RESUMEN
RNA therapies have recently taken a giant leap forward with the approval of Onpattro™, a siRNA therapy delivered using a lipid nanoparticle (LNP), and the LNP-enabled mRNA vaccines against COVID-19, which are the first mRNA drugs to reach the marketplace. The latter medicines have illustrated that stability is a significant challenge in the distribution of RNA drugs using non-viral delivery systems, particularly in areas without cold chain storage. Here, we describe a proof-of-concept study on the engineering of an LNP mRNA formulation suitable for spray drying. This process produced a dry powder formulation that maintained stability and preserved mRNA functionality with increased performance compared to liquid formulations stored two weeks at 4 °C. Intratracheal delivery of spray dried LNPs loaded with eGFP mRNA to rats resulted in the production of the eGFP protein in a range of cell types including bronchiolar epithelial cells, macrophages and type II pneumocytes; cell types involved in adaptive immunity and which would be valuable targets for inhaled vaccines against respiratory pathogens. Together, these data show that spray drying of LNPs enhances their stability and may enable RNA delivery to the lung for protein replacement therapy, gene editing, vaccination, and beyond.
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Vacunas contra la COVID-19 , Nanopartículas , Ratas , Animales , Humanos , ARN Mensajero , LiposomasRESUMEN
BACKGROUND: It is not known whether stromal cells in benign breast tissue can mediate risk of breast cancer. We recently described aldehyde dehydrogenase 1 A1 (ALDH1) positive (+) cells in morphologically normal breast stroma of premenopausal women, and the data indicated that their distribution is associated with clinical risk factors for breast cancer. The aim of the present study was to define the identities of these cells using histologic and immunohistologic methods, and to investigate associations between those cells and hormonal and genetic risk factors in pre- and postmenopausal women. METHODS: Stroma of morphologically normal tissue was analyzed in samples from 101 well-characterized women whose breasts had been operated. Morphology and immunolabeling were applied to determine cell identities based on the putative stem cell markers ALDH1 and stage-specific embryonic antigen-3 (SSEA3), and immunophenotypes indicating mast cells or stellate cells. The results were compared with the patients' risk factors using regression analysis (two-tailed). RESULTS: ALDH1+ round/oval cells were associated with low parity in BRCA1/2 carriers (p = 0.022), while in non-BRCA1/2-carriers they were negatively associated with nulliparity (p = 0.057). In premenopausal women ALDH1+ round/oval cells were associated with family history (p = 0.058). SSEA3+ round/oval cells were morphologically and immunohistologically consistent with multilineage stress-enduring (Muse) cells, and these cells were independently associated with the breast cancer risk factors low parity (p = 0.015), family history (p = 0.021), and hormone use after menopause (p = 0.032). ALDH1+ spindle-shaped/polygonal cells were immunohistologically consistent with stellate cells, and were negatively associated with family history of breast cancer (p = 0.001). CONCLUSION: This study identified novel stromal cell types in benign breast tissue that have a potential for stratifying women for breast cancer risk.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Isoenzimas/análisis , Células Madre Mesenquimatosas/patología , Retinal-Deshidrogenasa/análisis , Adulto , Anciano , Anciano de 80 o más Años , Familia de Aldehído Deshidrogenasa 1 , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Células Madre Mesenquimatosas/enzimología , Persona de Mediana Edad , Mutación , Factores de Riesgo , Adulto JovenRESUMEN
BACKGROUND: The gut microbiota plays an important role in the development of obesity and obesity-associated impairments such as low-grade inflammation. Lingonberries have been shown to prevent diet-induced obesity and low-grade inflammation. However, it is not known whether the effect of lingonberry supplementation is related to modifications of the gut microbiota. The aim of the present study was to describe whether consumption of different batches of lingonberries alters the composition of the gut microbiota, which could be relevant for the protective effect against high fat (HF)-induced metabolic alterations. METHODS: Three groups of C57BL/6J mice were fed HF diet with or without a supplement of 20% lingonberries from two different batches (Lingon1 and Lingon2) during 11 weeks. The composition and functionality of the cecal microbiota were assessed by 16S rRNA sequencing and PICRUSt. In addition, parameters related to obesity, insulin sensitivity, hepatic steatosis, inflammation and gut barrier function were examined. RESULTS: HF-induced obesity was only prevented by the Lingon1 diet, whereas both batches of lingonberries reduced plasma levels of markers of inflammation and endotoxemia (SAA and LBP) as well as modified the composition and functionality of the gut microbiota, compared to the HF control group. The relative abundance of Akkermansia and Faecalibacterium, genera associated with healthy gut mucosa and anti-inflammation, was found to increase in response to lingonberry intake. CONCLUSIONS: Our results show that supplementation with lingonberries to an HF diet prevents low-grade inflammation and is associated with significant changes of the microbiota composition. Notably, the anti-inflammatory properties of lingonberries seem to be independent of effects on body weight gain.
