RESUMEN
BACKGROUND: Infectious complications often occur in acute pancreatitis, related to impaired intestinal barrier function, with prolonged disease course and even mortality as a result. The bile salt nuclear receptor farnesoid X receptor (FXR), which is expressed in the ileum, liver and other organs including the pancreas, exhibits anti-inflammatory effects by inhibiting NF-κB activation and is implicated in maintaining intestinal barrier integrity and preventing bacterial overgrowth and translocation. Here we explore, with the aid of complementary animal and human experiments, the potential role of FXR in acute pancreatitis. METHODS: Experimental acute pancreatitis was induced using the CCK-analogue cerulein in wild-type and Fxr-/- mice. Severity of acute pancreatitis was assessed using histology and a semi-quantitative scoring system. Ileal permeability was analyzed in vitro by Ussing chambers and an in vivo permeability assay. Gene expression of Fxr and Fxr target genes was studied by quantitative RT-PCR. Serum FGF19 levels were determined by ELISA in acute pancreatitis patients and healthy volunteers. A genetic association study in 387 acute pancreatitis patients and 853 controls was performed using 9 tagging single nucleotide polymorphisms (SNPs) covering the complete FXR gene and two additional functional SNPs. RESULTS: In wild-type mice with acute pancreatitis, ileal transepithelial resistance was reduced and ileal mRNA expression of Fxr target genes Fgf15, SHP, and IBABP was decreased. Nevertheless, Fxr-/- mice did not exhibit a more severe acute pancreatitis than wild-type mice. In patients with acute pancreatitis, FGF19 levels were lower than in controls. However, there were no associations of FXR SNPs or haplotypes with susceptibility to acute pancreatitis, or its course, outcome or etiology. CONCLUSION: We found no evidence for a major role of FXR in acute human or murine pancreatitis. The observed altered Fxr activity during the course of disease may be a secondary phenomenon.
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Pancreatitis Aguda Necrotizante/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Animales , Estudios de Casos y Controles , Impedancia Eléctrica , Factores de Crecimiento de Fibroblastos/sangre , Técnicas de Inactivación de Genes , Humanos , Íleon/metabolismo , Mucosa Intestinal/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas/metabolismo , Páncreas/patología , Pancreatitis Aguda Necrotizante/genética , Polimorfismo de Nucleótido Simple , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Plasma B cells secrete immunoglobulinfree light chains (IgLC) which by binding to mast cells can mediate hypersensitivity responses and are involved in several immunological disorders. To investigate the effects of antigen-specific IgLC activation, intracellular recordings were made from cultured murine dorsal root ganglion (DRG) neurons, which can specifically bind IgLC. The neurons were sensitized with IgLC for 90min and subsequently activated by application of the corresponding antigen (DNP-HSA). Antigen application induced a decrease in the rate of rise of the action potentials of non-nociceptive neurons (MANOVA, p=2.10(-6)), without affecting the resting membrane potential or firing threshold. The action potentials of the nociceptive neurons (p=0.57) and the electrical excitability of both types of neurons (p>0.35) were not affected. We conclude that IgLC can mediate antigen-specific responses by reducing the rate of rise of action potentials in non-nociceptive murine DRG neurons. We suggest that antigen-specific activation of IgLC-sensitized non-nociceptive DRG neurons may contribute to immunological hypersensitivity responses and neuroinflammation.
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Potenciales de Acción/efectos de los fármacos , Ganglios Espinales/citología , Cadenas Ligeras de Inmunoglobulina/farmacología , Neuronas/efectos de los fármacos , Análisis de Varianza , Animales , Antígenos/metabolismo , Fenómenos Biofísicos/efectos de los fármacos , Biofisica , Células Cultivadas , Estimulación Eléctrica , Masculino , Ratones , Ratones Endogámicos BALB C , Neuronas/clasificación , Neuronas/fisiología , Factores de TiempoRESUMEN
The persistent organochlorine pesticide lindane is still abundantly found in the environment and in human and animal tissue samples. Lindane induces a wide range of adverse health effects, which are at least partially mediated via the known inhibition of GABA(A) and glycine receptors. Additionally, lindane has been reported to increase the basal intracellular Ca(2+) concentration ([Ca(2+)](i)). As Ca(2+) triggers many cellular processes, including cell death and vesicular neurotransmitter release (exocytosis), we investigated whether lindane affects exocytosis, Ca(2+) homeostasis, production of reactive oxygen species (ROS) and cytotoxicity in neuroendocrine PC12 cells. Amperometric recordings and [Ca(2+)](i) imaging experiments with fura-2 demonstrated that lindane (≥ 10 µM) rapidly increases basal exocytosis and basal [Ca(2+)](i). Additional imaging and electrophysiological recordings revealed that this increase was largely due to a lindane-induced membrane depolarization and subsequent opening of N- and P/Q-type voltage-gated Ca(2+) channels (VGCC). On the other hand, lindane (≥ 3 µM) induced a concentration-dependent but non-specific inhibition of VGCCs, thereby limiting the lindane-induced increase in basal [Ca(2+)](i) and exocytosis. Importantly, the non-specific inhibition of VGCCs also reduced stimulation-evoked exocytosis and Ca(2+) influx. Though lindane exposure concentration-dependently increased ROS production, cell viability was not affected indicating that the used concentrations were not acute cytotoxic. These combined findings indicate that lindane has two, partly counteracting effects. Lindane causes membrane depolarization, thereby increasing basal [Ca(2+)](i) and exocytosis. In parallel, lindane inhibits VGCCs, thereby limiting the basal effects and reducing stimulation-evoked [Ca(2+)](i) and exocytosis. This study further underlines the need to consider presynaptic, non-receptor-mediated effects in human risk assessment.
