitamin A deficiency (VAD) during pregnancy affects multiple central nervous system developmental processes during embryogenesis and fetal life. Here, we evaluated whether maternal diet-induced VAD during pregnancy alone can cause changes in the ENS that lead to GI dysfunction in rat offspring. MAIN METHODS: Rats were selected to construct animal models of normal VA, VA deficiency and VA supplementation. The fecal water content, total gastrointestinal transmission time and colonic motility were measured to evaluate gastrointestinal function of eight-week-old offspring rats. The expression levels of RARß, SOX10, cholinergic (ChAT) and nitrergic (nNOS) enteric neurons in colon tissues were detected through western blot and immunofluorescence. Primary enteric neurospheres were treated with retinoic acid (RA), infection with Ad-RARß and siRARß adenovirus, respectively. KEY FINDINGS: Our data revealed marked reductions in the mean densities of cholinergic and nitrergic enteric neurons in the colon and GI dysfunction evidenced by mild intestinal flatulence, increased fecal water content, prolonged total GI transit time and reduced colon motility in adult offspring of the VAD group. Interestingly, maternal VA supplementation (VAS) during pregnancy rescued these changes. In addition, in vitro experiments demonstrated that exposure to appropriate doses of RA promoted enteric neurosphere differentiation into cholinergic and nitrergic neurons, possibly by upregulating RARß expression, leading to enhanced SOX10 expression. SIGNIFICANCE: Maternal VAD during pregnancy is an environmental risk factor for GI dysfunction in rat offspring.

Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning.

BACKGROUND & AIMS: Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions. METHODS: Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions. RESULTS: We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4 × 5 mm2. Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm3 with 38,706 ± 5763 neuron/mm3 and 129,321 ± 25,356 glia/mm3. Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions. CONCLUSIONS: We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.

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.

High-Fat Diet During the Perinatal Period Induces Loss of Myenteric Nitrergic Neurons and Increases Enteric Glial Density, Prior to the Development of Obesity.

Diet-induced obesity induces peripheral inflammation accompanied by a loss of myenteric neurons. Few studies, however, have investigated the effects of a high-fat diet (HFD) on either the development of myenteric neurons or prior to the occurrence of obesity. The present study assessed the effects of maternal HFD on the density and neurochemical phenotype of myenteric ganglia in the upper gastrointestinal tract. Sprague-Dawley rats were fed either a control or HFD (14% or 60% kcal from fat, respectively) from embryonic day 13; the fundus, corpus and duodenum were fixed thereafter at postnatal 2, 4, 6 and 12 weeks of age for subsequent immunohistochemical studies. While myenteric ganglion size did not differ throughout the study, HFD exposure decreased the number of nitrergic neurons by 6 weeks of age in all regions. This decrease was accompanied by a loss of PGP-immunoreactive neurons, suggesting a decline in myenteric neuronal number. HFD also increased myenteric plexus glial cell density in all regions by 4 weeks of age. These changes occurred in the absence of an increase in serum or gastric inflammatory markers. The present study suggests that exposure to a HFD during the perinatal time period results in glial proliferation and loss of inhibitory nitrergic neurons prior to the onset of obesity, suggesting that dietary alterations may affect gastrointestinal functions independently of increased adiposity or glycemic dysregulation.

Differential effect of morphine on gastrointestinal transit, colonic contractions and nerve-evoked relaxations in Toll-Like Receptor deficient mice.

Toll-like receptors (TLRs) are expressed in enteric neurons, glia, gastrointestinal (GI) smooth muscle and mucosa, yet their functional roles in the GI tract are not fully understood. TLRs have been linked to many of the undesirable central effects of chronic opioid administration including hyperalgesia and dependence via activation of central microglia. Opioid-induced bowel dysfunction (OIBD) remains a primary reason for the reduction or withdrawal of opioid analgesics. Morphine-induced inhibition of colonic motility was assessed in vivo by GI transit studies and in vitro using isolated colons from wildtype (WT) and TLR deficient mice. Morphine slowed movement of ingested content in WT but this retardation effect was attenuated in TLR4 -/- and TLR2/4 -/- . In isolated colons, morphine reduced amplitude and frequency colonic migrating motor contractions in both WT and TLR2/4 -/- . Electrical field stimulation elicited distal colon relaxation that was potentiated by morphine in WT but not in TLR2/4 -/- . Inhibitory junction potentials were of similar amplitude and kinetics in WT and TLR2/4 -/- distal colon and not altered by morphine. Enteric nerve density and proportion of nitrergic nerves were similar in WT and TLR2/4 -/- distal colon. These data suggest an involvement of TLRs in opioid pharmacodynamics and thus a potential interventional target for OIBD.

