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Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKβ-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKβ inhibitors, and confirmed by the inability of IKBKB/IKKβ phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKβ's involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy. Abbreviations: AL: autolysosome; AP: acute pancreatitis; AV: autophagic vacuole; CCK: cholecystokinin; IKBKB/IKKβ: inhibitor of nuclear factor kappa B kinase subunit beta; SNAP23: synaptosome associated protein 23; SNARE: soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; STX: syntaxin; TAP: trypsinogen activation peptide; VAMP: vesicle associated membrane protein; ZG: zymogen granule. [ABSTRACT FROM AUTHOR]

Impaired macroautophagy/autophagy has been implicated in experimental and human pancreatitis. However, the transcriptional control governing the autophagy-lysosomal process in pancreatitis is largely unknown. We investigated the role and mechanisms of TFEB (transcription factor EB), a master regulator of lysosomal biogenesis, in the pathogenesis of experimental pancreatitis. We analyzed autophagic flux, TFEB nuclear translocation, lysosomal biogenesis, inflammation and fibrosis in GFP-LC3 transgenic mice, acinar cell-specific tfeb knockout (KO) and tfeb and tfe3 double-knockout (DKO) mice as well as human pancreatitis samples. We found that cerulein activated MTOR (mechanistic target of rapamycin kinase) and increased the levels of phosphorylated TFEB as well as pancreatic proteasome activities that led to rapid TFEB degradation. As a result, cerulein decreased the number of lysosomes resulting in insufficient autophagy in mouse pancreas. Pharmacological inhibition of MTOR or proteasome partially rescued cerulein-induced TFEB degradation and pancreatic damage. Furthermore, genetic deletion of tfeb specifically in mouse pancreatic acinar cells increased pancreatic edema, necrotic cell death, infiltration of inflammatory cells and fibrosis in pancreas after cerulein treatment. tfeb and tfe3 DKO mice also developed spontaneous pancreatitis with increased pancreatic trypsin activities, edema and infiltration of inflammatory cells. Finally, decreased TFEB nuclear staining was associated with human pancreatitis. In conclusion, our results indicate a critical role of impaired TFEB-mediated lysosomal biogenesis in promoting the pathogenesis of pancreatitis. Abbreviations: AC: acinar cell; AMY: amylase; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATP6V1B2: ATPase, H+ transporting, lysosomal V1 subunit B2; ATP6V1D: ATPase, H+ transporting, lysosomal V1 subunit D; ATP6V1H: ATPase, H+ transporting, lysosomal V1 subunit H; AV: autophagic vacuole; CDE: choline-deficient, ethionine-supplemented; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EM: electron microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H & E: hematoxylin and eosin; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: normal donor; NEU: neutrophil; PPARGC1A/PGC1α: peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha; RIPA: radio-immunoprecipitation; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TM: tamoxifen; WT: wild-type; ZG: zymogen granule [ABSTRACT FROM AUTHOR]

Acute pancreatitis (AP) is often accompanied by alterations in the acid-base balance, but how blood pH influences the outcome of AP is largely unknown. We studied the association between blood pH and the outcome of AP with meta-analysis of clinical trials, and aimed to discover the causative relationship between blood pH and AP in animal models. PubMed, EMBASE, and Cochrane Controlled Trials Registry databases were searched from inception to January 2017. Human studies reporting systemic pH status and outcomes (mortality rate, severity scores, and length of hospital stay) of patient groups with AP were included in the analyses. We developed a new mouse model of chronic metabolic acidosis (MA) and induced mild or severe AP in the mice. Besides laboratory blood testing, the extent of pancreatic edema, necrosis, and leukocyte infiltration were assessed in tissue sections of the mice. Thirteen studies reported sufficient data in patient groups with AP (n = 2,311). Meta-analysis revealed markedly higher mortality, elevated severity scores, and longer hospital stay in AP patients with lower blood pH or base excess (P < 0.001 for all studied outcomes). Meta-regression analysis showed significant negative correlation between blood pH and mortality in severe AP. In our mouse model, pre-existing MA deteriorated the pancreatic damage in mild and severe AP and, vice versa, severe AP further decreased the blood pH of mice with MA. In conclusion, MA worsens the outcome of AP, while severe AP augments the decrease of blood pH. The discovery of this vicious metabolic cycle opens up new therapeutic possibilities in AP.

Objective: Defective mucus production in the pancreas may be an important factor in the initiation and progression of chronic pancreatitis (CP), therefore we aimed to (i) investigate the qualitative and quantitative changes of mucus both in human CP and in an experimental pancreatitis model and (ii) to correlate the mucus phenotype with epithelial ion transport function.Design: Utilizing human tissue samples and a murine model of cerulein induced CP we measured pancreatic ductal mucus content by morphometric analysis and the relative expression of different mucins in health and disease. Pancreatic fluid secretion in CP model was measured in vivo by magnetic resonance cholangiopancreatography (MRCP) and in vitro on cultured pancreatic ducts. Time-changes of ductal secretory function were correlated to those of the mucin production.Results: We demonstrate increased mucus content in the small pancreatic ducts in CP. Secretory mucins MUC6 and MUC5B were upregulated in human, Muc6 in mouse CP. In vivo and in vitro fluid secretion was decreased in cerulein-induced CP. Analysis of time-course changes showed that impaired ductal ion transport is paralleled by increased Muc6 expression.Conclusion: Mucus accumulation in the small ducts is a combined effect of mucus hypersecretion and epithelial fluid secretion defect, which may lead to ductal obstruction. These results suggest that imbalance of mucus homeostasis may have an important role in the early-phase development of CP, which may have novel diagnostic and therapeutic implications.

