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Introduction. The trans population in Argentina represents 0.43%. Our objective was to describe the characteristics of trans and non-binary children and adolescents. Population and methods. A cross-sectional study was designed. The population was all trans or nonbinary persons under 24 years of age seen by an interdisciplinary team in a tertiary university hospital from January 2019 to May 2023. The sample was obtained from the database of patients seen by reviewing electronic medical records (EMR). Results. The EMRs of 107 individuals were analyzed; the average age at first consultation was 15.3 years, and the age of self-perceived transgender identity was 11.1 years. Seventy-two percent perceived themselves as having a trans male identity; in 89.7%, their gender expression was by their self-perception, and 46.3% had a bisexual sexual orientation. Seventy-six percent acknowledged having family support; 87.3%, school support; and 92.5%, peer support. 44.8% had a hormonal strategy, 14.1% had surgery, 57.1% had mental health intervention, and 29.1% received psychiatric medication. Only three patients (2.8%) detransitioned their gender identity. Conclusion. Most individuals were trans men and perceived good support from their environments. Almost half received a hormonal strategy; less than a quarter received a surgical intervention; more than half received a mental health intervention. The detransition was infrequent.
Introducción. La población trans en Argentina representa el 0,43 %. Nuestro objetivo fue describir las características de niñas, niños y adolescentes trans y no binarios. Población y métodos. Se diseñó un estudio de corte transversal. La población fueron todas las personas menores de 24 años trans o no binarias atendidas por un equipo interdisciplinario en un hospital universitario de tercer nivel desde enero de 2019 hasta mayo de 2023. La muestra se obtuvo de la base de datos de pacientes atendidos a través de la revisión de las historias clínicas electrónicas (HCE). Resultados. Se analizaron las HCE de 107 personas; el promedio de la primera consulta fue 15,3 años y la edad de autopercepción de identidad de género trans, 11,1 años. El 72 % se percibió con una identidad varón trans; en el 89,7 %, su expresión de género fue acorde a su autopercepción y el 46,3 % tuvo una orientación sexual bisexual. El 76 % reconoció tener contención familiar; el 87,3 %, contención escolar; y el 92,5 %, contención de sus pares. El 44,8 % realizó una estrategia hormonal; el 14,1 %, intervención quirúrgica; el 57,1 %, intervención con salud mental; y el 29,1 % recibió medicación psiquiátrica. Solo 3 pacientes (2,8 %) detransicionaron su identidad de género. Conclusión. La mayoría de las personas eran varones trans y percibieron una buena contención de sus entornos. Casi la mitad recibió una estrategia hormonal; menos de un cuarto, una intervención quirúrgica; más de la mitad, una intervención con salud mental. La detransición fue infrecuente.
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Aging induces cardiac remodeling, resulting in an increase in the risk of suffering heart diseases, including heart failure. Collagen deposition increases with age and, together with sarcomeric changes in cardiomyocytes, may lead to ventricular stiffness. Multiphoton (MP) microscopy is a useful technique to visualize and detect variations in cardiac structures in a label free fashion. Here, we propose a method based on MP imaging (both two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) modalities) to explore and objectively quantify age-related structural differences in various components of cardiac tissues. Results in transmural porcine left ventricle (LV) sections reveal significant differences when comparing samples from young and old animals. Collagen and myosin SHG signals in old specimens are respectively 3.8x and >6-fold larger than in young ones. Differences in TPEF signals from cardiomyocyte were â¼3x. Moreover, the increased amount of collagen in old specimens results in a more organized pattern when compared to young LV tissues. Since changes in collagen and myosin are associated with cardiac dysfunction, the technique used herein might be a useful tool to accurately predict and measure changes associated with age-related myocardium fibrosis, tissue remodeling and sarcomeric alterations, with potential implications in preventing heart disease.