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A few patients have been shown to develop severe abdominal pain and gastrointestinal dysmotility during treatment with gonadotropinreleasing hormone (GnRH) analogs. A rat model of enteric neuropathy has been developed by administration of the GnRH analog buserelin to rats. Loss of enteric neurons and ganglioneuritis throughout the gastrointestinal tract has been described, without other histopathological changes. The aim of the present study was to investigate the longterm effects of this rat model on body weight, and on morphology and inflammatory changes in the gastrointestinal tract. Rats were administered subcutaneous injections of buserelin or saline once daily for 5 days and allowed to recover for 3 weeks. This regimen was repeated four times. The rats were weighed weekly and were sacrificed 16 weeks after the fourth treatment. The bowel wall was measured by morphometry, and the presence of enteric neurons, mast cells, eosinophils and Tlymphocytes was evaluated. Buserelintreated rats were shown to have a lower body weight at sacrifice, as compared with the controls (P<0.05). Compared with controls, buserelin treatment caused loss of myenteric neurons in the ileum and colon (P<0.01), a thinner circular muscle layer in ileum (P<0.05) and longitudinal muscle layer in colon (P<0.05), increased number of eosinophils in the submucosa of the ileum (P<0.05), and an increased number of Tlymphocytes in the submucosa and circular muscle layer of the fundus (P<0.01 and P<0.05, respectively) and circular muscle layer of the colon (P<0.05). Mast cells were equally distributed in the two groups. Thus, longterm followup of buserelininduced enteric neuropathy reveals reduced body weight, loss of myenteric neurons, thinning of muscle layers, and increased numbers of eosinophils and Tlymphocytes in the gastrointestinal tract.
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Buserelina/toxicidad , Seudoobstrucción Intestinal/etiología , Animales , Colon/patología , Modelos Animales de Enfermedad , Eosinófilos/citología , Eosinófilos/patología , Femenino , Estudios de Seguimiento , Íleon/patología , Inyecciones Subcutáneas , Seudoobstrucción Intestinal/metabolismo , Seudoobstrucción Intestinal/patología , Mastocitos/citología , Mastocitos/patología , Neuronas/citología , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Estómago/patología , Linfocitos T/citología , Linfocitos T/patología , Factores de TiempoRESUMEN
BACKGROUND: The gonadotropin-releasing hormone (GnRH) analog buserelin causes enteric neuronal loss. Acute stress or injection of corticotropin-releasing factor (CRF) affects motility, secretion, and barrier function of the gastrointestinal tract. The aim of the study was to characterize the CRF immunoreactivity in enteric neurons after buserelin treatment, and to evaluate possible effects of enteric neuropathy on gut microbiota, intestinal permeability, and stress response behavior. RESULTS: Sixty rats were given buserelin (20 µg) or saline subcutaneously for 5 days, repeated four times with 3 weeks in-between. At the study end, enteric neuronal density, enteric expression of CRF, gut microbial composition, and plasma levels of adrenocorticotropic hormone (ACTH) and CRF were analyzed. Intestinal permeability was examined in Ussing chambers and the reaction to stressful events was measured by behavior tests. Buserelin treatment reduced the number of neurons along the entire gastrointestinal tract, with increased relative numbers of CRF-immunoreactive submucosal and myenteric neurons in colon (p < 0.05 and p < 0.01, respectively). The overall microbial diversity and relative abundance did not differ between groups, but Enterobacteriaceae was decreased in colon in buserelin-treated rats (p = 0.020). Basal intestinal permeability did not differ between groups, whereas carbachol stimulation increased ileum permeability in controls (p < 0.05), but not in buserelin-treated rats. Buserelin did not affect stress behavior. CONCLUSIONS: Although buserelin treatment leads to enteric neuronal loss along the gastrointestinal tract with an increased percentage of CRF-immunoreactive neurons in colon, the physiology is well preserved, with modest effects on colon microbiota and absence of carbachol-induced permeability in ileum as the only observed changes.
