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Tau protein is of primary importance for neuronal homeostasis and when hyperphosphorylated (PP-Tau), it tends to aggregate in neurofibrillary tangles, as is the case with tauopathies, a class of neurodegenerative disorders. Reversible PP-Tau accumulation occurs in the brain of hibernating rodents and it was recently observed in rats (a non-hibernator) during synthetic torpor (ST), a pharmacological-induced torpor-like condition. To date, the expression of PP-Tau in the rat enteric nervous system (ENS) is still unknown. The present study immunohistochemically investigates the PP-Tau expression in the myenteric plexus of the ileum and colon of normothermic rats (CTRL) and during ST, focusing on the two major subclasses of enteric neurons, i.e., cholinergic and nitrergic. Results showed that both groups of rats expressed PP-Tau, with a significantly increased percentage of PP-Tau immunoreactive (IR) neurons in ST vs. CTRL. In all rats, the majority of PP-Tau-IR neurons were cholinergic. In ST rats, the percentage of PP-Tau-IR neurons expressing a nitrergic phenotype increased, although with no significant differences between groups. In addition, the ileum of ST rats showed a significant decrease in the percentage of nitrergic neurons. In conclusion, our findings suggest an adaptive response of ENS to very low core body temperatures, with changes involving PP-tau expression in enteric neurons, especially the ileal nitrergic subpopulation. In addition, the high presence of PP-Tau in cholinergic neurons, specifically, is very interesting and deserves further investigation. Altogether, these data strengthen the hypothesis of a common cellular mechanism triggered by ST, natural hibernation and tauopathies occurring in ENS neurons. Electronic supplementary material The online version of this article (10.1007/s00441-020-03328-0) contains supplementary material, which is available to authorized users.

Background Prostatic radiation therapy (RT) often causes erectile dysfunction (ED) and the mechanisms governing RT-induced ED are unclear with a lack of therapeutic strategies. Aim To determine the effects of ex vivo RT on major pelvic ganglion (MPG) neuron survival, and neurite growth in whole vs dissociated culture. Methods MPGs were removed and irradiated (0 or 8 Gy) from male Sprague Dawley rats. For dissociated culture, MPG neurons were digested in collagenase/dispase and cultured on coverslips. Immunofluorescent staining for beta-tubulin III (TUBB3; neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick end labeling assessed neurite length, branching, autonomic neuron density, and apoptosis. For whole organ culture, MPGs were grown in Matrigel. Gene expression of apoptotic markers (caspase 1, 3), TUBB3, nNOS, TH, and Schwann cells (Sox10, Krox20, glial fibrillary acid protein) was measured in whole organ cultured MPGs by quantitative polymerase chain reaction. Outcomes After 72 hours, neurite length, branching, autonomic neuron density, and apoptosis were assessed, and gene expression was measured. Results RT increased apoptosis in dissociated neurons measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (P Clinical Translation Prostatic RT leads to increased neuronal cell death and less erectogenic nitrergic neurons contributing to ED. Strengths & Limitations The advantages of dissociated neuron culture include distinct neurites which are easily measured for apoptosis, length/branching, and specific neuron types. In contrast, whole MPG culture is advantageous as it contains all the supporting cells present in vivo. Conclusion The 2 different culture methods demonstrated opposing neurite growth after RT indicating the importance of supporting cell network to promote pelvic neuron neuritogenesis and survival following RT.

Enteric neuronal degeneration, as seen in inflammatory bowel disease, obesity, and diabetes, can lead to gastrointestinal dysmotility. Pyroptosis is a novel form of programmed cell death but little is known about its role in enteric neuronal degeneration. We observed higher levels of cleaved caspase-1, a marker of pyroptosis, in myenteric ganglia of overweight and obese human subjects compared with normal-weight subjects. Western diet–fed (WD-fed) mice exhibited increased myenteric neuronal pyroptosis, delayed colonic transit, and impaired electric field stimulation–induced colonic relaxation responses. WD increased TLR4 expression and cleaved caspase-1 in myenteric nitrergic neurons. Overactivation of nitrergic neuronal NF-κB signaling resulted in increased pyroptosis and delayed colonic motility. In caspase-11–deficient mice, WD did not induce nitrergic myenteric neuronal pyroptosis and colonic dysmotility. To understand the contributions of saturated fatty acids and bacterial products to the steps leading to enteric neurodegeneration, we performed in vitro experiments using mouse enteric neurons. Palmitate and lipopolysaccharide (LPS) increased nitrergic, but not cholinergic, enteric neuronal pyroptosis. LPS gained entry to the cytosol in the presence of palmitate, activating caspase-11 and gasdermin D, leading to pyroptosis. These results support a role of the caspase-11–mediated pyroptotic pathway in WD-induced myenteric nitrergic neuronal degeneration and colonic dysmotility, providing important therapeutic targets for enteric neuropathy.

Given the well-known antioxidant and neuroprotective properties of quercetin, the aim of this work was to evaluate the effects of quercetin stabilized by microencapsulation at two doses (10 mg kg−1 and 100 mg kg−1) on the oxidative/antioxidant status, number and morphological features of ICC, nitrergic neurons and M2-like macrophages in jejunum of diabetic rats. The rats were randomly distributed into six groups: normoglycemic control (N), diabetic control (D) and either normoglycemic or diabetic groups treated with quercetin-loaded microcapsules at a dose of 10 mg kg−1 (NQ10 and DQ10, respectively) or 100 mg kg−1 (NQ100 and DQ100, respectively). After 60 days, the jejunum was collected. Whole mounts were immunostained for Ano1, nNOS and CD206, and oxidative stress levels and total antioxidant capacity of the jejunum were measured. Diabetes led to a loss of ICC and nitrergic neurons, but increased numbers of M2-like macrophages and elevated levels of oxidative stress were seen in diabetic animals. High-dose administration of quercetin (100 mg kg−1) further aggravated the diabetic condition (DQ100) but this treatment resulted in harmful effects on healthy rats (NQ100), pointing to a pro-oxidant activity. However, low-dose administration of quercetin (10 mg kg−1) gave rise to antioxidant and protective effects on ICC, nNOS, macrophages and oxidative/antioxidant status in DQ100, but NQ100 displayed infrequent negative outcomes in normoglycemic animals. Microencapsulation of the quercetin may become promising alternatives to reduce diabetes-induced oxidative stress but antioxidant therapies should be careful used under healthy status to avoid toxic effects.

