RESUMEN
INTRODUCTION: Argyrophilic nucleolar organizer regions (AgNORs) are nonhistone argyrophilic nucleolar proteins associated with ribosomal genes found in the nucleolar organizer region that reflect cell proliferation and have an affinity for silver. AgNOR staining may be useful to evaluate prognosis in several neoplasms because higher AgNOR counts are related to higher grade tumors, metastases, and shorter survival times. OBJECTIVE: We aimed to report on a quick and practical technique to identify AgNORs adapted for use in routine cytology. MATERIALS AND METHODS: The cytopathologic diagnosis of mast cell tumor (MCT) in samples collected by fine-needle aspiration (FNA) was determined. Next, slides were impregnated with a solution containing silver nitrate; the main modification of our technique included incubation of these slides at a controlled temperature of 25 °C. Some slides were previously stained with Diff-Quik and others were only fixed with methanol. The slides were analyzed under a microscope, and the number of blackened intranuclear points (AgNORs) was counted. RESULTS: Slides prestained with Diff-Quik were easily counted compared with slides only fixed in methanol. Technical issues encountered with the methanol-fixed slides included insufficient cellularity, background precipitation, and an absence of silver impregnation. CONCLUSIONS: The technique reported in this study showed satisfactory results for AgNOR counting in cytologic smears from MCT, such as good impregnation and the elimination of background interferents. Further evaluation of this method comparing AgNOR counts with histologic examinations, tumor grades, other prognostic markers, and survival times are needed to fully evaluate the benefit of this technique.
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Enfermedades de los Perros , Neoplasias , Perros , Animales , Región Organizadora del Nucléolo/patología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/patología , Metanol , Tinción con Nitrato de Plata/veterinaria , Tinción con Nitrato de Plata/métodos , Neoplasias/patología , Neoplasias/veterinariaRESUMEN
The assessment of neuronal number, spatial organization and connectivity is fundamental for a complete understanding of brain function. However, the evaluation of the three-dimensional (3D) brain cytoarchitecture at cellular resolution persists as a great challenge in the field of neuroscience. In this context, X-ray microtomography has shown to be a valuable non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens, arisen as a new method for deciphering the cytoarchitecture and connectivity of the brain. In this work we present a method for imaging whole neurons in the brain, combining synchrotron-based X-ray microtomography with the Golgi-Cox mercury-based impregnation protocol. In contrast to optical 3D techniques, the approach shown here does neither require tissue slicing or clearing, and allows the investigation of several cells within a 3D region of the brain.
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Encéfalo/citología , Imagenología Tridimensional/métodos , Neuronas , Microtomografía por Rayos X/métodos , Animales , Encéfalo/diagnóstico por imagen , Imagenología Tridimensional/instrumentación , Cloruro de Mercurio/química , Ratones , Tinción con Nitrato de Plata/métodos , Sincrotrones , Fijación del Tejido/métodos , Microtomografía por Rayos X/instrumentaciónRESUMEN
One of the problems with 2DE is that proteins present in low amounts in a sample are usually not detected, since their signals are masked by the predominant proteins. The elimination of these abundant proteins is not a guaranteed solution to achieve the desired results. The main objective of this study was the comparison of common and simple methodologies employed for 2DE analysis followed by MS identification, focusing on a pre-purified sample using a wheat germ agglutinin (WGA) column. Adult male C57Black/Crj6 (C57BL/6) mice were chosen as the model animal in this study; the gastrocnemius muscles were collected and processed for the experiments. The initial fractionation with succinylated WGA was successful for the elimination of the most abundant proteins. Two quantification methods were employed for the purified samples, and bicinchoninic acid (BCA) was proven to be most reliable for the quantification of glycoproteins. The gel staining method, however, was found to be decisive for the detection of specific proteins, since their structures affect the interaction of the dye with the peptide backbone. The Coomassie Blue R-250 dye very weakly stained the gel with the WGA purified sample. When the same gel was stained with silver nitrate, however, MS could positively assign 12 new spots. The structure of the referred proteins was not found to be prone to interaction with Coomassie blue.
