RESUMEN
Inflammation is thought to play a pivotal role in the onset of term and some forms of preterm labour. Although, we recently found that myometrial inflammation is a consequence rather than a cause of term labour, there are several other reproductive tissues, including amnion, choriodecidua parietalis and decidua basalis, where the inflammatory stimulus to labour may occur. To investigate this, we have obtained amnion, choriodecidual parietalis and decidua basalis samples from women at various stages of pregnancy and spontaneous labour. The inflammatory cytokine profile in each tissue was determine by Bio-Plex Pro® cytokine multiplex assays and quantitative RT-PCR. Active motif assay was used to study transcription activation in the choriodecidua parietalis. Quantitative RT-PCR was use to study the pro-labour genes (PGHS-2, PGDH, OTR and CX43) in all of the tissues at the onset of labour and oxytocin (OT) mRNA expression in the choriodecidual parietalis and decidua basalis. Statistical significance was ascribed to a P value <0.05. In the amnion and choriodecidua parietalis, the mRNA levels of various cytokines decreased from preterm no labour to term no labour samples, but the protein levels were unchanged. The choriodecidua parietalis showed increase in the protein levels of IL-1ß and IL-6 in the term early labour samples. In the amnion and decidua basalis, the protein levels of several cytokines rose in term established labour. The multiples of the median derived from the 19-plex cytokine assay were greater in term early labour and term established labour samples from the choriodecidua parietalis, but only in term established labour for myometrium. These data suggest that the inflammatory stimulus to labour may begin in the choriodecidua parietalis, but the absence of any change in prolabour factor mRNA levels suggests that the cytokines may act on the myometrium where we observed changes in transcription factor activation and increases in prolabour gene expression in earlier studies.
Asunto(s)
Inflamación/fisiopatología , Trabajo de Parto/fisiología , Receptor 1 de Quimiocinas CX3C/metabolismo , Citocinas/metabolismo , Decidua/metabolismo , Femenino , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Miometrio/fisiología , Reacción en Cadena de la Polimerasa , EmbarazoRESUMEN
In our earlier work, we found that intrauterine (i.u.) and intraperitoneal (i.p.) injection of LPS (10-µg serotype 0111:B4) induced preterm labor (PTL) with high pup mortality, marked systemic inflammatory response and hypotension. Here, we used both i.u. and i.p. LPS models in pregnant wild-type (wt) and CCR2 knockout (CCR2-/-) mice on E16 to investigate the role played by the CCL2/CCR2 system in the response to LPS. Basally, lower numbers of monocytes and macrophages and higher numbers of neutrophils were found in the myometrium, placenta, and blood of CCR2-/- vs. wt mice. After i.u. LPS, parturition occurred at 14 h in both groups of mice. At 7 h post-injection, 70% of wt pups were dead vs. 10% of CCR2-/- pups, but at delivery 100% of wt and 90% of CCR2-/- pups were dead. Myometrial and placental monocytes and macrophages were generally lower in CCR2-/- mice, but this was less consistent in the circulation, lung, and liver. At 7 h post-LPS, myometrial ERK activation was greater and JNK and p65 lower and the mRNA levels of chemokines were higher and of inflammatory cytokines lower in CCR2-/- vs. wt mice. Pup brain and placental inflammation were similar. Using the IP LPS model, we found that all measures of arterial pressure increased in CCR2-/- but declined in wt mice. These data suggest that the CCL2/CCR2 system plays a critical role in the cardiovascular response to LPS and contributes to pup death but does not influence the onset of inflammation-induced PTL.
