Histopathological, ultrastructural, and immunohistochemical examination of changes in the placenta as a result of severe preeclampsia

Purpose: To investigate the role of hypoxia-inducible transcription factor-1 alpha (HIF-1α) and angiogenetic factor endothelin-1 (ET-1) expression in regulating hypoxia and placental development by routine histopathological methods. Methods: Twenty preeclamptic and normal placentas were used. Placenta tissue pieces were examined histopathologically after routine paraffin follow-ups. HIF-1α and ET-1 proteins were examined immunohistochemically, and placental tissues were examined ultrastructurally. Results: Increase in syncytial proliferation, endothelial damage in vessels, and increase in collagen were observed in preeclamptic placentas. As a result of preeclampsia, an increase was observed in HIF-1α and ET-1 protein levels in the placenta. Dilatation of endoplasmic reticulum and loss of cristae in mitochondria were observed in trophoblast cells in preeclamptic placental sections. Conclusion: High regulation of oxygen resulting from preeclampsia has been shown to be a critical determinant of placentagenesis and plays an important role in placental differentiation, changes in maternal and fetal blood circulation, trophoblastic invasion, and syncytial node increase. It has been thought that preeclampsia affects secretion by disrupting the endoplasmic reticulum structure and induces mitochondrial damage, and that ET-1 may potentially help in the induction of stress pathways as a result of hypoxia in preeclampsia.

Lysosomal storage disease induced by Sida planicaulis (Sin. Sida carpinifolia) (Malvaceae) in sheep in the state of Rio de Janeiro / Doença do armazenamento lisossomal induzida por Sida planicaulis (Sin. Sida carpinifolia) (Malvaceae) em ovinos no estado do Rio de Janeiro

Ingestion of S. planicaulis (Sin. S. carpinifolia) causes lysosomal storage disease in sheep. The main toxic compound of this plant, swainsonine, inhibits the enzymatic activity of α-mannosidase I and II, resulting in lysosomal storage of glycoproteins. We describe a case of spontaneous poisoning by S. planucaulis in Rio de Janeiro state, Brazil. Physical examination of affected animals revealed proprioceptive deficit, motor incoordination, staggering movement, and head tremors. Histopathological evaluation showed severe swelling/cytoplasmic vacuolization in Purkinje neurons, with a foamy appearance and occasional karyolysis or karyopyknosis, and intense vacuolization of acinar cells of the pancreas and, less markedly, thyroid follicular cells. The positive lectin-histochemistry labeling for Con A, WGA and sWGA lectins characterized the disease as a glycoproteinosis. The ultrastructural evaluation revealed numerous vacuoles up to 2.5µm in diameter bounded by membranes up to 20nm thick in pancreatic acinar cells. The diagnosis of S. planicaulis toxicity was established based on epidemiological data, clinicopathological, lectino-histochemical, and ultrastructural findings. This is the second report of spontaneous poisoning of sheep by S. planicaulis in Brazil, but the first in the Southeastern Brazil.

A ingestão de S. planicaulis (Sin. S. carpinifolia) tem sido responsabilizada por doença do armazenamento lisossomal em ovinos. O principal composto tóxico dessa planta, a swainsonina, inibe atividade enzimática da α-manosidase I e II, que redunda no armazenamento de glicoproteínas no interior de lisossomos. Descreveu-se um caso de intoxicação espontânea por S. planicaulis no estado do Rio de Janeiro, Brasil. O exame físico dos animais afetados revelou déficit proprioceptivo, incoordenação motora, cambaleio à movimentação e tremores de intenção e na cabeça. A avaliação histopatológica evidenciou severa tumefação/vacuolização citoplasmática em neurônios de Purkinje, com aspecto espumoso e eventual cariólise ou cariopicnose e intensa vacuolização de células acinares do pâncreas e, menos marcadamente, foliculares da tireoide. O exame lectino-histoquímico positivo para as lectinas Con A, WGA e sWGA foi capaz de caracterizar a enfermidade como uma glicoproteinose. A avaliação ultraestrutural revelou numerosos vacúolos de até 2,5µm de diâmetro, delimitados por membranas de até 20nm de espessura em células acinares do pâncreas. O diagnóstico da intoxicação por S. planicaulis foi estabelecido com base nos dados epidemiológicos, nos achados clínico-patológicos e confirmado pelas avaliações lectino-histoquímica e ultraestrutural. Esta é segunda descrição da intoxicação natural por S. planicaulis em ovinos no Brasil e a primeira na região Sudeste do país.