Background: Paralytic ileus is common in patients with septic shock, which may cause high morbidity and mortality. Enteric neurons and enteric glial cells (EGCs) participate in the regulation of intestinal motility, but little is known about their interaction in endotoxemia. We aim to investigate whether reactive EGCs have harmful effects on enteric neurons and participate in intestinal motility disorder in mice during endotoxemia. Methods: In this study, lipopolysaccharide (LPS) was used to induce endotoxemia in mice, and intraperitoneal injections of fluorocitrate (FC) before LPS injections to inhibit the reactive EGCs. The effects of reactive EGCs on intestinal motility were analyzed by motility assays in vivo and colonic migrating motor complexes (CMMCs) ex-vivo. The number of enteric neurons was evaluated by immunofluorescent staining HuCD, nNOS and CHAT in vivo. In addition, we stimulated EGCs with IL-1β and TNF-α in vitro and cultured the primary enteric neurons in the conditioned medium, detecting the apoptosis and morphology of neurons through staining TUNEL, cleaved Caspase-3 protein and Anti-β-III tubulin.Results: The expression of glial fibrillary acidic protein (GFAP) was significantly upregulated in LPS-injected animals, indicating EGCs were transformed into a reactive state. Inhibition of reactive EGCs improved intestinal motility and peristaltic reflex, and increased the density of the neuronal population in the colonic myenteric plexus significantly, especially the nitrergic neurons. In vitro, the apoptotic rate of enteric neurons significantly increased, and the dendritic complexity and the number of primary neurites were significantly reduced in the conditioned medium of reactive EGCs.Conclusion: Reactive enteric glial cells participated in paralytic ileus by damaging nitrergic neurons during endotoxemia. It may provide a novel therapeutic strategy for intestinal motility disorders during endotoxemia or sepsis.

Based on the bilateral relationship between the gut microbiota and formation/function of the enteric nervous system (ENS), we sought to determine whether antibiotics-induced dysbiosis might impact the expressivity of genetically-induced ENS abnormalities. To address this, we took advantage of the TashT mouse model of Hirschsprung disease, in which colonic aganglionosis and hypoganglionosis are both much more severe in males. These defects result into two male-biased colon motility phenotypes: either megacolon that is lethal around weaning age or chronic constipation in adults, the latter being also associated with an increased proportion of nitrergic neurons in the distal ENS. Induction of dysbiosis using a cocktail of broad-spectrum antibiotics specifically impacted the colonic ENS of TashTTg/Tg mice in a stage-dependent manner. It further decreased the neuronal density at post-weaning age and differentially modulated the otherwise increased proportion of nitrergic neurons, which appeared normalized around weaning age and further increased at post-weaning age. These changes delayed the development of megacolon around weaning age but led to premature onset of severe constipation later on. Finally, local inhibition of nitric oxide signaling improved motility and prevented death by megacolon. We thus conclude that exposure to antibiotics can negatively influence the expressivity of a genetically-induced enteric neuropathy.

Nitrergic neurons (NNs) are inhibitory neurons capable of releasing nitric oxide (NO) that are labeled with NADPHd histochemistry. The rat primary somatosensory (S1) and motor (M1) cortices are a favorable model to investigate NN populations by comparing their morphology, since these areas share the border of forepaw representation. The distribution of the type I NN of the forepaw representation in the S1 and M1 cortices of the rat in different laminar compartments and the morphological parameters related to the cell body and dendritic arborization were measured and compared. We observed that the neuronal density in the S1 (130 NN/mm3 ) was higher than the neuronal density in the M1 (119 NN/mm3 ). Most NN neurons were multipolar (S1 with 58%; M1 with 69%), and a minority of the NN neurons were horizontal (S1 with 6%; M1 with 12%). NN found in S1 had a higher verticality index than NN found in M1, and no significant differences were observed for the other morphological parameters. We also demonstrated significant differences in most of the morphological parameters of the NN between different cortical compartments of S1 and M1. Our results indicate that the NN of the forepaw in S1 and M1 corresponds to a neuronal population, where the functionality is independent of the different types of sensory and motor processing. However, the morphological differences found between the cortical compartments of S1 and M1, as well as the higher density of NNs found in S1, indicate that the release of NO varies between the areas. This article is protected by copyright. All rights reserved.

Background Constipation is commonly seen in patients with Parkinson's disease associated with a loss of dopaminergic neurons in both central and enteric nervous systems. However, the roles of enteric dopaminergic neurons in developing constipation remain to be elucidated. Here, we investigated the roles of enteric dopaminergic neurons in the generation of colonic peristalsis. Methods Cannulated segments of rat proximal colon were situated in the organ bath, abluminally perfused with physiological salt solution and luminally perfused with 0.9% saline. Drugs were applied in the abluminal solution. Changes in diameter along the length of the colonic segment were captured by a video camera and transformed into spatio-temporal maps. Fluorescence immunohistochemistry was also carried out. Key results Blockade of nitrergic neurotransmission prevented oro-aboral propagation of peristaltic waves and caused a colonic constriction without affecting ripples, non-propagating myogenic contractions. Blockade of cholinergic neurotransmission also prevented peristaltic waves but suppressed ripples with a colonic dilatation. Tetrodotoxin (0.6 μM) abolished peristaltic waves and increased ripples with a constriction. SCH 23390 (20 μM), a D1 -like dopamine receptor antagonist, slowed the peristaltic waves and caused a constriction, while GBR 12909 (1 μM), a dopamine reuptake inhibitor, diminished the peristaltic waves with a dilatation. Bath-applied dopamine (3 μM) abolished the peristaltic waves associated with a colonic dilation in an SCH 23390 (5 μM)-sensitive manner. D1 receptor immunoreactivity was co-localized to nitrergic and cholinergic neurons. Conclusions and inferences Dopaminergic neurons appear to facilitate nitrergic neurons via D1 -like receptors to stabilize asynchronous contractile activity resulting in the generation of colonic peristalsis.

