Dynamic features of duct epithelial cells in the mouse pancreas as shown by radioautography following continuous 3H-thymidine infusion.код для вставкиСкачать
THE ANATOMICAL RECORD 214:46-52 (1986) Dynamic Features of Duct Epithelial Cells in the Mouse Pancreas as Shown by Radioautography Following Continuous 3H-Thymidine Infusion SUSUMU TSUBOUCHI, EIICHI KANO, AND HARUMI SUZUKI Department of Experimental Radiology and Health Physics, Fukui Medical School, Matsuoka, Fukui 910-11, Japan (S.71, E.K.), and Laboratory of Pathology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464,Japan (H.S.) ABSTRACT The possibility of turnover of the epithelial duct cells was examined in the adult mouse pancreas by radioautography following continuous administration of 3H-thymidine for periods varying from 1 h to 60 days. One hour after an injection of 3H-thymidine, the label observed in small and large ducts was low but increased with the duration of the continuous infusion of 3H-thymidine and reached a level of about 67% cells labeled after 60 days. The rate of duct cell labeling was estimated from the regression line of the labeling index vs. time in four types of ducts classified according to their inner diameter and the presence of the adventitia and was given as 0.60% cells per day in small (adventitia-free) ducts (4 4-12 pm), 0.89%, 1.02%, and 1.23%)cells per day in large (adventitia-including) ducts (4 15-29, 30-49, and 50-160 pni respectively). In contrast, the labeling index of aciner cells after a 60-day infusion indicated an addition of only 0.02-0.07% per day, and that of islet cells 0.14-0.22% per day. It is known that most parenchymal cells belong to either expanding or renewing cell populations. The acinar cells of the pancreas have been shown to constitute an expanding population, a conclusion confirmed by the low addition of cells observed in the present work. However, the relatively high rate of cell addition in the duct epithelia indicates that they may turn over in a period of 2.7 months in the case of large ducts and 5.6 months in the case of small ducts. It is proposed that the added cells replace cells carried away by the flow of pancreatic juice. Even after adolescence, the total cell number slightly increases in the parenchymal cells of liver, kidney, pancreas, and many other rat tissues (Enesco and Leblond, 1962). In all such tissues, cells proliferate slowly but are retained thereafter without appreciable cell loss (Leblond et al., 1959; Enesco and Leblond, 1962; Cameron, 1970). Such cell populations have been referred to as “expanding” (Leblond, 1964). Although the evidence in support of the “expanding” status is good in some cases (e.g., hepatocytes), it is only suggestive in others. Thus, the cells of pancreatic ducts were examined in the mouse using radioautography after continuous infusion of 3Hthymidine. A high labeling of duct cells was observed. Whether duct cells undergo renewal or expansion was investigated. MATERIALS AND METHODS Continuous Infusion of 3H-Thymidine Male adult Swiss ICR/JCL mice, aged 3 months and weighing 32-35 g, were given 3H-thymidine (New England Nuclear Corporation, 6.7 Ci/mmole) in a continuous manner at the daily rate of 3.5 pCi (about 0.1 pCi/g body weight), using a procedure previously described (Tsubouchi and Leblond, 1979). 0 1986 ALAN R. LISS, INC Groups of 2-5 animals were killed after 1, 2, 4, 7, 14, 30, and 60 days of continuous infusion. In addition, two mice received 15 pCi of 3H-thymidine by IP injection 1 h prior to sacrifice. Fixation, Sectioning, and Labeling index of Duct Cells The animals, anesthetized by an IP injection of sodium pentobarbital (60 mgkg), were perfused for 15 min with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.3 through the left ventricle of the heart. Some pancreases were fixed with Bouin’s solution without perfusion. Whole pancreas was removed in as perfect condition as possible and embedded in paraffin. Sections of the pancreas were serially cut, leaving 3-5 pm between sections to