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Calcio/metabolismo , Exocitosis/efectos de los fármacos , Hexaclorociclohexano/toxicidad , Insecticidas/toxicidad , Animales , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo P/efectos de los fármacos , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/efectos de los fármacos , Canales de Calcio Tipo Q/metabolismo , Relación Dosis-Respuesta a Droga , Electrofisiología , Hexaclorociclohexano/administración & dosificación , Homeostasis/efectos de los fármacos , Insecticidas/administración & dosificación , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Alcohol consumption interferes with gastrointestinal transit causing symptoms in alcoholic patients. Nitric oxide (NO), synthesized by neuronal nitric oxide synthase (nNOS) plays an important role in the control of gastrointestinal motility. Our aim was to investigate whether chronic alcohol intake in a murine model induces gastrointestinal motility disturbances and affects the nitrergic myenteric neurons in the stomach and jejunum. Gastric emptying, small intestinal transit and geometric centre were measured in vivo after intragastric gavage of Evans blue. Nitrergic relaxations to electrical field stimulation (EFS) and exogenous NO were recorded in jejunal muscle strips in vitro. The proportion of nNOS-immunopositive myenteric neurons was assessed using PGP9.5 and nNOS immunostaining. After chronic alcohol consumption, gastric emptying and small intestinal transit were delayed compared with control mice, and the nitrergic nerve-mediated relaxations to EFS in the jejunum were decreased, whereas relaxations to exogenous NO did not differ. The proportion of nNOS-immunoreactive neurons did not change in the stomach, whereas in the jejunum the percentage decreased from 33% to 27% (P < 0.001) after chronic alcohol intake. The total number of myenteric neurons remained unchanged. These results suggest that chronic alcohol consumption disturbs gastric and small intestinal motility in vivo and in vitro and is associated with a decrease in the proportion of nNOS-immunoreactive myenteric neurons in the murine jejunum.
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Alcoholismo/fisiopatología , Motilidad Gastrointestinal , Yeyuno/inervación , Estómago/inervación , Animales , Modelos Animales de Enfermedad , Estimulación Eléctrica , Yeyuno/fisiopatología , Ratones , Neuronas Nitrérgicas/metabolismo , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Estómago/fisiopatologíaRESUMEN
INTRODUCTION: Cold hypoxia is a common factor in cold tissue preservation and mammalian hibernation. The purpose of this study was to determine the effects of cold preservation on the function of the retractor (RET) muscle of the hamster in the non-hibernating state and compare these with previously published data (van der Heijden et al., 2000) on the rat cutaneus trunci (CT) muscle. MATERIALS AND METHODS: After cold storage (16 h at 4 degrees C), muscles were stimulated electrically to measure maximum tetanus tension (P(0)) and histologically analyzed. The protective effects of addition of the antioxidants trolox and deferiprone and the calcium release inhibitor BDM to the storage fluid were determined. RESULTS: After storage, the twitch threshold current was increased (from 60 to 500 microA) and P(0) was decreased to 27% of control. RET morphology remained unaffected. RET muscle function was protected by trolox and deferiprone (P(0), resp., 43% and 59% of control). Addition of BDM had no effect on the RET. CONCLUSIONS: The observed effects of cold preservation and of trolox and deferiprone on the RET were comparable to those on CT muscle function, as reported in a previously published study (van der Heijden et al., 2000). Both hamster RET and rat CT muscles show considerable functional damage due to actions of reactive oxygen species. In contrast to the CT, in the RET cold preservation-induced functional injury could not be prevented by BDM and was not accompanied by morphological damage such as necrosis and edema. This suggests that the RET myocytes possess a specific adaptation to withstand the Ca(2+) overload induced by cold ischemia.
