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BRIEF OCCLUSION OF THE MAIN PANCREATIC DUCT RAPIDLY INITIATES SIGNALS WHICH LEAD TO INCREASED DUCT CELL PROLIFERATION IN THE RAT
Cell Biology International 2001, Vol. 25, No. 1, 113–117 doi:10.1006/cbir.2000.0683, available online at http://www.idealibrary.com on
BRIEF OCCLUSION OF THE MAIN PANCREATIC DUCT RAPIDLY INITIATES SIGNALS WHICH LEAD TO INCREASED DUCT CELL PROLIFERATION IN THE RAT W. F. FERRIS, C. W. WOODROOF, J. LOUW and S. A. WOLFE-COOTE Experimental Biology Programme, Medical Research Council, Tygerberg 7505, South Africa Received 23 June 2000; accepted 4 August 2000
In the course of investigating the signals associated with pancreas regeneration, we have developed a method to initiate pancreatic duct cell proliferation by brief occlusion of the main pancreatic duct. The resulting duct cell proliferation, induced by temporary partial main duct occlusion, was compared to that induced by firmly tying a cellophane strip around the head of the pancreas for longer periods of time. Both methods stimulated a biphasic increase in duct cell proliferation, with proliferation maxima at 3 and 14 days post operation. The short duration of temporary main duct occlusion (60 s) that was needed to stimulate duct cell proliferation, and the similar duct cell proliferation profiles that were observed after both the temporary and the longer term main duct occlusion, led us to conclude that the signals which initiate proliferation 2001 Academic Press occur rapidly at the beginning of each procedure. K: pancreas; duct cell; proliferation.
INTRODUCTION The incidence of diabetes is increasing significantly throughout the world and particularly in developing countries. In diabetes, the ability to maintain normal plasma glucose levels is either lost (type 1) or compromised (type 2) due, in simplistic terms, to either a destruction or impairment of the insulinproducing pancreatic cells. One of the key aims of diabetes research is to find ways to replace these lost or damaged cells in a diabetic subject. Pancreas regeneration has been initiated by a number of invasive protocols in animal models in which the pancreas is subjected to mechanical stress or resectioned. Such protocols include the ligation (Boquist and Edstrom, 1970; Edstrom and Falkmer, 1967; Isaksson et al., 1983; Wang et al., 1995), or partial occlusion with a cellophane strip (Rosenberg et al.,1983; Rosenberg, 1995, 1998; Vinik et al., 1997; Wolfe-Coote et al., 1996, 1998a,b) of the main pancreatic duct, or by subtotal (up to 90%) pancreatectomy (Bonner-Weir To whom correspondence should be addressed: Dr W. F. Ferris, EBP, MRC, PO Box 19070, Tygerberg 7505, South Africa. E-mail: [email protected] 1065–6995/01/010113+05 $35.00/0
et al., 1993; Brockenbrough et al., 1988). Since regeneration of pancreatic cells could ameliorate the deleterious effects of hyperglycaemia in patients with type 1 and type 2 diabetes we are investigating the process by which this regeneration occurs and whether a trophic factor could be identified to trigger it less invasively. All of the methods described above involve prolonged pertubation of the pancreas. In this brief communication we describe a method of temporary main duct occlusion and compare the cellular proliferation and pancreatitis initiated by this temporary occlusion with that initiated by the prolonged occlusion after firmly tying the head of the pancreas with a cellophane strip.
