Suda K (ed): Pancreas Pathological Practice and Research.

Basel, Karger, 2007, pp 178187

Regeneration of the Pancreas

Masanobu Eguchi
Department of Clinical Pathology, Juntendo University Nerima Hospital, Tokyo, Japan

Abstract
The potential for regeneration of pancreatic tissue in the adult human has generally been regarded as minimal. However, in chronic pancreatitis, isolated lobuli are frequently seen in fibrosis. These isolated lobuli have nodular architecture and bear resemblance to regenerative nodules of the cirrhotic liver. In experimental animals, regeneration of the acinar cells has been shown in the literature since Fitzgerald et al., followed by other experimental studies for pancreas exocrine and/or endocrine cell regeneration. Recently, expression of growth factors in pancreatic regeneration as platelet-derived growth factor-A (PDGF-A) and vascular endothelial growth factor (VEGF) was determined by immunohistochemical analysis, and a combination of epidermal growth factor and leukemia-inhibitor factor induces exocrine-endocrine transdifferentiation. Also, the morphological examinations of experimental animals clarified the potential endocrine and exocrine progenitors as tubular complex and acinoinsular and/or ductuloinsular transformation.
Copyright 2007 S. Karger AG, Basel

The regenerative potential of the human pancreas tissue has been regarded as minimal. However, with regard to the pancreatic duct epithelium, proliferative changes, including regeneration, hyperplasia and metaplasia, are common pathological features. In the damaged human pancreas, such as in acute or chronic pancreatitis, isolated lobuli are frequently seen in fibrosis (figs. 1, 2). These isolated lobuli have a nodular architecture and show a resemblance to regenerative nodules of the cirrhotic liver. Though sequential examination of regenerative changes in the human pancreas is almost impossible, regeneration of acinar cells has been shown in experimental animals by Fitzgerald et al. [1]. These authors reported regeneration of acinar cells in the rat pancreas after administration of DL-ethionine. In this chapter, I would like to show my previous experiment on the characteristic histological patterns of regeneration in the chemically-injured animal pancreas [2, 3], and the comparison between the

Fig. 1. Isolated lobuli in fibrosis in the case of chronic pancreatitis. HE.

Fig. 2. The mitotic figure was occasionally observed in such lobuli. HE.

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Days

14 (5)

21 24 28

34

56

(5)

DL-ethionine

(0.5%)

(ED) (10) (10)

DL-ethionine

and followed by normal diet

(END)

(5)

(5)

(5) Normal diet (control) (ND) ( )

(5)

Number of rats sacrificed per experimental period:

Fig. 3. Experimental design of pancreatic regeneration.

so-called cirrhotic human pancreas and the regenerative pancreas in experimental animals. Other experimental studies concerning pancreatic regeneration are also presented.

Experimental Pancreatic Regeneration

Methods in Brief Sixty young male Wistar rats were divided into two groups. Ten rats were fed a synthetic control diet (ND group), and fifty rats were fed an experimental diet, containing 0.5% DL-ethionine (ED group). On day 14, five rats from each group and on day 21, five rats of group ED were sacrificed. After affirmation of necrosis and destruction of the pancreas, the remaining rats of group ED were changed to the normal diet (END group). On days 24, 28, 50 and 70, i.e. 3, 7, 29 and 49 days after cessation of the DL-ethionine supplemental diet feeding, the END group rats and the remaining rats of the ND group were sacrificed (fig. 3). To observe the regeneration of pancreatic tissue, histological and immunohistochemical examinations were performed, and changes in the -glutamyl transpeptidase(-GTP) activity in the regenerating pancreatic tissues were demonstrated histochemically. A part of the pancreas was submitted for electron microscopy to examine the fine structures of the regenerative cells.

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Fig. 4. Degenerative pancreas in rat of ED group. Ductular aggregation with inter- and intra-lobular fibrosis were noted. HE.