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Frío , Contracción Muscular , Músculo Esquelético/fisiología , Preservación de Órganos , Animales , Cromanos/farmacología , Cricetinae , Deferiprona , Masculino , Mesocricetus , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/anatomía & histología , Soluciones Preservantes de Órganos/farmacología , Piridonas/farmacología , RatasRESUMEN
Immunoglobulin-free light chains (IgLC) secreted by B lymphocytes, have been shown to mediate hypersensitivity by inducing antigen-specific mast cell activation. Although both mast cells and sensory neurons contribute to the hypersensitivity response, the role of IgLC in relation to sensory neurons is unknown. We therefore aimed to investigate the effects of IgLC on cultures of murine dorsal root ganglion (DRG) neurons. Immunohistochemistry demonstrated that IgLC and IgE could specifically bind to DRG neurons, on which the presence of FcepsilonRI, the specific receptor for IgE, was demonstrated by western blotting. Further, optical recordings with Fluo-4 showed that application of the corresponding antigen to IgLC- or IgE-sensitized DRG neurons induces a sustained increase in intracellular Ca(2+) in about half of these neurons. These results show that IgLC and IgE can mediate antigen-specific responses in murine neurons. Our findings present a novel way of antigen-specific neuronal activation.
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Epítopos/inmunología , Ganglios Espinales/citología , Ganglios Espinales/inmunología , Cadenas Ligeras de Inmunoglobulina/fisiología , Células Receptoras Sensoriales/inmunología , Células Receptoras Sensoriales/metabolismo , Animales , Células Cultivadas , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Hipersensibilidad/patología , Inmunoglobulina E/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Células Receptoras Sensoriales/patologíaRESUMEN
BACKGROUND: Intestinal barrier failure during acute pancreatitis (AP) is associated with translocation of luminal bacteria, resulting in infectious complications. We examined the effects of multispecies probiotics on the intestinal barrier impairment in a murine model of AP. METHODS: Mice were injected with cerulein to induce AP and were sacrificed 11 (early AP) or 72 hours (late AP) after start of induction. AP and associated systemic effects were confirmed by histology of pancreas and lung. Animals received daily probiotics starting 2 days prior to AP induction (pretreatment) or at the moment of AP induction (treatment). Mucosal barrier function of the distal ileum was assessed in Ussing chambers by measurement of the epithelial electrical resistance and the permeability to Na-fluorescein. RESULTS: Histological analysis revealed pancreatic injury in both phases of AP, and lung damage in the early phase. Epithelial resistance of the ileum was reduced and permeability increased in both phases of AP, indicating impairment of the intestinal barrier. Pretreatment had no effect on resistance or permeability in the early phase of AP. In the late phase of AP, pretreatment but not treatment abolished the AP induced resistance decrease and permeability increase. Administration of probiotics as such (ie, without induction of AP) had no effect on intestinal barrier function. CONCLUSION: Pretreatment with multispecies probiotics for 2 days abolishes intestinal barrier dysfunction in the late phase of AP, while treatment does not. The effectiveness of probiotics in this model depends on the timing of administration. Clinical trials with probiotics should seek conditions where treatment can be started prior to onset of disease or elective surgical intervention.
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Traslocación Bacteriana , Enfermedades del Íleon/prevención & control , Pancreatitis/complicaciones , Probióticos/administración & dosificación , Animales , Bifidobacterium , Ceruletida , Enfermedades del Íleon/etiología , Lactobacillus acidophilus , Lacticaseibacillus casei , Lactococcus lactis , Pulmón/patología , Masculino , Ratones , Páncreas/patología , Pancreatitis/patologíaRESUMEN
Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal live cell imaging of physiological reactions in identified NEBs and surrounding epithelial cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated cells, Clara cells, and Clara-like cells in live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB cells and ciliated cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intracellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extracellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB cells, while Clara-like cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.
