Article
Changes in gene expression of tumor necrosis factor alpha and interleukin 6 in a canine model of caerulein-induced pancreatitis Ruhui Song, Dohyeon Yu, Jinho Park
Abstract Acute pancreatitis is an inflammatory process that frequently involves peripancreatic tissues and remote organ systems. It has high morbidity and mortality rates in both human and veterinary patients. The severity of pancreatitis is generally determined by events that occur after acinar cell injury in the pancreas, resulting in elevated levels of various proinflammatory mediators, such as interleukin (IL) 1b and 6, as well as tumor necrosis factor alpha (TNF-a). When these mediators are excessively released into the systemic circulation, severe pancreatitis occurs with systemic complications. This pathophysiological process is similar to that of sepsis; thus, there are many striking clinical similarities between patients with septic shock and those with severe acute pancreatitis. We induced acute pancreatitis using caerulein in dogs and measured the change in the gene expression of proinflammatory cytokines. The levels of TNF-a and IL-6 mRNA peaked at 3 h, at twice the baseline levels, and the serum concentrations of amylase and lipase also increased. Histopathological examination revealed severe hyperemia of the pancreas and hyperemia in the duodenal villi and the hepatic sinusoid. Thus, pancreatitis can be considered an appropriate model to better understand the development of naturally occurring sepsis and to assist in the effective treatment and management of septic patients.
Résumé La pancréatite aigüe est un processus inflammatoire qui implique fréquemment les tissus péri-pancréatiques et des systèmes organiques éloignés. Elle a des taux de morbidité et de mortalité élevés autant chez les humains que chez les animaux. La sévérité de la pancréatite est généralement déterminée par des évènements qui se produisent suite à des dommages aux cellules acinaires dans le pancréas, et qui induisent des niveaux élevés de différents médiateurs pro-inflammatoires, tels que l’interleukine (IL) 1b et 6, ainsi que le facteur nécrosant des tumeurs alpha (TNFa). Lorsque ces médiateurs sont libérés de manière excessive dans la circulation systémique, une pancréatite sévère se produit avec des complications systémiques. Ce processus pathophysiologique est similaire à celui d’un sepsis; donc, il y a plusieurs similarités cliniques entre des patients avec un choc septique et ceux avec une pancréatite aigüe sévère. Nous avons induit une pancréatite aigüe en utilisant de la caeruléine chez des chiens et avons mesuré le changement dans l’expression des gènes des cytokines pro-inflammatoires. Les niveaux d’ARNm de TNFa et d’IL-6 ont culminé après 3 h, atteignant le double des niveaux de base, et les concentrations sériques d’amylase et de lipase augmentèrent également. Un examen histopathologique a révélé une hyperémie sévère du pancréas et une hyperémie dans les villosités duodénales et les sinusoïdes hépatiques. Ainsi, la pancréatite peut être considérée un modèle approprié pour mieux comprendre le développement d’un sepsis naturel et aider dans le traitement efficace et la gestion de patients septiques. (Traduit par Docteur Serge Messier)
Introduction Acute pancreatitis is an inflammatory process of the pancreas that frequently involves peripancreatic tissues and remote organ systems (1) and has high morbidity and mortality rates in both human and veterinary patients. The mortality rate for humans with acute pancreatitis has been reported as just under 10% (2,3), but in severe cases it is as high as 20% to 30% (4,5). The mortality rate for dogs with acute pancreatitis ranges from 27% to 58% (6–8). The pathophysiological process of acute pancreatitis consists of activation of pancreatic enzymes within acinar cells, release of these
enzymes into the interstitium, autodigestion of the pancreas, and release of the enzymes and other factors into the circulation, which results in multiple organ dysfunction (9–13). Dogs with acute pancreatitis generally present with a sudden onset of anorexia, depression, abdominal pain, and vomiting (14). However, the findings on clinical examination vary considerably with the severity and stage of the pancreatitis and the degrees of associated dehydration and shock (8). Mild acute pancreatitis does not cause multisystem organ failure or a complicated recovery, whereas severe acute pancreatitis causes multisystem organ failure or development of severe complications (1). The severity of
Department of Veterinary Internal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 54896, Korea (Song, Park); Department of Veterinary Laboratory Medicine, College of Veterinary Medicine, Chonnam National University, Gwangju, 61186, Korea (Yu). Ruhui Song and Dohyeon Yu contributed equally to this work. Address all correspondence to Dr. Jinho Park; telephone: 182-63-850-0949; fax: 182-63-850-0910; e-mail: [email protected] Received August 28, 2015. Accepted March 1, 2016. 236
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pancreatitis is generally determined by the events that occur after acinar cell injury, when various proinflammatory mediators, such as interleukin (IL) 1b and 6, as well as tumor necrosis factor alpha (TNF-a), are produced (15,16). When these mediators are excessively released into the systemic circulation, severe pancreatitis occurs, with systemic complications. This pathophysiological process is similar to that of sepsis; thus, there are many striking clinical similarities between patients with septic shock and those with severe acute pancreatitis (17–19). In the present study, we used caerulein to induce acute pancreatitis in dogs. We examined the pancreas and adjacent organs histopathologically, measured the serum amylase and lipase levels and the gene expression of proinflammatory mediators, and evaluated the suitability of this pancreatitis model as a model of septic shock.
Materials and methods
Figure 2. Gross features of the canine abdominal cavity. Redness of the pancreas (white arrows) represents caerulein-induced hyperemia.
Animals Six healthy adult beagles weighing 7 to 8 kg each were hospitalized and fasted for 12 h before the study; they were provided with water during the fasting period. Eight hours after the first infusion of caerulein the dogs were fed a commercial diet and provided with tap water. The study was approved by the Committee on Bioethics of Chonbuk National University, Jeonju, Korea.
