Pancreatic Duct Ligation Research Articles

Effects of pancreatic duct ligation and aging on acute taurocholate-induced pancreatitis

When taurocholate was injected into the common bile duct, high ductal pressure due to ligation of the pancreatic duct did not produce more damage in the pancreas of both old rats and young adult rats, and levels of pancreatic enzymes in portal venous effluent were lower in old rats than in younger rats. The effects of ligation of the pancreatic duct and aging on acute pancreatitis caused by taurocholate are still unclear. Young adult and old male Wistar rats were used. Six hours after ligation of the common bile duct in both the duodenum and liver hilus, rats were killed and the pancreata were perfused. Taurocholate or normal saline was injected retrogradely into the common bile duct. The levels of amylase and lipase in the portal venous effluent were determined as markers of damage to the pancreas. The pancreas was also histologically examined after the perfusion experiments using an Image Analysis System. (1) A nonsignificant elevation of pancreatic enzymes was found in portal venous effluent by the retrograde injection of saline into the common bile duct. Injection of taurocholate caused a marked elevation of enzymes in the effluent for the first 30 min after injection, which then gradually decreased. (2) Basal levels of pancreatic enzymes were significantly higher in the ligation group than in the nonligation group. Injection of saline into the common bile duct had no apparent effect on enzymes in the effluent. In contrast, taurocholate injection into the common bile duct produced a marked increase in enzymes in the portal venous effluent. However, no significant difference was found between the ligation group and the nonligation group. (3) Similar findings were obtained when old rats were used. (4) Although basal levels of enzymes were almost the same in nonligated old and young adults rats, taurocholate injection into the pancreatic duct in old rats resulted in a significant depression of enzymes compared to that in young adult rats. In the ligation group, pancreatic enzymes in the portal venous effluent following taurocholate injection tended to be lower in old rats than in young adult rats. The results were histologically supported in that various degrees of fibrosis were found in the pancreata of old rats.

Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat

BACKGROUND & AIMS: Mechanisms of cell death in human and experimental pancreatitis remain poorly understood. The aim of this study was to determine the mechanisms of cell death, apoptosis vs. necrosis, in models of pancreatitis induced by pancreatic duct ligation in the opossum and rat. METHODS: Morphological changes were shown by light and electron microscopy, chromatin condensation and DNA breaks were assessed using Hoechst 33258 staining and DNA nickend labeling, and DNA fragmentation was characterized b gel electrophoresis. RESULTS: Cells with morphology of both necrosis and apoptosis were found in pancreata from duct-ligated animals; however, the apoptosis-necrosis ratio was different for the opossum and the rat. Both apoptotic and necrotic cells stained for breaks in DNA, suggesting that both apoptosis and necrosis are associated with DNA fragmentation and that DNA end labeling cannot specifically detect apoptotic cells. No significant oligonucleosomal DNA fragmentation was found in pancreas from duct- ligated animals, although the endonuclease responsible for oligonucleosomal DNA fragmentation was present in pancreatic nuclei. CONCLUSIONS: Duct ligation resulted predominantly in apoptosis in the rat and necrosis in the opossum. The differences in types of cell death in the two models may result from differences in inflammatory cell infiltration. (Gastroenterology 1996 Mar;110(3):875-84)

Ligation-Induced Acute Pancreatitis Increases Pancreatic and Circulating Trypsinogen Activation Peptides

Ligation of the common bile–pancreatic duct induces hyperamylasemia and acute pancreatitis in rats. Pancreatic morphologic changes include edema, acinar cell damage, and mild inflammation. The pathogenesis of acute pancreatitis in this model is not understood, but may involve altered secretion and intrapancreatic activation of acinar proteases. We hypothesized that trypsinogen activation, measured by the production of plasma and pancreatic trypsinogen activation peptides (TAP), occurs early in this model. We performed the following experiments: rats were prepared with (1) bile–pancreatic ducts ligated and (2) ducts dissected but not ligated (sham). Rats were killed after 6, 24, and 48 hr. Serum amylase was measured and histologic sections were analyzed for morphologic changes. TAP was measured in both serum and pancreatic tissue homogenates using a specific polyclonal anti-TAP antibody in an enzyme-linked immunosorbant assay. After 6, 24, and 48 hr of bile–pancreatic duct ligation, hyperamylasemia and acute morphologic changes of acute pancreatitis were observed. Evidence of acinar cell destruction was not evident until 48 hr after ligation. Levels of serum and pancreatic tissue TAP were significantly elevated at both 24 and 48 hr after ligation compared to those of sham. We conclude that increased intrapancreatic trypsinogen activation occurs early in this form of experimental acute pancreatitis and that it occurs prior to evidence of acinar cell destruction. These data and observations support the possibility that intrapancreatic protease activation contributes to the pathogenesis of ligation-induced acute pancreatitis.

