Mononuclear phagocytes in the blood of turtles characterized by ultrastructural and cytochemical analyses and by phagocytic activity.

Ultrastructural and cytochemical characteristics of mononuclear phagocyte cells in turtles are not well described in the literature, especially in Phrynops hilarii. Thus, the aim of this study was to evaluate these characteristics in the mononuclear phagocyte cells and their phagocytic activity "in vitro" using the turtle P. hilarii as an experimental animal model. The six turtles used in the study were observed in two seasons, spring and summer. Results showed that mononuclear phagocytes incubated only in diluted solution or with colloidal charcoal have cytoplasm phagolysosomes. The cells incubated with colloidal charcoal and further exposed to the cytochemical reaction for acid beta-glycerophosphatase, showed cytoplasm phagolysosomes filled by charcoal particles being digested and some positively stained lysosomes. Acid beta-glycerophosphatase positive reaction was present in lysosomes and inside the phagolysosomes, while acid cytidine 5-monophosphatase staining occurred in lysosome surroundings. A positive reaction for trimetaphosphatase was also found inside phagolysosomes. In conclusion, the presence of lysosomal enzymes like trimetaphosphatase and cytidine-5'-sodium monophosphate, in the circulating blood of P. hilarii indicate that mononuclear phagocytes participate in the phagocytic process by gathering many phagocytic cells and forming multinucleated giant cells, which probably have a role in the blood "clearance" process.

Radioautographic study of the seasonal distribution of leukocytes in turtles Phrynops hilarii (Chelonia Chelidae).

The aim of this study was to present a morphological description of the leukocytes of Phrynops hilarii turtles according to the seasonal distribution of these cells and to show their replacement in the blood circulation using a radioautographic method. Five animals of both sexes weighing 600-1200 g were used. The animal's blood was aspirated, smeared on glass slides, and stained with the Romanowsky stain, and 500 cells of each animal were counted during each season. The results obtained were analyzed statistically by analysis of variance followed by the Bonferroni test (NCSS), with the level of significance set at p<0.05. The radioautographic analysis of turtle blood exposed to 1000 microCi of (3)H-thymidine and developed after 30 days showed a large number of silver grains incorporated into the cells, except for basophils, with cell renewal occurring every seven days. Quantitative data demonstrated a seasonal influence on the distribution of some leukocyte types, with the following "p" values: heterophils (p=0.0007), basophils (p=0.0002), monocytes (p=0.0016), eosinophils (p=0.0073). However, using this statistical method, it was not possible to detect a significant difference related to seasonal influence on lymphocytes (p=0.16295) or thrombocytes (p=0.1046). Using this experimental animal model, a seasonal influence on the distribution of some leukocyte types was observed, and the radioautographic method revealed a cell renewal system occurring every seven days, except for basophils.

Morphological Characterization of the Leukocytes in Circulating Blood of the Turtle (Phrynops hilarii) / Caracterización Morfológica de Leucocitos Circulantes en la Sangre de la Tortuga (Phrynops hilarii)

The Phrynops hilarii specie of turtle has its characterization not well defined in the literature, it was proposed in this study the leukocyte characterization of the blood, stained by Leishman and analyzed under light and transmission electron microscope. It was not observe any cellular type with similar characteristics to neutrophils in mammalian group. We believed, based on the data obtained in this study that the heterophils have a morphofuncional analogy with another neutrophils belonged to mammalian group. This conclusion is being supported in many recent studies found in the literature.

La especie de tortuga Phrynops hilarii no ha sido aún bien descrita en la literatura. Fue propuesto en este estudio la caracterización de leucocitos de sangre de este animal coloreados con el método de Leishman y analizados con microscopías de luz y electrónica. No fue observado ningún tipo celular con características similares a los neutrófilos de mamíferos. Los resultados indican que los heterófilos tienen analogía morfofuncional con otros neutrófilos presentes en el grupo de los mamíferos. Esta conclusión es sustentada por varios estudios recientes encontrados en la literatura.

Microwave Fixation in Rat Fetuses Tissues: Histological and Immunohistochemical Analysis / Fijación de tejidos de Fetos de Ratas por Microondas: Análisis Histológico e Inmunohistoquímico

The aim of this study was to show the microwaves action in fixation of rat fetuses, dermal and cartilaginous tissues, using histological and immunohistochemistry methods for analysis. It was possible to conclude in this study using the rat as experimental model that the two methods for antibody retrieval, presented an excellent ways for the use of Ki67 antibody in the immunohistochemical analysis.

El objetivo de este estudio fue evaluar la acción de las microondas en la fijación de los tejidos dérmico y cartilaginoso de fetos de ratas, usando para el análisis métodos histológico e inmunohistoquímico. Fue posible concluir en este estudio usando la rata como modelo experimental, que los dos métodos empleados para la recuperación antigénica representan excelentes medios para el uso del anticuerpo Ki67, en el análisis inmunohistoquímico.

