Cellular and molecular effects of Baccharis dracunculifolia D.C. and Plectranthus barbatus Andrews medicinal plant extracts on retinoid metabolism in the human hepatic stellate cell LX-2.

BACKGROUND: Chronic hepatic diseases are serious problems worldwide, which may lead to the development of fibrosis and eventually cirrhosis. Despite the significant number of people affected by hepatic fibrosis, no effective treatment is available. In the liver, hepatic stellate cells are the major fibrogenic cell type that play a relevant function in chronic liver diseases. Thus, the characterization of components that control the fibrogenesis in the hepatic stellate cells is relevant in supporting the development of innovative therapies to treat and/or control liver fibrosis. The present study investigated the effects of Baccharis dracunculifolia D.C. and Plectranthus barbatus Andrews medicinal plant extracts in LX-2 transdifferentiation. METHODS: LX-2 is a human immortalized hepatic stellate cell that can transdifferentiate in vitro from a quiescent-like phenotype to a more proliferative and activated behavior, and it provides a useful platform to assess antifibrotic drugs. Then, the antifibrotic effects of hydroalcoholic extracts of Baccharis dracunculifolia and Plectranthus barbatus medicinal plants on LX-2 were evaluated. RESULTS: The results in our cellular analyses, under the investigated concentrations of the plant extracts, indicate no deleterious effects on LX-2 metabolism, such as toxicity, genotoxicity, or apoptosis. Moreover, the extracts induced changes in actin filament distribution of activated LX-2, despite not affecting the cellular markers of transdifferentiation. Consistent effects in cellular retinoid metabolism were observed, supporting the presumed activity of the plant extracts in hepatic lipids metabolism, which corroborated the traditional knowledge about their uses for liver dysfunction. CONCLUSION: The combined results suggested a potential hepatoprotective effect of the investigated plant extracts reinforcing their safe use as coadjuvants in treating imbalanced liver lipid metabolism.

An ultrastructural approach for gill responses after pollutants exposure / Un enfoque ultraestructural de las respuestas branquiales después de la exposición a contaminantes

SUMMARY: Morphological and physiological responses of gills exposed to environmental contaminants are widely described in the literature. Among the primary findings are the lamellar fusion, hyperplasia, aneurysms, proliferation of mitochondria-rich cells and mucous cells. This work has as main objectives, to show the main changes caused by a polluted lake in gills, and understand how each gill's cell type responds to pollutants' exposure. To those individuals of the species, Astyanax altiparanae were exposed to water from an urban lake, which receives such contaminants from various sources. The gills were analyzed under a TEM. Our results showed that prior to these changes, commonly found, only the mucous cells did not show structural changes. Among the main results, we observed the emergence of Rodlet cells in the group exposed to contaminants, as well as severe damage to the mitochondria-rich cells. The latter result was extremely relevant for demonstrating that the proliferation of this cell type, widely described as an alternative ionic regulation, may in some cases be completely inefficient because structurally these cells are no longer able to perform their basic metabolic functions.

RESUMEN: Las respuestas morfológicas y fisiológicas de las branquias expuestas a contaminantes ambientales se describen ampliamente en la literatura. Entre los hallazgos principales se encuentran la fusión laminar, la hiperplasia, los aneurismas, la proliferación de células ricas en mitocondrias y las células mucosas. Este trabajo tiene como objetivo principal, mostrar los principales cambios en las branquias, causados por un lago contaminado y comprender cómo responde el tipo de célula de las branquias a la exposición de los contaminantes. Individuos de la especie Astyanax altiparanae fueron expuestos al agua de un lago urbano, receptor de contaminantes de diversas fuentes. Las branquias se analizaron bajo un TEM. Nuestros resultados mostraron que antes de estos cambios, comúnmente encontrados, solo las células mucosas no mostraban cambios estructurales. Entre los resultados principales, observamos la aparición de células Rodlet en el grupo expuesto a contaminantes, así como el daño severo a las células ricas en mitocondrias. Este último resultado fue extremadamente relevante para demostrar que la proliferación de este tipo de células, ampliamente descrito como una regulación iónica alternativa, en algunos casos puede ser completamente ineficiente porque estructuralmente estas células ya no son capaces de realizar sus funciones metabólicas básicas.

