Acute necrotizing pancreatitis (ANP) is a common gastrointestinal cause of emergency admissions in dogs and humans and can lead to a systemic inflammatory response syndrome resulting in multiple organ dysfunction syndrome. Among the various complications associated with ANP, acute lung injury (ALI) or its more severe form, acute respiratory distress syndrome (ARDS), are major contributors leading to high mortality rates associated with severe acute pancreatitis (AP) in human patients. The incidence of ALI/ARDS in ANP dogs is not well characterized. However, signs of respiratory complications have been reported clinically in dogs suffering from AP. The pathophysiology of ANP and its systemic complications in dogs and humans are not well understood. Most of the data related to AP comes from rodent models of AP, which may not always represent the true mechanisms occurring in the lungs of dogs or humans with ANP. I decided to undertake evaluation of pancreas and lungs from dogs (N=21) that died of ANP. The cases were selected through the search of the medical records of the Veterinary Medical Center of the Western College of Veterinary Medicine (WCVM). Six healthy SPCA dogs were used as controls. The histology of pancreas was first graded to record the range of ANP severities within dog cases included in this study. Then, characterization of lung inflammation was done with histological grading and qualitative analysis of immunohistochemical staining for von Willebrand Factor (vWF), Toll-Like Receptor-4 (TLR4), interleukin-6 (IL6), and inducible nitric oxide synthase (iNOS). Quantification of the recruitment of septal macrophages in the lungs, designated as pulmonary intravascular macrophages (PIMs), in ANP dogs was achieved by counting the number of positive cells in alveolar septa using a macrophage antibody (MAC387). The results revealed that dogs suffering from ANP have variable lung inflammation, which was characterized by a significant infiltration of mononuclear phagocyte cells in the alveolar septa of all ANP dogs (median, 138; range 31-935) compared to control dogs (median: 1.5; range 0-16; p < 0.001), which suggested that PIMs are induced in ANP. In addition, robust staining for vWF in alveolar septal capillaries in lungs of ANP dogs suggested a strong microvascular inflammatory response. Finally, TLR4, IL6, and iNOS expression was increased in lungs of ANP dogs compared to control dogs. The second study was to investigate whether PIMs are induced in a mouse model of L-arginine-induced ANP. Therefore, lungs of L-arginine treated mice (n=7 per time point) were evaluated at various time points (24 hours, 72 hours and 120 hours) using histology and immunohistochemical staining for CD68 cells and vWF. Nine control mice were used. Counting of CD68-positive cells in the lungs of mice treated with L-arginine showed increased numbers of mononuclear phagocytes in alveolar septa at every time point (p<0.001). Also, the lung’s vasculature from L-arginine-treated mice showed increased vWF staining. Taken together, the data showed that ANP in dogs caused significant recruitment of PIMs, increased expression of vWF, TLR4, IL-6, and iNOS suggesting presence of lung inflammation. The mouse model of L-arginine-induced ANP also showed recruitment of PIMs and increased vascular expression of vWF suggesting that this model may be relevant to study the mechanisms of PIMs recruitment and their functions in lung physiology associated with ANP.