Asunto(s)
Presión Arterial/fisiología , Lipopolisacáridos/efectos adversos , Miometrio/metabolismo , Trabajo de Parto Prematuro/inducido químicamente , Placenta/metabolismo , Receptores CCR2/metabolismo , Animales , Presión Arterial/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Miometrio/efectos de los fármacos , Trabajo de Parto Prematuro/genética , Trabajo de Parto Prematuro/metabolismo , Parto/efectos de los fármacos , Parto/genética , Parto/metabolismo , Placenta/efectos de los fármacos , Embarazo , Receptores CCR2/genéticaRESUMEN
Preterm labor (PTL) is the predominant cause of childhood morbidity and mortality. It has several phenotypes, each with a distinct etiology often involving inflammation. Here, in samples of reproductive tissues obtained in early PTL from women with phenotypically defined PTL, we examined the presence and distribution of inflammation and its relationship with prolabor gene expression. In chorioamnionitis (CA-PTL), cytokine protein concentrations were increased across all tissues; in idiopathic (I-PTL), the inflammatory changes were limited to the choriodecidua; inflammation was not a feature of placental abruption (PA-PTL). CA-PTL was associated with activation of p65 in the myometrium and AP-1 in the choriodecidua, and PA-PTL with CREB in the choriodecidua. In the myometrium, PGHS-2 mRNA level was increased in CA- and I-PTL; in the amnion, PGHS-2 mRNA level was higher in PA- and I-PTL, while in CA-PTL, OT, OTR mRNA, and CX-43 expression were increased. In the choriodecidua, PGHS-2 mRNA level was unchanged, but in CA and I-PTL, OT mRNA level were increased and OTR was reduced. These data show that CA-PTL is associated with widespread inflammation and prolabor gene expression. In contrast, in I-PTL, inflammation is limited to the choriodecidua, with discrete increases in PGHS-2 in the amnion and OT in the choriodecidua. Inflammation is not a feature of PA-PTL, which is associated with increased OT and OTR in the amnion.
Asunto(s)
Corioamnionitis/metabolismo , Inflamación/metabolismo , Trabajo de Parto Prematuro , Contracción Uterina/fisiología , Adulto , Membranas Extraembrionarias/metabolismo , Femenino , Humanos , Embarazo , TranscriptomaRESUMEN
Although progesterone (P4) supplementation is the most widely used therapy for the prevention of preterm labor (PTL), reports of its clinical efficacy have been conflicting. We have previously shown that the anti-inflammatory effects of P4 can be enhanced by increasing intracellular cyclic adenosine monophosphate (cAMP) levels in primary human myometrial cells. Here, we have examined whether adding aminophylline (Am), a non-specific phosphodiesterase inhibitor that increases intracellular cAMP levels, to P4 might improve its efficacy using in vivo and in vitro models of PTL. In a mouse model of lipopolysaccharide (LPS)-induced PTL, we found that the combination of P4 and Am delayed the onset of LPS-induced PTL, while the same dose of P4 and Am alone had no effect. Pup survival was not improved by either agent alone or in combination. Myometrial prolabor and inflammatory cytokine gene expression was reduced, but the reduction was similar in P4 and P4/Am treated mice. There was no effect of the combination of P4 and Am on an ex vivo assessment of myometrial contractility. In human myometrial cells and myometrial tissue explants, we found that the combination had marked anti-inflammatory effects, reducing cytokine and COX-2 mRNA and protein levels to a greater extent than either agent alone. These data suggest that the combination of P4 and Am has a more potent anti-inflammatory effect than either agent alone and may be an effective combination in women at high-risk of PTL.
Asunto(s)
Aminofilina/farmacología , Endometritis/complicaciones , Trabajo de Parto Prematuro/etiología , Trabajo de Parto Prematuro/prevención & control , Progesterona/farmacología , Animales , Animales no Consanguíneos , Células Cultivadas , Modelos Animales de Enfermedad , Combinación de Medicamentos , Endometritis/patología , Femenino , Humanos , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/patología , Lipopolisacáridos , Ratones , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Miometrio/patología , Trabajo de Parto Prematuro/patología , Embarazo , Complicaciones Infecciosas del Embarazo/patología , Nacimiento Prematuro/etiología , Nacimiento Prematuro/prevención & controlRESUMEN
For inherited genetic diseases, fetal gene therapy offers the potential of prophylaxis against early, irreversible and lethal pathological change. To explore this, we studied neuronopathic Gaucher disease (nGD), caused by mutations in GBA. In adult patients, the milder form presents with hepatomegaly, splenomegaly and occasional lung and bone disease; this is managed, symptomatically, by enzyme replacement therapy. The acute childhood lethal form of nGD is untreatable since enzyme cannot cross the blood-brain barrier. Patients with nGD exhibit signs consistent with hindbrain neurodegeneration, including neck hyperextension, strabismus and, often, fatal apnea1. We selected a mouse model of nGD carrying a loxP-flanked neomycin disruption of Gba plus Cre recombinase regulated by the keratinocyte-specific K14 promoter. Exclusive skin expression of Gba prevents fatal neonatal dehydration. Instead, mice develop fatal neurodegeneration within 15 days2. Using this model, fetal intracranial injection of adeno-associated virus (AAV) vector reconstituted neuronal glucocerebrosidase expression. Mice lived for up to at least 18 weeks, were fertile and fully mobile. Neurodegeneration was abolished and neuroinflammation ameliorated. Neonatal intervention also rescued mice but less effectively. As the next step to clinical translation, we also demonstrated the feasibility of ultrasound-guided global AAV gene transfer to fetal macaque brains.
