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OBJECTIVES: Vagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human. DESIGN: Using Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1-5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI. RESULTS: EFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery. CONCLUSION: Enteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI. TRIAL REGISTRATION NUMBER: NCT02425774.
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Benzofuranos , Íleus , Intestino Delgado , Músculo Liso , Pancreaticoduodenectomia/efeitos adversos , Complicações Pós-Operatórias , Adulto , Animais , Benzofuranos/administração & dosagem , Benzofuranos/farmacologia , Modelos Animais de Doenças , Feminino , Motilidade Gastrointestinal/efeitos dos fármacos , Humanos , Íleus/etiologia , Íleus/imunologia , Íleus/fisiopatologia , Íleus/prevenção & controle , Inflamação/imunologia , Inflamação/prevenção & controle , Intestino Delgado/imunologia , Intestino Delgado/inervação , Intestino Delgado/patologia , Intestino Delgado/fisiopatologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Músculo Liso/efeitos dos fármacos , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Pancreaticoduodenectomia/métodos , Projetos Piloto , Complicações Pós-Operatórias/imunologia , Complicações Pós-Operatórias/fisiopatologia , Complicações Pós-Operatórias/prevenção & controle , Agonistas do Receptor 5-HT4 de Serotonina/administração & dosagem , Agonistas do Receptor 5-HT4 de Serotonina/farmacologia , Resultado do Tratamento , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
BACKGROUND: The vagus nerve has emerged as an important modulator of the intestinal immune system. Its anti-inflammatory properties have been previously shown in innate and Th1/Th17 predominant inflammatory models. To what extent the vagus nerve is of importance in Th2 inflammatory responses like food allergy is still unclear. In this study, we therefore aimed to investigate the effect of vagotomy (VGX) and vagus nerve stimulation (VNS), on the development and severity of experimental food allergy. METHODS: Balb/C mice were first sensitized with ovalbumin (OVA) in the presence of alum. Prior to oral challenges with OVA, mice were subjected to VGX or VNS. Disease severity was determined by assessing severity and onset of diarrhoea, OVA-specific antibody production, mast cell number and activity, inflammatory gene expression in duodenal tissue and lamina propria immune cells by flow cytometry analysis. RESULTS: When compared to control mice, VGX did not significantly affect the development and severity of the disease in our model of food allergy. VNS, on the other hand, resulted in a significant amelioration of the different inflammatory parameters assessed. This effect was independent of α7nAChR and is possibly mediated through the dampening of mast cells and increased phagocytosis of OVA by CX3CR1hi macrophages. CONCLUSIONS: These results underscore the anti-inflammatory properties of the vagus nerve and the potential of neuro-immune interactions in the intestine. Further insight into the underlying mechanisms could ultimately lead to novel therapeutic approaches in the treatment of not only food allergy but also other immune-mediated diseases.
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Hipersensibilidade Alimentar/etiologia , Hipersensibilidade Alimentar/metabolismo , Gastroenterite/etiologia , Gastroenterite/metabolismo , Estimulação do Nervo Vago , Alérgenos , Animais , Biomarcadores , Permeabilidade da Membrana Celular , Modelos Animais de Doenças , Hipersensibilidade Alimentar/diagnóstico , Gastroenterite/patologia , Imunofenotipagem , Macrófagos/imunologia , Macrófagos/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Mastocitose , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Ovalbumina/imunologia , Índice de Gravidade de Doença , Vagotomia , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
OBJECTIVE: Postoperative ileus (POI) is assumed to result from myeloid cells infiltrating the intestinal muscularis externa (ME) in patients undergoing abdominal surgery. In the current study, we investigated the role of infiltrating monocytes in a murine model of intestinal manipulation (IM)-induced POI in order to clarify whether monocytes mediate tissue damage and intestinal dysfunction or they are rather involved in the recovery of gastrointestinal (GI) motility. DESIGN: IM was performed in mice with defective monocyte migration to tissues (C-C motif chemokine receptor 2, Ccr2-/ - mice) and wild-type (WT) mice to study the role of monocytes and monocyte-derived macrophages (MΦs) during onset and resolution of ME inflammation. RESULTS: At early time points, IM-induced GI transit delay and inflammation were equal in WT and Ccr2 -/- mice. However, GI transit recovery after IM was significantly delayed in Ccr2 -/- mice compared with WT mice, associated with increased neutrophil-mediated immunopathology and persistent impaired neuromuscular function. During recovery, monocyte-derived MΦs acquire pro-resolving features that aided in the resolution of inflammation. In line, bone marrow reconstitution and treatment with MΦ colony-stimulating factor 1 enhanced monocyte recruitment and MΦ differentiation and ameliorated GI transit in Ccr2 -/- mice. CONCLUSION: Our study reveals a critical role for monocyte-derived MΦs in restoring intestinal homeostasis after surgical trauma. From a therapeutic point of view, our data indicate that inappropriate targeting of monocytes may increase neutrophil-mediated immunopathology and prolong the clinical outcome of POI, while future therapies should be aimed at enhancing MΦ physiological repair functions.
