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Pancreatic proteases are secreted in acute pancreatitis, but their contribution to associated lung injury is unclear. Applying models of mild edematous (intravenous caerulein) and severe necrotizing (intraductal glycodeoxycholic acid) pancreatitis in rats, we showed that both trypsinogen and trypsin concentrations in peripheral blood, as well as lung injury, correlate with the severity of the disease. To isolate the potential contribution of proteases to lung injury, trypsin or trypsinogen was injected into healthy rats or trypsinogen secreted in caerulein pancreatitis was activated by intravenous enterokinase. Pulmonary injury induced by protease infusions was dose dependent and was ameliorated by neutrophil depletion. Trypsinogen activation worsened lung injury in mild pancreatitis. In vitro incubation of leukocytes with trypsinogen showed that stimulated leukocytes can convert trypsinogen to trypsin. In conclusion, this study demonstrates that the occurrence and severity of pancreatitis-associated lung injury (PALI) corresponds to the levels of circulating trypsinogen and its activation to trypsin. Neutrophils are involved in both protease activation and development of pulmonary injury.

Background & Aims: Intracellular activation of trypsinogen is currently believed to initiate pancreatitis. Factors responsible for the progression of mild to necrotizing pancreatitis are poorly understood. This study evaluated the significance of interstitial protease release and activation in this process., Methods: In rats with cerulein-induced pancreatitis, concentrations of trypsinogen and its activation peptide TAP were measured in lymph and blood, and pancreatic injury was determined. Activation of extracellular trypsinogen was induced by intravenous infusion of enterokinase, which does not enter the acinar cell. Gabexate mesilate (acinar cell permeable) or soybean trypsin inhibitor (acinar cell nonpermeable) was administered to distinguish the effects of intracellular or extracellular protease activation., Results: In cerulein pancreatitis, trypsinogen levels increased prominently and were highest in lymph and portal vein blood, whereas TAP increments were modest. Combined cerulein/enterokinase infusions resulted in marked TAP increases in lymph and blood and in severe necrohemorrhagic pancreatitis. Gabexate mesilate as well as soybean trypsin inhibitor significantly decreased TAP levels in both lymph and blood and reduced pancreatic injury, with no significant differences between groups., Conclusions: In secretagogue-induced pancreatitis, large amounts of trypsinogen are present in the interstitium and drain via the portal and lymphatic circulation. Activation of this extracellular trypsinogen induces hemorrhagic necrosis in a setting of mild edematous pancreatitis. This phenomenon may be the central event in the progression to fulminant necrotizing pancreatitis.

Background: Ectopic protease activation, microcirculatory changes, and leucocyte activation are the main events in the pathogenesis of acute pancreatitis. Nitric oxide (NO) is known to be a key mediator in the normal and inflamed pancreas., Aims: To investigate the targets on which NO exerts its effect in caerulein induced pancreatitis., Methods: Acute pancreatitis was induced in rats which additionally received either the NO synthase substrate, L-arginine; the NO donor, sodium nitroprusside; or the NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME). At six hours, pancreatic injury (oedema, leucocyte content, ectopic trypsinogen activation) was analysed and pancreatic oxygenation and perfusion were determined. A direct influence of NO on amylase secretion and trypsinogen activation was evaluated separately in vitro., Results: Both NO donors reduced the grade of inflammation. L-NAME increased the severity of inflammation, while decreasing pancreatic tissue oxygenation. Although neither amylase secretion nor intracellular trypsinogen activation in caerulein stimulated pancreatic acini was influenced by either NO donors or inhibitors, both NO donors decreased intrapancreatic trypsinogen activation peptide (TAP) and pancreatic oedema in vivo, and L-NAME increased TAP., Conclusions: NO protects against injury caused by pancreatitis in the intact animal but has no discernible effect on isolated acini. It is likely that in pancreatitis NO acts indirectly via microcirculatory changes, including inhibition of leucocyte activation and preservation of capillary perfusion.