Acute pancreatitis (AP) is often accompanied by alterations in the acid-base balance, but how blood pH influences the outcome of AP is largely unknown. We studied the association between blood pH and the outcome of AP with meta-analysis of clinical trials, and aimed to discover the causative relationship between blood pH and AP in animal models. PubMed, EMBASE, and Cochrane Controlled Trials Registry databases were searched from inception to January 2017. Human studies reporting systemic pH status and outcomes (mortality rate, severity scores, and length of hospital stay) of patient groups with AP were included in the analyses. We developed a new mouse model of chronic metabolic acidosis (MA) and induced mild or severe AP in the mice. Besides laboratory blood testing, the extent of pancreatic edema, necrosis, and leukocyte infiltration were assessed in tissue sections of the mice. Thirteen studies reported sufficient data in patient groups with AP (n = 2,311). Meta-analysis revealed markedly higher mortality, elevated severity scores, and longer hospital stay in AP patients with lower blood pH or base excess (P < 0.001 for all studied outcomes). Meta-regression analysis showed significant negative correlation between blood pH and mortality in severe AP. In our mouse model, pre-existing MA deteriorated the pancreatic damage in mild and severe AP and, vice versa, severe AP further decreased the blood pH of mice with MA. In conclusion, MA worsens the outcome of AP, while severe AP augments the decrease of blood pH. The discovery of this vicious metabolic cycle opens up new therapeutic possibilities in AP. [ABSTRACT FROM AUTHOR]

Objective: Defective mucus production in the pancreas may be an important factor in the initiation and progression of chronic pancreatitis (CP), therefore we aimed to (i) investigate the qualitative and quantitative changes of mucus both in human CP and in an experimental pancreatitis model and (ii) to correlate the mucus phenotype with epithelial ion transport function. Design: Utilizing human tissue samples and a murine model of cerulein induced CP we measured pancreatic ductal mucus content by morphometric analysis and the relative expression of different mucins in health and disease. Pancreatic fluid secretion in CP model was measured in vivo by magnetic resonance cholangiopancreatography (MRCP) and in vitro on cultured pancreatic ducts. Time-changes of ductal secretory function were correlated to those of the mucin production. Results: We demonstrate increased mucus content in the small pancreatic ducts in CP. Secretory mucins MUC6 and MUC5B were upregulated in human, Muc6 in mouse CP. In vivo and in vitro fluid secretion was decreased in cerulein-induced CP. Analysis of time-course changes showed that impaired ductal ion transport is paralleled by increased Muc6 expression. Conclusion: Mucus accumulation in the small ducts is a combined effect of mucus hypersecretion and epithelial fluid secretion defect, which may lead to ductal obstruction. These results suggest that imbalance of mucus homeostasis may have an important role in the early-phase development of CP, which may have novel diagnostic and therapeutic implications. [ABSTRACT FROM AUTHOR]

Pharmacological studies conducted at the minimum possible number of animals in experiments based on the principles of humane attitude to laboratory animals. The selected animals in accordance with the type and status, depending on the program study, grown in the vivarium IFNMU that have been standardized by the physiological and biochemical indexes and were in accordance with requirements of sanitary - hygienic norms on a standard diet. During the experiment the animals treated according to international principles of the European Convention for the humane treatment of laboratory animals used for experimental and other scientific purposes (Strasbourg, 03.18.1986). The influence of water and water-alcohol Filipendula hexapetala extracts on the development of experimental pancreatitis have studied, and studied their acute toxicity, hepatoprotective and antitumor activity. [ABSTRACT FROM AUTHOR]

The literature indicates that acute pancreatitis is a complication of massive hemolysis with a prevalence of about 20%. We describe an experimental model of hemolysis-induced acute pancreatitis. Hemolytic anemia was induced in rats by a single ip injection of 60 mg/kg of 20 mg/ml acetylphenylhydrazine (APH) in 20% (v/v) ethanol on the first experimental day (day 0). One hundred and fifty Wistar albino rats weighing 180-200 g were divided into three groups of 50 animals each: groups 1, 2 and 3 were injected ip with APH, 20% ethanol, and physiological saline, respectively. Ten rats from each group were sacrificed on study days 1, 2, 3, 4 and 5. Serum amylase, lipase levels and pancreatic tissue tumor necrosis factor-alpha (TNF-alpha) and platelet-activating factor (PAF) contents were determined and a histological examination of the pancreas was performed. No hemolysis or pancreatitis was observed in any of the rats in groups 2 and 3. In group 1, massive hemolysis was observed in 35 (70%) of 50 rats, moderate hemolysis in seven (14%), and no hemolysis in eight (16%). Thirty-three of 35 (94.2%) rats with massive hemolysis had hyperamylasemia, and 29 of these rats (82.8%) had histologically proven pancreatitis. The most severe pancreatitis occurred on day 3, as demonstrated by histology. Tissue TNF-alpha and PAF levels were statistically higher in group 1 than in groups 2 and 3. Acute massive hemolysis induced acute pancreatitis, as indicated by histology, in almost 80% of cases. Hemolysis may induce acute pancreatitis by triggering the release of proinflammatory and immunoregulatory cytokines.

The endocannabinoid system has been shown to mediate beneficial effects on gastrointestinal inflammation via cannabinoid receptors 1 (CB1) and 2 (CB2). These receptors have also been reported to activate the MAP kinases p38 and c-Jun NH2-terminal kinase (JNK), which are involved in early acinar events leading to acute pancreatitis and induction of proinflammatory cytokines. Our aim was to examine the role of cannabinoid receptor activation in an experimental model of acute pancreatitis and the potential involvement of MAP kinases. Cerulein pancreatitis was induced in wild-type, CB1-/-, and MK2-/- mice pretreated with selective cannabinoid receptor agonists or antagonists. Severity of pancreatitis was determined by serum amylase and IL-6 levels, intracellular activation of pancreatic trypsinogen, lung myeloperoxidase activity, pancreatic edema, and histological examinations. Pancreatic lysates were investigated by Western blotting using phospho-specific antibodies against p38 and JNK. Quantitative PCR data, Western blotting experiments, and immunohistochemistry clearly show that CB1 and CB2 are expressed in mouse pancreatic acini. During acute pancreatitis, an upregulation especially of CB2 on apoptotic cells occurred. The unselective CB1/CB2 agonist HU210 ameliorated pancreatitis in wild-type and CB1-/- mice, indicating that this effect is mediated by CB2. Furthermore, blockade of CB2, not CB1, with selective antagonists engraved pathology. Stimulation with a selective CB2 agonist attenuated acute pancreatitis and an increased activation of p38 was observed in the acini. With use of MK2-/- mice, it could be demonstrated that this attenuation is dependent on MK2. Hence, using the MK2-/- mouse model we reveal a novel CB2-activated and MAP kinase-dependent pathway that modulates cytokine expression and reduces pancreatic injury and affiliated complications. [ABSTRACT FROM AUTHOR]