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Íleus/imunologia , Íleus/patologia , Macrófagos/imunologia , Monócitos/imunologia , Complicações Pós-Operatórias/imunologia , Complicações Pós-Operatórias/patologia , Receptores CCR2/imunologia , Animais , Diferenciação Celular , Movimento Celular , Modelos Animais de Doenças , Motilidade Gastrointestinal , Trânsito Gastrointestinal , Homeostase/imunologia , Inflamação/imunologia , Inflamação/patologia , Camundongos , Músculo Liso/patologiaRESUMO
Vagotomy (VGX) increases the susceptibility to develop colitis suggesting a crucial role for the cholinergic anti-inflammatory pathway in the regulation of the immune responses. Since oral tolerance and the generation of regulatory T cells (Tregs) are crucial to preserve mucosal immune homeostasis, we studied the effect of vagotomy and the involvement of α7 nicotinic receptors (α7nAChR) at the steady state and during colitis. Therefore, the development of both oral tolerance and colitis (induced by dextran sulfate sodium (DSS) or via T cell transfer) was studied in vagotomized mice and in α7nAChR-/- mice. VGX, but not α7nAChR deficiency, prevented oral tolerance establishment. This effect was associated with reduced Treg conversion in the lamina propria and mesenteric lymphnodes. To the same extent, vagotomized mice, but not α7nAChR-/- mice, developed a more severe DSS colitis compared with control mice treated with DSS, associated with a decreased number of colonic Tregs. However, neither VGX nor absence of α7nAChR in recipient mice affected colitis development in the T cell transfer model. In line, deficiency of α7nAChR exclusively in T cells did not influence the development of colitis induced by T cell transfer. Our results indicate a key role for the vagal intestinal innervation in the development of oral tolerance and colitis, most likely by modulating induction of Tregs independently of α7nAChR.
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The cholinergic anti-inflammatory pathway (CAIP) has been proposed as a key mechanism by which the brain, through the vagus nerve, modulates the immune system in the spleen. Vagus nerve stimulation (VNS) reduces intestinal inflammation and improves postoperative ileus. We investigated the neural pathway involved and the cells mediating the anti-inflammatory effect of VNS in the gut. The effect of VNS on intestinal inflammation and transit was investigated in wild-type, splenic denervated and Rag-1 knockout mice. To define the possible role of α7 nicotinic acetylcholine receptor (α7nAChR), we used knockout and bone marrow chimaera mice. Anterograde tracing of vagal efferents, cell sorting and Ca(2+) imaging were used to reveal the intestinal cells targeted by the vagus nerve. VNS attenuates surgery-induced intestinal inflammation and improves postoperative intestinal transit in wild-type, splenic denervated and T-cell-deficient mice. In contrast, VNS is ineffective in α7nAChR knockout mice and α7nAChR-deficient bone marrow chimaera mice. Anterograde labelling fails to detect vagal efferents contacting resident macrophages, but shows close contacts between cholinergic myenteric neurons and resident macrophages expressing α7nAChR. Finally, α7nAChR activation modulates ATP-induced Ca(2+) response in small intestine resident macrophages. We show that the anti-inflammatory effect of the VNS in the intestine is independent of the spleen and T cells. Instead, the vagus nerve interacts with cholinergic myenteric neurons in close contact with the muscularis macrophages. Our data suggest that intestinal muscularis resident macrophages expressing α7nAChR are most likely the ultimate target of the gastrointestinal CAIP.
