Proteção do dano oxidativo hepático induzido por ferro pelo extrato aquoso da planta Plectranthus barbatus / Protection against hepatic oxidative damage induced by iron by the aqueous extract of Plectranthus barbatus

Plectranthus barbatus Andrews (Lamiaceae) é uma planta muita utilizada na medicina popular para o tratamento de doenças gastrointestinais e hepáticas. O objetivo do presente trabalho foi estudar o efeito protetor do extrato aquoso de P. barbatus (EAPB) sobre os danos hepáticos causados pela sobrecarga de ferro provocada pelo ferro-dextran em ratos. O tratamento com ferro-dextran induziu uma redução significativa na concentração de glutationa reduzida nos animais tratados em relação ao grupo controle e o tratamento prévio dos animais com o EAPB protegeu o fígado do efeito provocado pelo ferro neste parâmetro. Com relação à lipoperoxidação, houve aumento significativo na concentração de malondialdeído (MDA) nos animais tratados em relação ao controle, entretanto, quando os animais receberam o tratamento prévio com o EAPB, houve redução significativa na concentração do MDA. A análise histopatológica mostrou que o grupo tratado com ferro-dextran apresentou grânulos de ferro no citoplasma das células de Kupffer com alargamento das mesmas e algumas com os núcleos hipertróficos. O tratamento prévio com EAPB resultou no desaparecimento dos sinais de danos às células de Kupffer sem nenhum núcleo hipertrófico, mas com a presença de grânulos de ferro totalmente fagocitados, o que demonstra uma aparência morfológica normal. Portanto, o EAPB pode ser útil na prevenção de danos hepáticos induzidos por sobrecarga de ferro.

The Plectranthus barbatus Andrews (Lamiaceae) is a plant largely used in folk medicine to treat gastrointestinal and liver diseases. The objective of this work was to study the protective effect of the aqueous extract of P. barbatus (EAPB) against damage caused by iron overload induced by iron dextran in rat liver. Treatment with iron-dextran induced a significant reduction in the glutathione levels in treated animals compared to control group, and the pretreatment of animals with EAPB protected the liver from the effects caused by iron in this parameter. With respect to lipid peroxidation, a significant increase in the malondialdehyde (MDA) levels in treated animals compared to control was observed; however, when the animals were pretreated with EAPB, there was a significant reduction in the MDA levels. Histopathological analysis showed that the group treated with iron-dextran showed iron granules in the cytoplasm of the Kupffer cells and some of them presented enlarged nuclei. The group previously treated with EAPB showed the disappearance of the signs of damage to the Kupffer cells with no nucleus hypertrophy but with the presence of iron granules completely phagocytosed by these cells, which showed a normal morphological appearance. Therefore, the EAPB may be useful in the prevention of liver damage induced by iron overload.

Modulation of maternal immune system during pregnancy in the cow.

There is a molecular crosstalk between the trophoblast and maternal immune cells of bovine endometrium. The uterine cells are able to secrete cytokine/chemokines to either induce a suppressive environment for establishment of the pregnancy or to recruit immune cells to the endometrium to fight infections. Despite morphological differences between women and cows, mechanisms for immune tolerance during pregnancy seem to be conserved. Mechanisms for uterine immunesuppression in the cow include: reduced expression of major histocompatability proteins by the trophoblast; recruitment of macrophages to the pregnant endometrium; and modulation of immune-related genes in response to the presence of the conceptus. Recently, an eGFP transgenic cloned embryo model developed by our group showed that there is modulation of foetal proteins expressed at the site of syncytium formation, suggesting that foetal cell can regulate not only by the secretion of specific factors such as interferon-tau, but also by regulating their own protein expression to avoid excessive maternal recognition by the local immune system. Furthermore, foetal DNA can be detected in the maternal circulation; this may reflect the occurrence of an invasion of trophoblast cells and/or their fragment beyond the uterine basement membrane in the cow. In fact, the newly description of exosome release by the trophoblast cell suggests that could be a new fashion of maternal-foetal communication at the placental barrier. Additionally, recent global transcriptome studies on bovine endometrium suggested that the immune system is aware, from an immunological point of view, of the presence of the foetus in the cow during early pregnancy.

Gene expression in placentation of farm animals: an overview of gene function during development.

