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Dynamic features of duct epithelial cells in the mouse pancreas as shown by radioautography following continuous 3H-thymidine infusion.

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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. Leblond (1974)Origin, differentiation and renewal
of the four main epithelial cell types in the mouse small intestine.
V. Unitarian theory of the origin of the four epithelial cell types.
Am. J. Anat., 141:537-562.
Enesco, M., and C.P. Leblond (1962)Increase in cell number as a factor
in the growth of the organs and tissues of the young male rat. J.
Embryol. Exp. Morphol., 10:530-564.
Leblond C.P., B. Messier, and B. Kopriwa (1959) Thymidine-H3 as a
tool for the investigation of the renewal of cell population. Lab.
Invest., 8:296-308.
Leblond C.P. (1964) Classification of cell population on the basis of
their proliferative behavior. Natl. Cancer Inst. Monogr., 14:119150.
Potten C.S., C. Chadwick, K., Ijiri, S. Tsubouchi, and W.R. Hanson
(1984)The recruitability and cellcycle state of intestinal stem cells
Int. J. Cell Cloning, 2r126-140.
Tsubouchi S., and C.P. Leblond (1979) Migration and turnover of entero-endocrine and caveolated cells in the epithelium of the descending colon, as shown by radioautography after continuous
infusion of 3H-thymidine into mice. Am. J. Anat., 156:431-452.
Tsubouchi S., and C.S. Potten (19851 Recruitment of cells in the small
intestine into rapid cell cycle by small doses of external Y or
internal 0-irradiation. Int. J. Radiat. Biol., 48:361-370.
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