Background & Aims: Clinical and experimental observations have associated acute and chronic hypercalcemia with pancreatitis. The aim of this study was to determine whether acute hypercalcemia can induce acute pancreatitis and, if so, whether the pathogenesis involves premature protease activation., Methods: Rats given bolus infusions of CaCl2 (200 mg/kg; n = 76) were compared with saline-treated controls (n = 40). Serum [Ca2+], serum amylase activity, trypsinogen activation peptide (TAP) concentration in serum and pancreatic tissue, pancreatic wet/dry weight ratio, and histology were assessed for 24 hours. For dose-response analysis, CaCl2 was injected at a dose of 50-200 mg/kg, and the aforementioned indices were assayed for 1 hour (n = 5 each)., Results: There were no significant changes in the controls. Calcium infusion increased serum [Ca2+] 3-fold after 5 minutes (P < 0.001). Within 1 hour, serum amylase (2.5-fold) and tissue TAP (3-fold) levels increased along with macroscopic and microscopic edema formation and leukocytic infiltration. The extent of the changes at 1 hour correlated with the calcium dose. Amylase and tissue TAP concentrations remained elevated until 24 hours when serum TAP concentration had increased (P < 0.001) and focal acinar necrosis became evident., Conclusions: Acute experimental hypercalcemia induces dose-dependent morphological alterations characteristic of acute pancreatitis, acute hyperamylasemia, and early ectopic trypsinogen activation. This supports the pathophysiological relevance of excess calcium and offers a possible pathogenetic mechanism for its association with clinical pancreatitis.

Objective: The potential of pancreatic ischemia to cause acute pancreatitis as indicated by morphologic changes and ectopic trypsinogen activation was investigated., Background: Experimental evidence has shown that pancreatic ischemia is important in the evolution of severe pancreatitis, but whether ischemia can initiate pancreatitis has been disputed., Methods: Pancreatic ischemia was induced in rats by hemorrhagic hypotension (30 mm Hg for 30 min; n = 64). Changes of pancreatic microcirculatory perfusion were studied using diffuse reflectance spectroscopy. Serum amylase, trypsinogen activation peptide (TAP) in serum and pancreatic tissue, wet/dry weight ratio, and histology were determined over 24 hours and compared with sham-operated control subjects (n = 35)., Results: In control animals, serum amylase (47.9 +/- 2.1 units/L), serum (7.9 +/- 0.7 nmol/L) and tissue TAP (63.0 +/- 5.4 nmol/L x g), wet/dry weight ratio (2.8 +/- 0.1), and histology remained unchanged. Temporary hypotension markedly decreased pancreatic perfusion with incomplete recovery after reperfusion. Pancreatic isoamylase activity increased within 1 hour (110 +/- 5 units/L, p < 0.05) and further to 151 +/- 18 units/L at 24 hours. Tissue TAP was elevated at 1 hour (134 +/- 16 nmol/L x g, p < 0.05) and increased to 341 +/- 43 nmol/L x g (p < 0.001) after 24 hours, whereas serum TAP remained unchanged (8.3 +/- 0.5 nmol/L). Morphologic alterations included elevated wet/dry weight ratio (4.1 +/- 0.3, p < 0.01) and increased histologic scores for edema (p < 0.05) and acinar necrosis (p < 0.05) at 24 hours. Trypsinogen activation preceded the development of pancreatic necrosis., Conclusions: In addition to its potentiating role, severe pancreatic ischemia can play a pathogenetic role in the initiation of acute pancreatitis.

Objective: To evaluate the factors thought to be involved in the pathogenesis of acute pancreatitis associated with alcohol., Background: The mechanism of alcohol-induced pancreatitis is believed to involve synergistic effects of various pathogenetic factors. The present study was designed to evaluate the possible contribution of pancreatic duct obstruction, physiologic exocrine stimulation, or secretory hyperstimulation to alcohol-induced pancreatic injury., Methods: Wistar rats were allocated randomly to a control group (group 1), or to a group with pancreatic duct obstruction (group 2), physiologic exocrine stimulation (group 3), ductal obstruction and exocrine stimulation (group 4), or exocrine hyperstimulation with the cholecystokinin analogue cerulein (group 5). Three hours after this pretreatment, animals in each experimental group were randomly divided into two subgroups for intragastric administration of either water (groups 1A through 5A) or beer (groups 1B through 5B). Test solutions were instilled over 9 hours (total amount of alcohol administered, 4.8 g/kg). Twenty-four hours after beginning the test infusion, animals were killed for histologic evaluation of pancreatic edema and determination of an acinar cell necrosis score. Serum amylase levels were determined at 3, 9, and 24 hours., Results: No increase in amylase levels or significant morphologic changes were found in control animals (group 1A) or in animals subjected to physiologic exocrine stimulation (group 2A). Pancreatic duct obstruction, with or without physiologic exocrine hyperstimulation (groups 3A and 4A), and exocrine hyperstimulation (group 5A) induced pancreatitis of similar severity with minor acinar cell damage. Alcohol superimposed on exocrine hyperstimulation (group 5B) increased acinar cell injury (group 5A, 0.4 +/- 0.1 points vs 5B, 1.0 +/- 0.2 points; P < .05) and serum amylase levels at 24 hours (group 5a, 41 +/- 6 U/L vs group 5B, 72 +/- 11 U/L; P < .05), whereas no differences between subgroups A and B (water vs beer) were found in groups 1 through 4., Conclusion: Our findings suggest that the pathogenesis of acute alcoholic pancreatitis may require a state of exocrine hyperstimulation, perhaps via cholecystokinin, but do not support a role for constriction or obstruction of Oddi's sphincter.