Objective. Cytokines initiate and modify systemic inflammatory response in early acute pancreatitis. The aim of this study was to analyze which cytokines are released from the pancreas to portal venous blood in the early phase of acute experimental necrotizing and oedematous pancreatitis and which of those cytokines are correlated with the more severe form of the disease. Material and methods. Fifteen pigs were randomized to develop mild oedematous pancreatitis (n = 5, saline infusion to pancreatic duct), severe necrotizing pancreatitis (n = 5, taurocholic acid infusion) along with a control group (n = 5). Arterial and venous blood samples were drawn and cytokine levels were measured from portal vein blood at 0, 120, 240 and 360 min after the induction of pancreatitis. Tissue samples from the pancreas were harvested at 0 and 360 min. Results. White blood cell count increased in necrotizing pancreatitis and the control group. The amount of neutrophils increased ( p < 0.001) and the lymphocyte and eosinophil counts decreased in all groups ( p < 0.001, p < 0.001). The monocyte count, as well as PDGF and IL-6 concentrations, increased only in necrotizing pancreatitis. IL-8 and eotaxin increased both in oedematous and necrotizing pancreatitis. MCP-1 increased in all groups. IL-9, IL-4, MIP-1α, IFN- γ concentrations did not change. Eotaxin and MCP-1 plasma levels from a previous series between portal venous and pulmonary arterial blood were not significantly different. Conclusions. The initial inflammatory process was diverse in oedematous and necrotizing pancreatitis. Increased monocyte count in combination with elevated PDGF and IL-6 are characteric of necrotizing pancreatitis in our model. [ABSTRACT FROM AUTHOR]

The pathomorphogenesis of experimental acute pancreatic necrosis induced by administration of phospholipase A2 into the pancreatic tissue is characterized by pathomorphological signs of hemorrhagic pancreatic necrosis (total necrosis and purulent fusion of some acini and massive hemorrhages in the interlobular and intralobular interstitial tissue) and fatty pancreatic necrosis (necrobiotic and necrotic changes in acinar cells that spread from the peripheral area and form the demarcation line). [ABSTRACT FROM AUTHOR]

Previously, we found that the University of North Carolina cystic fibrosis (UNC-CF) mouse had more severe experimental acute pancreatitis (AP) than wild-type (WT) mice characterized by exuberant pancreatic inflammation and impaired acinar apoptosis. Because exon 10 CFTR gene mutations exhibit different phenotypes in tissues such as the mouse lung, we tested the hypothesis that ΔF508-CF mice also develop severe AP associated with an antiapoptotic acinar phenotype, which requires indirect effects of the extracellular milieu. We used cerulein hyperstimulation models of AP. More severe pancreatitis occurred in cerulein-injected ΔF508-CF vs. WT mice based on histological severity (P < 0.01) and greater neutrophil sequestration [P < 0.0001; confirmed by myeloperoxidase activity (P < 0.005)]. In dispersed acini cerulein-evoked necrosis was greater in ΔF508-CF acini compared with WT (P < 0.05) and in WT acini pretreated with CFTRinh-172 compared with vehicle (P < 0.05). Cerulein-injected ΔF508-CF vs. WT mice had less apoptosis based on poly(ADP-ribose) polymerase (PARP) cleavage (P < 0.005), absent DNA laddering, and reduced terminal deoxynucleotidyltransferase biotin-dUTP nick end labeling (TUNEL) staining (P < 0.005). Unexpectedly, caspase-3 activation was greater in ΔF508-CF vs. WT acini at baseline (P < 0.05) and during AP (P < 0.0001). Downstream, ΔF508-CF pancreas overexpressed the X-linked inhibitor of apoptosis compared with WT (P < 0.005). In summary, the ΔF508-CF mutation, similar to the UNC-CF "null" mutation, causes severe AP characterized by an exuberant inflammatory response and impaired acinar apoptosis. Enhanced acinar necrosis in ΔF508-CF occurs independently of extracellular milieu and correlates with loss of CFTR-Cl conductance. Although both exon 10 models of CF inhibit acinar apoptosis execution, the ΔF508-CF mouse differs by increasing apoptosis signaling. Impaired transduction of increased apoptosis signaling in ΔF508-CF acini may be biologically relevant to the pathogenesis of AP associated with CFTR mutations. [ABSTRACT FROM AUTHOR]

Objective. It is unclear why pancreatitis progresses either to mild edematous disease or to severe necrotic disease. The aim of the study was to shed some light on this topic by investigating differences during the early stages of necrotic and edematous pancreatitis. Material and methods. Piglets were randomized into two groups. Necrotic pancreatitis was induced with retrograde injection of 20% taurocholic acid (1 ml/kg), and edematous pancreatitis was induced with 0.9% NaCl (1 ml/kg). Central hemodynamics was measured, and pancreatic microcirculation was directly examined by intravital microscopy. Vascular permeability to proteins and albumin was measured by microdialysis. Apoptosis and claudins 2, 3, 4, 5, and 7 were analyzed from pancreatic tissue samples. Blood samples were taken for analysis of blood cell counts, blood gases, lipase, and amylase. Results. Hemodynamic changes were similar in both groups, whereas microcirculatory impairment was more pronounced in necrotic pancreatitis. Necrosis was associated only with necrotic pancreatitis. Apoptosis increased only in edematous pancreatitis. The number of blood neutrophils and monocytes increased and lymphocyte and platelet counts decreased in both groups. Necrotic pancreatitis was associated with increased permeability to albumin and proteins. Expression of claudins 3, 4, 5, and 7 was not changed during pacreatitis, but in acinar cells, membranous expression of claudin-2 increased in both groups. Conclusions. The results show that acute edematous pancreatitis is characterized by induction of apoptosis, whereas full-blown pancreatitis is characterized by necrosis. Impaired vascular permeability to albumin and protein is related to the early phase of necrotic pancreatitis. Claudin-2 increases during acute necrotic and edematous pancreatitis and may be related to impaired permeability. [ABSTRACT FROM AUTHOR]

