Browsing by Author "Garcia, C."
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- Detection of apoptotic events, using different methods, in renal tissues after acute haemorrhagePublication . Vala, Helena; Cruz, R.; Venâncio, C.; Garcia, C.; Silva, A.; Mesquita, J.; Ortiz, A. L.; Ferreira, D.Introduction: Apoptosis plays an important role in cellular damage caused by acute renal hypoperfusion. The aim of this study was to detect apoptotic events in an animal model of acute haemorrhage, followed by volume replacement with different intravenous solutions. Materials and Methods: Renal samples were collected from animals submitted to passive arterial blood bleeding and reperfusion with a crystalloid (RL) (G1) and with a synthetic colloid (HES 130/0.4) (G2). All procedures were carried out under personal and project licenses approved by the Ethical Committee of the national regulatory office. Immunohistochemistry was performed, using cytochrome c antibody to detect mitochondrial activity and in situ TUNEL method to evaluate endonucleosomic cleavage of DNA by TdT. An immunofluorescence method, the M30 Cytodeath, was also used to detect early apoptotic events. Results: In all groups, apoptosis was detected in the epithelial tubular cells of the proximal and distal convoluted tubules, in the loop of Henle and in the collecting tubules. However, the percentage of apoptotic cells and the intensity of reaction was significantly higher in G2. Conclusions: Apoptosis plays an important role in the cellular damage in renal tubules following acute haemorrhage and volume replacement. The type of solution used for volume replacement may influence the extent of renal damage resulting from this clinical situation. In the present study, the use of RL was related with decreased cytosolic cytochrome c and a lower apoptotic index in the tubular renal cells.
- Evaluation and characterization of intestinal oedema in pigsPublication . Ortiz, A. L.; Vala, Helena; Silva, A.; Venâncio, C.; Garcia, C.; V, D.Introduction When characterizing small intestine early lesions in studies concerning intestinal hypoperfusion in pigs, authors faced a significant difficulty when trying to standard the oedema grading to allow an accurate comparison between groups. Therefore, specific alterations were assumed to characterize grade1 (G1), grade2 (G2) and grade3 (G3) oedema. Material and Methods Eighteen Large White pigs underwent total intravenous anaesthesia (TIVA) with propofol and remifentanil. 25 ml/kg of arterial blood were removed from the femoral artery over 20 minutes. Volume was replaced using Ringer Lactate in Gr1 (n=6) and Hydroxyethyl starch 130/0.4 in Gr2 (n=6), 20 minutes after bleeding. Animals of G3 (n=6) were under TIVA without any haemorrhage and volume replacement. One hour after, pigs were euthanized and small intestine samples taken for histopathological examination. Oedema was described and classified in a specific scale: G0-Normal–normal central lacteal G1-Mild–lacteal dilation G2-Moderate-lacteal dilation and/or oedema of lamina propria G3-Marked–lacteal dilation dilation, oedema of lamina propria, and/or presence of submucosal oedema Pictures were made to better characterize the scale used. Results G2 oedema was predominant in duodenum in Gr1 (n=3), while in Gr2 and Gr3 was G1 for the same intestinal segment (n=5, n=4, respectively). G1 was predominant in jejunum in Gr1 (n=4), while G2 and G0 was more observed in Gr2 (n=2, n=2, respectively) and G2 in Gr3 (n=3). G1 was predominant in ileum in both Gr1 (n=5) and Gr2 (n=4), being G0 the grade more often observed in this segment in Gr3 (n=3). Jejunum was the only intestinal segment were G3 oedema was observed, and only in Gr2 (n=1).The higher frequency of G0 oedema for all the intestinal segments was observed in Gr3 (n=8). Conclusion The characterization used for the different grades of oedema improved uniformity when deciding which grade to attribute in each case and may allow standardization of oedema grading in further studies.
- Evaluation and characterization of intestinal oedema in pigsPublication . Ortiz, A. L.; Vala, Helena; Venâncio, C.; Garcia, C.; Ferreira, D.Introduction When characterizing small intestine early lesions in studies concerning intestinal hypoperfusion in pigs, authors faced a significant difficulty when trying to standard the oedema grading to allow an accurate comparison between groups. Therefore, specific alterations were assumed to characterize grade1 (G1), grade2 (G2) and grade3 (G3) oedema. Material and Methods Eighteen Large White pigs underwent total intravenous anaesthesia (TIVA) with propofol and remifentanil. 25 ml/kg of arterial blood were removed from the femoral artery over 20 minutes. Volume was replaced using Ringer Lactate in Gr1 (n=6) and Hydroxyethyl starch 130/0.4 in Gr2 (n=6), 20 minutes after bleeding. Animals of G3 (n=6) were under TIVA without any haemorrhage and volume replacement. One hour after, pigs were euthanized and small intestine samples taken for histopathological examination. Oedema was described and classified in a specific scale: G0-Normal–normal central lacteal G1-Mild–lacteal dilation G2-Moderate-lacteal dilation and/or oedema of lamina propria G3-Marked–lacteal dilation dilation, oedema of lamina propria, and/or presence of submucosal oedema Pictures were made to better characterize the scale used. Results G2 oedema was predominant in duodenum in Gr1 (n=3), while in Gr2 and Gr3 was G1 for the same intestinal segment (n=5, n=4, respectively). G1 was predominant in jejunum in Gr1 (n=4), while G2 and G0 was more observed in Gr2 (n=2, n=2, respectively) and G2 in Gr3 (n=3). G1 was predominant in ileum in both Gr1 (n=5) and Gr2 (n=4), being G0 the grade more often observed in this segment in Gr3 (n=3). Jejunum was the only intestinal segment were G3 oedema was observed, and only in Gr2 (n=1).The higher frequency of G0 oedema for all the intestinal segments was observed in Gr3 (n=8). Conclusion The characterization used for the different grades of oedema improved uniformity when deciding which grade to attribute in each case and may allow standardization of oedema grading in further studies.