Induction of acute pancreatitis Four dogs received caerulein (Sigma–Aldrich, St. Louis, Missouri, USA) twice intravenously at a dose of 10 mg/kg body weight (BW) (20,21), with a 1-hour interval between infusions. The other 2 dogs were used as controls and were given 2 intravenous infusions, 1 h apart, of normal saline at a dose of 1 mL/kg BW. Body temperature, pulse rate, and respiratory rate were measured before the start of the injections and at 3, 6, 12, 24, and 48 h after the start. The dogs were euthanized at the end of the examination period, and necropsy was done immediately.
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Figure 3. Severe hyperemia of the pancreas in a dog exposed to caerulein. Necrosis of the pancreatic acinar cells was not observed. Hematoxylin and eosin (H&E); 3100.
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Figure 4. Severe hyperemia in the blood vessels of the duodenal villi in a dog exposed to caerulein. H&E; 3100.
Figure 5. Hyperemia in the hepatic sinusoid in a dog exposed to caerulein. H&E; 3100.
Sample collection
trophotometer (Epoch; Bio-Tek Instruments, Winooski, Vermont, USA). A 260:280 nm absorbance ratio of 1.8:2.0 was regarded as indicating pure RNA. Using the manufacturer’s protocol, we synthesized cDNA with the ImProm-II Reverse Transcription System (Promega) and stored it at −20°C until needed. Real-time polymerase chain reaction (RT-PCR) was done with gene-specific primers for canine TNF-a and IL-6. The 20-mL reaction solution contained 300 nM of each primer, 1 mL of cDNA, and 10 mL of 23 iQ SYBR Green Supermix (Bio-Rad, Hercules, California, USA). The CFX384 RT-PCR detection system (Bio-Rad) was used to quantify cytokine mRNA in the PBMCs. Samples were heated at 95°C for 3 min for i-Taq DNA polymerase activation, and then they underwent 40 cycles of denaturation at 95°C for 15 s, annealing for 15 s, and extension at 72°C for 15 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. All samples, controls, and standards were run in duplicate. Data collection and analysis were done with CFX Manager Software, version 1.0 (Bio-Rad). Relative quantification was analyzed by the 2−DDCt method. Sample values were averaged and calculated in relation to the quantity of GAPDH. These normalized values were used to calculate the expression of a given sequence relative to the control.
Blood (6 mL per dog divided into each of the 2 types of tube) was collected from the jugular vein into tubes treated with potassium ethylene diamine tetraacetic acid and plastic vacuum-filled tubes at time 0 (baseline; before the first infusion) and 3, 6, 12, 24, and 48 h after the first infusion. Serum was immediately separated by centrifugation of the blood at 875 3 g for 5 min and kept frozen at −70°C until needed.
Hematologic and biochemical analyses A complete blood (cell) count was done at each time point by means of an automatic impedance cell counter. The serum levels of alkaline phosphatase, alanine transaminase, amylase, lipase, cholesterol, urea nitrogen, creatinine, glucose, phosphate, bilirubin, total protein, and albumin were measured.
Separation of canine peripheral blood mononuclear cells (PBMCs) Centrifugation with Histopaque 1077 (Sigma–Aldrich) at 450 3 g for 45 min was used to isolate the PBMCs. Erythrocytes were lysed with an 83% ammonium chloride solution (pH 7.2) and washed twice with phosphate-buffered saline. Cells were counted manually with use of the trypan blue viability test. Cell purity was determined by a conventional Diff-Quik method after Cytospin centrifugation (Shandon Cytospin, Thermo Scientific, Waltham, Massachusetts, USA) at 250 3 g for 5 min. Viability was always more than 90%, and PBMCs constituted 95% to 98% of the cells.
Quantification of cytokine gene expression Isolated PBMCs were immediately placed in Buffer RLT (Qiagen, Hilden, Germany) and stored at −70°C. Total RNA was isolated with use of the RNeasy Mini Kit (Qiagen) with Qiacube (Qiagen). All RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, Wisconsin, USA) to remove any genomic DNA. The RNA concentration and purity of all samples were measured with a spec-
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Statistical analysis The results are expressed as mean 6 standard deviation (SD) or mean 6 standard error (SE). The significance of differences was evaluated by means of Student’s t-test. A P-value of less than 0.05 was accepted as statistically significant. The statistical analyses were done with the use of SPSS software, version 18.0 (SPSS, Chicago, Illinois, USA).
Results Within 1 h after the first dose of caerulein the dogs exhibited slight weakness and began having diarrhea. However, vomiting was not observed during the experiment. There were no significant changes in body temperature, heart rate, respiratory rate, or hematologic
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Figure 6. Gene expression of tumor necrosis factor alpha (TNF-a) (a) and interleukin (IL)-6 (b) in the 4 dogs exposed to caerulein (triangles) and the 2 control dogs (circles). The mRNA expression peaked 3 h after the first infusion of caerulein. Data presented as mean 6 standard error. *P , 0.05 compared with the control mean.
values within the study group. Of the various serum enzymes examined, only amylase and lipase, which are commonly used as markers of acute pancreatitis (8,22), showed significant changes over time (Figure 1). Canine pancreatic lipase immunoreactivity was measured at 3 h after the first dose of caerulein, with a commercial kit, and positive results were observed. On gross examination of the organs from the caerulein group, we observed redness of the pancreas and hemorrhagic inflammation of the small intestine (Figure 2). Histologic examination of the pancreas revealed severe hyperemia (Figure 3). Severe hyperemia was also found in the duodenal villi and the hepatic sinusoid (Figures 4 and 5, respectively). Caerulein-induced changes in cytokine mRNA abundance are shown in Figure 6. The levels of TNF-a (P , 0.05) and IL-6 mRNA peaked at 3 h, at twice the baseline levels, and then rapidly declined by 6 h.
