Heparin modulates the expression of genes encoding pro and anti-apoptotic proteins in endothelial cells exposed to intestinal ischemia and reperfusion in rats

PURPOSE: To investigate if expression of genes encoding pro and anti-apoptotic proteins in the rat enteric endothelial cells stimulated by intestinal ischemia followed by reperfusion (IR) can be modified by treatment with heparin (HP).METHODS: Eighteen adult Wistar rats were divided in three groups: sham group submitted to laparotomy only (SG), ischemia followed by reperfusion group (IRG); ischemia followed by reperfusion plus pretreatment with HP 100 mg.kg-1 (IRG+HP). Ischemia was performed by clamping of the superior mesenteric artery. After 60 min of ischemia, metal clamps were removed for reperfusion for 120 min. Gene expression of encoding pro (Casp1, Casp6, Casp3, Cflar, Fas and Pgl) and anti-apoptotic (Bcl2, Bcl2l1 and Naip2) proteins in rat enteric endothelial cells was evaluated by PCR microarray method.RESULTS: Compared to rat endothelial cells of SG, the expression of pro-apoptotic genes was up-regulated in IRG while anti-apoptotic genes were down-regulated. In contrast, the expression of anti-apoptotic genes in IRG+HP was up-regulated while pro-apoptotic genes was down-regulated compared to SG.CONCLUSION: The attenuation by heparin of intestinal ischemia-reperfusion previously demonstrated in rodents could be related with ability of this drug to stimulate and reduce gene expression of encoding anti and pro-apoptotic proteins, respectively.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Federal University of São Paulo Department of SurgeryUNIFESP Pharmacology DepartmentUNIFESP Gynecology DepartmentUniversidade Federal de São Paulo (UNIFESP) Department of BiochemistryUniversidade Federal de São Paulo (UNIFESP) Department of SurgeryUNIFESP, Department of SurgeryUNIFESP, Pharmacology DepartmentUNIFESP, Gynecology DepartmentUNIFESP, Department of BiochemistryUNIFESP, Department of SurgerySciEL

Heart injury following intestinal ischemia reperfusion in rats is attenuated by association of ischemic preconditioning and adenosine

PURPOSE: To investigate the effect of ischemic preconditioning (IPC) and adenosine as strategies to protect cardiac injury caused by intestinal IR in rats, based on increasing in adenosine bioavailability and improvement of cell energy state by IPC.METHODS:Male Wistar rats were submitted to 60 minutes of intestinal ischemia and 120 minutes of reperfusion. Intravenous injections of saline or Adenosine (AD) was administered five minutes before ischemia, five minutes before reperfusion and after 55 minutes reperfusion. Cardiac samples were obtained, fixed in formalin solution, embedded in paraffin, and sections of 5 μm were stained by hematoxylin-eosin. Histological analysis of myocardium was performed according occurrence of necrosis signs: piknosis, band contraction, eosinophilic cytoplasm, karyorrhexis and vacuolization (score - zero to 5).RESULTS:The groups submitted to ischemia alone (I=4.0), and reperfusion (IR=4.5) showed highest level of lesion compared to the others (I+IPC=3.3, IR+IPC=3.6, I+AD=3.0, IR+AD=3.8). The most interesting result was association of IPC and AD in IR model (IR+IPC+AD=1.2, p=0.002), showing preservation of the heart tissue, with fibers showing typical cross-striations and nuclei characteristics. Rare and small areas of tissue necrosis was observed and suggestion of capillaries congestion.CONCLUSION: Intestinal ischemia reperfusion promotes cardiac tissue injury. Ischemic preconditioning in association with adenosine is an efficient strategy to protect the heart against ischemia and reperfusion injury.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)ABC Medical SchoolFederal University of São PauloSão Paulo University Faculty of MedicineUniversidade Federal de São Paulo (UNIFESP) Department of SurgeryUNIFESP, Department of SurgerySciEL

The role of pneumoperitoneum in the respiratory and hemodynamic evaluation in anaesthetized rats, with or without intubation