Morphology and immunolocalization of intertubular steroidogenic cell in mesonephros of Podocnemis expansa during gonadal differentiation

Sex steroid hormones are critical in gonadal differentiation in turtles. The gonads are not the only organs responsible for producing these hormones during this phase. Mesonephros play an important role in steroidogenesis. The present study aimed to investigate the presence of steroidogenic cells in mesonephros of Podocnemis expansa during gonadal differentiation and to evaluate their morphology and ultrastructure. Ten embryos of P. expansa were collected from 5 nests on day 36 of incubation, during spawning period on an artificial beach. Embryos were extracted from eggs by slicing the shell and euthanized. They were dissected under a stereoscopic microscope to collect the gonad-mesonephro complex, in which were fixed and subsequently processed for light microscopy, immunohistochemistry and transmission electron microscopy analysis. During histological analysis was observed mesonephros has typical morphological structure. Immunohistochemistry showed immunoreaction to aromatase in cells of intertubular space. Confirming these findings, it was possible to observe a type of intertubular cell in several regions of mesonephro, being more predominant in region close to blood vessels, distal and proximal tubules. In ultrastructural analysis these cells were characterized by having a clear, large, and rounded nucleus with evident nucleolus and cytoplasm rich in electron-dense droplets. This study demonstrated for the first time the presence of cells with morphological, immunohistochemical and ultrastructural characteristics similar to steroid-producing cells in P. expansa mesonephrons, suggesting that this organ may contribute to gonadal differentiation in this species.(AU)

Intertubular morphometric and ultrastructural testes analyses in mdx mice

Duchenne Muscular Dystrophy (DMD) reproductive alterations and the influence of antioxidant treatments may aid in understanding morphometry testicular quantification. In this context, the aim of the present study was to characterize the intertubular compartment (ITC) morphometry of animal testes in mdx mice supplemented with ascorbic acid (AA). Sixteen mice were used, namely the C57BL/10 (non-dystrophic) and C57BL/10Mdx (dystrophic) lineages, distributed into the following groups: Control (C60), Dystrophic (D60), Control supplemented with AA (CS60), Dystrophic supplemented with AA (DS60). A total of 200 mg/kg of AA were administered to mice for 30 days. Subsequently, the testicles were collected, weighed, and fragmented. The obtained fragments were fixed in Karnovsky's solution (pH 7.2) and embedded in historesin for morphometric and transmission electron microscopy assessments. Leydig cells were hypertrophic in the D60 group, but was reverted by AA supplementation in the DS60 group. The DS60 group also exhibited increased intertubular volume compared to the CS60 group. The ultrastructural images identified multilamellar bodies in dystrophic animals (lipid storage) and telocyte cells (transport substances) in both control and dystrophic animals. Morphometric alterations were, therefore, noted in the intertubular compartment due to Duchenne muscular dystrophy (DMD), with AA administration capable of altering Leydig cells in this condition.(AU)

Ultrastructural study of the esophagus and stomach of Arapaima gigas (Schinz 1822), juvenile paiche, created excavated tank

Paiche (Arapaima gigas) belongs to the Kingdom Animalia, Phylum Chordata, Class Actinopterygii, Order Osteoglossiformes, Family Arapaimidae, Genus Arapaima, and its origin may date to the Jurassic period. The species has natural habitat in the Amazonian rivers, found mainly in marginal lakes, being considered an important fishing resource, with high market value and high demand for meat and leather in both Brazilian and international trade. This study aims to report the morphology of the esophagus and stomach by light microscopy and scanning electronics microscopy. The esophagus was presented as muscular, short, tubular and fan-shaped in the cranial portion, also presenting deep longitudinal folds, and the entire mucosa is covered by mucus secretory cells with distinct morphological characteristics. Pirarurcu's stomach has a J-shape divided into three regions: cardiac with a lighter aspect, fundus portion with few folds in the mucosa, and pyloric with deeper folds, also presenting gastroliths in fundus and pyloric portions. Both microscopy studies highlighted three glandular regions, composed by mucoid columnar epithelial cells, gastric crypts with different shapes and sizes depending on each portion, in which the different shapes of the mucosal folds in each region of the stomach were evident, and digitiform microsaliences were found in the cardiac region, and micro-orifices and desmosome in the fundus region. Also, fundus and pyloric portions produce more mucus than the cardiac. Then morphology found was consistent with the eating habits and management of distinct characteristics of the digestive tract.(AU)

Ultrastructural analysis of the antennae of Hemilucilia segmentaria (Diptera: Calliphoridae), a blowfly of forensic importance

Hemilucilia segmentaria (Fabricius, 1805) is a Neotropical blowfly species of forensic importance, with necrophagous and asynanthropic habits. In this study, the antennal ultrastructure of H. segmentaria was examined using scanning electron microscopy. The three antennal segments are covered by microtrichia. Sensilla chaetica were detected only on the scape and pedicel. Setiferous plaques and a pedicellar button were observed on the pedicel. Four types of sensilla were found on the postpedicel, including s. trichoidea, s. basiconica (subtype I, II and III), s. coeloconica subtype I, and sensory pits with s. coeloconica subtype II. This is the first time that the fine structure of the antennae of H. segmentaria was studied. Our results constitute a solid base for research on comparative and functional morphology in H. segmentaria and other blowflies.(AU)

Age-associated hemogram and ultrastructural leukocyte morphology in Pygocentrus nattereri (Characiformes: Serrasalmidae) from the Brazilian Pantanal