Hirschsprung disease (HSCR) is characterized by congenital absence of enteric neurons in distal portions of the gut. Although recent studies identified Schwann cell precursors (SCPs) as a novel cellular source of enteric neurons, it is unknown how SCPs contribute to the disease phenotype of HSCR. Using Schwann cell-specific genetic labeling, we investigated SCP-derived neurogenesis in two mouse models of HSCR; Sox10 haploinsufficient mice exhibiting distal colonic aganglionosis and Ednrb knockout mice showing small intestinal aganglionosis. We also examined Ret dependency in SCP-derived neurogenesis using mice displaying intestinal aganglionosis in which Ret expression was conditionally removed in the Schwann cell lineage. SCP-derived neurons were abundant in the transition zone lying between the ganglionated and aganglionic segments, although SCP-derived neurogenesis was scarce in the aganglionic region. In the transition zone, SCPs mainly gave rise to nitrergic neurons that are rarely observed in the SCP-derived neurons under the normal condition. Enhanced SCP-derived neurogenesis was also detected in the transition zone of mice lacking RET expression in the Schwann cell lineage. Increased SCP-derived neurogenesis in the transition zone suggests that reduction in the vagal neural crest-derived enteric neurons promotes SCP-derived neurogenesis. SCPs may adopt a neuronal subtype by responding to changes in the gut environment. Robust SCP-derived neurogenesis can occur in a Ret-independent manner, which suggests that SCPs are a cellular source to compensate for missing enteric neurons in HSCR.

Background We previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus. Methods We developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5. Key results In the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer. Conclusions and inferences The intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.

Simple Summary The gastrointestinal tract of the bottlenose dolphin (Tursiops truncatus) differs structurally and functionally from that of terrestrial mammals. In particular, the intestine does not show any macroscopic subdivision and lacks a caecum. In addition, the histological aspect of the intestine is relatively constant, without marked differences between the anterior and posterior parts. Although the intestine of these cetaceans presents differences in comparison with terrestrial mammals, little information is currently available on their enteric nervous system. The aim of the present study was to investigate the morphological and quantitative aspects of neurons immunoreactive (IR) for the neuronal nitric oxide synthase (nNOS) and Substance P (SP) in the intestine of bottlenose dolphins (Tursiops truncatus). In these dolphin specimens, a smaller number of nNOS-IR neurons in the submucosal plexus and a larger number of SP-IR neurons in the myenteric plexus were observed compared to other mammals. Interestingly, no co-localization between nNOS- and SP-IR neurons was detected in either of the plexuses, suggesting the existence of two completely distinct functional classes of neurons in the intestine of the bottlenose dolphin. Abstract Compared with other mammals, the digestive system of cetaceans presents some remarkable anatomical and physiological differences. However, the neurochemical features of the enteric nervous system (ENS) in these animals have only been described in part. The present study gives a description of the nitrergic and selected peptidergic systems in the myenteric plexus (MP) and submucosal plexus (SMP) of the intestine of the bottlenose dolphin (Tursiops truncatus). The distribution and morphology of neurons immunoreactive (IR) for the neuronal nitric oxide synthase (nNOS) and Substance P (SP) were immunohistochemically studied in formalin-fixed specimens from the healthy intestine of three animals, and the data were compared with those described in the literature on other mammals (human and non-human). In bottlenose dolphins, the percentages of nitrergic neurons (expressed as median and interquartile range—IQR) were 28% (IQR = 19–29) in the MP and 1% (IQR = 0–2) in the SMP, while the percentages of SP-IR neurons were 31% (IQR = 22–37) in the MP and 41% (IQR = 24–63) in the SMP. Although morphological features of nNOS- and SP-IR neurons were similar to those reported in other mammals, we found some noticeable differences in the percentages of enteric neurons. In fact, we detected a lower proportion of nNOS-IR neurons in the SMP and a higher proportion of SP-IR neurons in the MP compared to other mammals. To the best of the authors’ knowledge, this study represents the first description and quantification of nNOS-IR neurons and the first quantification of SP-IR neurons in the intestine of a cetacean species. As nNOS and SP are important mediators of intestinal functions and the nitrergic population is an important target for many neuroenteropathies, data obtained from a healthy intestine provide a necessary basis to further investigate and understand possible functional differences and motor intestinal dysfunctions/alterations in these special mammals.

Nitric oxide (NO) has been implicated as an important neurotransmitter in stress responses and sleep regulatory processes. However, the role of NO in the relationship between stress and sleep remains unclear. The medial septum (MS) and vertical diagonal band (VDB), regions of the basal forebrain involved in sleep regulation, contain nitric oxide synthase (NOS) producing neurons. Additionally, NOS neurons in the dorsal raphe nucleus (DRN) encode information about stress duration. The role of nitrergic neurons in these regions in subserving sex-specific responses to stress and sleep loss has yet to be elucidated. In this study, NADPH-d, an index of NOS activity, was used to examine the effects of acute restraint stress and sleep loss on NOS activity in the MS, VDB, and DRN. We show that NOS activity in response to restraint stress, total sleep deprivation (TSD), and partial sleep restriction (PSR) differs based on sex and region. Initial analysis showed no effect of restraint stress or TSD on NOS activity in the basal forebrain. However, investigation of each sex separately revealed that restraint stress and TSD significantly decrease NOS activity in the MS of females, but not males. Interestingly, the difference in NOS activity between restraint stress and TSD in females was not significant. Furthermore, PSR was not sufficient to affect NOS activity in males or females. These data suggest that restraint stress and sleep loss regulate NOS activation in a sex-dependent manner, and that the NOS stress response in females may be mediated by sleep loss.