Abstract: Premature activation of digestive enzymes within the pancreas which leads to autodigestion of the gland is an early step in the pathogenesis of pancreatitis. Pancreatic injury is followed by other manifestations of inflammation including plasma extravasation, edema, and neutrophil infiltration which constitute the features of pancreatitis. Recent studies indicate that neural innervation of the pancreas may play an important role in the initiation and maintenance of the inflammatory response to injury. The pancreas is innervated by vagal, sympathetic and parasympathetic neurons, as well as sensory neurons. Activation of pancreatic primary sensory neurons causes the release of inflammatory neuropeptides both in the spinal cord to signal pain and in the pancreas itself where they produce plasma extravasation and neutrophil infiltration. Recent studies indicate that primary sensory neurons of the pancreas express transient receptor potential V1 (TRPV1) channels whose activation induces pancreatic inflammation. Moreover, blockade of these TRP channels significantly ameliorates experimental pancreatitis. This review describes our current understanding of the role of TRPV1 channels in pancreatitis and illustrates how this mechanism might be used to direct future treatments of pancreatic diseases. [Copyright &y& Elsevier]

Abstract: Clusterin is overexpressed in pancreas during the acute phase of pancreatitis. We intended to clarify the role of clusterin expression in stressed exocrine pancreas. We performed in vitro experiments in transfected AR4-2J cells with modified expression levels of clusterin and in vivo studies in clusterin-deficient mice. AR4-2J cells were exposed to agents mimicking cell-stress during pancreatitis (cerulein, hydrogen peroxide, staurosporine or lysophosphatidylcholine). Clusterin-overexpressing AR4-2J cells showed higher viability after cell stress and accordingly reduced rates of apoptosis and lessened caspase-3 activation. Blockage of endogenous clusterin expression reduced viability and enhanced apoptosis. Presence of clusterin reduced NF-κB activation and expression of the NF-κB target genes TNF-α and MOB-1 under cell stress. Clusterin-deficient mice showed a more severe course of acute experimental pancreatitis with enhanced rates of apoptosis and inflammatory cell infiltration. We concluded that clusterin was protective during inflammation of exocrine pancreas because of its anti-apoptotic and anti-inflammatory functions. [Copyright &y& Elsevier]

Background: Research aimed at elucidating the pathogenesis of pancreatitis-associated lung injury and evaluating novel strategies for preventing respiratory complications in acute pancreatitis (AP) has not yet involved intravital microscopic (IVM) studies of pulmonary microcirculation in animals with severe disease. Objective: To characterize and compare pulmonary microcirculation in severe/necrotizing (NP) and mild/edematous pancreatitis (EP) in the rat. Methods: EP was induced by intravenous cerulein infusion (n = 10) and NP by a standardized intraductal infusion of glycodeoxycholic acid followed by intravenous cerulein (n = 10). After 24 h a left-sided thoracotomy was performed for IVM examination of pulmonary capillary blood flow, permeability, leukocyte sticking and the thickness of alveolar septi. Further measurements included monitoring of arterial blood gases and histological evaluation of lung injury. Results: In animals with NP, histology revealed severe pulmonary edema together with clustering of polymorphonuclear leukocytes in pulmonary microvessels and alveoli. IVM showed a greater number (n) of leukocytes sticking the endothelium of pulmonary capillaries (9.4 ± 0.7 1.8 ± 0.2 in healthy control animals) and increased capillary permeability (260 ± 14 vs. 136 ± 6% relative fluorescein intensity) while capillary blood flow was decreased (0.41 ± 0.05 vs. 0.57 ± 0.03 mm/s). In comparison, changes in EP were significantly less pronounced flow 0.5 ± 0.04 mm/s, permeability 156 ± 4%, leukocyte sticking n = 4.6 ± 0.7). Conclusions: These findings suggest that deterioration of pulmonary microcirculation in AP correlates with disease severity and that a model featuring NP may therefore be more suitable to further study pancreatitis-associated pulmonary injury. [ABSTRACT FROM AUTHOR]

Objective: To investigate pancreatic tissue perfusion and oxygenation in severe and mild experimental acute pancreatitis in pigs. Design: Randomised controlled experiment. Setting: Animal laboratory, Finland. Animals: 24 domestic pigs weighing 21-27 kg. Interventions: 24 pigs were randomised into severe acute pancreatitis, mild acute pancreatitis and control groups (n = 8 in each). The pancreatic duct of eight anaesthetised and mechanically ventilated pigs was cannulated and taurocholic acid was infused into the pancreatic duct to induce severe acute pancreatitis. Eight animals received intraductally infused saline and developed mild acute pancreatitis. Eight pigs had their ducts cannulated alone, and served as controls. Main outcome measurements: Pancreatic tissue oxygenation, laser Doppler red cell flux, central haemodynamics. Results: Intraductally infused taurocholic acid rapidly induced macroscopically and histologically proven severe necrotising acute pancreatitis. Histological changes characterising mild acute pancreatitis were seen in animals after intraductal saline infusion. Pancreatic tissue oxygen tension decreased in the severe group and increased in the mild group during the six-hour study period. Laser Doppler red cell flux decreased in the severe group. Central haemodynamics, arterial blood gases, and acid base balances were stable throughout the study period in all groups. Conclusion: The present model of severe acute pancreatitis significantly impairs pancreatic oxygenation in the early phase. In mild acute pancreatitis, pancreatic oxygenation increases. [ABSTRACT FROM AUTHOR]