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Electroforesis en Gel Bidimensional/métodos , Glicoproteínas/análisis , Músculos/química , Animales , Cromatografía de Afinidad/métodos , Colorantes/análisis , Glicoproteínas/aislamiento & purificación , Glicosilación , Masculino , Ratones Endogámicos C57BL , Proteínas Musculares/análisis , Proteínas Musculares/aislamiento & purificación , Colorantes de Rosanilina/análisis , Tinción con Nitrato de Plata/métodos , Espectrometría de Masas en Tándem/métodos , Aglutininas del Germen de Trigo/químicaRESUMEN
Introdução: As próteses totais visam conservar a função do sistema estomatognático do paciente totalmente edêntulo. Porém, na mucosa bucal podem aparecer manifestações cuja principal causa são as próteses totais mal adaptadas. Objetivo: o presente estudo objetiva investigar a proliferação tecidual das lesões causadas por próteses totais removíveis através do método de impregnação pela prata (AgNOR), com isso facilitando o tratamento e a determinação do prognóstico das lesões a serem estudadas. Metodologia: foram selecionados todos os casos das lesões bucais mais comumente associadas à utilização de próteses totais registradas no Serviço de Diagnóstico Histopatológico do ICBUPF nos anos de 2012 e 2013, tendo sido encontrados 5 casos de granuloma piogênico, 5 casos de hiperplasia de fundo de sulco, 5 casos de fibroma de irritação e 2 casos de fibroma ossificante periférico. Os cortes histopatológicos das lesões foram impregnados pela prata (método AgNOR), tendo sido obtido, com auxílio do programa Image Tool®, o número de NORs de 100 células de cada caso, resultando numa média de NORs em cada grupo de lesões. Resultados: os resultados obtidos foram tabulados em planilha eletrônica e a comparação do número médio de NORs de cada grupo foi realizado por meio do teste estatístico ANOVA, 5% de significância. Resultados: o grupo das hiperplasias de fundo de sulco mostrou média de 2,41 NORs por núcleo, o grupo dos granulo mas piogênicos mostrou 2,44, o fibroma de irritação 2,22, e o fibroma ossificante periférico mostrou média de 1,89 NORs por núcleo celular, diferindo estatisticamente esta lesão das anteriormente mencionadas (p = 0,002). Conclusão: o fibroma ossificante periférico mostrou ser a lesão causada por prótese total removível com a menor atividade proliferativa celular. Tal estudo precisa ser complementado por futuros estudos clínicos.
Introduction: the total dentures are aimed at preserving the function of the stomatognathic system of the fully edentulous patient. However, in the oral mucosa may appear manifestations whose main cause are the totally unsuitable dentures. Objective: this study aims to investigate the proliferation of tissue lesions caused by removable dentures by impregnation method for silver (AgNOR), thereby facilitating the treatment and determining the prognosis of the lesions to be studied. Methodology: we selected all cases of oral lesions most commonly associated with the use of dentures recorded in Histopathological Diagnostic Service ICB-UPF in the years 2012 and 2013, having been found 5 cases of pyogenic granuloma, 5 cases of hyperplasia, 5 cases of irritation fibroma and 2 cases of peripheral ossifying fibroma. Histopathological lesions cuts were impregnated by silver (AgNOR method), having been obtained with the aid of the program Image Tool®, the number of NOR cells 100 in each case, resulting in an average NORs in every group of lesions. Results: the results were tabulated in a spreadsheet and comparing the average number of NORs of each group was conducted through ANOVA, 5% significance level. Results: The group of hyperplasias showed average of 2.41 NORs per nucleus, the group of pyogenic granulomas showed 2.44, the irritation fibroma 2.22, and peripheral ossifying fibroma showed average of 1.89 NORs for cell nucleus, differing significantly from that of the aforementioned lesions (p = 0.002). Conclusion: the peripheral ossifying fibroma proved the injury caused by removable dentures with lower cell proliferative activity. This study needs to be complemented by future clinical studies.