MATERIALS AND METHODS Temporary main duct occlusion Thirty female Wistar rats, 16 weeks old (weight 200 g), were anaesthetized and kept under surgical anaesthesia using 2% fluothane in oxygen. Twenty-four animals were subjected to a midline 2001 Academic Press
114
laparotomy and the pancreas isolated with minimal disturbance to internal organs. The main pancreatic duct was then partially occluded by gently squeezing the head of the pancreas between the experimenter’s forefinger and thumb for 60 s. The pancreas was then released, gently relocated in the abdomen and the wound was closed. Animals (n=6) were then terminated at 3, 7, 14 and 21 days post-operation. For controls, six animals were subjected to sham operations and then terminated at 14 days post-operation. The pancreata were removed, fixed using buffered formalin (pH 7.0) and embedded in paraffin wax. Serial sections (4 m) were mounted on glass slides for immunocytochemistry (ICC) and histology. Cellophane wrapping Cellophane wrapping was achieved using the same protocol as that described by Rosenberg (Rosenberg et al., 1983); Twenty-four female Wistar rats, 16 weeks old (weight 200 g), were anaesthetized as for temporary main duct occlusion. Each animal was subjected to a midline laparotomy and the pancreas isolated with minimal disturbance to internal organs. A cellophane strip was wrapped around the head of the pancreas, causing partial occlusion of the main pancreatic duct and the wound closed. Animals (n=6) were then terminated by administration of a lethal dose of sodium pentabarbitone (Kyron Laboratories, Johannesburg Gauteng, R.S.A.) at 3, 7, 14 and 21 days post-operation. The pancreata were removed, fixed using buffered formalin (pH 7.0) and embedded in paraffin wax. Serial sections (4 m) were mounted on glass slides for ICC and histology. Immunocytochemistry; antigen retrieval method for the proliferation marker Ki-67 (Gerdes et al., 1984) Serial unstained dewaxed sections were incubated for 5 min in 3% H2O2 in distilled water to block endogenous peroxidase. After a 50 m Trisbuffered saline (TBS) wash the sections were placed in a glass staining jar filled with 10 m citrate buffer, pH 6.0. The sections were irradiated in a microwave oven at 750 W six times for 5 min each. Between each treatment the jar was refilled with distilled water to the orginal level. After 30 min of irradiation the sections were removed and left on the bench in citrate buffer for 20 min and the immunolabelling was continued.
Cell Biology International, Vol. 25, No. 1, 2001
Immunolabelling In brief, the sections were incubated as follows: (1) 1/20 diluted normal horse serum (NHS) (Delft Animal Holding Facility, Delft, Cape Province, R.S.A.) for 20 min, (2) 1/100 diluted primary monoclonal anti-MIB-5 antibody (Dianova GmBh, Hamburg, Germany, catalog no. dia 5055) overnight at 4C, (3) 10 min wash in 50 m TBS, (4) horse anti-mouse biotinylated antiserum (Vectastain) for 30 min, (5) 10 min wash in 50 m TBS, (6) 60 min in Avidin D-biotinylated horseradish peroxidase H complex (Vectastain, Vector Laboratories Inc., Burlingame, California, U.S.A.). The peroxidase marker was finally revealed by incubating the sections for 5 min in a 0.05% enzyme substrate solution of diaminobenzidine tetrahydrochloride (DAB; Sigma, Saint Louis, Missouri, U.S.A.) containing 0.01% hydrogen peroxide. The sections were counterstained with haematoxylin for 30 s. Counting of proliferating duct cells The proliferation index was calculated as the number of MIB 5-labelled cells expressed as a percentage of the total number of cells in each area counted. All cells were counted in the field of view, with a minimum of 500 cells counted per cell type. Fasting serum glucose and amylase levels Serum amylase and glucose were measured in animals fasted overnight. Amylase levels were assayed using Beckman amylase reagent and a Beckman synchron LX system (Beckman, Pinelands, Cape town, R.S.A.). Glucose was assayed using Technicon glucose (Glu-cinet) reagent and a Technicon RA-1000 analyser (Technicon, Bayer Diagnostics, Isando, Gauteng, R.S.A.). RESULTS Temporary main duct occlusion and cellophane wrapping of the main pancreatic duct stimulate both duct (Fig. 1) and endocrine cell proliferation (Table 1). Duct cell proliferation peaked in the rat pancreas, at 3 and 14 days after temporary occlusion and cellophane wrapping. Animals subjected to either procedure exhibited high fasting plasma glucose and amylase levels (Table 2). In the animals subjected to temporary occlusion, plasma glucose peaked at 7 days and
Cell Biology International, Vol. 25, No. 1, 2001
115
DISCUSSION
Fig. 1. Ducts showing proliferating endothelial cells. Temporary main duct occlusion. 14 Days. MIB5, ABC labelled. Original magnification 250.