Background: The efficacy of octreotide, the synthetic analogue of the hormone somatostatin, for the treatment of acute pancreatitis is controversial. Octreotide has been commonly administered in subcutaneous bolus injections; however, continuous intravenous infusion may be advantageous for acute conditions. Methods: Acute experimental pancreatitis was induced in rats by intraparenchymal injections of 1 ml 10% sodium taurocholate, and octreotide (1 µg/kg/h, dissolved in physiological solution, intravenously was started 4 h later and continuously infused for 48 h. Physiological solution infusions, in identical volumes, were used in the controls. The following parameters were examined: mortality; macroscopic and histological damage; hematocrit; plasma pH; acid-base balance; serum glucose; calcium, and amylase. Results: Octreotide treatment had a striking effect on mortality: 8.3 versus 91.6% in the treatment and control groups, respectively (p < 0.001). Octreotide also ameliorated pancreatic edema and intestinal dilatation, and had significant beneficial effects on histopathological damage and the biochemical alterations which are associated with acute pancreatitis. Conclusions: Continuous intravenous octreotide infusion is a potentially efficacious therapeutic method for acute pancreatitis. [ABSTRACT FROM AUTHOR]

Hyperstimulation with cholecystokinin analogue cerulein induces a mild edematous pancreatitis in rats. There is evidence for a diminished energy metabolism of acinar cells in this experimental model. The aim of this study was to demonstrate permeability transition of the mitochondrial inner membrane as an early change in mitochondrial function and morphology. As functional parameters, the respiration and membrane potential of mitochondria isolated from control and cerulein-treated animals were measured, and changes in volume and morphology were investigated by swelling experiments and electron microscopy. Five hours after the first injection of cerulein, the leak respiration was nearly doubled and the resting membrane potential was decreased by about 17 mV. These alterations were reversed by extramitochondrial ADP or did not occur when cyclosporin A was added to the mitochondrial incubation. A considerable portion of the mitochondria isolated from cerulein-treated animals was swollen and showed dramatic changes in morphology such as a wrinkled outer membrane and the loss of a distinct cristae structure. These data provide evidence for the opening of the mitochondrial permeability transition pore at an early stage of cerulein induced pancreatitis. This suggests that the permeability transition is an initiating event for lysis of individual mitochondria and the initiation of apoptosis and/or necrosis, as had been shown to occur in this experimental model. [ABSTRACT FROM AUTHOR]

While hypovolemia or hypovolemic shock is dominant in the early stage of severe acute pancreatitis, there have been few studies on the effects of hypertonic solutions in the management of this disease. We conducted this study to evaluate the therapeutic effects of hypertonic saline solutions (HS) on the course of severe acute pancreatitis in rats. Pancreatitis was induced in male Wistar rats by injecting a 5% solution of sodium taurocholate into the biliopancreatic duct. The effective circulating plasma volume (ECPV) was measured using radioiodinated [125I]bovine serum albumin. Samples of blood and of ascitic fluid were obtained 3, 6, and 12h after the onset of pancreatitis. Lactated Ringer's solution (LR) and HS were administered consecutively for 3h beginning 3h after the induction of pancreatitis. ECPV was measured 6h after the onset of pancreatitis. The survival rates were investigated for up to 10 days. The mean ECPV decreased significantly from 24.9±1.1ml/kg before disease onset to 11.5±1.3 ml/kg 6h postoperatively. LR failed to achieve a normal value for ECPV even following a 150 ml/kg infusion. HS200 and HS300 restored the ECPV to the normal level, and with smaller volumes infused. All rats in the untreated group died within 3 days. LR and HS improved the survival rates, with the infusion of HS200, 100 ml/kg, thus attaining a 45% survival at 10 days. [ABSTRACT FROM AUTHOR]

In l-arginine (Arg)-induced pancreatitis, evidence of acute inflammation was observed on d 1–3. Continuous tissue atrophy became visible at the sites of previous pancreatic necrosis, with simultaneous regeneration of the pancreas, mainly around the Langerhans islets. Administration of low doses of cholecystokinin-octapeptide (CCK-8) increased the inflammatory signs of pancreatitis in the early phase, but subsequently diminished the level of atrophy and accelerated the processes of regeneration in this model of pancreatitis. The aim of this work was to study the regenerative processes following Arg-induced pancreatitis in rats. Besides the spontaneous regeneration, the effects of low doses of CCK-8 on the laboratory and morphologic parameters in this type of experimental pancreatitis were investigated. Male Wistar rats were divided into three groups. In group I, the rats received 200 mg/100 g body weight of Arg ip twice, at an interval of 1 h, and 0.5 mL saline was administered sc twice daily. In group II, besides the same amount of Arg, the rats received 1 μg/kg of CCK-8 sc in 0.5-mL saline twice daily (7 am and 7 pm). In the control animals (group III), an identical amount of glycine was administered ip instead of Arg at the same times. The rats were examined on d 1, 3, 7, 14, and 28 after pancreatitis induction. The pancreatic weight/body weight ratio (pw/bw) was calculated in each case. The serum levels of amylase, and glucose and the pancreatic contents of soluble protein, trypsin, amylase and DNA were determined, and histologic examinations were performed. In groups I and II, both pw/bw (3.5±0.2 mg/g and 4.1±0.28 mg/g, respectively) and the serum amylase level (8900±560 IU/L and 11100±1390 IU/L, respectively) were significantly elevated on d 1 vs group III (2.1±0.06 mg/g and 5562±373 IU/L, respectively). Pw/bw was significantly decreased in groups I (0.96±0.12 mg/g, 0.8±0.1 mg/g, and 1.8±0.1 mg/g, respectively) and II (1.4±0.15 mg/g, 1.7±0.2 mg/g, and 1.95±0.1 mg/g, respectively) on d 7, 14, and 28 vs group III (2.6±0.3 mg/g, 3.1±0.15 mg/g, and 2.7 ±0.1 mg/g, respectively), whereas in group II it was significantly elevated vs. group I on d 7 and 14. The pancreatic contents of soluble protein, DNA, trypsin and amylase were significantly decreased on d 3–14 in groups I and II vs group III. The pancreatic DNA level was significantly elevated in group II (1.23±0.2 mg/pancreas) vs group I (0.7±0.1 mg/pancreas) on d 7. In group II, the soluble protein (73.1±15.5 mg/pancreas) and amylase (1104±160 IU/pancreas) levels were significantly elevated on d 14 as was that of trypsin (27.2±3.1 IU/pancreas) on d 28, vs group I (26.4±5.3 mg/p, 525±111 IU/pancreas, and 16.3±1.1 IU/pancreas, respectively). On histologic sections, the signs of acute inflammation of the pancreas were more pronounced in group II than in group I on d 1–3. After that time, in spite of the progressive atrophy of the pancreas, the signs of tissue repair were more expressed in group II. [ABSTRACT FROM AUTHOR]