[Changes in spatial relationships between catecholamine- and nitroxidergic neurons in the nuclei of the caudal brain stem in the development of hypertension]. / Izmenenie prostranstvennykh vzaimootnoshenii katekholamin- i nitroksidergicheskikh neironov v iadrakh kaudal'noi chasti stvola mozga pri razvitii arterial'noi gipertenzii.

AIM: To obtain the data on the spatial relationships between catecholamine (TH-positive) and nitroxidergic (nNOS-positive) neurons in vasomotor nuclei of the medulla in different periods of hypertension development. MATERIAL AND METHODS: The experiment was performed on male Wistar rats (n=45) with induced renovascular hypertension (RVH). TH and nNOS in neurons of solitary tract nuclei, reticular small-and giant cell nuclei were detected using immunohistochemical methods. RESULTS AND CONCLUSION: The most early and severe changes in the intensity of reaction and amount of nNOS-positive neurons were noted in the solitary tract nucleus. Significant changes in the quantitative parameters of TH-positive neurons in RVH were identified only in the reticular giant cell nucleus but they appeared later and were less expressed compared to nNOS-positive cells. This resulted in the changes of spatial relationships between two types of neurons and remodeling of the bulbar region of the cardiovascular center.

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.

Acute infection with an avirulent strain of Toxoplasma gondii causes decreasing and atrophy of nitrergic myenteric neurons of rats.

In the enteric nervous system (ENS), nitrergic neurons produce and use nitric oxide (NO) as an inhibitory motor neurotransmitter in response to parasitic infections, including those caused by Toxoplasma gondii. However, damage to the host caused by NO has been reported by various authors, and the role of NO in protection or cytotoxicity continues to be extensively studied. In this study, nitrergic neurons were investigated in the myenteric plexus of the jejunum and the distal colon of rats infected with 500 oocysts of the M7741 strain of T. gondii. Ten rats were randomly assigned into a control group (CG) and infected group (IG; received 500 sporulated oocysts of T. gondii orally). After 24h, the rats were euthanized, and samples of the jejunum and distal colon were obtained and processed for NADPH-diaphorase histochemical analysis. Quantitative and morphometric analysis of the nitrergic neurons in whole mounts containing the myenteric plexus was performed. There was a numeric reduction of nitrergic neurons per mm2 in both jejunum and distal colon. The remaining nitrergic neurons suffered atrophy in the areas of the cell body and nucleus, which resulted in a decrease in cytoplasm. Thus, we conclude that an avirulent strain of T. gondii in a short time causes neuroplastic changes in the small and large intestine of rats.

Inflammatory bowel disease affects density of nitrergic nerve fibers in the mucosal layer of the canine gastrointestinal tract.

The objective of this study was to determine the effect of inflammatory bowel disease (IBD) on the density of nitrergic nerve fibers in the mucosal layer of different sections of the gastrointestinal tract of dogs. Twenty-eight German shepherd hybrid dogs of both sexes, weighing from 15 to 25 kg and aged 6 to 10 y, were studied. The dogs were divided into 4 groups with 7 animals in each group: healthy animals, as well as dogs suffering from mild, moderate, and severe IBD. Immunoreactivity to neuronal isoform of nitric oxide synthase, which is a marker of nitrergic neurons, in samples of the mucosal layer in the duodenum, jejunum, and descending colon was studied using the single immunofluorescence method and the number of nerve fibers was evaluated in each observation field. The obtained results showed that IBD causes an increase in the number of nitrergic nerve fibers in all intestinal segments studied and these changes are directly proportional to the intensity of the disease process. These observations may be useful in diagnostic evaluation of the stage of canine inflammatory bowel disease in veterinary practice. The pathological mechanisms of these observed changes and the specific reasons for them are still not completely explained, however, and further study is required.

