Br. J. Surg. 1992, Vol. 79, August, 803-1306
T. Hirano, T. Manabe, K. lmanishi and K. Ando First Department of Surgery, Faculty of Medicine, Kyoto University, 54 ShogoinKawaracho, Sakyoku, Kyoto 606, Japan CorresDondence to: Dr T. Manabe
Direct surface cooling of the exocrine pancreas in the rat A study was carried out to evaluate the eflects of direct cooling on the exocrine pancreas. Changes in amylase and cathqsin B release, and in the subcellular distribution of amylase and cathepsin B were measured after 1, 2 and 3 h of direct pancreatic cooling in rats. Cooling f o r 2 and 3 h caused sign$cant hyperamylasaemia and increased pancreatic amylase content, but minimal histological change. Furthermore, 3 h of cooling caused marked redistribution of cathepsin B activity from the lysosomal fraction to the heavier zymogen fraction, and co-localization of lysosomal and digestive enzymes; amylase and cathepsin B output into pancreatic juice after caerulein stimulation were also reduced. These results show that direct pancreatic cooling impairs exocrine function and implicate lysosomal enzymes in the pathogenesis of pancreatic injury, in agreement with results f r o m other models of experimental pancreatitis.
Acute pancreatitis is common after accidental hypothermia' , and almost half of hypothermic patients have high serum amylase level^^.^. Several reports have noted high serum amylase levels and suggest a role for hypothermia in pancreatic injury after major abdominal operations4.'. In these situations, hypothermia may act by systemic effects or by causing local pancreatic injury. In accidental hypothermia, ischaemia as a result of microcirculatory failure is considered to be a likely causative factor in pancreatic damage, but it is not clear whether local pancreatic hypothermia has an effect on the exocrine pancreas. If this is the case, it may have implications for pancreatic transplantation, in which the preservation solution is always very cold, and the donor pancreas is kept cold before transplantation. In this study the effects of local pancreatic cooling on the exocrine pancreas were evaluated by determining serum amylase levels, pancreatic water content, pancreatic amylase and cathepsin B content, and the distribution of lysosomal enzymes in acinar cells.
Materials and methods A group of 56 male Wistar rats weighing about 350g (Shizuoka Experimental Animals, Shizuoka, Japan ) were maintained throughout the study in accordance with the guidelines of the Animal Care Committee of Kyoto University. After a 16-h fast and under general anaesthesia with intraperitoneal pentobarbital (25 mg kg-' ), a venous line was inserted into the superior vena cava via the right external jugular vein. Another catheter was placed in the right carotid artery for blood pressure monitoring. A temperature probe was inserted into the rectum. All animals were kept on heating pads at 40°C under overhead lamps. Anaesthesia was continued by intermittent intravenous administration of pentobarbital (10 mg kg- ' ). Animals were divided into two groups. Group 1 (32 rats) underwent pancreas cooling. The abdomen was opened by an upper midline incision, and 30 ml ice-cold saline in a plastic bag placed on the pancreas from the duodenal loop to the gastric and splenic lobes. This bag was changed every 30 min to keep the pancreas cold. During cooling, the abdomen was closed with small haemostats. Group 2 (24 rats) underwent sham cooling. The abdomen was opened and a bag containing warm saline (38°C) placed in the same position as in group 1. During the experiments, all the animals received intravenous heparin-saline (30 units ml- ' ) at 0.58ml h - ' . After 1, 2 or 3 h (group 1 , eight rats at each stage; group 2, six rats at each stage) the animals were killed by a large dose of pentobarbital, and the abdomen reopened. After blood sampling for the determination of serum amylase levels, portions (approximately 300mg) of the pancreas were removed quickly. One specimen was weighed immediately after excision (wet weight) and again after desiccation at 150°C for 48 h (dry weight). Other portions were homogenized in 4 ml cold phosphate-buffered
0007-1323/92/08080344 0 1992 Butterworth-Heinemann Ltd