Pygocentrus nattereri is a carnivorous fish widely distributed in the Brazilian wetland and occupies different river strata. Based on the sexual maturity, the standard length (SL) has been used to categorize age and physiological attributes. However, hematological parameters and their relationship with age and growth features are unknow. Here, the aim was to evaluate the hematological profile and leukocyte data of juvenile and adult specimens, associating it with SL. Specimens of both sexes were weighted, and SL were measured for classify as juveniles (SL ≤16.0 cm) and adults (SL ≥16.1 cm). Blood was collected to perform the hemogram, for leukocyte quantification and ultrastructural descriptions. In general, hemogram values and leukocyte, lymphocyte, and monocyte counts were high in juveniles. Hemogram variables were negatively correlated with SL. Thrombocyte and neutrophil counts were higher in adults than in juveniles. SL was correlated with leukogram variables. Ultrastructural analysis showed that granular leukocyte count in P. nattereri was similar another fishes. PAS-positive granular leukocytes are slightly smaller than neutrophils and are characterized by dense elongated and semi-lunar shapes in the cytoplasm. Our results suggest that age-based SL may be useful for the analysis of the relationship between P. nattereri health status and wetland aquatic environments.(AU)

Pygocentrus nattereri é um peixe carnívoro amplamente distribuído no pantanal brasileiro e ocupa diferentes estratos de rios. Com base na maturidade sexual, o comprimento padrão (CP) tem sido usado para categorizar a idade e os atributos fisiológicos. No entanto, os parâmetros hematológicos e sua relação com a idade e características de crescimento são desconhecidos. O objetivo foi avaliar o perfil hematológico e leucocitário de espécimes juvenis e adultos, associando-o ao CP. Espécimes de ambos os sexos foram pesados e os CPs foram medidos para classificação em juvenis (CP ≤16,0 cm) e adultos (CP ≥16,1 cm). Foi coletado sangue para realização do hemograma, perfil e morfologia dos leucócitos. Em geral, os valores do hemograma e as contagens de leucócitos, linfócitos e monócitos foram maiores nos jovens. A contagem de trombócitos e neutrófilos foi maior em adultos do que em jovens. A análise ultraestrutural mostrou que a contagem de leucócitos granulares em P. nattereri foi semelhante a outros peixes. Os leucócitos granulares PAS-positivos são menores do que os neutrófilos, sendo caracterizados por formas alongadas e semilunares densas no citoplasma. Nossos resultados sugerem que CP é útil para a análise da relação entre o estado de saúde de P. nattereri em ambientes aquáticos alagados.(AU)

Age-associated hemogram and ultrastructural leukocyte morphology in Pygocentrus nattereri (Characiformes: Serrasalmidae) from the Brazilian Pantanal

Pygocentrus nattereri is a carnivorous fish widely distributed in the Brazilian wetland and occupies different river strata. Based on the sexual maturity, the standard length (SL) has been used to categorize age and physiological attributes. However, hematological parameters and their relationship with age and growth features are unknow. Here, the aim was to evaluate the hematological profile and leukocyte data of juvenile and adult specimens, associating it with SL. Specimens of both sexes were weighted, and SL were measured for classify as juveniles (SL ≤16.0 cm) and adults (SL ≥16.1 cm). Blood was collected to perform the hemogram, for leukocyte quantification and ultrastructural descriptions. In general, hemogram values and leukocyte, lymphocyte, and monocyte counts were high in juveniles. Hemogram variables were negatively correlated with SL. Thrombocyte and neutrophil counts were higher in adults than in juveniles. SL was correlated with leukogram variables. Ultrastructural analysis showed that granular leukocyte count in P. nattereri was similar another fishes. PAS-positive granular leukocytes are slightly smaller than neutrophils and are characterized by dense elongated and semi-lunar shapes in the cytoplasm. Our results suggest that age-based SL may be useful for the analysis of the relationship between P. nattereri health status and wetland aquatic environments.(AU)

Pygocentrus nattereri é um peixe carnívoro amplamente distribuído no pantanal brasileiro e ocupa diferentes estratos de rios. Com base na maturidade sexual, o comprimento padrão (CP) tem sido usado para categorizar a idade e os atributos fisiológicos. No entanto, os parâmetros hematológicos e sua relação com a idade e características de crescimento são desconhecidos. O objetivo foi avaliar o perfil hematológico e leucocitário de espécimes juvenis e adultos, associando-o ao CP. Espécimes de ambos os sexos foram pesados e os CPs foram medidos para classificação em juvenis (CP ≤16,0 cm) e adultos (CP ≥16,1 cm). Foi coletado sangue para realização do hemograma, perfil e morfologia dos leucócitos. Em geral, os valores do hemograma e as contagens de leucócitos, linfócitos e monócitos foram maiores nos jovens. A contagem de trombócitos e neutrófilos foi maior em adultos do que em jovens. A análise ultraestrutural mostrou que a contagem de leucócitos granulares em P. nattereri foi semelhante a outros peixes. Os leucócitos granulares PAS-positivos são menores do que os neutrófilos, sendo caracterizados por formas alongadas e semilunares densas no citoplasma. Nossos resultados sugerem que CP é útil para a análise da relação entre o estado de saúde de P. nattereri em ambientes aquáticos alagados.(AU)

