RESUMO
Highly organized circuits of enteric neurons are required for the regulation of gastrointestinal functions, such as peristaltism or migrating motor complex. However, the factors and molecular mechanisms that regulate the connectivity of enteric neurons and their assembly into functional neuronal networks are largely unknown. A better understanding of the mechanisms by which neurotrophic factors regulate this enteric neuron circuitry is paramount to understanding enteric nervous system (ENS) physiology. EphB2, a receptor tyrosine kinase, is essential for neuronal connectivity and plasticity in the brain, but so far its presence and function in the ENS remain largely unexplored. Here we report that EphB2 is expressed preferentially by enteric neurons relative to glial cells throughout the gut in rats. We show that in primary enteric neurons, activation of EphB2 by its natural ligand ephrinB2 engages ERK signaling pathways. Long-term activation with ephrinB2 decreases EphB2 expression and reduces molecular and functional connectivity in enteric neurons without affecting neuronal density, ganglionic fiber bundles, or overall neuronal morphology. This is highlighted by a loss of neuronal plasticity markers such as synapsin I, PSD95, and synaptophysin, and a decrease of spontaneous miniature synaptic currents. Together, these data identify a critical role for EphB2 in the ENS and reveal a unique EphB2-mediated molecular program of synapse regulation in enteric neurons.
Assuntos
Sistema Nervoso Entérico/enzimologia , Sistema de Sinalização das MAP Quinases , Plasticidade Neuronal , Neurônios/enzimologia , Receptor EphB2/metabolismo , Sinapses/metabolismo , Animais , Feminino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The type 3 adenylyl cyclase (AC3) enzyme is involved in the synthesis of cyclic adenosine monophosphate (cAMP). It is primarily expressed in the central nervous system (CNS) and plays a crucial role in neurogenesis and neural dendritic arborization. However, the AC3's functional role in the gastrointestinal tract remains ambiguous. METHODS: AC3 expression in enteric tissue of AC3+/+ mice was investigated using immunohistochemistry and RT-PCR. AC3 knock-out mice (AC3-/- ) were used to examine the effect of AC3 on the enteric nervous system (ENS) function and the number of cilia and apoptotic cells. Additionally, total gastrointestinal transit time and colonic motility were compared between the AC3-/- and AC3+/+ groups of mice. KEY RESULTS: AC3 was predominately expressed in the myenteric plexus of the large intestine. Colonic-bead expulsion analysis showed accelerated propulsion in the large intestine of the AC3-/- mice. The AC3-/- mice demonstrated reduced nerve fibers and enteric glial cells count in colonic mucosa compared to the AC3+/+ mice. Furthermore, AC3-/- mice exhibited increased cellular apoptosis and reduced ARL13B+ cilium cells in the colonic lamina propria compared to the AC3+/+ mice. CONCLUSIONS: In AC3-/- mice, innervation of the lamina propria in the colonic mucosa was reduced and colonic propulsion was accelerated. AC3 is crucial for the development and function of the adult neural network of ENS. AC3 deficiency caused atrophy in the colonic mucosal neural network of mice.
Assuntos
Adenilil Ciclases/metabolismo , Sistema Nervoso Entérico/enzimologia , Mucosa Intestinal/inervação , Animais , Motilidade Gastrointestinal/fisiologia , Camundongos , Camundongos KnockoutRESUMO
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.
Assuntos
Caspases Iniciadoras/metabolismo , Caspases/metabolismo , Colo , Dieta Ocidental/efeitos adversos , Sistema Nervoso Entérico , Motilidade Gastrointestinal , Neurônios , Piroptose , Animais , Caspases/genética , Caspases Iniciadoras/genética , Colo/enzimologia , Colo/inervação , Colo/patologia , Sistema Nervoso Entérico/enzimologia , Sistema Nervoso Entérico/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/enzimologia , Neurônios/patologiaRESUMO
Early-life adversity is a major risk factor for the development of diseases later in life. Maternal protein restriction (MPR) is associated with morbidities in offspring affecting multiple organs, but its impact on the gastrointestinal (GI) tract remains poorly studied. Using a rat model, we examined the consequences of MPR on GI function and on the enteric nervous system (ENS) in the offspring at postnatal d 35 under basal state and following a water avoidance stress (WAS). Compared with control rats, MPR rats exhibited greater colonic motility, permeability, and corticosteronemia. In contrast to controls, MPR rats presented a blunted functional and corticosteronemic response to WAS. Furthermore, MPR rats showed an increased proportion of choline acetyltransferase-immunoreactive (ChAT-IR) neurons and a reduced level of autophagy in colonic myenteric neurons. In ENS cultures, corticosterone treatment increased the proportion of ChAT-IR neurons and reduced autophagy level in enteric neurons. Inhibition of autophagy in ENS cultures resulted in a higher vulnerability of enteric neurons to a cellular stress. Altogether, this study suggests that MPR induced GI dysfunction and ENS alterations in offspring rats and that MPR-induced increased corticosteronemia might be involved in ENS remodeling and altered responsiveness of the gut to stressors later in life.-Aubert, P., Oleynikova, E., Rizvi, H., Ndjim, M., Le Berre-Scoul, C., Grohard, P. A., Chevalier, J., Segain, J.-P., Le Drean, G., Neunlist, M., Boudin, H. Maternal protein restriction induces gastrointestinal dysfunction and enteric nervous system remodeling in rat offspring.
