Morphological and morphometric study of pre-ovigerous and post-ovigerous adults of Tanaisia (Paratanaisia) bragai (Santos, 1934) (Digenea, Eucotylidae)

The aim of this study was to obtain data on the morphology and morphometry of pre-ovigerous and post-ovigerous adults of the species Tanaisia (Paratanaisia) bragai, using confocal laser scanning microscopy to obtain tomographic images of the suckers and tegument. For morphometric analysis, 45 specimens (30 pre-ovigerous adults and 15 post-ovigerous adults) were measured with the aid of an ocular micrometer coupled to the objective of a photonic microscope. Pre-ovigerous and post-ovigerous adult individuals, stained with Mair carmalumen and mounted in permanent preparations, were analyzed by means of confocal laser scanning microscopy. Positive correlation was detected between the body length and ovary length of postovigerous adults (rs: 0.774; p< 0.05), the body length and the length of uterus (rs: 0.839; p< 0,01) and between the ovary width and egg length (rs: 0.777; p p<0.01). Morphological study of the pre-ovigerous adults demonstrated that the ovary and testes develop simultaneously before the development of the uterus and vitelline glands. The acetabulum was detected in pre-ovigerous adults stained with hematoxilin and observed using light microscopy. In these specimens, the acetabulum measured 36.7 ± 6.9 µm (25-50 µm) in width and 39.91 ± 6.8 µm (25-55 µm) in length. The acetabulum was not detected in post-ovigerous adults observed with light microscopy. However, this structure was detected using confocal miscrocopy. In the post-ovigerous specimens, the acetabulum presented a reduced size compared to the pre-ovigerous adults. This may imply that this structure has more functional significance in the larval and pre-ovigerous stages.(AU)

Morphological and morphometric study of pre-ovigerous and post-ovigerous adults of Tanaisia (Paratanaisia) bragai (Santos, 1934) (Digenea, Eucotylidae)

The aim of this study was to obtain data on the morphology and morphometry of pre-ovigerous and post-ovigerous adults of the species Tanaisia (Paratanaisia) bragai, using confocal laser scanning microscopy to obtain tomographic images of the suckers and tegument. For morphometric analysis, 45 specimens (30 pre-ovigerous adults and 15 post-ovigerous adults) were measured with the aid of an ocular micrometer coupled to the objective of a photonic microscope. Pre-ovigerous and post-ovigerous adult individuals, stained with Mair carmalumen and mounted in permanent preparations, were analyzed by means of confocal laser scanning microscopy. Positive correlation was detected between the body length and ovary length of postovigerous adults (rs: 0.774; p< 0.05), the body length and the length of uterus (rs: 0.839; p< 0,01) and between the ovary width and egg length (rs: 0.777; p p<0.01). Morphological study of the pre-ovigerous adults demonstrated that the ovary and testes develop simultaneously before the development of the uterus and vitelline glands. The acetabulum was detected in pre-ovigerous adults stained with hematoxilin and observed using light microscopy. In these specimens, the acetabulum measured 36.7 ± 6.9 µm (25-50 µm) in width and 39.91 ± 6.8 µm (25-55 µm) in length. The acetabulum was not detected in post-ovigerous adults observed with light microscopy. However, this structure was detected using confocal miscrocopy. In the post-ovigerous specimens, the acetabulum presented a reduced size compared to the pre-ovigerous adults. This may imply that this structure has more functional significance in the larval and pre-ovigerous stages.

ATIVIDADE ANTI-HELMÍNTICA in vitro DE QUIMIOTIPOS DO ÓLEO ESSENCIAL DE Lippia alba SOBRE Haemonchus contortus