declined. Fasting glucose levels remained high at 21 days after cellophane wrapping. Plasma amylase levels showed the same profile after both procedures, peaking at 14 days and then beginning to decrease at 21 days post-operation. Budding of islets from ductal epithelium was suggested from observational analysis of sections taken 14 days post-operation from pancreata that were subjected to either procedure (Fig. 2). This was not seen in the sham operated 14 day control pancreata, or normal control animals. Furthermore, although amylase levels were high at termination (Table 2), only slight pancreatitis was observed infrequently in tissue sections (Fig. 3).
Previously other workers as well as ourselves have shown that duct cell proliferation precedes pancreatic islet regeneration after firmly tying a cellophane strip around the head of the pancreas (Rosenberg et al., 1983; Rosenberg, 1995; Vinik et al., 1997; Wolfe-Coote et al., 1996, 1998a,b). Evidence has also been reported that regeneration during neogenesis may occur, as in organogenesis, by ductal epithelia budding off to produce new islets (Bouwens, 1998; Vinik et al., 1997; WolfeCoote et al., 1998a). Although islet number and area are not yet available for this study, we also observe islets that appear to be budding from pancreatic ducts (Fig. 2), 14 days after both cellophane wrapping and temporary occlusion, in a manner similar to that described above (Bouwens, 1998; Vinik, 1997; Wolfe-Coote et al., 1998a). Furthermore this apparent budding was not observed in control, sham-operated pancreata, or normal controls. Since we have observed apparent budding after both cellophane wrapping and temporary occlusion, it is assumed that both procedures result in similar phenotypic changes in the pancreas. Also, as both procedures result in similar levels of duct cell proliferation and similar proliferation profiles, it may be concluded that the signals that promote regeneration are transmitted rapidly after manipulations affecting the main pancreatic duct. In contrast to the work of Rosenberg et al. on the Syrian golden hamster (Rosenberg et al., 1983), signs of pancreatitis had been observed in the
Table 1. Proliferation indices and standard error of duct, endocrine and acinar cells after cellophane wrapping (cw) and temporary main duct occlusion (tmdo)
Duct cells (cw) Duct cells (tmdo)
Control
3 Days
7 Days
14 Days
21 Days
0.7260.237 0.7250.237
2.0940.564 1.8670.953
1.2950.471 1.0430.247
1.7900.577 1.8530.570
1.5850.419 1.3490.518
Table 2. Serum glucose (mmol/l) and amylase (IU/l) concentrations after cellophane wrapping (cw) and temporary main duct occlusion (tmdo)
Glucose (cw) Glucose (tmdo) Amylase (cw) Amylase (tmdo)
Control
3 Days
7 Days
14 Days
21 Days
4.070.07 4.070.07 491.448.9 491.448.9
8.661.47 7.70.64 646.045.6 601.444.2
8.350.94 9.981.14 778.852.3 728.756.8
9.980.51 9.400.50 978.061.0 885.288.5
10.100.73 8.750.61 820.763.4 696.775.9
116
Fig. 2. Group of endocrine cells adjacent to small duct. Temporary main duct occlusion. 14 Days. H&E. Original magnification 250.
Fig. 3. Mild pancreatitis with inflammatory cell (arrow) and eosinophils (arrowheads). Temporary main duct occlusion. 14 Days. H&E. Original magnification 250.
monkey (Wolfe-Coote et al., 1996) and were observed, in the present study, in the rat pancreata after both procedures. This was confirmed by raised fasting serum glucose and amylase levels, the former indicating a disruption of glucose homeostasis by pancreatic injury and the latter, disruption of pancreatic cellular integrity. Furthermore, at 21 days post-operation, cellophane wrapping was found to result in slightly higher mean serum amylase levels and prolonged elevation of fasting glucose levels than after temporary pancreatic duct occlusion. This might be due to the chronic partial occlusion of the main duct in cellophane wrapping, which does not allow, or delays, pancreatic recovery.