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Humanos , Masculino , Femenino , Dentadura Completa/efectos adversos , Dentadura Completa/estadística & datos numéricos , Boca/lesiones , Estudios Epidemiológicos , Tinción con Nitrato de Plata/métodosRESUMEN
En la investigación biológica sigue siendo necesaria la demostración de la inervación periférica en numerosos tejidos y órganos. El objetivo de este trabajo fue rescatar y modernizar uno de los métodos más constantes que hemos probado para demostrar la inervación periférica. La técnica de Llombart para fibras nerviosas se adaptó en cortes por parafina de 7 µm en diferentes tejidos animales. La impregnación argéntica se hizo por goteo en cámara húmeda. Se demostraron en forma constante, precisa y seriada terminaciones nerviosas y corpúsculos sensoriales, neuronas y fibras nerviosas periféricas. A pesar de la alta especificidad para fibras nerviosas, la técnica no compromete el panorama tisular por lo que da bellas imágenes de conjunto. Sin ser una técnica para argentafinidad, demuestra claramente dos tipos de células argentafines en las glándulas adrenales. La adición de los reactivos metálicos en gotas y en cámara húmeda, ofrece una variante sumamente económica.
In Biological research is still necessary for the demonstration of the peripheral innervation in numerous tissues and organs. The aim of this study was to rescue and modernize one of the most consistent methods that we have tried to demonstrate peripheral innervation. Llombart's technique for nerve fibers was adapted by paraffin cuts of 7 µm in different animal tissue. The silver impregnation was done by dripping in a moist chamber. It was demonstrated in a constant, precise and serial form, nerve terminations, and sensorial corpuscles, neurons, and peripheral nerve fibers. Despite being highly specific to nerve fibers, the technique does not sacrifice tissue panorama so it gives beautiful images set. Without being a technique to argentaffin structures, it clearly shows two types of argentaffin cells in the adrenal glands. The addition of the metal reactive in droplets and in a humid chamber provides a very economical variant.
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Animales , Nervios Periféricos , Tinción con Nitrato de Plata/métodos , Células Enterocromafines , Fibras NerviosasRESUMEN
INTRODUCTION: Luting posts inside the root canal is still a challenge because of the difficulty of bonding adhesive materials in the apical third of roots. This study evaluated the effect of the application mode of 3 simplified etch-and-rinse adhesives on the push-out bond strength (PBS), nanoleakage (NL), and in situ degree of conversion (DC) of fiber posts in the root canal. METHODS: The roots of human premolars were endodontically prepared and divided into 6 groups according to the combination of the main factors: adhesive (Ambar, FGM, Joinville, SC, Brazil; Adper Single Bond 2, 3MESPE, St Paul, MN; and XP Bond+self-cure activator, DeTrey Dentsply, Konstanz, Germany) and application mode (manual or sonic). The posts were cemented and the PBS tested at 0.5 mm/min. The NL was evaluated by scanning electron microscopy after the immersion of specimens in 50% silver nitrate. Micro-Raman spectroscopy was used to measure the in situ DC. Root third was also considered in the statistical evaluation. Data were analyzed by 3-way repeated measures analysis of variance and Tukey tests (5%). RESULTS: Under sonic application, the PBS and the in situ DC increased, whereas NL decreased significantly for all groups in the middle and apical thirds (P < .05). CONCLUSIONS: The application of simplified adhesives by sonic mode in the root canal is a feasible tool to increase the fiber post bond to root canals.
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Recubrimiento Dental Adhesivo/métodos , Cavidad Pulpar/ultraestructura , Recubrimientos Dentinarios/química , Técnica de Perno Muñón/instrumentación , Cementos de Resina/química , Sonicación/métodos , Bisfenol A Glicidil Metacrilato/química , Cementación/métodos , Cementos Dentales/química , Filtración Dental/clasificación , Análisis del Estrés Dental/instrumentación , Humanos , Ensayo de Materiales , Metacrilatos/química , Microscopía Electrónica de Rastreo , Polietilenglicoles/química , Polimerizacion , Ácidos Polimetacrílicos/química , Distribución Aleatoria , Preparación del Conducto Radicular/métodos , Tinción con Nitrato de Plata/métodos , Espectrometría Raman , Estrés Mecánico , Ápice del Diente/ultraestructura , Diente no Vital/patologíaRESUMEN