Assuntos
Proteínas Alimentares/administração & dosagem , Sistema Nervoso Entérico/fisiopatologia , Trato Gastrointestinal/fisiopatologia , Exposição Materna , Animais , Autofagia , Tamanho Corporal , Peso Corporal , Colina O-Acetiltransferase/metabolismo , Colo/fisiopatologia , Corticosterona/sangue , Sistema Nervoso Entérico/enzimologia , Feminino , Absorção Intestinal , Modelos Animais , Neurônios/enzimologia , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Ratos Sprague-DawleyRESUMO
Enteric nervous system neuropathy causes a wide range of severe gut motility disorders. Cell replacement of lost neurons using enteric neural stem cells (ENSC) is a possible therapy for these life-limiting disorders. Here we show rescue of gut motility after ENSC transplantation in a mouse model of human enteric neuropathy, the neuronal nitric oxide synthase (nNOS-/-) deficient mouse model, which displays slow transit in the colon. We further show that transplantation of ENSC into the colon rescues impaired colonic motility with formation of extensive networks of transplanted cells, including the development of nNOS+ neurons and subsequent restoration of nitrergic responses. Moreover, post-transplantation non-cell-autonomous mechanisms restore the numbers of interstitial cells of Cajal that are reduced in the nNOS-/- colon. These results provide the first direct evidence that ENSC transplantation can modulate the enteric neuromuscular syncytium to restore function, at the organ level, in a dysmotile gastrointestinal disease model.
Assuntos
Colo/enzimologia , Sistema Nervoso Entérico/citologia , Pseudo-Obstrução Intestinal/cirurgia , Células-Tronco Neurais/transplante , Óxido Nítrico Sintase/deficiência , Animais , Colo/fisiopatologia , Sistema Nervoso Entérico/enzimologia , Feminino , Motilidade Gastrointestinal , Humanos , Pseudo-Obstrução Intestinal/enzimologia , Pseudo-Obstrução Intestinal/genética , Pseudo-Obstrução Intestinal/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/transplante , Óxido Nítrico Sintase/genéticaRESUMO
OBJECTIVE: Recent studies have suggested that the enteric nervous system can modulate gut immunity. Ecto-nucleoside triphosphate diphosphohydrolases [E-NTPDases] regulate purinergic signalling by sequential phosphohydrolysis of pro-inflammatory extracellular adenosine 5'-triphosphate [ATP]. Herein, we test the hypothesis that E-NTPDases modulate gut inflammation via neuro-immune crosstalk. DESIGN: We determined expression patterns of NTPDase2 and NTPDase3 in murine and human colon. Experimental colitis was induced by dextran sodium sulphate [DSS] in genetically engineered mice deficient in NTPDase2 or NTPDase3. We compared plasma adenosine diphosphatase [ADPase] activity from Crohn's patients and healthy controls, and linked the enzyme activity to Crohn's disease activity. RESULTS: NTPDase2 and -3 were chiefly expressed in cells of the enteric nervous system in both murine and human colon. When compared with wild type, DSS-induced colitis was exacerbated in Entpd2, and to a lesser extent, Entpd3 null mice as measured by disease activity score and histology, and marked anaemia was seen in both. Colonic macrophages isolated from Entpd2 null mice displayed a pro-inflammatory phenotype compared with wild type. In human plasma, Crohn's patients had decreases in ADPase activity when compared with healthy controls. The drop in ADPase activity was likely associated with changes in NTPDase2 and -3, as suggested by inhibitor studies, and were correlated with Crohn's disease activity. CONCLUSIONS: NTPDase2 and -3 are ecto-enzymes expressed in the enteric nervous system. Both enzymes confer protection against gut inflammation in experimental colitis and exhibit alterations in Crohn's disease. These observations suggest that purinergic signalling modulated by E-NTPDases governs neuro-immune interactions that are relevant in Crohn's disease.