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Acetilcolina/metabolismo , Buserelina/efectos adversos , Hormona Liberadora de Corticotropina/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Hormona Liberadora de Gonadotropina/análogos & derivados , Enfermedades Intestinales/inducido químicamente , Enfermedades del Sistema Nervioso/inducido químicamente , Animales , Conducta Animal/efectos de los fármacos , Colon/efectos de los fármacos , Enterobacteriaceae/efectos de los fármacos , Femenino , Hormona Liberadora de Gonadotropina/efectos adversos , Íleon/efectos de los fármacos , Neuronas/efectos de los fármacos , Permeabilidad , RatasRESUMEN
Weight loss is an important complication of Huntington's disease (HD), however the mechanism for weight loss in HD is not entirely understood. Mutant huntingtin is expressed in the gastrointestinal (GI) tract and, in HD mice, mutant huntingtin inclusions are found within the enteric nervous system along the GI tract. A reduction of neuropeptides, decreased mucosal thickness and villus length, as well as gut motility impairment, have also been shown in HD mice. We therefore set out to study gastric mucosa of patients with HD, looking for abnormalities of mucosal cells using immunohistochemistry. In order to investigate possible histological differences related to gastric acid production, we evaluated the cell density of acid producing parietal cells, as well as gastrin producing cells (the endocrine cell controlling parietal cell function). In addition, we looked at chief cells and somatostatin-containing cells. In gastric mucosa from HD subjects, compared to control subject biopsies, a reduced expression of gastrin (a marker of G cells) was found. This is in line with previous HD mouse studies showing reduction of GI tract neuropeptides.
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BACKGROUND: Clinical observations have suggested repeated gonadotropin-releasing hormone (GnRH) exposure to cause intestinal dysfunction and loss of enteric neurons. This has been further studied and confirmed in a rat in vivo model involving iterated GnRH treatments. Mechanisms behind are enigmatic since no GnRH receptors are found to be expressed in enteric neurons neither in man nor rat. Both species, however, harbor substantial subpopulations of luteinizing hormone (LH) receptor-immunoreactive myenteric neurons which suggests that intestinal GnRH-induced neuropathy may be mediated by LH release. AIMS: To reveal if exposures of GnRH or LH to rat myenteric neurons in vitro cause neuronal loss. METHODS: Primary cultured adult rat myenteric neurons were exposed to single or repeated treatments of the GnRH analog buserelin or the LH analog lutrotropin alpha, and neuronal survival was determined by cell counting. Possible presence of GnRH- or LH receptor -immunoreactive neurons was determined by immunocytochemistry. RESULTS: Exposure to the LH, but not the GnRH, analog caused significantly reduced neuronal survival. LH, but not GnRH, receptors were found to be expressed on cultured myenteric neurons. CONCLUSION: Myenteric neurons express LH receptors in vitro and LH exposure causes reduced neuronal survival. This suggests that GnRH-induced enteric neuropathy in vivo is mediated by way of LH release and activation of enteric neuronal LH receptors.
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Plexo Mientérico/fisiología , Neuronas/fisiología , Receptores de HL/metabolismo , Animales , Buserelina/farmacología , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormonas/farmacología , Íleon/inervación , Inmunohistoquímica , Hormona Luteinizante/análogos & derivados , Masculino , Plexo Mientérico/efectos de los fármacos , Neuronas/efectos de los fármacos , Ratas Sprague-Dawley , Receptores LHRH/metabolismoRESUMEN
BACKGROUND: Sporadic cases of abdominal pain and dysmotility has been described after treatment with gonadotropin-releasing hormone (GnRH) analogs. The aim of the present study was to scrutinize for patients with severe gastrointestinal complaints after treatment with GnRH analogs, to describe the expression of antibodies against progonadoliberin-2, GnRH1, GnRH receptor (GnRHR), luteinizing hormone (LH), and LH receptor in serum in these patients, and to search for possible triggers and genetic factors behind the development of this dysmotility. METHODS: Patients suffering from prolonged gastrointestinal complaints after treatment with GnRH analogs at the Department of Gastroenterology, Skåne University Hospital, were included. GnRHR and LH receptor (LHCGR) genes were exome-sequenced. Serum was analyzed by enzyme-linked immune sorbent assays for the presence of antibodies. Healthy blood donors and women treated with GnRH analogs because of in vitro fertilization (IVF) were used as controls. RESULTS: Seven patients with severe gastrointestinal complaints after GnRH treatment were identified, of whom six suffered from endometriosis. Several variants were found within the 11 exons of LHCGR. The minor allele G, at the single nucleotide polymorphism rs6755901, was detected in homozygosity in two patients (28.5%) who had developed chronic intestinal pseudo-obstruction and in 5.5% of the IVF controls. Three patients expressed IgM antibodies against progonadoliberin-2 and three against GnRH1 (42.9%) when cut off was set to a titer >97.5th percentile in blood donors. CONCLUSION: A high prevalence of endometriosis, polymorphism in the LHCGR and GnRH1 and progonadoliberin-2 antibodies in serum was found among the patients with severe dysmotility after treatment with GnRH analogs.