OBJECTIVE: To evaluate the effects of simulated aging in bond strength and nanoleakage of class II restorations using three different restorative techniques. MATERIALS AND METHODS: Class II preparations (n = 12) were restored using: FS - composite resin Filtek Supreme Plus (3M/ESPE); RMGIC + FS - resin-modified glass ionomer cement Vitrebond Plus (3M/ESPE) + FS; and FFS + FS - flowable composite resin Filtek Supreme Plus Flowable (3M ESPE) + FS. The teeth were assigned into two groups: Control and Simulated Aging - Thermal/Mechanical cycling (3,000 cycles, 20-80 °C/500,000 cycles, 50 N). From each tooth, two slabs were assessed to microtensile bond strength test (µTBS) (MPa), and two slabs were prepared for nanoleakage assessment, calculated as penetration along the restoration margin considering the penetration length (%) and as the area of silver nitrate particle deposition (µm(2)). Data were analyzed by two-way analysis of variance (ANOVA) followed by Tukey's post hoc test (p < 0.05). RESULTS: FS presented the highest µTBS to dentin (22.39 ± 7.55 MPa) after simulated aging, while the presence of flowable resin significantly decreased µTBS (14.53 ± 11.65 MPa) when compared to no aging condition. Both control and aging groups of RMGIC + FS presented the highest values of silver nitrate penetration (89.90 ± 16.31 % and 97.14 ± 5.76 %) and deposition area (33.05 ± 12.49 and 28.08 ± 9.76 µm(2)). Nanoleakage was not affected by simulated aging. CONCLUSIONS: FS presented higher bond strength and lower nanoleakage and was not affected by simulated aging. Use of flowable resin compromised the bond strength after simulated aging. CLINICAL RELEVANCE: The use of an intermediate layer did not improve the dentin bond strength neither reduced nanoleakage at the gingival margins of class II restorations under simulated aging conditions.
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Recubrimiento Dental Adhesivo , Preparación de la Cavidad Dental/clasificación , Filtración Dental/clasificación , Restauración Dental Permanente/métodos , Bisfenol A Glicidil Metacrilato/química , Resinas Compuestas/química , Preparación de la Cavidad Dental/instrumentación , Restauración Dental Permanente/clasificación , Cementos de Ionómero Vítreo/química , Humanos , Curación por Luz de Adhesivos Dentales , Ensayo de Materiales , Cementos de Resina/química , Tinción con Nitrato de Plata/métodos , Estrés Mecánico , Temperatura , Resistencia a la Tracción , Factores de Tiempo , Agua/químicaRESUMEN
The medial nucleus (Me) is a superficial component of the amygdaloid complex. Here we assessed the density and morphology of the neurons and glial cells, the glial fibrillary acidic protein (GFAP) immunoreactivity, and the ultrastructure of the synaptic sites in the human Me. The optical fractionator method was applied. The Me presented an estimated mean neuronal density of 1.53 × 105 neurons/mm³ (greater in the left hemisphere), more glia (72% of all cells) than neurons, and a nonneuronal:neuronal ratio of 2.7. Golgi-impregnated neurons had round or ovoid, fusiform, angular, and polygonal cell bodies (10-30 µm in diameter). The length of the dendrites varied, and pleomorphic spines were found in sparsely spiny or densely spiny cells (1.5-5.2 spines/dendritic µm). The axons in the Me neuropil were fine or coarsely beaded, and fibers showed simple or notably complex collateral terminations. The protoplasmic astrocytes were either isolated or formed small clusters and showed GFAP-immunoreactive cell bodies and multiple branches. Furthermore, we identified both asymmetrical (with various small, clear, round, electron-lucent vesicles and, occasionally, large, dense-core vesicles) and symmetrical (with small, flattened vesicles) axodendritic contacts, also including multisynaptic spines. The astrocytes surround and may compose tripartite or tetrapartite synapses, the latter including the extracellular matrix between the pre- and the postsynaptic elements. Interestingly, the terminal axons exhibited a glomerular-like structure with various asymmetrical contacts. These new morphological data on the cellular population and synaptic complexity of the human Me can contribute to our knowledge of its role in health and pathological conditions.