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Colorantes Fluorescentes , Cuerpos Neuroepiteliales/fisiología , Mucosa Respiratoria/fisiología , Compuestos de Anilina , Animales , Animales Recién Nacidos , Bencimidazoles , Calcio/metabolismo , Carbocianinas , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Cationes Bivalentes/metabolismo , Cationes Monovalentes/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Cuerpos Neuroepiteliales/ultraestructura , Potasio/metabolismo , Compuestos de Piridinio , Mucosa Respiratoria/ultraestructura , XantenosRESUMEN
ALS (amyotrophic lateral sclerosis) is an adult-onset and deadly neurodegenerative disease characterized by a progressive and selective loss of motoneurons. Transgenic mice overexpressing a mutated human gene (G93A) coding for the enzyme SOD1 (Cu/Zn superoxide dismutase) develop a motoneuron disease resembling ALS in humans. In this generally accepted ALS model, we tested the electrophysiological properties of individual embryonic and neonatal spinal motoneurons in culture by measuring a wide range of electrical properties influencing motoneuron excitability during current clamp. There were no differences in the motoneuron resting potential, input conductance, action potential shape, or afterhyperpolarization between G93A and control motoneurons. The relationship between the motoneuron's firing frequency and injected current (f-I relation) was altered. The slope of the f-I relation and the maximal firing rate of the G93A motoneurons were much greater than in the control motoneurons. Differences in spontaneous synaptic input were excluded as a cause of increased excitability. This finding identifies a markedly elevated intrinsic electrical excitability in cultured embryonic and neonatal mutant G93A spinal motoneurons. We conclude that the observed intrinsic motoneuron hyperexcitability is induced by the SOD1 toxic gain-of-function through an aberration in the process of action potential generation. This hyperexcitability may play a crucial role in the pathogenesis of ALS as the motoneurons were cultured from presymptomatic mice.
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Esclerosis Amiotrófica Lateral/patología , Neuronas Motoras/fisiología , Médula Espinal/patología , Potenciales de Acción/fisiología , Alanina/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Impedancia Eléctrica , Estimulación Eléctrica , Embrión de Mamíferos , Glicina/genética , Técnicas In Vitro , Modelos Lineales , Ratones , Ratones Transgénicos , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.
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Colon/inervación , Íleon/inervación , Interleucina-1/farmacología , Neuroglía/fisiología , Neuronas/fisiología , Animales , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Sistema Nervioso Entérico/metabolismo , Inducción Enzimática , Cobayas , Inmunohistoquímica , Indometacina/farmacología , Isoenzimas/metabolismo , Masculino , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Several studies have suggested an age-related reduction in the number of myenteric neurons in the lower gastrointestinal (GI) tract linked to changes in GI neuromuscular functions. The present study, combining protein gene product 9.5 immunostaining and NADPH-diaphorase histochemistry, aimed at quantifying the proportion of nitrergic neurons compared to the overall number of enteric neurons in the esophagus of young (3-4.5 months) and aged (18-20 months) Sprague-Dawley and Wistar rats. In both strains, the neuron numbers per ganglion in the cervical region were almost twice as high as in the other esophageal regions. Irrespective of age or strain, the esophagus harbored a very high proportion of intrinsic nitrergic neurons (greater than approximately 65%). Both strains showed with aging an overall neuronal loss of approximately 27%. While a significant increase (young: 64-71%; aged: 82-89%) was observed in all esophageal regions in the Wistar strain, the proportion of nitrergic neurons remained stable with aging in the Sprague-Dawley strain (range: 72-82%). In conclusion, the age-related reduction in the overall number of myenteric, nitrergic, and non-nitrergic neurons observed in the rat esophagus, appears to be highly region- and strain-dependent. Therefore, a protective mechanism against neuronal cell loss, selectively present in specific (nitrergic) enteric subpopulations, as suggested in earlier reports, cannot be put forward as a general phenomenon throughout the entire GI tract.
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Envejecimiento , Esófago/inervación , Plexo Mientérico/citología , Neuronas Nitrérgicas/citología , Animales , Recuento de Células , Femenino , Masculino , Plexo Mientérico/enzimología , NADPH Deshidrogenasa/metabolismo , Neuronas Nitrérgicas/enzimología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Especificidad de la Especie , Ubiquitina Tiolesterasa/metabolismoRESUMEN
The aim of this study was to investigate if the preservation of isolated skeletal muscles for 16 h at 4 degrees C could be improved by pre-storage perfusion (PSP). Two rat muscle models were used: the soleus (SOL) and a posterior strip of the cutaneous trunci (CT). The effects of a 10 min PSP (at 25 degrees C) with University of Wisconsin solution (UW) or HTK-Bretschneider solution (HTK) on muscle function were analysed. The perfusion model was validated by the demonstration that the SOL and CT could be perfused with donor blood, UW and HTK at a flow rate of 0.2 ml x min(-1) x g(-1) muscle for 10 min without any immediate adverse effects on muscle weight, function (maximum tetanus tension) and cytoarchitecture (multivariate analysis of variance, P >0.05; n =6). For each muscle type and for each solution, six perfused and six non-perfused muscles were stored for 16 h at 4 degrees C. In the perfused groups, the storage and perfusion solution were matched. For both muscle types, the function (maximum tetanus tension), weight and cytoarchitecture of pre-storage perfused muscles was not preserved any better than that of non-perfused muscles, irrespective of the solution used (multivariate analysis of variance, P >0.05). We conclude that PSP for 10 min with UW and HTK does not improve the preservation of function of rat skeletal muscles during storage for 16 h at 4 degrees C.