Assuntos
Adenosina Trifosfatases/metabolismo , Colite/enzimologia , Colo/enzimologia , Doença de Crohn/enzimologia , Sistema Nervoso Entérico/enzimologia , Adolescente , Adulto , Animais , Apirase/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/patologia , Doença de Crohn/imunologia , Doença de Crohn/patologia , Sulfato de Dextrana , Sistema Nervoso Entérico/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Adulto JovemRESUMO
AIM: To determine if expression of colonic tryptophan hydroxylase-2 (TPH2), a surrogate marker of neuronal 5-hydroxytryptamine, is altered in Hirschsprung's-associated enterocolitis. METHODS: Entire resected colonic specimens were collected at the time of pull-through operation in children with Hirschsprung's disease (HSCR, n = 12). Five of these patients had a history of pre-operative Hirschsprung's-associated enterocolitis (HAEC). Controls were collected at colostomy closure in children with anorectal malformation (n = 10). The distribution of expression of TPH2 was evaluated using immunofluorescence and confocal microscopy. Protein expression of TPH2 was quantified using western blot analysis in the deep smooth muscle layers. RESULTS: TPH2 was co-expressed in nitrergic and cholinergic ganglia in the myenteric and submucosal plexuses in ganglionic colon in HSCR and healthy controls. Co-expression was also seen in submucosal interstitial cells of Cajal and PDGFRα(+) cells. The density of TPH2 immuno-positive fibers decreased incrementally from ganglionic bowel to transition zone bowel to aganglionic bowel in the myenteric plexus. Expression of TPH2 was reduced in ganglionic bowel in those affected by pre-operative HAEC compared to those without HAEC and healthy controls. However, expression of TPH2 was similar or high compared to controls in the colons of children who had undergone diverting colostomy for medically refractory HAEC. CONCLUSION: Altered TPH2 expression in colonic serotonergic nerves of patients with HSCR complicated by HAEC may contribute to intestinal secretory and motor disturbances, including recurrent HAEC.
Assuntos
Colo/inervação , Sistema Nervoso Entérico/enzimologia , Enterocolite/enzimologia , Doença de Hirschsprung/enzimologia , Neurônios Serotoninérgicos/enzimologia , Triptofano Hidroxilase/análise , Anoctamina-1 , Biomarcadores/análise , Western Blotting , Estudos de Casos e Controles , Canais de Cloreto/análise , Colo/patologia , Colo/cirurgia , Sistema Nervoso Entérico/patologia , Sistema Nervoso Entérico/fisiopatologia , Enterocolite/patologia , Enterocolite/fisiopatologia , Enterocolite/cirurgia , Feminino , Imunofluorescência , Doença de Hirschsprung/patologia , Doença de Hirschsprung/fisiopatologia , Doença de Hirschsprung/cirurgia , Humanos , Lactente , Masculino , Microscopia Confocal , Proteínas de Neoplasias/análise , Receptores do Fator de Crescimento Derivado de Plaquetas/análiseRESUMO
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines and TH immunoreactivity is indicative of cells synthesising either adrenaline/noradrenaline or dopamine. In this study, the distribution of TH immunoreactivity was examined in two distantly related teleost species, zebrafish (Danio rerio) and shorthorn sculpin (Myoxocephalus scorpius). In both species, TH-immunoreactive nerve cell bodies and varicose nerve fibres were common in the myenteric plexus of the intestine. However, no TH-immunoreactive nerve cell bodies were seen in the sculpin stomach. The TH-immunoreactive nerve cell bodies seemed to constitute a larger proportion of the total enteric population in shorthorn sculpin (50 ± 5 %, n = 3067 cells) compared with zebrafish (14 ± 2 %, n = 10,163 cells). In contrast, in sculpin, the TH-immunoreactive cells were smaller than the average enteric nerve cell bodies, whereas in zebrafish, the relationship was the opposite. In developing zebrafish larvae, TH-immunoreactive nerve cell bodies were common (approx. 75 % of the total population) at 3 days post-fertilization (dpf), but decreased in numbers between 3 and 7 dpf. In conclusion, in contrast to previous studies, TH-immunoreactive intrinsic neurons are common in the fish gut. Their role and function need to be further characterized in order to understand the potential importance of this enteric subpopulation in controlling various gut functions.