avoid observing the same cells in successive sections. The first two sections of every six successive sections from two different positions were collected on a glass slide for radioautography until a total of 12 sections per Received April 24, 1985; accepted September 5, 1985. Dr. H. Suzuki’s present address is Department of Clinical Pathology, Shizuoka General Hospital, Shizuoka, 104 Japan. Address reprint requests to Dr. S. Tsubouchi. RENEWING CHARACTERISTICS OF PANCREATIC DUCT CELLS animal were available. The unstained slides were radioautographed by coating with Kodak NTB2 emulsion, developed 14-28 days later, and stained with hematoxylin and eosin. The first section of each set was examined in the light microscope. Any nucleus overlaid by five or more silver grains was recorded as labeled. The labeing index-i.e., the ratio of labeled to total number of nuclei-was recorded. Classification of Pancreatic Duct The pancreatic ducts were classified into the following four types, one small and three large ones, using both the internal lumen size of the ducts and the presence of the adventitia. First, the ducts considered small were those devoid of adventitia. Those with distinguishable adventitia were considered large ducts. These were arbitrarily classified into three types according to their inner diameter: 15-29, 30-49, and 50-160 pm, respectively. In general, the small ducts corresponded to the classifically described intercalated and very small intralobular ducts, whereas the three groups of large ducts approximately corresponded to intralobular, interlobular, and main ducts. RESULTS Observation of Pancreatic Duct and Epithelial Lining The pancreatic ducts were easily identified with their eosinophilic material in the lumen and in the characteristic epithelial lining. In the perfused pancreas, acini were separated from one another (Figs. 2-61, whereas they were packed in section fixed by immersion (Fig. 1). Perfusion was preferable to immersion for the identification of small ducts (Fig. 3). Generally, the mouse pancreatic ducts seen in ordinary paraffin sections were composed of a sheet of one or sometimes two layers of columnar, simple cuboidal, flattened, or pseudosquamous epithelial cells except in the vicinity of the branches (i.e., neck region as seen in Fig. 2). In these areas, occasionally a group of duct cells were clustered together. Their lumen was not visible probably because of being cut obliquely. Therefore, the labeling index was obtained by scanning only the one or two layers of the epithelial cell sheet facing the lumen. In the vicinity of the duodenum, the orifice portion of the main ducts is occasionally lined with typical columnar cells with intervening scattered mucous cells (Fig. 6). These were excluded from the counts because of too few observed cases. The majority of so-called large ducts with distinguishable adventitia were lined with cuboidal or flattened epithelial cells. In the small ducts without adventitia, the epithelium was composed of flattened cells. Centroacinar cells were not considered. Number of Labeled Pancreatic Duct Cells One hour after ’H-thymidine administration, label was occasionally seen in the large duct with thick adventitia as well as in the acinus and islet, but not in small ducts (Fig. 1, Table 1).These findings indicate that cells of duct, acinus, and islet acquire label independently. During the first few days of continuous infusion of 3Hthymidine, labeled cells were rare in small ducts. After 7 days, labeled cells gradually increased in number, so that their labeling index averaged 37% at 60 days (Table 1).In contrast, the labeled calls of the large ducts fairly 47 rapidly increased in number during the first few days of continuous ‘H-thymidine infusion, and the labeling index was 67% a t 60 days. When large ducts were separated into three types according to diameter, it was found that the larger the duct, the greater the labeling index (Table 2 and Fig. 7): 48% in ducts 15-29 pm, 59% in ducts 30-49 pm, and 71% in ducts 