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Amígdala del Cerebelo/citología , Astrocitos/ultraestructura , Neuronas/ultraestructura , Sinapsis/ultraestructura , Anciano , Anciano de 80 o más Años , Astrocitos/metabolismo , Axones/ultraestructura , Recuento de Células , Forma de la Célula , Colorantes , Dendritas/ultraestructura , Espinas Dendríticas/ultraestructura , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Fenotiazinas , Tinción con Nitrato de Plata/métodos , Vesículas Sinápticas/ultraestructuraRESUMEN
The aim of this study was to apply immunohistochemistry (IHC) for the diagnosis of canine sporotrichosis and to compare this method with the Grocott's silver stain (GSS) and periodic acid Schiff (PAS) techniques. Eighty-seven dogs with sporotrichosis (group 1) and 35 with American tegumentary leishmaniosis (ATL) (group 2) were studied. The fungus was detected in group 1 by GSS, PAS and IHC. IHC was also applied to group 2 to evaluate the occurrence of cross-reactions. PAS, GSS and IHC detected yeast cells in 19.5%, 43.7% and 65.5% of the group 1 cases, respectively. The detection of intracellular antigens of Sporothrix schenckii by IHC increased the sensitivity of the histological diagnosis to 80.5%. No positive reaction was observed in ATL lesions. The results suggest that IHC may be indicated for the diagnosis of sporotrichosis because of its higher diagnostic sensitivity.
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Enfermedades de los Perros/diagnóstico , Inmunohistoquímica/veterinaria , Esporotricosis/veterinaria , Animales , Antígenos Bacterianos/aislamiento & purificación , Reacciones Cruzadas , Perros , Inmunohistoquímica/métodos , Leishmaniasis Cutánea/veterinaria , Reacción del Ácido Peryódico de Schiff/métodos , Reacción del Ácido Peryódico de Schiff/veterinaria , Sensibilidad y Especificidad , Tinción con Nitrato de Plata/métodos , Tinción con Nitrato de Plata/veterinaria , Piel/microbiología , Sporothrix/inmunología , Esporotricosis/diagnósticoRESUMEN
The presence of lingual papillae and the nerve endings in the middle region of the tongue mucosa of collared peccary (Tayassu tajacu) were studied using scanning electron microscopy and light microscopy, based upon the silver impregnation method. The middle region of tongue mucosa revealed numerous filiform and fungiform papillae. The thick epithelial layer showed epithelial cells and a dense connective tissue layer containing nerve fibre bundles and capillaries. The sensory nerve endings, intensely stained by silver impregnation, were usually non-encapsulated and extended into the connective tissue of the filiform and fungiform papillae very close to the epithelial cells. In some regions, the sensory nerves fibres formed a dense and complex network of fine fibrils. The presence of these nerve fibrils may characterize the mechanisms of transmission of sensitive impulses to the tongue mucosa.
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Encía/ultraestructura , Mamíferos/anatomía & histología , Terminaciones Nerviosas/ultraestructura , Lengua/ultraestructura , Animales , Tejido Conectivo/irrigación sanguínea , Tejido Conectivo/inervación , Células Epiteliales , Femenino , Encía/inervación , Masculino , Microscopía Electrónica de Rastreo , Tinción con Nitrato de Plata/métodos , Lengua/inervaciónRESUMEN
The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The "single-section" Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, "sandwiched" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the "single-section" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results.
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Encéfalo/citología , Microtomía/métodos , Neuroanatomía/métodos , Tinción con Nitrato de Plata/métodos , Fijación del Tejido/métodos , Anciano , Artefactos , Encéfalo/fisiología , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Formaldehído/química , Humanos , Masculino , Persona de Mediana Edad , Neuroglía/citología , Neuroglía/fisiología , Neuronas/citología , Neuronas/fisiología , Tetróxido de Osmio/química , Cambios Post Mortem , Dicromato de Potasio/química , Nitrato de Plata/química , Factores de TiempoRESUMEN
beta-Amyloid plays an important role in the neurodegeneration process of Alzheimer's disease (AD), but its neurotoxic mechanisms are not clear. It has been associated with the increase of oxidative stress and cognitive impairment because the beta-amyloid peptide 25-35 (Abeta((25-35))) has the critical neurotoxic properties of the full-length Abeta(1-42). Our present study shows the role of Abeta((25-35)) when injected into the temporal cortex on the nitric oxide pathways, 3-nitrotyrosine, neuronal death, and the spatial memory of rats 1 month after the injection. Our data showed that Abeta((25-35)) increases oxidative stress, causes neuronal damage, and decreases spatial memory in rats. Notably, the injection of the fraction Abeta((25-35)) caused an increase of nNOS and iNOS immunoreactivity in the temporal cortex and hippocampus. We demonstrated a significant increase of reactive astrocytosis, which was accompanied by neuronal damage in the temporal cortex and hippocampus of rats injected with Abeta((25-35)). These data suggest that the fraction Abeta((25-35)) injected into the temporal cortex might contribute to understanding the role of nitric oxide on the biological changes related to the neuropathological progression and the memory impairment in AD.