Bile-pancreatic juice replacement not cholinergic-and cholecystokinin-receptor blockade reverses acinar cell hyperstimulation after bile-pancreatic duct ligation

Acinar cell inhibitors (eg, atropine) fail to ameliorate clinical and experimental acute pancreatitis. We hypothesized that amelioration of pancreatic acinar cell hyperstimulation after bile and pancreatic duct ligation is better with gut replacement of bile and pancreatic juice than with cholinergic- and cholecystokinin (CCK)-receptor blockade. Using acinar cell amylase activity as an index of hyperstimulation, we studied 63 rats in two sets of experiments. Bile-pancreatic juice exclusion from gut--without (set one) and with (set two) bile and pancreatic duct obstruction--was treated with atropine and CCK-receptor antagonist L-364,718, or with enteral replacement of bile-pancreatic juice. In the set one experiment, acinar cell hyperstimulation after bile-pancreatic juice exclusion was reversed by combined L-364,718 and atropine pretreatment. In set two, acinar cell hyperstimulation after bile and pancreatic duct ligation was reversed by enteral bile and pancreatic juice replacement, but not by combined L-364,718 and atropine pretreatment. According to this experimental corollary of early gallstone impaction, prevention of acinar cell hyperstimulation after duct occlusion should be aimed at the source of the response to bile-pancreatic juice exclusion, namely, the gut, rather than at the target of the response, the pancreatic acinar cell.

Enterostatin in gut endocrine cells--immunocytochemical evidence.

The presence of enterostatin, a pentapeptide acting as a potential satiety signal in rats, was investigated in rat intestine by immunocytochemical methods. Using antibodies directed against the C-terminal part of enterostatin, the peptide was identified in endocrine cells in the antral part of the stomach and in the small intestine of rat. The immunoreactive cells were more frequent in the antrum and duodenum and became gradually fewer towards the distal small intestine. In some of the labeled endocrine cells, a coexistence of enterostatin with serotonin was revealed by immunocytochemical double staining, implying that the cells were enterochromaffin cells. In the pancreas, no enterostatin-immunoreactive cells were detected, indicating enterostatin to be included in its parent molecule, procolipase. In addition, the existence of procolipase in the gastrointestinal tract, including the pancreas, was investigated. Procolipase immunoreactivity was also identified, except in the pancreas, in chief cells in the fundus region of the stomach. The number of labeled cells declined distally in the stomach, finally being absent in the intestine. Immunoreactive enterostatin was measured with a specific ELISA method. Intestinal content and serum were found to average 540 +/- 70 and 50 +/- 4 nM, respectively. Pancreatic duct ligation strongly reduced the levels of enterostatin in intestinal content to 5.4 +/- 1.5 nM (p < 0.001), and also reduced the serum enterostatin level to 35 +/- 5 nM (p < 0.05). It is concluded that the peptide enterostatin in the rat is produced both in the exocrine pancreas, as part of pancreatic procolipase, and in gut endocrine cells, both sources of peptide being important for the circulating enterostatin.

Apoptosis of acinar cells in rat pancreatic duct ligation.

Apoptosis of acinar cells in rat pancreatic duct ligation.

Effect of kinin inhibition in experimental acute pancreatitis.