Cell Biology International, Vol. 25, No. 1, 2001
The observed pancreatitis after both procedures suggests that there is an inflammatory response and it may be hypothesized that inflammatory signals may initiate the proliferative process. Evidence in support of this hypothesis comes from research where transgenic mice, expressing IFN- under the control of the insulin promoter, undergo -cell regeneration (Gu and Sarvetnick, 1993). Alternatively, pancreatic ischaemia, caused by temporarily squeezing the pancreas, followed by reperfusion at the termination of squeezing, may initiate the observed duct cell proliferation, with the inflammatory response being subsidiary to this process. This is currently being investigated in our laboratory. To summarize, we had previously found that partial occlusion of the main pancreatic duct of the monkey, by cellophane wrapping the head of the pancreas, leads to a biphasic duct cell proliferative response (Wolfe-Coote et al., 1998a). These results could suggest that the initial proliferationinducing-signals originate at a specific time point after occlusion. In this study we have shown that an increased duct cell biphasic proliferative response is also induced by only a transient stimulation of the rat pancreas as a result of brief partial occlusion of the main pancreatic duct. These results indicate that the signals that induce duct cell proliferation are rapidly initiated. Both prolonged and brief occlusion methods induced slight pancreatitis and elevated serum glucose levels, indicating pancreatic dysfunction, in addition to similar stimulated levels of duct cell proliferation. In conclusion, duct cell proliferative signals would appear to be rapidly transduced after both types of pancreatic manipulation. It follows that the prolonged stimulation of cellophane wrapping is superfluous to the initiating signals which occur with both cellophane wrapping and temporary duct occlusion of the pancreas. REFERENCES B-W S, B LA, S GT, S FE, 1993. A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 42: 1715–1720. B L, E C, 1970. Ultrastructure of pancreatic acinar and islet parenchyma in rats at various intervals after duct ligation. Virchows Arch A Pathol Pathol Anat 349: 69–79. B L, 1998. Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Microsc Res Tech 43: 332–336. B JS, W GC, B-W S, 1988. Discordance of exocrine and endocrine growth after 90% pancreatectomy in rats. Diabetes 37: 232–236.
Cell Biology International, Vol. 25, No. 1, 2001
E C, F S, 1967. Qualitative and quantitative morphology of rat pancreatic islet tissue five weeks after ligation of the pancreatic ducts. Acta Soc Med Ups 72: 376–390. G J, L H, B H, W HH, S U, S H, 1984. Cell cycle analysis of a cell proliferationassociated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133: 1710–1715. G D, S N, 1993. Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice. Development 118: 33–46. I G, I I, L I, 1983. Influence of pancreatic duct ligation on endocrine and exocrine rat pancreas. Acta Physiol Scand 117: 281–286. R L, 1995. In vivo cell transformation: neogenesis of beta cells from pancreatic ductal cells. Cell Transplant 4: 371–383. R L, 1998. Induction of islet cell neogenesis in the adult pancreas: the partial duct obstruction model. Microsc Res Tech 43: 337–346. R L, B RA, D WP, 1983. A new approach to the induction of duct epithelial hyperplasia and
117
nesidioblastosis by cellophane wrapping of the hamster pancreas. J Surg Res 35: 63–72. V A, R R, P G, R L, D W, 1997. Induction of pancreatic islet neogenesis. Horm Metab Res 29: 278–293. W RN, K G, B L, 1995. Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 38: 1405–1411. W-C S, L J, W C, T D, 1996. The non-human primate endocrine pancreas: development, regeneration potential and metaplasia. Cell Biol Int 20: 95–101. W-C S, L J, W C, T D, 1998a. Development, differentiation, and regeneration potential of the Vervet monkey endocrine pancreas. Microsc Res Tech 43: 322–331. W-C SA, L J, W CW, H JJ, T D, 1998b. Induction of cell proliferation and differentiation in the pancreas of the adult Vervet monkey (Cercopithecus aethiops): preliminary results. Pancreas 16: 129–133.