saline ( p H 7.4) containing 0.5 per cent Triton X-100 (Fisher Scientific, Fairlawn, New Jersey, USA) in a Polytron (Brinkmann Instruments, Westbury, New York, USA). The amylase6 and cathepsin B 7 activity and DNA concentration* in the resulting supernatant were measured after low-speed centrifugation ( 1509, 15 min, 4°C). Pancreatic amylase and cathepsin B contents were expressed as units per mg DNA. Pancreas from each group at each stage was stained with haematoxylin and eosin. The sections were examined by an observer unaware of the treatment. Interstitial oedema, acinar cell vacuolation and inflammatory cell infiltration were graded on a scale of 0-4. Portions (approximately 700 mg) of the remaining pancreas were homogenized in 6 ml ice-cold 5 mmol I - ' 3-(N-morpholino)propanesulphonic acid buffer ( p H 6.5) containing 1 mmol I-' MgSO, and 250 mmol I - ' sucrose (Sigma Chemicals, St Louis, Missouri, USA). After low-speed centrifugation (150g, P C , 15 min) the supernatant was centrifuged. The homogenate was centrifuged twice, at 4°C for 15 min, at 150g and 13009, t o yield a zymogen granule pellet and again for 12 min at 120009 to yield a lysosomal and mitochondria1 pellet and the microsomal and soluble fraction. The amylase and cathepsin B activity in each fraction was expressed as a percentage of the total activity to give an index of the subcellular distribution of digestive and lysosomal enzymes in pancreatic acinar cell^^^'^. In a fresh and separate group of rats, after 3 h cooling (eight rats) or sham cooling (six animals), the abdomen was opened, and the hepatic duct catheterized t o divert bile. The pancreatobiliary duct was cannulated just distal to the duodenum for the collection of pancreatic juice. After 30 min stabilization, caerulein 0.2 pg kg- ' was infused for 1 h and pancreatic juice collected in preweighed microfuge tubes. Amylase and cathepsin B activity in the pancreatic juice was expressed as units kg-' h-'. The results are reported as mean(s.e.m. ). Differences between groups were assessed using Student's r test. For evaluating the histological changes, the Wilcoxon rank sum test was used. Significance was defined as P < 0.05.
Results All rats survived the experiment. Rectal temperatures in the pancreas cooling group (group 1 ) tended to be lower than in the sham cooling group (group 2), but the differences were not significant at any stage. The mean arterial blood pressure (MABP) showed the same trend, without significant differences and no systemic hypotension. No rats were excluded because of extreme abnormalities of rectal temperature or MABP. Serum amylase levels in group 1 increased gradually and after 3 h of cooling were significantly higher (mean(s.e.m.) 12(2) units ml-') than in group 2 (7( 1 ) units m1-I; Figure l a ) . Pancreatic water content rose slightly with cooling time, but there were no significant differences between the two groups (Figure I b ) . The pancreatic amylase content in group 1 increased gradually and significantly more so than in group 2, particularly after 2 h (579(46) uersus 452(39) units per mg DNA) and 3 h
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(632(53) uersus 471(42) units per mg DNA) of cooling. The cathepsin B content in group 1 was not significantly higher than that in group 2 (Figure 2 ) . Direct cooling of the pancreas induced no significant changes in interstitial oedema and acinar cell vacuolation. Oedema ranged from 0 to 1 (median 1 ) at 3 h in group 1 and from 0 to 1 (median 0 ) in group 2. Acinar cell vacuolation (median 1 (range 0-1 ) in group 1 ) and inflammatory infiltration (median 0 (range 0- 1 ) in group 1 ) were not seen in group 2. Direct cooling of the pancreas for 3 h caused a significant decrease of amylase activity in the zymogen fraction (29(2) uersus 36(2) per cent) and a significant increase in the microsomal and soluble fraction (57(2) versus 48(2) per cent; Figure 3 ) , indicating increased fragility of amylase-containing organelles (zymogen granules) in the process of subcellular fractionation. Cooling of the pancreas for 2 or 3 h caused a significant increase of cathepsin B activity in the zymogen fraction (32(2) and 38(2) per cent versus 26(2) and
804
27(2) per cent) and a significant decrease in the lysosomal fraction (48(2) and 42(2) per cent versus 56(2) and 54(2) per cent; Figure 4 ) . Cooling of the pancreas for 3 h caused a significant decrease in the volume of pancreatic juice after caerulein (0.85(0.12) uersus 1 '6( 0.18 ) ml kg ' h - ) and a significant decrease in both amylase and cathepsin B output (Figure 5 ). ~
Discussion There have been several reports of the close relationship between hypothermia and acute pancreatic injury'-3, but the exact mechanism of this injury is not clear. In this study, direct pancreatic surface cooling with external whole-body warming was used to evaluate the effect of local pancreatic hypothermia on the exocrine pancreas. This method has less systemic effect than does accidental hypothermia because systemic blood pressure and rectal temperature remain almost normal. Direct
Br. J. Surg., Vol. 79, No. 8. August 1992
Surface cooling of the exocrine pancreas: T. Hirano et al.