BACKGROUND: To investigate hemodynamic and respiratory changes in rats submitted to CO2 pneumoperitoneum or not, under anesthesia with and without endotracheal intubation. METHODS: Male albino rats (n = 40), average weight of 300g, three months aged, were randomized: GA - anesthesia with endotracheal intubation for one hour with CO2 pneumoperitoneum (4mmHg); GB - anesthesia without intratracheal intubation for one hour with CO2 pneumoperitoneum (4mmHg); GC - anesthesia without endotracheal intubation for one hour without pneumoperitoneum; GD - anesthesia with endotracheal intubation for one hour without pneumoperitoneum. Median arterial pressure (MAP- mmHg), heart rate (HR-beat/min), respiratory frequency (RF - rm/min), central venous pressure (CVP - cmH2O), peripheral oxygen saturation (STO2), partial CO2 pressure (PaCO2), arterial bicarbonate (HCO3-), oxygen saturation (SO2) and pH were recorded. Measurements were performed at the begining of the procedure (MO) and after, 30 minutes (M1) and 60 minutes (M2). RESULTS: In GA and GC (groups with CO2 pneumoperitoneum) there was an increase of MAP, PaCO2, HCO3-, HR, CVP and decrease of pH, CO2, STO2 in comparison with those without pneumoperitoneum. On the other hand, endotracheal intubation attenuated these changings during the first hour of anesthesia. CONCLUSION: General anesthesia with endotracheal intubation, associated to CO2 pneumoperitoneum (4 mmHg) during one hour, proved advantageous in hemodynamic and respiratory systems, in relation to the animals with pneumoperitoneum without endotraqueal intubation, and to those within four hour of anesthesia, in spite of intubation.OBJETIVO: Estudar as alterações hemodinâmicas e respiratórias em ratos submetidos ou não ao pneumoperitônio com CO2, sob anestesia com ou sem intubação intratraqueal. MÉTODO: Ratos machos (n = 40), albinos, com peso médio de 300g, idade de três meses, foram randonizados em quatro grupos: GA - anestesia com intubação endotraqueal por uma hora e com pneumoperitônio de 4mmHg de CO2; GB - anestesia sem intubação endotraqueal por uma hora e com pneumoperitônio de 4mmHg de CO2; GC - anestesia sem intubação endotraqueal por uma hora e sem pneumoperitônio; GD - anestesia com intubação endotraqueal por uma hora, sem pneumoperitônio. Foram registrados os valores da pressão arterial média (PAM), pressão parcial de gás carbônico (pCO2), freqüência cardíaca (FC), freqüência respiratória (FR), pressão venosa central (PVC), potencial hidrogeniônico acidose (pH), saturação de oxigênio periférico (spO2), bicarbonato (HCO3-) e saturação de oxigênio no sangue arterial (SO2,). Os dados foram coletados no início do experimento (M0), após 30 minutos (M1) e após 60 minutos (M2). RESULTADOS: Em GA e GC (grupos com pneumoperitônio) ocorreu aumento da PAM, PCO2, HCO3-, FR, PVC e uma diminuição do pH, SO2, spO2 e da FC em relação aos animais sem pneumoperitônio. Por outro lado a intubação intratraqueal demonstrou atenuar estas alterações nos animais com até uma hora de anestesia. CONCLUSÃO: A anestesia geral com intubação endotraqueal associada ao pneumoperitônio de 4mmHg, por um período de uma hora, mostrou vantagens do ponto de vista respiratório e hemodinâmico, em relação aos animais com pneumoperitônio e sem intubação e também sobre aqueles com até quatro horas de anestesia, apesar da intubação.UNIFESP-EPMUNIFESP-EPM Departamento de CirurgiaUniversidade de Santo AmaroUNIFESP, EPM, Depto. de CirurgiaSciEL