The developmental variations of the sagitta otolith in the young and mature male of a hermaphrodite polynemidae fish, Eleutheronema tetradactylum (Shaw, 1804)

Abstract The Eleutheronema tetradactylum is a protandrous, hermaphrodite, marine perciformes fish. The body length of this fish acts as an important diagnostic marker for male and female discrimination. The present study describes for the first time the ultrastructural characteristics on the medial surface of the sagitta otolith in different body size groups of males of E. tetradactylum (Polynemidae: Perciformes) using scanning electron microscopy. The sagitta is a spindle-shaped structure that includes a well-developed rostrum and a poorly developed antirostrum. The sulcus is ostio-pseudocaudal type, almost straight and devoid of the collum. The ostium is a well-developed, vase-shaped structure. The cauda includes the colliculum and a well-developed caudal bulb with several distinct growth stripes. The length of the caudal bulb is significantly correlated to the growth of the body size of the fish. The excisura major is indistinct and the excisura minor is absent. The cristae are distinct on both sides of the sulcus. The one-way ANOVA test revealed that the development of several sagitta features shows significant differences in various body size groups of E. tetradactylum. The growth of the sagitta length is more closely related to the fork length than the sagitta width. Therefore, the sagitta length and the caudal bulb length can be used as important predictors to evaluate the fish size. The cauda region of the sagitta in E. tetradactylum is unique as well as more decorative than those of another Polynemidae fish and other hermaphrodite, marine perciformes fishes. The sagitta characteristics of E. tetradactylum might be advantageous in the identification of the sex and the taxonomy of the hermaphrodite fish species.

The developmental variations of the sagitta otolith in the young and mature male of a hermaphrodite polynemidae fish, Eleutheronema tetradactylum (Shaw, 1804)

Abstract The Eleutheronema tetradactylum is a protandrous, hermaphrodite, marine perciformes fish. The body length of this fish acts as an important diagnostic marker for male and female discrimination. The present study describes for the first time the ultrastructural characteristics on the medial surface of the sagitta otolith in different body size groups of males of E. tetradactylum (Polynemidae: Perciformes) using scanning electron microscopy. The sagitta is a spindle-shaped structure that includes a well-developed rostrum and a poorly developed antirostrum. The sulcus is ostio-pseudocaudal type, almost straight and devoid of the collum. The ostium is a well-developed, vase-shaped structure. The cauda includes the colliculum and a well-developed caudal bulb with several distinct growth stripes. The length of the caudal bulb is significantly correlated to the growth of the body size of the fish. The excisura major is indistinct and the excisura minor is absent. The cristae are distinct on both sides of the sulcus. The one-way ANOVA test revealed that the development of several sagitta features shows significant differences in various body size groups of E. tetradactylum. The growth of the sagitta length is more closely related to the fork length than the sagitta width. Therefore, the sagitta length and the caudal bulb length can be used as important predictors to evaluate the fish size. The cauda region of the sagitta in E. tetradactylum is unique as well as more decorative than those of another Polynemidae fish and other hermaphrodite, marine perciformes fishes. The sagitta characteristics of E. tetradactylum might be advantageous in the identification of the sex and the taxonomy of the hermaphrodite fish species.

Anatomopathological, ultrastructural, immunohistochemical and molecular characterization of infectious laryngotracheitis outbreaks in poultry farms in Egypt (2018–2020)

Infectious laryngotracheitis (ILT) is a severe respiratory disease, which causes high morbidity and mortality in affected birds. In our study, ILT were reported in 42 farms from nine governates over two years (2018–2020) that showed clinical signs of ILT including dyspnea, blood expectoration of, excessive lacrimation, rattling, conjunctivitis. The disease affected different chicken breeds and age groups despite vaccination with licensed and commonly used vaccines. Samples of larynx, trachea, lungs and air sacs were examined and collected for histopathological, ultrastructural, immunohistochemical examination and molecular detection. Gross examination of laryngeal and tracheal lumen revealed different types of exudate varied from catarrhal to fibrinonecotric, also pneumonia and airsacculitis were detected. Histopathological examination showed different alternation in larynx, trachea, lung and air sac as characteristic syncytial cells containing intranuclear inclusion body hanged in fibrinoheterphilic exudate that precent in laryngeal, tracheal, bronchial and parabronchial lumen and air sacs. Tracheal lesion scoring system was used to categorize the severity of lesion in different governates. Tracheal lesion score showed that 6.02%, 26.5%, 43.3% of the birds exhibited mild, moderate, and severe changes, respectively, while 24.18% of the birds exhibited very severe changes. Furthermore, severe cases were related to the Qalyubia , Fayoum then Sharkia Governorate. Moreover, immunohistochemistry was used to detect viral particles in syncytial cells, inflammatory cells beside epithelium of trachea and lung. Transmission electron microscopy enabled the detection of virus particles and demonstrated that heterophils could be infected. PCR targeting a region in the thymidine kinase gene and glycoprotein gJ gene confirmed the presence of infectious laryngotracheitis ILT virus-specific DNA. In conclusion, anatomopathological, immunohistochemical, molecular and ultrastructural findings showed increased of ILTV severity in Egypt. Larynx, trachea, lungs and air sac should be collected and examined that aid in diagnosis. Importance of good biosecurity level to be considered.(AU)