The long-term effects of octreotide, the synthetic analog of the hormone somatostatin, on acute experimental pancreatitis were studied. Acute pancreatitis was induced in rats by intraparenchymal injections of 0.5 ml 5% or 10% sodium taurocholate. Octreotide (10 mg/kg/day, subcutaneously), or saline injections as controls, were started four hours later, and their effects were assessed 30, 60, and 90 days after the induction of pancreatitis. Neither intrapancreatic saline injections nor octreotide administration without the induction of pancreatitis caused any biochemical or histological abnormalities. Taurocholate-induced pancreatitis was followed by remarkable hyperglycemia, which was ameliorated by octreotide. Thirty days after induction of pancreatitis, glucose levels were 269+/-21 mg/100 ml and 153+/-17 mg/100 ml in the control and octreotide treated animals, respectively (P < 0.02). Octreotide administration was associated with increased pH values after 60 and 90 days (P < 0.05 for the 90 days group). The levels of hematocrit, calcium, and amylase were already within the normal ranges after 30 days and were unaffected by octreotide. There were no signs of chronic exocrine insufficiency and all the surviving rats gained weight during the follow-up. However, the relative weights of the pancreases of the octreotide-treated animals were higher than those of the controls 30 days after IOP. Histopathological evaluation demonstrated regeneration of the pancreatic tissue, and increased number and hypertrophy of the islets of Langherhans. There were no significant differences whether the octreotide treatment was given for only 48 or 96 hr. Survival was significantly improved by octreotide; only one octreotide-treated rat (2.5%) with 10% taurocholate-induced pancreatitis died, while six (15%) of the control animals succumbed (P < 0.05). These studies provided data on the sequelae of acute pancreatitis and showed that octreotide may have long-term beneficial effects in this disease. [ABSTRACT FROM AUTHOR]

The inhibitory effects of somatostatin (SMS) and glucagon (Gn) on acute pancreatitis were evaluated in an experimental acute pancreatitis model in male Wistar rats. The effects of these agents were compared with those of nafamostat mesilate (NM). The acute pancreatitis was induced by four serial subcutaneous injections of caerulein. The rats were divided into four groups. The first group ( n=28) received SMS daily, the second group ( n=28) received Gn daily, and the third group ( n=28) received NM daily after the first injection of caerulein. The fourth group ( n=42) received caerulein alone and served as the control group. Animals were sacrificed 4, 6, 8, 12, and 24 h, and 3 and 7 days after the first administration of caerulein and the degree of severity of the acute pancreatitis was evaluated by serial morphological and histological examinations of pancreatic tissues, as well as in terms of the serum concentrations of amylase and lipase. The characteristic findings of acute pancreatitis in the animals of all groups treated with SMS, Gn, or NM were markedly attenuated at all time points after the treatments compared with findings in the controls (caerulein alone) in terms of wet weight of pancreas, serum concentrations of amylase and lipase, formation of intracellular vacuoles in acinar cells, interstitial edema, and infiltration of an inflammatory cell component. The inhibitory effects of SMS, Gn, and NM on acute pancreatitis were similar at the doses used. These results suggest that SMS and Gn are as useful as NM, they may be of value for the treatment of acute pancreatitis. [ABSTRACT FROM AUTHOR]

This study was conducted to elucidate the possible influence of long-term peroral administration of alcohol on the repair process of acute necrotizing pancreatitis. Male Wistar rats fed with balanced diet were divided into two groups. The first group had free access to 15% ethanol, and the second group, the control group, has access to water instead. After fifty weeks, acute necrotizing pancreatitis was induced in rats by infusing 0.4% lysolecithin into their pancreatic duct. In the course of pancreatitis, pancreatic enzymes in serum, enzymes and protein in pancreas, and DNA synthesis in pancreas in both groups, changed in the same way. Histologically, interstitial edema, necrosis of parenchyma, infiltration of inflammatory cells, and formation of tubular complex were observed. Most of these histological changes of pancreas in both groups disappeared in twenty days and pancreas was repaired almost completely. These findings suggest that the repair process of acute necrotizig pancreatitis is not affected by preceding long-term intake of alcohol. [ABSTRACT FROM AUTHOR]

To elucidate the pathophysiological process of alcoholic pancreatitis, chronic alcohol intoxication was made in Wistar rats on balanced diet giving 20% ethanol freely for 60 weeks. The control rats received water. Histological picture of the pancreas, hormonal activity in the mucosa of upper digestive tract and the nature of pancreatic juice were examined in every 15th week. The results were as follows. 1) No histological changes were noted in the pancreas of control group. In the ethanol group, morphological abnormalities of the pancreas appeared after 30 weeks. Of the histological findings, the changes on the ductal system such as dilatation of pancreatic duct, plug formation in the ductal lumen and periductal fibrosis were significant. 2) The long term ethanol administration tended to decrease the amounts of gastrin, secretin and cholecystokinin contained in the gastrointestinal mucosa. 3) Regardless of the histological changes of the pancreas, almost no changes were noted in the bicarbonate and protein concentration during the experimental period of 60 weeks. From the above results, a mechanism obstructing pancreatic ductal system is considered to be important in the pancreatic lesions by alcohol rather than a mechanism of stimulating pancreatic exocrine secretion. [ABSTRACT FROM AUTHOR]