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Péptidos beta-Amiloides , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/patología , Óxido Nítrico/metabolismo , Fragmentos de Péptidos , Lóbulo Temporal/enzimología , Animales , Conducta Animal/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ratas , Ratas Wistar , Tinción con Nitrato de Plata/métodos , Percepción Espacial/efectos de los fármacos , Lóbulo Temporal/efectos de los fármacosRESUMEN
In recent years, we have used a variety of tau immunological markers combined with the dye thiazin red (TR), an accurate marker to differentiate the fibrillar from the nonfibrillar state of both amyloid-beta and tau in Alzheimer's disease (AD). In this study, we used TR as a potential diagnostic marker of AD in frozen-thawed (F-T) brain tissue and imprint cytology. Control experiments included the use of Thioflavin-S staining, fixed tissue, and some double-labeled material with TR and selected tau markers, including AT100, MC1, Alz-50, TG-3, Tau-C3, and S396. Our results indicate that TR retains its strong affinity for both tangles and plaques in unfixed F-T tissue and imprint cytology. This information provides a potential use of TR as an accurate diagnostic tool for the rapid postmortem diagnosis of AD neuropathology. This study shows the advantages of TR on cytology mainly because tools for the fast postmortem diagnosis of AD are practically nonexistent. In addition, we observed Tau-C3 immunoreactivity in extracellular tangles, suggesting that the Tau-C3 epitope is characteristically stable. Moreover, this study demonstrates that chemical fixation is not necessarily required for tau immunoreactivity on histological sections.
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Enfermedad de Alzheimer/diagnóstico , Encéfalo/patología , Neuronas/patología , Placa Amiloide/patología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Benzotiazoles , Encéfalo/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Persona de Mediana Edad , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Neuronas/metabolismo , Compuestos Orgánicos/metabolismo , Placa Amiloide/metabolismo , Cambios Post Mortem , Tinción con Nitrato de Plata/métodos , Tiazoles/metabolismo , Proteínas tau/metabolismoRESUMEN
The anterodorsal (MeAD) and posteroventral (MePV) subnuclei would form the proposed "ventral" division of the rat medial nucleus of the amygdala (MeA). These parts receive chemosensorial inputs, have gonadal hormone receptors and modulate hypothalamic neuroendocrine secretion and defensive/reproductive behaviors. The aims of this study were: (1) to provide further data on the morphology of Golgi-impregnated dendrites from the MeAD and the MePV of adult rats; and (2) to compare the results obtained for dendritic branching and predominant dendritic spatial distribution in both these subnuclei in males and diestrus females. Dendritic arborization levels, number of branches in each level, distribution of dendrites around the cell body and distally from it, and the preferred spatial distribution of dendritic branches were studied using different techniques and compared between sexes. MeAD and MePV multipolar neurons had spiny dendrites with sparse ramifications. The main statistically significant differences were found in the predominant dendritic spatial distribution in the MeAD (rather medially and laterally in males and ventromedially in females, p<0.02) and in the MePV (rather medially and mediodorsally in males and ventrally in females, p<0.01). Results suggest that synaptic information might be processed and integrated differently in the dendrites of males and females in these sex steroid-responsive MeA subnuclei. The inclusion of the MeAD and the MePV in one single "ventral" MeA division is further discussed.