Introdução: Vários são os mecanismos envolvidos no desenvolvimento da resposta local e sistêmico na pancreatite aguda. O sistema cardiovascular pode ser afetado durante todo o curso clínico da pancreatite aguda. O objetivo deste estudo foi avaliar a produção local de citocinas pelo miocárdio, assim como, as alterações funcionais e histológicas do miocárdio na pancreatite aguda grave. Métodos: Os animais foram divididos em três grupos: Grupo 1: controle; Grupo 2: controle operado; Grupo 3: pancreatite aguda grave. Foram medidos os níveis séricos de amilase e de citocinas (TNF-alfa IL-6 e IL-10), expressão de RNAm de TNF-alfa, IL-6 e TGF-beta e ecocardiograma com avaliação da função cardíaca. Alterações do tecido cardíaco foram analisadas pelo exame histológico. Resultados: Os níveis séricos de TNF-alfa e IL-10 foram significativamente maiores no grupo pancreatite aguda 2h. Os níveis de RNAm de IL-6 do grupo pancreatite aguda 2h foram estatisticamente superiores. Os níveis de RNAm do TNF-alfa do grupo controle operado e pancreatite aguda 2h foram estatisticamente menores. Mudanças significativas no diâmetro do ventrículo esquerdo foram encontradas nos grupos pancreatite aguda 2h e 12h. Houve alterações estatísticas para a degeneração vacuolar, picnose e perda de núcleo, e os linfócitos. Conclusão: Encontramos alterações cardíacas e histológicas compatíveis com o processo inflamatório desencadeado por pancreatite aguda grave com o incremento da produção de citocinas pelo miocárdio / Background: Several mechanisms are involved in the development of the local and systemic response in acute pancreatitis. Cardiovascular system may be affected throughout the clinical course of acute pancreatitis. The aim was to evaluate local myocardial cytokine production, as well as, functional and histological myocardial alterations in severe acute pancreatitis. Methods: The animals were divided into three groups: Group 1: control; Group 2: sham; Group 3: severe acute pancreatitis. Echocardiographic assessment of cardiac function, serum levels of amylase and cytokines (TNF-alfa, IL-6 and IL-10), and mRNA expression of TNF-alfa, IL-6 and TGF-beta were measured. Myocardial tissue alterations were analysed by histological examination. Results: The serum TNF-alfa, and IL-10 levels were significant higher in acute pancreatitis 2h group. The mRNA IL-6 levels from acute pancreatitis 2h group were statistically higher. The mRNA TNF-alfa levels from sham group and acute pancreatitis 2h group were statistically lower. Significant changes in the left ventricular diameter were found in acute pancreatitis 2h and 12h groups. There were statistical changes for vacuolar degeneration, picnosis and loss of nucleus, and lymphocytes. Conclusion: We found cardiac and histological changes compatible with the inflammatory process triggered by severe acute pancreatitis with the promotion of local myocardial cytokine production

Introdução: Vários são os mecanismos envolvidos no desenvolvimento da resposta local e sistêmico na pancreatite aguda. O sistema cardiovascular pode ser afetado durante todo o curso clínico da pancreatite aguda. O objetivo deste estudo foi avaliar a produção local de citocinas pelo miocárdio, assim como, as alterações funcionais e histológicas do miocárdio na pancreatite aguda grave. Métodos: Os animais foram divididos em três grupos: Grupo 1: controle; Grupo 2: controle operado; Grupo 3: pancreatite aguda grave. Foram medidos os níveis séricos de amilase e de citocinas (TNF-alfa IL-6 e IL-10), expressão de RNAm de TNF-alfa, IL-6 e TGF-beta e ecocardiograma com avaliação da função cardíaca. Alterações do tecido cardíaco foram analisadas pelo exame histológico. Resultados: Os níveis séricos de TNF-alfa e IL-10 foram significativamente maiores no grupo pancreatite aguda 2h. Os níveis de RNAm de IL-6 do grupo pancreatite aguda 2h foram estatisticamente superiores. Os níveis de RNAm do TNF-alfa do grupo controle operado e pancreatite aguda 2h foram estatisticamente menores. Mudanças significativas no diâmetro do ventrículo esquerdo foram encontradas nos grupos pancreatite aguda 2h e 12h. Houve alterações estatísticas para a degeneração vacuolar, picnose e perda de núcleo, e os linfócitos. Conclusão: Encontramos alterações cardíacas e histológicas compatíveis com o processo inflamatório desencadeado por pancreatite aguda grave com o incremento da produção de citocinas pelo miocárdio / Background: Several mechanisms are involved in the development of the local and systemic response in acute pancreatitis. Cardiovascular system may be affected throughout the clinical course of acute pancreatitis. The aim was to evaluate local myocardial cytokine production, as well as, functional and histological myocardial alterations in severe acute pancreatitis. Methods: The animals were divided into three groups: Group 1: control; Group 2: sham; Group 3: severe acute pancreatitis. Echocardiographic assessment of cardiac function, serum levels of amylase and cytokines (TNF-alfa, IL-6 and IL-10), and mRNA expression of TNF-alfa, IL-6 and TGF-beta were measured. Myocardial tissue alterations were analysed by histological examination. Results: The serum TNF-alfa, and IL-10 levels were significant higher in acute pancreatitis 2h group. The mRNA IL-6 levels from acute pancreatitis 2h group were statistically higher. The mRNA TNF-alfa levels from sham group and acute pancreatitis 2h group were statistically lower. Significant changes in the left ventricular diameter were found in acute pancreatitis 2h and 12h groups. There were statistical changes for vacuolar degeneration, picnosis and loss of nucleus, and lymphocytes. Conclusion: We found cardiac and histological changes compatible with the inflammatory process triggered by severe acute pancreatitis with the promotion of local myocardial cytokine production