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Enfermedades Gastrointestinales/inducido químicamente , Motilidad Gastrointestinal/efectos de los fármacos , Hormona Liberadora de Gonadotropina/efectos adversos , Seudoobstrucción Intestinal/diagnóstico , Dolor Abdominal , Adulto , Anticuerpos/sangre , Estudios de Casos y Controles , Endometriosis/tratamiento farmacológico , Femenino , Fertilización In Vitro , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/inmunología , Humanos , Inmunoglobulina M/sangre , Persona de Mediana Edad , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Receptores de HL/genética , Receptores de HL/inmunología , Receptores LHRH/genética , Receptores LHRH/inmunología , Adulto JovenRESUMEN
BACKGROUND: Women treated with gonadotropin-releasing hormone (GnRH) analogs may develop enteric neuropathy and dysmotility. Administration of a GnRH analog to rats leads to similar degenerative neuropathy and ganglioneuritis. The aim of this study on rat was to evaluate the early GnRH-induced enteric neuropathy in terms of distribution of neuronal subpopulations and gastrointestinal (GI) function. METHODS: Forty rats were given the GnRH analog buserelin (20 µg, 1 mg/ml) or saline subcutaneously, once daily for 5 days, followed by 3 weeks of recovery, representing one treatment session. Two weeks after the fourth treatment session, the animals were tested for GI transit time and galactose absorption, and fecal weight and fat content was analyzed. After sacrifice, enteric neuronal subpopulations were analyzed. Blood samples were analyzed for zonulin and antibodies against GnRH and luteinizing hormone, and their receptors. RESULTS: Buserelin treatment transiently increased the body weight after 5 and 9 weeks (p < 0.001). Increased estradiol in plasma and thickened uterine muscle layers indicate high estrogen activity. The numbers of both submucous and myenteric neurons were reduced by 27%-61% in ileum and colon. The relative numbers of neurons containing calcitonin gene-related peptide (CGRP), cocaine- and amphetamine-related transcript (CART), galanin, gastrin-releasing peptide (GRP), neuropeptide Y (NPY), nitric oxide synthase (NOS), serotonin, substance P (SP), vasoactive intestinal peptide (VIP) or vesicular acetylcholine transporter (VAchT), and their nerve fiber density, were unchanged after buserelin treatment, but the relative number of submucous neurons containing somatostatin tended to be increased (p = 0.062). The feces weight decreased in buserelin-treated rats (p < 0.01), whereas feces fat content increased (p < 0.05), compared to control rats. Total GI transit time, galactose absorption, zonulin levels in plasma, and antibody titers in serum were unaffected by buserelin treatment. CONCLUSIONS: A marked enteric neuronal loss with modest effects on GI function is found after buserelin treatment. Increased feces fat content is suggested an early sign of dysfunction.