Physical training reverses changes in hepatic mitochondrial diameter of Alloxan-induced diabetic rats.

OBJECTIVE: To investigate the effects of physical training on metabolic and morphological parameters of diabetic rats. METHODS: Wistar rats were randomized into four groups: sedentary control, trained control, sedentary diabetic and trained diabetic. Diabetes mellitus was induced by Alloxan (35mg/kg) administration for sedentary diabetic and Trained Diabetic Groups. The exercise protocol consisted of swimming with a load of 2.5% of body weight for 60 minutes per day (5 days per week) for the trained control and Trained Diabetic Groups, during 6 weeks. At the end of the experiment, the rats were sacrificed and blood was collected for determinations of serum glucose, insulin, albumin and total protein. Liver samples were extracted for measurements of glycogen, protein, DNA and mitochondrial diameter determination. RESULTS: The sedentary diabetic animals presented decreased body weight, blood insulin, and hepatic glycogen, as well as increased glycemia and mitochondrial diameter. The physical training protocol in diabetic animals was efficient to recovery body weight and liver glycogen, and to decrease the hepatic mitochondrial diameter. CONCLUSION: Physical training ameliorated hepatic metabolism and promoted important morphologic adaptations as mitochondrial diameter in liver of the diabetic rats.

Physical training reverses changes in hepatic mitochondrial diameter of Alloxan-induced diabetic rats / Treinamento físico reverte alterações no diâmetro de mitocôndrias hepáticas de ratos diabéticos induzidos pela Aloxana

ABSTRACT Objective To investigate the effects of physical training on metabolic and morphological parameters of diabetic rats. Methods Wistar rats were randomized into four groups: sedentary control, trained control, sedentary diabetic and trained diabetic. Diabetes mellitus was induced by Alloxan (35mg/kg) administration for sedentary diabetic and Trained Diabetic Groups. The exercise protocol consisted of swimming with a load of 2.5% of body weight for 60 minutes per day (5 days per week) for the trained control and Trained Diabetic Groups, during 6 weeks. At the end of the experiment, the rats were sacrificed and blood was collected for determinations of serum glucose, insulin, albumin and total protein. Liver samples were extracted for measurements of glycogen, protein, DNA and mitochondrial diameter determination. Results The sedentary diabetic animals presented decreased body weight, blood insulin, and hepatic glycogen, as well as increased glycemia and mitochondrial diameter. The physical training protocol in diabetic animals was efficient to recovery body weight and liver glycogen, and to decrease the hepatic mitochondrial diameter. Conclusion Physical training ameliorated hepatic metabolism and promoted important morphologic adaptations as mitochondrial diameter in liver of the diabetic rats.

RESUMO Objetivo Investigar os efeitos do treinamento físico nos parâmetros morfológicos e metabólicos de ratos diabéticos. Métodos Ratos Wistar foram randomizados para quatro grupos: controle sedentário, controle treinado, diabético sedentário e diabético treinado. Diabetes mellitus foi induzido por administração de Aloxana (35mg/kg) nos Grupos Diabético Sedentário e diabético treinado. O protocolo de treinamento físico incluiu natação com carga de 2,5% do peso corporal, por 60 minutos por dia (5 dias por semana) para os Grupos Controle Treinado e diabético treinado, durante 6 semanas. Ao final do experimento, os ratos foram sacrificados, e o sangue foi coletado para determinação das concentrações séricas de glicose, insulina, albumina e proteínas totais. Amostras do fígado foram coletadas para determinação do glicogênio, proteínas, DNA e diâmetro mitocondrial. Resultados O Grupo Sedentário Diabético apresentou redução no peso corporal, insulinemia e glicogênio hepático, além de maior glicemia e diâmetro mitocondrial hepático. O protocolo de treinamento físico em animais diabéticos foi eficiente para restaurar o peso corporal e o glicogênio hepático, além de reduzir o diâmetro mitocondrial hepático. Conclusão O treinamento físico melhorou o metabolismo hepático e promoveu importantes adaptações morfológicas, como no diâmetro mitocondrial no fígado de animais diabéticos.

Quantifying structural modifications of gills of two fish species Astyanax altiparanae (Lambari) and Prochilodus lineatus (Curimbatá) after exposure to biodegradable detergents in urban lake water.

