Browsing by Author "Ortiz, A. L."
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- Apoptosis as a prognostic marker in prediction of renal injury, after acute bleeding and volume replacement with HES 130/0.4 or Ringer solution, in a pig modelPublication . Cruz, R.; Vala, Helena; Machado, A.; Venâncio, C.; Mesquita, J.; Silva, A.; Ortiz, A. L.; Ferreira, D.Objectives: The aim of this study is to identify and quantify apoptosis in renal tissue, using a biochemical marker (TUNEL) in a pig haemorrhagic model, after intravascular volume replacement with Ringer's lactate RL) or Hydroxyethylstarch (HES) 130/0.4) solutions. Methods: 18 Large White pigs underwent total intravenous anaesthesia (TIVA) with propofol and remifentanil. 25 ml/kg of arterial blood were removed from the femoral artery. Volume was replaced, RL, in group1 (n=6) and HES 130/0.4, in group2 (n=6), 20 min after bleeding. The control group did not face bleeding and volume reposition. One hour after volume replacement, pigs were euthanized with intravenous KCl, and renal tissue samples were taken for several studies, including immunohistochemically with in situ TUNEL method for apoptosis detection. ANOVA was used to compare data between groups. Results: In all groups apoptosis was, as expected, mainly detected in epithelial tubular cells of the proximal and distal convoluted tubules, in the loop of Henle and in the epithelium of collecting tubules, however the number of apoptotic cells per mm2 was lower in group 1 (35.68 apoptotic cells/mm2), when compared with group 2 (67.94 apoptotic cells/mm2) and the control group (146.34 apoptotic cells/mm2). The level of apoptosis was significantly higher in Ringer Lactate group, comparing with controls group, for the epithelial tubular cells (P<0.05). Conclusion: The median apoptotic levels were significantly lower in pigs, subjected to fluid replacement with RL, when compared with HES 130/0.4. Ringer lactate might promote better renal perfusion in the presence of severe hypovolaemia following acute haemorrhage.
- 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.
- Hepatic histopathological lesions in acute controled haemorrhage followed by volume replacement with a crystalloid or colloid solutionPublication . Vala, Helena; Pina, R.; Cruz, R.; Venâncio, C.; Esteves, Fernando; Silva, A.; Mesquita, João; Ortiz, A. L.; Ferreira, D.Introduction: severe hemorrhage remains the major cause of morbidity and mortality in trauma victims or surgical intervened animals, despite of all the advances in the therapeutic approach. The resulting injuries, or even death, are due to the deficit in intravascular volume and subsequent development of hypovolaemic fluid state, leading to poor tissue perfusion and consequent decreased oxygen delivery to the tissues, often with compromise of organ function. Objective: the aim of this work was to evaluate the effect of different intravenous solutions used for volume replacement following acute controlled haemorrhage, one crystalloid (RL) and one colloid (HES130/0.4) in the integrity of the hepatic tissue in a pig animal model. Material and methods: hepatic 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). Samples were also collected from animals that were not subjected to acute bleeding nor volume replacement (G3, control group). All procedures were carried out under personal and project licenses approved by the Ethical Committee of the national regulatory office. Samples were collected and fixed in 10% neutral-buffered formalin, for a maximum of 24hours, embedded in paraffin wax and 3µm sections were stained for routine histopathology with haematoxylin and eosin. Results: the histopathological assessment revealed no statistically significant differences between the three groups. However, some lesions were more often expressed in some groups. More severe hepatocellular hydropic degeneration and hepatocellular steatosis was seen in G1, which is the only group in which haemorrhage was observed and within which oedema was not present. Hyperaemia was only observed in G2 and G3. Necrosis was not present in any of the groups. Conclusion: hepatic histopathological lesions following controlled bleeding and intravenous volume replacement with RL or HES130/0.4 were subtle. However, more pronounced hydropic degeneration and hepatocellular steatosis was seen in G1 (RL), which suggests that HES130/04 may be associated with better hepatic perfusion when used for intravenous volume replacement when compared to RL.
- Histological evaluation of the spleen after acute bleeding followed by blood replacement with two different physiologic solutionsPublication . Cabral, M.; Venâncio, C.; Ortiz, A. L.; Mesquita, J.; Nóbrega, C.; Silva, A.; Vala, Helena; Ferreira, D.Background: The spleen is important for many hematopoietic and immunological functions, namely hemodynamic compensation during hemorrhagic shock, with a crucial role in the compensation of hypovolaemia, on restoring blood volume in situations of acute haemorrhage. Also, the administration of physiological solutions is of great importance for the correction of circulating volume, avoiding the complications of hypovolaemia. Objectives: To evaluate spleen’s histopathological changes in pigs associated with volume replacement using two different physiologic solutions, after acute bleeding. Methods: 31 Large White pigs under general anaesthesia with propofol and remifentanil. A total of 25ml/kg of blood was passively removed during 20 minutes. Intravascular volume was replaced using Ringer Lactate 25 ml/kg (Group 1- thirteen pigs) and HES 130/0.4 20 ml/kg (Group 2- eleven pigs). Spleen samples were processed for routine hystologic evaluation. Congestion, inflammatory infiltrate, ellipsoids enlargement and follicular lymphoid hyperplasia were evaluated in a semi-quantitative score. Results: Follicular lymphoid hyperplasia was exacerbated in group 1 (84.6 %; grade III), when comparing with group 2 (36.4 %; grade III). The remaining lesions did not show differences between group 1 and group 2. Conclusion: Volume replacement with HES 130/0.4 may reduce follicular lymphoid hyperplasia when compared with volume replacement with Ringer Lactate.