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Figure 4 Effect of a I-h, b 2-h and c 3-h direct cooling of the pancreas on subcellular cathepsin B distribution. 13, Pancreas cooling group ( n = 8 ) ; 0,sham cooling group (n = 6 ) . * P < 0.05; t P < 0.02 (Student's t test)
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Br. J. Surg., Vol. 79, No. 8, August 1992
805
Surface cooling of the exocrine pancreas: T. Hirano et al. 8. surface cooling for 3 h caused moderate hyperamylasaemia, a moderate increase in pancreatic water content, moderate 9. accumulation of amylase in acinar cells and minimal histological changes. Cooling caused redistribution of lysosomal enzymes 10. from the lysosomal fraction to the heavier zymogen fraction, indicating co-localization of lysosomal and digestive enzymes. Pancreatic digestive enzymes and lysosomal hydrolases are generally transported separately from the Golgi apparatus to their own subcellular compartments, condensing vacuoles and lysosomes' '-I4, and theoretically there should be no co-localization of these two types of enzyme in the same subcellular compartment of acinar cells. However, colocalization of pancreatic digestive and lysosomal enzymes has been reported in caerulein-induced' ' . I 6 , diet-induced'73'8, 13. and d ~ c t - o b s t r ~ c t e dpancreatitis. '~~~~ In these types of experimental pancreatitis, although the ultimate degree of 14. pancreatic injury differs considerably, co-localization of digestive enzymes and lysosomal hydrolases seems to be an 15. important triggering event in the development of acute pancreatitis2'.22. Cathepsin B can activate t r y p ~ i n o g e n ~ ~ - ~ ~ and trypsin can activate many other key enzymes in acute pancreatitis. In the present study, direct cooling also led to 16. redistribution and co-localization of lysosomal and digestive enzymes. In recent studies in this unit, lysosomal enzymes were secreted into pancreatic juice both in the basal state and after 17. stimulation by secretin or caerulein. Lysosomal enzyme secretion seems to be important for the maintenance of the normal organization of acinar cell^'^^^^. Direct cooling of the 18. pancreas reduced both cathepsin B and amylase output into the pancreatic juice, which indicates possible accumulation of lysosomal enzymes in the acinar cells. Since the microcirculation or the core temperature of 19. the pancreas was not evaluated, it is impossible to explain clearly how direct surface cooling injures the exocrine pancreas, which is still innervated and vascularized during the cooling 20. period. Pancreas preserved for transplantation is completely denervated and devascularized and preserved in hyper21. tonic solution. However, there have been reports of hyperamylasaemia or pancreatitis after pancreas transplant22. a t i ~ n ' ' - ~ ~and , also ofthe adverse effects on both the exocrine3' and endocrine3* pancreas of too low preservation temperatures. 23. This study calls attention to methods of organ preservation for pancreas transplantation, because very low temperatures 24. might lead to malfunction of the exocrine pancreas before transplantation, or may predispose the graft to acute 25. pancreatitis.
Acknowledgements This study was supported by a grant, Scientific Research B-03454319, from the Ministry of Education, Science and Culture, and a grant from the Ministry of Health and Welfare of Japan. The authors thank Ms Yoko Manabe for typing the manuscript and Mrs Kimiko Hirano for its preparation.
References Foulis AK. Morphological study of the relation between accidental hypothermia and acute pancreatitis. J Clin Patliol 1982;35: 1244-8. 2. Duguid H, Simpson RG, Stomers JM. Accidental hypothermia. Lancet 1961;ii: 1213-19. 3. Maclean D, Murison J, Griffiths PD. Acute pancreatitis and diabetes ketoacidosis in accidental hypothermia and hypothermic rnyoedema. BMJ 1973;iv: 757-61. 4. White TT, Morgan A, Hopton D . Postoperative pancreatitis. A study of seventy cases. Surgery 1970; 120: 132-7. 5. Bragg LE,Thompson JS, Burnett DA, Hodgson PE, Rikkers LF. Increased incidence of pancreas-related complications in patients with postoperative pancreatitis. Ain J Surg 1985; 150:694-7. 6. Bernfeld P. Amylase a and b. Methods Enzymoll955;1 : 149-58. 7. McDonald JK, Ellis S. On the substrate specificity of cathepsin B, and B, including a new fluorogenic substrate for cathepsin B,. Life Sci 1975;17: 1269-76.