Anthropic actions in rivers and urban lakes are a cause for concern to our ecosystem. The effects on fauna and flora of substances discharged into waterways have become a focus for investigations globally. Biodegradable detergents are widely used in residences and small industries, but little is known regarding the consequences on fish fauna. The objective of the present study was to identify modifications in gill structure in two fish species, Astyanax altiparanae and Prochilodus lineatus, after treatment with water obtained from an urban lake and an exposure to 1 ppm diluted biodegradable detergents (linear alkylbenzene sulfonate). Data demonstrated exposure to urban lake produced various alterations in gill functions such as lamellar fusions, aneurysms, mucous, and chlorine cell proliferation, which may be attributed to the presence of detergents in the water but may also be a consequence of synergetic actions of detergents with other pollutants. Results showed that the levels of NO-2, Na, F-, Cl-, and Fe were significantly higher in urban lake water but in the presence of detergents Ni was also detected. Evidence indicates that biodegradable detergents produce damage to gill functions, which subsequently alters the fish physiology and reduces the ability to cope with stress and survival.

Effects of biodegradable detergents in the accumulation of lipofuscin (age pigment) in gill and liver of two neotropical fish species / Efectos de detergentes biodegradables en la acumulación de lipofuscina (pigmento de envejecimiento) en branquias e hígado de dos espécies de peces neotropicales

Currently there is a growing concern, in both population and governments, to identify the effects of substances commonly disposed of into rivers and lakes, on aquatic fauna and flora. Thus the objective of the present study was to verify effects of biodegradable detergents and water from an urban lake on gills and liver of two neotropical fish species of great economic importance, Astyanax altiparanae and Prochilodus lineatus. In order to do so, lipofuscin, also called the ageing pigment, was used as bioindicator. After one and five months of experiment both tissues accumulated this pigment. These data are discussed from physiological points of view, related with lipid peroxidation and mitochondrial damage.

Existe una preocupación creciente de la población y los gobiernos para identificar los efectos de substancias comúnmente arrojadas en ríos y lagos, sobre la fauna y flora acuática. El objetivo fue verificar los efectos de detergentes biodegradables y agua de un lago urbano sobre las branquias e hígado de dos especies de peces neo-tropicales de gran importancia económica, Astyanax altiparanae y Prochilodus lineatus. Analizamos los pigmentos de lipofuscina, también llamado pigmento de envejecimiento, el que fue utilizado como biomarcador. Después de uno y cinco meses de experimento, ambos tejidos acumulados con el pigmento fueron analizados. Los datos fueron discutidos desde el punto de vista fisiológico, relacionado con la peroxidación lipídica y daño mitocondrial.

Morphological analysis of colon goblet cells and submucosa in type I diabetic rats submitted to physical training.

Colon layers, especially the submucosa, as well as the secretion of goblet cells are extremely important for the functioning and transit of substances in this organ. However, the damages arising from type I diabetes and the effects of physical training, which plays crucial role in the treatment of this disease, are not yet known in these regions. To analyze the changes in colon submucosa and goblet cells of diabetic rats, as well as the effects of physical training, Wistar rats were divided into four groups: sedentary control, trained control, sedentary diabetic (SD), and trained diabetic (TD). The training protocol consisted of swimming for 60 min a day, 5 days per week, during 8 weeks. Colon samples were collected, processed, and evaluated by histochemical and ultrastructural techniques. Goblet and submucosa cells did not show alterations in shape, size, protein and carbohydrate content, in all treatment groups. Decreased amount of collagen fibers, however, was observed in the submucosa and lamina propria of SD rats, but this alteration was recovered in TDs. The ultrastructural analysis, in turn, revealed greater quantity of Golgi apparatus cisterns in SDs, distinctly than TDs, which showed improvement in this diabetic condition. Thus, physical training was responsible for the recovery of some important diabetic alterations, possibly improving the motility of substances in the large intestine. Nevertheless, it cannot be considered alone in the treatment of this disease, requiring the combined practice of other methods.

Effects of exposition to polluted environments on blood cells of the fish Prochilodus lineatus.