L'objectif de la présente étude était de déterminer l'effet de la maladie inflammatoire de l'intestin (MII) sur la densité des fibres nerveuses nitrergiques dans la couche muqueuse de différentes sections du tractus gastro-intestinal de chiens. Vingt-huit chiens de race Berger Allemand croisée des deux sexes, pesant entre 15 et 25 kg et âgés de 6 à 10 ans, ont fait partie de l'étude. Les chiens ont été répartis en quatre groupes de 7 chiens dans chaque groupe : des animaux en santé, aussi bien que des chiens souffrant de MII légère, modérée et sévère. L'immunoréactivité à un isoforme neuronal de l'oxyde nitrique synthase, qui est un marqueur des neurones nitrergiques, dans des échantillons de la couche muqueuse du duodénum, jéjunum, et côlon descendant a été étudiée en utilisant la méthode d'immunofluorescence unique et le nombre de fibres nerveuses a été évalué dans chaque champs observé. Les résultats obtenus montrent que la MII cause une augmentation des fibres nerveuses nitrergiques dans tous les segments intestinaux étudiés et que ces changements sont directement proportionnels à l'intensité du processus pathologique. Ces observations pourraient être utiles pour l'évaluation diagnostique des stades de la MII en pratique vétérinaire. Par contre, les mécanismes pathologiques des changements observés et leurs raisons spécifiques ne sont pas encore complètement élucidés et des études supplémentaires sont requises.(Traduit par Docteur Serge Messier).

Loss of nitrergic and cholinergic neurons in the enteric nervous system of APP/PS1 transgenic mouse model.

Alzheimer's disease (AD) is an age-related neurodegenerative brain disorder characterized by aggregation of amyloid-ß (Aß) peptide, formation of neurofibrillary tangles, synaptic loss, and neuronal cell death; however, the characteristic pathological alterations taking place in the "second brain"-enteric nervous system is still largely undefined. In this study, we aimed to detect the pathological changes in the APP/PS1 mice ileum by a novel whole mount technique. The deposition of Aß plaque and the overexpression of phosphorylated Tau (pTau) protein were observed in the myenteric neurons of APP/PS1 mice. Compared to the control mice, the proportions of neuronal nitric oxide synthase (nNOS)+and choline acetyltransferase (ChAT)+neurons in the myenteric plexus of APP/PS1 mice significantly decreased (p<0.05). Moreover, whole mount preparations and paraffin sections both demonstrated that the number of CD68+ macrophages in the APP/PS1 mice ileum significantly increased (p<0.05). But, there was no significant difference (p>0.05) in the number of enteric HuC/D+ neurons and the density of Tuj1 between the APP/PS1 and wild type mice, which may be due to the compensatory function of enteric nervous system. These results suggest that the deposition of Aß plaque and pTau might activate the enteric resident macrophages, further leading to the loss of myenteric nitrergic and cholinergic neurons in the enteric nervous system.

Aspirin prevents atrophy of esophageal nitrergic myenteric neurons in a mouse model of chronic Chagas disease.

The consequences of using aspirin (ASA) for the pathogenesis of Chagas disease are unclear. This study evaluated the effects of treatment of Chagas disease with ASA on the esophageal nitrergic myenteric neuron population and esophageal wall in mice. We observed that treatment of chagasic infection with ASA protects the esophageal myenteric neurons from the atrophy caused by the Trypanosoma cruzi infection. The mice were infected with 1300 trypomastigotes of Y strain T. cruzi intraperitoneally. Part of infected mice was treated with ASA from fifth to twelfth day after inoculation. Our data support the hypothesis that eicosanoids given during the acute phase of the chagasic infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. Besides, ASA treatment did not provoke alterations in the esophageal wall and the myenteric neurons in infected mice.

Bowel dysfunction following pullthrough surgery is associated with an overabundance of nitrergic neurons in Hirschsprung disease.