O controle de nematoides gastrintestinais de pequenos ruminantes é realizado convencionalmente com a administração de anti-helmínticos sintéticos e semissintéticos. Entretanto, o uso inadequado de determinado anti-helmíntico, seleciona indivíduos que possuem a capacidade de sobreviver a esse quimioterápico tornando essencial a busca por métodos alternativos. Os produtos derivados de plantas têm sido amplamente investigados quanto ao seu potencial anti-helmíntico sobre Haemonchus contortus. O objetivo do presente estudo foi avaliar os efeitos dos óleos essenciais de quimiotipos carvona (CV) e citral (CT) de Lippia alba sobre H. contortus. A caracterização química foi feita por meio de cromatografia gasosa acoplada a espectrometria de massa (CG/MS). O efeito anti-helmíntico dos óleos essenciais foi avaliado no teste de eclosão de ovos (TEO) e teste de motilidade de nematoides adultos (TMA), utilizando um isolado de H. contortus multirresistente, Kokstad. Foi realizada microscopia confocal de varredura a laser (MCVL) dos ovos e adultos de H. contortus e microscopia eletrônica de varredura (MEV) dos adultos após tratamento com os óleos para observações qualitativas de seus efeitos. Os resultados foram submetidos à análise de variância (ANOVA) e comparados pelo teste Tukey (P>0,05). A concentração inibitória de 50% (CE50) para o TEO foi calculada a partir do programa SPSS 17.0 program for Windows. Os resultados demonstraram a existência de dois quimiotipos, CV e CT do óleo essencial de L. alba (La). O quimiotipo CV do óleo essencial de L. alba (LaCV) apresentou 70,2% de CV e o quimiotipo CT do óleo essencial de L. alba (LaCT) estava constituído por 20,40% e 29,49% de neral e geranial, respectivamente. No TEO, a CE50 de LaCV e LaCT foi de 0,2 e 0,3mg/mL, respectivamente. No TMA, após 12h de exposição ao LaCV e LaCT, 100% dos nematoides adultos estavam imóveis. A MCVL evidenciou alterações na motilidade da larva dentro do ovo provocada por LaCV, enquanto que LaCT impediu a formação da larva. Nos adultos expostos aos dois óleos foram observadas aparentes alterações na porção anterior, em nível de esôfago. Na MEV foram observadas alterações morfológicas na porção anterior, cápsula bocal e na porção medial. Conclui-se que os dois óleos essenciais de L. alba causaram aparentes alterações morfológicas e inibiram a eclosão de ovos, assim como a motilidade de nematoides adultos de H. contortus. Porém é importante avaliar o efeito in vivo, para identificar níveis seguros de eficácia e toxicidade antes de confirmar sua utilização como antihelmíntico.

The control of gastrointestinal nematodes in small ruminants is carried out conventionally with the administration of synthetic and semi-synthetic anthelmintics. However, the inappropriate use of a certain anthelmintic, selects individuals who have the capacity to survive this chemotherapy, making the search for alternative methods essential. Plant derived products have been extensively investigated for their anthelmintic potential on H. contortus. The aim of the present study was to evaluate the effects of the essential oils of Lippia alba chemotypes carvone (CV) and citral (CT) on H. contortus. Chemical characterization was done by means of gas chromatography coupled to mass spectrometry (CG/MS). The anthelmintic effect of essential oils was assessed in the egg hatch test (EHT) and adult nematode motility test (ANMT), using a multidrug-resistant H. contortus isolate, Kokstad. Confocal laser scanning microscopy (CLSM) of eggs and adults of H. contortus and scanning electron microscopy (SEM) of adults was performed after treatment with oils for qualitative observations of their effects. The results were submitted to analysis of variance (ANOVA) and compared by the Tukey test (P> 0.05). The 50% inhibitory concentration (EC50) for EHT was calculated using the SPSS 17.0 program for Windows. The results demonstrated the existence of two chemotypes, CV and CT of the essential oil of L. alba (La). The chemotype CV of the essential oil of L. alba (LaCV) presented 70.2% of CV and the chemotype CT of the essential oil of L. alba (LaCT) was constituted by 20.40% and 29.49% of neral and geranial, respectively. In the EHT, the EC50 of LaCV and LaCT was 0.2 and 0.3 mg/mL, respectively. In ANMT, after 12h of exposure to LaCV and LaCT, 100% of adult nematodes were immobile. CLSM showed changes in the larval motility inside the egg caused by LaCV, while LaCT promoted changes in the larva formation. In adults exposed to both oils, apparent changes were observed in the anterior portion, at the level of the esophagus. In SEM, morphological changes were observed in the anterior portion, mouthpiece and medial portion. It is concluded that the two oils of L. alba, chemotypes CV and CT, caused apparent morphological changes and inhibited the hatching of eggs, as well as the motility of adult H. contortus nematodes. However, it is important to evaluate the effect in vivo to identify safe levels of efficacy and toxicity before confirming its use as an anthelmintic.