In this study, we investigated the effects of contaminated water on the blood parameters of the fish Prochilodus lineatus exposed during 7 and 20 days. Blood was collected with heparinized syringes, and blood smears were prepared and stained with Leishman stain. Slides were examined and photographed using a Leica light microscope. Total white blood cell counts and differential counts of thrombocytes and leukocytes were obtained for statistical analysis. The group exposed to water samples from Lago Azul exhibited an increase in the number of leukocytes and in the total number of white blood cells, suggesting that the chemical contaminants in this environment were acting similarly to antigens in this fish species, causing the proliferation of defense cells. In the group exposed to detergent during 20 days, the number of thrombocytes decreased. These results suggest that the variations in the number of leukocytes were indicators of environmental pollution and that biodegradable detergents may, after a certain time of exposure, affect vital functions in fish, such as coagulation and prevention of infections, which directly involves thrombocytes.

Histology, histochemistry and stereology of the adipose fin of Prochilodus lineatus.

The adipose fin is small, nonpared, and usually located medially between the dorsal and caudal fin. Its taxonomic occurrence is very restrict; thus, it represents an important trace for taxon distinction. As it does not play a known vital physiological roll and it is easily removed, it is commonly used in marking and recapture studies. The present study characterizes the adipose fin of Prochilodus lineatus, as it is poorly explored by the literature. The adipose fin consists basically of a loose connective core, covered by a stratified epithelium supported by collagen fibers. At the epithelium, pigmented cells and alarm substance cells are found. Despite the name, adipocytes or lipid droplets are not observed on the structure of the fin.

Ultramorphological features of the egg of Telmatoscopus albipunctatus (Williston) (Diptera, Psychodidae)

Ultramorphological features of the egg of Telmatoscopus albipunctatus (Williston) (Diptera, Psychodidae). Psychodidae flies, also known as sewage, sand and filter flies are important for medical and veterinary purposes. General information about life cycle and adult habits is available, but few species are known about the egg morphology. Therefore, in this study, the egg ultramorphology of Telmatoscopus albipunctatus (Williston, 1893) was analyzed by scanning electron microscopy to describe its structure, generating data for further comparison between different fly species and genera. General aspects of T. albipunctatus egg are similar to other Psychodidae; egg measuring approximately 0.4 mm in length and 0.1 mm in width. However, based on the continuous and discontinuous longitudinal ridge sculptures observed on the exochorion, which can be species-specific, we can infer that T. albipunctatus eggs can survive under dry or moist conditions, making their control much more difficult. Our data emphasize the advantages of the electron microscope approach in the study of the exochorion patterns. Eggshell morphology of T. albipunctatus can be used as basis for further studies and as a tool to compare different species of Psychodidae flies.

Aspectos ultramorfológicos do ovo de Telmatoscopus albipunctatus (Williston) (Diptera, Psychodidae). As moscas da família Psychodidae, também conhecidas como moscas de banheiro, são de importância médica e veterinária. Informações gerais sobre ciclo de vida e hábitos do adulto são facilmente encontradas, mas pouco se sabe sobre a morfologia do ovo. Dessa forma, neste estudo, a ultramofologia do ovo de Telmatoscopus albipunctatus (Williston, 1893) foi analisada por microscopia eletrônica de varredura com o objetivo de descrever sua estrutura, provendo importantes dados para futuras comparações entre moscas de diferentes espécies e gêneros. O aspecto geral do ovo de T. albipunctatus é similar a outros Psychodidae, medindo aproximadamente 0.4 mm de comprimento e 0.1 mm de largura. Entretanto, baseado nas esculturas contínuas e descontínuas do exocórion, as quais podem ser espécie-específicas, podemos inferir que os ovos de T. albipunctatus podem sobreviver sob condições de muita umidade ou seca, dificultando o controle da espécie. Nossos resultados ressaltam a importância do uso da microscopia eletrônica como uma ferramenta no estudo dos padrões do exocórion. A morfologia externa do ovo de T. albipunctatus pode ser usada como base para futuros estudos e como ferramenta para comparação de diferentes espécies de moscas do gênero Psychodidae.

Rhyncophorus palmarum L. (Linnaeus, 1758): a morphological and histological study of the female reproductive system.