26.
27. 28.
1.
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29. 30. 31.
32.
LaBarca C, Paigen K. A simple, rapid and sensitive DNA assay procedure. Anal Biochem 1980; 102: 334-52. Tartakoff A, Jamieson JE. Fractionation of guinea pig pancreas. Methods Enzymol 1974;31: 41-59. DeLisle R, Schultz I, Tyrakowski T, Haase W, Hopfer U. Isolation of stable pancreatic zymogen. Am J Physiol 1984;246: G411 - 18. Rosenfeld MG, Kreibich G , Popov D, Kato K , Sabatini DD. Biosynthesis of lysosomal hydrolases; their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution. J Cell Biol 1982; 93: 135-43. Sly W, Fischer D. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochcm 1982; 18: 67-85. Kornfeld S. Trafficking of lysosomal enzymes in normal and disease states. J Clin Invest 1986;77: 1-6. Palade G . Intracellular aspects of the process of protein synthesis. Science 1975;189:437-58. Watanabe 0,Baccino F M , Steer ML, Meldolesi J. Supramaximal cazrulein stimulation and ultrastructure of rat pancreatic acinar cell; early morphological changes during development of experimental pancreatitis. A m J Physiol 1984; 246: G457-67. Saluja A, Hashimoto S, Saluja M, Powers RE, Meldolesi J , Steer ML. Subcellular redistribution of lysosomal enzymes during caerulein supramaximal stimulation. Am J Physiol 1987; 253: G508-16. Koike H, Steer ML, Meldolesi J. Pancreatic effects of ethionine; blockage of exocytosis and appearance of crinophagy and autophagy precede cellular necrosis. A m J Physiol 1982; 242: G297-307. Ohshio G, Saluja AK, Leli U, Sengputa A, Steer ML. Esterase inhibitors prevent lysosomal enzyme redistribution in two noninvasive models of experimental pancreatitis. GastroentcrOlogy 1989;96: 853-9. Hirano T, Saluja A, Ramarao P, Lerch M M , Saluja M , Steer ML. Apical secretion of lysosomal enzymes in rabbit pancreas occurs via a secretagogue regulated pathway and is increased after pancreatic duct obstruction. J Clin Invest 1991;87:865-9. Ohshio G,Saluja A, Steer ML. Effects of short-term pancreatic duct obstruction. Gastroenterology 1991;100: 196-202. Steer ML, Meldolesi J , Figarella C. Pancreatitis. The role of lysosomes. Dig Dis Sci 1984;29: 934-8. Steer ML, Meldolesi J. The cell biology of experimental pancreatitis. N Engl J Med 1987;316: 144-50. Greenbaum LM, Hirshkowitz A, Schoichet I. The activation of trypsinogen by cathepsin B. J Biol Cheni 1959;234: 2885-90. Greenbaum LM, Hirshkowitz A. Endogenous cathepsin activation of trypsinogen in extracts of dog pancreas. Proc Soc E.xp Biol Med 1961; 107: 74-6. Rinderknecht H . Activation of pancreatic zymogens. Normal activation, premature intrapancreatic activation, protective mechanism against inappropriate activation. Dig Dis Sci 1986; 31: 314-21. Figarella C, Miszczuk-Jamska B, Barrett AJ. Possible lysosomal activation of pancreatic zymogens. Activation of both human trypsinogen by cathepsin B and spontaneous acid activation of human trypsinogen I. Biol Chem Hoppe-Se!,ler 1988;369(Suppl): 293-8. Hirano T, Manabe T, Tobe T. Pancreatic lysosomal enzyme secretion is changed by hepatectomy in rats. Senrid J Gastroenterol 1990; 25: 1274-80. Tyden G , Wilczek H, Lundgren G et a/. Experience with 21 intraperitoneal segmental pancreatic transplants with enteric or gastric exocrine diversion in humans. Tramplorit Proc 1985; 17: 331-5. Mittal VK, Toledo-Pereyra LH, Prough D. Fiantzis P. Effect of graft pancreatitis on the outcome ofwhole pancreatic transplants. Trunsplunt Proc, 1989;21: 2856-7. Castoldi R, Staudacher C, Ferrari G et nl. Early postoperative surgical complications after combined segmental duct-occluded pancreas transplantation. Transplan/ Proc 1990;22: 582-4. Busing M, Hopt UT, Schareck WD, Quacken M, Morgenroth K. Ultrastructural changes of human pancreatic allografts after cold ischemia and reperfusion. Transplant Proc 1990;22: 612-13. Lakey JRT, Wang LCH, Rajotte R V . Optimal temperature in short-term hypothermic preservation of rat pancreas. Transplantation 1991; 51: 977-81.