50-160 pm. In spite of large individual variation in labeling index, particularly in the animals with short continuous infusion of 3H-thymidine, the regression equation was calculated (Fig. 7). The coefficient x yielded the increase rate of labeled cells per day, and the time required for 100% of cell population to become labeled was the inverse of x. From such calculation, it was estimated that pancreatic duct cells acquired label at the rate of 0.6% and 1.18%per day in small and large ducts, respectively. The calculated times to reach 100% labeling were 166.7 and 84.7 days, respectively. The half-lives of the cells (obtained by multiplying by 0.693) were 115 and 59 days. When large ducts were classified according to size, the cells of larger ducts were found to proliferate more rapidly than those of smaller ones (Table 3). These results imply that there is a decreasing gradient of cell proliferation from major to minor duct-i.e., interlobular, intralobular, and intercalated ducts in that order. Increase in Labeled Acinar and Langerhans ’ Islet Cells in Two Animals Labeled cells were few in acini and islets of Langerhans as compared to the ducts. Also the distribution of labeled cells in the acini did not appear to be random. Preliminary measurements of labeling index were carried out in two animals continually infused with ‘Hthymidine for 60 days. The labeling index of islet cells reached 8.4% (161/1954) and 13.4% (207/1542), respectively, and that of acinar cells was 4.2% (78/1850) and 1.4% (29/2073), respectively, in the two animals. These preliminary data indicate that both acinar and islet cells acquire label much more slowly than duct cells. In addition, the variation in acinar labeling from region to region and from animal to animal was considerable. Occasionally, most of the cells in some acini were labeled. These labeling patterns of acinus cells seem to indicate that a few acini enlarged or new acini developed during the continuous infusion of 3H-thymidine, confirming the expanding characteristics of the acinar cell population. DISCUSSION Validity of 3H-ThymidineAdministration for the Identification of Newly Formed Cells Data presented here were obtained using low doses of ’H-thymidine (0.1 pCilglday for continuous infusion or 0.5 pCUg for single injection) as compared to the doses used by most investigators (above 1 pCi/g/day or above 1 pCi/g for single injection). With such rather large doses, there was a possibility of preferential labeling of long cycling cells as a result of the cytotoxic effect on other cells (Cheng and Leblond, 1974; Potten et al., 1984; Tsubouchi and Potten, 1985). In the present work, no sign of cell death was observed. It was therefore concluded that the doses used here did not significantly disturb normal cell kinetics, even in the animals receiving a 60-day continuous infusion. 48 S. TSUBOUCHI, E. KANO, AND H. SUZUKI Figs. 1-5. Radioautography of duct cells in mouse pancreas given single or continuous 3H-thymidineinfusion. The sections were stained with hematoxylin and eosin. large duct with thick adventitia is shown in the center. Most duct cells are labeled, whereas acinar cells are not. A small branch of the duct (neck region) is indicated by leftward horizontal arrow. ~ 5 4 0 . Fig. 1 . One hour after single 3H-thymidine injection. Labeled duct, acinus, and islet cells are shown by leftward horizontal, downward vertical, and upward vertical arrows, respectively. ~ 2 7 0 . Fig. 3. Thirty days after continuous infusion of 3H-thymidine. A labeled duct cell, corresponding to a cell of intercalated duct, is indicated by the downward vertical arrow. Three labeled duct cells of the larger duct are shown by upward oblique arrows. Acinar cells are not labeled. X540. Fig. 2. Thirty days after continuous infusion of 3H-thymidine. A RENEWING CHARACTERISTICS OF PANCREATIC DUCT CELLS Fig. 4. Thirty days after continuous infusion of 3H-thymidine. In a large duct (L), several labeled cells are depicted. Cells of intercalated duct (downward oblique arrow) and small duct (S) do not show label. x540. Fig. 5. Sixty days of continuous infusion of 3H-thymidine. In a large 49 a), duct almost all the cells are labeled. Cells of small duct (S) are also labeled. In a small islet, a label appears in a cell (arrow). x540. Fig. 6. A main duct (D) in the vicinity of the duodenum (nonradioautographed section fixed with Bouin’s fixative). The lumen (L) is lined up with typical columnar cells and mucous cells. x270. 