The enteric nervous system is responsible for controlling the gastrointestinal tract (GIT) functions. Enteric neuropathies are highly correlated to the development of several intestinal disturbances. Fluoride (F) is extensively applied for dental health improvement and its ingestion can promote systemic toxicity with mild to severe GIT symptomatology and neurotoxicity. Although F harmful effects have been published, there is no information regarding noxiousness of a high acute F exposure (25 mg F/kg) on enteric neurons and levels of expression of intestinal proteins in the duodenum. Quantitative proteomics of the duodenum wall associated to morphometric and quantitative analysis of enteric neurons displayed F effects of a high acute exposure. F-induced myenteric neuroplasticity was characterized by a decrease in the density of nitrergic neurons and morphometric alterations in the general populations of neurons, nitrergic neurons, and substance P varicosities. Proteomics demonstrated F-induced alterations in levels of expression of 356 proteins correlated to striated muscle cell differentiation; generation of precursor metabolites and energy; NADH and glutathione metabolic process and purine ribonucleoside triphosphate biosynthesis. The neurochemical role of several intestinal proteins was discussed specially related to the modulation of enteric neuroplasticity. The results provide a new perspective on cell signaling pathways of gastrointestinal symptomatology promoted by acute F toxicity.

Quercetin-loaded microcapsules (QLM) promote controlled release and higher bioavailability of quercetin, an antioxidant and neuroprotective agent. We evaluated the antioxidant effect of QLM on enteric innervation and in the oxidative status of the ileum of diabetic rats. Wistar adult rats (Rattus norvegicus) were used in six groups containing normoglycemic (N), diabetic (D) and either normoglycemic or diabetic groups treated with QLM at a dose of 10 mg/kg (NQ10 and DQ10, respectively) or 100 mg/kg (NQ100 and DQ100, respectively). DQ10 e DQ100 did not prevent overall neuronal loss in the total and cholinergic populations. Nitrergic population showed differences regarding the treatments: DQ10 preserved neurons in the nitrergic population whilst DQ100 increased nitrergic loss. Evaluation of the redox status showed pro-oxidant effects in NQ100 by t-butyl-induced chemiluminescence analysis. We observed a reduction in the carbonylic content and an increase of low molecular weight antioxidants for DQ10 e DQ100. Therefore, QLM treatment at a dose of 10 mg/kg acted positively on nitrergic neurons reducing oxidative damage induced by diabetes.

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 RARβ, 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-RARβ and siRARβ 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 RARβ expression, leading to enhanced SOX10 expression. Significance Maternal VAD during pregnancy is an environmental risk factor for GI dysfunction in rat offspring.

T2 toxin synthetized by Fusarium spp. negatively affects various internal organs and systems, including the digestive tract and the immune, endocrine, and nervous systems. However, knowledge about the effects of T2 on the enteric nervous system (ENS) is still incomplete. Therefore, during the present experiment, the influence of T2 toxin with a dose of 12 &micro, g/kg body weight (b.w.)/per day on the number of enteric nervous structures immunoreactive to neuronal isoform nitric oxide synthase (nNOS&mdash, used here as a marker of nitrergic neurons) in the porcine duodenum was studied using the double immunofluorescence method. Under physiological conditions, nNOS-positive neurons amounted to 38.28 &plusmn, 1.147%, 38.39 &plusmn, 1.244%, and 35.34 &plusmn, 1.151 of all enteric neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively. After administration of T2 toxin, an increase in the number of these neurons was observed in all types of the enteric plexuses and nNOS-positive cells reached 46.20 &plusmn, 1.453% in the MP, 45.39 &plusmn, 0.488% in the OSP, and 44.07 &plusmn, 0.308% in the ISP. However, in the present study, the influence of T2 toxin on the intramucosal and intramuscular nNOS-positive nerves was not observed. The results obtained in the present study indicate that even low doses of T2 toxin are not neutral for living organisms because they may change the neurochemical characterization of the enteric neurons.

Nitrergic neurons (NN) are inhibitory neurons capable of releasing nitric oxide (NO) that are labeled with NADPHd histochemistry, allowing the study of their distribution and morphology. The rat primary somatosensory (S1) and motor (M1) cortices are a favorable model to investigate the morphology of the NN population. The distribution of the type I NN of the forepaw representation in the primary somatosensory (S1) and motor (M1) cortices of the rat in different laminar compartments and the morphological parameters related to the cell body and dendritic arborization were measured and compared. We found that the neuronal density in the S1 (130 NN/mm3) was higher than in the M1 (119 NN/mm3). Most NN neurons are multipolar (S1 with 58%; M1 with 69%) and a minority are horizontal (S1 with 6%; M1 with 12%). NN found in the S1 had a higher verticality index than those of the M1, and no statistical differences was found for the others morphological parameters. We also demonstrated statistical differences for most of the morphological parameters of the NN between different cortical compartments of the S1 and M1. Our results indicate that the NN of the forepaw in the S1 and M1 correspond to a single neuronal population whose functionality is independent of the different types of sensory and motor processing. However, the morphological differences found between cortical compartments of S1 and M1, as well as the higher density of NN found in the S1 indicate that the release of NO varies along and between the areas. Keywords: NADPH Diaphorase, Nitric Oxide, Primary Somatosensory Cortex, Primary Motor Cortex, GABAergic Neurons, Interneurons.