BRIEF OCCLUSION OF THE MAIN PANCREATIC DUCT RAPIDLY INITIATES SIGNALS WHICH LEAD TO INCREASED DUCT CELL PROLIFERATION IN THE RAT W. F. FERRIS, C. W. WOODROOF, J. LOUW and S. A. WOLFE-COOTE Experimental Biology Programme, Medical Research Council, Tygerberg 7505, South Africa Received 23 June 2000; accepted 4 August 2000
In the course of investigating the signals associated with pancreas regeneration, we have developed a method to initiate pancreatic duct cell proliferation by brief occlusion of the main pancreatic duct. The resulting duct cell proliferation, induced by temporary partial main duct occlusion, was compared to that induced by firmly tying a cellophane strip around the head of the pancreas for longer periods of time. Both methods stimulated a biphasic increase in duct cell proliferation, with proliferation maxima at 3 and 14 days post operation. The short duration of temporary main duct occlusion (60 s) that was needed to stimulate duct cell proliferation, and the similar duct cell proliferation profiles that were observed after both the temporary and the longer term main duct occlusion, led us to conclude that the signals which initiate proliferation 2001 Academic Press occur rapidly at the beginning of each procedure. K: pancreas; duct cell; proliferation.
INTRODUCTION The incidence of diabetes is increasing significantly throughout the world and particularly in developing countries. In diabetes, the ability to maintain normal plasma glucose levels is either lost (type 1) or compromised (type 2) due, in simplistic terms, to either a destruction or impairment of the insulinproducing pancreatic cells. One of the key aims of diabetes research is to find ways to replace these lost or damaged cells in a diabetic subject. Pancreas regeneration has been initiated by a number of invasive protocols in animal models in which the pancreas is subjected to mechanical stress or resectioned. Such protocols include the ligation (Boquist and Edstrom, 1970; Edstrom and Falkmer, 1967; Isaksson et al., 1983; Wang et al., 1995), or partial occlusion with a cellophane strip (Rosenberg et al.,1983; Rosenberg, 1995, 1998; Vinik et al., 1997; Wolfe-Coote et al., 1996, 1998a,b) of the main pancreatic duct, or by subtotal (up to 90%) pancreatectomy (Bonner-Weir To whom correspondence should be addressed: Dr W. F. Ferris, EBP, MRC, PO Box 19070, Tygerberg 7505, South Africa. E-mail: [email protected] 1065–6995/01/010113+05 $35.00/0
et al., 1993; Brockenbrough et al., 1988). Since regeneration of pancreatic cells could ameliorate the deleterious effects of hyperglycaemia in patients with type 1 and type 2 diabetes we are investigating the process by which this regeneration occurs and whether a trophic factor could be identified to trigger it less invasively. All of the methods described above involve prolonged pertubation of the pancreas. In this brief communication we describe a method of temporary main duct occlusion and compare the cellular proliferation and pancreatitis initiated by this temporary occlusion with that initiated by the prolonged occlusion after firmly tying the head of the pancreas with a cellophane strip.