Discussion Caerulein is a 10-peptide molecule that structurally resembles gastrin and the C-terminal octapeptide of cholecystokinin. It has various biologic functions, such as stimulating gallbladder contraction, gastric acid secretion, pancreatic enzyme secretion, and hepatic bile flow in a number of species, including humans (23–26). Caerulein can stimulate pancreatic acinar cells to excrete a large amount of digestive enzymes and pancreatic fluid, resulting in mild edematous pancreatitis characterized by a high serum amylase concentration, interstitial edema, leukocyte infiltration, and vacuolation of the acinar cells (20,27,28). It has been used successfully to induce acute pancreatitis in various animals (11,21,29–32). The dogs in the present study that were treated with caerulein showed distinct signs of mild acute pancreatitis, including higher serum concentrations of amylase and lipase compared with the control group at 3 and 6 h after the first infusion of caerulein. These are the factors primarily used to diagnose pancreatitis, so we assumed that caerulein damaged the canine pancreas through increases in these serum amylase and lipase levels. In addition, microscopic
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examination showed severe hyperemia in the pancreas and adjacent organs (duodenum and liver) of the dogs treated with caerulein, indicating significant acute pancreatitis and inflammation. Also, the gene expression of TNF-a and IL-6 peaked 3 h after the first infusion of caerulein, which is similar to the results of our previous endotoxemia experiment (33). It is unknown how the initial insult to the pancreas generates the inflammatory reaction, but it has been proposed that the pancreatic tissue is capable of producing a range of proinflammatory cytokines. The pancreas can produce large quantities of kinins, which might also play a key role in the inflammatory cascade (34). In a number of human medical and experimental studies (35–37) the concentration of TNF has been elevated in severe acute pancreatitis, which correlates with the outcome of our study. Also, the IL-6 level is of major prognostic significance in human acute pancreatitis (10). In addition, IL-6 plays an important role is the induction of hepatic synthesis of acute-phase proteins such as C-reactive protein (CRP) (38,39), and there is a close relationship between production and serum concentrations of IL-6 and CRP (40). In particular, IL-6 is a very sensitive predictor of the severity of illness 24 h after the onset of acute pancreatitis (41). However, there have been only a few studies of these cytokines in canine acute pancreatitis. There are many striking clinical and physiological similarities between patients with septic shock and those with severe acute pancreatitis (17–19). The complications that develop in the most critically ill patients are very similar, and evidence suggests that the proinflammatory cascade is activated in the same way in each of these patient groups (42). In addition, according to the changing patterns of TNF-a and IL-6 in this study, it is possible that this pancreatitis model could be used as a model of septic shock. However, in future studies it will be necessary to supplement the caeruleininduction model since the acute pancreatitis was mild in this study. Ding, Li, and Jin (20) induced pancreatitis in mice with a combination of caerulein and lipopolysaccharide (LPS). The pancreas was so severely damaged that it resulted in an inflammatory reaction in the entire body and systemic organ dysfunction. Ding, Li, and Jin (20) also reported that the model was almost stable when the
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experiment was duplicated. Furthermore, the pancreatic injury was much more severe, with a predominance of necrosis, after prolonged and sustained exposure to caerulein. According to Jacob et al (43) caerulein administration for 4 h in mice caused acute pancreatitis with apoptosis and significantly milder systemic injury than 8 injections of caerulein. We demonstrated that caerulein injection resulted in mild acute pancreatitis in dogs and confirmed that the changes in several proinflammatory cytokines are similar to those in sepsis (21,44). Further studies are needed to evaluate the cause of more severe pancreatitis in dogs by modifying the method of inducing pancreatitis. None-the-less, our results aid in the development of pancreatitis models that can be used to further study sepsis in dogs.
Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (grant NRF-2013R1A1A2057479).
References 1. Windsor JA, Hammodat H. Metabolic management of severe acute pancreatitis. World J Surg 2000;24:664–672. 2. Kim YT. [Medical management of acute pancreatitis and complications]. Korean J Gastroenterol 2005;46:339–344. 3. Larvin M. Circulating mediators in acute pancreatitis as predictors of severity. Scand J Gastroenterol Suppl 1996;219:16–19. 4. Banks PA. Modern concepts in pancreatitis. Mt Sinai J Med 1993; 60:170–174. 5. Rau B, Uhl W, Buchler MW, Beger HG. Surgical treatment of infected necrosis. World J Surg 1997;21:155–161. 6. Maxie MG, Jubb KVF. Pathology of Domestic Animals. 5th ed. Edinburgh, UK: Elsevier Saunders, 2007:389–408. 7. Ruaux CG, Atwell R. General practice attitudes to the treatment of spontaneous canine acute pancreatitis. Aust Vet Pract 1998;28:67–74. 8. Mansfield C. Acute pancreatitis in dogs: Advances in understanding, diagnostics, and treatment. Top Companion Anim Med 2012;27:123–132. e-pub 2012 May 30. 9. Steer ML, Meldolesi J. Pathogenesis of acute pancreatitis. Annu Rev Med 1988;39:95–105. 10. Frossard JL, Hadengue A, Pastor CM. New serum markers for the detection of severe acute pancreatitis in humans. Am J Respir Crit Care Med 2001;164:162–170. 11. Su KH, Cuthbertson C, Christophi C. Review of experimental animal models of acute pancreatitis. HPB (Oxford) 2006;8: 264–286. 12. Yadav D, Agarwal N, Pitchumoni CS. A critical evaluation of laboratory tests in acute pancreatitis. Am J Gastroenterol 2002;97:1309–1318. 13. Ruben DS, Scorpio DG, Gabrielson KL, Simon BW, Buscaglia JM. Refinement of canine pancreatitis model: Inducing pancreatitis by using endoscopic retrograde cholangiopancreatography. Comp Med 2009;59:78–82. 14. Hess RS, Saunders HM, Van Winkle TJ, Shofer FS, Washabau RJ. Clinical, clinicopathologic, radiographic, and ultrasonographic