There are no studies that characterize the enteric nervous system (ENS) bats. The organization and density of myenteric neurons may vary according to the animal species, as well as the segment of the digestive tube considered. The nitric oxide is one of the key neurotransmitters present in the myenteric neurons, acting as a mediator in the smooth muscle relaxation. These neurons are evidenced by immunohistochemistry of nitric oxide synthase (NOS) or by NADPH-diaphorase histochemistry. In this sense, this study aimed to characterize the total neuronal population and subpopulation NADPH-d+ of the myenteric plexus present in the jejunum of the insectivore species Molossus rufus quantitatively. Five specimens were collected of M. rufus in a buffer area of the “Reserva Biológica das Perobas” in the microregion of Cianorte/PR. After the euthanasia, in a chamber saturated with isoflurane, segments were collected from the small intestine corresponding to the jejunum intended for two techniques for neuronal marking, Giemsa and NADPH-diaphorase, and a fragment to the histological technique of hematoxylin-eosin and Masson’s trichrome. All the procedures were approved by the “Comitê de Ética no Uso de Animais Unipar” (CEUA - protocol No. 34347/2017) and the “Instituto Chico Mendes de Conservação da Biodiversidade” (ICMBio - protocol No. 60061-1) The histological sections allowed to highlight the location of the myenteric plexus between the longitudinal and circular layers of the muscular tunic. The myenteric plexus had an average of total neuronal population (neurons Giemsa+) of 279.23 neurons/mm2, being the nitrergic neurons (neurons NADPH-d+) represented 20.4% of this total population, with an average of 58.14 neuron/mm2. Therefore, the collected data are consistent with previous studies in other mammalian species concerning the location of the myenteric plexus, as well as the neural myenteric proportion NADPH-d+ compared with the population of neurons Giemsa+. The gaps in the knowledge of ENS of bats limits comparative intraspecific and interspecific studies.

INTRODUCTION AND OBJECTIVE:Erectile dysfunction is induced after cavernous nerve (CN) injury by inflammation in pelvic ganglia. We examined levels of tumor necrosis factor alpha (TNFA), after bilat...

BACKGROUND AND PURPOSE: The enteric neurotransmitter nitric oxide (NO) regulates gastrointestinal motility by relaxing smooth muscle. Pharmacological cAMP induction also relaxes gastrointestinal smooth muscle, but it is uncertain whether cAMP augments or suppresses enteric NO signalling. In other organ systems, cAMP can increase neuronal NO production by stimulating protein kinase A (PKA) to phosphorylate neuronal NOS (nNOS) Serine‐1412 (S1412). We hypothesized that cAMP also increases nNOS S1412 phosphorylation by PKA in enteric neurons to augment nitrergic relaxation of mouse ileum. EXPERIMENTAL APPROACH: We measured contractile force and nNOS S1412 phosphorylation in ileal rings suspended in an organ bath. We used forskolin to induce cAMP‐dependent relaxation of wild type, nNOS(S1412A) knock‐in and nNOSα‐null ileal rings in the presence or absence of PKA, protein kinase B (Akt) and NOS inhibitors. KEY RESULTS: Forskolin stimulated phosphorylation of nNOS S1412 in mouse ileum. Forskolin relaxed nNOSα‐null and nNOS(S1412A) ileal rings less than wild‐type ileal rings. PKA inhibition blocked forskolin‐induced nNOS phosphorylation and attenuated relaxation of wild type but not nNOS(S1412A) ileum. Akt inhibition did not alter nNOS phosphorylation with forskolin but did attenuate relaxation of wild type and nNOS(S1412A). NOS inhibition with l‐NAME eliminated the effects of PKA and Akt inhibitors on relaxation. CONCLUSION AND IMPLICATIONS: PKA phosphorylation of nNOS S1412 augments forskolin‐induced nitrergic ileal relaxation. The relationship between cAMP/PKA and NO is therefore synergistic in enteric nitrergic neurons. Because NO regulates gut motility, selective modulation of enteric neuronal cAMP synthesis may be useful for the treatment of gastrointestinal motility disorders.

Nitric oxide (NO) plays an important role in regulating gut motility, mucosal barrier function and secretions in the enteric nervous system. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining has been used to identify nitrergic neurons of the enteric nervous system in different species. However, NADPH-d staining lacks specificity because it also reflects the presence of enzymes other than nitric oxide synthase (NOS). Therefore, NOS immunohistochemistry techniques are needed to test for nitrergic neurons in the avian gut. In the present work, the morphology, density and size of NOS-positive neurons in the duodenum, jejunum, ileum, caecum and rectum myenteric plexus of adult pigeons were investigated using NOS immunohistochemistry and whole-mount preparations techniques. The density of NOS-positive ganglion was highest in the ileum, similar to the caecum and rectum, and the lowest staining levels were observed in the duodenum. The staining intensity of NOS-positive neurons in the duodenum, jejunum and ileum was dark, followed by the rectal regions, with weak staining in the caecum. These results suggested that NOS immunohistochemistry and whole-mount preparation techniques provide an effective assessment method of the ganglia in the pigeon intestinal myenteric nerve plexus and are more accurate for cell counting compared with conventional sections.

Background Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is commonly found in cereals ingested by humans and animals. Its ingestion is correlated with hepatic, hematologic, renal, splenic, cardiac, gastrointestinal, and neural damages, according to dose, duration of exposure and species. In this work, the effects of the ingestion of DON-contaminated diet at concentrations considered tolerable for human and animal intake were assessed. Methods Male Wistar rats aging 21 days were allotted to five groups that were given, for 42 days, diets contaminated with different concentrations of DON (0, 0.2, 0.75, 1.75, and 2 mg kg-1 of chow). Food ingestion, bodyweight, oxidative status and morphometric analyses of gliocytes, and neurons of jejunal myenteric ganglia were recorded. Key results At these concentrations, there was no food rejection, decrease in bodyweight gain, changes in oxidative status, or loss of either neurons or gliocytes. However, DON decreased gliocyte area, general neuronal population, nitrergic, cholinergic and NADH-diaphorase positive subpopulations and, as a result, ganglion area. Conclusions & inferences It was concluded that, even in the absence of visible effect, DON exposure reduces cell body area of gliocytes and neurons of the myenteric plexus of the rat jejunum.

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.