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Tracto Gastrointestinal/fisiopatología , Seudoobstrucción Intestinal/patología , Seudoobstrucción Intestinal/fisiopatología , Neuronas/patología , Animales , Buserelina , Colon/patología , Modelos Animales de Enfermedad , Estradiol/sangre , Heces/química , Femenino , Tránsito Gastrointestinal , Íleon/patología , Seudoobstrucción Intestinal/inducido químicamente , Lípidos/análisis , Neuronas/química , Ratas Sprague-Dawley , Estómago/patología , Útero/anatomía & histologíaAsunto(s)
Buserelina/efectos adversos , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/patología , Neuritis/tratamiento farmacológico , Animales , Buserelina/administración & dosificación , Buserelina/uso terapéutico , Femenino , Neuritis/patología , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Obese and/or diabetic patients have elevated levels of free fatty acids and increased susceptibility to gastrointestinal symptoms. Since the enteric nervous system is pivotal in regulating gastrointestinal functions alterations or neuropathy in the enteric neurons are suspected to occur in these conditions. Lipid induced intestinal changes, in particular on enteric neurons, were investigated in vitro and in vivo using primary cell culture and a high fat diet (HFD) mouse model. DESIGN: Mice were fed normal or HFD for 6 months. Intestines were analyzed for neuronal numbers, remodeling and lipid accumulation. Co-cultures of myenteric neurons, glia and muscle cells from rat small intestine, were treated with palmitic acid (PA) (0 - 10(-3) M) and / or oleic acid (OA) (0 - 10(-3) M), with or without modulators of intracellular lipid metabolism. Analyses were by immunocyto- and histochemistry. RESULTS: HFD caused substantial loss of myenteric neurons, leaving submucous neurons unaffected, and intramuscular lipid accumulation in ileum and colon. PA exposure in vitro resulted in neuronal shrinkage, chromatin condensation and a significant and concentration-dependent decrease in neuronal survival; OA exposure was neuroprotective. Carnitine palmitoyltransferase 1 inhibition, L-carnitine- or alpha lipoic acid supplementation all counteracted PA-induced neuronal loss. PA or OA alone both caused a significant and concentration-dependent loss of muscle cells in vitro. Simultaneous exposure of PA and OA promoted survival of muscle cells and increased intramuscular lipid droplet accumulation. PA exposure transformed glia from a stellate to a rounded phenotype but had no effect on their survival. CONCLUSIONS: HFD and PA exposure are detrimental to myenteric neurons. Present results indicate excessive palmitoylcarnitine formation and exhausted L-carnitine stores leading to energy depletion, attenuated acetylcholine synthesis and oxidative stress to be main mechanisms behind PA-induced neuronal loss.High PA exposure is suggested to be a factor in causing diabetic neuropathy and gastrointestinal dysregulation.
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Dieta Alta en Grasa/efectos adversos , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/patología , Ácido Palmítico/efectos adversos , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ceramidas/biosíntesis , Técnicas de Cocultivo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Intestino Delgado/citología , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Miocitos del Músculo Liso/citología , Neuroglía/citología , Neurotoxinas/efectos adversos , Ácido Oléico/efectos adversos , Palmitoilcarnitina/biosíntesis , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) regulate the reproductive axis. Their analogs have been found to influence gastrointestinal activity and enteric neuronal survival. The aims of the study were to investigate expression and cellular distribution of GnRH, LH, and FSH and their receptors in human and rat gastrointestinal tract. METHODS: Bioinformatic analysis of publicly available microarray gene expression data and Real-Time PCR mRNA quantification were used to study mRNA expression levels of hormones and receptors in human intestinal tissue. Full-thickness sections of human ileum and colon, and rat stomach, ileum, and colon, were used for immunocytochemistry. Antibodies against human neuronal protein HuC/D (HuC/D) were used as general neuronal marker. LH and FSH, and GnRH-, LH-, and FSH receptor immunoreactive (IR) neurons were evaluated. RESULTS: GnRH1 mRNA was detected in both small and large intestine, whereas GnRH2 was mainly expressed in small intestine. Approximately 20% of both submucous and myenteric neurons displayed LH receptor immunoreactivity in human ileum and colon. In rat, 4%-9% of all enteric neurons in fundus and ileum, and 13% of submucous neurons and 21% of myenteric neurons in colon were LH receptor-IR. Neither mRNA (man) nor the fully expressed proteins (man and rat) of LH and FSH, or GnRH and FSH receptors, could be detected. CONCLUSIONS: GnRH1 and GnRH2 mRNA are expressed in human intestine. LH receptor-IR enteric neurons are found along the entire gastrointestinal tract in both man and rat.