Results Chronological Changes in the Exocrine Pancreas On day 14, the pancreata of the rats fed the ethionine-supplemented diet (ED group) showed diffuse and/or focal necrosis and destruction of the acinar cells. On day 21, interstitial edema became gradually prominent and mild interand intra-lobular fibrosis appeared in the ED group (figs. 4, 5). Three days after the end of the ethionine-supplemented diet, most of the acinar cells in the END group had large oval-shaped nuclei with prominent nucleoli and frequent mitotic figures (fig. 6). Under the electron microscope, these acinar cells with basophilic cytoplasma were seen to be filled with compact rough endoplasmic reticuli and had large irregular shaped nuclei. On the day 7 after the end of DLethionine administration, the END group showed no edema, mononuclear cell infiltration or degeneration of the acinar cells. The mitotic index of the acinar cells on day 14 showed the same value as in the ED and ND groups. The peak of the mitotic index was observed in the END group. The index then gradually decreased, and consequently showed almost the same value in the END and ND groups on day 49 day after the end of DL-ethionine administration (fig. 7). Chronological Changes in the Endocrine Pancreas In the END groups, proliferation of small groups or isolated endocrine cells and the irregular-shaped hyperplastic islets (fig. 8) were occasionally found in

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Fig. 5. Degenerative pancreas in rat of ED group. Most acinar cell cytoplasmas showed marked hydropic change and decrease of zymogen granules. HE.

Fig. 6. Acinar cells in regenerated pancreatic tissue had large oval-shaped nuclei with prominent nucleoli, and mitotic figures were recognized. HE.

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DL-ethionine

administrated diet

(3 weeks) and followed by normal diet Normal diet (M SD)

DL-ethionine

14

21

24

28 Days

50

70

Fig. 7. Mitotic index (number of mitoses per 1,000 acinar cells; M SD).

Fig. 8. Irregular hyperplastic islets were noted in regenerative pancreas. HE.

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Fig. 9. Insulin-producing cells in islets, isolated in acinus and in ductular epithelium. Immunostain for insulin.

the regenerating pancreas. The endocrine cells in these hyperplastic and/or small islets were directly connected with acinar cells and ductular epithelia. Immunohistochemical examinations showed that such small groups or isolated endocrine cells in acini and ductular epithelia coincided with insulinproducing cells (fig. 9). The Activity of Pancreatic -GTP The histochemically and electronmicroscopically observed activity of -GTP was positive in all the acinar cells of the ED group during the experiment. The -GTP was located in the cytoplasmic membrane and a part of the apical portion of the acinar cells (fig. 10), as well as in the apical portion of the ductular epithelium, but not in the endocrine cells. Electron-microscopic examination revealed that the -GTP activity was located along with the cytoplasmic membrane and demonstrated as electron dense material (fig. 11). On the other hand, the ED group showed reduced -GTP activity in all acinar cells along with cell degeneration and necrosis. On day 28 (7 days after the end of DL-ethionine administration), the acinar cells in the END group showed a greater -GTP activity than in the END group on day 24. Histochemically, on days 34 and 56, the acinar cells in the END group showed -GTP activity of almost the same intensity as in the ND group.

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Fig. 10. Localization of -GTP in the acinar cells.Histochemical -GTP.

Fig. 11. Electron microscopic cytochemical demonstration of -GTP activity in the rat pancreas.