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: an ultrastructural and cytochemical study.

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4 degrees C during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/+/-37 degrees C. In the first day, the specimens were irradiated 9 times and stored at 40 degrees C overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

Caracterización de los fagocitos mononucleares en la sangre de phrynops hilarii (Chelonia chelidae) / Characterization of blood mononuclear phagocytes in Phrynops hilarii (Chelonia chelidae)

The localization of peroxidase activity in different cell regions is used as a criterion for the classification of the stage of maturation of mammalian mononuclear phagocytes with a positive peroxidase reaction indicating the presence of monoblasts, promonocytes, monocytes and macrophages. In this study it was evaluated the peroxidase activity of blood mononuclear phagocytes of this turtle detected at different stages of differentiation. The present observations suggest that, in turtles, the differentiation of mononuclear phagocytes occur in the blood circulation, in contrast to animals, where only are monocytes in circulating blood and macrophage differentiation occurs in other body compartments.

La localization de la actividad de la peroxidasa en diversas regiones de la célula se utiliza como criterio para la clasificación de la etapa de maduración de fagocitos mononucleares. Una reacción positiva de peroxidasa indica la presencia de monoblastos, promonocitos, monocitos y macrófagos. En este estudio fue evaluada la actividad de la peroxidasa de los fagocitos mononucleares de la sangre de la tortuga Phrynops Hilarii detectada en diversas etapas de la diferenciación. Las actuales observaciones sugieren que, en tortugas, la diferenciación de fagocitos mononucleares ocurre en la circulación de la sangre, en contraste a los mamíferos, donde están solamente los monocitos en la sangre circulante y la diferenciación de los macrófagos ocurre en otras partes del cuerpo.

Dinámica de la descalcificación de tejido mineralizado de perros a través de microondas / Decalcification dynamic of dog mineralized tissue by microwaves

The aim of this article is to present the decalcification process dynamic of mineralized tissue in dogs, teeth and jaw, comparing the traditional decalcification method, immersion, and microwave, immersion followed by irradiation using a domestic microwave oven, accompanying the liberation of calcium through spectrophotometer of atomic absorption. It was used as decalcified agent, EDTA solution or nitric acid. The results showed that with the use of nitric acid (5 percent), after 15 days, the irradiated fragments could be processed for histological analysis, otherwise the tooth not irradiated need to be submerged for 65 days. The EDTA decalcified action was slower than the nitric acid. The histological observations of the irradiated samples showed an excellent preservation of the morphological characteristics, independently of the decalcified agent used.

El objetivo de este artículo fue presentar, un método de descalcificación dinámico de tejido mineralizado de perros, dientes y mandíbula, comparando el método de descalcificación tradicional e irradiación en horno de microondas, analizando la liberación de calcio en espectro fotómetro de absorción atómica. Usamos como agente descalcificante, solución de EDTA y acido nítrico. Los resultados mostraron que los fragmentos descalcificados con ácido nítrico después de 15 días, ya podían ser preparados para análisis histológico, el diente al ser irradiado tardó 65 días para ser descalcificado. El EDTA descalcificó lentamente, en relación al ácido nítrico. Las observaciones histológicas de las muestras irradiadas mostraron una excelente preservación de las características morfológicas independiente del agente descacificador usado.

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: an ultrastructural and cytochemical study

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4ºC during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/±37ºC. In the first day, the specimens were irradiated 9 times and stored at 40ºC overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

A preservação da estrutura de ossos é dependente da qualidade e da velocidade em que ocorre o processo de desmineralização. Neste estudo foi observada a ultraestrutura de maxila de rato descalcificada utilizando microondas. Ratos Wistar sofreram perfusão com paraformaldeído e o segmento de maxila retirado e fixado em glutaraldeído. Após esta etapa algumas amostras foram descalcificadas por imersão em solução de Warshawsky durante 45 dias a 4(0)C. Outras amostras foram submetidas a irradiação por microondas (forno de microondas doméstico 700 Watts de potência), durante 20 s/350 W/ ± 37ºC. No primeiro dia foram realizadas um total de 9 irradiações e os espécimes foram deixadas posteriormente a 4ºC por 12 h na solução descalcificadora sem agitação. No segundo dia, os fragmentos foram submetidos à nova irradiação totalizando 20 banhos, trocando-se a solução e o gelo a cada banho. A seguir algumas amostras foram pós-fixadas com tetróxido de ósmio e outras com tetróxido de ósmio e piroantimonato de potássio. As amostras foram observadas em microscópio eletrônico de transmissão. Os resultados mostraram que o processo de descalcificação ativado por microondas reduziu para 48 h o período de descalcificação, o qual pelo método tradicional ocorre em 45 dias.

Phagocytosis of PLGA microparticles in rat peritoneal exudate cells: a time-dependent study.