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Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action potential and lower cell-to-cell coupling than adult-like CMs. These immature electrophysiological properties favor arrhythmias due to the generation of electrophysiological gradients when hiPSC-CMs are injected in the cardiac tissue. Culturing hiPSC-CMs on three-dimensional (3D) scaffolds can promote their maturation and influence their alignment. However, it is still uncertain how on-scaffold culturing influences the overall electrophysiology of the in vitro and implanted EHTs, as it requires expensive and time consuming experimentation. Here, we computationally investigated the impact of the scaffold design on the EHT electrical depolarization and repolarization before and after engraftment on infarcted tissue. We first acquired and processed electrical recordings from in vitro EHTs, which we used to calibrate the modeling and simulation of in silico EHTs to replicate experimental outcomes. Next, we built in silico EHT models for a range of scaffold pore sizes, shapes (square, rectangular, auxetic, hexagonal) and thicknesses. In this setup, we found that scaffolds made of small (0.2 mm2), elongated (30° half-angle) hexagons led to faster EHT activation and better mimicked the cardiac anisotropy. The scaffold thickness had a marginal role on the not engrafted EHT electrophysiology. Moreover, EHT engraftment on infarcted tissue showed that the EHT conductivity should be at least 5% of that in healthy tissue for bidirectional EHT-myocardium electrical propagation. For conductivities above such threshold, the scaffold made of small elongated hexagons led to the lowest activation time (AT) in the coupled EHT-myocardium. If the EHT conductivity was further increased and the hiPSC-CMs were uniformly oriented parallel to the epicardial cells, the total AT and the repolarization time gradient decreased substantially, thus minimizing the likelihood for arrhythmias after EHT transplantation.
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Células Madre Pluripotentes Inducidas , Infarto del Miocardio , Humanos , Ingeniería de Tejidos/métodos , Miocitos Cardíacos/fisiología , Miocardio , Arritmias CardíacasRESUMEN
The procedures used routinely for collagen and lipofuscin evaluation are, in many cases, qualitative, observer dependent, and disregard spatial distribution. Here, we present a protocol for automatic quantification and spatial characterization of collagen and lipofuscin from label-free microscopy images of human ventricular tissues. We describe the steps for sample collection, tissue processing, image acquisition, and quantification of collagen and lipofuscin. This protocol avoids discrepancies between observers and can be adapted to other tissues and species. For complete details on the use and execution of this protocol, please refer to García-Mendívil et al. (2022).1.
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Lipofuscina , Microscopía , Humanos , Ventrículos Cardíacos/diagnóstico por imagen , Miocardio , ColágenoRESUMEN
Connexin 43 (CX43) is one of the major components of gap junctions, the structures responsible for the intercellular communication and transmission of the electrical impulse in the left ventricle. There is limited information on the histological changes of CX43 with age and their effect on electrophysiology, especially in humans. Here, we analyzed left ventricular biopsies from living donors starting at midlife to characterize age-related CX43 remodeling. We assessed its quantity, degree of lateralization, and spatial heterogeneity together with fibrotic deposition. We observed no significant age-related remodeling of CX43. Only spatial heterogeneity increased slightly with age, and this increase was better explained by biological age than by chronological age. Importantly, we found that CX43 features varied considerably among individuals in our population with no relevant relationship to age or fibrosis content, in contrast to animal species. We used our experimental results to feed computational models of human ventricular electrophysiology and to assess the effects of interindividual differences in specific features of CX43 and fibrosis on conduction velocity, action potential duration, and arrhythmogenicity. We found that larger amounts of fibrosis were associated with the highest arrhythmic risk, with this risk being increased when fibrosis deposition was combined with a reduction in CX43 amount and/or with an increase in CX43 spatial heterogeneity. These mechanisms underlying high arrhythmic risk in some individuals were not associated with age in our study population. In conclusion, our data rule out CX43 remodeling as an age-related arrhythmic substrate in the population beyond midlife, but highlight its potential as a proarrhythmic factor at the individual level, especially when combined with increased fibrosis.
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Age-related fibrosis in the left ventricle (LV) has been mainly studied in animals by assessing collagen content. Using second-harmonic generation microscopy and image processing, we evaluated amount, aggregation and spatial distribution of LV collagen in young to old pigs, and middle-age and elder living donors. All collagen features increased when comparing adult and old pigs with young ones, but not when comparing adult with old pigs or middle-age with elder individuals. Remarkably, all collagen parameters strongly correlated with lipofuscin, a biological age marker, in humans. By building patient-specific models of human ventricular tissue electrophysiology, we confirmed that amount and organization of fibrosis modulated arrhythmia vulnerability, and that distribution should be accounted for arrhythmia risk assessment. In conclusion, we characterize the age-associated changes in LV collagen and its potential implications for ventricular arrhythmia development. Consistency between pig and human results substantiate the pig as a relevant model of age-related LV collagen dynamics.