KIBRA has been recognized as a memory-related gene, which is abundant in the brain and kidney of mammals. However, the expression pattern of KIBRA in the “second brain”-enteric nervous system (ENS) is still unknown, especially in neurodegenerative disorders. In this study, we aimed to investigate the detailed expression pattern of KIBRA in the intestinal myenteric nerve plexus of APP/PS1 and wild type mice by whole mount staining technology. The deposition of Aβ and increased levels of phosphorylated Tau (p-Tau) and total Tau (T-Tau) protein were observed in the intestinal myenteric nerve plexus of APP/PS1 mice. Interestingly, the amount of Tuj+ cells remained unchanged between these two groups. Compared to the control mice, the protein levels of KIBRA significantly increased in the jejunal myenteric plexus of APP/PS1 mice, and the proportion of KIBRA+ GABAergic neurons in both the jejunal myenteric nerve plexus and the cortex was much higher in the APP/PS1 mice. But there was no significant difference in the number of KIBRA+ cholinergic neurons and KIBRA+ nitrergic neurons between APP/PS1 and wild type mice. In summary, our study further confirmed that typical pathology features of Alzheimer’s disease (AD) not only existed in the central nervous system but also in the ENS.

Introduction The present study evaluated the effects of different inocula of Toxoplasma gondii oocysts on the ileal myenteric plexus in rats. Materials and Methods Male Wistar rats, 60 days old, were distributed into four groups: control group (CG; which received saline solution) and groups that were infected with 100 (TG100), 1000 (TG1000), and 5000 (TG5000) T. gondii oocysts. Thirty days after infection, the rats were sacrificed, and the ileum was collected to make whole-mount preparations that were subjected to immunofluorescence staining to observe the general neuronal population (HuC/D), nitrergic neurons (nNOS), and enteric glial cells (S100). Morphometric and quantitative analyses of myenteric neurons were performed. Results The infections with different T. gondii inocula did not cause neuronal or glial loss, but cause neuronal hypertrophy in general population and nitrergic subpopulation in infected groups. Conclusion: Changes in neuronal morphology were observed in the TG5000 group, including the presence of vacuoles, translocation of Hu protein to the nucleus, and dendritic distortions, suggesting functional alterations in these cells.

Duchenne-like muscular dystrophy (canine dystrophinopathy) is a hereditary degenerative disease characterized by muscle changes similar to those described for Duchenne muscular dystrophy (DMD) and by alterations in the smooth muscles of the gastrointestinal tract. Some authors have suggested that these abnormalities may be associated with intestinal motility. This study analyzed the nitrergic and cholinergic neurons and P2X7 receptor expression in the myenteric plexus of the ileum and distal colon of dogs with muscular dystrophy. Immunohistochemical techniques were used to detect nitric oxide synthase (NOS) and acetylcholine transferase (ChAT) expression and to label all HuC/D- and P2X7 receptor-immunoreactive (IR) neurons. Transmission electron microscopy and basic histology were performed for further analysis. The results showed that nitrergic neurons exhibited a Dogiel type I morphology in the ileum and distal colon. The neuronal profile results showed that there were fewer NOS-, ChAT-, and HuC/D-IR neurons in the ileum than in the distal colon in the dystrophic (DT) dogs. Additionally, there were more NOS-, ChAT- and HuC/D-IR neurons per ganglion in the distal colon than in the ileum. The P2X7 receptor-expressing neurons colocalized with nitrergic and cholinergic neurons. Transmission and light microscopy revealed collagen between the muscle fibers, between the circular and longitudinal muscle layers and within the myenteric ganglia of dogs with muscular dystrophy. These findings provide a morphological description of the myenteric neurons in the ileum and distal colon of these DT dogs and may contribute to a better understanding of the gastrointestinal disorders found in patients with DMD. Anat Rec, 301:673-685, 2018. © 2017 Wiley Periodicals, Inc.

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.

Background/Aims Neuronal degeneration and changes in interstitial cells of Cajal (ICCs) are important mechanisms of age-related constipation. This study aims to compare the distribution of ICCs and neuronal nitric oxide synthase (nNOS) with regard to age-related changes between the ascending colon (AC) and descending colon (DC) in 6-, 31-, and 74-week old and 2-year old male Fischer-344 rats. Methods The amount of fecal pellet and the bead expulsion times were measured. Fat proportion in the muscle layer of the colon was analyzed by hematoxylin and eosin staining. Proto-oncogene receptor tyrosine kinase (KIT) and neuronal nitric oxide synthase (nNOS) expression were analyzed with Western blotting and immunohistochemistry. Isovolumetric contractile measurements and electrical field stimulation were used to assess smooth muscle contractility. Results Colon transit and bead expulsion slowed with senescence. Fat in the muscle layer accumulated with age in the AC, but not in the DC. The proportion of KIT-immunoreactive ICCs in the submucosal and myenteric plexus was higher in the DC than in the AC, and it declined with age, especially in the AC. In contrast, the proportion of NOS-immunoreactive neurons in the myenteric plexus was higher in the AC than in the DC, and both decreased in older rats. Nitric oxide levels declined with age in the DC. Muscle strip experiments showed that the inhibitory response mediated by nitric oxide in the circular direction of the DC was reduced in 2-year old rats. Conclusion The AC and DC differ in their distribution of ICCs and nNOS, and age-related loss of nitrergic neurons more severely affects the DC than the AC.

The present study aimed to characterize the effects of Salmonella Heidelberg challenge, as well as its changes in the inhibitory neuron subpopulation in the myenteric plexus of broilers. At the age of 21 days, after euthanasia of birds, duodenum fragments were collected from 5 male chicken broilers Cobb 500 linage from the challenged group and the control group for the quantification from the subpopulation of myenteric plexus inhibitory neurons by the histochemical method of NADPH-diaphorase. The membrane preparations obtained after neuronal marking were used for quantifying the neurons from micrographs obtained under light microscope, corresponding to 60 microscope fields per animal. Regarding the location of the neurons, they were grouped into those forming ganglia and those isolated between the fibers connecting the neuron groupings. The average number of neurons obtained was 24.38 neurons per mm2 in the control group, and 9.57 neurons per mm2 in the challenged group, with such values presenting statistical variation. This reduction in the number of neurons is perhaps a sign of the deleterious effects of Salmonella Heidelberg on positive NADPH-diaphorase neurons. Such reduction may promote changes in the functional characteristics of the digestive tube, reducing or slowing down the processes of digestion and absorption of nutrients, thereby limiting the animals’ productive performance. Key words: Gallus gallus, NADPH-diaphorase, inhibitory neurons, enteric nervous system, Salmonella sp.

