Suppression of L-arginine-induced acute necrotizing pancreatitis in rats by metformin associated with the inhibition of myeloperoxidase and activation of interleukin-10 / Supresión de la pancreatitis necrotizante aguda inducida por L-arginina en ratas por metformina asociada con la inhibición de mieloperoxidasa y activación de interleucina-10

SUMMARY: Acute pancreatitis is a frequent life-threatening inflammatory disease of the pancreas characterized by severe abdominal pain that lasts for days to weeks. We sought to determine whether the antidiabetic and anti-inflammatory drug, metformin can substantially protect against acute pancreatitis in an animal model of L-arginine-induced acute pancreatitis, and whether this is associated with the augmentation of the anti-inflammatory cytokine interleukin-10 (IL-10) and inhibition of the enzyme that promotes tissue damage, myeloperoxidase (MPO). Rats were either injected with two doses of the amino acid L-arginine (2.5 gm/kg; i.p., at one-hour intervals) before being sacrificed after 48 hours (model group) or were pretreated with metformin (50 mg/kg) daily for two weeks prior to L- arginine injections and continued receiving metformin until the end of the experiment (protective group). Using microscopic examination of the pancreas and blood chemistry, we observed that L-arginine induced acute pancreatic injury. This is demonstrated by an enlarged pancreas with patchy areas of haemorrhage, vacuolated cytoplasm and pyknotic nuclei in the acini, disorganized lobular architecture with infiltration of inflammatory cells within the interlobular connective tissue (CT) septa, and the presence of congested blood vessels that were substantially ameliorated by metformin. Metformin also significantly (p<0.05) inhibited L-arginine-induced MPO, lactate dehydrogenase (LDH), and the inflammatory biomarker tumor necrosis factor alpha (TNF-α). Whereas, metformin significantly (p<0.05) increased IL-10 levels that were inhibited by pancreatitis induction. We further demonstrated a significant (p<0.001) correlation between the scoring of the degree of pancreatic lobules damage tissue damage and the blood levels of TNF-α, IL-10, LDH, and MPO. Thus, metformin effectively protects against L-arginine-induced acute pancreatitis, which is associated with the inhibition of MPO and augmentation of IL-10.

RESUMEN: La pancreatitis aguda es una enfermedad inflamatoria del páncreas que amenaza la vida y se caracteriza por un dolor abdominal intenso que dura de días a semanas. Buscamos determinar si la metformina, fármaco antidiabético y antiinflamatorio, puede proteger contra la pancreatitis aguda en un modelo animal de pancreatitis aguda inducida por L-arginina. Además se estudió la asociación con el aumento de la citocina antiinflamatoria interleucina-10. (IL-10) e inhibición de la enzima que promueve el daño tisular, mieloperoxidasa (MPO). Las ratas se inyectaron con dos dosis del aminoácido L-arginina (2,5 g / kg; ip, a intervalos de una hora) antes de ser sacrificadas des- pués de 48 horas (grupo modelo) o se pre trataron con metformina (50 mg / kg) durante dos semanas antes del tratamiento de L- arginina y continuaron recibiendo metformina hasta el final del experimento (grupo protector). Mediante el examen microscópico del páncreas y la química sanguínea, se observó que la L- arginina inducía una lesión pancreática aguda. Se observó un aumento significativo de tamaño del páncreas con áreas hemorrágicas, citoplasma vacuolado y núcleos picnóticos en los acinos, arquitectura desorganizada con infiltración de células inflamatorias dentro de los tabiques del tejido conjuntivo interlobulillar (TC) y la presencia de vasos sanguíneos congestionados mejorados por metformina. Se observó que la metformina inhibió significativamente (p <0,05) la MPO inducida por L- arginina, la lactato deshidrogenasa (LDH) y el factor de necrosis tumoral alfa (TNF-α). Además, demostramos una correlación significativa (p <0,001) entre la puntuación del grado de daño tisular de los lóbulos pancreáticos y los niveles sanguíneos de TNF-α, IL-10, LDH y MPO. Por tanto, la metformina protege eficazmente contra la pancreatitis aguda inducida por L-arginina, que se asocia con la inhibición de MPO y el aumento de IL-10.