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Aging is the main risk factor for cardiovascular diseases. In humans, cardiac aging remains poorly characterized. Most studies are based on chronological age (CA) and disregard biological age (BA), the actual physiological age (result of the aging rate on the organ structure and function), thus yielding potentially imperfect outcomes. Deciphering the molecular basis of ventricular aging, especially by BA, could lead to major progresses in cardiac research. We aim to describe the transcriptome dynamics of the aging left ventricle (LV) in humans according to both CA and BA and characterize the contribution of microRNAs, key transcriptional regulators. BA is measured using two CA-associated transcriptional markers: CDKN2A expression, a cell senescence marker, and apparent age (AppAge), a highly complex transcriptional index. Bioinformatics analysis of 132 LV samples shows that CDKN2A expression and AppAge represent transcriptomic changes better than CA. Both BA markers are biologically validated in relation to an aging phenotype associated with heart dysfunction, the amount of cardiac fibrosis. BA-based analyses uncover depleted cardiac-specific processes, among other relevant functions, that are undetected by CA. Twenty BA-related microRNAs are identified, and two of them highly heart-enriched that are present in plasma. We describe a microRNA-gene regulatory network related to cardiac processes that are partially validated in vitro and in LV samples from living donors. We prove the higher sensitivity of BA over CA to explain transcriptomic changes in the aging myocardium and report novel molecular insights into human LV biological aging. Our results can find application in future therapeutic and biomarker research.
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Envejecimiento/genética , Biomarcadores/metabolismo , Ventrículos Cardíacos/metabolismo , MicroARNs/genética , Femenino , Humanos , MasculinoRESUMEN
La aparición de aparatología preadjustada ha colaborado en la efectividad de los tratamientos de ortodoncia, pero para que la expresión de esta aparatología se logre, es necesario una correcta colocación de los brackets y la permanencia de estos en boca durante todo el tratamiento. La precisión en la colocación mejora con la técnica de cementado indirecta, ya que permite el acceso a las zonas posteriores, a lugares donde se ve disminuida la visión y además disminuye la condensación de aliento y contaminación salival. Si bien esta técnica requiere tiempo extra de laboratorio, es más rápida en la etapa clínica (AU)
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Humanos , Masculino , Femenino , Adolescente , Adulto , Elastómeros de Silicona , Cementación/métodos , Soportes Ortodóncicos , Ácidos Fosfóricos/química , Proyectos de Investigación , Facultades de Odontología , Grabado Ácido Dental/instrumentación , Eficacia , Técnica de Impresión Dental , Recubrimiento Dental Adhesivo/instrumentación , Resinas Compuestas , Modelos DentalesRESUMEN
GABAB-receptors (GABAB-Rs) are metabotropic, G protein-coupled receptors for the neurotransmitter GABA. Their activation induces slow inhibitory control of the neuronal excitability mediated by pre- and postsynaptic inhibition. Presynaptically GABAB-Rs reduce GABA and glutamate release inhibiting presynaptic Ca2+ channels in both inhibitory and excitatory synapses while postsynaptic GABAB-Rs induce robust slow hyperpolarization by the activation of K+ channels. GABAB-Rs are activated by non-synaptic or volume transmission, which requires high levels of GABA release, either by the simultaneous discharge of GABAergic interneurons or very intense discharges in the thalamus or by means of the activation of a neurogliaform interneurons in the cortex. The main receptor subunits GABAB1a, GABAB1b and GABAB2 are strongly expressed in neurons and glial cells throughout the central nervous system and GABAB-R activation is related to many neuronal processes such as the modulation of rhythmic activity in several brain regions. In the thalamus, GABAB-Rs modulate the generation of the main thalamic rhythm, spindle waves. In the cerebral cortex, GABAB-Rs also modulate the most prominent emergent oscillatory activity-slow oscillations-as well as faster oscillations like gamma frequency. Further, recent studies evaluating the complexity expressed by the cortical network, a parameter associated with consciousness levels, have found that GABAB-Rs enhance this complexity, while their blockade decreases it. This review summarizes the current results on how the activation of GABAB-Rs affects the interchange of information between brain areas by controlling rhythmicity as well as synaptic plasticity.