Anatomopathological, ultrastructural, immunohistochemical and molecular characterization of infectious laryngotracheitis outbreaks in poultry farms in Egypt (2018–2020)

Infectious laryngotracheitis (ILT) is a severe respiratory disease, which causes high morbidity and mortality in affected birds. In our study, ILT were reported in 42 farms from nine governates over two years (2018–2020) that showed clinical signs of ILT including dyspnea, blood expectoration of, excessive lacrimation, rattling, conjunctivitis. The disease affected different chicken breeds and age groups despite vaccination with licensed and commonly used vaccines. Samples of larynx, trachea, lungs and air sacs were examined and collected for histopathological, ultrastructural, immunohistochemical examination and molecular detection. Gross examination of laryngeal and tracheal lumen revealed different types of exudate varied from catarrhal to fibrinonecotric, also pneumonia and airsacculitis were detected. Histopathological examination showed different alternation in larynx, trachea, lung and air sac as characteristic syncytial cells containing intranuclear inclusion body hanged in fibrinoheterphilic exudate that precent in laryngeal, tracheal, bronchial and parabronchial lumen and air sacs. Tracheal lesion scoring system was used to categorize the severity of lesion in different governates. Tracheal lesion score showed that 6.02%, 26.5%, 43.3% of the birds exhibited mild, moderate, and severe changes, respectively, while 24.18% of the birds exhibited very severe changes. Furthermore, severe cases were related to the Qalyubia , Fayoum then Sharkia Governorate. Moreover, immunohistochemistry was used to detect viral particles in syncytial cells, inflammatory cells beside epithelium of trachea and lung. Transmission electron microscopy enabled the detection of virus particles and demonstrated that heterophils could be infected. PCR targeting a region in the thymidine kinase gene and glycoprotein gJ gene confirmed the presence of infectious laryngotracheitis ILT virus-specific DNA. In conclusion, anatomopathological, immunohistochemical, molecular and ultrastructural findings showed increased of ILTV severity in Egypt. Larynx, trachea, lungs and air sac should be collected and examined that aid in diagnosis. Importance of good biosecurity level to be considered.

Ultrastructural and morphometric description of the ear skin and cartilage of two South American wild histricognate rodents (Dasyprocta leporina and Galea spixii) / Descrição ultraestrutural e morfométrica da pele e cartilagem auricular de dois roedores histricognatos silvestres da América do Sul (Dasyprocta leporina e Galea spixii)

Skin and cartilage have been the main source for the recovery of somatic cells to be used in conservation strategies in wild mammals. In this sense, an important step for the cryopreservation of these samples is to recognize the properties of the skin and cartilage. Thus, knowing that the skin may differ among species and aiming to contribute to the establishment of cryobanks, the study examined the differences in the ear skin and cartilage of wild rodents from South America, agouti (Dasyprocta leporina) and spix's yellow-toothed cavy (Galea spixii). Ultrastructural and quantitative methods were used to measure skin and cartilage thickness, density of collagen and elastic fibers, cell type number and distribution, and proliferative activity. Although ultrastructural analysis revealed a similar pattern between species, morphometric analysis of the skin and cartilage showed differences between agoutis and cavies regarding thickness of epidermis layers (corneum: 5.3±2.5μm vs. 3.9±0.6μm; intermediate: 16.4±6.2μm vs. 23.4±8.1μm; basal: 9.9±2.1μm vs. 4.8±0.5μm), dermis (183.1±44.0μm vs. 258.2±22.9μm), total skin (211.8±46.0μm vs. 290.3±23.7μm) and perichondrium (27.6±6.1μm vs. 10.5±1.8μm). A greater number of epidermal cells (61.7±15.2 vs. 24.8±7.6) and chondrocytes (32.7±9.0 vs. 27.5±4.7) were observed in agouti, while the cavy presented a greater number of melanocytes (12.6±4.7 vs. 29.9±6.2), keratinocytes (14.7±4.2 vs. 29.8±7.6), and fibroblasts (103.6±24.7 vs. 112.2±11.3). Moreover, a higher percentage of collagen fibers and proliferative activity was observed in the skin of cavies, when compared to the skin of agoutis. Therefore, there are differences between agouti and cavy for ear skin and cartilage, requiring the establishment of species-specific cryopreservation protocols.(AU)