Asunto(s)
Feto/metabolismo , Terapia Genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/terapia , Animales , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/terapia , Humanos , Lactante , Inyecciones Intravenosas , Inyecciones Intraventriculares , Ratones Endogámicos C57BLRESUMEN
The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2), and the onset of parturition was examined in (i) naïve mice delivering at term; (ii) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and (iii) in mice treated with P4 to prevent term parturition. In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43, and COX-2 increased with, but not before, parturition. With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9 h post-RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition. In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented, and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased. These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabor factor synthesis via mRNA-dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation.
Asunto(s)
Miometrio/efectos de los fármacos , Parto/efectos de los fármacos , Progesterona/farmacología , Animales , Quimiocinas/metabolismo , Conexina 43/metabolismo , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Femenino , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Mifepristona/farmacología , Monocitos/metabolismo , Miometrio/inmunología , Miometrio/metabolismo , Neutrófilos/metabolismo , Parto/inmunología , Parto/metabolismoRESUMEN
Myometrial inflammation is thought to have a pivotal role in the onset of term and some forms of preterm labour. This is based on the comparison of samples taken from women undergoing term elective CS prior to the onset of labour with those taken from women in established labour. Consequently, it is not clear whether myometrial inflammation is a cause or a consequence of labour. Our objective is to test the hypothesis that myometrial inflammation is a consequence of the onset of labour. To test this hypothesis, we have obtained myometrial samples from women at various stages of pregnancy and spontaneous labour and studied the activation of the AP-1 (c-Jun) and NFκB (p65) systems, cytokine mRNA expression and protein levels and inflammatory cell infiltration and activation. We found that the activation of p65 declined from preterm to term not in labour samples and thereafter increased in early and established labour. Cytokine mRNA expression and protein levels increased in established labour only. Using flow cytometry of myometrial tissue, we found that the number of neutrophils did increase with the onset of labour, but on tissue section, these were seen to be intravascular and not infiltrating into the myometrium. These data suggest that myometrial inflammation is a consequence rather than a cause of term labour.
Asunto(s)
Miometrio/inmunología , Trabajo de Parto Prematuro/inmunología , Adulto , Citocinas/genética , Citocinas/inmunología , Femenino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Neutrófilos/inmunología , Embarazo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Adulto JovenRESUMEN
Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 µg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta.
Asunto(s)
Inflamación/inmunología , Leucocitos/inmunología , Lipopolisacáridos/farmacología , Miometrio/inmunología , Trabajo de Parto Prematuro/inmunología , Útero/efectos de los fármacos , Animales , Quimiocinas/metabolismo , Citocinas/metabolismo , Femenino , Expresión Génica , Inflamación/inducido químicamente , Inflamación/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Miometrio/efectos de los fármacos , Miometrio/metabolismo , FN-kappa B/metabolismo , Trabajo de Parto Prematuro/inducido químicamente , Trabajo de Parto Prematuro/metabolismo , Embarazo , Transducción de Señal/fisiología , Útero/inmunología , Útero/metabolismoRESUMEN
In Western countries heart disease is the leading cause of maternal death during pregnancy. The effect of pregnancy on the heart is difficult to study in patients with preexisting heart disease. Since experimental studies are scarce, we investigated the effect of pressure overload, produced by transverse aortic constriction (TAC) in mice, on the ability to conceive, pregnancy outcome, and maternal cardiac structure and function. Four weeks of TAC produced left ventricular (LV) hypertrophy and dysfunction with marked interstitial fibrosis, decreased capillary density, and induced pathological cardiac gene expression. Pregnancy increased relative LV and right ventricular weight without affecting the deterioration of LV function following TAC. Surprisingly, the TAC-induced increase in relative heart and lung weight was mitigated by pregnancy, which was accompanied by a trend towards normalization of capillary density and natriuretic peptide type A expression. Additionally, the combination of pregnancy and TAC increased the cardiac phosphorylation of c-Jun, and STAT1, but reduced phosphoinositide 3-kinase phosphorylation. Finally, TAC did not significantly affect conception rate, pregnancy duration, uterus size, litter size, and pup weight. In conclusion, we found that, rather than exacerbating the changes associated with cardiac pressure overload, pregnancy actually attenuated pathological LV remodeling and mitigated pulmonary congestion, and pathological gene expression produced by TAC, suggesting a positive effect of pregnancy on the pressure-overloaded heart.