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Receptores de GABA-B , Sinapsis , Plasticidad Neuronal , Neuronas/metabolismo , Receptores de GABA-A , Receptores de GABA-B/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica , Ácido gamma-AminobutíricoRESUMEN
Cardiac tissue slices preserve the heterogeneous structure and multicellularity of the myocardium and allow its functional characterization. However, access to human ventricular samples is scarce. We aim to demonstrate that slices from small transmural core biopsies collected from living donors during routine cardiac surgery preserve structural and functional properties of larger myocardial specimens, allowing accurate electrophysiological characterization. In pigs, we compared left ventricular transmural core biopsies with transmural tissue blocks from the same ventricular region. In humans, we analyzed transmural biopsies and papillary muscles from living donors. All tissues were vibratome-sliced. By histological analysis of the transmural biopsies, we showed that tissue architecture and cellular organization were preserved. Enzymatic and vital staining methods verified viability. Optically mapped transmembrane potentials confirmed that action potential duration and morphology were similar in pig biopsies and tissue blocks. Action potential morphology and duration in human biopsies and papillary muscles agreed with published ranges. In both pigs and humans, responses to increasing pacing frequencies and ß-adrenergic stimulation were similar in transmural biopsies and larger tissues. We show that it is possible to successfully collect and characterize tissue slices from human myocardial biopsies routinely extracted from living donors, whose behavior mimics that of larger myocardial preparations both structurally and electrophysiologically.
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Potenciales de Acción , Electrofisiología Cardíaca , Fenómenos Electrofisiológicos , Ventrículos Cardíacos/fisiopatología , Donadores Vivos , Potenciales de la Membrana , Animales , Humanos , PorcinosRESUMEN
KEY POINTS: We confirm that GABAB receptors (GABAB -Rs) are involved in the termination of Up-states; their blockade consistently elongates Up-states. GABAB -Rs also modulate Down-states and the oscillatory cycle, thus having an impact on slow oscillation rhythm and its regularity. The most frequent effect of GABAB -R blockade is elongation of Down-states and subsequent decrease of oscillatory frequency, with an increased regularity. In a quarter of cases, GABAB -R blockade shortened Down-states and increased oscillatory frequency, changes that are independent of firing rates in Up-states. Our computer model provides mechanisms for the experimentally observed dynamics following blockade of GABAB -Rs, for Up/Down durations, oscillatory frequency and regularity. The time course of excitation, inhibition and adaptation can explain the observed dynamics of the network. This study brings novel insights into the role of GABAB -R-mediated slow inhibition on the slow oscillatory activity, which is considered the default activity pattern of the cortical network. ABSTRACT: Slow wave oscillations (SWOs) dominate cortical activity during deep sleep, anaesthesia and in some brain lesions. SWOs are composed of periods of activity (Up states) interspersed with periods of silence (Down states). The rhythmicity expressed during SWOs integrates neuronal and connectivity properties of the network and is often altered under pathological conditions. Adaptation mechanisms as well as synaptic inhibition mediated by GABAB receptors (GABAB -Rs) have been proposed as mechanisms governing the termination of Up states. The interplay between these two mechanisms is not well understood, and the role of GABAB -Rs controlling the whole cycle of the SWO has not been described. Here we contribute to its understanding by combining in vitro experiments on spontaneously active cortical slices and computational techniques. GABAB -R blockade modified the whole SWO cycle, not only elongating Up states, but also affecting the subsequent Down state duration. Furthermore, while adaptation tends to yield a rather regular behaviour, we demonstrate that GABAB -R activation desynchronizes the SWOs. Interestingly, variability changes could be accomplished in two different ways: by either shortening or lengthening the duration of Down states. Even when the most common observation following GABAB -Rs blocking is the lengthening of Down states, both changes are expressed experimentally and also in numerical simulations. Our simulations suggest that the sluggishness of GABAB -Rs to follow the excitatory fluctuations of the cortical network can explain these different network dynamics modulated by GABAB -Rs.