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Conclusion and Summary

DL-Ethionine is the antagonist of methionine, which is one of the essential amino acids, and is known to cause pancreatic acinar cell necrosis in rats and other experimental animals by either intraperitoneal injection or oral administration. The mechanism of pancreatic damage is presumed to be abnormal metabolism in protein synthesis. There are also other chemical agents, such as 1-aminocyclopentane carboxylic acid (ACPC) and puromycin, which can cause pancreatic acinar cell necrosis and degeneration. These chemical agents have therefore been used for experiments on pancreatic acinar cell regeneration. On the other hand, the chemical agents which produce pancreatic endocrine cell destruction, such as alloxan [4] and streptozotocin [57], have been used for various experiments on pancreatic endocrine regeneration, and it is also known that these chemical agents produce diabetic states in rats and guinea-pigs. Furthermore, studies on regeneration of pancreatic acinar cells and endocrine cells after partial or subtotal pancreatectomy of some experimental animals, have been reported [8]. My previous experiment on the characteristic histological patterns of regeneration in the chemically-injured animal pancreas suggested that pancreatic acinar cell regeneration followed by necrosis and destruction by DL-ethionine administration occurred as early as 3 days after the termination of DL-ethionine administration. Pancreatic endocrine cells also regenerated in an early phase, and these cells were found in ductal and/or ductular epithelia, as well as in isolated cases in the interstitium. Histochemically recognized -GTP activity in such pancreata was restored rapidly to the control value in parallel with the histopathological restoration. Electronmicroscopic observations supported this view, suggesting that functional restoration of pancreatic exocrine cells begins at an early stage and finishes within a shorter period. Recently, expression of some growth factors was proposed in close relation to pancreatic regeneration. Expression of plateletderived growth factor-A (PDGF-A) and vascular endothelial growth factor (VEGF) in regeneration of rat pancreas was determined by immunohistochemical analysis [9], and epidermal growth factor and leukemia-inhibitor factor induced exocrine-endocrine transdifferentiation in vitro [10]. Furthermore, Renuka et al. [11] suggested that the increased muscarinic M1 and M3 receptor subtypes stimulated insulin secretion and islet cell proliferation during pancreas regeneration. Also, morphological examinations of experimental animals clarified the potential endocrine and exocrine progenitors as tubular complex [12] and acinoinsular and/or ductuloinsular transformation.

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References
1 2 Fitzgerald PJ, Alvizouri M: Rapid restitution of the rat pancreas following acinar cell necrosis subsequent to ethione. Nature 1952;170:929930. Eguchi M, Suzuki F, Matsumoto M, et al: Regeneration of pancreatic acinar and endocrine cell after DL-ethionine administration in the rat; in Sato T, Yamauchi H (eds): Pancreatitis. Tokyo, University of Tokyo Press, 1985, pp 317325. Eguchi M, Matsumoto M, Shirai T, et al: Electronmicroscopic examination of localization of -GTP activity in regenerative rat pancreas. J Clin Electron Microsc 1991;24:740741. Jacob S: Regeneration of the islets of Langerhans in the guinea pig. Cell Tissue Res 1977;181: 277286. Buchanan KD, Mawhinney WAA: Glucagon release from isolated pancreas in streptozotocintreated rats. Diabetes 1973;22:797800. Patel YO, Cameron DP, Brinkier A: Changes in somatostatin concentration in pancreas and other tissues of streptozotocin diabetic rats. Endocrinology 1978;103:917923. Cantenys D, Portha B, Dutrillaux MC, et al: Histogenesis of the endocrine pancreas in new born rats after destruction by streptozotocin. Virchows Arch B Cell Pathol Incl Mol Pathol 1981;35: 109122. Pearson KW, Scott D, Torrance B: Effect of partial surgical pancreatectomy in rats. Gastroenterology 1977;72:469473. Dembinski A, Warzecha Z, Ceranowicz P, et al: Effect of ischemic precondition on pancreatic regeneration and pancreatic expression of vascular endothelial growth factor and platelet-derived growth factor-A in ischemia/reperfusion-induced pancreatitis. J Physiol Pharmacol 2006;57: 3958. Lardon J, Bouwens L: Metaplasia in the pancreas. Differentiation 2005;73:278286. Renuka TR, Savitha B, Paulose CS: Muscarinic M1 and M3 receptor binding alterations in pancreas during pancreatic regeneration of young rats. Endocr Res 2005;31:259270. Wang GS, Rosenberg L, Scott FW: Tubular complexes as a source for islet neogenesis in the pancreas of diabetes-prone BB rats. Lab Invest 2005;85:675688.

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Masanobu Eguchi Department of Clinical Pathology Juntendo University, Nerima Hospital Takanodai 3-10-10, Nerima, Tokyo 177-8521 (Japan) Tel 81 3 5923 3111, E-Mail m.egichi@juntendo-nerima.jp

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