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Macrófagos/metabolismo , Músculo Liso/citologia , Estimulação do Nervo Vago , Nervo Vago/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Denervação Autônoma , Citocinas/genética , Enterite/metabolismo , Trânsito Gastrointestinal , Expressão Gênica , Macrófagos/citologia , Camundongos , Camundongos Knockout , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Nicotina/farmacologia , Peroxidase/metabolismo , Transdução de Sinais , Baço/inervação , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Background: We previously showed increased susceptibility to dextran sulfate sodium (DSS)-induced colitis in vagotomized mice. Here, we evaluated whether vagus nerve stimulation (VNS) is able to reduce the severity of DSS colitis and aimed to unravel the mechanism involved. Methods: Colitis was induced in wild type mice by 2.5% DSS administration in drinking water for 5 days. VNS (5 Hz, 1 ms, 1 mA) was applied 1 day prior to and after 4 days of DSS administration to evaluate changes in epithelial integrity and inflammatory response, respectively. Epithelial integrity was assessed using TUNEL and Ki67 staining. Monocytes, immature and mature macrophages were sorted from colonic samples and gene expression levels of pro-inflammatory cytokines were studied. Results: VNS applied prior to DSS administration (i.e., prophylactic VNS) reduced disease activity index (VNS 0.8 ± 0.6 vs. sham 2.8 ± 0.7, p < 0.001, n = 5) and tended to improve histology score. Prophylactic VNS significantly increased epithelial cell proliferation and diminished apoptosis compared to sham stimulation. VNS applied at day 4 during DSS administration (i.e., therapeutic VNS) decreased the influx of monocytes, monocyte-derived macrophages and neutrophils, and significantly reduced pro-inflammatory cytokine expression (i.e., Tnfα and Cxcl1) in immature macrophages compared to sham stimulation. Conclusions: A single period of VNS applied prior to DSS exposure reduced DSS-induced colitis by an improvement in epithelial integrity. On the other hand, VNS applied during the inflammatory phase of DSS colitis reduced cytokine expression in immature macrophages. Our data further underscores the potential of VNS as novel therapeutic approach for inflammatory bowel diseases.
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Impairment in gallbladder emptying, increase in residual volume, and reduced smooth muscle contractility are hallmarks of acute acalculous cholecystitis and seem to be related to ischemia/reperfusion (I/R). This study was designed to determine the effects of tempol, a general antioxidant, on I/R-induced changes in gallbladder contractile capacity, the mechanisms involved in the contractile process, and the level of inflammatory mediators. Experimental gallbladder I/R was induced in male guinea pigs by common bile duct ligation for 2 days, then a deligation of the duct was performed and after 2 days the animals were sacrificed. A group of animals was treated with tempol, administered in the drinking water at 1 mmol/l for 10 days prior the bile duct ligation and until animal sacrifice. Isometric tension recordings showed that KCl and cholecystokinin-induced contractions were impaired by I/R, which correlated with decreased F-actin content and detrimental effects on Ca(2+) influx. In addition, I/R depolarized mitochondrial membrane potential, as indicated by the reduction of the heterogeneity of the rhodamine123 fluorescence signal, and increased the expression of NF-kappaB, COX-2, and iNOS. Tempol treatment improved contractility via normalization of Ca(2+) handling and improvement of F-actin content. Moreover, the antioxidant ameliorated mitochondrial polarity and normalized the expression levels of the inflammatory mediators. These results show that antioxidant treatment protects the gallbladder from I/R, indicating the potential therapeutic benefits of tempol in I/R injury.