Assuntos
Sistema Nervoso Entérico/enzimologia , Trato Gastrointestinal/imunologia , Fibras Nervosas/imunologia , Perciformes/imunologia , Tirosina 3-Mono-Oxigenase/imunologia , Peixe-Zebra/imunologia , Animais , Dopamina/metabolismo , Sistema Nervoso Entérico/imunologia , Epinefrina/metabolismo , Imuno-Histoquímica , Plexo Mientérico/metabolismo , Norepinefrina/metabolismoRESUMO
Mutations that impair the proliferation of enteric neural crest-derived cells (ENCDC) cause Hirschsprung disease, a potentially lethal birth defect where the enteric nervous system (ENS) is absent from distal bowel. Inosine 5' monophosphate dehydrogenase (IMPDH) activity is essential for de novo GMP synthesis, and chemical inhibition of IMPDH induces Hirschsprung disease-like pathology in mouse models by reducing ENCDC proliferation. Two IMPDH isoforms are ubiquitously expressed in the embryo, but only IMPDH2 is required for life. To further understand the role of IMPDH2 in ENS and neural crest development, we characterized a conditional Impdh2 mutant mouse. Deletion of Impdh2 in the early neural crest using the Wnt1-Cre transgene produced defects in multiple neural crest derivatives including highly penetrant intestinal aganglionosis, agenesis of the craniofacial skeleton, and cardiac outflow tract and great vessel malformations. Analysis using a Rosa26 reporter mouse suggested that some or all of the remaining ENS in Impdh2 conditional-knockout animals was derived from cells that escaped Wnt1-Cre mediated DNA recombination. These data suggest that IMPDH2 mediated guanine nucleotide synthesis is essential for normal development of the ENS and other neural crest derivatives.
Assuntos
Sistema Nervoso Entérico/irrigação sanguínea , Sistema Nervoso Entérico/embriologia , Face/embriologia , IMP Desidrogenase/metabolismo , Crista Neural/embriologia , Crista Neural/enzimologia , Crânio/embriologia , Alelos , Animais , Bromodesoxiuridina/metabolismo , Sistema Nervoso Entérico/enzimologia , Sistema Nervoso Entérico/patologia , Feminino , Feto/anormalidades , Feto/embriologia , Deleção de Genes , Genes Reporter , Doença de Hirschsprung/patologia , IMP Desidrogenase/deficiência , Marcação In Situ das Extremidades Cortadas , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Especificidade de Órgãos , RNA não Traduzido/metabolismo , Recombinação Genética/genética , Crânio/metabolismo , Proteína Wnt1/metabolismoRESUMO
Background electroacupuncture (EA) at acupoint ST-36 (Zusanli) has been used to alleviate gastrointestinal symptoms and improve gastrointestinal motility, but the effects and mechanisms of EA on enteric nervous system (ENS) have scarcely been investigated. SD rats were randomly divided into eight groups: normal control group, diabetes mellitus group (DM), chronic high-frequency EA (C-HEA), chronic low-frequency EA (C-LEA), chronic sham stimulation group (C-SEA), acute high-frequency EA group (A-HEA), acute low-frequency EA group (A-LEA), and diabetic with acute sham stimulation group (A-SEA). The parameters of HEA included a frequency of 100 Hz and an amplitude of 1 mA, while the parameters for LEA were 10 Hz and 1 mA. The expressions of PGP9.5, neuronal nitric oxide synthase neurons, CHAT neurons, glia cell line-derived neurotrophic factor (GDNF) and p-Akt were measured by immunofluorescence or immunohistochemistry, real-time PCR, and Western blotting methods in colon tissues of each rat. The total neurons and the two types of enteric neurons (neuronal nitric oxide synthase and choline acetyl transferase neurons), together with GDNF and p-Akt in the mRNA and protein level were significantly decreased in DM group compared with the normal control group in colon (P < 0.01). Compared with DM or all other DM with EA groups, the chronic HEA could induce a more significant quantitative increase in the mRNA and protein level of the enteric neurons and GDNF and p-Akt in colon (P < 0.01). EA with high-frequency and long-term stimuli at acupoint ST-36 can induce regeneration of lost enteric neurons in diabetic rats, and GDNF and PI3K/Akt signal pathway may play an important role in EA-induced regeneration of impaired enteric neurons.