Ameliorative effect of certolizumab on experimentally induced acute necrotic pancreatitis in rats

SUMMARY OBJECTIVE: The effects of Certolizumab, a pegylated monoclonal antibody to tumor necrosis factor α, on experimentally induced acute pancreatitis (AP) were examined. METHODS: Thirty-six Wistar Albino male rats were randomly divided into four groups. Group I was the control group and no medication administered to this group. Group II was the Certolizumab group, and 100 ml/kg serum physiologic administered into the biliopancreatic duct and a single dose of 10 μg Certolizumab was simultaneously administered intraperitoneally. Acute pancreatitis was induced with a retrograde injection of 3% Na taurocholate into the common biliopancreatic duct in the study (Group III) and treatment (Groups IV) groups. Rats were sacrificed 72 hours later. Serum amylase, lipase, lactate dehydrogenase activities, along with pancreatic histopathology, were examined. RESULTS: Certolizumab treatment significantly decreased serum amylase, lipase, and LDH levels; histopathologically edema, hemorrhage, parenchymal necrosis, fat necrosis, and infiltration scores; immunohistochemically MDA, MPO, TNF-α and Caspase-3 activity. CONCLUSION: The results support the idea that certolizumab might be beneficial for the severity of AP.

RESUMO OBJETIVO: Os efeitos de Certolizumab, um anticorpo monoclonal pegilado para o fator de necrose tumoral α, na pancreatite aguda induzida experimentalmente (PA) foram examinados. MÉTODO: Trinta e seis ratos Wistar Albino foram divididos aleatoriamente em quatro grupos. O Grupo I foi considerado o grupo controle e não recebeu medicação; o Grupo II foi o grupo Certolizumab e recebeu 100 ml/kg de soro fisiológico administrado no ducto biliopancreático e dose única de 10 mg Certolizumab administrada por via intraperitoneal simultaneamente. A pancreatite aguda foi induzida com uma injeção retrógrada de uma solução de 3% taurocolato de sódio aplicada no ducto biliopancreático comum nos grupos de estudo (Grupo III) e tratamento (Grupos IV). Os ratos foram sacrificados 72 horas depois. As atividades séricas de amilase, lipase, lactato desidrogenase, juntamente com a histopatologia pancreática, foram examinadas. RESULTADOS: O tratamento com Certolizumab diminuiu significativamente os níveis séricos de amilase, lipase e LDH; edema histopatológico, hemorragia, necrose paranquimatosa, necrose gordurosa e escores de infiltração; atividade imuno-histoquímica de MDA, MPO, TNF-α e Caspase-3. CONCLUSÃO: Estes resultados suportam a ideia de que o Certolizumab pode ser benéfico para a gravidade da PA.

Pancreatic Necrosis and Gas in the Retroperitoneum: Treatment with Antibiotics Alone

OBJECTIVE: To present our experience in the management of patients with infected pancreatic necrosis without drainage. METHODS: The records of patients with pancreatic necrosis admitted to our facility from 2011 to 2015 were retrospectively reviewed. RESULTS: We identified 61 patients with pancreatic necrosis. Six patients with pancreatic necrosis and gas in the retroperitoneum were treated exclusively with clinical support without any type of drainage. Only 2 patients had an APACHE II score >8. The first computed tomography scan revealed the presence of gas in 5 patients. The Balthazar computed tomography severity index score was >9 in 5 of the 6 patients. All patients were treated with antibiotics for at least 3 weeks. Blood cultures were positive in only 2 patients. Parenteral nutrition was not used in these patients. The length of hospital stay exceeded three weeks for 5 patients; 3 patients had to be readmitted. A cholecystectomy was performed after necrosis was completely resolved; pancreatitis recurred in 2 patients before the operation. No patients died. CONCLUSIONS: In selected patients, infected pancreatic necrosis (gas in the retroperitoneum) can be treated without percutaneous drainage or any additional surgical intervention. Intervention procedures should be performed for patients who exhibit clinical and laboratory deterioration.