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abnormalities in dogs with fatal acute pancreatitis: 70 cases (1986–1995). J Am Vet Med Assoc 1998;213:665–670. 15. Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg 1998;175:76–83. 16. Lane JS, Todd KE, Gloor B, et al. Platelet activating factor antagonism reduces the systemic inflammatory response in a murine model of acute pancreatitis. J Surg Res 2001;99:365–370. 17. Beger HG, Bittner R, Buchler M, Hess W, Schmitz JE. Hemo dynamic data pattern in patients with acute pancreatitis. Gastroenterology 1986;90:74–79. 18. Ito K, Ramirez-Schon G, Shah PM, Agarwal N, Delguercio LR, Reynolds BM. Myocardial function in acute pancreatitis. Ann Surg 1981;194:85–88. 19. Shoemaker WC, Appel PL, Kram HB, Bishop MH, Abraham E. Temporal hemodynamic and oxygen transport patterns in medical patients. Septic shock. Chest 1993;104:1529–1536. 20. Ding SP, Li JC, Jin C. A mouse model of severe acute pancreatitis induced with caerulein and lipopolysaccharide. World J Gastroenterol 2003;9:584–589. 21. Renner IG, Wisner JR, Jr. Ceruletide-induced acute pancreatitis in the dog and its amelioration by exogenous secretin. Int J Pancreatol 1986;1:39–49. 22. Van den Bossche I, Paepe D, Daminet S. Acute pancreatitis in dogs and cats: Pathogenesis, clinical signs and clinicopathologic findings. Vlaams Diergeneeskd Tijdschr 2010;79:13–22. 23. Erspamer V, Bertaccini G, De Caro G, Endean R, Impicciatore M. Pharmacological actions of caerulein. Experientia 1967;23:702–703. 24. Bertaccini G, Braibanti T, Uva F. Cholecystokinetic activity of the new peptide caerulein in man. Gastroenterology 1969;56:862–867. 25. Bertaccini G, De Caro G, Endean R, Erspamer V, Impicciatore M. The action of caerulein on pancreatic secretion of the dog and biliary secretion of the dog and the rat. Br J Pharmacol 1969;37:185–197. 26. Erspamer V. Progress report: Caerulein. Gut 1970;11:79–87. 27. Frossard JL, Bhagat L, Lee HS, et al. Both thermal and non-thermal stress protect against caerulein induced pancreatitis and prevent trypsinogen activation in the pancreas. Gut 2002;50:78–83. 28. Wagner AC, Mazzucchelli L, Miller M, Camoratto AM, Göke B. CEP-1347 inhibits caerulein-induced rat pancreatic JNK activation and ameliorates caerulein pancreatitis. Am J Physiol Gastrointest Liver Physiol 2000;278:G165–172. 29. Lampel M, Kern HF. Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Archiv A Pathol Anat Histol 1977;373:97–117. 30. Watanabe O, Baccino FM, Steer ML, Meldolesi J. Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: Early morphological changes during development of experimental pancreatitis. Am J Physiol 1984;246:G457–G467. 31. Niederau C, Ferrell LD, Grendell JH. Caerulein-induced acute necrotizing pancreatitis in mice: Protective effects of proglumide, benzotript, and secretin. Gastroenterology 1985;88:1192–1204. 32. Chan YC, Leung PS. Acute pancreatitis: Animal models and recent advances in basic research. Pancreas 2007;34:1–14. 33. Song R, Kim J, Yu D, Park C, Park J. Kinetics of IL-6 and TNFalpha changes in a canine model of sepsis induced by endotoxin. Vet Immunol Immunopathol 2012;146:143–149.
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34. Orstavik TB, Brandtzaeg P, Nustad K, Pierce JV. Immunohis tochemical localization of kallikrein in human pancreas and salivary glands. J Histochem Cytochem 1980;28:557–562. 35. Exley AR, Leese T, Holliday MP, Swann RA, Cohen J. Endotox aemia and serum tumour necrosis factor as prognostic markers in severe acute pancreatitis. Gut 1992;33:1126–1128. 36. Grewal HP, Kotb M, el Din AM, et al. Induction of tumor necrosis factor in severe acute pancreatitis and its subsequent reduction after hepatic passage. Surgery 1994;115:213–221. 37. Norman JG, Franz MG, Fink GS, et al. Decreased mortality of severe acute pancreatitis after proximal cytokine blockade. Ann Surg 1995;221:625–631; discussion 631–624. 38. Mayer AD, McMahon MJ, Bowen M, Cooper EH. C reactive protein: An aid to assessment and monitoring of acute pancreatitis. J Clin Pathol 1984;37:207–211. 39. Wilson C, Heads A, Shenkin A, Imrie CW. C-reactive protein, antiproteases and complement factors as objective markers of severity in acute pancreatitis. Br J Surg 1989;76:177–181.
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40. Leser HG, Gross V, Scheibenbogen C, et al. Elevation of serum interleukin-6 concentration precedes acute-phase response and reflects severity in acute pancreatitis. Gastroenterology 1991;101:782–785. 41. Heath DI, Cruickshank A, Gudgeon M, Jehanli A, Shenkin A, Imrie CW. Role of interleukin-6 in mediating the acute phase protein response and potential as an early means of severity assessment in acute pancreatitis. Gut 1993;34:41–45. 42. Wilson PG, Manji M, Neoptolemos JP. Acute pancreatitis as a model of sepsis. J Antimicrob Chemother 1998;41 Suppl A:51–63. 43. Jacob TG, Raghav R, Kumar A, Garg PK, Roy TS. Duration of injury correlates with necrosis in caerulein-induced experimental acute pancreatitis: Implications for pathophysiology. Int J Exp Pathol 2014;95:199–208. 44. Jung WS, Chae YS, Kim DY, et al. Gardenia jasminoides protects against cerulein-induced acute pancreatitis. World J Gastroenterol 2008;14:6188–6194.
