Browsing by Author "Ferreira, D."
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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.
- Characterization of a grading system to evaluate splenic congestion.Publication . Oliveira, A. L.; Vala, Helena; Silva, A.; Venâncio, C.; Ferreira, D.Background: Frequently, the spleen may be targeted by active hyperaemia, or acute or active congestion, common in acute systemic infections and in some acute bacterial intoxications, and also by passive congestion, or chronic congestion, due to disturbances in the systemic and portal circulation and, most commonly, due to the use of barbiturate drugs. An acutely congested spleen is enlarged, moderately turgid and cyanotic, with the capsule blue-black. The normal architecture is not discernible on the cut surface and the pulp is red-black and exudes blood. Microscopically, the red pulp sinusoids are dilated filled with packed red cells, the germinal centres are widely separated and trabeculae are thinned. With chronic congestion, the spleen becomes firm, the capsule and trabeculae thickened; there is lymphoid atrophy, sinus proliferation, with a marked increase in fixed cells in the red pulp and heavy sinus colonization with macrophages and hemosiderin, following RBC destruction in dilated sinusoids. Extend of the acute congestion may make histological examination difficult. In some species there is still some difficulty on determine the severity of spleen congestion and rule out other conditions like primary neoplastic lesions, metastatic disease or manifestation of systemic involvement, due to it specific circulation system. Objectives: The authors present an illustrated system to evaluate splenic congestion, using the pig as a model. Methods: Samples of the spleen of Large White Pigs were fixed in 10% neutral-buffered formalin, for a maximum of 48 hours, embedded in paraffin wax, and 3µm sections were stained for routine histopathological diagnosis with hematoxylin and eosine. All samples were examined by light microscopy, using a Microscope Zeiss Mod. Axioplan 2. Results: In this study, fourteen spleen samples from Large White pigs were observed and classified. The semi-quantitative parameter congestion was evaluated by two independent examiners and classified in a specific scale from 0 (minimal) to 3 (severe and extensive damage), from which exemplificative pictures will be presented, in order to better characterize the grading system used. Conclusion: Based on the description and pictures presented, the authors conclude that it was possible to clarify a classification grading system to evaluate splenic congestion, using the pig as a model, which can be used to evaluate splenic congestion in other domestic animals, as dogs and cats, which can be of great importance to characterize lesions in specific diseases and conditions.
- 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; 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
- 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 assessment of small bowel hypoperfusion lesions in the pigPublication . Oliveira, A. L.; Ferreira, D.; Vala, HelenaBackground Authors propose the use of a quantitative morphological assessment for helping in studies concerning intestinal hypoperfusion. The method was applied to the small intestine mucosa stained with the standard hematoxylin and eosin, from pigs that underwent severe hypotension due to acute hemorrhage.,. Methods Six Large White pigs underwent total intravenous anesthesia with propofol and remifentanil. 25 ml/kg of arterial blood were passively removed from the femoral artery over 20 minutes. Volume was replaced using Ringer Lactate in Group 1 and Hydroxyethyl starch 130/0.4 in Group 2, with a delay of 20 minutes after the bleeding. One hour after the volume replacement, pigs were euthanized and the small intestine samples were taken for histopathological examination. Parameters were classified using two specific scales (Chiu, 1970; Çetin, 1995; Kaplan, 2007). Mucosal loss (ML) percentage and crypt:interstitium ratio were obtained (Faleiros, 2001). Results Inflammatory infiltrate was present in all animals, varying from grade 2 to grade 3. Hydropic cellular degeneration and epithelial detachment were more pronounced in duodenum and more noticeable in Group 1. In Group 1, ML percentage was 2.18±0.46 % in duodenum, 0.62±1,07% in jejunum and 0.45±0.77% in ileum. In Group 2 ML percentage was 0.75±1.30 in duodenum and 0±0% in the other intestinal segments. In the whole small intestine, was 1.08±0.78% in Group 1 and 0.25±0.35% in Group 2. Crypt:interstitium ratio did not presented significant differences between the groups. Conclusions The quantitative morphological assessment may be useful in quantifying the degree of mucosal lost in small intestine stained with hematoxylin and eosin, from pigs submitted to acute severe bleeding.
- 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.
- Histological evaluation of the spleen after acute bleeding followed by volume replacement with two different physiologic solutionsPublication . Cabral, M.; Ortiz, A.; Venâncio, C.; Mesquita, J.; Nóbrega, C.; Silva, A.; Vala, Helena; Ferreira, D.Spleen is important for many hematopoietic and immunological functions, namely in hemodynamic compensation during hemorrhagic shock, with a crucial role in 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 complications due to hypovolemia. The aim of this study was to evaluate spleen`s histopathological changes, associated with volume replacement using two different physiologic solutions (Hydroxyethyl Starch (HES) 130/0.4 and Ringer Lactate), after acute controlled bleeding, in a pig model.
- 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.