Hypercalcemia causes acute pancreatitis in humans, a phenomenon reproduced experimentally in cats and guinea pigs. Because the rat is the most frequently used animal for the study of experimental pancreatitis, the present studies were performed to evaluate the effects of hypercalcemia in the rat In in vitro studies, pancreatic lobules were prepared from fasted Wistar rats (200-250 g) and incubated in HEPES bicarbonate-buffered medium (pH 7.4) containing 0, 0.6, 1.2, 2.5, 5, and 10 mM CaCL with or without carbachol l0M. Amylase was measured in the medium after 30 min to 3 h, and expressed as percent of total amylase. In in vivo studies, fasted male Wistar rats (300-400 g) received calcium (CaCl; 0.6 mmol/kgh) into the tail vein for 12 h. Control animals received NaCl 0.9% infusion. Histologic slides (H&E-stained) were evaluated in a blinded fashion. Pancreatic lobules showed a higher basal amylase output when incubated in higher calcium medium. The largest, significant difference (2.6-fold) was between 0.6 and 5 mM medium CaCl ( < 0.05). Carbachol-stimulated amylase release was again higher with increasing medium calcium with the most pronounced difference (1.3-fold) between 0.6 and 2.5 mM CaCl, ( < 0.05). In vivo calcium-treated animals showed accumulation of zymogen granules in the cytoplasm, cytoplasmic vacuolization, focal acinar cell depolarization, acinar necrosis, and edema. Calcium causes amylase release from rat pancreatic lobules in vitro. Higher medium calcium levels both significantly increase amylase release from unstimulated and carbachol stimulated lobules. Twelve-hour in vivo calcium infusion leads to accumulation of zymogen granules in acinar cells and acinar injury. These findings are consistent with pancreatitis evolving from calciuminduced hypersecretion to a secretion block lesion in the rat. [ABSTRACT FROM AUTHOR]

The effect of allopurinol, a xanthine oxidase inhibitor, on canine experimental ischemic pancreatitis was studied. The animals were divided into nine groups: Serum amylase was determined in all animals. In Groups 1 and 5, following the ischemic period, hyperamylasemia developed and a peak was reached 24 h after ischemia. In Group 2, a significant decrease of amylase levels was found, compared to matched controls immediately after ischemia and then rose, reaching on the fifth day a peak that was less than the controls at 24 h. In Group 3, the serum amylase level increased immediately to values similar to controls; later, there was a drop to levels lower than those found in controls, followed by a peak on the fifth day. In Group 4, there was no significant elevation in the amylase values. Groups 6 and 7 showed no changes of amylasemia. In this experimental model, allopurinol blocked or ameliorated significantly cellular injury, as shown by a decrease of amylase levels in blood, and of histopathological changes, depending on dose and time of administration. These results offer the possibility of a prophylactic therapy for chronic relapsing and idiopathic pancreatitis. [ABSTRACT FROM AUTHOR]

The present study investigates the effect of CCK-receptor blockade on taurocholate-induced pancreatitis in rats using the potent antagonist loxiglumide. Intraperitoneal administration (50 mg/kg) of loxiglumide began 3 h before, or 10 min or 3 h after induction of pancreatitis. Mean survival times of the experimental groups were 31.2, 23.6, and 20.5 h, respectively, compared to 18.2 h for controls. Survival for 24 h after induction of pancreatitis was significantly improved when the antagonist was given 3 h before, but not in the time periods after induction. After 72 h, survival time was not significantly altered in any of the groups. Furthermore, amylase and lipase levels quantified 10 h after induction of pancreatitis in ascites, blood, or tissue did not indicate a significant difference, nor was improvement in survival seen when the CCK-antagonist was tested in rats receiving a basal treatment with intravenous volume substitution, peritoneal lavage, and protease inhibition. We conclude that CCK-receptor blockade does not improve the final outcome of bile-induced pancreatitis in the rat, even if treatment is started before induction of pancreatitis. [ABSTRACT FROM AUTHOR]

To evaluate the in vivo effects of ischemia on the pancreas, 13 dogs had all blood vessels to the pancreas divided at laparotomy, except for the pancreatical duodenal artery and vein. In seven of the 13 dogs, ischemia was induced by clamping the pancreaticoduodenal artery for 5 h. In six dogs, pancreatitis was induced by injection of autologous bile (1 mL/kg body wt) into the pancreatic duct. Following ischemia or bile injection, the gland was reperfused and pancreatic blood flow was measured for the subsequent 4 h. Serum amylase was measured in all animals prior to operation, before arterial occlusion or bile injection, and after pancreatic perfusion. Pancreatic exudate was collected for trypsin assay. Gross and histologic examination of the pancreas and measurement of pancreatic water content was done on all specimens. Mean pancreaticoduodenal artery blood flow in ischemic animals showed reperfusion hyperemia immediately after removal of the arterial clamp, followed by a gradual return to baseline flow. Bile injection caused pancreatic blood flow to gradually decline to levels approximately one-half of baseline. Serum amylase rose significantly from baseline levels following bile injection (p<.05), but there was no significant change in serum amylase following 5 h of total ischemia and 4 h of reperfusion. Pancreatic water content increased with both ischemia and bile injection, but this increase reached statistical significance only with ischemia. Microscopically, edema was seen in the ischemic pancreas, but cellular architecture was preserved. Bile injection regularly revealed hemorrhage, necrosis, and disruption of acinar architecture. Trypsin activity was present in the exudate of four of six bile-injected dogs, but only one of seven ischemic dogs ( p<.05). In conclusion, ischemia did not elevate amylase, active trypsin, or cause hemorrhage and disruption of acinar architecture. Ischemia, unlike bile injection, did not initiate a process similar to severe clinical pancreatitis. [ABSTRACT FROM AUTHOR]