Paper accepted 22 February 1992
Br. J. Surg.. Vol. 79, No. 8, August 1992
T. Hirano, T. Manabe, K. lmanishi and K. Ando First Department of Surgery, Faculty of Medicine, Kyoto University, 54 ShogoinKawaracho, Sakyoku, Kyoto 606, Japan CorresDondence to: Dr T. Manabe
Direct surface cooling of the exocrine pancreas in the rat A study was carried out to evaluate the eflects of direct cooling on the exocrine pancreas. Changes in amylase and cathqsin B release, and in the subcellular distribution of amylase and cathepsin B were measured after 1, 2 and 3 h of direct pancreatic cooling in rats. Cooling f o r 2 and 3 h caused sign$cant hyperamylasaemia and increased pancreatic amylase content, but minimal histological change. Furthermore, 3 h of cooling caused marked redistribution of cathepsin B activity from the lysosomal fraction to the heavier zymogen fraction, and co-localization of lysosomal and digestive enzymes; amylase and cathepsin B output into pancreatic juice after caerulein stimulation were also reduced. These results show that direct pancreatic cooling impairs exocrine function and implicate lysosomal enzymes in the pathogenesis of pancreatic injury, in agreement with results f r o m other models of experimental pancreatitis.
Acute pancreatitis is common after accidental hypothermia' , and almost half of hypothermic patients have high serum amylase level^^.^. Several reports have noted high serum amylase levels and suggest a role for hypothermia in pancreatic injury after major abdominal operations4.'. In these situations, hypothermia may act by systemic effects or by causing local pancreatic injury. In accidental hypothermia, ischaemia as a result of microcirculatory failure is considered to be a likely causative factor in pancreatic damage, but it is not clear whether local pancreatic hypothermia has an effect on the exocrine pancreas. If this is the case, it may have implications for pancreatic transplantation, in which the preservation solution is always very cold, and the donor pancreas is kept cold before transplantation. In this study the effects of local pancreatic cooling on the exocrine pancreas were evaluated by determining serum amylase levels, pancreatic water content, pancreatic amylase and cathepsin B content, and the distribution of lysosomal enzymes in acinar cells.
Materials and methods A group of 56 male Wistar rats weighing about 350g (Shizuoka Experimental Animals, Shizuoka, Japan ) were maintained throughout the study in accordance with the guidelines of the Animal Care Committee of Kyoto University. After a 16-h fast and under general anaesthesia with intraperitoneal pentobarbital (25 mg kg-' ), a venous line was inserted into the superior vena cava via the right external jugular vein. Another catheter was placed in the right carotid artery for blood pressure monitoring. A temperature probe was inserted into the rectum. All animals were kept on heating pads at 40°C under overhead lamps. Anaesthesia was continued by intermittent intravenous administration of pentobarbital (10 mg kg- ' ). Animals were divided into two groups. Group 1 (32 rats) underwent pancreas cooling. The abdomen was opened by an upper midline incision, and 30 ml ice-cold saline in a plastic bag placed on the pancreas from the duodenal loop to the gastric and splenic lobes. This bag was changed every 30 min to keep the pancreas cold. During cooling, the abdomen was closed with small haemostats. Group 2 (24 rats) underwent sham cooling. The abdomen was opened and a bag containing warm saline (38°C) placed in the same position as in group 1. During the experiments, all the animals received intravenous heparin-saline (30 units ml- ' ) at 0.58ml h - ' . After 1, 2 or 3 h (group 1 , eight rats at each stage; group 2, six rats at each stage) the animals were killed by a large dose of pentobarbital, and the abdomen reopened. After blood sampling for the determination of serum amylase levels, portions (approximately 300mg) of the pancreas were removed quickly. One specimen was weighed immediately after excision (wet weight) and again after desiccation at 150°C for 48 h (dry weight). Other portions were homogenized in 4 ml cold phosphate-buffered
0007-1323/92/08080344 0 1992 Butterworth-Heinemann Ltd