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Changes in gene expression of tumor necrosis factor alpha and interleukin 6 in a canine model of caerulein-induced pancreatitis Ruhui Song, Dohyeon Yu, Jinho Park
Abstract Acute pancreatitis is an inflammatory process that frequently involves peripancreatic tissues and remote organ systems. It has high morbidity and mortality rates in both human and veterinary patients. The severity of pancreatitis is generally determined by events that occur after acinar cell injury in the pancreas, resulting in elevated levels of various proinflammatory mediators, such as interleukin (IL) 1b and 6, as well as tumor necrosis factor alpha (TNF-a). When these mediators are excessively released into the systemic circulation, severe pancreatitis occurs with systemic complications. This pathophysiological process is similar to that of sepsis; thus, there are many striking clinical similarities between patients with septic shock and those with severe acute pancreatitis. We induced acute pancreatitis using caerulein in dogs and measured the change in the gene expression of proinflammatory cytokines. The levels of TNF-a and IL-6 mRNA peaked at 3 h, at twice the baseline levels, and the serum concentrations of amylase and lipase also increased. Histopathological examination revealed severe hyperemia of the pancreas and hyperemia in the duodenal villi and the hepatic sinusoid. Thus, pancreatitis can be considered an appropriate model to better understand the development of naturally occurring sepsis and to assist in the effective treatment and management of septic patients.
Résumé La pancréatite aigüe est un processus inflammatoire qui implique fréquemment les tissus péri-pancréatiques et des systèmes organiques éloignés. Elle a des taux de morbidité et de mortalité élevés autant chez les humains que chez les animaux. La sévérité de la pancréatite est généralement déterminée par des évènements qui se produisent suite à des dommages aux cellules acinaires dans le pancréas, et qui induisent des niveaux élevés de différents médiateurs pro-inflammatoires, tels que l’interleukine (IL) 1b et 6, ainsi que le facteur nécrosant des tumeurs alpha (TNFa). Lorsque ces médiateurs sont libérés de manière excessive dans la circulation systémique, une pancréatite sévère se produit avec des complications systémiques. Ce processus pathophysiologique est similaire à celui d’un sepsis; donc, il y a plusieurs similarités cliniques entre des patients avec un choc septique et ceux avec une pancréatite aigüe sévère. Nous avons induit une pancréatite aigüe en utilisant de la caeruléine chez des chiens et avons mesuré le changement dans l’expression des gènes des cytokines pro-inflammatoires. Les niveaux d’ARNm de TNFa et d’IL-6 ont culminé après 3 h, atteignant le double des niveaux de base, et les concentrations sériques d’amylase et de lipase augmentèrent également. Un examen histopathologique a révélé une hyperémie sévère du pancréas et une hyperémie dans les villosités duodénales et les sinusoïdes hépatiques. Ainsi, la pancréatite peut être considérée un modèle approprié pour mieux comprendre le développement d’un sepsis naturel et aider dans le traitement efficace et la gestion de patients septiques. (Traduit par Docteur Serge Messier)
Introduction Acute pancreatitis is an inflammatory process of the pancreas that frequently involves peripancreatic tissues and remote organ systems (1) and has high morbidity and mortality rates in both human and veterinary patients. The mortality rate for humans with acute pancreatitis has been reported as just under 10% (2,3), but in severe cases it is as high as 20% to 30% (4,5). The mortality rate for dogs with acute pancreatitis ranges from 27% to 58% (6–8). The pathophysiological process of acute pancreatitis consists of activation of pancreatic enzymes within acinar cells, release of these
enzymes into the interstitium, autodigestion of the pancreas, and release of the enzymes and other factors into the circulation, which results in multiple organ dysfunction (9–13). Dogs with acute pancreatitis generally present with a sudden onset of anorexia, depression, abdominal pain, and vomiting (14). However, the findings on clinical examination vary considerably with the severity and stage of the pancreatitis and the degrees of associated dehydration and shock (8). Mild acute pancreatitis does not cause multisystem organ failure or a complicated recovery, whereas severe acute pancreatitis causes multisystem organ failure or development of severe complications (1). The severity of
Department of Veterinary Internal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 54896, Korea (Song, Park); Department of Veterinary Laboratory Medicine, College of Veterinary Medicine, Chonnam National University, Gwangju, 61186, Korea (Yu). Ruhui Song and Dohyeon Yu contributed equally to this work. Address all correspondence to Dr. Jinho Park; telephone: 182-63-850-0949; fax: 182-63-850-0910; e-mail: [email protected] Received August 28, 2015. Accepted March 1, 2016. 236
The Canadian Journal of Veterinary Research
2016;80:236–241
Amylase
Lipase 7000
2000 1800
6000
1400
Mean value (U/L)
Mean value (U/L)
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400 1000
200 0
0
3
6
12
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48
Time (h)
0
0
3
6
12
24
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Figure 1. Changes in serum amylase (a) and lipase (b) concentrations in 4 dogs exposed to caerulein (black bars) and 2 control dogs (grey bars). Data presented as mean 6 standard deviation.
pancreatitis is generally determined by the events that occur after acinar cell injury, when various proinflammatory mediators, such as interleukin (IL) 1b and 6, as well as tumor necrosis factor alpha (TNF-a), are produced (15,16). When these mediators are excessively released into the systemic circulation, severe pancreatitis occurs, with systemic complications. This pathophysiological process is similar to that of sepsis; thus, there are many striking clinical similarities between patients with septic shock and those with severe acute pancreatitis (17–19). In the present study, we used caerulein to induce acute pancreatitis in dogs. We examined the pancreas and adjacent organs histopathologically, measured the serum amylase and lipase levels and the gene expression of proinflammatory mediators, and evaluated the suitability of this pancreatitis model as a model of septic shock.
Materials and methods
Figure 2. Gross features of the canine abdominal cavity. Redness of the pancreas (white arrows) represents caerulein-induced hyperemia.
Animals Six healthy adult beagles weighing 7 to 8 kg each were hospitalized and fasted for 12 h before the study; they were provided with water during the fasting period. Eight hours after the first infusion of caerulein the dogs were fed a commercial diet and provided with tap water. The study was approved by the Committee on Bioethics of Chonbuk National University, Jeonju, Korea.