Activation of the endogenous kinin system is a consistent observation in acute pancreatitis and has repeatedly been implicated in the pathophysiology of the disease. We have studied the effect of a potent bradykinin antagonist on the onset and development of acute pancreatitis in four unrelated animal models. Pancreatitis was induced in rats by either supramaximal stimulation with caerulein, intraductal injection of sodium taurocholate, or pancreatic duct ligation with secretin infusion, and in mice by feeding a choline-deficient, ethionine-supplemented diet. The potent, long-acting bradykinin antagonist HOE-140 was administered subcutaneously (0.1 mg/kg every 5 h). Effective kinin inhibition had no effect on pancreatitis-associated mortality, the extent of morphological damage and inflammation, or the intracellular distribution of lysosomal hydrolases. Pancreatic edema was only reduced in caerulein-induced pancreatitis, the only model in which edema formation was paralleled by increased vascular permeability. We conclude that, contrary to previous suggestions, kinins do not play a predominant role in the development of acute pancreatitis. Their participation is strictly limited to vascular events and does not involve the early cell biological alterations in pancreatic acinar cells.

Luminal endocytosis and intracellular targeting by acinar cells during early biliary pancreatitis in the opossum.

Cell necrosis in acute experimental pancreatitis is preceded by a redistribution of digestive enzymes into a lysosomal subcellular compartment. We have investigated whether endocytosis from the acinar cell lumen might contribute to this disturbance of intracellular compartmentation. In an animal model of pancreatitis involving pancreatic bile duct ligation in opossums, we have studied in vivo endocytosis of dextran 40 and [14C]dextran 70, cationized ferritin, and horseradish peroxidase from the apical surface of acinar cells before the onset of necrosis. Marker solutions were instilled into the pancreatic duct of anesthetized animals at physiological pressure. Tissue samples obtained at intervals of up to 60 min after instillation of markers were studied by electron microscopy and electron microscope autoradiography. All markers were taken up by acinar cells in control animals and in animals with obstructed pancreatic bile ducts. Markers for membrane-mediated endocytosis (cationated ferritin and horseradish peroxidase) were transported to lysosomes in both groups. In contrast, the fluid-phase tracer dextran was transported to the secretory pathway in controls but to lysosomes after duct ligation. Since dextran and luminally present secretory proteins can be expected to follow the same route after endocytosis, our findings suggest that altered intracellular targeting of endocytosed proteases might be one mechanism by which digestive zymogens reach an intracellular compartment in which premature activation can occur. This phenomenon may be a critical and early event in the pathogenesis of biliary pancreatitis.

Apoptosis is the dominant form of acinar cell death after pancreatic duct ligation in rats

Apoptosis is the dominant form of acinar cell death after pancreatic duct ligation in rats

Changes in the mouse exocrine pancreas after pancreatic duct ligation: a qualitative and quantitative histological study.

The pancreatic duct from the splenic lobe, the largest lobe of the pancreas in the mouse, was ligated at 6 weeks of age, with histological and cytological changes in the organ examined 1 day to 16 weeks after the ligation. Changes in the volumes of the pancreatic lobe, exocrine tissue, and interstitial tissue as well as relative total numbers of each cellular element in the organ after duct ligation were stereologically obtained using serial sections of the whole pancreas. Cell sizes, degenerated cell and mitotic cell indices, and nuclear densities of the acinar and ductal cells were also obtained. After duct ligation, the volume of the pancreas increased by interstitial edema in the first 2 days but rapidly decreased thereafter due to atrophy of the exocrine tissue, amounting to 10% or less of normal volume by 7 days. The acinar cells showed an accumulation of the zymogen granules, cytoplasmic condensation and a pyknotic figure of the nucleus; they then were thoroughly deleted with appearance of numerous macrophages. This cell death was suggested to be due to apoptosis. On the other hand, the ductal cells remained in the atrophic pancreas and proliferated with mitotic figures to two times the normal frequency at 3 days, and then formed duct-like structures lacking in the acinar cells. After 2 week, the ductal cells slowly decreased in number also due to cell death, but the pancreas became gradually enlarged by intralobular fatty replacement, to reach a volume approximating that of normal 8 weeks after duct ligation. The stereological method serves for the correct evaluation of cell dynamics including the deletion and proliferation of the cells in the whole organ.