A pele e cartilagem têm sido uma importante fonte de recuperação de células somáticas a serem utilizadas em estratégias de conservação em mamíferos silvestres. Nesse contexto, uma importante etapa para criopreservação é conhecer, inicialmente, as propriedades que compõem a pele e cartilagem. Sabendo, então, que a pele pode diferir-se entre espécies e com o objetivo de contribuir para o estabelecimento de criobancos, o estudo evidenciou as diferenças da pele e da cartilagem do pavilhão auricular apical de cutias (Dasyprocta leporina) e preás (Galea spixii) que são roedores silvestres presentes na América do Sul. Para tanto, métodos ultraestruturais e quantitativos foram utilizados para mensurar a espessura da pele e da cartilagem, densidade de fibras colágenas e elásticas, número e distribuição dos tipos celulares e atividade proliferativa. Embora as propriedades ultraestruturais em cutias e preás tenham se mostrado semelhantes, avaliações acerca da morfometria da pele e da cartilagem demonstrou diferenças, especialmente nas camadas epidérmicas (córnea: 5,3±2,5μm vs. 3,9±0,6μm; espinhosa: 16,4±6,2μm vs. 23,4±8,1μm; basal: 9,9±2,1μm vs. 4,8±0,5μm), derme (183,1±44,0μm vs. 258,2±22,9μm), pele total (211,8±46,0μm vs. 290,3±23,7μm) e pericôndrio (27,6±6,1μm vs. 10,5±1,8μm). Além disso, um número maior de células epidérmicas (61,7±15,2 vs. 24,8±7,6) e condrócitos (32,7±9,0 vs. 27,5±4,7) foram observados em cutias, enquanto em preás um maior número de melanócitos (12,6±4,7 vs. 29,9±6,2), queratinócitos (14,7±4,2 vs. 29,8±7,6) e fibroblastos (103,6±24,7 vs. 112,2±11,3) foram evidenciados. Ainda, em preás, uma maior porcentagem de fibras colágenas e da atividade proliferativa foram observadas quando comparadas a pele de cutias. Portanto, existem diferenças entre cutias e preás para pele e cartilagem do pavilhão auricular, exigindo desta forma um estabelecimento de protocolos de criopreservação específica para cada uma destas espécies.(AU)

Ultrastructural and morphometric description of the ear skin and cartilage of two South American wild histricognate rodents (Dasyprocta leporina and Galea spixii) / Descrição ultraestrutural e morfométrica da pele e cartilagem auricular de dois roedores histricognatos silvestres da América do Sul (Dasyprocta leporina e Galea spixii)

Skin and cartilage have been the main source for the recovery of somatic cells to be used in conservation strategies in wild mammals. In this sense, an important step for the cryopreservation of these samples is to recognize the properties of the skin and cartilage. Thus, knowing that the skin may differ among species and aiming to contribute to the establishment of cryobanks, the study examined the differences in the ear skin and cartilage of wild rodents from South America, agouti (Dasyprocta leporina) and spix's yellow-toothed cavy (Galea spixii). Ultrastructural and quantitative methods were used to measure skin and cartilage thickness, density of collagen and elastic fibers, cell type number and distribution, and proliferative activity. Although ultrastructural analysis revealed a similar pattern between species, morphometric analysis of the skin and cartilage showed differences between agoutis and cavies regarding thickness of epidermis layers (corneum: 5.3±2.5μm vs. 3.9±0.6μm; intermediate: 16.4±6.2μm vs. 23.4±8.1μm; basal: 9.9±2.1μm vs. 4.8±0.5μm), dermis (183.1±44.0μm vs. 258.2±22.9μm), total skin (211.8±46.0μm vs. 290.3±23.7μm) and perichondrium (27.6±6.1μm vs. 10.5±1.8μm). A greater number of epidermal cells (61.7±15.2 vs. 24.8±7.6) and chondrocytes (32.7±9.0 vs. 27.5±4.7) were observed in agouti, while the cavy presented a greater number of melanocytes (12.6±4.7 vs. 29.9±6.2), keratinocytes (14.7±4.2 vs. 29.8±7.6), and fibroblasts (103.6±24.7 vs. 112.2±11.3). Moreover, a higher percentage of collagen fibers and proliferative activity was observed in the skin of cavies, when compared to the skin of agoutis. Therefore, there are differences between agouti and cavy for ear skin and cartilage, requiring the establishment of species-specific cryopreservation protocols.(AU)

A pele e cartilagem têm sido uma importante fonte de recuperação de células somáticas a serem utilizadas em estratégias de conservação em mamíferos silvestres. Nesse contexto, uma importante etapa para criopreservação é conhecer, inicialmente, as propriedades que compõem a pele e cartilagem. Sabendo, então, que a pele pode diferir-se entre espécies e com o objetivo de contribuir para o estabelecimento de criobancos, o estudo evidenciou as diferenças da pele e da cartilagem do pavilhão auricular apical de cutias (Dasyprocta leporina) e preás (Galea spixii) que são roedores silvestres presentes na América do Sul. Para tanto, métodos ultraestruturais e quantitativos foram utilizados para mensurar a espessura da pele e da cartilagem, densidade de fibras colágenas e elásticas, número e distribuição dos tipos celulares e atividade proliferativa. Embora as propriedades ultraestruturais em cutias e preás tenham se mostrado semelhantes, avaliações acerca da morfometria da pele e da cartilagem demonstrou diferenças, especialmente nas camadas epidérmicas (córnea: 5,3±2,5μm vs. 3,9±0,6μm; espinhosa: 16,4±6,2μm vs. 23,4±8,1μm; basal: 9,9±2,1μm vs. 4,8±0,5μm), derme (183,1±44,0μm vs. 258,2±22,9μm), pele total (211,8±46,0μm vs. 290,3±23,7μm) e pericôndrio (27,6±6,1μm vs. 10,5±1,8μm). Além disso, um número maior de células epidérmicas (61,7±15,2 vs. 24,8±7,6) e condrócitos (32,7±9,0 vs. 27,5±4,7) foram observados em cutias, enquanto em preás um maior número de melanócitos (12,6±4,7 vs. 29,9±6,2), queratinócitos (14,7±4,2 vs. 29,8±7,6) e fibroblastos (103,6±24,7 vs. 112,2±11,3) foram evidenciados. Ainda, em preás, uma maior porcentagem de fibras colágenas e da atividade proliferativa foram observadas quando comparadas a pele de cutias. Portanto, existem diferenças entre cutias e preás para pele e cartilagem do pavilhão auricular, exigindo desta forma um estabelecimento de protocolos de criopreservação específica para cada uma destas espécies.(AU)