Asunto(s)
Estenosis de la Válvula Aórtica/fisiopatología , Hipertrofia Ventricular Izquierda/fisiopatología , Complicaciones Cardiovasculares del Embarazo/fisiopatología , Disfunción Ventricular Izquierda/fisiopatología , Animales , Animales Recién Nacidos , Estenosis de la Válvula Aórtica/complicaciones , Factor Natriurético Atrial/genética , Peso al Nacer , Capilares/patología , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Fibrosis , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , Tamaño de la Camada , Ratones , Ratones Endogámicos C57BL , Miocardio/patología , Cadenas Pesadas de Miosina/genética , Péptido Natriurético Encefálico/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Embarazo , Complicaciones Cardiovasculares del Embarazo/etiología , Complicaciones Cardiovasculares del Embarazo/genética , Complicaciones Cardiovasculares del Embarazo/patología , Índice de Embarazo , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT1/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Factores de Tiempo , Transcriptoma , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/patologíaRESUMEN
OBJECTIVES: We tested the hypothesis that there is a positive association between maternal body mass index (BMI) and the concentration of appetite-regulating hormones leptin, insulin, ghrelin and resistin in breast milk. We also aimed to describe the change in breast milk hormone concentration within each feed, and over time. SETTING: Mothers were recruited from the postpartum ward at a university hospital in London. Breast milk samples were collected at the participants' homes. PARTICIPANTS: We recruited 120 healthy, primiparous, breastfeeding mothers, aged over 18â years. Mothers who smoked, had multiple births or had diabetes were excluded. Foremilk and hindmilk samples were collected from 105 women at 1â week postpartum and 92 women at 3â months postpartum. PRIMARY AND SECONDARY OUTCOME MEASURES: We recorded maternal and infant anthropometric measurements at each sample collection and measured hormone concentrations using a multiplex assay. RESULTS: The concentration of leptin in foremilk correlated with maternal BMI at the time of sample collection, at 7â days (r=0.31, p=0.02) and 3â months postpartum (r=0.30, p=<0.00). Foremilk insulin correlated with maternal BMI at 3â months postpartum (r=0.22, p=0.04). Breast milk ghrelin and resistin were not correlated with maternal BMI. Ghrelin concentrations at 3â months postpartum were increased in foremilk compared with hindmilk (p=0.01). Concentrations of ghrelin were increased in hindmilk collected at 1â week postpartum compared with samples collected at 3â months postpartum (p=0.03). A trend towards decreased insulin concentrations in hindmilk was noted. Concentrations of leptin and resistin were not seen to alter over a feed. CONCLUSIONS: A positive correlation between maternal BMI and foremilk leptin concentration at both time points studied, and foremilk insulin at 3â months postpartum was observed. This may have implications for infant appetite regulation and obesity risk.