Inhibition of mouse and rat lymphoproliferation by gangliosides

Our previous study have demonstrated that Glycosphingolipids (GSLs) have an immunosuppressive effect on murine lymphoproliferation and IL-2 production. In the present study we examined the effect of a pool of Gangliosides (Gang) on spleen lymphocyte proliferation from either isogeneic strains of Wistar rats or BALB/c mice. Two hundred-fifty grams adult female isogeneic Wistar rats and 8-week-old BALB/c mice were used. The animals were sacrificed and the spleen harvested aseptically for cellular assays. Spleen cells suspensions were obtained by homogenization in RPMI 1640 with a loose tissue grinder. After washing, the cells were suspended in RPMI 1640 supplemented. Cell viability was measured by Trypan blue exclusion. Cells were cultured in triplicate using increasing concentrations of Gang (1; 2; 5; 10; 15; 20 mug/well) and in the presence of Concanavalin A. The cells were incubated for 48 hours and were pulsed with [³H] thymidine 18 hours prior to harvesting on glass fiber paper for counting in a beta-counter. Data were presented as rate of inhibition, as previously described. At concentrations 1 and 2 mug/well, Gang stimulated lymphoproliferation (30% and 50%, rats and mice respectively), while at concentration from 5 to 20 mug/well an increasing inhibition was observed for spleen cells from both mouse and rat (from 40% up to 80%). In preliminary studies we observed inhibition of mixed lymphocyte reaction on spleen cells from rats treated with Gang for 10 days (data not shown). Our data suggest that Gang may be investigated as a immunosuppressive drug in organ transplantation.UNIFESP-EPM Departamento de CirurgiaUNIFESP-EPM Disciplina de Técnica Operatória e Cirurgia ExperimentalUNIFESP-EPM Disciplina de ParasitologiaUNIFESP, EPM, Depto. de CirurgiaUNIFESP, EPM Disciplina de Técnica Operatória e Cirurgia ExperimentalUNIFESP, EPM Disciplina de ParasitologiaSciEL

One layer end to side choledochusduodenal anastomosis with evertion of the choledochus wall in swines

PURPOSE: This article aims to study the one layer end to side choledochusduodenal anastomosis creating a neopapila which, because of the mucosa recovering it, would prevent stenosis. METHODS: Thirty male pigs were distributed within 3 equal groups = Group I (control) - conventional choledochusduodenal anastomosis. Group II (experiment)- one layer end to side anastomosis with evertion of the choledochus wall (neopapila). Group III (sham)- isolation and mobilization of the biliar tract with no choledocotomy. We got the biliar flow and pressure (pre and postoperative) monitorized on the 15 PO day the animals went trough an endoscopy and on the 30 PO day they were operated and the tissues were analysed under macro and microscopic view. RESULTS: Regarding to biliar flow and manometry there were differences between all groups but the III. Animals in groups I and II showed intestinal metaplasia and diminished elastic fibers in the choledochus. All animals showed choledoco dilation. CONCLUSION: The proposed technique allowed the same results as the standard technique.OBJETIVO: Estudar anastomose coledocoduodenal término-lateral em plano único, com a eversão da mucosa do colédoco e sua implantação em segmento do intestino, confeccionando-se uma neopapila que estando recoberta por mucosa afastaria a possibilidade de estenose. MÉTODOS: Foram estudados 30 porcos, machos, distribuídos em 3 grupos de 10 animais: Grupo I- controle-colédocoduodeno anastomose convencional. Grupo II- experimento-coledocoduodeno-anastomose com eversão da parede do colédoco (neopapila). Grupo III - simulado - isolamento e mobilização das vias biliares sem coledocotomia. Os parâmetros analisados foram o fluxo biliar e manometria pré e pós - operatória, endoscopia com 15 dias de PO, e com 30 dias os animais reoperados e avaliados do ponto de vista macro e microscópicos. RESULTADOS: A fluxometria e a manometria mostraram diferenças entre o pré e o pós-operatória nos grupos I e II, mas não no III. Nos animais dos grupos I e II,ocorreu metaplasia intestinal e diminuição das fibras elásticas do colédoco. Todos os animais apresentaram dilatação coledociana. CONCLUSÃO: A técnica de realização de neopapila mostrou resultado semelhante à técnica convencional.UNIFESP-EPM Departamento de CirurgiaUNIFESP, EPM, Depto. de CirurgiaSciEL