Abstract Acute infected necrotizing pancreatitis (ANP) is a very complex disease with a high risk of complications and death. ANP is difficult to treat and is often associated with poor outcomes. Despite the increasing data on the technical details required to perform a mini-invasive necrosectomy for walled-off necrosis (WON), relatively few studies have focused on the presence and consequences of pancreatic duct disruption in the context of APN. Moreover, the long-term prognosis of patients with acute pancreatitis (AP) is scant. The aim of this study was to examine the diagnosis, treatment and prophylaxis of pancreatic fistulas (PFs) associated with APN. In addition, the long-term prognosis of AP was evaluated. The study population consists of the patients with AP treated at Oulu University Hospital, Finland (Studies I–IV) and Copenhagen University Hospital, Denmark (Study II) during 1995–2015. In the first part of the study, all consecutive patients following open necrosectomy for infected ANP were demonstrated to have PF. Endoscopic transpapillary pancreatic stenting (ETPS) was attempted and proven to be an effective and safe treatment for patients with PF. In Study II, prophylactic pancreatic stenting in the early stage of the disease was tested in a randomized controlled trial to the patients with ANP to prevent PFs associated with the disease. However, the study showed that the patients with ANP did not benefit from early prophylactic pancreatic ductal stenting (PPDS); instead, it seemed to be harmful for the patients. The results of Study III showed that single drain amylase level measurement after surgical necrosectomy is unreliable. According to this study, serial measurements are recommended to diagnose PFs after necrosectomy. Study IV including 1644 patients showed that AP, especially alcohol AP, was associated with a high long-term mortality. On the other hand, AP without an alcohol aetiology had a minimal impact on survival. In conclusion, in patients with infected ANP, a PF has to be considered in treatment, but the prevention of ductal leak with PPDS is not recommended. In addition, the poor long-term outcome among alcohol AP patients was due to alcohol-related diseases. / Tiivistelmä Akuutti nekrotisoiva haimatulehdus ja erityisesti siihen liittyvä bakteeri-infektio on sairaus, johon liittyy korkea komplikaatio- ja kuolleisuusriski. Tautia usein komplisoi infektion lisäksi nekroosiin liittyvä haimafisteli, joka tekee hoidosta entistä haasteellisemman. Viime aikaisissa tutkimuksissa on käsitelty runsaasti mini-invasiivista nekrosektomiaa, mutta suhteellisen vähän on tutkimuksia nekrotisoivaan haimatulehdukseen liittyvästä fisteliongelmasta. Haimatulehdus-potilaiden pitkäaikaisennuste on myös epäselvä. Tämän väitöskirjatutkimuksen tavoitteena oli selvittää nekrotisoivaan haimatulehdukseen liittyvän haimafistelin yleisyyttä, diagnostiikkaa, ehkäisyä ja hoitoa. Lisäksi tarkasteltiin akuuttiin haimatulehdukseen sairastuneiden potilaiden pitkäaikaisennustetta. Ensimmäisessä osatyössä ilmeni, että kaikille potilaille, joille suoritettiin haiman nekrosektomia kehittyi fisteli ja endoskooppinen transpapillaarinen haimateiden stenttaus (ETPS) osoittautui hyväksi ja turvalliseksi hoidoksi fistelin hoidossa. Toisessa prospektiivisessa randomoidussa kontrolloidussa osatyössä tutkittiin profylaktista haimateiden stenttausta nekrotisoivassa haimatulehduksessa. Tutkimus osoitti, etteivät potilaat hyötyneet stenttauksesta: toimenpiteestä oli enemmän haittaa kuin hyötyä. Tämän tutkimuksen mukaan protetisointia ei suositella tehtäväksi taudin alkuvaiheessa. Kolmannessa osatyössä selvitettiin haiman nekrosektomian jälkeisen haimafistelin diagnosointia. Tutkimustuloksen mukaan haimafistelin osoittamiseksi dreenieritteen amylaasitasoa mittaamalla tarvitaan useita mittauskertoja, koska yksittäisen mittauksen sensitiivisyys on matala. Neljännessä osatyössä analysoitiin Oulun yliopistollisessa sairaalassa 1995–2012 akuutin haimatulehduksen sairastaneiden työikäisten potilaiden pitkäaikaisennustetta ja kuolinsyitä. Noin kymmenen vuoden seurannassa tutkimusryhmän (n = 1 644) kuolleisuus oli yli nelinkertainen verrattuna ikä- ja sukupuolivakioituihin verrokeihin (n = 8 220). Merkittävin kuolleisuutta lisäävä tekijä oli alkoholi. Tutkimuksemme osoitti, että infektoituneen haimanekroosiin liittyvä haimafisteli on huomioitava hoidossa. Varhaisesta profylaktisesta haimateiden protetisoinnista ei tutkimuksessa osoitettu olevan hyötyä. Alkoholin aiheuttaman haimatulehduksen pitkäaikaisennusteen mortaliteetti on korkea johtuen alkoholin käytöstä ja siihen liittyvistä sairauksista.