PURPOSE: Recent evidence suggests that patients with Hirschsprung disease (HD) have abnormal neurotransmitter expression in the ganglionated proximal colon. These alterations may cause persistent bowel dysfunction even after pullthrough surgery. We sought to quantify the proportion of nitrergic neurons in the ganglionic colon of HD patients and relate these findings to functional outcome. METHODS: The proximal resection margin from 17 patients with colonic HD who underwent a pullthrough procedure and colorectal tissue from 4 age-matched controls were immunohistochemically examined to quantify the proportion of nitrergic neurons. The incidence of constipation, incontinence, and enterocolitis in HD patients was assessed retrospectively and correlated with the proportion of nitric oxide synthase (NOS) expressing neurons. Neuronal subtypes in the ganglionic colon of the Edrnb-/- mouse model of HD were also studied. RESULTS: Mice with HD had a significantly higher proportion of NOS+ neurons in ganglionic colon than normal littermates (32.0±5.6% vs. 19.8±1.2%, p<0.01). Patients with HD also had significantly more NOS+ neurons than controls (18.4±4.6% vs. 13.1±1.9%, p<0.01). Patients who experienced constipation or enterocolitis postoperatively tended toward a higher proportion of NOS+ neurons (21.4±3.9% vs. 17.1±4.1%, p=0.06). Furthermore, patients with a proportion of NOS+ neurons above the median of all HD patients (18.3%) were significantly more likely to have constipation than those below the median (75% vs. 14%, p<0.05). CONCLUSION: An overabundance of nitrergic neurons in the proximal resection margin is associated with HD and may predict bowel dysfunction following pullthrough surgery.

Resveratrol Reduces Morphologic Changes in the Myenteric Plexus and Oxidative Stress in the Ileum in Rats with Ischemia/Reperfusion Injury.

BACKGROUND: Intestinal ischemia/reperfusion injury can be caused by surgical procedures and inflammatory bowel disease. It is normally associated with the increased production of free radicals and changes in the enteric nervous system. AIMS: Given the antioxidant and neuroprotective properties of resveratrol, the present study assessed its influence on oxidative stress in the intestinal wall and the morphology of myenteric neurons in the ileum of rats subjected to ischemia/reperfusion. METHODS: Resveratrol was orally administered daily at a dose of 10 mg/kg for 5 days. Changes in the ileum response to ischemia after 45 min were investigated followed by 3 h reperfusion. Lipoperoxide and carbonylated protein levels, and the activity of the antioxidant enzymes glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured following ischemia/reperfusion injury. RESULTS: The density and morphometry of the general neuronal population, nitrergic neurons and glial cells, and morphometry of VIP varicosities in the ileum were also studied. Lipoperoxide and carbonylated protein levels were 171 and 40% higher during the ischemia/reperfusion, respectively, compared to control cohorts, and resveratrol attenuated these values. The glutathione ratio was 64% lower during ischemia/reperfusion, compared to control cohorts. Resveratrol increased the reduced/oxidized glutathione ratio, attenuated the changes in the activity of the antioxidant enzymes and the detrimental morphologic changes caused by ischemia/reperfusion in the general neuronal population and nitrergic neurons. CONCLUSIONS: Oral treatment with resveratrol reduced the oxidative stress in the ileum and attenuated the morphologic changes that occurred in the myenteric plexus of the ileum in rats subjected to ischemia/reperfusion.

Intestinal and neuronal myenteric adaptations in the small intestine induced by a high-fat diet in mice.

BACKGROUND: The prevalence of obesity has increased at alarming rates, particularly because of the increased consumption of high-fat diets (HFDs). The influence of HFDs on intrinsic innervation and the intestinal wall has not been fully characterized. The aim of this study was to investigate the morpho-quantitative aspects of myenteric neurons and the wall of the small intestine in mice fed a HFD. METHODS: Swiss mice were fed a HFD (59% kcal from fat) or standard chow (9% Kcal from fat) for 8 weeks. Segments of the duodenum, jejunum, and ileum were subjected to histological processing for morpho-quantitative examination of the intestinal wall and mucosal cells, and immunohistochemistry was performed to evaluate myenteric neurons. The data for each segment were compared between the groups using an unpaired Student's t-test or an equivalent nonparametric test. RESULTS: The HFD increased body weight and visceral fat and decreased the length of the small intestine and the circumference of the ileum. In the duodenum, the HFD increased the density of the nitrergic subpopulation and decreased the area of nitrergic neurons and vasoactive intestinal peptide (VIP) varicosities. In the jejunum, the density of the nitrergic subpopulation was increased and the neuronal areas of the general population, nitrergic subpopulation and (VIP) varicosities were reduced. In the ileum, the density of the general population and nitrergic subpopulation were increased and the neuronal areas of the general population, nitrergic subpopulation and (VIP) varicosities were reduced. The morphometric parameters of the villi, crypts, muscular layer and total wall generally increased in the duodenum and jejunum and decreased in the ileum. In the duodenum and jejunum, the HFD promoted a decreased in the proportion of intraepithelial lymphocytes. In the ileum, the proportion of intraepithelial lymphocytes and goblet cells reduced, and the enteroendocrine cells increased. CONCLUSIONS: The high-fat diet induces changes in the myenteric innervation of the small intestine, intestinal wall and mucosal cells responsible for the secretion of hormones and maintenance of the protective intestinal barrier. The morpho-quantitative data provide a basis for further studies to clarify the influence of HFD in the motility, digestive and absorptive capacity, and intestinal barrier.