Preserved bovine peritoneum for ventral hernia repair in rats: a biological surgical mesh alternative

OBJECTIVE: The aim of this study is to assess the tensile strength of the preserved bovine peritoneum (PBP) as a surgical mesh in rats ventral hernia. METHODS: Using 57 rats, two mesh materials, the PBP and polypropylene (PP), were compared after implantation in a standardized ventral hernia model (full-thickness abdominal wall defects). After 7 (subgroup A) and 28 (subgroup B) days of implantation, they were removed and tensile strength assessment in vitro was performed. One group, with no procedure, just normal abdominal wall, was used as control for tensile strength. The Mann-Whitney and Kruskal- Wallis tests were applied for statistical analysis. RESULTS: All animals survived until the end point, but one (PBP group, subgroup B). No recurrence was seen during the period of observation. The mean load required to disrupt the interface were similar in PBP and PP groups, and both were weaker than normal group (p = 0,003) in all periods of observation. CONCLUSION: Preserved bovine peritoneum is a biological mesh that can be used for ventral hernia repair in rat model.OBJETIVO: Verificar a possibilidade de implantação e a capacidade de resistência tênsil do peritônio parietal bovino como tela cirúrgica na correção de hérnia ventral em um modelo animal de experimentação. MÉTODO: Utilizando 57 ratos machos Wistar, comparou-se o implante do peritônio bovino com a tela de polipropileno na correção de um defeito provocado na parede abdominal do animal. Após sete (sub-grupo A) e 28 (sub-grupo B) dias de observação, as peças foram retiradas e procedeu-se a avaliação da resistência à tração em Máquina Universal de Ensaios. Um grupo sem implante de material protético foi utilizado como controle nos testes de força tênsil. Os testes de Mann-Whitney e de Kruskal-Wallis foram utilizados e estabeleceu-se em 0,05 o nível para rejeição da hipótese de nulidade. RESULTADOS: Os testes de resistência à tração, com valores expressos em Newton, não mostraram diferenças estatísticas entre os grupos estudados, tanto no 7º quanto no 28º dia de pós-operatório, e ambos foram menos resistentes que a parede abdominal normal (p = 0,003). CONCLUSÃO: O peritônio parietal bovino apresentou resistência tênsil semelhante a da tela de polipropileno em um modelo de correção de hérnia ventral em ratos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Faculdade de Medicina de MaríliaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUNIFESP-EPM Departamento de CirurgiaUniversidade de Santo Amaro Departamento de Saúde ColetivaFAMEMAUNIFESP, EPM, Depto. de CirurgiaSciEL

Ischemic preconditioning and the gene expression of enteric endothelial cell biology of rats submitted to intestinal ischemia and reperfusion

PURPOSE: To investigate the effects of ischemic preconditioning (IPC) on the expression of pro and anti-apoptotic genes in rat endothelial cells undergoing enteric ischemia (I) and reperfusion (R). METHODS: Thirty rats underwent clamping of the superior mesenteric vessels. Sham group (GS) laparotomy only; Ischemia (GI): intestinal ischemia (60 min); Ischemia and Reperfusion (GIR): ischemia (60 min) and reperfusion (120 min); Ischemia and intestinal ischemic preconditioning (GI + IPC) : 5 minutes of ischemia followed by 10 min of reperfusion before sustained ischemia (60 min) ischemia and reperfusion and IPC (GIR + IPC): 5 min ischemia followed by 10 min of reperfusion before sustained ischemia (60min) and reperfusion (120 min). Rat Endothelial Cell Biology (PCR array) to determine the expression of genes related to endothelial cell biology. RESULTS: Gene expression of pro-apoptotic markers (Casp1, Casp6, Cflar, Fas, and Pgl) was down regulated in GI+IPC and in GIR + IPC. In contrast, the expression of anti-apoptotic genes (Bcl2 and Naip2), was up-regulated in GI + IPC and in GIR + IPC. CONCLUSION: Ischemic preconditioning may protect against cell death caused by ischemia and reperfusion.UNIFESPUNIFESPSciEL