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Neuronas , Receptores de GABA-B , Simulación por Computador , Periodicidad , Ácido gamma-AminobutíricoRESUMEN
The 3xTg-AD mouse model reproduces the main features associated with the etiology of familial Alzheimer's disease (AD). To investigate whether these features imply functional cortical network alterations and their evolution with age, we studied spontaneous slow oscillations, activity that integrates cellular and network properties. We quantified different parameters of the emergent slow oscillations-alternating Up and Down states-and of the embedded beta-gamma rhythms of 3xTg-AD and wild-type mice at 7 and 20 months of age. Most group differences occurred at 20 months of age: 3xTg-AD mice presented lower oscillatory frequency, higher cycle variability, and reduced relative (Up/Down) firing rate with respect to controls. The high-frequency analysis revealed a shift toward lower frequencies in older 3xTg-AD animals, reminiscent of one of the electroencephalography hallmarks of patients with AD. This first systematic characterization of the cortical emergent rhythms in 3xTg-AD strain provides insights into the network mechanisms underlying associated network activity alterations.
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Enfermedad de Alzheimer/fisiopatología , Anestesia , Electroencefalografía , Neocórtex/fisiopatología , Animales , Ritmo beta , Modelos Animales de Enfermedad , Femenino , Ritmo Gamma , Masculino , Ratones TransgénicosRESUMEN
Liver damage induces hepatic stellate cells (HSC) activation, characterised by a fibrogenic, proliferative and migratory phenotype. Activated HSC are mainly regulated by transforming growth factor ß 1 (TGFß1), which increases the production of extracellular matrix proteins (e.g. collagen-I) promoting the progression of hepatic fibrosis. AGAP2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 2) is a GTPase/GTP-activating protein involved in the actin remodelling system and receptor recycling. In the present work the role of AGAP2 in human HSC in response to TGFß1 was investigated. LX-2 HSC were transfected with AGAP2 siRNA and treated with TGFß1. AGAP2 knockdown prevented to some extent the proliferative and migratory TGFß1-induced capacities of LX-2 cells. An array focused on human fibrosis revealed that AGAP2 knockdown partially prevented TGFß1-mediated gene expression of the fibrogenic genes ACTA2, COL1A2, EDN1, INHBE, LOX, PDGFB, TGFΒ12, while favored the expression of CXCR4, IL1A, MMP1, MMP3 and MMP9 genes. Furthermore, TGFß1 induced AGAP2 promoter activation and its protein expression in LX-2. Moreover, AGAP2 protein levels were significantly increased in liver samples from rats with thioacetamide-induced fibrosis. In addition, AGAP2 silencing affected TGFß1-receptor 2 (TGFR2) trafficking in U2OS cells, blocking its effective recycling to the membrane. AGAP2 silencing in LX-2 cells prevented the TGFß1-induced increase of collagen-I protein levels, while its overexpression enhanced collagen-I protein expression in the presence or absence of the cytokine. AGAP2 overexpression also increased focal adhesion kinase (FAK) phosphorylated levels in LX-2 cells. FAK and MEK1 inhibitors prevented the increase of collagen-I expression caused by TGFß1 in LX-2 overexpressing AGAP2. In summary, the present work shows for the first time, that AGAP2 is a potential new target involved in TGFß1 signalling, contributing to the progression of hepatic fibrosis.
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Proteínas de Unión al GTP/fisiología , Proteínas Activadoras de GTPasa/fisiología , Células Estrelladas Hepáticas/metabolismo , Factor de Crecimiento Transformador beta1/fisiología , Animales , Línea Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Colágeno Tipo I/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Expresión Génica , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/enzimología , Células Estrelladas Hepáticas/fisiología , Humanos , Cirrosis Hepática/metabolismo , Masculino , Ratas Sprague-Dawley , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismoRESUMEN
NADPH oxidase (Nox) variants Nox1, Nox2 and Nox4 are implicated in the progression of liver fibrosis. However, the role of Nox5 is not yet known, mainly due to the lack of this enzyme in rat and mouse genomes. Here we describe the expression and functional relevance of Nox5 in the human cell line of hepatic stellate cells (HSC) LX-2. Under basal conditions, three long (Nox5-L: Nox5α, -ß, and -δ) and a short (Nox5-S or Nox5ε) splice variants were detected, which were silenced with specific siRNAs for Nox5. The most abundant isoform was Nox5-S, accounting for more than 90% of Nox5 protein. Overexpression of Nox5ß generated reactive oxygen species (ROS) in the presence of calcium, as judged by the production of hydrogen peroxide, L-012 luminescence and cytochrome c reduction. Nox5ε did not generated ROS under these conditions, and a reduced ROS production was observed when co-expressed with Nox5ß. In contrast, dihydroethidium oxidation was increased by Nox5ß or Nox5ε, suggesting that Nox5ε induced intracellular oxidative stress by an unknown mechanism. Functional studies showed that both Nox5ß and Nox5ε stimulated the proliferation of LX-2 cells and the collagen type I levels, while Nox5 siRNAs inhibited these effects. Interestingly, TGF-ß and angiotensin II upregulated Nox5 expression, which was reduced in cells pre-incubated with catalase. Further studies silencing Nox5 in TGF-ß-treated cells resulted in a reduction of collagen levels via p38 MAPK. Collectively, these results show for the first time that Nox5 can play a relevant role in the proliferation and fibrosis on human HSC.