Induction of acute pancreatitis Four dogs received caerulein (Sigma–Aldrich, St. Louis, Missouri, USA) twice intravenously at a dose of 10 mg/kg body weight (BW) (20,21), with a 1-hour interval between infusions. The other 2 dogs were used as controls and were given 2 intravenous infusions, 1 h apart, of normal saline at a dose of 1 mL/kg BW. Body temperature, pulse rate, and respiratory rate were measured before the start of the injections and at 3, 6, 12, 24, and 48 h after the start. The dogs were euthanized at the end of the examination period, and necropsy was done immediately.
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Figure 3. Severe hyperemia of the pancreas in a dog exposed to caerulein. Necrosis of the pancreatic acinar cells was not observed. Hematoxylin and eosin (H&E); 3100.
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Figure 4. Severe hyperemia in the blood vessels of the duodenal villi in a dog exposed to caerulein. H&E; 3100.
Figure 5. Hyperemia in the hepatic sinusoid in a dog exposed to caerulein. H&E; 3100.
Sample collection
trophotometer (Epoch; Bio-Tek Instruments, Winooski, Vermont, USA). A 260:280 nm absorbance ratio of 1.8:2.0 was regarded as indicating pure RNA. Using the manufacturer’s protocol, we synthesized cDNA with the ImProm-II Reverse Transcription System (Promega) and stored it at −20°C until needed. Real-time polymerase chain reaction (RT-PCR) was done with gene-specific primers for canine TNF-a and IL-6. The 20-mL reaction solution contained 300 nM of each primer, 1 mL of cDNA, and 10 mL of 23 iQ SYBR Green Supermix (Bio-Rad, Hercules, California, USA). The CFX384 RT-PCR detection system (Bio-Rad) was used to quantify cytokine mRNA in the PBMCs. Samples were heated at 95°C for 3 min for i-Taq DNA polymerase activation, and then they underwent 40 cycles of denaturation at 95°C for 15 s, annealing for 15 s, and extension at 72°C for 15 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. All samples, controls, and standards were run in duplicate. Data collection and analysis were done with CFX Manager Software, version 1.0 (Bio-Rad). Relative quantification was analyzed by the 2−DDCt method. Sample values were averaged and calculated in relation to the quantity of GAPDH. These normalized values were used to calculate the expression of a given sequence relative to the control.
Blood (6 mL per dog divided into each of the 2 types of tube) was collected from the jugular vein into tubes treated with potassium ethylene diamine tetraacetic acid and plastic vacuum-filled tubes at time 0 (baseline; before the first infusion) and 3, 6, 12, 24, and 48 h after the first infusion. Serum was immediately separated by centrifugation of the blood at 875 3 g for 5 min and kept frozen at −70°C until needed.
Hematologic and biochemical analyses A complete blood (cell) count was done at each time point by means of an automatic impedance cell counter. The serum levels of alkaline phosphatase, alanine transaminase, amylase, lipase, cholesterol, urea nitrogen, creatinine, glucose, phosphate, bilirubin, total protein, and albumin were measured.
Separation of canine peripheral blood mononuclear cells (PBMCs) Centrifugation with Histopaque 1077 (Sigma–Aldrich) at 450 3 g for 45 min was used to isolate the PBMCs. Erythrocytes were lysed with an 83% ammonium chloride solution (pH 7.2) and washed twice with phosphate-buffered saline. Cells were counted manually with use of the trypan blue viability test. Cell purity was determined by a conventional Diff-Quik method after Cytospin centrifugation (Shandon Cytospin, Thermo Scientific, Waltham, Massachusetts, USA) at 250 3 g for 5 min. Viability was always more than 90%, and PBMCs constituted 95% to 98% of the cells.
Quantification of cytokine gene expression Isolated PBMCs were immediately placed in Buffer RLT (Qiagen, Hilden, Germany) and stored at −70°C. Total RNA was isolated with use of the RNeasy Mini Kit (Qiagen) with Qiacube (Qiagen). All RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, Wisconsin, USA) to remove any genomic DNA. The RNA concentration and purity of all samples were measured with a spec-
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Statistical analysis The results are expressed as mean 6 standard deviation (SD) or mean 6 standard error (SE). The significance of differences was evaluated by means of Student’s t-test. A P-value of less than 0.05 was accepted as statistically significant. The statistical analyses were done with the use of SPSS software, version 18.0 (SPSS, Chicago, Illinois, USA).
Results Within 1 h after the first dose of caerulein the dogs exhibited slight weakness and began having diarrhea. However, vomiting was not observed during the experiment. There were no significant changes in body temperature, heart rate, respiratory rate, or hematologic
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Figure 6. Gene expression of tumor necrosis factor alpha (TNF-a) (a) and interleukin (IL)-6 (b) in the 4 dogs exposed to caerulein (triangles) and the 2 control dogs (circles). The mRNA expression peaked 3 h after the first infusion of caerulein. Data presented as mean 6 standard error. *P , 0.05 compared with the control mean.
values within the study group. Of the various serum enzymes examined, only amylase and lipase, which are commonly used as markers of acute pancreatitis (8,22), showed significant changes over time (Figure 1). Canine pancreatic lipase immunoreactivity was measured at 3 h after the first dose of caerulein, with a commercial kit, and positive results were observed. On gross examination of the organs from the caerulein group, we observed redness of the pancreas and hemorrhagic inflammation of the small intestine (Figure 2). Histologic examination of the pancreas revealed severe hyperemia (Figure 3). Severe hyperemia was also found in the duodenal villi and the hepatic sinusoid (Figures 4 and 5, respectively). Caerulein-induced changes in cytokine mRNA abundance are shown in Figure 6. The levels of TNF-a (P , 0.05) and IL-6 mRNA peaked at 3 h, at twice the baseline levels, and then rapidly declined by 6 h.