Asunto(s)
Ghrelina/metabolismo , Insulina/metabolismo , Leptina/metabolismo , Leche Humana/metabolismo , Sobrepeso/metabolismo , Resistina/metabolismo , Adulto , Índice de Masa Corporal , Estudios de Cohortes , Femenino , Ghrelina/análisis , Humanos , Lactante , Recién Nacido , Insulina/análisis , Leptina/análisis , Estudios Longitudinales , Masculino , Leche Humana/química , Resistina/análisis , Manejo de Especímenes/métodosRESUMEN
BACKGROUND: Preterm birth is now recognized as the primary cause of infant mortality worldwide. Interplay between hormonal and inflammatory signaling in the uterus modulates the onset of contractions; however, the relative contribution of each remains unclear. In this study we aimed to characterize temporal transcriptome changes in the uterus preceding term labor and preterm labor (PTL) induced by progesterone withdrawal or inflammation in the mouse and compare these findings with human data. METHODS: Myometrium was collected at multiple time points during gestation and labor from three murine models of parturition: (1) term gestation; (2) PTL induced by RU486; and (3) PTL induced by lipopolysaccharide (LPS). RNA was extracted and cDNA libraries were prepared and sequenced using the Illumina HiSeq 2000 system. Resulting RNA-Seq data were analyzed using multivariate modeling approaches as well as pathway and causal network analyses and compared against human myometrial transcriptome data. RESULTS: We identified a core set of temporal myometrial gene changes associated with term labor and PTL in the mouse induced by either inflammation or progesterone withdrawal. Progesterone withdrawal initiated labor without inflammatory gene activation, yet LPS activation of uterine inflammation was sufficient to override the repressive effects of progesterone and induce a laboring phenotype. Comparison of human and mouse uterine transcriptomic datasets revealed that human labor more closely resembles inflammation-induced PTL in the mouse. CONCLUSIONS: Labor in the mouse can be achieved through inflammatory gene activation yet these changes are not a requisite for labor itself. Human labor more closely resembles LPS-induced PTL in the mouse, supporting an essential role for inflammatory mediators in human "functional progesterone withdrawal." This improved understanding of inflammatory and progesterone influence on the uterine transcriptome has important implications for the development of PTL prevention strategies.
Asunto(s)
Mediadores de Inflamación/metabolismo , Trabajo de Parto/metabolismo , Trabajo de Parto Prematuro/metabolismo , Progesterona/metabolismo , Transcriptoma/fisiología , Útero/fisiología , Animales , Femenino , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/fisiopatología , Trabajo de Parto/efectos de los fármacos , Trabajo de Parto/genética , Lipopolisacáridos/toxicidad , Ratones , Modelos Animales , Miometrio/efectos de los fármacos , Miometrio/fisiología , Trabajo de Parto Prematuro/inducido químicamente , Trabajo de Parto Prematuro/genética , Parto/efectos de los fármacos , Parto/genética , Parto/metabolismo , Embarazo , Progesterona/genética , Transcriptoma/efectos de los fármacos , Útero/efectos de los fármacosRESUMEN
RATIONALE: Continuous performance tests (CPTs) are widely used to assess attentional processes in a variety of disorders including Alzheimer's disease and schizophrenia. Common human CPTs require discrimination of sequentially presented, visually patterned 'target' and 'non-target' stimuli at a single location. OBJECTIVES: The aims of this study were to evaluate the performance of three popular mouse strains on a novel rodent touchscreen test (rCPT) designed to be analogous to common human CPT variants and to investigate the effects of donepezil, a cholinesterase inhibitor and putative cognitive enhancer. METHODS: C57BL/6J, DBA/2J and CD1 mice (n = 15-16/strain) were trained to baseline performance using four rCPT training stages. Then, probe tests assessed the effects of parameter changes on task performance: stimulus size, duration, contrast, probability, inter-trial interval or inclusion of flanker distractors. rCPT performance was also evaluated following acute administration of donepezil (0-3 mg/kg, i.p.). RESULTS: C57BL/6J and DBA/2J mice showed similar acquisition rates and final baseline performance following rCPT training. On probe tests, rCPT performance of both strains was sensitive to alteration of visual and/or attentional demands (stimulus size, duration, contrast, rate, flanker distraction). Relative to C57BL/6J, DBA/2J mice exhibited (1) decreasing sensitivity (d') across the 45-min session, (2) reduced performance on probes where the appearance of stimuli or adjacent areas were changed (size, contrast, flanking distractors) and (3) larger dose- and stimulus duration-dependent changes in performance following donepezil administration. In contrast, CD1 mice failed to acquire rCPT (stage 3) and pairwise visual discrimination tasks. CONCLUSIONS: rCPT is a potentially useful translational tool for assessing attention in mice and for detecting the effects of nootropic drugs.