Regular consumption of a silicic acid-rich water prevents aluminium-induced alterations of nitrergic neurons in mouse brain: histochemical and immunohistochemical studies.

Silicon is not generally considered an essential nutrient for mammals and, to date, whether it has a biological role or beneficial effects in humans is not known. The results of a number of studies suggest that dietary silicon supplementation might have a protective effect both for limiting aluminium absorption across the gut and for the removal of systemic aluminium via the urine, hence, preventing potential accumulation of aluminium in the brain. Since our previous studies demonstrated that aluminium exposure reduces the number of nitrergic neurons, the aim of the present study was to compare the distribution and the morphology of NO-containing neurons in brain cortex of mice exposed to aluminium sulphate dissolved in silicic acid-rich or poor drinking water to assess the potential protective role of silicon against aluminium toxicity in the brain. NADPH-d histochemistry and nNOS immunohistochemistry showed that high concentrations of silicon in drinking water were able to minimize the impairment of the function of nitrergic neurons induced by aluminium administration. We found that silicon protected against aluminium-induced damage to the nitrergic system: in particular, we demonstrated that silicon maintains the number of nitrergic neurons and their expression of nitrergic enzymes at physiological levels, even after a 12 and 15 month exposure to aluminium.

The vulnerability of nitrergic neurons to transient spinal cord ischemia: a quantitative immunohistochemical and histochemical study.

Spinal cord ischemia belongs to serious and relatively frequent diseases of CNS. The aim of the present study was to find out the vulnerability of nitrergic neurons to 15 min transient spinal cord ischemia followed by 1 and 2 weeks of reperfusion. We studied neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in structural elements of lumbosacral spinal cord along its rostrocaudal axis. In addition, a neurological deficit of experimental animals was evaluated. Spinal cord ischemia, performed on the rabbit, was induced by abdominal aorta occlusion using Fogarty catheter introduced into the right femoral artery for a period of 15 min. After surgical intervention the animals survived for 7 and 14 days. nNOS-immunoreactivity (nNOS-IR) was measured by immunohistochemical and NADPHd-positivity by histochemical method, and both immunohistochemical and histochemical stainings were quantified by densitometric analyses. Neurological deficit was evaluated according Zivin's criteria. The number of nNOS-IR and/or NADPH-d positive neurons and the density of neuropil were markedly increased in superficial dorsal horn (laminae I-III) after 15 min ischemia and 7 days of reperfusion. However, ischemia followed by longer time of survival (14 days) returned the number of nNOS-IR and NADPH-d positive neurons to control. In the pericentral region (lamina X) containing interneurons and crossing fibers of spinal tracts, than in lamina VII and in dorsomedial part of the ventral horn (lamina VIII) we recorded a decreased number of nNOS-IR and NADPH-d positive neurons after both ischemia/reperfusion periods. In the medial portion of lamina VII and dorsomedial part of the ventral horn (lamina VIII) we observed many necrotic loci. This area was the most sensitive to ischemia/reperfusion injury. Fifteen minute ischemia caused a marked deterioration of neurological function of hind limbs, often developing into paraplegia. A quantitative immunohistochemical and histochemical study have shown a strong vulnerability of nitrergic neurons in intermediate zone to transient spinal cord ischemia.

Obstructive jaundice activates nitroxidergic neurons of the vago-vagal neural circuit that regulates the hepatobiliary system in rabbits.

