Effect of age on cell division, 3H-thymidine
incorporation, and diurnal rhythm in
the lens epithelium of rats
Ludtvig von Sallmann and Patricia Grimes
The rapid growth of the rat lens during the first year of life is accompanied by only small
changes in the size of the epithelial population. The cell layer adapts to the great expansion
of lens surface area by spreading of the cells. Cell proliferation as represented in the mitotic
and SH-labeled cell indices decreases during this period of life. Animals killed in the forenoon
showed a reduction of 3H-thymidine incorporation ivith age tohich surpassed the fall of
mitotic activity. Age-dependent changes in the ratio of labeled cells to mitosis can be explained in part by a shift in the timing of diurnal fluctuations of mitosis in older animals.
Variation of 3H-thymidine incorporation associated with the diurnal rhythm in mitotic activity
could not be detected with 6 hour sampling intervals.
S
Methods and materials
tudies of mitotic activity in the lens
epithelium have shown that the rate of cell
division decreases with age. 13 Daily rhythmical fluctuations of the number of mitoses
have also been recorded in young rats.4
The present work deals with the correlation of cell proliferation and population
expansion in the growing lens and the
effect of age on diurnal variations of both
mitosis and 3H-thymidine incorporation.
The results are of interest for comparing
data obtained under different experimental
conditions; also, they have a bearing on the
reliability of data relating to the life cycle
of cells in the population under investigation.
Male rats of the Osborn-Mendel strain were
used. Their ages ranged from 1 day to 1 year.
The effect of age on cell proliferation was studied
in groups of animals killed in the morning between 9 and 11 A.M. Diurnal variations in mitotic
activity and 3H-thymidine incorporation were
examined in groups of rats 6 weeks, 6 months,
and 1 year of age. These animals were killed
at 6 A.M., 12 noon, 6 P.M., and 12 midnight and
for approximately 1 week prior to use they were
maintained under normal laboratory conditions
with food and water freely available. The lighting
regimen followed the natural cycle of day and
night with the addition of artificial illumination
during the working hours from 8 A.M. to 5 P.M.
The experiments were conducted during the
winter and spring seasons, from November to
May.
One hour before the rats were killed, they
were injected intraperitoneally with a solution of
3
H-thymidine in a dose of 1 /iC per gram of body
weight. The specific activity of the tracer compound was 3.0 C per millimole (Schwartz Bioresearch, Inc.). Feulgen-stained whole mounts of
the lens epithelium were prepared and mitoses
and labeled cells counted according to the criteria
From the Ophthalmology Branch, National Institute of Neurological Diseases and Blindness,
National Institutes of Health, Public Health
Service, United States Department of Health,
Education, and Welfare, Bethesda, Md.
560
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Effect of age on cell division of lens epithelium 561
Volume 5
Number 6
described previously/' To calculate the size of the
epithelial population the preparations first were
projected on a sheet of paper at a linear enlargement of 30 times. The equatorial, pre-equatorial,
and central areas were traced and measured with
a planimeter. The region of the meridional rows
where no cell division occurs was omitted from
the measurements. Then, in each preparation, the
number of nuclei in small microscopic fields outlined by an. Ehrlich stop were counted. Magnification of 1,500 times was used for the densely populated equatorial zone and a magnification of 960
times for the pre-equatorial and central areas.
Counts of the nuclei in 10 fields of each zone
were averaged and the population density per
zone calculated according to the following formula: Zone population = number of cells/field
area x zone area. The sum of the three zone
populations gave the total cell population.
number of cells and area of the epithelium
are compared in Fig. 1. Between 2 weeks
and 8 weeks of age the epithelial area
increased by 65 per cent but the cell population grew by only 14 per cent. At 6
months of age the epithelial area was 80
per cent larger without further change of
the population size. The area measured at
1 year was slightly greater than that at 6
months, but the number of epithelial cells
had fallen during this interval so that the
population equaled that in 2-week-old rats.
Within this 1 year period, the cell nuclei became progressively more separated from
Results
Table I. Number of cells in the epithelial
population and area covered by the lens
epithelium in rats of different ages
Changes in the size of the epithelial
population in rats from 2 weeks to 1 year
of age are listed in Table I. Preparations
from animals younger than 2 weeks had
to be excluded because they were usually
incomplete, and multilayering of cells in
the crowded equatorial zone prevented
accurate cell counts. The measurements
showed that the number of epithelial cells
increased only slightly in the growing lens
though the area covered by the epithelium
enlarged rapidly. The relative gains in the
Age
(wk.)