Development of experimental in vitro burn model

To propose an experimental burn model in NIH-3T3 cell line. Induction of thermal injury in cultures of mouse fibroblast - NIH-3T3- cell line and determination of cell viability by MTT and imunofluorescence. The heating of the Petri dish increased proportionally to the temperature of the base and the time of exposure to microwave. In this in vitro burn model, using the cell line NIH-3T3 was observed drastic cellular injury with significant changes in cell viability and activity. It showed drastically modified cell morphology with altered membrane, cytoskeleton and nucleus, and low cellularity compared to the control group. The burn model in vitro using the cell line NIH-3T3 was reproductive and efficient. This burn model was possible to determine significant changes in cell activity and decreased viability, with drastic change in morphology, cell lysis and death.(AU)

VITRIFICAÇÃO DE TECIDO OVARIANO DE FETOS BOVINOS ASSOCIADA AO RESVERATROL

Objetivou-se com este estudo analisar a morfologia e a viabilidade tecidual de folículos ovarianos pré-antrais de fetos bovinos por meio de análise histológica, sondas fluorescentes em microscopia confocal, antes e após os procedimentos de vitrificação/ aquecimento com ou sem o antioxidante resveratrol. Foram fragmentados 18 ovários e os fragmentos distribuídos aos grupos controle (C), vitrificado sem resveratrol (SR) e vitrificado com resveratrol (CR) na concentração de 20 µM. No experimento 1 foi realizada análise morfológica dos folículos pré-antrais inclusos em fragmentos de tecidos ovarianos por histologia clássica. Os folículos pré-antrais foram quantificados e classificados de acordo com a fase de desenvolvimento. O percentual médio de folículos normais foi maior (P < 0,05) no grupo de folículos vitrificados com resveratrol em relação aos vitrificados sem a adição do antioxidante. No grupo de folículos vitrificados com resveratrol a classe de folículos primordiais apresentou maior percentual (P < 0,05) de folículos normais. Em contrapartida a classe de folículos secundários apresentou o menor percentual (P < 0,05) de folículos normais em todos os tratamentos. Adicionalmente, foi observada uma associação negativa entre a proporção de folículos viáveis e o estágio de desenvolvimento folicular. A probabilidade de se encontrar folículos viáveis foi superior (P < 0,05) no grupo de folículos vitrificados na presença do resveratrol e os folículos primordiais do grupo com resveratrol apresentaram 2,5 vezes mais chance de viabilidade após a vitrificação. A chance de se observar folículos normais foi maior (P < 0,05) nas fases iniciais de desenvolvimento folicular. O diâmetro dos folículos de transição e de seus respectivos ovócitos foi menor quando submetidos ao processo de vitrificação com resveratrol (P < 0,05). Na classe de folículos primários, os diâmetros foliculares e ovocitários foram semelhantes (P > 0,05) entre os grupos estudados. No experimento 2 foi realizada a análise de viabilidade tecidual por meio da técnica de microscopia confocal que avaliou os níveis de emissão de espécies reativas de oxigênio (EROs- DCF) e de viabilidade tecidual (iodeto de propídeo- IP). Os níveis de degeneração tecidual no grupo de fragmentos vitrificados com resveratrol foram semelhantes (P > 0,05) aos do grupo controle. Os níveis de EROs foram semelhantes entre os grupos vitrificados e vitrificados com resveratrol e inferior em relação ao grupo controle. A intensidade de fluorescência emitida com a utilização de ambas probes diminuiu (P < 0,05) com o aumento da profundidade tecidual analisada. Além disso, a análise de regressão linear apresentou correlação negativa entre intensidade de fluorescência e a profundidade tecidual. Observou-se uma associação positiva (P < 0,01) entre os níveis de células degeneradas e as taxas de EROs produzidas nos tratamentos em diferentes profundidades do tecido ovariano, independentemente do tratamento e da profundidade tecidual os grupos apresentaram similaridade para os baixos EROs. A proporção de fragmentos com alto nível de EROs foi superior no grupo controle comparado aos grupos vitrificados. Em conclusão, o resveratrol favoreceu a preservação de folículos ovarianos pré-antrais em fases iniciais de desenvolvimento quando submetidas ao processo de vitrificação/ aquecimento, além de proporcionar redução na produção de EROs e maior viabilidade celular.