Alzheimer's disease (AD) is an age-related neurodegenerative brain disorder characterized by aggregation of amyloid-β (Aβ) 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 Aβ 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 (p0.05). Moreover, whole mount preparations and paraffin sections both demonstrated that the number of CD68+ macrophages in the APP/PS1 mice ileum significantly increased (p0.05). But, there was no significant difference (p0.05) 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 Aβ 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.

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.

Introduction: Tulp’s valve is also known as ileocaecal valve which is situated at ileocaecal junction. Tulp’s valve is a diverse anatomical valve where the ileum makes an access into the caecum. Earlier researchers have observed that there is occurrence of high densities of neurons in the Tulp’s valve, though meticulous anatomical depiction about constitution of the myenteric plexus in and around the Tulp’s valve has thus far to be obtained in humans. In array to add contribution to this vicinity of explore, the present work was performed to appreciate the anatomical composition of myenteric plexus in and around the Tulp’s valve in human foetuses. Materials and Methods: The research was performed on ileocaecal sections obtained from human foetuses (n=17) of various gestational ages after obtaining ethical authorization from the ethical clearance committee of Hind Institute of Medical Sciences, Sitapur. Cresyl Violet staining and NADPH- diaphorase enzyme histochemistry was performed over the obtained ileocaecal sections. Study Group-1(G-1) included foetuses of 9 to 14 week of gestational age group while Group-2(G-2) comprised of foetuses of 15 to 33 week of gestational age group. The Neuronal cell profiles (area, perimeter, ferret diameter) and Neuronal cell density were assessed in and around the Tulp’s valve in human foetuses. Results: In Cresyl violet stained sections, the difference in the neuronal cell profile area between G-1 (50.10±2.26 µm2 )and G-2 (61.25±4.07 µm2 ) was found to be statistically significant (p=0.0028).The perimeter of myenteric neuronal cells in G-1 (26.32±0.51 µm ) was significantly (p=0.0028) lower than G-2 (29.17±0.85 µm). The differences in Feret diameter of neuronal cells between G-1 (9.49±0.27 µm) and G-2 (10.53±0.21 µm) was statistically significant (p=0.0028). The neuronal cell density in Myenteric Ganglia (MG) in G-1 and G-2 was 60.01±9.77 and 52.27±6.54 respectively, which was statistically insignificant (p=0.1367) difference. The difference in the nitrergic neuronal cell area between G-1 (66.35±8.43 µm2) and G-2 (74.93±0.53 µm2) was statistically significant (p=0.0038). The mean perimeter of nitrergic neuronal cells in G-1 (32.01±2.57µm) was insignificantly (p=0.2001) lower than G-2 (33.83±0.73 µm). The mean Feret diameter of G-1 (11.97±1.26) was also insignificantly (p=0.2624) lower than G-2 (12.91±0.75). The nitrergic neuronal cell density in Myenteric Ganglia in G-1 was 22.41±4.13 and in G-2 it was 18.61±2.09, which was statistically insignificant (p=0.0983). acquaintance of the innervations of the Tulp’s valve in human foetuses may help in restoration of it for improved movement of ileum inside into the caecum, as well as to check retrospective run of caecal inside into the ileum. Keywords: Tulp’s Valve, Cresyl Violet, NADPH- diaphorase, Nitrergic Neurons

Gonadotrophin-releasing hormone (GnRH) is the main controller of the reproductive axis and stimulates the synthesis and secretion of gonadotrophins. Estrogen is the main peripheral factor controlling GnRH secretion, and this action is mainly mediated by the transsynaptic pathway through nitric oxide, kisspeptin, leptin, among other factors. Kisspeptin is the most potent factor known to induce GnRH release. Nitric oxide and leptin also promote GnRH release; however, neurons expressing GnRH do not express the leptin receptor (OB-R). Leptin seems to modulate the expression of genes and proteins involved in the kisspeptin system. However, few kisspeptin-synthesizing cells in the arcuate nucleus (ARC) and few cells, if any, in the preoptic area (POA) express OB-R; this indicates an indirect mechanism of leptin action on kisspeptin. Nitric oxide is an important intermediate in the actions of leptin in the central nervous system. Thus, this work aimed to verify the numbers of nNOS cells were activated by leptin in different hypothalamic areas; the modulatory effects of the nitrergic system on the kisspeptin system; and the indirect regulatory effect of leptin on the kisspeptin system via nitric oxide. Ovariectomized rats were treated with estrogen or a vehicle and received an intracerebroventricular (i.c.v.) injection of a nitric oxide donor, leptin or neuronal nitric oxide synthase (nNOS) enzyme inhibitor. Thirty minutes after the injection, the animals were decapitated. Leptin acts directly on nitrergic neurons in different hypothalamic regions, and the effects on the ventral premammillary nucleus (PMV) and ventral dorsomedial hypothalamus (vDMH) are enhanced. The use of a nitric oxide donor or the administration of leptin stimulates the expression of the kisspeptin mRNA in the ARC of animals with or without estrogenic action; however, these changes are not observed in the POA. In addition, the action of leptin on the expression of the kisspeptin mRNA in the ARC is blocked by a nitric oxide synthesis inhibitor. We concluded that the effects of leptin on the central nervous system are at least partially mediated by the nitrergic system. Also, nitric oxide acts on the kisspeptin system by modulating the expression of the kisspeptin mRNA, and leptin at least partially modulates the kisspeptin system through the nitrergic system, particularly in the ARC.