INTRODUÇÃO: Relatos conflitantes têm dificultado para se estabelecer o potencial benefício da glutamina (GLN) no tratamento de condições inflamatórias agudas. Nós avaliamos o efeito da infusão parenteral de GLN, prévia à pancreatite aguda (PA) experimental, nos mediadores inflamatórios, morbidade e mortalidade. MÉTODOS: Ratos Lewis (n = 131) receberam glutamina parenteral (grupo GG), solução salina (grupo SS ou controle), ou permaneceram sem infusão parenteral (grupo Sham) por 48h. Após este período, foi induzida PA por meio da injecção retrógrada de taurocolato de sódio no ducto pancreático. Sangue, amostras de pulmão, fígado, pâncreas e líquido ascítico foram colhidos a partir de 2, 12 e 24 horas após PA para avaliação das variáveis propostas (citocinas, hsp, histologia, amilase). Sessenta animais permaneceram vivos após PA para a análise da mortalidade em sete dias. RESULTADOS: A análise entre grupos não mostrou diferenças significativas nos níveis de citocinas (p > 0,05). Análise cinética dentro de cada grupo ao longo do tempo mostrou maior INF-y no grupo Sham e SS às 2h do que em 12h e 24h, maior IL-2 e inferior IL-10 no Sham, às 24h do que em 2h e 12h, e menor IL-10 no SS e GG em 24 h do que no tempo de 2h (p <= 0.05). O grupo GG exibiu maior expressão de HSP 90 no pulmão e no fígado do que no grupo Sham nos tempos de 2h e 12h, respectivamente; e maior expressão no fígado de HSP90 e HSP70 no grupo SS no tempo 12 horas (p < 0,01). O grupo Sham apresentou maior expressão de HSP 70 no pulmão e HSP 90 no fígado do que os outros grupos no tempo de 24h. Não ocorreram alterações na taxa de mortalidade. CONCLUSÕES: Em modelo de PA experimental induzida por taurocolato de sódio, o pré-tratamento com GLN parenteral melhorou o perfil dos mediadores inflamatórios, sem afetar a mortalidade / INTRODUCTION: Conflicting reports have hindered establish the potential glutamine (GLN) benefit in treating acute inflammatory conditions. We evaluated the effect of parenteral GLN infusion before experimental acute pancreatitis (AP), as systemic inflammation-reproducing model, on inflammatory mediators and mortality. METHODS: Lewis rats (n=131) received parenteral glutamine (GG group), saline (SS or Control group), or remained without parenteral infusion ( Sham group) for 48h. Thereafter, AP was induced by retrograde injection of sodium taurocholate into pancreatic duct. Blood, lung, liver and pancreas samples were collected from 2, 12 and 24h post-AP to assess serum cytokines levels, tissue HSP expression, histology and amylase. Sixty animals remained alive post-PA for seven-day mortality analysis. RESULTS: Punctual between-groups analysis did not show differences in cytokine levels (p > 0.05). Intragroup analysis over time showed higher INF-y in Sham and SS at 2h than at 12h and 24h, higher IL-2 and lower IL-10 in Sham at 24h than at 2h and 12h, and lower IL-10 in SS and GG at 24h than at 2h timepoint (p <= 0.05). GG group exhibited higher lung and liver HSP90 than Sham at 2h and 12h timepoints, respectively; and higher liver HSP90 and HSP70 than SS at 12h timepoint (p < 0.01). Sham group presented higher lung HSP70 and liver HSP90 than the others at 24h timepoint (p < 0.02). No changes occurred on mortality rate. CONCLUSIONS: In sodium taurocholate-induced PA model, pretreatment with parenteral GLN improved inflammatory mediator\'s profile, without affecting mortality