The aim of this study was to analyze the morphology and tissue viability of preantral follicles of bovine fetuses by means of histological analysis, fluorescence probes diacetate diclodihydrofluocein and propidium iodide in confocal microscopy, before and after vitrification / warning procedures without or with antioxidant resveratrol. The ovaries (n=18) were fragmented and the fragments distributed to the control (C) groups, vitrified without resveratrol (SR) and vitrified with resveratrol (CR) at the concentration of 20 M. The experiment 1, morphological analysis of the preantral follicles included in fragments of ovarian tissues by classical histology was performed. The preantral follicles were quantified and classified according to the developmental stage. The mean percentage of normal follicles was higher (P <0.05) in the group of follicles vitrified with resveratrol than in the vitrified without the addition of the antioxidant. In the group of follicles vitrified with resveratrol the class of primordial follicles had a higher percentage (P <0.05) of normal follicles. In contrast, the secondary follicle class had the lowest percentage (P <0.05) of normal follicles in all treatments. In addition, a negative association was observed between the proportion of viable follicles and the stage of follicular development. The probability of finding viable follicles was higher (P <0.05) in the group of vitrified follicles in the presence of resveratrol and follicles of the group with resveratrol presented 2.5 times more chance of viability after vitrification. The chance of observing normal follicles was higher (P <0.05) in the early stages of follicular development. The diameter of the transitional follicles and their respective oocytes was lower when submitted to the vitrification process with resveratrol (P <0.05). In the primary follicle class, the follicular and oocyte diameters were similar (P> 0.05) between the studied groups. The experiment 2, the tissue viability analysis was performed using a confocal microscopy technique that evaluated the fluorescence levels emitted by DCF and IP. The fluorescence levels emitted by PI in the group of fragments vitrified with resveratrol were similar (P> 0.05) to the control group. The levels of EROs were similar between the vitrified and vitrified groups with resveratrol and lower in relation to the control group. The intensity of fluorescence emitted with the use of both probes decreased (P <0.05) with the increased tissue depth analyzed. In addition, linear regression analysis showed a negative correlation between fluorescence intensity and tissue depth. A positive association (P <0.01) was observed between the degenerate cell levels and the ROS rates produced in the treatments with different depths of ovarian tissue, regardless of treatment and tissue depth, groups showed similarity to low ROS.. The proportion of fragments with high level of EROs was higher in the control group compared to the vitrified groups. In conclusion, resveratrol favored the preservation of preantral follicles in the early stages of development when submitted to the vitrification / warning process, as well as to reduce the production of ROS and greater cell viability

Morphological and cytochemical characterization of spores and gills of Lepista sordida (Fungi: Basidiomycota)

Some ultrastructural and cytochemical aspects of Lepista sordida are described. Basidiospores with verruculose and irregular ornamentations were observed through scanning electron microscopy. Confocal microscopy indicated that most of them exhibited a single large lipid body, no acidic vesicles and nucleus localized in the cell periphery.

Este trabalho descreve aspectos ultraestruturais e citoquímicos de Lepista sordida. Basidiósporos recobertos com ornamentações verruculosas e irregulares foram observados através de microscopia eletrônica de varredura. Através de microscopia confocal verificou-se que a maioria apresentava um grande corpo lipídico, ausência de vesículas ácidas e núcleo localizado na periferia.