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Hormona Folículo Estimulante/metabolismo , Tracto Gastrointestinal/metabolismo , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Luteinizante/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Hormona Folículo Estimulante/genética , Expresión Génica , Perfilación de la Expresión Génica , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Inmunohistoquímica , Hormona Luteinizante/genética , Masculino , Especificidad de Órganos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de HFE/genética , Receptores de HFE/metabolismo , Receptores de HL/genética , Receptores de HL/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismo , Adulto JovenRESUMEN
Gonadotropin-releasing hormone (GnRH) analogs are given to women undergoing in vitro fertilization. Case reports describing the development of chronic intestinal pseudo-obstruction and auto-antibodies against GnRH after such treatment suggest a strong association between intestinal dysfunction and GnRH analogs. No experimental model for studying such a relationship is currently at hand. Our main goal was to investigate possible enteric neurodegeneration and titers of GnRH antibodies in response to repeated administration of the GnRH analog buserelin in rat. Rats were treated for 1-4 sessions with daily subcutaneous injections of buserelin or saline for 5 days, followed by 3 weeks of recovery. Buserelin treatment caused significant loss of submucous and myenteric neurons in the fundus, ileum, and colon. The loss of enteric neurons can, at least partly, be explained by increased apoptosis. No GnRH- or GnRH-receptor-immunoreactive (IR) enteric neurons but numerous luteinizing hormone (LH)-receptor-IR neurons were detected. After buserelin treatment, the relative number of enteric LH-receptor-IR neurons decreased, whereas that of nitric-oxide-synthase-IR neurons increased. No intestinal inflammation or increased levels of circulating interleukins/cytokines were noted in response to buserelin treatment. Serum GnRH antibody titers were undetectable or extremely low in all rats. Thus, repeated administrations of buserelin induce neurodegeneration in rat gastrointestinal tract, possibly by way of LH-receptor hyperactivation. The present findings suggest that enteric neurodegenerative effects of GnRH analog treatment in man can be mimicked in rat. However, in contrast to man, no production of GnRH auto-antibodies has been noted in rat.
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Buserelina/farmacología , Tracto Gastrointestinal/citología , Hormona Liberadora de Gonadotropina/análogos & derivados , Neuronas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Colon/citología , Femenino , Inmunohistoquímica , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de HL/metabolismo , Receptores LHRH/metabolismo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
BACKGROUND: Neuropathy is believed to be a common feature of functional and inflammatory intestinal diseases. Vasoactive intestinal peptide (VIP) is an acknowledged neuroprotective agent in peripheral, including enteric, and central neurons. The proglucagon-like hormones glucagon-like peptide 1 and 2 (GLP1 and GLP2) belong to the secretin/glucagon/VIP superfamily of peptides and GLP1 and GLP2 receptors are expressed in enteric neurons. Possible neuroprotective effects of these peptides were investigated in the present study. METHODS: GLP1, GLP2 and VIP were added to cultured myenteric neurons from rat small intestine or to co-cultures of myenteric neurons and rat peritoneal mast cells. Receptor selectivity was tested by the simultaneous presence of a GLP1 receptor antagonist (exendin (9-39) amide) or a VIP receptor antagonist (hybrid of neurotensin 6-11 and VIP 7-28). Neuronal survival was examined using immunocytochemistry and cell counting. RESULTS: GLP1, GLP2 and VIP significantly and concentration-dependently enhanced neuronal survival. In addition the peptides efficiently counteracted mast cell-induced neuronal cell death in a concentration-dependent manner. Exendin(9-39)amide reversed GLP1-induced neuroprotection while GLP2- and VIP-induced enhanced neuronal survival were unaffected. The VIP receptor antagonist reversed GLP1- and VIP-induced neuroprotection while the GLP2-induced effect on neuronal survival was unaffected. CONCLUSIONS: By activating separate receptors VIP, GLP1 and GLP2 elicit neuroprotective effects on rat myenteric neurons cultured with or without mast cells. This implies a powerful therapeutic potential of these peptides in enteric neuropathies with a broad spectrum of applications from autoimmunity to functional disorders.