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Óxidos N-Cíclicos/uso terapêutico , Vesícula Biliar/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Animais , Cálcio/metabolismo , Ducto Colédoco/cirurgia , Vesícula Biliar/irrigação sanguínea , Vesícula Biliar/metabolismo , Esvaziamento da Vesícula Biliar/efeitos dos fármacos , Cobaias , Ligadura , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Marcadores de SpinRESUMO
BACKGROUND: Vagus nerve (VN) stimulation is currently evaluated as a novel approach to treat immune-mediated disorders. The optimal stimulation parameters, however, largely depend on the VN composition potentially impacting on its clinical translation. Hence, we evaluated whether morphological differences exist between the cervical and abdominal VNs across different species. MATERIALS AND METHODS: The cervical and abdominal VNs of mouse, pig, and humans were stained for major basic protein and neurofilament F to identify the percentage and size of myelinated and non-myelinated fibers. RESULTS: The percentage of myelinated fibers was comparable between species, but was higher in the cervical VN compared with the abdominal VN. The cervical VN contained 54 ± 4%, 47 ± 7%, and 54 ± 7% myelinated fibers in mouse, pig, and humans, respectively. The myelinated fibers consisted of small-diameter (mouse: 71%, pig: 80%, and humans: 63%), medium-diameter (mouse: 21%, pig: 18%, and humans: 33%), and large-diameter fibers (mouse: 7%, pig: 2%, and humans: 4%). The abdominal VN predominantly contained unmyelinated fibers (mouse: 93%, pig: 90%, and humans: 94%). The myelinated fibers mainly consisted of small-diameter fibers (mouse: 99%, pig: 85%, and humans: 74%) and fewer medium-diameter (mouse: 1%, pig: 13%, and humans: 23%) and large-diameter fibers (mouse: 0%, pig: 2%, and humans: 3%). CONCLUSION: The VN composition was largely similar with respect to myelinated and unmyelinated fibers in the species studied. Human and porcine VNs had a comparable diameter and similar amounts of fibrous tissue and contained multiple fascicles, implying that the porcine VN may be suitable to optimize stimulation parameters for clinical trials.
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Bainha de Mielina/metabolismo , Nervo Vago/metabolismo , Animais , Humanos , Camundongos , Fibras Nervosas/metabolismo , SuínosRESUMO
In order to control cell functions, extracellular agents, such as hormones or neurotransmitters among others, generate a diversity of calcium (Ca(2+)) signals in target cells. Here, we review the components involved in Ca(2+) handling and effectors, both members of the known calcium signaling pathways. In the pancreas, Ca(2+) signal appears as local increases, global elevations or Ca(2+) oscillations. Ca(2+) plays a key role in the pancreatic cells, regulating secretion in exocrine cells, a widely used model for studying the coupling between Ca(2+) signaling and secretion, and the release of insulin, glucagon and somatostatin in the exocrine pancreas. Interestingly, Ca(2+) deregulations have been related to pancreatitis and aging of the pancreas, and treatment with melatonin has shown beneficial effects suggesting that melatonin could be an adequate therapeutic approach.
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Sinalização do Cálcio/fisiologia , Pâncreas/fisiologia , Pancreatite/fisiopatologia , Envelhecimento/fisiologia , Animais , Cálcio/metabolismo , HomeostaseRESUMO
INTRODUCTION: It is unknown if ANO-1 is expressed in the heart, though the presence of a calcium-activated chloride current has been proposed to mediate some cardiac dysrhythmias. Furthermore, a specific cell type termed telocytes, morphologically mimicking Cajal cells which use ANO-1 to modulate their pacemaker activity in the gut, have been described in the heart. We therefore sought to determine whether this channel is expressed in the canine heart. METHODS: Myocardium was sampled from the ventricles of five canines. Sections were labeled with anti-Kit and anti-ANO-1 antibodies. Slides were reviewed by four investigators looking at cell morphology, distribution, and co-localization. Identification of telocytes was based on criteria including morphology, Kit positivity (+), and ANO-1 positivity (+). RESULTS: Clusters of cells meeting criteria for telocytes were seen in the epicardium, sub-epicardium, and mid-myocardium. A small subset of cells that were morphologically similar to myocytes was ANO-1 (+) but Kit (-). In total, three different cell classes were found: (i) Kit (+), ANO-1 (+) cells with the appearance of telocytes; (ii) Kit (+), ANO-1 (-) cells; and (iii) Kit (-), ANO-1 (+) cells with the morphologic appearance of cardiac myocytes. CONCLUSIONS: Telocytes are present in the canine ventricle and express ANO-1. These data merit further study to elucidate the functional expression of these channels in the heart and whether they may be targets for cardiac arrhythmias.