MATERIALS AND METHODS Temporary main duct occlusion Thirty female Wistar rats, 16 weeks old (weight 200 g), were anaesthetized and kept under surgical anaesthesia using 2% fluothane in oxygen. Twenty-four animals were subjected to a midline 2001 Academic Press
114
laparotomy and the pancreas isolated with minimal disturbance to internal organs. The main pancreatic duct was then partially occluded by gently squeezing the head of the pancreas between the experimenter’s forefinger and thumb for 60 s. The pancreas was then released, gently relocated in the abdomen and the wound was closed. Animals (n=6) were then terminated at 3, 7, 14 and 21 days post-operation. For controls, six animals were subjected to sham operations and then terminated at 14 days post-operation. The pancreata were removed, fixed using buffered formalin (pH 7.0) and embedded in paraffin wax. Serial sections (4 m) were mounted on glass slides for immunocytochemistry (ICC) and histology. Cellophane wrapping Cellophane wrapping was achieved using the same protocol as that described by Rosenberg (Rosenberg et al., 1983); Twenty-four female Wistar rats, 16 weeks old (weight 200 g), were anaesthetized as for temporary main duct occlusion. Each animal was subjected to a midline laparotomy and the pancreas isolated with minimal disturbance to internal organs. A cellophane strip was wrapped around the head of the pancreas, causing partial occlusion of the main pancreatic duct and the wound closed. Animals (n=6) were then terminated by administration of a lethal dose of sodium pentabarbitone (Kyron Laboratories, Johannesburg Gauteng, R.S.A.) at 3, 7, 14 and 21 days post-operation. The pancreata were removed, fixed using buffered formalin (pH 7.0) and embedded in paraffin wax. Serial sections (4 m) were mounted on glass slides for ICC and histology. Immunocytochemistry; antigen retrieval method for the proliferation marker Ki-67 (Gerdes et al., 1984) Serial unstained dewaxed sections were incubated for 5 min in 3% H2O2 in distilled water to block endogenous peroxidase. After a 50 m Trisbuffered saline (TBS) wash the sections were placed in a glass staining jar filled with 10 m citrate buffer, pH 6.0. The sections were irradiated in a microwave oven at 750 W six times for 5 min each. Between each treatment the jar was refilled with distilled water to the orginal level. After 30 min of irradiation the sections were removed and left on the bench in citrate buffer for 20 min and the immunolabelling was continued.
Cell Biology International, Vol. 25, No. 1, 2001
Immunolabelling In brief, the sections were incubated as follows: (1) 1/20 diluted normal horse serum (NHS) (Delft Animal Holding Facility, Delft, Cape Province, R.S.A.) for 20 min, (2) 1/100 diluted primary monoclonal anti-MIB-5 antibody (Dianova GmBh, Hamburg, Germany, catalog no. dia 5055) overnight at 4C, (3) 10 min wash in 50 m TBS, (4) horse anti-mouse biotinylated antiserum (Vectastain) for 30 min, (5) 10 min wash in 50 m TBS, (6) 60 min in Avidin D-biotinylated horseradish peroxidase H complex (Vectastain, Vector Laboratories Inc., Burlingame, California, U.S.A.). The peroxidase marker was finally revealed by incubating the sections for 5 min in a 0.05% enzyme substrate solution of diaminobenzidine tetrahydrochloride (DAB; Sigma, Saint Louis, Missouri, U.S.A.) containing 0.01% hydrogen peroxide. The sections were counterstained with haematoxylin for 30 s. Counting of proliferating duct cells The proliferation index was calculated as the number of MIB 5-labelled cells expressed as a percentage of the total number of cells in each area counted. All cells were counted in the field of view, with a minimum of 500 cells counted per cell type. Fasting serum glucose and amylase levels Serum amylase and glucose were measured in animals fasted overnight. Amylase levels were assayed using Beckman amylase reagent and a Beckman synchron LX system (Beckman, Pinelands, Cape town, R.S.A.). Glucose was assayed using Technicon glucose (Glu-cinet) reagent and a Technicon RA-1000 analyser (Technicon, Bayer Diagnostics, Isando, Gauteng, R.S.A.). RESULTS Temporary main duct occlusion and cellophane wrapping of the main pancreatic duct stimulate both duct (Fig. 1) and endocrine cell proliferation (Table 1). Duct cell proliferation peaked in the rat pancreas, at 3 and 14 days after temporary occlusion and cellophane wrapping. Animals subjected to either procedure exhibited high fasting plasma glucose and amylase levels (Table 2). In the animals subjected to temporary occlusion, plasma glucose peaked at 7 days and
Cell Biology International, Vol. 25, No. 1, 2001
115
DISCUSSION
Fig. 1. Ducts showing proliferating endothelial cells. Temporary main duct occlusion. 14 Days. MIB5, ABC labelled. Original magnification 250.