- Evaluation of small intestine apoptosis after haemorrhage followed by volume replacement – a preliminary studyPublication . Ortiz, A.; Vala, Helena; Garcia, C.; Cruz, R.; Venâncio, C.; Silva, A.; Ferreira, D.Apoptosis plays a key role in intestinal injury, as response to ischemia. Furthermore, reperfusion exacerbates abnormal intestinal apoptosis. In this study, the apoptotic index (AI) in the small intestine after haemorrhage and volume replacement with Ringer´s solution was evaluated, in a pig model
- Main difficulties encountered in implementation of techniques for apoptosis detection in animal tissuesPublication . Garcia, C.; Cruz, R.; Mega, A.; Ortiz, A. L.; Teixeira de Lemos, E.; Ferreira, D.; Reis, F.; Vala, HelenaIntroduction: The implementation of methodologies capable of identifying apoptotic cells is a valuable tool in many biomedical studies. Much of the knowledge acquired about the mechanisms regulating apoptosis comes from observations in isolated cells; however, the properties shown by these assays do not always apply to the study of tissue samples. For detection of apoptosis in tissues, many investigators recommend the TUNEL method, nonetheless, other histochemical methodologies are available, and their choice must be carefully evaluated, taking into account the morphologic alterations which they intend to detect. Objective: To share encountered difficulties in the implementation of three different methods for the detection of apoptotic cells in animal tissue. Methods: Animal tissues, fixed in 10% neutral-buffered formalin and embedded in paraffin were submitted to immunohistochemical techniques using the TUNEL method (In situ Cell Death Kit, Roche) and polyclonal Bax and Bcl-2 antibodies (Santa Cruz Biotechnology). Results: After a long period of tests and costs, in which unspecific reactions, as well as several artifacts were obtained, the persistence to overcome these obstacles made all methods effective in the detection of apoptosis. Observed differences in immunolabeling were related to the type of tissues tested, which will be documented in the present study. Conclusions: Immunohistochemical methods are an approach to consider in the detection of apoptosis in animal tissues. Sharing the challenging difficulties found in our work, might obviate many tests and costs in its implementation in other laboratory facilities.
- Métodos imunológicos e suas aplicações em diagnóstico. ImunofluorescênciaPublication . Capela, I.; Almeida, D.; Moreira, R.; Garcia, C.; Cruz, R.; Vala, HelenaINTRODUÇÃO: Os testes laboratoriais imunológicos podem fornecer informações importantes para o diagnóstico e cuidado clínico de pacientes, sendo de extrema importância no diagnóstico de doenças relacionadas com o envolvimento directo do sistema imune, como para doenças de etiologia independente do sistema imunológico. Os testes estabelecidos e clássicos detectam a presença de anticorpos contra parasitas, fungos, bactérias, vírus, indicando a presença de uma resposta imune contra o agente. Testes mais modernos e sensíveis podem detectar a presença de antigénios destes agentes, indicando directamente a sua presença no hospedeiro. Os testes imunológicos podem, ainda, ser utilizados para a detecção de hormonas ou outras substâncias (Hames, 1999). A imunofluorescência combina técnicas histológicas, imunológicas e bioquímicas com o objectivo de identificar componentes celulares, denominados antigénios (Ag), em células e tecidos através da reacção com anticorpos específicos. A técnica utiliza anticorpos (Ac) que se ligam covalentemente a moléculas relevadoras denominadas fluorocromos, permitindo a visualização dos complexos antigénio-anticorpo, sem que as suas propriedades imunitárias se modifiquem (Meira, 2010).
- Routine histological technique applied to a biological model for environmental studiesPublication . Vala, Helena; Rodriguez-Seijo, A.; Lourenço, J.; Garcia, C.; Francisco, A.; Rocha-Santos, T.; Costa, J.; Pereira, R.Introduction and aim Earthworms are an important test organisms used in several standard ecotoxicological tests (e.g. ISO, 2008, 2012; OECD, 2004, 1094) and they are also model organisms to test soil structure and permeability, as well as for important soil services as the degradation organic matter. Although histopathological changes have been demonstrated to be good biomarkers to assess the exposure of these animals to different physical and chemical stress agents, studies with clear and high quality images describing normal tissue conditions are scarce in the literature, so the aim of this work was to better characterize this biological model. Material and Methods Eight adult earthworms exposed to an artificial standard soil (OECD, 2004) for 28 days, were extracted and placed in a plastic box to depurate their gut content, fixed in 10% neutral-buffered formalin and processed for routine histopathological diagnosis. Results and discussion Satisfactory histological sections were obtained. Some difficulties were faced related with microtome sectioning, resulting in artefacts, namely lines across sections produced by a nick in the cutting edge of the microtome knife cutting tear, motivated by the presence of sand and other solid particles that persisted in the gut of earthworms. Nevertheless, it was possible to obtain representative figures from different earthworm sections. Conclusion Routine histological technique was effective for obtaining satisfactory histological sections and the knowledge of the histology of earthworms could be very useful for future application in environmental studies, using this biological model.
- Small intestine apoptosis evaluation after haemorrhage followed by volume replacement with colloid and crystalloid solution in a pig modelPublication . Ortiz, A.; Vala, Helena; Garcia, C.; Cruz, R.; Venâncio, C.; Silva, A.; Ferreira, D.Apoptosis plays a key role in intestinal injury, as response to ischemia. Furthermore, reperfusion exacerbates abnormal intestinal apoptosis. In this study, the apoptotic index (AI) in the small intestine after haemorrhage and volume replacement with Ringer´s solution was evaluated, in a pig model