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 7-glutamyl transpeptidase(7-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.
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 inter-and 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 dl-ethionine 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).
In the END groups, proliferation of small groups or isolated endocrine cells and the irregular-shaped hyperplastic islets (fig. 8) were occasionally found in
Fig. ZMitotic index (number of mitoses per 1,000 acinar cells; M ± SD).
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 insulin-producing cells (fig. 9).
The histochemically and electronmicroscopically observed activity of 7-GTP was positive in all the acinar cells of the ED group during the experiment. The 7-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 7-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 7-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 7-GTP activity than in the END group on day 24. Histochemically, on days 34 and 56, the acinar cells in the END group showed 7-GTP activity of almost the same intensity as in the ND group.
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