declined. Fasting glucose levels remained high at 21 days after cellophane wrapping. Plasma amylase levels showed the same profile after both procedures, peaking at 14 days and then beginning to decrease at 21 days post-operation. Budding of islets from ductal epithelium was suggested from observational analysis of sections taken 14 days post-operation from pancreata that were subjected to either procedure (Fig. 2). This was not seen in the sham operated 14 day control pancreata, or normal control animals. Furthermore, although amylase levels were high at termination (Table 2), only slight pancreatitis was observed infrequently in tissue sections (Fig. 3).
Previously other workers as well as ourselves have shown that duct cell proliferation precedes pancreatic islet regeneration after firmly tying a cellophane strip around the head of the pancreas (Rosenberg et al., 1983; Rosenberg, 1995; Vinik et al., 1997; Wolfe-Coote et al., 1996, 1998a,b). Evidence has also been reported that regeneration during neogenesis may occur, as in organogenesis, by ductal epithelia budding off to produce new islets (Bouwens, 1998; Vinik et al., 1997; WolfeCoote et al., 1998a). Although islet number and area are not yet available for this study, we also observe islets that appear to be budding from pancreatic ducts (Fig. 2), 14 days after both cellophane wrapping and temporary occlusion, in a manner similar to that described above (Bouwens, 1998; Vinik, 1997; Wolfe-Coote et al., 1998a). Furthermore this apparent budding was not observed in control, sham-operated pancreata, or normal controls. Since we have observed apparent budding after both cellophane wrapping and temporary occlusion, it is assumed that both procedures result in similar phenotypic changes in the pancreas. Also, as both procedures result in similar levels of duct cell proliferation and similar proliferation profiles, it may be concluded that the signals that promote regeneration are transmitted rapidly after manipulations affecting the main pancreatic duct. In contrast to the work of Rosenberg et al. on the Syrian golden hamster (Rosenberg et al., 1983), signs of pancreatitis had been observed in the
Table 1. Proliferation indices and standard error of duct, endocrine and acinar cells after cellophane wrapping (cw) and temporary main duct occlusion (tmdo)
Duct cells (cw) Duct cells (tmdo)
Control
3 Days
7 Days
14 Days
21 Days
0.7260.237 0.7250.237
2.0940.564 1.8670.953
1.2950.471 1.0430.247
1.7900.577 1.8530.570
1.5850.419 1.3490.518
Table 2. Serum glucose (mmol/l) and amylase (IU/l) concentrations after cellophane wrapping (cw) and temporary main duct occlusion (tmdo)
Glucose (cw) Glucose (tmdo) Amylase (cw) Amylase (tmdo)
Control
3 Days
7 Days
14 Days
21 Days
4.070.07 4.070.07 491.448.9 491.448.9
8.661.47 7.70.64 646.045.6 601.444.2
8.350.94 9.981.14 778.852.3 728.756.8
9.980.51 9.400.50 978.061.0 885.288.5
10.100.73 8.750.61 820.763.4 696.775.9
116
Fig. 2. Group of endocrine cells adjacent to small duct. Temporary main duct occlusion. 14 Days. H&E. Original magnification 250.
Fig. 3. Mild pancreatitis with inflammatory cell (arrow) and eosinophils (arrowheads). Temporary main duct occlusion. 14 Days. H&E. Original magnification 250.
monkey (Wolfe-Coote et al., 1996) and were observed, in the present study, in the rat pancreata after both procedures. This was confirmed by raised fasting serum glucose and amylase levels, the former indicating a disruption of glucose homeostasis by pancreatic injury and the latter, disruption of pancreatic cellular integrity. Furthermore, at 21 days post-operation, cellophane wrapping was found to result in slightly higher mean serum amylase levels and prolonged elevation of fasting glucose levels than after temporary pancreatic duct occlusion. This might be due to the chronic partial occlusion of the main duct in cellophane wrapping, which does not allow, or delays, pancreatic recovery.