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Impaired Ca2+ homeostasis and smooth muscle contractility co-exist in acute cholecystitis (AC) leading to gallbladder dysfunction. There is no pharmacological treatment for this pathological condition. Our aim was to evaluate the effects of melatonin treatment on Ca2+ signaling pathways and contractility altered by cholecystitis. [Ca2+]i was determined by epifluorescence microscopy in fura-2 loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. Malondialdehyde (MDA) and reduced glutathione (GSH) contents were determined by spectrophotometry and cycloxygenase-2 (COX-2) expression was quantified by western blot. Melatonin was tested in two experimental groups, one of which underwent common bile duct ligation for 2 days and another that was later de-ligated for 2 days. Inflammation-induced impairment of Ca2+ responses to cholecystokinin and caffeine were recovered by melatonin treatment (30 mg/kg). This treatment also ameliorated the detrimental effects of AC on Ca2+ influx through both L-type and capacitative Ca2+ channels, and it was effective in preserving the pharmacological phenotype of these channels. Despite its effects on Ca2+ homeostasis, melatonin did not improve contractility. After de-ligation, Ca2+ influx and contractility were still impaired, but both were recovered by melatonin. These effects of melatonin were associated to a reduction of MDA levels, an increase in GSH content and a decrease in COX-2 expression. These findings indicate that melatonin restores Ca2+ homeostasis during AC and resolves inflammation. In addition, this indoleamine helps in the subsequent recovery of functionality.
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Colecistite Acalculosa/tratamento farmacológico , Colecistite Acalculosa/fisiopatologia , Cálcio/fisiologia , Colecistite Aguda/tratamento farmacológico , Homeostase/efeitos dos fármacos , Melatonina/uso terapêutico , Animais , Cafeína/farmacologia , Colecistite Aguda/fisiopatologia , Ducto Colédoco , Modelos Animais de Doenças , Vesícula Biliar/efeitos dos fármacos , Vesícula Biliar/fisiologia , Cobaias , Ligadura , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Contração Muscular/efeitos dos fármacos , Nitrendipino/farmacologia , Pinacidil/farmacologia , Tapsigargina/farmacologiaRESUMO
Information regarding age-induced Ca(2+) signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca(2+) signals and mitochondrial potential, a parameter involved in both Ca(2+) signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca(2+) homeostasis and mitochondrial potential in exocrine cells. Pancreatic acinar cells were obtained from adult (3 months old) and aged (22-24 months old) mice by collagenase dispersion. Ca(2+) signals, in situ mitochondrial potential and in vitro amylase secretion were determined. Secretion in response to increasing levels of the secretagogues, acetylcholine and cholecystokinin (CCK), were impaired in aged pancreatic acini. This decrease was accompanied by an inhibition in the amplitude of the peak response to maximal concentrations of the agonists, and by a decrease in the pattern of Ca(2+) oscillations induced by postprandial levels of CCK. Both the size of the calcium pools, assessed by low levels of ionomycin, and capacitative calcium entry, induced by depletion of the stores with thapsigargin, were diminished in aged cells. These changes in Ca(2+) homeostasis were associated with depolarization of intracellular mitochondria. Oral administration of melatonin for 3 months to aged mice restored the secretory response, the amplitude and frequency of Ca(2+) responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca(2+) signals and mitochondrial potential of aged exocrine cells.
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Envelhecimento/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Glândulas Exócrinas/efeitos dos fármacos , Melatonina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Acetilcolina/farmacologia , Amilases/metabolismo , Animais , Antioxidantes/farmacologia , Células Cultivadas , Colecistocinina/farmacologia , Glândulas Exócrinas/citologia , Glândulas Exócrinas/metabolismo , CamundongosRESUMO
Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin-treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca(2+)](i)) was estimated by epifluorescence microscopy of fura-2-loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca(2+)](i) in response to the stimuli, suggesting a reduced Ca(2+)sensitivity. Indeed, the agonist-induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca(2+) sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI-17 and MYPT1, and with the absence of CPI-17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca(2+) handling and Ca(2+) sensitizations pathways. Moreover, the indoleamine restored age-induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging-related urinary bladder contractile impairment.