Eutherian mammals share a common ancestor that evolved into two main placental types, i.e., hemotrophic (e.g., human and mouse) and histiotrophic (e.g., farm animals), which differ in invasiveness. Pregnancies initiated with assisted reproductive techniques (ART) in farm animals are at increased risk of failure; these losses were associated with placental defects, perhaps due to altered gene expression. Developmentally regulated genes in the placenta seem highly phylogenetically conserved, whereas those expressed later in pregnancy are more species-specific. To elucidate differences between hemotrophic and epitheliochorial placentae, gene expression data were compiled from microarray studies of bovine placental tissues at various stages of pregnancy. Moreover, an in silico subtractive library was constructed based on homology of bovine genes to the database of zebrafish - a nonplacental vertebrate. In addition, the list of placental preferentially expressed genes for the human and mouse were collected using bioinformatics tools (Tissue-specific Gene Expression and Regulation [TiGER] - for humans, and tissue-specific genes database (TiSGeD) - for mice and humans). Humans, mice, and cattle shared 93 genes expressed in their placentae. Most of these were related to immune function (based on analysis of gene ontology). Cattle and women shared expression of 23 genes, mostly related to hormonal activity, whereas mice and women shared 16 genes (primarily sexual differentiation and glycoprotein biology). Because the number of genes expressed by the placentae of both cattle and mice were similar (based on cluster analysis), we concluded that both cattle and mice were suitable models to study the biology of the human placenta.

IFPA Meeting 2010 Workshop Report I: Immunology; ion transport; epigenetics; vascular reactivity; epitheliochorial placentation; proteomics.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.

Transplacental transfer of iron in the water buffalo (Bubalus bubalis): uteroferrin and erythrophagocytosis.

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental-maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl's reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal-placental tissues in buffalo throughout pregnancy.

Morpho-physical recording of bovine conceptus (Bos indicus) and placenta from days 20 to 70 of pregnancy.

The study is based on 141 pregnant Bos indicus cows, from days 20 to 70 post-insemination. First, special attention was given to the macroscopically observable phenomena of attachment of the conceptus to the uterus, i.e. the implantation, from about days 20 to 30 post-insemination up to day 70, and placentome development by growth, vascularization and increase in the number of cotyledons opposite to the endometrial caruncles. Secondly, as for the conceptuses, semiquantitative, statistical analyses were performed of the lengths of chorio-allantois, amnion and yolk sac; and the different parts of the centre and two extremes of the yolk sacs were also analysed. Thirdly, the embryos/foetuses corresponding to their membranes were measured by their greatest length and by weight, and described by the appearance of external developmental phenomena during the investigated period like neurulation, somites, branchial arcs, brain vesicles, limb buds, C-form, pigmented eye and facial grooves. In conclusion, all the data collected in this study from days 20 to 70 of bovine pregnancy were compared extensively with corresponding data of the literature. This resulted in an 'embryo/foetal age-scale', which has extended the data in the literature by covering the first 8 to 70 days of pregnancy. This age-scale of early bovine intrauterine development provides model for studies, even when using slaughtered cows without distinct knowledge of insemination or fertilization time, through macroscopic techniques. This distinctly facilitates research into the cow, which is now being widely used as 'an experimental animal' for testing new techniques of reproduction like in vitro fertilization, embryo transfer and cloning.

Initial development of bovine placentation (Bos indicus) from the point of view of the allantois and amnion.

The aim of this study was to perform a morphological characterization of the initial bovine placental development, between 20 and 70 days post-insemination (p.i.), with emphasis on the differentiation of the allantois and amnion. After collection, the conceptuses were dissected, macroscopically measured and photographically documented. The extraembryonic membranes were cut into fragments measuring 5 cm(2), and then fixed in 4% paraformaldehyde for analysis using light microscopy, and in 2.5% glutaraldehyde for use in scanning and transmission electron microscopy. The extraembryonic and fetal membranes presented variable degrees of development throughout the periods analysed. The macroscopic appearance of vascularization of the allantois, its attempt to merge with the chorium and the effective appearance of the first cotyledons in development were the events observed from 30 to 40 days of pregnancy. The measurements of the amnion increased gradually as gestation developed. The allantoic epithelia presented cellular dimorphism from 20 to 25 days of pregnancy, but was shown to be immature from 60 to 70 days of pregnancy.

Placentation in cloned cattle: structure and microvascular architecture.