Cell Biology International, Vol. 25, No. 1, 2001
The observed pancreatitis after both procedures suggests that there is an inflammatory response and it may be hypothesized that inflammatory signals may initiate the proliferative process. Evidence in support of this hypothesis comes from research where transgenic mice, expressing IFN- under the control of the insulin promoter, undergo -cell regeneration (Gu and Sarvetnick, 1993). Alternatively, pancreatic ischaemia, caused by temporarily squeezing the pancreas, followed by reperfusion at the termination of squeezing, may initiate the observed duct cell proliferation, with the inflammatory response being subsidiary to this process. This is currently being investigated in our laboratory. To summarize, we had previously found that partial occlusion of the main pancreatic duct of the monkey, by cellophane wrapping the head of the pancreas, leads to a biphasic duct cell proliferative response (Wolfe-Coote et al., 1998a). These results could suggest that the initial proliferationinducing-signals originate at a specific time point after occlusion. In this study we have shown that an increased duct cell biphasic proliferative response is also induced by only a transient stimulation of the rat pancreas as a result of brief partial occlusion of the main pancreatic duct. These results indicate that the signals that induce duct cell proliferation are rapidly initiated. Both prolonged and brief occlusion methods induced slight pancreatitis and elevated serum glucose levels, indicating pancreatic dysfunction, in addition to similar stimulated levels of duct cell proliferation. In conclusion, duct cell proliferative signals would appear to be rapidly transduced after both types of pancreatic manipulation. It follows that the prolonged stimulation of cellophane wrapping is superfluous to the initiating signals which occur with both cellophane wrapping and temporary duct occlusion of the pancreas. REFERENCES B-W S, B LA, S GT, S FE, 1993. A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 42: 1715–1720. B L, E C, 1970. Ultrastructure of pancreatic acinar and islet parenchyma in rats at various intervals after duct ligation. Virchows Arch A Pathol Pathol Anat 349: 69–79. B L, 1998. Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Microsc Res Tech 43: 332–336. B JS, W GC, B-W S, 1988. Discordance of exocrine and endocrine growth after 90% pancreatectomy in rats. Diabetes 37: 232–236.
Cell Biology International, Vol. 25, No. 1, 2001
E C, F S, 1967. Qualitative and quantitative morphology of rat pancreatic islet tissue five weeks after ligation of the pancreatic ducts. Acta Soc Med Ups 72: 376–390. G J, L H, B H, W HH, S U, S H, 1984. Cell cycle analysis of a cell proliferationassociated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133: 1710–1715. G D, S N, 1993. Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice. Development 118: 33–46. I G, I I, L I, 1983. Influence of pancreatic duct ligation on endocrine and exocrine rat pancreas. Acta Physiol Scand 117: 281–286. R L, 1995. In vivo cell transformation: neogenesis of beta cells from pancreatic ductal cells. Cell Transplant 4: 371–383. R L, 1998. Induction of islet cell neogenesis in the adult pancreas: the partial duct obstruction model. Microsc Res Tech 43: 337–346. R L, B RA, D WP, 1983. A new approach to the induction of duct epithelial hyperplasia and
117
nesidioblastosis by cellophane wrapping of the hamster pancreas. J Surg Res 35: 63–72. V A, R R, P G, R L, D W, 1997. Induction of pancreatic islet neogenesis. Horm Metab Res 29: 278–293. W RN, K G, B L, 1995. Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 38: 1405–1411. W-C S, L J, W C, T D, 1996. The non-human primate endocrine pancreas: development, regeneration potential and metaplasia. Cell Biol Int 20: 95–101. W-C S, L J, W C, T D, 1998a. Development, differentiation, and regeneration potential of the Vervet monkey endocrine pancreas. Microsc Res Tech 43: 322–331. W-C SA, L J, W CW, H JJ, T D, 1998b. Induction of cell proliferation and differentiation in the pancreas of the adult Vervet monkey (Cercopithecus aethiops): preliminary results. Pancreas 16: 129–133.