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Envelhecimento/metabolismo , Antioxidantes/farmacologia , Contração Isométrica/efeitos dos fármacos , Melatonina/farmacologia , Doenças da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Envelhecimento/patologia , Amidas/farmacologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Cobaias , Indóis/farmacologia , Maleimidas/farmacologia , Fosfatase de Miosina-de-Cadeia-Leve/antagonistas & inibidores , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Piridinas/farmacologia , Bexiga Urinária/patologia , Doenças da Bexiga Urinária/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Oxazolone-induced colitis has been frequently used in literature as a model of IBD, but insights into the underlying immune response and pathological features are surprisingly still very limited. Vagus nerve stimulation (VNS) has proven to be effective in innate and Th1/Th17 predominant inflammatory models, including pre-clinical models of colitis, however to what extent VNS is also effective in ameliorating Th2-driven colitis remains to be studied. In the present study, we therefore further characterized the immune response in oxazolone-induced colitis and investigated the potential therapeutic effect of VNS. METHODS: Colitis was induced in Balb/c mice by cutaneous sensitization with 3% oxazolone followed by intracolonic administration of 1% oxazolone 7 days later. To evaluate the effect of VNS on the development of Th2-driven colitis, VNS and sham-treated mice were challenged with 1% oxazolone. RESULTS: Intracolonic oxazolone administration resulted in a severe destruction of the colonic mucosa and a rapid drop in body temperature leading to a 65% mortality rate at day 5. Severe infiltration of neutrophils and monocytes was detected 6h after oxazolone administration which was associated with a Th2-type inflammatory response. VNS significantly improved survival rate which correlated with decreased levels of HMGB1 and reduced colonic (il6 and cxcl1 mRNA) and serum cytokine levels (IL-6, TNFα and CXCL1) compared to sham treated mice. CONCLUSIONS: Oxazolone-induced colitis rather represents a model of sepsis and, at best, may resemble a severe type of ulcerative colitis, associated with early and severe mucosal damage and a high mortality rate. VNS reduces colonic inflammation and improves survival in this model, supporting the anti-inflammatory properties of VNS, even in an aggressive model as oxazolone-induced colitis.
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Colite/fisiopatologia , Colite/terapia , Estimulação do Nervo Vago , Animais , Colite/induzido quimicamente , Colite/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Hipotermia/complicações , Hipotermia/imunologia , Hipotermia/patologia , Hipotermia/fisiopatologia , Inflamação/complicações , Inflamação/patologia , Mucosa Intestinal/patologia , Camundongos Endogâmicos BALB C , Células T Matadoras Naturais/imunologia , Oxazolona , Análise de SobrevidaRESUMO
We investigated the effects of aging in Ca(2+) extrusion mechanisms in smooth muscle bladder cells from 4 and 20-24-month-old guinea pigs using fluorescence microscopy and fura-2. Cells were challenged with a pulse of KCl immediately before perfusion with a Ca(2+) free solution containing no inhibitors (control, untreated cells) or inhibitors of plasma membrane Ca(2+) pump (PMCA, 1mM La(3+)), Na(+)/Ca(2+) exchanger (NCX, 1 microM SEA0400) or the sarcoendoplasmic Ca(2+) pump (SERCA, 1 microM thapsigargin). Treatment of young adult cells with the inhibitors allowed estimating a relative contribution of 55% for NCX, 27% for PMCA and 31% for SERCA. Combination of two inhibitors at the same time showed the presence of interaction between extrusion mechanisms. In aged cells the [Ca(2+)](i) extrusion was impaired due to decrease of PMCA activity, as revealed by the loss of effect of La(3+), and to inhibitory interactions between NCX and SERCA activities, indicated by acceleration of decay in response to their respective inhibitors. In conclusion, in smooth muscle cells aging decreases the overall Ca(2+) extrusion activity and modifies the interactions between the activities of the main Ca(2+) removing mechanisms.