To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies established by somatic nucleus transfer (NT), the umbilical cord and fetal membranes were edematous. Placentomal fusion was common, resulting in increased size and a decreased number of placentomes. Extensive areas of the chorioallantoic membrane were devoid of placentomes. An increased number of functional or accessory microcotyledons (<1 cm) were present at the maternally oriented surface of fetal membranes. Extensive areas of extravasated maternal blood were present within the placentomes and in the interplacentomal region. The crypts on the caruncular surface were dilated and accommodated complexes of more than one primary villus, as opposed to a single villus in non-cloned placentae. Scanning electron microscopy of blood vessel casts revealed that there was also more than one stem artery per villous tree and that the ramification of the vessels failed to form dense complexes of capillary loops and sinusoidal dilations as in normal pregnancies. At the materno-fetal interface, however, the trophoblast and uterine epithelium had normal histology. In conclusion, the NT placentas had a range of pathomorphological changes; this was likely associated with the poor clinical outcome of NT pregnancies.

Fator de crescimento fibroblástico básico e seus receptores em relação à atividade proliferativa na placenta bubalina em diferentes fases da gestação / Basic fibloblast growth factor and its receptors in relation to proliferative activity in the buffalo placenta during gestation

Estudou-se a distribuição espaço-temporal do fator de crescimento fibroblástico básico (bFGF), do receptor 1 do fator de crescimento fibroblástico (FGFR1) e do receptor 2 do fator de crescimento fibroblástico (FGFR2) na placenta bubalina, correlacionando-a à proliferação celular. Para a detecção do bFGF, FGFR1, FGFR2 e antígeno Ki-67, colheram-se 12 placentas de búfalas nos terços inicial, médio e final da gestação, em abatedouros, e realizaram-se testes de imunoistoquímica. Detectou-se e avaliou-se a expressão do bFGF, do FGFR1, do FGFR2 e do antígeno Ki-67 ao longo da gestação. No compartimento fetal da placenta, observaram-se correlações positivas entre a expressão do bFGF e Ki-67, entre FGFR1 e Ki-67 e entre FGFR2 com Ki-67 (r=0,313, 0,358 e 0,384, respectivamente). No epitélio e estroma maternos observaram-se altas correlações entre FGFR1 e Ki-67 (r=0,739 e r=0,511, respectivamente). Os resultados sugerem envolvimento do bFGF, FGFR1 e FGFR2 na proliferação do trofoblasto enquanto no compartimento materno da placenta bubalina apenas o FGFR1 atuaria como modulador dessa atividade.

The space-temporal expression of basic fibroblast growth factor (bFGF), fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor receptor 2 (FGFR2) in buffalo placenta and correlation to proliferative activity was studied. For the localization of bFGF, FGFR1, FGFR2 and Ki-67, 12 buffalo placentas from initial, middle and final gestational thirds were collected and immunohistochemistry tests were performed. Expression of bFGF and its receptors was detected and analyzed from the initial third until the end of gestation. In the fetal compartment, positive correlations were observed between the expression of bFGF and Ki-67, FGFR1 and Ki-67, besides FGFR2 and Ki-67 (r=0.313, 0.358 and 0.384, respectively). High correlations were found between FGFR1 and Ki-67 in maternal epithelium and stroma (r=0.789 and r=0.511, respectively). The results suggest that bFGF, FGFR1 and FGFR2 may be involved in the modulation of trophoblast proliferation, whereas maternal compartment proliferation in the buffalo placenta would only be modulated by FGFR1.

Binucleate trophoblast giant cells in the water buffalo (Bubalus bubalis) placenta.

The binucleate trophoblast giant cells (BNC) of the water buffalo, Bubalus bubalis, placenta were studied, with emphasis on the synthesis of BNC-specific proteins. Placentomal tissues of 27 water buffalos (2-10 months of pregnancy) were processed for light and electron microscopy. The frequency of BNCs was 20% of the trophoblastic cells in 2-3-month placentas and increased to 27% in the later stages. Ultrastructurally, binucleate cells displayed a prominent granular endoplasmic reticulum and Golgi apparatus, typical of cells involved with protein synthesis and exportation. The buffalo BNCs contained periodic acid-Schiff (PAS)-positive granules and reacted with antisera against bovine placental lactogen, prolactin-related protein-I, and pregnancy-associated glycoproteins. Lectin histochemistry with Dolichos biflorus agglutinin, Vicia villosa agglutinin, and Phaseolus vulgaris leucoagglutinin showed specific staining of BNCs. Different stages of BNC migration and fusion with uterine epithelial cells were observed. Trinucleate feto-maternal hybrid cells were the typical outcome of cell fusions. These cells underwent degeneration, with typical morphological features of apoptosis. The results revealed a strong homology between water buffalo and cattle BNCs concerning cell morphology, protein expression, glycosylation pattern, and characteristics of cell migration and fusion.