50 S. TSUBOUCHI, E. KANO, AND H. SUZUKI TABLE 1. Labeling index of pancreatic duct cells after continuous infusion of ‘H-thymidine for various periods of time Cells of large ducts’ Total Percent labeled number labeled Number Time 3 6 1,982 1,972 5 4 1,399 1,379 4 6 1,037 1,371 17 22 1,073 1,647 3 28 810 818 191 14 1,267 1,070 339 1,392 1,275 2,864 1,152 179 407 1,361 902 1,320 326 508 2,368 1,600 l h Total 1day Total 2 days Total 4 days Total 7 days Total 14 days Total 30 days Total 60 days ~~ Total 0.151 0.304 0.228 0.357 0.290 0.324 0.386 0.438 0.412 1.584 1.336 1.460 0.370 3.42 1.895 15.07 1.31 8.19 26.6 48.60 37.60 87.27 54.9 80.1 57.47 56.38 67.22 Cells of small ducts’ Total Percent labeled number labeled Number 0 0 1,957 2,196 2 1 3,434 2.637 0 0 2,125 2,314 0 0 2,032 2,116 0 5 1,405 1,204 22 2 1,834 1,544 52 71 700 618 211 53 114 95 127 277 224 253 423 717 0 0 0 0.06 0.04 0.05 0 0 0 0 0 0 0 0.42 0.21 1.20 0.13 0.67 7.43 11.5 9.47 76.2 23.7 45.1 22.5 17.7 37.0 ‘Main interlobular and intralobular ducts with large or moderate amount of adventitia and internal lumen size 15-150 pm thick. ‘Small intralobular and intercalated ducts with sparse or undetected amount of adventitia and internal lumen size 4-20 r m thick. Proliferative Behavior of Duct Cells in Comparison to Acinar Cells, Islet Cells, and Hepatocytes According to Leblond (1964), the cell populations in adult tissues may be classified on the basis of their proliferative behavior as follows: 1)static, as in neurons that do not proliferate; 2) expanding, as in parenchymal cells of liver, kidney, pancreas, and muscle fibers, in which cells added by mitosis are retained; and 3) renewing, as in cells of epidermis, intestinal epithelium, thymus, etc., in which a high mitotic activity is balanced by a cell loss to maintain the steady-state condition of the cell population. The acinar cells of pancreas have been assigned to expanding populations, since they show a low labeling index (Enesco and Leblond, 1962; Leblond, 1964). In the present work, a n acinus was occasionally observed in which most cells were labeled, indicating that localized growth could occur during the period of continuous 3Hthymidine infusion. On the whole, however, labeling of acinar cells was rare. Thus, over a 60-day period, the labeling in two animals was 4.2% and 1.4 %, implying an addition of 0.07% and 0.02% cells per day, respectively. Such rate of growth was of the order observed in “expanding” cell populations (Enesco and Leblond, 1962).According to Cameron (1970), the cell of pancreas, liver, kidney, and salivary gland may be categorized as slowly renewing populations. Our results indicate, how- ever, that a t least acinar pancreatic cells form a truly expanding population. This is also the case of islet cells which, in the two 60-day animals, increased by 8.4%and 13.4% (0.14% and 0.2% per day, respectively). Such an increase is in line with direct measurements of increase with age until about 6 months (Bunnag, 1966). It is concluded that both acinar and islet cells of pancreas constitute expanding populations. In contrast to the slow proliferative behavior of acinar and islet cells, the data on the pancreatic duct epithelium show a relatively high rate of proliferation with daily labeling increases of 0.6% cells in small ducts and 1.18% in large ducts (Fig. 7). If these increases were due to expansion of the duct cell populations, there should be a doubling of the number of cells in 166 days (5.6 months) in small ducts and 84 days (2.7 months) in large ducts, respectively. Yet there was no