Effects of diazoxide in experimental acute necrotizing pancreatitis

OBJECTIVE: We aimed to assess the effects of diazoxide on the mortality, pancreatic injury, and inflammatory response in an experimental model of acute pancreatitis. METHODS: Male Wistar rats (200-400 g) were divided randomly into two groups. Fifteen minutes before surgery, animals received physiological (0.9%) saline (3 mL/kg) (control group) or 45 mg/kg diazoxide (treatment group) via the intravenous route. Acute pancreatitis was induced by injection of 2.5% sodium taurocholate via the biliopancreatic duct. Mortality (n=38) was observed for 72 h and analyzed by the Mantel-Cox Log-rank test. To study pancreatic lesions and systemic inflammation, rats (10 from each group) were killed 3 h after acute pancreatitis induction; ascites volume was measured and blood as well as pancreases were collected. Pancreatic injury was assessed according to Schmidt's scale. Cytokine expression in plasma was evaluated by the multiplex method. RESULTS: Mortality at 72 h was 33% in the control group and 60% in the treatment group (p=0.07). Ascites volumes and plasma levels of cytokines between groups were similar. No difference was observed in edema or infiltration of inflammatory cells in pancreatic tissues from either group. However, necrosis of acinar cells was lower in the treatment group compared to the control group (3.5 vs. 3.75, p=0.015). CONCLUSIONS: Treatment with diazoxide can reduce necrosis of acinar cells in an experimental model of acute pancreatitis, but does not affect the inflammatory response or mortality after 72 h.

Ethyl-pyruvate reduces lung injury matrix metalloproteinases and cytokines and improves survival in experimental model of severe acute pancreatitis

PURPOSE: To investigate if the ethyl-pyruvate solution could reduce mortality in AP and/or diminish the acute lung injury. METHODS: Forty male rats, weighing between 270 to 330 grams were operated. An experimental model of severe AP by injection of 0.1ml/100g of 2.5% sodium taurocholate into the bilio-pancreatic duct was utilized. The rats were divided into two groups of ten animals each: CT - control (treatment with 50ml/kg of Ringer's solution, intraperitoneal) and EP (treatment with 50ml/kg of Ringer ethyl- pyruvate solution, intra-peritoneal), three hours following AP induction. After six hours, a new infusion of the treatment solution was performed in each group. Two hours later, the animals were killed and the pulmonary parenchyma was resected for biomolecular analysis, consisting of: interleukin, myeloperoxidase, MDA, nitric oxide, metalloproteinases and heat shock protein. In the second part of the experiment, another, 20 rats were randomly divided into EP and CT groups, in order to evaluate a survival comparison between the two groups. RESULTS: There were no significant differences in IL-1B,IL-10, MMP-9, HSP70, nitric oxide, MPO, MDA (lipidic peroxidation) concerning both groups. The levels of IL-6 were significantly diminished in the EP group. Furthermore, the MMP-2 levels were also reduced in the EP group (p<0.05). The animals from the EP treatment groups had improved survival, when compared to control group (p<0.05). CONCLUSION: The ethyl-pyruvate diminishes acute lung injury inflammatory response in acute pancreatitis and ameliorates survival when compared to control group, in the experimental model of necrotizing acute pancreatitis.

Is there a therapeutic window for pentoxifylline after the onset of acute pancreatitis? / Existe uma janela terapêutica para a pentoxifilina após o início da pancreatite aguda?

PURPOSE: To investigate the effects of pentoxifylline (PTX) in experimental acute pancreatitis (AP) starting drug administration after the induction of the disease. METHODS: One hundred male Wistar rats were submitted to taurocholate-induced AP and divided into three groups: Group Sham: sham-operated rats, Group Saline: AP plus saline solution, and Group PTX: AP plus PTX. Saline solution and PTX were administered 1 hour after induction of AP. At 3 hours after AP induction, peritoneal levels of tumor necrosis factor (TNF)-α, and serum levels of interleukin (IL)-6 and IL-10 levels were assayed by Enzyme-Linked Immunosorbent Assay (ELISA). Determinations of lung myeloperoxidase activity (MPO), histological analysis of lung and pancreas, and mortality study were performed. RESULTS: PTX administration 1 hour after induction of AP caused a significant decrease in peritoneal levels of TNF-α and in serum levels of IL-6 and IL-10 when compared to the saline group. There were no differences in lung MPO activity between the two groups with AP. A decrease in mortality was observed in the PTX treatment compared to the saline group. CONCLUSIONS: Administration of PTX after the onset of AP decreased the systemic levels of proinflammatory cytokines, raising the possibility that there is an early therapeutic window for PTX after the initiation of AP.