INTRODUÇÃO: Relatos conflitantes têm dificultado para se estabelecer o potencial benefício da glutamina (GLN) no tratamento de condições inflamatórias agudas. Nós avaliamos o efeito da infusão parenteral de GLN, prévia à pancreatite aguda (PA) experimental, nos mediadores inflamatórios, morbidade e mortalidade. MÉTODOS: Ratos Lewis (n = 131) receberam glutamina parenteral (grupo GG), solução salina (grupo SS ou controle), ou permaneceram sem infusão parenteral (grupo Sham) por 48h. Após este período, foi induzida PA por meio da injecção retrógrada de taurocolato de sódio no ducto pancreático. Sangue, amostras de pulmão, fígado, pâncreas e líquido ascítico foram colhidos a partir de 2, 12 e 24 horas após PA para avaliação das variáveis propostas (citocinas, hsp, histologia, amilase). Sessenta animais permaneceram vivos após PA para a análise da mortalidade em sete dias. RESULTADOS: A análise entre grupos não mostrou diferenças significativas nos níveis de citocinas (p > 0,05). Análise cinética dentro de cada grupo ao longo do tempo mostrou maior INF-y no grupo Sham e SS às 2h do que em 12h e 24h, maior IL-2 e inferior IL-10 no Sham, às 24h do que em 2h e 12h, e menor IL-10 no SS e GG em 24 h do que no tempo de 2h (p <= 0.05). O grupo GG exibiu maior expressão de HSP 90 no pulmão e no fígado do que no grupo Sham nos tempos de 2h e 12h, respectivamente; e maior expressão no fígado de HSP90 e HSP70 no grupo SS no tempo 12 horas (p < 0,01). O grupo Sham apresentou maior expressão de HSP 70 no pulmão e HSP 90 no fígado do que os outros grupos no tempo de 24h. Não ocorreram alterações na taxa de mortalidade. CONCLUSÕES: Em modelo de PA experimental induzida por taurocolato de sódio, o pré-tratamento com GLN parenteral melhorou o perfil dos mediadores inflamatórios, sem afetar a mortalidade / INTRODUCTION: Conflicting reports have hindered establish the potential glutamine (GLN) benefit in treating acute inflammatory conditions. We evaluated the effect of parenteral GLN infusion before experimental acute pancreatitis (AP), as systemic inflammation-reproducing model, on inflammatory mediators and mortality. METHODS: Lewis rats (n=131) received parenteral glutamine (GG group), saline (SS or Control group), or remained without parenteral infusion ( Sham group) for 48h. Thereafter, AP was induced by retrograde injection of sodium taurocholate into pancreatic duct. Blood, lung, liver and pancreas samples were collected from 2, 12 and 24h post-AP to assess serum cytokines levels, tissue HSP expression, histology and amylase. Sixty animals remained alive post-PA for seven-day mortality analysis. RESULTS: Punctual between-groups analysis did not show differences in cytokine levels (p > 0.05). Intragroup analysis over time showed higher INF-y in Sham and SS at 2h than at 12h and 24h, higher IL-2 and lower IL-10 in Sham at 24h than at 2h and 12h, and lower IL-10 in SS and GG at 24h than at 2h timepoint (p <= 0.05). GG group exhibited higher lung and liver HSP90 than Sham at 2h and 12h timepoints, respectively; and higher liver HSP90 and HSP70 than SS at 12h timepoint (p < 0.01). Sham group presented higher lung HSP70 and liver HSP90 than the others at 24h timepoint (p < 0.02). No changes occurred on mortality rate. CONCLUSIONS: In sodium taurocholate-induced PA model, pretreatment with parenteral GLN improved inflammatory mediator\'s profile, without affecting mortality