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Células Estrelladas Hepáticas/enzimología , Cirrosis Hepática/enzimología , NADPH Oxidasa 5/genética , Isoformas de Proteínas/genética , Línea Celular , Proliferación Celular/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Cirrosis Hepática/fisiopatología , NADPH Oxidasa 5/metabolismo , Oxidación-Reducción , Estrés Oxidativo/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
NMDA receptor (NMDAr) hypofunction has been widely used as a schizophrenia model. Decreased activation of NMDAr is associated with a disrupted excitation/inhibition balance in the prefrontal cortex and with alterations in gamma synchronization. Our aim was to investigate whether this phenomenon could be reproduced in the spontaneous oscillatory activity generated by the local prefrontal network in vitro and, if so, to explore the effects of antipsychotics on the resulting activity. Extracellular recordings were obtained from prefrontal cortex slices bathed in in vivo-like ACSF solution. Slow (<1â¯Hz) oscillations consisting of interspersed Up (active) and Down (silent) states spontaneously emerged. Fast-frequency oscillations (15-90â¯Hz) occurred during Up states. We explored the effects of the NMDAr antagonist MK-801 on the spontaneously generated activity. Bath-applied MK-801 induced a dose-dependent decrease in Up-state duration and in the frequency of Up states. However, the beta/gamma power during Up states significantly increased; this increase was in turn prevented by the antipsychotic drug clozapine. The increased beta/gamma power with NMDAr blockade implies that NMDAr activation in physiological conditions prevents hypersynchronization in this frequency range. High-frequency hypersynchronization following NMDAr blockade occurring in cortical slices suggests that-at least part of-the underlying mechanisms of this schizophrenia feature persist in the local cortical circuit, even in the absence of long-range cortical or subcortical inputs. The observed action of clozapine decreasing hypersynchronization in the local circuit may be one of the mechanisms of action of clozapine in preventing schizophrenia symptoms derived from NMDA hypofunction.
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Ritmo beta/fisiología , Ritmo Gamma/fisiología , Corteza Prefrontal/fisiología , Esquizofrenia , Animales , Ritmo beta/efectos de los fármacos , Clozapina/farmacología , Modelos Animales de Enfermedad , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hurones , Ritmo Gamma/efectos de los fármacos , Técnicas In Vitro , Masculino , Técnicas de Cultivo de Órganos , Corteza Prefrontal/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
The senescence-accelerated mouse prone 8 (SAMP8) model is characterized by accelerated, progressive cognitive decline as well as Alzheimer's disease (AD)-like neurodegenerative changes, and resembles the etiology of multicausal, sporadic late-onset/age-related AD in humans. Our aim was to find whether these AD-like pathological features, together with the cognitive deficits present in the SAMP8 strain, are accompanied by disturbances in cortical network activity with respect to control mice (SAM resistance 1, SAMR1) and, if so, how the alterations in cortical activity progress with age. For this purpose, we characterized the extracellular spontaneous oscillatory activity in different regions of the cerebral cortex of SAMP8 and SAMR1 mice under ketamine anesthesia at 5 and 7 months of age. Under these conditions, slow oscillations and fast rhythms generated in the cortical network were recorded and different parameters of these oscillations were quantified and compared between SAMP8 and their control, SAMR1 mice. The average frequency of slow oscillations in SAMP8 mice was decreased with respect to the control mice at both studied ages. An elongation of the silent periods or Down states was behind the decreased slow oscillatory frequency while the duration of active or Up states remained stable. SAMP8 mice also presented increased cycle variability and reduced high frequency components during Down states. During Up states, the power peak in the gamma range was displaced towards lower frequencies in all the cortical areas of SAMP8 with respect to control mice suggesting that the spectral profile of SAMP8 animals is shifted towards lower frequencies. This shift is reminiscent to one of the principal hallmarks of electroencephalography (EEG) abnormalities in patients with Alzheimer's disease, and adds evidence in support of the suitability of the SAMP8 mouse as a model of this disease. Although some of the differences between SAMP8 and control mice were emphasized with age, the evolution of the studied parameters as SAMR1 mice got older indicates that the SAMR1 phenotype tends to converge with that of SAMP8 animals. To our knowledge, this is the first systematic characterization of the cortical slow and fast rhythms in the SAMP8 strain and it provides useful insights about the cellular and synaptic mechanisms underlying the reported alterations.