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Cálcio/metabolismo , Senescência Celular , Miócitos de Músculo Liso/fisiologia , Bexiga Urinária/citologia , Animais , Transporte Biológico , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Fura-2 , Cobaias , Microscopia de Fluorescência , Miócitos de Músculo Liso/metabolismoRESUMO
BACKGROUND: Electrical stimulation of the vagus nerve suppresses intestinal inflammation and normalizes gut motility in a mouse model of postoperative ileus. The exact anatomical interaction between the vagus nerve and the intestinal immune system remains however a matter of debate. In the present study, we provide additional evidence on the direct and indirect vagal innervation of the spleen and analyzed the anatomical evidence for neuroimmune modulation of macrophages by vagal preganglionic and enteric postganglionic nerve fibers within the intestine. METHODS: Dextran conjugates were used to label vagal preganglionic (motor) fibers projecting to the small intestine and spleen. Moreover, identification of the neurochemical phenotype of the vagal efferent fibers and enteric neurons was performed by immunofluorescent labeling. F4/80 antibody was used to label resident macrophages. RESULTS: Our anterograde tracing experiments did not reveal dextran-labeled vagal fibers or terminals in the mesenteric ganglion or spleen. Vagal efferent fibers were confined within the myenteric plexus region of the small intestine and mainly endings around nNOS, VIP and ChAT positive enteric neurons. nNOS, VIP and ChAT positive fibers were found in close proximity of intestinal resident macrophages carrying α7 nicotinic receptors. Of note, VIP receptors were found on resident macrophages located in close proximity of VIP positive nerve fibers. CONCLUSION: In the present study, we show that the vagus nerve does not directly interact with resident macrophages in the gut or spleen. Instead, the vagus nerve preferentially interacts with nNOS, VIP and ChAT enteric neurons located within the gut muscularis with nerve endings in close proximity of the resident macrophages.
Assuntos
Intestino Delgado/inervação , Macrófagos/fisiologia , Baço/inervação , Nervo Vago/fisiologia , Acetilcolina/metabolismo , Animais , Vias Eferentes , Feminino , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Plexo Mientérico/citologia , Plexo Mientérico/fisiologia , Pescoço , Fatores de Crescimento Neural/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Baço/citologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
INTRODUCTION: Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3(Cre/+) , devoid of mast cells but with intact Kit signaling. DESIGN: The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. Kit(W-sh/W-sh) and Cpa3(Cre/+) mice, and by use of the mast cell stabilizer cromolyn. RESULTS: Kit(W-sh/W-sh) mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3(Cre/+) mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3(Cre/+) mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3(+/+) ). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI. CONCLUSIONS: Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.
Assuntos
Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Trânsito Gastrointestinal , Íleus/patologia , Mastócitos/citologia , Complicações Pós-Operatórias/patologia , Animais , Carboxipeptidases A/genética , Carboxipeptidases A/metabolismo , Degranulação Celular , Movimento Celular , Cromolina Sódica/farmacologia , Modelos Animais de Doenças , Expressão Gênica , Humanos , Íleus/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Células Intersticiais de Cajal/metabolismo , Células Intersticiais de Cajal/patologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Transgênicos , Monócitos/metabolismo , Monócitos/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Especificidade da EspécieRESUMO
BACKGROUND: Postoperative ileus (POI) is characterized by impaired gastrointestinal motility resulting from intestinal handling-associated inflammation. The introduction of laparoscopic surgery has dramatically reduced the duration of POI. However, it remains unclear to what extent this results in a reduction of intestinal inflammation. The aim of the present study is to compare the degree of intestinal inflammation and gastrointestinal transit following laparoscopic surgery and open abdominal surgery. METHODS: Mice were subjected to laparoscopic surgery or laparotomy alone or, in combination with standardized intestinal manipulation of the small bowel (IM). Gastrointestinal transit and intestinal inflammation were assessed 24 h after surgery by the number of myeloperoxidase (MPO) positive cells and the level of cytokine expression. The recovery time and the degree of inflammation were also analyzed in patients subjected to colectomy under open conditions (laparotomy) or laparoscopic conditions. KEY RESULTS: Mice undergoing IM by laparotomy (open IM), but not by laparoscopy (Lap IM) developed a significant delay in gastrointestinal transit compared to laparotomy or laparoscopy alone. In addition, there was significant intestinal inflammation only after open IM. Similarly, cytokine levels in peritoneal lavage fluid were lower while recovery time was faster in patients subjected to colectomy under laparoscopic conditions compared to open colectomy. CONCLUSIONS & INFERENCES: Our data confirms that intestinal inflammation is underlying the delayed gastrointestinal transit observed after open surgery. Most importantly, we demonstrate that intestinal inflammation under laparoscopic conditions is significantly lower compared to open surgery, most likely explaining the faster recovery following laparoscopic surgery.