Asunto(s)
Atención/efectos de los fármacos , Condicionamiento Operante/efectos de los fármacos , Nootrópicos/farmacología , Desempeño Psicomotor/efectos de los fármacos , Animales , Inhibidores de la Colinesterasa/farmacología , Aprendizaje Discriminativo/efectos de los fármacos , Discriminación en Psicología/efectos de los fármacos , Donepezilo , Evaluación Preclínica de Medicamentos , Humanos , Indanos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Piperidinas/farmacología , Especificidad de la Especie , Percepción Visual/efectos de los fármacosRESUMEN
OBJECTIVE: Uterine overdistention is thought to induce preterm labor in women with twin and multiple pregnancies, but the pathophysiology remains unclear. We investigated for the first time the pathogenesis of preterm birth associated with rapid uterine distention in a pregnant nonhuman primate model. STUDY DESIGN: A nonhuman primate model of uterine overdistention was created using preterm chronically catheterized pregnant pigtail macaques (Macaca nemestrina) by inflation of intraamniotic balloons (N = 6), which were compared to saline controls (N = 5). Cesarean delivery was performed due to preterm labor or at experimental end. Microarray, quantitative reverse transcriptase polymerase chain reaction, Luminex (Austin, TX), and enzyme-linked immunosorbent assay were used to measure messenger RNA (mRNA) and/or protein levels from monkey (amniotic fluid, myometrium, maternal plasma) and human (amniocytes, amnion, myometrium) tissues. Statistical analysis employed analysis of covariance and Wilcoxon rank sum. Biomechanical forces were calculated using the law of Laplace. RESULTS: Preterm labor occurred in 3 of 6 animals after balloon inflation and correlated with greater balloon volume and uterine wall stress. Significant elevations of inflammatory cytokines and prostaglandins occurred following uterine overdistention in an "inflammatory pulse" that correlated with preterm labor (interleukin [IL]-1ß, tumor necrosis factor [TNF]-α, IL-6, IL-8, CCL2, prostaglandin E2, prostaglandin F2α, all P < .05). A similar inflammatory response was observed in amniocytes in vitro following mechanical stretch (IL1ß, IL6, and IL8 mRNA multiple time points, P < .05), in amnion of women with polyhydramnios (IL6 and TNF mRNA, P < .05) and in amnion (TNF-α) and myometrium of women with twins in early labor (IL6, IL8, CCL2, all P < .05). Genes differentially expressed in the nonhuman primate after balloon inflation and in women with polyhydramnios and twins are involved in tissue remodeling and muscle growth. CONCLUSION: Uterine overdistention by inflation of an intraamniotic balloon is associated with an inflammatory pulse that precedes and correlates with preterm labor. Our results indicate that inflammation is an early event after a mechanical stress on the uterus and leads to preterm labor when the stress is sufficiently great. Further, we find evidence of uterine tissue remodeling and muscle growth as a common, perhaps compensatory, response to uterine distension.
Asunto(s)
Inflamación/metabolismo , Trabajo de Parto Prematuro/fisiopatología , Estrés Mecánico , Útero/fisiopatología , Amnios/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Dinoprost/genética , Dinoprost/metabolismo , Dinoprostona/genética , Dinoprostona/metabolismo , Femenino , Humanos , Macaca nemestrina , Modelos Animales , Miometrio/metabolismo , Polihidramnios/metabolismo , Embarazo , Embarazo Múltiple/fisiología , ARN Mensajero/metabolismoRESUMEN
Intrauterine inflammation is recognized as a key mediator of both normal and preterm birth but is also associated with neonatal neurological injury. Lipopolysaccharide (LPS) is often used to stimulate inflammatory pathways in animal models of infection/inflammation-induced preterm labor; however, inconsistencies in maternal and neonatal responses to LPS are frequently reported. We hypothesized that LPS serotype-specific responses may account for a portion of these inconsistencies. Four different Escherichia coli LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection at gestational day 16. Although control animals delivered at term 60 ± 15 hours postinjection (p.i.), those administered with O111:B4 delivered 7 ± 2 hours p.i., O55:B5 delivered 10 ± 3 hours p.i., O127:B8 delivered 16 ± 10 hours p.i., and O128:B12 delivered 17 ± 2 hours p.i. (means ± SD). A correlation between the onset of preterm labor and myometrial activation of the inflammatory transcription factor, activator protein 1, but not NF-κB was observed. Specific LPS serotypes induced differential activation of downstream contractile and inflammatory pathways in myometrium and neonatal pup brain. Our findings demonstrate functional disparity in inflammatory pathway activation in response to differing LPS serotypes. Selective use of LPS serotypes may represent a useful tool for targeting specific inflammatory response mechanisms in these models.