indication of such increase in size. It is therefore likely that the added cells replaced cells lost, in which case there would be turnover of the epithelia. Assuming the latter to be the case, turnover of pancreatic duct cells would occur in 2.7-5.6 months, with the higher rate taking place in large ducts; such cell production in adult animals under “steadystate” condition would imply a corresponding cell loss. In fact, sections of the animals continuously infused with 3H-thymidine for 60 days showed silver grains occasionally present over basophilic debris in the lumen of large ducts. These observations suggest that the cells 51 RENEWING CHARACTERISTICS OF PANCREATIC DUCT CELLS TABLE 2. Labeling index of pancreatic duct cells in three different sizes of the large duct with the adventitia after continuous infusion of 3H-thymidine for various periods of time Time l h Number labeled 50-160 pm' Total number 2 4 1,409 1,457 2 2 313 510 2 3 546 822 15 6 607 485 2 18 422 544 102 6 644 274 184 1,024 866 1,974 109 53 284 1,212 706 143 87 334 1,780 1,056 Percent labeled 0.14 0.27 0.21 0.64 0.39 0.52 0.37 0.36 0.37 2.47 1.24 1.86 0.47 3.25 1.86 15.84 2.18 9.01 21.24 51.87 36.56 76.22 60.9 85.03 68.09 66.86 71.42 Total 1 day Total 2 days Total 4 days Total 7 days Total 14 days Total 30 days Total 60 days Number labeled Total 30-49 pml Total number 1 1 473 689 2 2 729 574 2 2 285 341 2 3 249 165 1 4 274 123 70 5 395 221 315 244 1,227 622 587 28 107 102 135 634 47 137 437 336 Percent labeled Number labeled 0.21 0.14 0.18 0.27 0.35 0.31 0.70 0.59 0.65 15-29 pm' Total number 0 1 90 196 1 0 357 279 206 208 199 564 0.80 1.82 1.31 0.36 3.25 1.81 17.72 2.26 9.99 25.67 39.23 32.45 92.59 59.6 78.10 23.34 40.12 58.75 0 6 114 138 14 3 192 575 182 122 703 262 409 98 16 47 59 494 197 37 151 182 Percent labeled 0 0.5 0.25 0.28 0 0.14 0 0.48 0.24 0 0.70 0.35 0 4.34 2.17 7.29 0.52 3.91 25.89 46.56 36.23 82.79 49.7 43.2 31.1 32.4 47.84 'Pancreatic ducts with large or moderate amount of the adventitia were divided arbitrarily into three groups depending on internal lumen size of their minor axis. %cells ';Ted / 60. 50. 40. 30. 20. 10. 0 0 10 20 30 40 50 60 b Y S Fig. 7. Regression lines of the percent cells labeled (labeling index) vs. time in days for the five classified ducts according to their size and the condition of the adventitia (see Tables 1 and 2 and text). S. TSUBOUCHI, E. KANO, AND H. SUZUKI 52 TABLE 3. Regression lines of the percent cells labeled (labeling index) vs. time (days) in various size of the pancreatic ducts Size of duct Large duct with adventitia Classification of the large duct t@ 50-160 pm 30-49 pm 15-29 pm Small duct with sparse or undetected adventitia (4-20 pm) Equation of regression line Correlation coefficient X intercept (days) Turnover time (days) 2.74 0.99 2.32 84.7 Y = 1.23X - 2.98 Y = 1.02X - 1.96 Y = 0.89X - 1.71 Y = 0.60X - 2.94 0.99 0.99 0.96 0.96 2.42 1.92 1.92 4.90 81.3 98.0 112.3 166.7 Y = 1.18X - of pancreatic duct epithelia desquamate into the lumen. Such lost cells would then be replaced by new ones. In conclusion, it is proposed that duct cells belong to a renewing rather than a n expanding cell population. The overpopulation of cells would compensate for the loss of cells to the lumen. Perhaps the flow of secretion carries away some of the duct cells, which are eventually transported to the duodenum and lost. Cell proliferation would replace the cell dying in this manner. ACKNOWLEDGMENTS The authors are indebted to Dr. C.P. Leblond for his critical reading of this paper. This work was performed with the support of grants from the ministry of Education, Science and Culture in Japan. The assistance of Miss F. Kotani for the histological work is acknowledged. LITERATURE CITED Bunnag S.C. (1966)Postnatal neogenesis of islets of Langerhans in the mouse. Diabetes, 15r480-491. Cameron I.L. (1970) Cell renewal in the organs and tissues of the nongrowing adult mouse. Texas Rep. Biol. Med., 28203-248. Chqng H., and C.P. 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