OBJETIVO: Investigar os efeitos da pentoxifilina (PTX) na pancreatite aguda (PA) experimental administrando a droga após a indução da doença. MÉTODOS: Cem ratos machos Wistar foram submetidos à indução da PA através da infusão de taurocolato de sódio e divididos em três grupos: Grupo Sham: sham-operated ratos, Grupo Salina: AP e solução salina, e Grupo PTX: AP e PTX. Solução salina e PTX foram administradas 1 hora após a indução da PA. Três horas após indução da PA os níveis de fator de necrose tumoral (TNF)-α no líquido peritoneal e os níveis séricos de interleucina (IL)-6 e IL-10 foram analisados pelo método de Enzima Imunoensaio (ELISA). A atividade da mieloperoxidase (MPO) foi analisada no pulmão e foram realizadas análises histológicas do pulmão e pâncreas, além do estudo da mortalidade. RESULTADOS: A administração de PTX 1 hora após a indução da PA reduziu significativamente os níveis de TNF-α peritoneal e os níveis séricos de IL-6 e IL-10 quando comparado ao grupo salina. Redução na mortalidade foi observado após o tratamento com PTX comparado ao grupo salina. CONCLUSÃO: A administração de PTX após a indução da PA diminuiu os níveis sistêmicos de citocinas pró-inflamatórias, sugerindo a possibilidade de que existe uma janela terapêutica para PTX após o início do PA.

Effects of infliximab on bacterial translocation in experimental acute necrotizing pancreatitis.

Background & objectives: Translocation of bacteria from the gut is an important factor in the development of septic complications and mortality in acute pancreatitis (AP). The present study was designed to assess the effects of infliximab treatment on bacterial translocation (BT) in experimental acute necrotizing pancreatitis. Methods: Male Sprague-Dawley rats (n=45) were allocated into three groups. AP was induced in group II (positive control, n=15) and group III (Infliximab; n=15) by retrograde injection of taurocholate into the common biliopancreatic duct. Group I rats (Sham; n=15) received normal saline infusion into the common biliopancreatic duct as placebo. Groups I and II were treated by normal saline and group III was treated with infliximab intraperitoneally on 6, 30 and 54 h after induction of pancreatitis. All surviving animals were killed 60 h after the induction of pancreatitis, and specimens were collected for amylase measurement as well as histopathologic and microbiologic examinations. Results: Oedema, acinar cell necrosis, inflammatory infiltration, haemorrhage, fat necrosis and perivascular inflammation in group III rats were decreased with infliximab treatment when compared with group II (P<0.001). BT to mesentery lymph node in groups I, II and III were 20, 100 and 46 per cent, respectively. BT to peritoneum and pancreas in group III was lower than group II (P<0.05). Interpretation & conclusions: Infliximab administration resulted in beneficial effects on BT and histopathologic changes in the experimental necrotizing pancreatitis. Whether anti-TNF therapy has a role in prevention of complications of ANP needs to be established.

Uso de antibióticos profilácticos en pancreatitis aguda grave: ¿es útil? / Prophylactic antibiotics in severe acute pancreatitis: are they useful?

The use in intravenous antibiotics in prophylaxis of acute necrotizing pancreatitis is still an issue under active debate, and consensus have not been accomplished yet. The main goal of this prophylaxis is the impregnation of pancreatic tissue with antibiotics, in order to prevent the bacterial colonization, ensuing the microbial translocation from the gut. Different experts have developed different approaches to this issue, each one with argumentation for the defense or the rejection of prophylactic use of antibiotics. Our review reveled strong evidence that nobody has demonstrated yet a real benefit, in terms of lower mortality or decreased rate of infections, with the prophylactic use of antibiotics in pancreatic necrosis. Many groups have reported a decrease in numbers of infections or mortality, but without statistical significance. In some series the use of prophylactic antibiotics decreased the length of stay, but no correlation with changes in mortality or infection rate in pancreatic necrosis was established. The indication of antibiotic prophylaxis in a sterile necrotic pancreatic tissue is a decision that involves several factors as: the individual clinical assessment of every case, the knowledge about the pathogenic flora and of the bacterial spectrum and tissue penetration of the antimicrobial agent, and, also, its cost-effectiveness and toxicity. The criteria used by some hospitals in the country is the antibioprophylaxis is a cases whose pancreatic necrosis exceeds 30 percent in abdominal CT scan with contrast medium, as in these cases severity increases twofold, but there has not been shown significant reduction of morbility in these cases corresponding to an evidence type II. Consequently and according to the available reports we dont't recommend the prophylactic use of antibiotics in pancreatic necrosis. A promising field, shown in a dutch study by Luiten & cols is the selective decontamination of the gut with non absorbable antibiotics...