Rhyncophorus palmarum (Coleoptera) is a pest of great economic importance because of the damage caused in the plants of the Palmae family. This study showed that the female reproductive system of this beetle is composed of two ovaries of the telotrophic meroistic type invariably containing two ovarioles per ovary. Each ovariole is home to about 50 oocytes at different stages of maturation. The proximal region of the ovary is rather peculiar because it houses inside it a large quantity of bacilliform structures immersed in a glycolipoprotein substance, which has never before been described in the literature. The function of these structures is still unknown. However, it is suggested that they can function as a method of neutralizing the resistance of the plant because these insects are responsible for the transmission of the red ring disease to the palm plants.

Efeitos em curto prazo da aplicação de aloxana para indução de diabetes em ratos Wistar / Effects in short-term of alloxan application to diabetes induction in Wistar rats

O presente estudo teve como objetivo verificar a ação da aloxana em parâmetros metabólicos e do sistema imune 24 e 192 horas após sua aplicação em ratos Wistar. Para isso, oito ratos foram mantidos em jejum de 12 horas e receberam aloxana monoidratada, Sigma (32 mg/kg de peso corporal) via endovenosa. Foram registrados os valores de massa corporal, ingestão hídrica e alimentar e realizada a contagem de leucócitos totais previamente, 24 e 192 horas após a aplicação da aloxana. As análises de glicemia e trigliceridemia foram realizadas previamente e 192 horas após a aplicação da aloxana. Após 24 horas, a aplicação da aloxana causou aumento da ingestão hídrica e redução da massa corporal, ingestão alimentar e contagem de leucócitos. Na avaliação realizada 192 horas após a aplicação da aloxana houve recuperação da ingestão alimentar e contagem de leucócitos. Por outro lado, neste período houve aumento da glicemia e ingestão hídrica além de redução da massa corporal. Isso indica que parte dos sinais de diabetes causados pela aloxanamanifesta-se em curto prazo da administração da droga.

The present study aimed to verify action of alloxan in metabolic and immune parameters after 24 and 192 hours of the injection in Wistar rats. Thus, eight rats were fasted and received monohidrated alloxan Sigma (32 mg/kg body weight) via endovenous. Glycemia and trglyceridemia analyzes were performed before and 192 hours after alloxan application. After 24 hours, alloxan application increased water intake and decreased body mass, food intake and leucocytes counting. 192 hours after alloxan application, there was a recuperation in food intake and leucocytes counting. On the other hand, in this period there was an increase of glycemia and water intake and reduction of body mass. These results indicate that some of diabetic signs caused by alloxan occur in short-term after drug administration.

Histochemical technique for the detection of chloride cells in fish.

Chloride cells are responsible for ionic exchanges in the fish gill. These cells have been widely studied, considering its importance in vital functions of the gill, and because they proliferate when exposed to unfavorable environments. One of the main characteristic of these cells is an acidic cytoplasm, which has been used for identification through histochemical techniques with dyes such as Toluidine Blue and Hematoxylin and Eosin. However, these techniques can be problematic, since epithelial cells can, in certain situations, acquire acidic characteristics similar to those of chloride cells, thus staining in a similar way. Among other functions, chloride cells play a role in calcium uptake from the environment, and therefore have a high concentration of this element. Based on this information, this study aims at developing a specific protocol for the identification of chloride cells. With this purpose, the Von Kossa method specific for calcium was used combined with Hematoxylin counterstaining. Chloride cells had cytoplasm slightly stained with Hematoxylin and the presence of dark stained granules dispersed in the cytoplasm resulted from the Von Kossa reaction due to the calcium present in these cells. This was not found in any other gill cell. Thus, the technique used in this study was specific and efficient to identify chloride cells in fish gills.