Assuntos
Pontos de Acupuntura , Colo/inervação , Diabetes Mellitus Experimental/terapia , Eletroacupuntura/métodos , Sistema Nervoso Entérico/enzimologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Regeneração Nervosa , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Colina O-Acetiltransferase/metabolismo , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Sistema Nervoso Entérico/fisiopatologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Masculino , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Intestinal inflammation affects the enteric nervous system (ENS) that lies adjacent to the smooth muscle layers. Previously, we showed that the loss of ENS neurons in animal models such as tri-nitrobenzene sulphonic acid (TNBS)-induced colitis was a limited and early event despite progressive worsening of inflammation. Here, we demonstrated that the rapid appearance of activated immune cells in the intestinal wall is selectively neurotoxic via iNOS-derived NO, using TNBS-induced colitis in both rats and mice, and a co-culture model of ENS neurons and smooth muscle. An influx of neutrophils and macrophages occurred within hours of initiation of rat colitis, correlating with iNOS expression, acutely elevated NO and neuronal death. In vitro, chemical donors of NO selectively caused axonal damage and neuronal death. These outcomes were similar to those seen with combined culture with either activated peritoneal immune cells or the immune cell lines RAW-264 and RBL-2H3. Immune cell-mediated neurotoxicity was blocked by the iNOS inhibitor L-NIL, and neuronal death was inhibited by the RIP-1 kinase inhibitor necrostatin. In a mouse model, the stereotypic loss of myenteric neurons by Day 4 post-TNBS was abrogated by the selective iNOS inhibitors L-NIL or 1400W without effect on other parameters of intestinal inflammation. Preservation of ENS neurons also ameliorated the hyperplasia of smooth muscle that is characteristic of intestinal inflammation, in line with prior work showing neural regulation of smooth muscle phenotype. This identifies a predominant pathway of immune cell damage to the ENS, where early, acute elevation of NO from iNOS can be cytotoxic to myenteric neurons.
Assuntos
Colite/enzimologia , Sistema Nervoso Entérico/enzimologia , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura , Colite/patologia , Modelos Animais de Doenças , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/patologia , Feminino , Hiperplasia/tratamento farmacológico , Hiperplasia/patologia , Hiperplasia/fisiopatologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Macrófagos/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Músculo Liso/imunologia , Músculo Liso/patologia , Neuroimunomodulação/efeitos dos fármacos , Neuroimunomodulação/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Neurônios/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Neutrófilos/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Ratos Sprague-Dawley , Ácido TrinitrobenzenossulfônicoRESUMO
AIM: To investigate the relationship between neuronal nitric oxide synthase (nNOS) expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin (STZ)-induced diabetic mice. METHODS: Diabetic mice were induced by injection of STZ solution. Immunofluorescence labeling of HuC/D, nNOS and natriuretic peptide receptor-A, B, C (NPRs) in the gastric fundus (GF) was used to observe nNOS expression and whether NPRs exist on enteric neurons. The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting. An isometric force transducer recorded the electric field stimulation (EFS)-induced relaxation and contraction in the diabetic GF. An intracellular recording method assessed EFS-induced inhibitory junction potentials (IJP) on the GF. GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide (CNP) for 24 h, after which their nNOS expressions were detected by western blotting. RESULTS: Eight weeks after injection, 43 diabetic mice were obtained from mouse models injected with STZ. Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF. The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced, while the contraction was enhanced in the diabetic GF. EFS-induced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice. However, both NPR-A and NPR-B were detected on enteric neurons, and their expression levels were upregulated in the diabetic GF. The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP. CONCLUSION: These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Sistema Nervoso Entérico/enzimologia , Fundo Gástrico/inervação , Músculo Liso/inervação , Peptídeos Natriuréticos/metabolismo , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/fisiopatologia , Regulação para Baixo , Estimulação Elétrica , Sistema Nervoso Entérico/fisiopatologia , Masculino , Camundongos Endogâmicos ICR , Contração Muscular , Óxido Nítrico/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Transdução de Sinais , Estreptozocina , Técnicas de Cultura de Tecidos , Regulação para CimaRESUMO
Enteric glial cells are increasingly acknowledged as important partners of enteric neurons in the control of gastrointestinal function. They share morphological features and expression of antigenic markers with astrocytes of the central nervous system. Recently, aldehyde dehydrogenase 1 family member L1 (Aldh1L1) has been proposed as a novel and specific marker for astrocytes. Taking the known similarities between astrocytes and enteric glia into account, we sought to investigate whether enteric glial cells also express Aldh1L1. To this end, we performed immunostaining on preparations of myenteric plexus obtained from adult Aldh1L1-eGFP bacterial artificial chromosome (BAC) transgenic mice and found that the Aldh1L1 promoter is indeed active in enteric glia, albeit mainly in cells residing outside the myenteric ganglia. Apart from enteric glia, we also observed eGFP expression in interstitial cells of Cajal. Furthermore, myenteric ganglia of the large intestine contained eGFP positive neurons. Taken together, our data indicate that Aldh1L1 is not a suitable marker for enteric glial cells.