2
4
6
8
10
14
18
26
52
Average cell
No. of
prepara- population ±
S.E.
tions
113,160+3,131
5
121,120+5,134
5
24
124,004 + 1,344
5
128,460 ± 2,879
128,780 + 5,313
5
5
128,180 ± 2,584
4
129,550 ± 6,700
4
128,630 ±1,581
5
113,640 + 2,633
Average
Area ± S.E.
(sq.mm.)
10.0 + 0.2
14.0 + 0.1
15.4 ± 0.3
16.5 + 0.5
19.2 + 0.5
21.6 + 0.3
22.9 ± 0.3
24.1 ±0.3
24.4+1.2
l50r
22
26
AGE (weeks)
Fig. 1. Comparison of the increases in size of the cell population and epithelial area of rats
from two weeks to one year of age.
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Inoestigat-ivc Ophthalmology
December 1966
562 von Sallmann and Grimes
each other and the average nuclear area
increased demonstrating that the epithelial
layer adapted to the growth of lens surface
area by spreading and flattening of the cells
(Table II). This process affected all three
zones to a similar extent, and the proportional distribution of cells was not influenced by growth. Approximately 37 per
cent of the cells were in the equatorial
zone, 37 per cent in the pre-equatorial
zone, and 26 per cent in the central area.
Data on the rate and extent of decline
in cell proliferation during the first year
of life are entered in Table III. These include the average number of mitoses and
3
H-labeled cells per preparation for groups
Table II. Average cell area calculated for
each of the three zones of the lens
epithelium in rats of different ages0
Age
(tok.)
PreNo. of
Prepara- Equatorial equatorial Central
tions
(sq. (i)
(sq. (i)
(sq. n)
169.9
38.7
111.1
205.1
47.9
132.5
47.9
141.2
216.2
220.1
49.7
141.2
171.4
58.4
244.8
258.0
212.3
65.2
263.7
206.8
70.9
70.3
198.3
342.8
265.1
84.5
333.3
"Calculated from the average number of nuclei counted
in fields of known area assuming that one nucleus represents one cell.
2
4
6
8
10
14
18
24
52
5
5
24
5
5
5
4
4
5
of rats at nine different ages killed in the
morning between 9 and 11 A.M. Mitotic
counts fell rapidly from birth until the
age of 6 weeks and thereafter decreased
more gradually up to 6 months of age. No
further decline of mitotic activity was seen
at 1 year. The mitotic index (mitoses per
100 cells) calculated on the basis of population size for each age group pointed to
an even more rapid reduction of mitotic
activity during the first 6 weeks of life because this value takes into account the
simultaneous growth of the population.
When the number of epithelial cells became constant after 6 weeks of age, the
mitotic index varied only with the number
of mitoses and both expressions of cell
proliferation decreased with age at the same
rate. The equatorial region, which always
contains the greatest number of mitoses,
showed the most pronounced fall in mitotic
activity. The low rate of cell division in the
central zone, in contrast, was almost unaffected by age after 2 weeks (Fig. 2).
An unusually high incidence of nuclear
fragmentation was observed in preparations from very young animals. From the
day of birth to 7 days an average of 101
clumps of Feulgen-positive material per
preparation were counted. This figure
dropped to 18 at 2 weeks and to 3 at 4
weeks of age. In the older animals of this
series nuclear fragmentation was rarely observed. Many Feulgen-positive clumps
Table III. Effect of age on mitotic activity and "H-thymidine incorporation in
rat lens epithelium (9 A.M.-11 A.M.)
Age
(wk.)
1 day
1
2
4
6
8
10
14
18
26*
52°
No. of
Mitoses ± S.E.
756
452
309
237
202
182
175
162
128
104
149
± 252
± 24
±6
±7
±4
+7
+7
±8
±9
±5
+5
Mitotic
Index (%)
No. of
preps.
—
—
0.27
0.20
0.16
0.14
0.14
0.13
0.10
0.08
0.13
3
6
10
14
77
14
9
8
6
10
9
No. of
m-labeled
cells ± S.E.
—
—
3,672 + 183
2,983 + 95
1,729 ±37
1,498 + 96
1,098 + 62
872 ± 61
608 + 20
583 ± 27
566 + 39
3
H-labeling
index (%)
No. of
preps.
—
—
3.25
2.46
1.40
1.17
0.85
0.68
0.47
0.45
0.50
—
—
3
14
77
14
9
8
6
8
9
•Animals killed at noon.