Morphometry of bovine blastocysts produced in vitro in culture media with antioxidants cysteamine or oily extract of Lippia origanoides / [Morfometria de blastocistos bovinos produzidos in vitro em meio de cultura com antioxidantes cisteamina ou extrato oleoso de Lippia origanoides]

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)

Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Antifungal efficacy of Moringa oleifera leaf and seed extracts against Botrytis cinerea causing gray mold disease of tomato (Solanum lycopersicum L. ) / Eficácia antifúngica de extratos de folha e de semente de Moringa oleifera contra Botrytis cinerea causador da doença do molde cinza de tomate (Solanum lycopersicum L. )

Drawbacks associated with the use of chemical fungicides to control plant pathogenic fungi such as Botrytis cinerea stimulate the need for alternatives. Therefore, the present study was carried out to determine the antifungal potentials of Moringa oleifera extracts against B. cinerea. Phytochemical analysis using qualitative chemical tests revealed the presence of huge amount of crucial phytochemicals compounds like phenolic compounds, alkaloids and saponins in the M. oleifera leaf extract. Antifungal bioassay of the crude extracts indicated better mycelial growth inhibition by methanol leaf extract (99%). The minimum inhibitory concentration (MIC) was 5 mg/ml with 100% spore germination inhibition and minimum fungicidal concentration (MFC) was 10 mg/ml with 98.10% mycelial growth inhibition using broth micro dilution and poisoned food techniques. Gas chromatographymass spectrometry (GC-MS) analysis led to the identification of 67 volatile chemical compounds in the leaf extract with 6-decenoic acid (Z)- (19.87%) was the predominant compound. Further chemical elucidation of the crude extracts performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) showed the presence of non-volatile chemical compounds, mostly flavones, flavonoids and phenolic acids (i.e. quercetin and kaempferol). Scanning electron microscopy and transmission electron microscopy analysis showed positive effect of M. oleifera leaf extract on the treated conidia and mycelium of B. cinerea. Findings revealed that irreversible surface and ultra-structural changes with severe detrimental effects on conidia and mycelium morphology compared to control treatment. Overall findings suggested that M. oleifera leaf extract is a promising candidate for biological control of fungal pathogens, thus limiting overdependence on chemical fungicides.(AU)

As desvantagens associadas ao uso de fungicidas químicos para controlar fungos fitopatogênicos, como Botrytis cinerea, estimulam a necessidade de alternativas. Portanto, o presente estudo foi realizado para determinar o potencial antifúngico de extratos de Moringa oleifera contra B. cinerea. A análise fitoquímica usando testes químicos qualitativos revelou a presença de uma grande quantidade de compostos fitoquímicos cruciais, como compostos fenólicos, alcaloides e saponinas no extrato da folha de M. oleifera. O bioensaio antifúngico dos extratos brutos indicou melhor inibição do crescimento micelial pelo extrato de folhas de metanol (99%). A concentração inibitória mínima (MIC) foi de 5 mg/mL com 100% de inibição da germinação de esporos e a concentração fungicida mínima (MFC) foi de 10 mg/mL com 98,10% de inibição do crescimento micelial usando microdiluição em caldo e técnicas de alimentos envenenados. A análise por cromatografia gasosa-espectrometria de massa (GC-MS) levou à identificação de 67 compostos químicos voláteis no extrato da folha, sendo o ácido 6-decenoico (Z) (19,87%) o composto predominante. Elucidação química adicional dos extratos brutos realizada por cromatografia líquida com espectrometria de massa em tandem (LC-MS/MS) mostrou a presença de compostos químicos não voláteis, principalmente flavonas, flavonoides e ácidos fenólicos (ou seja, quercetina e kaempferol). As análises de microscopia eletrônica de varredura e microscopia eletrônica de transmissão mostraram efeito positivo do extrato de folhas de M. oleifera sobre os conídios e micélios tratados de B. cinerea. Os resultados revelaram a superfície irreversível e alterações ultraestruturais com graves efeitos prejudiciais sobre os conídios e a morfologia micelial, em comparação com o tratamento de controle. Os resultados gerais sugeriram que o extrato da folha de M. oleifera é um candidato promissor para o controle biológico de patógenos fúngicos, limitando assim a dependência excessiva de fungicidas químicos.(AU)