Background: Bacterial infection of pancreatic necrosis is thought to be a major determinant of outcome in acute necrotizing pancreatitis. The determinants and possibilities for prophylaxis are unknown and difficult to study in humans., Objective: The time course of bacterial infection of the pancreas in a rodent model of acute necrotizing pancreatitis was characterized. The authors ascertained if there is a correlation with the degree of necrosis., Methods: Acute pancreatitis (AP) of graded severity was induced under sterile conditions by an intravenous infusion of cerulein (5 micrograms/kg/hr) for 6 hours (mild AP), or a combination of intravenous cerulein with an intraductal infusion of 10-mM glycodeoxycholic acid (0.2 mL for 2 min for moderate AP, 0.5 mL for 10 min for severe AP). Sham-operated animals (intravenous and intraductal NaCl 0.9%) served as controls. Ninety-six hours after induction, animals were killed for quantitative bacterial examination and histologic scoring of necrosis. In addition, groups of animals with severe AP were investigated at 12, 24, 48, 96, and 144 hours., Results: No significant pancreatic necrosis was found in control animals (0.3 +/- 0.1) or animals with mild AP (0.6 +/- 0.1) killed at 96 hours. Necrosis scores were 1.1 +/- 0.2 for animals with moderate AP and 1.9 +/- 0.2 for animals with severe AP. Control animals did not develop significant bacterial infection of the pancreas (> or = 10(3) CFU/g). At 96 hours, the prevalence of infection was 37.5% in animals with mild AP and 50% in animals with moderate AP. In animals with severe AP, infection of the pancreas increased from 33% in the first 24 hours to 75% between 48 and 96 hours (p < 0.05). The bacterial counts and the number of different species increased with time and was maximal (> 10(11) CFU/g) at 96 hours., Conclusion: Bacterial infection of the pancreas in rodent AP increases during the first several days, and its likelihood correlates with the severity of the disease. This model, which closely mimics the features of human acute pancreatitis, provides a unique opportunity to study the pathogenesis of infected necrosis and test therapeutic strategies.

Background: Inappropriate extraluminal activation of trypsin is assumed to play a part in the pathogenesis of acute pancreatitis (AP), but proof has been elusive because active trypsin is transient and difficult to measure. We have previously shown increased levels of trypsinogen activation peptides (TAP), a direct measure of trypsin activation, to correlate with severity of AP, tissue necrosis, and survival in a rodent model induced by cerulein hyperstimulation and bile salt infusion. The present study seeks to show that increased trypsinogen activation also characterizes three other models of experimental AP in rodents to give credence to the generality of the phenomenon and to its potential relevance to human AP., Methods: Experimental AP was induced in mice by a choline-deficient diet supplemented with ethionine and in rats by creation of a closed duodenal loop or by ligation of the biliopancreatic duct plus physiologic stimulation. TAP were quantified by an immunoassay in tissue and plasma at various time points after onset of AP., Results: In the group with choline-deficient diet supplemented with ethionine a significant increase in tissue and plasma TAP was found at 48 and 72 hours, respectively. In the group with closed duodenal loop significant TAP elevations were found in plasma as early as 6 hours and in the group with ligation of the biliopancreatic duct plus physiologic stimulation at 24 hours., Conclusions: These experiments provide further evidence that extraluminal protease activation is a pathophysiologic event common to the evolution of various models of experimental acute pancreatitis and therefore increase the likelihood that this phenomenon is important in the human disease as well.