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Amígdala del Cerebelo/citología , Dendritas/ultraestructura , Neuronas/ultraestructura , Caracteres Sexuales , Tinción con Nitrato de Plata/métodos , Animales , Femenino , Masculino , Ratas , Ratas WistarRESUMEN
Successful reproduction requires that changes in plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), oxytocin (OT), estrogen (E(2)) and progesterone (P(4)) occur together with the display of maternal behaviors. Ovarian steroids and environmental stimuli can affect the dendritic spines in the rat hippocampus. Here, studying Wistar rats, it is described: (a) the sequential and concomitant changes in the hormonal profile of females at postpartum days (PP) 4, 8, 12, 16, 20 and 24, comparing to estrous cycle referential values; (b) the dendritic spine density in the stratum radiatum of CA1 (CA1-SR) Golgi-impregnated neurons in virgin females across the estrous cycle and in multiparous age-matched ones; and (c) the proportion of different types of spines in the CA1-SR of virgin and postpartum females, both in diestrus. Plasma levels of gonadotrophins and ovarian hormones remained low along PP while LH increased and PRL decreased near the end of the lactating period. The lowest dendritic spine density was found in virgin females in estrus when compared to diestrus and proestrus phases or to postpartum females in diestrus (p<0.03). Other comparisons among groups were not statistically significant (p>0.4). There were no differences in the proportions of the different spine types in nulliparous and postpartum females (p>0.2). Results suggest that medium layer CA1-SR spines undergo rapid modifications in Wistar females across the estrous cycle (not quite comparable to Sprague-Dawley data or to hormonal substitutive therapy following ovariectomy), but persistent effects of motherhood on dendritic spine density and morphology were not found in this area.
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Dendritas/fisiología , Hipocampo/citología , Hormonas/sangre , Neuronas/citología , Periodo Posparto/sangre , Periodo Posparto/fisiología , Animales , Dendritas/clasificación , Dendritas/ultraestructura , Ciclo Estral/sangre , Femenino , Hipocampo/fisiología , Procesamiento de Imagen Asistido por Computador , Embarazo , Ratas , Ratas Wistar , Tinción con Nitrato de Plata/métodos , Factores de TiempoRESUMEN
OBJECTIVE: To determine the cell proliferation rate and possible effects of cigarette smoking on the oral mucosa lining through analysis of silver-stained nucleolar organizer regions (AgNORs) in exfoliative cytology specimens. STUDY DESIGN: Exfoliative cytology was performed on the left side of the border of the tongue and of the floor of the mouth in 25 smoking patients and 25 nonsmoking patients. The inclusion criterion for smokers was the consumption of more than 20 cigarettes per day for a minimum of 30 years. RESULTS: The slides were stained by histochemical AgNOR method. In the nonsmoking group the mean number of AgNORs per nucleus was 2.732 +/- 0.236 in the tongue border and 2.918 +/- 0.195 in the floor of the mouth. In smoking patients the mean number of AgNORs per nucleus was 3.372 +/- 0.375 in the tongue border and 3.245 +/- 0.237 in the floor of the mouth. CONCLUSION: The results suggest higher cell proliferation quantified by the histochemical AgNOR technique in exfoliative cytology specimens obtained from the oral mucosa lining of smokers presenting no clinical alterations.
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Antígenos Nucleares/análisis , Citodiagnóstico/métodos , Mucosa Bucal/química , Mucosa Bucal/citología , Región Organizadora del Nucléolo/química , Tinción con Nitrato de Plata/métodos , Fumar , Adulto , Anciano , Proliferación Celular , Humanos , Persona de Mediana Edad , Región Organizadora del Nucléolo/ultraestructura , Fumar/epidemiologíaRESUMEN
The posterodorsal portion of the medial amygdalar nucleus (MePD) contains receptors for gonadal hormones and modulates the function of a social behavior network in rodents. The aims of this study were: to provide further data about the morphology of Golgi-impregnated dendrites of neurons from the MePD of adult rats; and, to compare the results obtained for dendritic branching and predominant dendritic spatial distribution in the MePD of males and diestrus females. MePD neurons were classified as bitufted or stellate, their spiny dendrites showed variable lengths, divided sparingly and decreased the number of branches with the distance from the soma. Dendritic arborization levels, number of branches in each level, distribution of the dendrites around the cell body and away from it, and the preferred spatial distribution of dendritic branches were studied according to different techniques and compared between sexes. Statistically significant differences were found in the predominant dendritic spatial distribution in the MePD, males with branches more oriented medially and dorsolaterally and females with more dorsally and ventromedially ones (p< or =0.05 in all cases). This result adds another clue to understand how information is processed and integrated in the MePD and within functionally dynamic sex steroids-responsive circuits relevant for reproduction in both sexes.