Discussion Caerulein is a 10-peptide molecule that structurally resembles gastrin and the C-terminal octapeptide of cholecystokinin. It has various biologic functions, such as stimulating gallbladder contraction, gastric acid secretion, pancreatic enzyme secretion, and hepatic bile flow in a number of species, including humans (23–26). Caerulein can stimulate pancreatic acinar cells to excrete a large amount of digestive enzymes and pancreatic fluid, resulting in mild edematous pancreatitis characterized by a high serum amylase concentration, interstitial edema, leukocyte infiltration, and vacuolation of the acinar cells (20,27,28). It has been used successfully to induce acute pancreatitis in various animals (11,21,29–32). The dogs in the present study that were treated with caerulein showed distinct signs of mild acute pancreatitis, including higher serum concentrations of amylase and lipase compared with the control group at 3 and 6 h after the first infusion of caerulein. These are the factors primarily used to diagnose pancreatitis, so we assumed that caerulein damaged the canine pancreas through increases in these serum amylase and lipase levels. In addition, microscopic
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examination showed severe hyperemia in the pancreas and adjacent organs (duodenum and liver) of the dogs treated with caerulein, indicating significant acute pancreatitis and inflammation. Also, the gene expression of TNF-a and IL-6 peaked 3 h after the first infusion of caerulein, which is similar to the results of our previous endotoxemia experiment (33). It is unknown how the initial insult to the pancreas generates the inflammatory reaction, but it has been proposed that the pancreatic tissue is capable of producing a range of proinflammatory cytokines. The pancreas can produce large quantities of kinins, which might also play a key role in the inflammatory cascade (34). In a number of human medical and experimental studies (35–37) the concentration of TNF has been elevated in severe acute pancreatitis, which correlates with the outcome of our study. Also, the IL-6 level is of major prognostic significance in human acute pancreatitis (10). In addition, IL-6 plays an important role is the induction of hepatic synthesis of acute-phase proteins such as C-reactive protein (CRP) (38,39), and there is a close relationship between production and serum concentrations of IL-6 and CRP (40). In particular, IL-6 is a very sensitive predictor of the severity of illness 24 h after the onset of acute pancreatitis (41). However, there have been only a few studies of these cytokines in canine acute pancreatitis. There are many striking clinical and physiological similarities between patients with septic shock and those with severe acute pancreatitis (17–19). The complications that develop in the most critically ill patients are very similar, and evidence suggests that the proinflammatory cascade is activated in the same way in each of these patient groups (42). In addition, according to the changing patterns of TNF-a and IL-6 in this study, it is possible that this pancreatitis model could be used as a model of septic shock. However, in future studies it will be necessary to supplement the caeruleininduction model since the acute pancreatitis was mild in this study. Ding, Li, and Jin (20) induced pancreatitis in mice with a combination of caerulein and lipopolysaccharide (LPS). The pancreas was so severely damaged that it resulted in an inflammatory reaction in the entire body and systemic organ dysfunction. Ding, Li, and Jin (20) also reported that the model was almost stable when the
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experiment was duplicated. Furthermore, the pancreatic injury was much more severe, with a predominance of necrosis, after prolonged and sustained exposure to caerulein. According to Jacob et al (43) caerulein administration for 4 h in mice caused acute pancreatitis with apoptosis and significantly milder systemic injury than 8 injections of caerulein. We demonstrated that caerulein injection resulted in mild acute pancreatitis in dogs and confirmed that the changes in several proinflammatory cytokines are similar to those in sepsis (21,44). Further studies are needed to evaluate the cause of more severe pancreatitis in dogs by modifying the method of inducing pancreatitis. None-the-less, our results aid in the development of pancreatitis models that can be used to further study sepsis in dogs.
Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (grant NRF-2013R1A1A2057479).
References 1. Windsor JA, Hammodat H. Metabolic management of severe acute pancreatitis. World J Surg 2000;24:664–672. 2. Kim YT. [Medical management of acute pancreatitis and complications]. Korean J Gastroenterol 2005;46:339–344. 3. Larvin M. Circulating mediators in acute pancreatitis as predictors of severity. Scand J Gastroenterol Suppl 1996;219:16–19. 4. Banks PA. Modern concepts in pancreatitis. Mt Sinai J Med 1993; 60:170–174. 5. Rau B, Uhl W, Buchler MW, Beger HG. Surgical treatment of infected necrosis. World J Surg 1997;21:155–161. 6. Maxie MG, Jubb KVF. Pathology of Domestic Animals. 5th ed. Edinburgh, UK: Elsevier Saunders, 2007:389–408. 7. Ruaux CG, Atwell R. General practice attitudes to the treatment of spontaneous canine acute pancreatitis. Aust Vet Pract 1998;28:67–74. 8. Mansfield C. Acute pancreatitis in dogs: Advances in understanding, diagnostics, and treatment. Top Companion Anim Med 2012;27:123–132. e-pub 2012 May 30. 9. Steer ML, Meldolesi J. Pathogenesis of acute pancreatitis. Annu Rev Med 1988;39:95–105. 10. Frossard JL, Hadengue A, Pastor CM. New serum markers for the detection of severe acute pancreatitis in humans. Am J Respir Crit Care Med 2001;164:162–170. 11. Su KH, Cuthbertson C, Christophi C. Review of experimental animal models of acute pancreatitis. HPB (Oxford) 2006;8: 264–286. 12. Yadav D, Agarwal N, Pitchumoni CS. A critical evaluation of laboratory tests in acute pancreatitis. Am J Gastroenterol 2002;97:1309–1318. 13. Ruben DS, Scorpio DG, Gabrielson KL, Simon BW, Buscaglia JM. Refinement of canine pancreatitis model: Inducing pancreatitis by using endoscopic retrograde cholangiopancreatography. Comp Med 2009;59:78–82. 14. Hess RS, Saunders HM, Van Winkle TJ, Shofer FS, Washabau RJ. Clinical, clinicopathologic, radiographic, and ultrasonographic