Ligation-Induced Acute Pancreatitis in Opossums: Acinar Cell Necrosis in the Absence of Colocalization

Acute necrotizing pancreatitis in opossums after bile and pancreatic duct ligation (BPDL) is a useful experimental corollary of gallstone-induced acute pancreatitis in humans. In experimental and human acute pancreatitis, a loss of segregation of the lysosomal enzyme cathepsin B and the zymogen proenzyme trypsinogen (colocalization) is implicated as the triggering event of disease pathogenesis, as cathepsin B can activate trypsinogen. The object of this study was to quantitate acinar cell necrosis and to study subcellular distribution of cathepsin B in BPDL-induced acute necrotizing pancreatitis in opossums. Bile and pancreatic ducts were ligated separately (no bile reflux) in four opossums, while ducts were dissected in four sham controls. Opossums were killed 24 hr after operation. Three equidistant cross-sectional portions of each opossum pancreas were submitted to histologic examination. In blinded fashion, each focus of acinar cell necrosis was photographed and quantitated with digitizing morphometry. Numerical density (foci/cm2) and areal density (×103 μm2/cm2) of focal acinar cell necrosis were determined. Differentially centrifuged pancreatic homogenates were assayed for cathepsin B, the lysosomal marker enzyme N -acetylglucosaminidase, and amylase. Morphometric quantitation of acinar cell necrosis confirmed development of acute necrotizing pancreatitis after 24 hr of BPDL in opossums. However, colocalization was not observed after BPDL, as evidenced by an absence of subcellular shift of cathepsin B activity (and N-acetyl-glucosaminidase activity) from the lysosome-enriched to the zymogen-enriched subcellular fraction. Amylase activity was increased in subcellular fractions after BPDL. In this experimental corollary of early gallstone-induced acute pancreatitis, colocalization is not the triggering event of acute necrotizing pancreatitis pathogenesis, as acinar cell necrosis occurs even in the absence of colocalization.

Apoptosis of mouse pancreatic acinar cells after duct ligation.

It has been established that pancreatic exocrine acinar cells disappear after pancreatic duct obstruction. This study aimed to examine the relationship between the disappearance of the acinar cells and apoptosis after pancreatic duct ligation of the splenic lobe in dd-mice, six weeks of age. In some mice, the ligature was removed after two or three days. In addition to general light and electron microscopic examinations on the pancreatic tissues, paraffin sections stained with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method were observed to detect nuclear DNA fragmentation. Pancreatic acinar cells underwent apoptosis initiated with nuclear DNA damages three days after duct ligation and were completely deleted by seven days. Due to the elevation of the intraluminal pressure, the acinar cells showed interrupted secretion of their zymogen granules and disorganization of their rough endoplasmic reticulum, causing the cessation of granule formation before apoptosis started. These cytoplasmic changes prior to apoptosis are reversible, as observed after removal of the ligation. Apoptosis of the acinar cells was indentified by TUNEL-labeling of the nuclei, the condensation and margination of nuclear chromatin, and round fragmentation of cell bodies, all irreversible changes. Apoptosis of acinar cells seemed to stimulate the proliferation of duct cells, which comprised the main cell components in the exocrine pancreas after the disappearance of acinar cells.

Effect of protein derivatives on pancreatic secretion and release of secretin and CCK in rats.

We investigated the effect of intraduodenal administration of oligopeptide and a mixed amino acid solution, which contains the same amino acid composition as oligopeptide, on pancreatic exocrine secretion and the release of secretin and cholecystokinin (CCK). Anesthetized rats were prepared with pyloric ligation and cannulation of pancreatic duct and bile duct. Protein derivatives in three different doses (oligopeptide: 25, 100, and 400 mg/h; and mixed amino acid solution: 70, 140, and 280 mg/h, pH 7.0) were infused into the duodenum for 1 h. Pancreatic juice was collected, and plasma concentrations of secretin and CCK were measured by radioimmunoassay. In addition, the effect of intravenous injection of an antisecretin serum or a CCK antagonist, loxiglumide, on pancreatic secretion stimulated by oligopeptide or mixed amino acid solution was also studied. Oligopeptide produced a significant dose-related increase in pancreatic secretion including volume, HCO3-, amylase, and trypsin output, plasma secretin (r = 0.792, P < 0.001), and plasma CCK (r = 0.421, P < 0.01). Similarly, mixed amino acid solution produced a dose-related increase in pancreatic juice volume, HCO3-, amylase, and trypsin output. Compared with CCK, the percentage increase in plasma secretin was 7.3x and 2.8x higher in response to oligopeptide (400 mg/h) and mixed amino acid solution (280 mg/h), respectively. An antisecretin serum almost completely inhibited volume flow and HCO3- output stimulated by oligopeptide as well as mixed amino acid solution, but not amylase and trypsin output. In contrast, loxiglumide significantly suppressed amylase and trypsin output stimulated by protein derivatives, but did not affect volume flow or HCO3- output.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of cholecystokinin receptor antagonist (CR1505) on recovery of experimental pancreatitis after pancreatic duct occlusion in rats.