Assuntos
Enterite/etiologia , Íleus/etiologia , Doenças do Jejuno/etiologia , Laparoscopia/efeitos adversos , Animais , Modelos Animais de Doenças , Enterite/metabolismo , Feminino , Trânsito Gastrointestinal , Humanos , Interleucinas/metabolismo , Doenças do Jejuno/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: The cholinergic anti-inflammatory pathway is an endogenous mechanism by which the autonomic nervous system attenuates macrophage activation via nicotinic acetylcholine receptors (nAChR). This concept has however not been demonstrated at a cellular level in intact tissue. To this end, we have studied the effect of nicotine on the activation of resident macrophages in a mouse stomach preparation by means of calcium imaging. METHODS: Calcium transients ([Ca(2+)]i) in resident macrophages were recorded in a mouse stomach preparation containing myenteric plexus and muscle layers by Fluo-4. Activation of macrophages was achieved by focal puff administration of ATP. The effects of nicotine on activation of macrophages were evaluated and the nAChR involved was pharmacologically characterized. The proximity of cholinergic nerves to macrophages was quantified by confocal microscopy. Expression of ß2 and α7 nAChR was evaluated by ß2 immunohistochemistry and fluorophore-tagged α-bungarotoxin. RESULTS: In 83% of macrophages cholinergic varicose nerve fibers were detected at distances <900 nm. The ATP induced [Ca(2+)]i increase was significantly inhibited in 65% or 55% of macrophages by 100 µM or 10 µM nicotine, respectively. This inhibitory effect was reversed by the ß2 nAChR preferring antagonist dihydro-ß-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (α3ß4 nAChR-preferring antagonist), α-bungarotoxin or methyllycaconitine (both α7 nAChR-preferring antagonist). Macrophages in the stomach express ß2 but not α7 nAChR at protein level, while those in the intestine express both receptor subunits. CONCLUSION: This study is the first in situ demonstration of an inhibition of macrophage activation by nicotine suggesting functional signaling between cholinergic neurons and macrophages in the stomach. The data suggest that the ß2 subunit of the nAChR is critically involved in the nicotine-induced inhibition of these resident macrophages.
Assuntos
Macrófagos/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/metabolismo , Estômago/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Compostos de Anilina/metabolismo , Animais , Cálcio/metabolismo , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/metabolismo , Estimulantes Ganglionares/farmacologia , Mucosa Gástrica/metabolismo , Imuno-Histoquímica , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estômago/inervação , Xantenos/metabolismoRESUMO
Calcium sensitization is an important physiological process in agonist-induced contraction of smooth muscle. In brief, calcium sensitization is a pathway that leads to smooth muscle contraction independently of changes in [Ca(2+)](i) by mean of inhibition of myosin light chain phosphatase. Aging has negative impacts on gallbladder contractile response due to partial impairment in calcium signaling and alterations in the contractile machinery. However, information regarding aging-induced alterations in calcium sensitization is scanty. We hypothesized that the calcium sensitization system is negatively affected by age. To investigate this, gallbladders were collected from adult (4 months old) and aged (22-24 months old) guinea pigs. To evaluate the contribution of calcium sensitization pathways we assayed the effect of the specific inhibitors Y-27632 and GF109203X on the "in vitro" isometric gallbladder contractions induced by agonist challenges. In addition, expression and phosphorylation (as activation index) of proteins participating in the calcium sensitization pathways were quantified by Western blotting. Aging reduced bethanechol- and cholecystokinin-evoked contractions, an effect associated with a reduction in MLC20 phosphorylation and in the effects of both Y-27632 and GF109203X. In addition, there was a drop in ROCK I, ROCK II, MYPT-1 and PKC expression and in the activation/phosphorylation of MYPT-1, PKC and CPI-17 in response to agonists. Interestingly, melatonin treatment for 4 weeks restored gallbladder contractile responses due to re-establishment of calcium sensitization pathways. These results demonstrate that age-related gallbladder hypocontractility is associated to alterations of calcium sensitization pathways and that melatonin treatment exerts beneficial effects in the recovery of gallbladder contractility.