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Supervivencia Celular/efectos de los fármacos , Péptido 1 Similar al Glucagón/farmacología , Péptido 2 Similar al Glucagón/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Péptido Intestinal Vasoactivo/farmacología , Análisis de Varianza , Animales , Células Cultivadas , Femenino , Intestino Delgado/inervación , Mastocitos , Plexo Mientérico , Neurotensina/farmacología , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
UNLABELLED: Aims of the present study were to describe the distribution of corticotropin releasing factor (CRF) immunoreactivity in rat small and large intestines, to quantify the percentage of CRF-immunoreactive (CRF-IR) enteric neurons, to reveal possible CRF immunoreactivity in cultured myenteric neurons from rat ileum and to examine if additions of CRF, urocortin 1 (Ucn1), CRF antagonist or vasoactive intestinal peptide (VIP) affect neuronal survival in vitro. Co-localization of CRF- and VIP-immunoreactivity was examined, as well as a possible interplay between CRF and VIP in neuroprotection. Further we wanted to elucidate if mast cells affect neuronal survival via CRF signaling. Networks of CRF-containing nerve cell bodies and fibers were detected in rat intestine. CRF-IR neurons contained to a high degree also VIP. A low number of cultured myenteric neurons was CRF-IR. CRF, Ucn1 or CRF-antagonist did not promote neuronal survival of cultured myenteric neurons, while VIP significantly enhanced neuronal survival. Simultaneous presence of CRF attenuated the VIP mediated increase in neuronal survival. Co-culturing neurons and mast cells resulted in a marked reduction in neuronal survival, not executed via CRF signaling pathways. CONCLUSION: CRF is present in enteric neurons and counteracts the neuroprotective effect of VIP in vitro.
Asunto(s)
Hormona Liberadora de Corticotropina/metabolismo , Mucosa Intestinal/metabolismo , Neuronas/metabolismo , Animales , Apoptosis , Técnicas de Cocultivo , Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Hormona Liberadora de Corticotropina/farmacología , Femenino , Íleon/citología , Íleon/inervación , Intestinos/inervación , Mastocitos/fisiología , Plexo Mientérico/efectos de los fármacos , Plexo Mientérico/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/antagonistas & inhibidores , Fármacos Neuroprotectores/metabolismo , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Urocortinas/farmacología , Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Mast cell-nerve interactions play a key role in intestinal inflammation and irritable bowel disease. Loss of enteric neurons has been reported in inflammatory conditions but the contribution of mast cells in this event is unknown. To study neuronal survival and plasticity of myenteric neurons in contact with mast cells a co-culture system using myenteric neurons from rat small intestine and peritoneal mast cells was set up. Dissociated myenteric neurons were cultured for 4 days before addition of mast cells isolated by peritoneal lavage. Neuronal survival and expression of vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) were studied by immunocytochemistry and neuronal cell counting. Myenteric neurons cultured without mast cells were used to study the rate of neuronal survival after the addition of various mast cell mediators, proteinase-activated receptor(2) (PAR(2)) agonist, VIP or corticosteroid. A striking mast cell-induced neuronal cell death was found after co-culturing. It was counteracted by the addition of mast cell stabiliser doxantrazole, protease inhibitors, PAR(2) antagonist FSLLRY-amide, corticosteroid or VIP. In myenteric neurons cultured without mast cells the PAR(2) agonist SLIGRL-amide, prostaglandin D(2) and interleukin (IL) 6 reduced neuronal survival while histamine, serotonin, heparin, IL1beta and tumour necrosis factor alpha had no effect; corticosteroid and VIP enhanced neuronal survival. The relative numbers of VIP-, but not NOS-expressing myenteric neurons increased after co-culturing. Mast cell-induced neuronal cell death is suggested to be mediated via PAR(2) activation, IL6 and prostaglandin D(2). Corticosteroid and VIP are neuroprotective and able to prevent cell death of myenteric neurons in co-culture.
Asunto(s)
Mastocitos/fisiología , Plexo Mientérico/citología , Neuronas/fisiología , Animales , Benzamidinas , Recuento de Células/métodos , Supervivencia Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo/métodos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Femenino , Guanidinas/farmacología , Histamina/metabolismo , Histamina/farmacología , Mastocitos/efectos de los fármacos , Neuronas/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Prolina/análogos & derivados , Prolina/metabolismo , Ratas , Ratas Sprague-Dawley , Inhibidores de Serina Proteinasa/farmacología , Serotonina/farmacología , Factores de Tiempo , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Intestinal ischemia as well as mastocytosis occur in patients with inflammatory bowel disease and irritable bowel syndrome. Our aim was to clarify how ischemia with reperfusion (I/R) affects the structure, enteric neurons, and immune cells in the colon. Rats were subjected to colon ischemia for 1 h and reperfused for 1 day up to 20 weeks; sham-operated rats were used as controls. No structural remodeling of the intestinal segment was detected after I/R. The number and distribution of eosinophils were not affected by I/R. Local areas containing numerous mast cells were detected in the muscle layers, the serosa, and in and around the myenteric ganglia 4-20 weeks post ischemia. It was notable that myenteric ganglionic formations within mast-cell-rich areas virtually lacked neurons. Mast cells were rarely found in controls. In conclusion, I/R of the colon attracts mast cells, and death of myenteric neurons occurs in such locations.