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This viewpoint highlights major, partly controversial concepts about the pathogenesis of pemphigus. The monopathogenic theory explains intra-epidermal blistering through the "desmoglein (Dsg) compensation" hypothesis, according to which an antibody-dependent disabling of Dsg 1- and/or Dsg 3-mediated cell-cell attachments of keratinocytes (KCs) is sufficient to disrupt epidermal integrity and cause blistering. The multipathogenic theory explains intra-epidermal blistering through the "multiple hit" hypothesis stating that a simultaneous and synchronized inactivation of the physiological mechanisms regulating and/or mediating intercellular adhesion of KCs is necessary to disrupt epidermal integrity. The major premise for a multipathogenic theory is that a single type of autoantibody induces only reversible changes, so that affected KCs can recover due to a self-repair. The damage, however, becomes irreversible when the salvage pathway and/or other cell functions are altered by a partnering autoantibody and/or other pathogenic factors. Future studies are needed to (i) corroborate these findings, (ii) characterize in detail patient populations with non-Dsg-specific autoantibodies, and (iii) determine the extent of the contribution of non-Dsg antibodies in disease pathophysiology.
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Pénfigo/etiología , Animales , Desmogleínas/inmunología , HumanosRESUMEN
Unfolded protein response (UPR) triggered as a consequence of ER stress has been shown to be involved in the development of different pathologies, including fibrotic disorders. In the present paper we explore the role played by UPR on a key fibrogenic parameter in the liver: collagen type I levels in activated hepatic stellate cells (HSC). Using Brefeldin A (BFA) as an ER stress inducer we found that UPR correlated with enhanced mRNA and protein levels of collagen type I in a cell line of immortalized non-tumoral rat HSC. Analysis of the three branches of UPR revealed the activation of IRE1α, PERK and ATF6 in response to BFA, although PERK activation was shown not to be involved in the fibrogenic action of BFA. BFA also activated p38 MAPK in an IRE1α-dependent way and the p38 MAPK inhibitor SB203580 prevented the increase in collagen type I mRNA and protein levels caused by BFA, suggesting the involvement of this kinase on this effect. Analysis of Smad activation showed that phosphorylated nuclear levels of Smad2 and 3 were increased in response to BFA treatment. Inhibition of Smad3 phosphorylation by SIS3 prevented the enhancement of collagen type I levels caused by BFA. Pretreatment with IRE1α and p38 MAPK inhibitors also prevented the increased p-Smad3 accumulation in the nucleus, suggesting an IRE1α-p38 MAPK-Smad pathway to be responsible for the fibrogenic action of BFA on HSC.
Asunto(s)
Brefeldino A/farmacología , Colágeno Tipo I/biosíntesis , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/fisiología , Células Estrelladas Hepáticas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Complejos Multienzimáticos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteína smad3/fisiología , Respuesta de Proteína Desplegada/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Línea Celular , Colágeno Tipo I/genética , Estrés del Retículo Endoplásmico/fisiología , Endorribonucleasas/antagonistas & inhibidores , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Imidazoles/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Respuesta de Proteína Desplegada/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (H(2)DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H(2)DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H(2)DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H(2)DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.
Asunto(s)
Antioxidantes/administración & dosificación , Apigenina/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Radicales Libres/metabolismo , Glutatión/metabolismo , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción/efectos de los fármacos , Oxígeno/metabolismo , RatasRESUMEN
The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX-2 based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease-9 (MMP-9) expression in human monocytes.