saline ( p H 7.4) containing 0.5 per cent Triton X-100 (Fisher Scientific, Fairlawn, New Jersey, USA) in a Polytron (Brinkmann Instruments, Westbury, New York, USA). The amylase6 and cathepsin B 7 activity and DNA concentration* in the resulting supernatant were measured after low-speed centrifugation ( 1509, 15 min, 4°C). Pancreatic amylase and cathepsin B contents were expressed as units per mg DNA. Pancreas from each group at each stage was stained with haematoxylin and eosin. The sections were examined by an observer unaware of the treatment. Interstitial oedema, acinar cell vacuolation and inflammatory cell infiltration were graded on a scale of 0-4. Portions (approximately 700 mg) of the remaining pancreas were homogenized in 6 ml ice-cold 5 mmol I - ' 3-(N-morpholino)propanesulphonic acid buffer ( p H 6.5) containing 1 mmol I-' MgSO, and 250 mmol I - ' sucrose (Sigma Chemicals, St Louis, Missouri, USA). After low-speed centrifugation (150g, P C , 15 min) the supernatant was centrifuged. The homogenate was centrifuged twice, at 4°C for 15 min, at 150g and 13009, t o yield a zymogen granule pellet and again for 12 min at 120009 to yield a lysosomal and mitochondria1 pellet and the microsomal and soluble fraction. The amylase and cathepsin B activity in each fraction was expressed as a percentage of the total activity to give an index of the subcellular distribution of digestive and lysosomal enzymes in pancreatic acinar cell^^^'^. In a fresh and separate group of rats, after 3 h cooling (eight rats) or sham cooling (six animals), the abdomen was opened, and the hepatic duct catheterized t o divert bile. The pancreatobiliary duct was cannulated just distal to the duodenum for the collection of pancreatic juice. After 30 min stabilization, caerulein 0.2 pg kg- ' was infused for 1 h and pancreatic juice collected in preweighed microfuge tubes. Amylase and cathepsin B activity in the pancreatic juice was expressed as units kg-' h-'. The results are reported as mean(s.e.m. ). Differences between groups were assessed using Student's r test. For evaluating the histological changes, the Wilcoxon rank sum test was used. Significance was defined as P < 0.05.
Results All rats survived the experiment. Rectal temperatures in the pancreas cooling group (group 1 ) tended to be lower than in the sham cooling group (group 2), but the differences were not significant at any stage. The mean arterial blood pressure (MABP) showed the same trend, without significant differences and no systemic hypotension. No rats were excluded because of extreme abnormalities of rectal temperature or MABP. Serum amylase levels in group 1 increased gradually and after 3 h of cooling were significantly higher (mean(s.e.m.) 12(2) units ml-') than in group 2 (7( 1 ) units m1-I; Figure l a ) . Pancreatic water content rose slightly with cooling time, but there were no significant differences between the two groups (Figure I b ) . The pancreatic amylase content in group 1 increased gradually and significantly more so than in group 2, particularly after 2 h (579(46) uersus 452(39) units per mg DNA) and 3 h
803
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(632(53) uersus 471(42) units per mg DNA) of cooling. The cathepsin B content in group 1 was not significantly higher than that in group 2 (Figure 2 ) . Direct cooling of the pancreas induced no significant changes in interstitial oedema and acinar cell vacuolation. Oedema ranged from 0 to 1 (median 1 ) at 3 h in group 1 and from 0 to 1 (median 0 ) in group 2. Acinar cell vacuolation (median 1 (range 0-1 ) in group 1 ) and inflammatory infiltration (median 0 (range 0- 1 ) in group 1 ) were not seen in group 2. Direct cooling of the pancreas for 3 h caused a significant decrease of amylase activity in the zymogen fraction (29(2) uersus 36(2) per cent) and a significant increase in the microsomal and soluble fraction (57(2) versus 48(2) per cent; Figure 3 ) , indicating increased fragility of amylase-containing organelles (zymogen granules) in the process of subcellular fractionation. Cooling of the pancreas for 2 or 3 h caused a significant increase of cathepsin B activity in the zymogen fraction (32(2) and 38(2) per cent versus 26(2) and
804
27(2) per cent) and a significant decrease in the lysosomal fraction (48(2) and 42(2) per cent versus 56(2) and 54(2) per cent; Figure 4 ) . Cooling of the pancreas for 3 h caused a significant decrease in the volume of pancreatic juice after caerulein (0.85(0.12) uersus 1 '6( 0.18 ) ml kg ' h - ) and a significant decrease in both amylase and cathepsin B output (Figure 5 ). ~
Discussion There have been several reports of the close relationship between hypothermia and acute pancreatic injury'-3, but the exact mechanism of this injury is not clear. In this study, direct pancreatic surface cooling with external whole-body warming was used to evaluate the effect of local pancreatic hypothermia on the exocrine pancreas. This method has less systemic effect than does accidental hypothermia because systemic blood pressure and rectal temperature remain almost normal. Direct
Br. J. Surg., Vol. 79, No. 8. August 1992
Surface cooling of the exocrine pancreas: T. Hirano et al.