Asunto(s)
Movimiento Celular/fisiología , Colon/patología , Mastocitos/patología , Daño por Reperfusión/patología , Animales , Muerte Celular/fisiología , Colon/inervación , Colon/metabolismo , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/patología , Eosinófilos/patología , Femenino , Tracto Gastrointestinal/patología , Músculo Liso/patología , Neuronas/patología , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Factores de Tiempo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
The pharmacology of tachykinin NK receptors varies greatly among species. The aim of the present study was to assess the role of NK(1) and NK(2) receptors in mediating colorectal distension-evoked nociception and psychological stress-induced defecation in gerbils, a species with human-like NK receptor pharmacology. The effects of the selective NK(1) and NK(2) receptor antagonists, aprepitant and saredutant, on acute (1 h) restraint stress-evoked defecation and plasma adenocorticotropin (ACTH) levels in gerbils were assessed. The effects of antagonists alone or in combination on colorectal distension-evoked visceral pain in conscious gerbils were evaluated using the visceromotor response as a surrogate marker of pain. Restraint stress increased fecal pellet output 2-3-fold and plasma ACTH levels 9-fold. Aprepitant inhibited the defecatory and endocrine responses to stress by 50%, while saredutant completely normalized the same parameters. Visceral pain responses during colorectal distension were attenuated by both compounds, but aprepitant (19+/-6% inhibition, P<0.01) was slightly more effective than saredutant (10+/-9% inhibition, P<0.05). A combination of both compounds resulted in an additive effect (30+/-10% inhibition, P<0.01). The results demonstrate that NK(1) and NK(2) receptors are involved in stress-related colonic motor alterations and visceral pain responses in gerbils and that combined antagonism provides enhanced inhibition of visceral pain responses. This suggests that for therapeutic use in for instance functional gastrointestinal disorders, dual NK(1)/NK(2) receptor antagonists may provide better clinical outcome than selective compounds.
Asunto(s)
Colon/fisiopatología , Defecación/efectos de los fármacos , Receptores de Neuroquinina-1/fisiología , Receptores de Neuroquinina-2/fisiología , Estrés Psicológico/fisiopatología , Hormona Adrenocorticotrópica/sangre , Animales , Aprepitant , Benzamidas/farmacología , Colon/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Motilidad Gastrointestinal/efectos de los fármacos , Gerbillinae , Masculino , Morfolinas/farmacología , Antagonistas del Receptor de Neuroquinina-1 , Dolor/fisiopatología , Piperidinas/farmacología , Receptores de Neuroquinina-2/antagonistas & inhibidoresRESUMEN
UNLABELLED: The role of the enteric nervous system in intestinal inflammation is not fully understood and the plethora of cellular activities concurrently ongoing in vivo renders intelligible studies difficult. In order to explore possible effects of bacterial lipopolysaccharide (LPS) on enteric neurons we utilised cultured myenteric neurons from rat small intestine. Exposure to LPS caused markedly reduced neuronal survival and increased neuronal expression of vasoactive intestinal peptide (VIP), while the expression of Toll-like receptor 4 (TLR4) was unchanged. TLR4 was expressed in approximately 35% of all myenteric neurons irrespective of if they were cultured in the presence or absence of LPS. In neurons cultured in medium, without LPS, 50% of all TLR4-immunoreactive neurons contained also VIP. Addition of LPS to the neuronal cultures markedly increased the proportion of TLR4-immunoreactive neurons also expressing VIP, while the proportion of TLR4 neurons devoid of VIP decreased. Simultaneous addition of LPS and VIP to the neuronal cultures resulted in a neuronal survival comparable to controls. CONCLUSIONS: LPS recognition by myenteric neurons is mediated via TLR4 and causes neuronal cell death. Presence of VIP rescues the neurons from LPS-induced neurodegeneration.