In this study, we investigated the expression of neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), two specific enzymes for nitric oxide (NO) synthesis, in the development of liver fibrosis induced by chronic bile duct ligation (BDL) in the rabbit. We specifically studied the liver-innervated nitroxidergic neurons that originate in the nodose ganglion (NG), nucleus of the solitary tract (NTS) and dorsal motor vagal nucleus (DMV). Our data showed that BDL resulted in overexpression of NADPH-d/nNOS in the NG, NTS and DMV neurons. Using densitometric analysis, we found a significant increase in NADPH-d expression as a result of BDL in the NG, NTS and DMV (72.6, 79.4 and 57.4% increase, respectively). These findings were corroborated by serum biochemistry and hepatic histopathological examination, which were influenced by NADPH-d/nNOS-generated NO in the liver following BDL. Upregulation of NADPH-d/nNOS expression may have important implications, including (1) facilitation of extrahepatic biliary parasympathetic tone that promotes gallbladder emptying of excess stagnant bile; (2) relaxation of smooth muscles of bile canaliculi thus participating in the pathogenesis of cholestasis; (3) dilation of hepatic sinusoids to counter BDL-induced intrahepatic portal hypertension in which endothelia may be damaged, and (4) alterations in hepatic metabolism, such as glycogenesis, bile formation and secretion, and bilirubin clearance.

Differential regional and subtype-specific vulnerability of enteric neurons to mitochondrial dysfunction.

Mitochondrial dysfunction is a central mediator of disease progression in diverse neurodegenerative diseases that often present with prominent gastrointestinal abnormalities. Gastrointestinal dysfunction in these disorders is related, at least in part, to defects in the enteric nervous system (ENS). The role of mitochondrial deficits in ENS neurodegeneration and their relative contribution to gastrointestinal dysfunction, however, are unclear. To better understand how mitochondrial abnormalities in the ENS influence enteric neurodegeneration and affect intestinal function, we generated mice (Tfam-ENSKOs) with impaired mitochondrial metabolism in enteric neurons and glia through the targeted deletion of the mitochondrial transcription factor A gene (Tfam). Tfam-ENSKO mice were initially viable but, at an early age, they developed severe gastrointestinal motility problems characterized by intestinal pseudo-obstruction resulting in premature death. This gastrointestinal dysfunction was caused by extensive, progressive neurodegeneration of the ENS involving both neurons and glia. Interestingly, mitochondrial defects differentially affected specific subpopulations of enteric neurons and regions of the gastrointestinal tract. Mitochondrial deficiency-related neuronal and glial loss was most prominent in the proximal small intestine, but the first affected neurons, nitrergic inhibitory neurons, had the greatest losses in the distal small intestine. This regional and subtype-specific variability in susceptibility to mitochondrial defects resulted in an imbalance of inhibitory and excitatory neurons that likely accounts for the observed phenotype in Tfam-ENSKO mice. Mitochondrial dysfunction, therefore, is likely to be an important driving force of neurodegeneration in the ENS and contribute to gastrointestinal symptoms in people with neurodegenerative disorders.

Loss of NRF2 impairs gastric nitrergic stimulation and function.

Emerging research suggests that antioxidant gene expression has the potential to suppress the development of gastroparesis. However, direct genetic evidence that definitively supports this concept is lacking. We used mice carrying a targeted disruption of Nfe2l2, the gene that encodes the transcription factor NRF2 and directs antioxidant Phase II gene expression, as well as mice with a targeted disruption of Gclm, the modifier subunit for glutamate-cysteine ligase, to test the hypothesis that defective antioxidant gene expression contributes to development of gastroparesis. Although expression of heme oxygenase-1 remained unchanged, expression of GCLC, GCLM, SOD1, and CAT was down-regulated in gastric tissue from Nrf2(-/-) mice compared to wild-type animals. Tetrahydrobiopterin oxidation was significantly elevated and nitrergic relaxation was impaired in Nrf2(-/-) mouse gastric tissue. In vitro studies showed a significant decrease in NO release in Nrf2(-/-) mouse gastric tissue. Nrf2(-/-) mice displayed delayed gastric emptying. The use of Gclm(-/-) mice demonstrated that the loss of glutamate-cysteine ligase function enhanced tetrahydrobiopterin oxidation while impairing nitrergic relaxation. These results provide genetic evidence that loss of antioxidant gene expression can contribute to the development of gastroparesis and suggest that NRF2 represents a potential therapeutic target.