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Amígdala del Cerebelo/citología , Dendritas/ultraestructura , Neuronas/citología , Caracteres Sexuales , Tinción con Nitrato de Plata/métodos , Animales , Femenino , Masculino , RatasRESUMEN
Induction of status epilepticus (SE) with kainic acid results in a large reorganization of neuronal brain circuits, a phenomenon that has been studied primarily in the hippocampus. The neurotrophin BDNF, by acting through its receptor TrkB, has been implicated in such reorganization. In the present work we investigated, by Western blot and immunohistochemistry, whether regional changes of TrkB expression within the rat brain cortex are correlated with altered neuronal morphology and/or with apoptotic cell death. We found that the full-length TrkB protein decreased within the cortex when measured 24 h to 1 week after induction of SE. Analysis by immunohistochemistry revealed that TrkB staining diminished within layer V of the retrosplenial granular b (RSGb) and motor cortices, but not within the auditory cortex. In layer II/III, differential changes were also observed: TrkB decreased in the motor cortex, did not change within the RSGb but increased within the auditory cortex. Reduced TrkB was associated with dendritic atrophy and decreased spine density in pyramidal neurons within layer V of the RSGb. No correlation was observed between regional and cellular changes of TrkB protein and apoptosis, measured by the TdT-mediated dUTP nick end labeling (TUNEL) method. The global decrease of TrkB within the neocortex and the associated dendritic atrophy may counteract seizure propagation in the epileptic brain but may also underlie cognitive impairment after seizures.
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Corteza Cerebral/patología , Espinas Dendríticas/patología , Neuronas/patología , Receptor trkB/metabolismo , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Animales , Espinas Dendríticas/ultraestructura , Modelos Animales de Enfermedad , Etiquetado Corte-Fin in Situ/métodos , Ácido Kaínico , Masculino , Ratas , Ratas Sprague-Dawley , Tinción con Nitrato de Plata/métodos , Estadísticas no Paramétricas , Estado Epiléptico/inducido químicamente , Factores de TiempoRESUMEN
Silver stain offers the possibility to stain comets permanently, but up to now it was impossible to measure the majority of the comet parameters, because the distinction between head and tail was not recognised by software. Here, we report a silver staining protocol that allows the measurement of comet parameters using the free Internet software CASP. We validated the silver stain protocol by comparing the behaviour of the parameter '% DNA in tail' in silver and fluorescent stained comets. The range of % DNA in tail for different visual categories of damage in silver stained comets was similar to that reported with fluorescence staining. The range was for category 0 (no damage), <1%; category 1 (low damage), 1-25%; category 2 (medium damage), >25-45%; category 3 (high damage), >45-70%; category 4 (very high damage), >70%. The mean of % DNA in tail in silver stained comets was also similar to that reported with fluorescence staining. The mean was for category 0, 0.4+/-0.34%; category 1, 12+/-7%; category 2, 37+/-4%; category 3, 57+/-5% and category 4, 83+/-6%. Others comet parameters such as tail length, tail moment and Olive tail moment can be also measured. The silver staining protocol reported here opens new opportunities for those working in the assay without fluorescent microscope as the measurement of comet parameters using free Internet software and conventional microscope becomes possible.
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Ensayo Cometa/métodos , Daño del ADN/genética , Tinción con Nitrato de Plata/métodos , Programas Informáticos , Internet , Proyectos de Investigación , Sensibilidad y EspecificidadRESUMEN
Specimens of Leptodactylus mystacinus from Brazil were karyotyped with conventional and differential staining. The 2n = 22 karyotype is similar to that found for the majority of the Leptodactylus, the karyotypic conservatism also confirmed by the similarity of the replication banding patterns with those previously described. L. mystacinus has a small amount of C-banded heterochromatin, located mainly at the centromeres, although telomeric or interstitial bands have also been noticed. With DA/CMA(3) some chromosome regions showed slightly bright fluorescence, and with DA/DAPI, no particular AT-rich repetitive region was observed. Silver staining showed an extensive inter- and intraindividual variation in the number and position of Ag-positive regions, in 1p, 4p, 8p, 8q, and 11p. Nevertheless, FISH using rDNA probes confirmed only the signals on the short arms of chromosomes 4 and 8 as true NORs. The remaining silver stained regions are probably due to the heterochromatin with some affinity to the Ag-staining. Phylogenetic analysis based on partial cytochrome b sequence revealed that L. mystacinus forms a basal branch, so that the presence of multiple NORs in pairs 4 and 8 in this species indicates an autapomorphy.