Assuntos
Aldeído Desidrogenase/metabolismo , Astrócitos/enzimologia , Sistema Nervoso Entérico/enzimologia , Neuroglia/enzimologia , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Aldeído-Desidrogenase Mitocondrial , Animais , Biomarcadores/metabolismo , Sistema Nervoso Entérico/citologia , Feminino , Masculino , Camundongos Transgênicos , Plexo Mientérico/citologia , Plexo Mientérico/enzimologia , Neurônios/enzimologia , Regiões Promotoras GenéticasRESUMO
The distribution of interstitial cells of Cajal (ICC), the probable pacemakers in gastrointestinal motility, was investigated using an antigenic marker of gastric ICC known as C-Kit. Antiserum raised against the general neuronal marker protein gene peptide 9.5 (PGP) as well as the nitrergic neuronal marker neuronal nitric oxide synthase (nNOS) were used to investigate the distribution of gastric nerves. Polyclonal goat anti-human C-Kit was reliable in labelling ICC in the stomach. Two classes of ICC were identified according to their distribution: ICC-MY distributed around the periphery of myenteric ganglia and ICC-IM in the circular and longitudinal muscle layers. The neuronal marker PGP was reliably consistent in revealing the density and distribution of the enteric nervous system. Density of nerve fibres was higher in circular smooth muscle than in longitudinal smooth muscle. From nNOS immunohistochemistry, it is evident that inhibitory (nitrergic) nerves constitute a substantial fraction of the enteric nervous system.
Assuntos
Cães/anatomia & histologia , Sistema Nervoso Entérico/citologia , Motilidade Gastrointestinal/fisiologia , Células Intersticiais de Cajal/enzimologia , Estômago/inervação , Animais , Sistema Nervoso Entérico/enzimologia , Humanos , Imuno-Histoquímica/veterinária , Células Intersticiais de Cajal/patologia , Músculo Liso/inervação , Músculo Liso/fisiologia , Vias Neurais/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Estômago/citologiaRESUMO
Enteric neural dysfunction leads to increased mucous production and dysmotility in inflammatory bowel disease (IBD). Prior studies have shown that tissue eosinophilia is related to disease activity. We hypothesized that interactions between eosinophils and nerves contribute to neural dysfunction in IBD. Tissue from patients with intractable IBD, endoscopic biopsies from patients with steroid responsive IBD, both when active and quiescent, and control tissue were studied. Immunohistochemical studies showed that eosinophils localize to nerves in the mucosal layer of patients with Crohn's disease (CD) (p<0.001) and ulcerative colitis (UC), (p<0.01). Eosinophils localized to substance P and choline acetyltransferase (ChAT) immunostained nerves. Real time PCR of laser capture micro-dissected enteric ganglia demonstrated Intercellular Adhesion Molecule 1 (ICAM-1) mRNA was increased 7-fold in UC (nâ=â4), (pâ=â0.03), and 10-fold in CD (nâ=â3), (pâ=â0.05). Compared with controls, eotaxin-3 (CCL-26) mRNA was increased 9-fold in UC (pâ=â0.04) and 15-fold in CD (pâ=â0.06). Eosinophil numbers correlated with disease activity, while deposition of major basic protein (MBP) and eosinophil Transforming Growth Factor ß-1 (TGFß-1) expression were seen in therapeutically responsive disease. These data indicate a significant localization of eosinophils to nerves in IBD, mediated through neurally expressed ICAM-1 and eotaxin-3. This cell/neural interaction may influence the function of nerves and contribute to symptoms in IBD.