The effect of long term administration of a synthetic cholecystokinin (CCK) receptor antagonist CR1505 (loxiglumide) on pancreatitis was examined in rats after pancreatic duct ligation (PL) with an internal bile fistula. All rats were given both 6 mg/day of CR1505 continuously infused intraduodenally with an osmotic pump and 32 mg/day introduced into the stomach with an orogastric tube. Rats were killed 7, 14, and 28 days after PL, and changes of body weight, pancreatic wet weight, daily food intake, pancreatic protein content, and histology, plasma amylase concentration, and both plasma and duodenal CCK concentrations were examined. Administration of CR1505 for 7 days from immediately after PL, resulted in decrease of body weight, increase of daily food intake, and significant increases of intestinal CCK concentration and the level of CCK mRNA. However, its administration from day 7 to day 14 or 28 did not improve any of the parameters examined except inflammatory infiltration of the pancreas on the 14th postoperative day. These results suggest that CR1505 may have a beneficial effect on recovery from pancreatitis when administered during the early stage, but not when administered during a later stage.

Colonic proteolysis following pancreatic duct ligation in the rat.

Luminal proteolytic activity (PA) of different colonic segments was ascertained in animals subjected to pancreatic duct ligation (PDL) and in control rats. The PDL rats revealed a significant PA reduction in the cecum, proximal colon (P < 0.01), and distal colon (P < 0.005). Proteolytic activity, trypsin, and chymotrypsin activity in control rats diminished progressively from the cecum to the distal colon. Conversely in PDL rats, we found maximal PA in distal colon. The conclusion is drawn that a significant proportion of colonic proteolytic activity can be attributed to pancreatic proteases with a maximal contribution at cecum level.

Ligation-Induced Acute Pancreatitis in Rats and Opossums: A Comparative Morphologic Study of the Early Phase

Bile and pancreatic duct ligation (BPDL) in rats does not induce severe acute pancreatitis but only mild inflammation, which is self-limiting and eventually leads to pancreatic atrophy. However, BPDL in opossums induces severe acute necrotizing pancreatitis which uniformly leads to death within 14 days. We compared pancreatic morphologic changes after 24 hr of BPDL in rats and opossums. Pancreatitis histology score and acinar cell ultrastructural changes were evaluated. In both species, BPDL was associated with significant increases in histology score compared to sham controls (5.0 ± 0.3 vs 1.5 ± 0.3 in rats, 5.3 ± 0.4 vs 1.1 ± 0.1 in opossums; mean ± SEM, ANOVA, P < 0.05). However, there was no significant difference in histology score between rats and opossums following BPDL; histologic changes, such as white blood cell infiltration, acinar cell vacuolation, and focal acinar cell necrosis, were similar. Acinar cell ultrastructural changes after BPDL in both species included dilated endoplasmic reticulum and autophagic vacuole formation. These findings indicate that the early morphologic changes after BPDL in rats are quite similar to those seen early in the course of BPDL-induced acute necrotizing pancreatitis in opossums. As the rat is a more economical and convenient model to study than the opossum, this study supports the use of the rat model to conduct pilot studies of early events in the development of BPDL-induced acute pancreatitis. This study also suggests the potential for investigating mechanisms that may be present in the rat which protect against progressive and fatal acute necrotizing pancreatitis as observed in opossums after longer periods of BPDL.

Gut ischaemia.