- Histopathological assessment of renal lesions after volume replacement with HES 130/0.4 or Ringer solution, following acute bleedingPublication . Cruz, R.; Vala, Helena; Venâncio, C.; Mesquita, J.; Mega, A. C.; Silva, A.; Ortiz, A. L.; Ferreira, D.Background: Surgical blood loss can lead to ineffective tissue perfusion of vital organs, including the kidney. Physiologic solution chosen for blood volume replacement may be determinant for preserving renal integrity. Objectives: Study kidney histopathological changes in a hemorrhagic model, followed by intravascular volume replacement with Ringer's lactate or Hydroxyethylstarch 130/0.4 solutions. Methods: Thirty one pigs under general anaesthesia with propofol and remifentanil underwent haemorrhage at a volume of 25ml kg-1, over 20 minutes. After a waiting period, intravascular volume was replaced using HES 130/0.4 (group 1) and Ringer’s lactate (group 2). One hour after, pigs were euthanized and sixty two renal samples were taken for histopathological examination, using PAS staining. Renal damage was assessed for glomerular, tubulointerstitial and vascular lesions. Contingency tables for frequency analysis were used. Results: Mean arterial pressure values (MAP) reached 40 mmHg after bleeding, and recovered for values above 60 mmHg in both groups after volume replacement. Histopathological lesions observed in Group 2 were more frequent than those in Group 1. Conclusion: HES 130/0.4 may reduce the incidence of histopathological lesions secondary to renal hypoperfusion after severe bleeding when compared with Ringer Lactate. Results suggest that reestablishment of intravascular volume with HES 130/0.4 may preserve renal integrity secondary to blood loss.
- Importance of the canine norovirus in the etiopathogenesis of enteritis like IBDPublication . Vala, Helena; Mesquita, J.; Ortiz, A. L.; Nascimento, M. S. J.Background: Recent studies stated that canine norovirus (caNoV) infection is an endemic and highly contagious disease in the dog population in Portugal, with peak shedding in winter months. Previous studies reported caNoV as a potentially important zoonotic virus with increased importance since dogs are an integral part of household, potentially, affecting both owners and small animal veterinarians. However, despite the impact of norovirus-induced disease, the pathogenic features of infection are not well understood. The aim of this study was to evaluate the histopathological lesions in gastroenteritis, associated to CaNoV, in order to contribute to the knowledge of its pathogenic features. Material and methods: Samples of small intestine from 2 diarrheic dogs housed in a kennel, previously diagnosed with caNoV, were collected and fixed (10% neutral buffered formalin) for a maximum of 48 hours, embedded in paraffin wax, and 3µm sections were stained for routine histopathological evaluation. Results: The microscopic findings revealed a more notorious mononuclear inflammatory infiltrate within lamina propria of small intestine proximal segments, with increasing numbers of lymphocytes and plasma cells, changing from severe to moderate, from the duodenum to jejunum, respectively, like IBD pattern. Discussion and Conclusions: The histopathological features led to the hypothesis that caNoV could be significantly involved in the complex aetiopathogenesis of IBD, as reported with murine norovirus and human norovirus, the later having an important role exacerbating or complicating other diseases, like human Crohn disease - an example of human IBD, which highlights the relevance of norovirus, in its role of indirect infectious agent.
- 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.
- Presence of Kimmelstiel-Wilson like lesions in canine renal faillurePublication . Vala, Helena; Mega, A. C.; Mesquita, J.; Nóbrega, C.; Cruz, R.; Esteves, F.; Ortiz, A. L.; Santos, C.; Teixeira de Lemos, E.; Reis, F.Background: Nodular increases in mesangial matrix is the definition of Kimmelstiel-Wilson (K-W) lesions. In man, focal mesangiolyses and K-W nodules were positively associated with diabetes, proteinuria and hyalinization of afferent and efferent arterioles. Although, nodular sclerotic lesions may also occur in the absence of diabetic nephropathy and are clinically related to hypertension, smoking, hypercholesterolemia, among others. This study aimed to assess the presence of nodular glomerulosclerosis in dogs in non-diabetic chronic kidney disease. Materials and Methods A retrospective study including dogs with non-diabetic chronic kidney disease (n=27) was performed. Samples were stained with haematoxylin-eosin and periodic acid of Schiff and classified according to Maxie (2007). Lesions were classified as absent or present and the later, as non-convincing and convincing K-W like lesions. Univariate logistic regression models were performed to measure the association between a positive histological diagnosis of K-W lesions and the accessed histological features (variables membranes, inflammation, atrophy and fibrosis) by using statistical software R with Epicalc package. Results Nodular glomerulosclerosis was present in 66.66% of evaluated cases (n=18) of which, 38.88% (n=7) were classified as convincing and 61.11% (n=11) as non-convincing. The convincing K-W like lesions observed, revealed similar microscopic aspects to the descriptions in literature related to man. The presence of fibrosis was positively associated to the diagnosis of K-W lesions (OR=6; p<0.05). The remaining variables were not statistically associated (p>0.05). Conclusion Presence of K-W like lesions, in dogs with renal failure exposed a possible association with end-stage renal disease. Therefore, the diagnosis of K-W like lesions could be helpful to specify and define renal biopsies.