240
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abnormalities in dogs with fatal acute pancreatitis: 70 cases (1986–1995). J Am Vet Med Assoc 1998;213:665–670. 15. Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg 1998;175:76–83. 16. Lane JS, Todd KE, Gloor B, et al. Platelet activating factor antagonism reduces the systemic inflammatory response in a murine model of acute pancreatitis. J Surg Res 2001;99:365–370. 17. Beger HG, Bittner R, Buchler M, Hess W, Schmitz JE. Hemo dynamic data pattern in patients with acute pancreatitis. Gastroenterology 1986;90:74–79. 18. Ito K, Ramirez-Schon G, Shah PM, Agarwal N, Delguercio LR, Reynolds BM. Myocardial function in acute pancreatitis. Ann Surg 1981;194:85–88. 19. Shoemaker WC, Appel PL, Kram HB, Bishop MH, Abraham E. Temporal hemodynamic and oxygen transport patterns in medical patients. Septic shock. Chest 1993;104:1529–1536. 20. Ding SP, Li JC, Jin C. A mouse model of severe acute pancreatitis induced with caerulein and lipopolysaccharide. World J Gastroenterol 2003;9:584–589. 21. Renner IG, Wisner JR, Jr. Ceruletide-induced acute pancreatitis in the dog and its amelioration by exogenous secretin. Int J Pancreatol 1986;1:39–49. 22. Van den Bossche I, Paepe D, Daminet S. Acute pancreatitis in dogs and cats: Pathogenesis, clinical signs and clinicopathologic findings. Vlaams Diergeneeskd Tijdschr 2010;79:13–22. 23. Erspamer V, Bertaccini G, De Caro G, Endean R, Impicciatore M. Pharmacological actions of caerulein. Experientia 1967;23:702–703. 24. Bertaccini G, Braibanti T, Uva F. Cholecystokinetic activity of the new peptide caerulein in man. Gastroenterology 1969;56:862–867. 25. Bertaccini G, De Caro G, Endean R, Erspamer V, Impicciatore M. The action of caerulein on pancreatic secretion of the dog and biliary secretion of the dog and the rat. Br J Pharmacol 1969;37:185–197. 26. Erspamer V. Progress report: Caerulein. Gut 1970;11:79–87. 27. Frossard JL, Bhagat L, Lee HS, et al. Both thermal and non-thermal stress protect against caerulein induced pancreatitis and prevent trypsinogen activation in the pancreas. Gut 2002;50:78–83. 28. Wagner AC, Mazzucchelli L, Miller M, Camoratto AM, Göke B. CEP-1347 inhibits caerulein-induced rat pancreatic JNK activation and ameliorates caerulein pancreatitis. Am J Physiol Gastrointest Liver Physiol 2000;278:G165–172. 29. Lampel M, Kern HF. Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Archiv A Pathol Anat Histol 1977;373:97–117. 30. Watanabe O, Baccino FM, Steer ML, Meldolesi J. Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: Early morphological changes during development of experimental pancreatitis. Am J Physiol 1984;246:G457–G467. 31. Niederau C, Ferrell LD, Grendell JH. Caerulein-induced acute necrotizing pancreatitis in mice: Protective effects of proglumide, benzotript, and secretin. Gastroenterology 1985;88:1192–1204. 32. Chan YC, Leung PS. Acute pancreatitis: Animal models and recent advances in basic research. Pancreas 2007;34:1–14. 33. Song R, Kim J, Yu D, Park C, Park J. Kinetics of IL-6 and TNFalpha changes in a canine model of sepsis induced by endotoxin. Vet Immunol Immunopathol 2012;146:143–149.
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34. Orstavik TB, Brandtzaeg P, Nustad K, Pierce JV. Immunohis tochemical localization of kallikrein in human pancreas and salivary glands. J Histochem Cytochem 1980;28:557–562. 35. Exley AR, Leese T, Holliday MP, Swann RA, Cohen J. Endotox aemia and serum tumour necrosis factor as prognostic markers in severe acute pancreatitis. Gut 1992;33:1126–1128. 36. Grewal HP, Kotb M, el Din AM, et al. Induction of tumor necrosis factor in severe acute pancreatitis and its subsequent reduction after hepatic passage. Surgery 1994;115:213–221. 37. Norman JG, Franz MG, Fink GS, et al. Decreased mortality of severe acute pancreatitis after proximal cytokine blockade. Ann Surg 1995;221:625–631; discussion 631–624. 38. Mayer AD, McMahon MJ, Bowen M, Cooper EH. C reactive protein: An aid to assessment and monitoring of acute pancreatitis. J Clin Pathol 1984;37:207–211. 39. Wilson C, Heads A, Shenkin A, Imrie CW. C-reactive protein, antiproteases and complement factors as objective markers of severity in acute pancreatitis. Br J Surg 1989;76:177–181.
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40. Leser HG, Gross V, Scheibenbogen C, et al. Elevation of serum interleukin-6 concentration precedes acute-phase response and reflects severity in acute pancreatitis. Gastroenterology 1991;101:782–785. 41. Heath DI, Cruickshank A, Gudgeon M, Jehanli A, Shenkin A, Imrie CW. Role of interleukin-6 in mediating the acute phase protein response and potential as an early means of severity assessment in acute pancreatitis. Gut 1993;34:41–45. 42. Wilson PG, Manji M, Neoptolemos JP. Acute pancreatitis as a model of sepsis. J Antimicrob Chemother 1998;41 Suppl A:51–63. 43. Jacob TG, Raghav R, Kumar A, Garg PK, Roy TS. Duration of injury correlates with necrosis in caerulein-induced experimental acute pancreatitis: Implications for pathophysiology. Int J Exp Pathol 2014;95:199–208. 44. Jung WS, Chae YS, Kim DY, et al. Gardenia jasminoides protects against cerulein-induced acute pancreatitis. World J Gastroenterol 2008;14:6188–6194.
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