The developmental variations of the sagitta otolith in the young and mature male of a hermaphrodite polynemidae fish, Eleutheronema tetradactylum (Shaw, 1804)

The Eleutheronema tetradactylum is a protandrous, hermaphrodite, marine perciformes fish. The body length of this fish acts as an important diagnostic marker for male and female discrimination. The present study describes for the first time the ultrastructural characteristics on the medial surface of the sagitta otolith in different body size groups of males of E. tetradactylum (Polynemidae: Perciformes) using scanning electron microscopy. The sagitta is a spindle-shaped structure that includes a well-developed rostrum and a poorly developed antirostrum. The sulcus is ostio-pseudocaudal type, almost straight and devoid of the collum. The ostium is a well-developed, vase-shaped structure. The cauda includes the colliculum and a well-developed caudal bulb with several distinct growth stripes. The length of the caudal bulb is significantly correlated to the growth of the body size of the fish. The excisura major is indistinct and the excisura minor is absent. The cristae are distinct on both sides of the sulcus. The one-way ANOVA test revealed that the development of several sagitta features shows significant differences in various body size groups of E. tetradactylum. The growth of the sagitta length is more closely related to the fork length than the sagitta width. Therefore, the sagitta length and the caudal bulb length can be used as important predictors to evaluate the fish size. The cauda region of the sagitta in E. tetradactylum is unique as well as more decorative than those of another Polynemidae fish and other hermaphrodite, marine perciformes fishes. The sagitta characteristics of E. tetradactylummight be advantageous in the identification of the sex and the taxonomy of the hermaphrodite fish species.(AU)

Anabolic steroids and their effects of on neuronal density in cortical areas and hippocampus of mice / Esteroides anabolizantes e seus efeitos na densidade neuronal em áreas corticais e no hipocampo de camundongos

Anabolic substances have been increasingly used by bodybuilders and athletes with the goal of improving performance and aesthetics. However, this practice has caused some concern to physicians and researchers because of unknowledge of consequences that the indiscriminate and illicit use of these substances can cause. Thus, this study analyzed the effects of two commercially available anabolic steroids (AS), Winstrol Depot® (Stanozolol) and Deposteron® (Testosterone Cypionate), in the neuronal density of limbic, motor and sensory regions on the cerebral cortex and in CA1, CA2, CA3 regions of the hippocampus. A total of 60 Swiss mice were used (30 males and 30 females), separated into three groups: control and two experimental groups, which received the AAS. From each brain, homotypic and semi-serial samples were taken in frontal sections from areas established for the study. The results showed that females treated with testosterone cypionate presented a reduction in all regions tested and the ones treated with Stanozolol showed a decrease in some hippocampal areas. Regarding male animals, stanozolol led to a decrease in neuron number in one hippocampal region. These data allow us to conclude that supra-physiological doses of steroids used in this study, can cause considerable damage to nervous tissue with ultrastructural and consequently behavioral impairment. These changes could interfere with the loss of physical yield and performance of athletes and non-athletes and may cause irreparable damage to individuals making irresponsible use of anabolic steroids.(AU)

As substancias anabólicas tem sido cada vez mais utilizadas por fisiculturistas e atletas com o objetivo de melhorar o desempenho e a estética. No entanto, essa prática tem causado algumas preocupações aos médicos e pesquisadores, devido ao desconhecimento das consequencias que o uso indiscriminado e ilícito dessas substâncias podem causar. Diante disso, este estudo analisou os efeitos de dois esteroides anabolizantes (EA) comercialmente disponíveis, Winstrol Depot® (Stanozolol) e Deposteron® (cipionato de testosterona), na densidade neuronal das regiões corticais límbica, motora e sensitive bem como das áreas CA1, CA2, CA3 do hipocampo. Foram utilizados 60 camundongos Swiss (30 machos e 30 fêmeas), separados em três grupos: controle e dois grupos experimentais, que receberam o EA. De cada cérebro, foram coletadas amostras homotípicas e semi-seriadas em cortes frontais das áreas estabelecidas para o estudo. Os resultados mostraram que as fêmeas tratadas com cipionato de testosterona apresentaram uma redução em todas as regiões analisadas já as fêmeas tratadas com Stanozolol mostraram uma diminuição em algumas áreas do hipocampo. Em relação aos animais machos, o stanozolol levou a uma diminuição na densidade neuronal em uma região do hipocampo. Estes dados nos permitem concluir que doses supra fisiológicas de esteroides utilizadas neste estudo podem causar danos consideráveis ao tecido nervoso com comprometimento ultraestrutural e consequentemente comportamental. Essas alterações podem interferir na perda de rendimento físico e no desempenho de atletas e não atletas e podem causar danos irreparáveis a indivíduos que fazem uso irresponsável destes EA.(AU)