Intestinal mucosal injury that results from local ischaemia can be detected by early increases in gut permeability, followed by later morphological, histological, and biochemical abnormalities. Local adaptive mechanisms (for example increased oxygen extraction) can cope with reductions in blood flow of up to 50%, as may occur during episodes of septic shock or cardiac tamponade. Why then does hypoxic injury develop? The peculiar vascular anatomy of the villi allows for oxygen short circuiting to occur at their base, when blood flow is low. Although overall oxygen extraction efficiency may be high, regional hypoxia at the villus tip may, paradoxically occur. The severity of reperfusion injury depends on the duration of preceding hypoxia. Free radical generation through the hypoxanthine xanthine oxidase system is important in mediating cellular damage. In addition, luminal aggressive factors (for example, pancreatic proteases) may cause mucosal damage, as suggested by earlier studies. More recent studies in pigs suggests that pancreatic duct ligation merely delays, but does not prevent development of gut reperfusion injury. Enteral nutrition should benefit patients with the ischaemic intestine because in comparison with total parental nutrition, it stimulates regional blood flow, and attenuates mucosal injury. There are no randomised trials to verify this, but use of tonometry to monitor local ischaemia may help resolve the issue.

In situ kinetics of acinar, duct, and inflammatory cells in duct ligation-lnduced pancreatitis in rats

Background:Acute pancreatitis remains a disease of uncertain pathogenesis. The resolution of the cellular events that underlie the development of pancreatitis may allow for significant advances in therapy. Methods: A new experimental approach to acute pancreatitis in the rat, i.e., selective ligation of pancreatic ducts and immunohistochemical analysis of cellular kinetics using a panel of monoclonal antibodies, was devised. Results: Immunostaining showed that the majority of the infiltrating cells bore macrophage-specific antigens (ED1-positive). Some neutrophils were also among the cellular infiltrate, but only a few T (OX19-positive) or B (His14-positive) lymphocytes were seen. At a later stage, more than half of the mononuclear cells in the stroma were neither macrophages nor lymphocytes, but possibly fibroblasts. In vivo labeling of proliferating cells with 5-bromo-2′-deoxyuridine and immunostaining showed active proliferation of duct epithelial and wall cells of the interlobular pancreatic duct on days 1 and 2, proliferation of nonacinar cells on days 1–4, and proliferation of acinar cells on days 2–28. Conclusions: These results indicate an important role of macrophages and the presence of complex processes of degeneration and regeneration in duct ligation-induced pancreatitis.

A PROSPECTIVE STUDY OF THE USEFULNESS OF FIBRIN GLUE TO PREVENT POSTOPERATIVE PANCREATIC FISTULA

The clinical courses of 37 consecutive patients undergone distal pancreatectomy between January 1990 and December 1991 were prospectively studied for the usefulness of fibrin glue to prevent postoperative pancreatic fistula. The 37 cases were allocated to fibrin glue used group (18 cases) and without fibrin glue group (19). There was no significant difference in background factors between the both groups. In all patients pancreatic ducts were ligated with a 3-0 silk thread and the stumps of the pancreas were sutured continuously with a 3-0 prolene thread. Pancreatic fistula occurred postoperatively in 13 out of 37 patients (35.1%). Ten pancreatic fistulas (52.6%) occurred among 19 patients without fibrin glue, versus 3 pancreatic fistulas (16.7%) among 18 patients with fibrin glue, with a significant difference (p<0.05). These results suggest that, in patients undergone distal pancreatectomy, application of fibrin glue to the stumps of the pancreas after ligation of pancreatic duct might be useful to prevent from postoperative pencreatic fistula.

How and where does acute pancreatitis begin?

Circumstantial evidence suggests that gallstone-induced pancreatitis is triggered by obstruction of the pancreatic duct. In this report I will review the results of studies conducted during the last decade that have employed the diet-induced, secretagogue-induced, and duct obstruction-induced models of experimental pancreatitis to investigate the early events that lead to the development of acute pancreatitis. In each of these models, digestive enzyme zymogens and the lysosomal hydrolase cathepsin B were found to become colocalized. These observations have led to the hypothesis that intra-acinar cell activation of digestive enzyme zymogens by lysosomal hydrolases may be an important critical event in the development of acute pancreatitis. Recent morphologic studies evaluating the initial 24 hours after ligation of the opossum pancreatic duct indicate that the earliest lesions in this model of hemorrhagic pancreatitis occur in acinar cells.