Trypsinogen activation peptides (TAP) can be found in patients with acute pancreatitis (AP) as well as in different models of experimental AP. First experience has suggested that early elevation of TAP indicates development of necrotizing AP and that the amount of TAP correlates with the extent of acinar cell necrosis and mortality. It is however unknown whether TAP similarly assess severity of AP in the later course of the disease. The present study monitores TAP in plasma, urine and ascites during the initial development of pancreatic injury and correlates the amount of TAP and the extent of pancreatic necrosis over 48 h in a rodent model of AP. While there was no elevation of TAP in control animals or animals with edemantous AP, significant amounts of TAP im plasma were found as early as 30 min following induction of severe necrotizing AP. Serum amylase returned to normal values within 24 h, TAP maintained at high levels until the end of the 48 h observation period. During the first 24 h TAP paralleled the development of acinar cell necrosis, but decreased thereafter despite further progression of pancreatic injury. Our results provide further evidence suggesting that TAP initially precede morphological changes in AP. Early in the course of the disease TAP parallel development of pancreatic injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Background/aims: Contrast-enhanced computed tomography (CECT) is used to show areas of decreased pancreatic perfusion in severe acute pancreatitis (AP). To evaluate possible adverse effects of the contrast medium (CM) on the course of AP, the impact of intravenous CM in AP of graded severity in the rat was studied., Methods: Pancreatitis of three levels of severity was induced in Sprague-Dawley rats with intravenous cerulein hyperstimulation plus time- and pressure-controlled intraductal infusion of saline or glycodeoxycholic acid. At 7 hours, control and pancreatitis animals received intravenous ionic CM, nonionic CM, or saline. The principal outcome measures were 24-hour survival, trypsinogen activation peptides (TAP) in ascites, and histological acinar necrosis score., Results: There was no measurable effect of CM on the index features in control animals or animals with mild or moderate AP. In severe AP, CM caused a significant increase in mortality, ascites TAP, and necrosis score., Conclusions: Intravenous CM increases pancreatic injury when administered early in the course of severe experimental AP. Because CM may convert borderline ischemia to irreversible necrosis, CECT performed early in pancreatitis to show poor perfusion and predict areas of necrosis may depict a self-fulfilling prophecy. Early CECT should be reconsidered and perhaps avoided.

Acinar necrosis in patients with acute pancreatitis can be due to enzymatic injury, ischemia, or both. We hypothesized that novel therapy aimed at an improvement of pancreatic microcirculation early in the course of pancreatitis may reduce the lethality and acinar damage. Forty-six dextran-resistant rats received controlled intraductal infusion of glycodeoxycholic acid (10 mmol/L), followed by intravenous cerulein (5 micrograms/kg/h) for 6 hours. Beginning 30 minutes after the induction of pancreatitis, all animals were resuscitated with Ringer's lactate (RL) (8 mL/kg/h intravenously for 9 hours). In addition, they were given intra-aortic bolus infusions (2 mL/kg at 30, 60, 90, and 150 minutes) of either RL, sodium chloride (NaCl) (7.5%) and dextran 60,000 (10%) (HHS-60), NaCl (7.5%) and dextran 500,000 (10%) (HHS-500), or NaCl (0.9%) and dextran 500,000 (10%) (DEX-500). Despite high-volume fluid resuscitation in the groups that received RL and HHS-60, 70% of the animals in each of these groups died within 24 hours. In contrast, the mortality rates in the groups of animals that received HHS-500 and DEX-500 were dramatically reduced to 0% and 10%, respectively (p = 0.005, p = 0.02). Histopathologic scores for acinar necrosis were significantly lower in the group of animals that received DEX-500 (p < 0.009) compared with those that received RL and HHS-60. Finally, total amounts of trypsinogen activation peptides in ascites were significantly lower in the animals that received HHS-500 (p < 0.004) and DEX-500 (p < 0.02) compared with those that received RL and HHS-60. Rapid bolus infusion of hyperoncotic ultrahigh molecular weight dextran solution with or without hypertonic saline but not RL or hypertonic-hyperoncotic saline-dextran significantly reduced pathologic trypsinogen activation, prevented acinar necrosis, and improved survival in acute experimental pancreatitis. We speculate that a sustained improvement of pancreatic microcirculation by ultrahigh molecular weight dextran is the mechanism of action.