Assuntos
Sistema Nervoso Entérico/imunologia , Eosinófilos/imunologia , Doenças Inflamatórias Intestinais/sangue , Sequência de Bases , Colina O-Acetiltransferase/metabolismo , Primers do DNA , Sistema Nervoso Entérico/enzimologia , Sistema Nervoso Entérico/metabolismo , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Substância P/metabolismo , Fator de Crescimento Transformador betaRESUMO
BACKGROUND: During development of the enteric nervous system, a subpopulation of enteric neuron precursors transiently expresses catecholaminergic properties. The progeny of these transiently catecholaminergic (TC) cells have not been fully characterized. METHODS: We combined in vivo Cre-lox-based genetic fate-mapping with phenotypic analysis to fate-map enteric neuron subtypes arising from tyrosine hydroxylase (TH)-expressing cells. KEY RESULTS: Less than 3% of the total (Hu(+) ) neurons in the myenteric plexus of the small intestine of adult mice are generated from transiently TH-expressing cells. Around 50% of the neurons generated from transiently TH-expressing cells are calbindin neurons, but their progeny also include calretinin, neurofilament-M, and serotonin neurons. However, only 30% of the serotonin neurons and small subpopulations (<10%) of the calbindin, calretinin, and neurofilament-M neurons are generated from TH-expressing cells; only 0.2% of nitric oxide synthase neurons arise from TH-expressing cells. CONCLUSIONS & INFERENCES: Transiently, catecholaminergic cells give rise to subpopulations of multiple enteric neuron subtypes, but the majority of each of the neuron subtypes arises from non-TC cells.
Assuntos
Catecolaminas/biossíntese , Mapeamento Cromossômico/métodos , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/enzimologia , Tirosina 3-Mono-Oxigenase/biossíntese , Animais , Catecolaminas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
BACKGROUND: Ca(2+)/calmodulin-dependent protein kinases (CaMKs) are major downstream mediators of neuronal calcium signaling that regulate multiple neuronal functions. CaMKII, one of the key CaMKs, plays a significant role in mediating cellular responses to external signaling molecules. Although calcium signaling plays an essential role in the enteric nervous system (ENS), the role of CaMKII in neurogenic intestinal function has not been determined. In this study, we investigated the function and expression pattern of CaMKII in the ENS across several mammalian species. METHODOLOGY/PRINCIPAL FINDINGS: CaMKII expression was characterized by immunofluorescence analyses and Western Blot. CaMKII function was examined by intracellular recordings and by assays of colonic contractile activity. Immunoreactivity for CaMKII was detected in the ENS of guinea pig, mouse, rat and human preparations. In guinea pig ENS, CaMKII immunoreactivity was enriched in both nitric oxide synthase (NOS)- and calretinin-containing myenteric plexus neurons and non-cholinergic secretomotor/vasodilator neurons in the submucosal plexus. CaMKII immunoreactivity was also expressed in both cholinergic and non-cholinergic neurons in the ENS of mouse, rat and human. The selective CaMKII inhibitor, KN-62, suppressed stimulus-evoked purinergic slow EPSPs and ATP-induced slow EPSP-like response in guinea pig submucosal plexus, suggesting that CaMKII activity is required for some metabotropic synaptic transmissions in the ENS. More importantly, KN-62 significantly suppressed tetrodotoxin-induced contractile response in mouse colon, which suggests that CaMKII activity is a major determinant of the tonic neurogenic inhibition of this tissue. CONCLUSION: ENS neurons across multiple mammalian species express CaMKII. CaMKII signaling constitutes an important molecular mechanism for controlling intestinal motility and secretion by regulating the excitability of musculomotor and secretomotor neurons. These findings revealed a fundamental role of CaMKII in the ENS and provide clues for the treatment of intestinal dysfunctions.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sistema Nervoso Entérico/enzimologia , Animais , Calmodulina/metabolismo , Sistema Nervoso Entérico/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Motilidade Gastrointestinal/fisiologia , Cobaias , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/citologia , Neurônios Motores/enzimologia , Plexo Mientérico/citologia , Plexo Mientérico/enzimologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Especificidade da EspécieRESUMO
Gastrointestinal disorders, constipation in particular, are the most common non-motor dysfunctions affecting Parkinson's disease (PD) patients. We have previously reported that rats bearing unilateral nigrostriatal lesion caused by 6-hydroxydopamine (6-OHDA) stereotaxic injection develop severe constipation together with a region-specific decrease of neuronal nitric oxide synthase (nNOS) in enteric neurons of the lower intestinal tract. Here, we extend these observations on other enteric neuronal subpopulations, investigating also the propulsive activity of isolated colonic specimens. Four weeks post 6-OHDA injection, lesioned rats showed a significant increase of vasoactive intestinal polypeptide (VIP) concomitant with the reduced expression of nNOS in the myenteric plexus of distal ileum and proximal colon; in particular VIP increased in a subpopulation of neurons actively expressing nNOS. On the other hand, choline acetyltransferase (ChAT) was not modified in any of the intestinal segments analyzed. Interestingly, we found a reduced expression of dopamine receptor type 2 (D2R) in proximal (-66.8%) and distal (-54.5%) colon, together with reduced peristalsis efficiency (decrease in intraluminal pressure and frequency of peristaltic events) in the 6-OHDA-lesioned rats. The selective depletion of dopaminergic nigrostriatal neurons is associated with changes in the expression of enteric inhibitory neurotransmitters, as well as of the D2R in intestinal specific regions. Moreover, 6-OHDA-lesioned rats demonstrated altered colon propulsive activity referable to the D2R decrease. Our findings unveil subtle mechanisms underlying the enteric neurochemical plasticity events evoked by disruption of the normal brain-gut cross-talk, giving a peculiar point of view on the pathophysiology of the severe constipation that frequently affects PD patients.