Efeitos do treinamento de natação em aspectos metabólicos e morfológicos de ratos diabéticos / Effects of swimming training on metabolic and morphologic aspects in diabetic rats

Fundamento: O exercício físico aeróbio influencia vários aspectos da patologia diabética, incluindo o desenvolvimento de cardiomiopatias. O modelo de diabetes induzido experimentalmente em ratos tem contribuído para avanços nesta linha de pesquisa. Objetivo: Estudar os efeitos de um protocolode treinamento físico aeróbio moderado em meio aquático sobre alguns aspectos metabólicos, histológicos e ultra-estruturais do miocárdio de ratos diabéticos experimentais. Métodos: Ratos da linhagem Wistar foram distribuídos em 4 grupos; controle sedentário (CS), controle treinado (CT), diabético sedentário (DS) e diabético treinado (DT). Para indução do diabetes experimental, os animais receberam Aloxana monoidratada Sigma (35mg/Kg de peso corporal i.v.). O programa de treinamento aeróbio consistiu em natação por 60 minutos diários, 5 dias/semana, durante 4 semanas consecutivas, com cargas equivalentes à 2,5% do peso corporal. Após o sacrifício dos animais, o sangue foi utilizado para dosagem de glicose e ácidos graxos livres (AGL). Amostras do miocárdio foram coletadas para análises histológicas, histoquímicas e ultra-estruturais. Resultados: O diabetes experimental causou aumento da glicemia e AGL séricos e redução da insulinemia. Os ratos diabéticos apresentaram aumento da glicose e da concentração de AGL séricos e alterações histológicas e histoquimicas no miocárdio. Por outro lado, o treinamento físico aeróbio restaurou algumas destas alterações. Conclusão: A análise dos resultados mostrou que a indução do diabetes por meio da droga aloxana promove alterações metabólicas, histoquímicas e ultra-estruturais no miocárdio de ratos e que o treinamento físico aeróbio moderado em meio aquático restaurou algumas destas alterações.

Background: Aerobic physical exercise influences several aspects of diabetic pathology, including cardiomyopathy development. The experimentally-induced diabetic rat model has contributed to advances in this line of research. Objective: The aim of the present study was to examine the effects of moderate aerobic physical training on the metabolic, histological and ultra structural aspects of myocardium in experimental diabetic rats. Methods: Wistar rats were divided into 4 groups, sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). Diabetes was induced by Alloxan (35 mg/kg b.w. i.v.). The aerobic training program consisted by swimming 5 days/week, 1 h/day, supportinga load of 2.5 % body weight for 4 weeks. At the end of the training period, the rats were sacrificed and theblood was collected for determinations of serum glucose and free fat acids (FFA). The myocardium wascollected for determinations of histological, histochemistry and ultra structural aspects. Results: Diabetes increased glucose and FFA concentrations in blood and induced histological and histoquimical alterations in myocardium. In the other hand, aerobic physical training was able to restore this alterations. Conclusion: Inconclusion experimental alloxan-induced diabetes promoted some metabolic, histological and ultra structural alterations in myocardium of rats and moderate aerobic physical training recuperates some ofthese alterations.

Ultramorphological characteristics of Chrysomya megacephala (Diptera, Calliphoridae) eggs and its eclosion.

Chrysomya blowflies are originally from Africa and Australasia and were introduced in the American Continent, probably as a sheep parasite, in the 1970s. These flies are extremely important for medical-sanitary purposes and are useful to forensic entomology being used as an indicative of decomposition time in human corpses. The morphology of the larval and pupal stages of some species has been already studied by different authors demonstrating that it is possible to identify them in premature stages. In this study, Chrysomya megacephala egg ultramorphology was analyzed to describe its structure, generating data for further comparison between different species and genera. The blowflies were collected in Rio Claro city, SP, Brazil. Flies were attracted by fish carcasses and maintained in net cages; small portions of minced meat were placed in plastic pots for egg laying. Eggs were collected, fixed in alcoholic Bouin, prepared according to SEM routine and observed under Philips scanning electron microscope. C. megacephala eggs are oval with one flat face and another convex, measuring approximately 0.52 (+/-0.03) mm of length and 0.12 (+/-0.02) mm of width. The micropyle is circular in shape, with two perforations enclosed by countless small projections and is located in one of the extremities in the interface between the flat and convex faces. On the convex surface hexagonal imprints were observed, this surface is impermeable to water and gases. The flat surface has numerous round projections with different sizes, creating a permeable surface with a thinner chorion from which the larvae hatches. The larvae presents itself all wrinkled and with numerous cuticular projections with a thorn shape facing the posterior region. The cuticle projections with a thorn shape from C. megacephala larvae are probably a vestige of the thorns found in larvae of parasite Diptera.

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.