Sepsis is the most comon cause of late death in pancreatitis. The presence of early bacterial infection has been correlated with the severity of the disease. A choline-deficient ethionine-supplemented (CDE) diet given to young female mice produces severe necrotizing pancreatitis that has morphologic and biochemical similarities to the human disease. We therefore searched for bacterial pancreatic infection in female CD-1 mice given the CDE diet. The mortality rate was 47.5% in mice fed the CDE diet. All of these mice had severe pancreatitis with inflammation, edema, and necrosis on histologic examination. Bacterial infection was present in 1/12 pancreatica among nonsurvivors and in 1/32 pancreatica in surviving animals (p not significant). Histologic examination showed edema to be more pronouced in surviving mice, although the overall seventy of morphologic changes was not significantly different between survivors and nonsurvivors. We conclude that bacterial infection is not a determinant of the severity or lethality of experimental pancreatitis induced by the CDE diet. [ABSTRACT FROM AUTHOR]

The effects of hemorrhagic shock, aspirin, and ethanol on the biochemical and morphologic changes of experimental pancreatitis were evaluated. Pancreatitis was induced by infusing rats with a supramaximally stimulating dose (5 µg/kg/h) of caerulein. Hemorrhagic shock was established by removing sufficient blood to reduce mean arterial pressure by 30%, where it was maintained for 30 min. Aspirin (25 mg/kg) and ethanol (2 g/kg) were administered through an orogastric tube at 8-h intervals for 48 h. Hemorrhagic shock did not alter the degree of hyperamylasemia, pancreatic edema, cathepsin subcellular redistribution, or in vitro LDH leakage that characterize this model of pancreatitis. Hemorrhagic shock did, however, worsen the morphologic evidence of pancreatic injury. Administration of aspirin with ethanol did not alter the degree of hyperamy-lasemia, pancreatic edema, or subcellular cathepsin redistribution. Aspirin-ethanol pretreatment also did not alter the morphologic severity of pancreatitis. These observations indicate that hemorrhagic shock worsens the microscopic evidence of pancreatitis induced by supramaximal secretagogue stimulation. In contrast, aspirin-ethanol pretreatment, which might have been expected to increase pancreatic ductal permeability, did not alter the severity of this model of experimental pancreatitis. [ABSTRACT FROM AUTHOR]

Experimental acute necrotizing pancreatitis was induced retrogradely in dogs with sunflower oil injected intraductally. Then, a zipper was sutured into the abdominal wound. From the first postoperative day, three different treatments were started: first group: only conservative therapy was used; second group: removal of necrotized tissue and single peritoneal lavage were aplied; and third group: the necrotic part of the pancreas was marsupialized into the stomach. Through the systematic opening of the zipper, the abdominal cavity could easily be explored and the temporal course of disease could be observed in all animals. During this regular procedure, the amylase concentration and the amount of peritoneal exudate were determined. The blood amylase and glucose levels were also measured. On the first postoperative day, the amylase level and the amount of peritoneal exudate were high in all groups. The dogs of the pancreatogastrostomized group showed a dramatic decrease of the exudate and the most advantageous temporal course of the blood amylase level. The survival rate similarly was advantageous in the pancreatogastrostomized group. [ABSTRACT FROM AUTHOR]

The effect of somatostatin on the course and severity of experimental pancreatitis was tested. Acute pancreatitis was induced in 210 Sprague-Dawely rats by injecting a 4.3% sodium taurocholate solution, saturated with trypsin, into a temporarily closed duodenal loop. Immediately after the end of the surgical procedure somatostatin or, alternatively, normal saline were administered as a bolus followed by continuous subcutaneous infusion for 9 h. Ninety rats (30 untreated, 30 saline-treated and 30 somatostatin-treated) were sacrificed 10 h after the induction of pancreatitis to assess the histologic severity of pancreatic lesions, the amount of peritoneal exudate and the circulating levels of amylase. In another 120 rats (40 untreated, 40 saline-treated and 40 drug-treated) the mortality rate was evaluated so that the histologic examination of the pancreas followed spontaneous death. In sacrificed animals somatostatin treatment lowered serum amylase levels and definitely improved pancreatic histopathology (edema, leucocyte infiltration and necrosis). The drug prevented the occurrence of severe necrosis in all treated animals. Somatostatin did not affect the mortality rate of pancreatitic rats (70%) although post-mortem histologic examination revealed significantly less pancreatic histopathology in drug-treated rats than in their controls. [ABSTRACT FROM AUTHOR]

High concentrations of lipid peroxidation (free-radical oxidation) products have been found in bile from patients with recurrent pancreatitis, and the principal component, after hydrolysis, has been identified as an isomerised form of linoleic acid -typical concentration 25 mmol/l, compared with 4 mmol/l in controls. Chromatographically identical products can be generated by peroxidising linoleic acid using an ultraviolet (UV) source in the presence of albumin, whereas peroxidation by lipoxidase without albumin results in a constellation of products that bear no resemblance to those in biological fluids. These facts, and the suspicion that reflux of abnormal bile may be an initiating mechanism in acute pancreatitis, led us to investigate the effects of linoleic acid peroxidation products in the rat pancreas. Two concentrations of ultraviolet-peroxidised linoleic acid were used (3.6 mmol/l or 25 mmol/l, in a 2.09% solution of bile salts containing albumin 10 g/l) to simulate the human findings and, for comparison, the effects of lipoxidase-peroxidised linoleic acid, 25 mmol/l (in the 2.09% bile salt solution but without albumin), were also studied. 100 μl of test solution was infused retrogradely into the pancreatic duct using a syringe pump. The results were assessed microscopically at 3-h intervals, and histologically at 12 h: if the animal died before the end of the experiment, the time of death was recorded. Both forms of peroxidised linoleic acid, 25 mmol/l, caused a greater degree of pancreatic injury than that produced by bile salts alone (e.g., macroscopic score at 3 h: ultraviolet, P<0.01; lipoxidase, P<0.05). Non-peroxidised linoleic acid 25 mmol/l caused less damage than ultraviolet-peroxidised linoleic acid 25 mmol/l, both macroscopically (3 h: P<0.01; 12 h: P<0.05) and on histology ( P<0.01). Pancreatic haemorrhage was not a feature. [ABSTRACT FROM AUTHOR]