With the purpose of enhancing the efficacy of microparticle-encapsulated therapeutic agents, in this study we evaluated the phagocytic ability of rat peritoneal exudate cells and the preferential location of poly(d,l-lactide-co-glycolic acid) (PLGA) microparticles inside these cells. The microparticles used were produced by a solvent evaporation method and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Size distribution analysis using DLS and SEM showed that the particles were spherical, with diameters falling between 0.5 and 1.5 mum. Results from cell adhesion by SEM assay, indicated that the PLGA microparticles are not toxic to cells and do not cause any distinct damage to them as confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Among the large variety of cell populations found in the peritoneal exudates (neutrophils, eosinophils, monocytes, and macrophages), TEM showed that only the latter phagocytosed PLGA microparticles, in a time-dependent manner. The results obtained indicate that the microparticles studied show merits as possible carriers of drugs for intracellular delivery.

Estudo populacional relacionado à saúde geral e uso de medicamentes em idosos não institucionalizados e desdentados totais / Population survey related to general health and use of medication in one group of non-institutionalized complete edentulous elderly

O objetivo deste estudo foi conhecer o perfil de saúde geral, uso de medicamentos e sintomas bucais auto-referidos em um grupo de 307 idosos desdentados totais. Foram entrevistados 215 mulheres (70%) e 92 (30%) homens com idades entre 60 e 91 anos(média de 69) utilizando-se um questionário de saúde. A maioria dos idosos (88%) apresentava pelo menos uma doença pesquisada, sendo a hipertensão (56%) a mais comum. Outros problemas prevaleceram como artropatias (32%), osteoporose (28%),cardiopatias (27%) e diabetes (17%). Na amostra, 85% dos idosos consumiam algum tipo de medicamento com média de 2,92 tipos por indivíduo. Destacaram-se o uso dos anti-hipertensivos (38%), diuréticos (28%), complementos vitamínicos (20%), antiinflamatórios não-esteroidais (18%), hormônios de reposição (12%), vasodilatadoresperiféricos (11%), anticoagulantes (11%), hipoglicemiantes (11%), ansiolíticos (9%) e antidepressivos (7%) respectivamente. A mulheres tenderam a consumir mais medicamentos que os homens (88% vs. 76%). O grupo apresentou alta freqüência de doenças crônicas predominando as condições cardiovasculares e reumáticas, sendotambém freqüente a necessidade do tratamento medicamentoso cardiovascular e de controle da dor ou inflamação. A incidência de doenças crônicas, uso de medicamentos e politerapia medicamentosa foi maior nas mulheres que predominaram neste segmento populacional. A xerostomia foi o sintoma bucal mais comum, relatado por 93% dos usuários de medicamentos e sua possível causa era geralmente desconhecida pelo idoso

Cytochemical characterization of eosinophilic leukocytes circulating in the blood of the turtle (Chrysemys dorbignih).

Eosinophils and neutrophils are granulocytic leukocytes that are present in the blood of most vertebrates. Studies have been performed on lower vertebrates to understand the biological roles of the cells in defense mechanisms and to establish phylogenetic studies and new experimental models. Whether these 2 cell types exist in reptiles is a matter of controversy. In the blood of turtles there are 2 types of granulocytes that exhibit eosinophilia, one of them with round cytoplasmic granules and the other with elongated cytoplasmic granules. It has been suggested that these cells may be eosinophils in different stages of maturation but they also may be distinct cell types, i.e. eosinophils and neutrophils. In the present study, we characterized the 2 types of granulocytes that are present in the blood of Chrysemys dorbignih, using cytochemical techniques. Type I eosinophils showed activity of nonspecific esterase, peroxidase activity that is resistant to KCN, and basic proteins. Type II eosinophils exhibited activity of trimetaphosphatase, alkaline phosphatase, nonspecific esterase, peroxidase that is sensitive to KCN, and basic proteins. These observations indicate the existence of 2 distinct cell types in the blood of Chrysemys dorbignih, type I and type II eosinophils, that correspond to eosinophils and heterophils (neutrophils) of mammals and other vertebrates.

Apoptosis process in mouse Leydig cells during postnatal development.

The development of Leydig cells in mammals has been widely described as a biphasic pattern with two temporally mature Leydig cell populations, fetal stage followed by the adult generation beginning at puberty. In the present study, mouse Leydig cells were examined for apoptosis during postnatal testis development using electron microscopy and in situ DNA fragmentation by terminal deoxynucleotidyl transferase staining (TdT). Both the morphological study and the DNA fragmentation analysis showed that cellular death by apoptosis did not occur in Leydig cells during the neonatal, prepubertal, puberty, and adult periods. From these results, we suggest that the remaining fetal Leydig cells in the neonatal testis are associated with the involution or degeneration processes. In contrast, in the prepubertal and puberty stages, fragmentation of apoptotic DNA was detected in germ cells present in some seminiferous tubules.