BACKGROUND: Few studies regarding arthritic diseases have been performed to verify the presence of the neurodegeneration. Given the increased oxidative stress and extra-articular effects of the rheumatoid arthritis, the gastrointestinal studies should be further investigated aiming a better understanding of the systemic effects the disease on enteric nervous system. OBJECTIVE: To determine whether the rheumatoid arthritis affects the nitrergic density and somatic area of the nNOS- immunoreactive (IR) myenteric neurons, as well as the morphometric areas of CGRP and VIP-IR varicosities of the ileum of arthritic rats. METHODS: Twenty 58-day-old male Holtzmann rats were distributed in two groups: control and arthritic. The arthritic group received a single injection of the Freund’s Complete Adjuvant in order to induce arthritis model. The whole-mount preparations of ileum were processed for immunohistochemistry to VIP, CGRP and nNOS. Quantification was used for the nitrergic neurons and morphometric analyses were performed for the three markers. RESULTS: The arthritic disease induced a reduction 6% in ileal area compared to control group. No significant differences were observed in nitrergic density comparing both groups. However, arthritic group yielded a reduction of the nitrergic neuronal somatic area and VIP-IR varicosity areas. However, an increase of varicosity CGRP-IR areas was also observed. CONCLUSION: Despite arthritis resulted in no alterations in the number of nitrergic neurons, the retraction of ileal area and reduction of nitrergic somatic and VIP-IR varicosity areas may suggest a negative impact the disease on the ENS. RESUMO CONTEXTO: Poucos estudos sobre doenças artríticas têm sido realizados para verificar a presença de neurodegeneração. Diante do aumento do estresse oxidativo e dos efeitos extra-articulares da artrite reumatoide, estudos gastrointestinais devem ser investigados visando uma melhor compreensão dos efeitos sistêmicos da doença no sistema nervoso entérico. OBJETIVO: Determinar se a artrite reumatoide afeta a densidade nitrérgica e a área somática dos neurônios mioentéricos imunorreativos ao nNOS (nNOS-IR), bem como para as áreas morfométricas das varicosidades CGRP-IR e VIP-IR do íleo de ratos artríticos. MÉTODOS: Vinte ratos Holtzmann, com 58 dias de idade, foram distribuídos em dois grupos: controle e artrítico. O grupo artrítico recebeu uma única injeção do adjuvante completo de Freund para induzir o modelo de artrite. Os preparados totais de íleo foram processados para imuno-histoquímica ao VIP, CGRP e nNOS. A quantificação foi utilizada para os neurônios nitrérgicos e as análises morfométricas foram realizadas para os três marcadores. RESULTADOS: A doença artrítica induziu uma redução de 6% na área ileal em relação ao grupo controle. Não foram observadas diferenças significativas na densidade nitrérgica comparando os dois grupos. No entanto, o grupo artrítico produziu uma redução da área somática neuronal nitrérgica e da área das varicosidades do VIP-IR. Entretanto, foi observado um aumento das áreas das viricosidades CGRP-IR. CONCLUSÃO: Apesar da artrite não resultar em alterações no número de neurônios nitrérgicos, a retração da área ileal e a redução das áreas somática nitrérgica e das varicosidades do VIP-IR podem sugerir um impacto negativo da doença no sistema nervoso entérico.

High fat diet can result in the loss of nitrergic neurons in the myenteric plexus. The study aimed at finding out the effect of high fat diet on the adaptation of nitrergic nerve of rat intestine. It used 15 male rats (Rattus norvegicus) of a month of age with mean body weight of 53.73 gr. The rats were adapted for 7 days to individual cages with ad libitum feeding. After random adaptation, all of the rats were assigned to 3 groups of five rats, namely K-7, K-10, and K-13 groups. Feed and drinking water were given ad libitum. The treatment of the high fat diet lasted for 7 weeks. After the treatment, all of the rats were fasted for 12 hours and then killed. Subsequently, small intestine segments (duodenum, jejunum, and ileum) were taken. The intestinal segments were prepared by using NADPH-d histopathological technique to determine the morphometric changes of nitrergic myenteric neurons. During the treatment the rats were weighed every week and at the end of the study orbitalis vein blood measurement was carried out to see its glucose, cholesterol and cholecystokinin (CCK) plasma levels. The data of body weight, glucose, cholesterol, CCK levels, the total number of the nippergenic myenteric segments of the small intestine were statistically analyzed using Anova. The results of the study showed that the treatment of 7% to 13% fat diets for 7 weeks did not indicate any weight gain and any increase in cholescystokinin level, and any decrease in glucose level. However, it indicated significant increase in cholesterol level. The treatment of 10% and13% fat diets increased the total number of neurons in the jejunum and the ileum. Thus, it was concluded that the treatments of the high-fat diet of the rats (K-7, K-10, and K-13) for 7 weeks had significant effect on the adaptation of the neurons of the jejunum and the ileum.

Diabetic autonomic peripheral neuropathy (PN) involves a broad spectrum of organs. One of them is the gastrointestinal (GI) tract. The molecular mechanisms underlying the pathogenesis of digestive complications are not yet fully understood. Digestion is controlled by the central nervous system (CNS) and the enteric nervous system (ENS) within the wall of the GI tract. Enteric neurons exert regulatory effects due to the many biologically active substances secreted and released by enteric nervous system (ENS) structures. These include nitric oxide (NO), produced by the neural nitric oxide synthase enzyme (nNOS). It is a very important inhibitory factor, necessary for smooth muscle relaxation. Moreover, it was noted that nitrergic innervation can undergo adaptive changes during pathological processes. Additionally, nitrergic neurons function may be regulated through the synthesis of other active neuropeptides. Therefore, in the present study, using the immunofluorescence technique, we first examined the influence of hyperglycemia on the NOS- containing neurons in the porcine small intestine and secondly the co-localization of nNOS with vasoactive intestinal polypeptide (VIP), galanin (GAL) and substance P (SP) in all plexuses studied. Following chronic hyperglycaemia, we observed a reduction in the number of the NOS-positive neurons in all intestinal segments studied, as well as an increased in investigated substances in nNOS positive neurons. This observation confirmed that diabetic hyperglycaemia can cause changes in the neurochemical characteristics of enteric neurons, which can lead to numerous disturbances in gastrointestinal tract functions. Moreover, can be the basis of an elaboration of these peptides analogues utilized as therapeutic agents in the treatment of GI complications.