El uso de antibióticos endovenosos en la profilaxis de la pancreatitis aguda necrótica, es un tema vigente en plena discusión. No existe un consenso con respecto a su utilidad. La profilaxis antibiótica tiene como objetivo primordial impregnar el tejido pancreático con antibióticos que prevengan luego la colonización a partir de la translocación bacteriana del tubo digestivo. Distintos grupos de expertos han tomado diferentes posturas al respecto, utilizando diversos argumentos para defenderse, o rechazar el uso de antibióticos profilácticos. Nuestra revisión muestra de manera bastante contundente que ningún grupo ha logrado demostrar que el uso de antibióticos entregue beneficios en cuanto a prevenir la mortalidad o disminuir la infección de la necrosis pancreática o de su mortalidad. En algunas series el uso de antibióticos disminuyó la permanencia hospitalaria, pero esto no se relacionó con cambios en la mortalidad ni en la infección de la necrosis. La indicación de antibioprofilaxis en tejido pancreático necrótico estériles es una decisión que implica considerar varios factores tales como: evaluación clínica individual de cada caso; conocimiento de la flora patógena así como del espectro y penetración del antimicrobiano, pero también su costo-efectividad y su toxicidad. Una conducta utilizada en algunos centros hospitalarios del país consiste en la indicación de antibióticos profilácticos en necrosis pancreática mayores del 30 por ciento estimadas al TAC abdominal con contraste, valor sobre el cual la gravedad se duplica, pero la experiencia no avala una reducción significativa de la mortalidad en estos casos que corresponden a un nivel de evidencia tipo II. En consecuencia y en base a los estudios disponibles no recomendamos el uso de antibióticos profilácticos en la necrosis pancreática. Un aspecto promisorio, demostrado por el estudio holandés de Luiten y cols es el uso de la descontaminación selectiva del tracto digestivo con antibióticos...

The role of allopurinol in experimental acute necrotizing pancreatitis.

BACKGROUND & OBJECTIVES: Acute pancreatitis (AP) in its severe form can lead to severe complications and death. Translocation of bacteria from the gut is one of the most important factors in the development of septic complications and mortality in acute pancreatitis. Oxygen-derived free radicals have been suggested to play a major role in the pathogenesis of AP. Xanthine oxidase enzyme is an important source of reactive oxygen metabolites. We undertook this study to evaluate the effect of allopurinol on bacterial translocation, oxidative stress and the course of AP in a rat model. METHODS: Male Sprague-Dawley rats (n=48) were randomly allocated into three equal groups. Acute pancreatitis (AP) was induced in group II (AP+Saline), and group III (AP+allopurinol) by retrograde infusion of taurocholate into the common biliopancreatic duct. Group I rats (Sham) received normal saline infusion into the common biliopancreatic duct for mimicking pressure effect. Group III rats were treated with allopurinol intraperitoneally for 48 h after induction of pancreatitis. Blood samples were drawn from all animals for biochemical analyses and pancreatic tissues were examined for bacterial translocation. RESULTS: Acute pancreatitis was developed in all groups, but not in group I (Sham), as indicated by microscopic parenchymal necrosis, fat necrosis and abundant turbid peritoneal fluid. Pathologic score of the pancreatitis in the allopurinol group (14.0 +/- 0.5) was lower when compared with group II (19.2 +/- 0.6) (P<0.001). Bacterial translocation to pancreas in group treated with allopurinol was significantly lower when compared with control group (p<0.02). Plasma glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels were higher and malondialdehyde (MDA) levels were lower in allopurinol group when compared with those in control groups. INTERPRETATION & CONCLUSION: Our findings suggested that addition of allopurinol to the treatment protocol in the acute pancreatitis might improve the pathologic score, bacterial translocation and oxidative stress parameters. However, more studies need to be done to confirm these findings.

Necrosis pancreática infectada: 2 casos de tratamiento médico exclusivo / Infected pancreatic necrosis: 2 cases of exclusive medical treatment

La necrosis pancreática infectada ha sido tradicionalmente tratada con cirugía de desbridamiento y drenaje. Se presentan dos pacientes en los cuales se demostró esta patología. Los enfermos no presentaban signos de insuficiencias sistémicas y fueron tratados con antibióticos, según sensibilidad, logrando una recuperación completa de su cuadro clínico

Pancreatitis aguda: valor pronóstico y tratamiento / Acute pancreatitis: prognostic value and treatment

Presentamos 14 casos de pancreatitis aguda ingresados en el Hospital Docente General Calixto García de La Habana, durante el primer trimestre de 1994. Efectuamos valoración pronóstica en base a parámetros clínicos, humorales, imagenológicos y terapéuticos con el empleo de 5 Fluorouracilo; con el que se obtuvo resultados satisfactorios y estadía hospitalaria acepatable