Asunto(s)
Lipopolisacáridos/farmacología , Miometrio/efectos de los fármacos , Trabajo de Parto Prematuro/inducido químicamente , Nacimiento Prematuro/metabolismo , Animales , Modelos Animales de Enfermedad , Escherichia coli/efectos de los fármacos , Femenino , Inflamación/inducido químicamente , Inflamación/complicaciones , Ratones , Miometrio/metabolismo , Embarazo , Complicaciones del Embarazo/inducido químicamente , Factor de Transcripción AP-1/metabolismoRESUMEN
Activation of uterine inflammatory pathways leads to preterm labor (PTL), associated with high rates of neonatal mortality and morbidity. The transcription factors nuclear factor κB (NFκB) and activator protein 1 (AP-1) regulate key proinflammatory and procontractile genes involved in normal labor and PTL. Here we show that NFκB activation normally occurs in the mouse myometrium at gestation day E18, prior to labor, whereas AP-1 and JNK activation occurs at labor onset. Where labor was induced using the progesterone receptor antagonist RU486, NFkB and AP-1/JNK activation both occurred at the time of labor (20 h compared to 60 h in DMSO-treated controls). Using an LPS (Escherichia coli: serotype O111)-induced PTL model that selectively activates AP-1 but not NFkB, we show that myometrial AP-1 activation drives production of cytokines (Il-6, Il-8, and Il-1ß), metalloproteinases (Mmp3 and Mmp10), and procontractile proteins (Cox-2 and Cx43) resulting in PTL after 7 h. Protein levels of CX43 and IL-1ß, and IL-1ß cleavage, were increased following LPS-induced activation of AP-1. Inhibition of JNK by SP600125 (30 mg/kg) delayed PTL by 6 h (7.5 vs. 13.5 h P<0.05). Our data reveal that NFκB activation is not a functional requirement for infection/inflammation-induced preterm labor and that AP-1 activation is sufficient to drive inflammatory pathways that cause PTL.
Asunto(s)
Subunidad p50 de NF-kappa B/metabolismo , Trabajo de Parto Prematuro/metabolismo , Preñez , Factor de Transcripción AP-1/metabolismo , Animales , Antracenos , Citocinas/metabolismo , Femenino , Inflamación , Trabajo de Parto/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Miometrio/metabolismo , FN-kappa B/metabolismo , Embarazo , Progesterona/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores de Progesterona/metabolismo , Factores de Tiempo , Receptor Toll-Like 4/metabolismo , Útero/metabolismoRESUMEN
We have previously demonstrated that the anti-inflammatory prostaglandin 15-deoxy-Δ 12,14-prostaglandin J(2) (15dPGJ(2)) delays inflammation-induced preterm labour in the mouse and improves pup survival through the inhibition of nuclear factor-κB (NF-κB) by a mechanism yet to be elucidated. 15dPGJ(2) is an agonist of the second prostaglandin D(2) receptor, chemoattractant receptor homologous to the T helper 2 cell (CRTH2). In human T helper cells CRTH2 agonists induce the production of the anti-inflammatory interleukins IL-10 and IL-4. We hypothesized that CRTH2 is involved in the protective effect of 15dPGJ(2) in inflammation-induced preterm labour in the murine model. We therefore studied the effects of a specific small molecule CRTH2 agonist on preterm labour and pup survival. An intrauterine injection of lipopolysaccharide (LPS) was administered to CD1 mice at embryonic day 16, ± CRTH2 agonist/vehicle controls. Mice were killed at 4.5 hr to assess fetal wellbeing and to harvest myometrium and pup brain for analysis of NF-κB, and T helper type 1/2 interleukins. To examine the effects of the CRTH2 agonist on LPS-induced preterm labour, mice were allowed to labour spontaneously. Direct effects of the CRTH2 agonist on uterine contractility were examined ex vivo on contracting myometrial strips. The CRTH2 agonist increased fetal survival from 20 to 100% in LPS-treated mice, and inhibited circular muscle contractility ex vivo. However, it augmented LPS-induced labour and significantly increased myometrial NF-κB, IL-1ß, KC-GRO, interferon-γ and tumour necrosis factor-α. This suggests that the action of 15dPGJ(2) is not via CRTH2 and therefore small molecule CRTH2 agonists are not likely to be beneficial for the prevention of inflammation-induced preterm labour.