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Figure 3 Effect ofa I-h, b 2-h and c 3-h direct cooling of the pancreas on subcellular amylase distribution. 0. Pancreas cooling group ( n = 8 ) ; 0, sham cooling group ( n = 6 ) . * P < 0.05 (Student's t test)
Figure 4 Effect of a I-h, b 2-h and c 3-h direct cooling of the pancreas on subcellular cathepsin B distribution. 13, Pancreas cooling group ( n = 8 ) ; 0,sham cooling group (n = 6 ) . * P < 0.05; t P < 0.02 (Student's t test)
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Br. J. Surg., Vol. 79, No. 8, August 1992
805
Surface cooling of the exocrine pancreas: T. Hirano et al. 8. surface cooling for 3 h caused moderate hyperamylasaemia, a moderate increase in pancreatic water content, moderate 9. accumulation of amylase in acinar cells and minimal histological changes. Cooling caused redistribution of lysosomal enzymes 10. from the lysosomal fraction to the heavier zymogen fraction, indicating co-localization of lysosomal and digestive enzymes. Pancreatic digestive enzymes and lysosomal hydrolases are generally transported separately from the Golgi apparatus to their own subcellular compartments, condensing vacuoles and lysosomes' '-I4, and theoretically there should be no co-localization of these two types of enzyme in the same subcellular compartment of acinar cells. However, colocalization of pancreatic digestive and lysosomal enzymes has been reported in caerulein-induced' ' . I 6 , diet-induced'73'8, 13. and d ~ c t - o b s t r ~ c t e dpancreatitis. '~~~~ In these types of experimental pancreatitis, although the ultimate degree of 14. pancreatic injury differs considerably, co-localization of digestive enzymes and lysosomal hydrolases seems to be an 15. important triggering event in the development of acute pancreatitis2'.22. Cathepsin B can activate t r y p ~ i n o g e n ~ ~ - ~ ~ and trypsin can activate many other key enzymes in acute pancreatitis. In the present study, direct cooling also led to 16. redistribution and co-localization of lysosomal and digestive enzymes. In recent studies in this unit, lysosomal enzymes were secreted into pancreatic juice both in the basal state and after 17. stimulation by secretin or caerulein. Lysosomal enzyme secretion seems to be important for the maintenance of the normal organization of acinar cell^'^^^^. Direct cooling of the 18. pancreas reduced both cathepsin B and amylase output into the pancreatic juice, which indicates possible accumulation of lysosomal enzymes in the acinar cells. Since the microcirculation or the core temperature of 19. the pancreas was not evaluated, it is impossible to explain clearly how direct surface cooling injures the exocrine pancreas, which is still innervated and vascularized during the cooling 20. period. Pancreas preserved for transplantation is completely denervated and devascularized and preserved in hyper21. tonic solution. However, there have been reports of hyperamylasaemia or pancreatitis after pancreas transplant22. a t i ~ n ' ' - ~ ~and , also ofthe adverse effects on both the exocrine3' and endocrine3* pancreas of too low preservation temperatures. 23. This study calls attention to methods of organ preservation for pancreas transplantation, because very low temperatures 24. might lead to malfunction of the exocrine pancreas before transplantation, or may predispose the graft to acute 25. pancreatitis.
Acknowledgements This study was supported by a grant, Scientific Research B-03454319, from the Ministry of Education, Science and Culture, and a grant from the Ministry of Health and Welfare of Japan. The authors thank Ms Yoko Manabe for typing the manuscript and Mrs Kimiko Hirano for its preparation.
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Paper accepted 22 February 1992
Br. J. Surg.. Vol. 79, No. 8, August 1992