Assuntos
Colo/fisiopatologia , Sistema Nervoso Entérico/fisiopatologia , Motilidade Gastrointestinal , Doença de Parkinson/fisiopatologia , Animais , Colina O-Acetiltransferase/metabolismo , Colo/enzimologia , Modelos Animais de Doenças , Sistema Nervoso Entérico/enzimologia , Íleo/enzimologia , Íleo/fisiopatologia , Intestino Delgado/enzimologia , Intestino Delgado/fisiopatologia , Masculino , Neurônios/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Doença de Parkinson/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
Colostrum is an indispensable source of antibodies (IgG) protecting the newborn pig against infection. We studied the effect of feeding colostrum and purified IgG on early structure and development of the gastrointestinal tract (GIT). Newborn littermate pigs were fed either colostrum, an elemental diet (ED), or an ED supplemented with purified serum IgG (ED + IgG) for 24 h or then only ED up to 72 h. Afterwards, pigs were slaughtered. Colostrum-fed pigs or ED supplemented with IgG (ED + IgG) increased thickness (P < 0.001) of stomach mucosa and muscularis (P < 0.05) compared to the ED group not receiving IgG. Feeding an ED supplemented with IgG improved morphology of the GIT towards that of colostrum-fed piglets and indicates a beneficial effect of IgG on GIT development in neonatal pigs. Immunohistochemical studies indicate that ED feeding may influence the expression of nitric oxide synthase in jejunal myenteric (but not submucous) neurons of newborn pigs.
Assuntos
Ração Animal/análise , Colostro , Dieta/veterinária , Trato Gastrointestinal/anatomia & histologia , Imunoglobulina G/farmacologia , Suínos , Fenômenos Fisiológicos da Nutrição Animal , Animais , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/enzimologia , Sistema Nervoso Entérico/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismoRESUMO
The purpose of this study was to analyze the nicotinamide adenine dinucleotide phosphate - diaphorase (NADPH-d) activity in the rat jejunum after a mesenteric ischemia/reperfusion injury. Nitric oxide, synthetised from L-arginine by the enzyme nitric oxide synthase, is a nonadrenergic noncholinergic relaxant neurotransmitter of the intestinal smooth muscle. It plays an important role in the process of plasticity after the ischemia/reperfusion injury. Experimental animals were divided in two groups: the control group and the ischemic/reperfusion group, with different period of the reperfusion. The NADPH-d histochemical method has been used as a marker for the nitric oxide synthase. NADPH-d activity has been rapidly decreased in the neurons of both enteric nervous systems in plexuses of the jejunum after 1 h mesenteric ischemia and 1 h reperfusion. Differences were predominantly detected in the myenteric plexus; they were seen in change of the neuronal shape, in the arrangement of neurons and in intensity of their staining. The NADPH-d positivity was absent in the intestinal crypts. After 1 h ischemia and 24 h reperfusion, the NADPH-d activity was gradually increased, but it was lower in comparison with the control group. On the 30th day following the ischemia/reperfusion there were no changes in NADPH-d positivity compared with the control animals. These results indicated that the jejunal ischemia/reperfusion has affected the neurons of the enteric nervous system of adult rats and resulted in the early decrease of NADPH-d positivity 1 h of the reperfusion insult. The gradual increasing of NADPH-d activity in 24 h following the reperfusion could be considered as a result of the plasticity process. On the 30(th) day after the ischemia/reperfusion all histochemical changes were returned to the control levels.