Asunto(s)
Muerte Fetal/inducido químicamente , Lipopolisacáridos/administración & dosificación , Trabajo de Parto Prematuro/inducido químicamente , Péptidos/administración & dosificación , Receptores Inmunológicos/agonistas , Receptores de Prostaglandina/agonistas , Animales , Antiinflamatorios/agonistas , Antiinflamatorios/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Factores Inmunológicos/agonistas , Factores Inmunológicos/metabolismo , Inflamación , Lipopolisacáridos/farmacología , Ratones , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Trabajo de Parto Prematuro/inmunología , Trabajo de Parto Prematuro/prevención & control , Embarazo , Prostaglandina D2/agonistas , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismoRESUMEN
Fetal gene transfer has been studied in various animal models, including rabbits, guinea pigs, cats, dogs, and nonhuman primate; however, the most common model is the rodent, particularly the mouse. There are numerous advantages to mouse models, including a short gestation time of around 20 days, large litter size usually of more than six pups, ease of colony maintenance due to the small physical size, and the relatively low expense of doing so. Moreover, the mouse genome is well defined, there are many transgenic models particularly of human monogenetic disorders, and mouse-specific biological reagents are readily available. One criticism has been that it is difficult to perform procedures on the fetal mouse with suitable accuracy. Over the past decade, accumulation of technical expertise and development of technology such as high-frequency ultrasound have permitted accurate vector delivery to organs and tissues. Here, we describe our experiences of gene transfer to the fetal mouse with and without ultrasound guidance from mid to late gestation. Depending upon the vector type, the route of delivery and the age of the fetus, specific or widespread gene transfer can be achieved, making fetal mice excellent models for exploratory biodistribution studies.
Asunto(s)
Terapia Genética/métodos , Modelos Animales , Atención Prenatal/métodos , Anestesia , Crianza de Animales Domésticos , Animales , Recolección de Muestras de Sangre , Cruzamiento , Embrión de Mamíferos/diagnóstico por imagen , Femenino , Técnicas de Transferencia de Gen , Humanos , Inyecciones , Masculino , Ratones , Cuidados Preoperatorios , Ultrasonografía , Toma de Muestras de Orina , Membrana Vitelina/irrigación sanguíneaRESUMEN
OBJECTIVE: To produce transgenic mice expressing the D374Y variant of the human proprotein convertase subtilisin/kexin type 9 (PCSK9) gene at physiological levels to investigate the mechanisms causing hypercholesterolemia and accelerated atherosclerosis. METHODS AND RESULTS: A bacterial artificial chromosome containing PCSK9 and its flanking regions was modified to introduce the D374Y mutation and a C-terminal myc(2) tag. Transgenic mice that expressed 1 copy of the mutant or wild-type (WT) PCSK9 bacterial artificial chromosome were produced. Human PCSK9 mRNA was expressed at levels comparable to endogenous pcsk9 and with the same tissue specificity. The expression of D374Y or WT human PCSK9 increased the serum cholesterol level and reduced hepatic low-density lipoprotein receptor protein levels in the transgenic mice compared with bacterial artificial chromosome-negative controls; however, the effects were more marked in D374Y mice. The effect of a high-cholesterol diet on increasing serum cholesterol level was greater in D374Y mice, and atherosclerotic plaques after 15 weeks were more extensive in mice expressing D374Y than in WT PCSK9. D374Y mice secreted more triglyceride-rich lipoproteins into the circulation than WT mice. CONCLUSIONS: The expression of human D374Y PCSK9 at physiological levels produced a phenotype that closely matched that found in heterozygous D374Y patients and suggested that reduced low-density lipoprotein receptor activity is not the sole cause of their hypercholesterolemia.