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H/M
—
—
12.0
12.6
8.6
8.2
6.3
5.4
4.8
5.9
3.8
Voiiime 5
Number 6
1
Effect of age on cell division of lens epithelium 563
300
200
100
24
Fig. 2. Zone distribution of mitoses in the lens
epithelium of rats of different ages. The average
numbers of mitoses in each zone were obtained
from counts of the preparations listed in Table III.
were also seen in the lens epithelium of
newborn kittens and puppies."
S
H-Thymidine incorporation fell much
more drastically than mitotic activity between the ages of 2 weeks and 1 year
when measured in the forenoon. The relatively greater reduction of 3H-thymidine
uptake was shown clearly in the change
of the ratio of HH-labeled cells to mitoses
(H/M) from 12.0 for the 2 week age
group to 3.8 at 1 year (Table III). Diurnal
variations in proliferative activity were
considered to be a possible cause of the
age-related change in H/M ratios for rats
killed during the late morning since daily
fluctuation in mitotic activity had been
described in 6-week-old rats.1 It was
thought that alteration or exaggeration of
the diurnal rhythm with age induces a
shift in the number of labeled cells or
mitoses at a given time of the day thereby
modifying the H/M ratio.
Diurnal fluctuations in mitotic activity
and "H-thymidine incorporation were examined in 6 week-, 6 month-, and 1-yearold rats; in 3-month-old rats rhythmical
changes in mitosis alone were investigated.
The pattern of time-dependent periodicity
in mitotic activity in 6-week-old animals
was the same as that reported previously:'1
mitotic counts in animals killed at midnight and 6 A.M. were significantly higher
than in the noon and 6 P.M. groups (Table
IV). The highest average count (6 A.M.)
was 1.6 times the lowest figure (6 P.M.).
No statistically significant difference was
detected between either the two "high"
counts or the two "low" counts. Mitotic
activity in the three groups of older
animals, however, did not follow the same
pattern of diurnal variations. Though the
amplitude and duration of the fluctuations
were similar to those of the 6-week-old
rat, a 6 hour shift in the time relation occurred. Three month-, 6 month-, and 1year-old rats had higher numbers of
mitoses when killed at 6 A.M. and noon
and significantly lower numbers of mitoses
at 6 P.M. and midnight. As in the 6-weekold animals there was no statistically significant difference between the two "high"
values or the two "low" values.
The number of :sH-labeled cells in these
preparations did not correlate with the
well-defined changes in mitotic activity at
different times of the day. Little variation
was found in any of the age groups (Table
V). In 6-week-old rats the noon value was
significantly lower than the values at other
times. At 6 months of age the 6 A.M. count
was lowest, while in 1-year-old animals
the 6 P.M. count was higher than those at
other intervals. The changes were not obviously related to the diurnal pattern of
mitotic activity nor did they indicate any
consistent rhythmical fluctuation of :tHlabeled cells independent of the variations
in mitosis.
Changes in mitotic counts over the 24
hour period were sufficient to alter the
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Investigative Ophthalmology
December 1966
564 von Sallmann and Grimes
Table IV. Number of mitoses at different times of day in rats of different ages
Age
6 wk.
3 mo.
6 mo.
1 yr.
6 A.M.
301 ± 11 (12)'
228 ± 10 (10)
109 ± 3 (10)
148 ± 13 (8)
Mitoses ± S .£.
12 Noon
6 P.M.
212 ± 5 (19)
193 ± 10 (9)
108 ± 5 (10)
185 ± 9 (10)
61 ± 5 (10)
104 ± 5 (10)
95 ± 9 (10)
149 ± 5 (9)
12
293
138
72
78
Midnight
± 8 (10)
± 7 (10)
± 3 (12)
± 10 (8)
"The boldfaced values are significantly higher than others of the same age group at a probability level of P £ 0.01.
The number of preparations in each group is listed in parentheses.
Table V. Number of ;<H-labeled cells at different times of day in rats
of different ages
3
H-celh ± S.E.
Age
6 vvk.
6 mo.
1 yr.
6
A.M
2,281 ± 35 (12)°
463 ± 20 (8)
571 ± 32 (8)
12
1,804 ±
583 ±
566 ±
Noon
42 (18)
27 ( 8 )
39 ( 9 )
6 P.M.
2,262 ± 47 (7)
621 ± 37 (10)
866 ± 82 (10)
1
12 Midnight
2,316 ± 73 (10)
577 ± 19 (12)
491 ± 78 (8)
°The boldfaced values are significantly higher than others of the same age group at a probability level of P g 0.01.
The number of preparations in each group is listed in parentheses.
Table VI. Ratio of 3H-labeled cells to mitoses at different times of day in rats
of different ages
H/M ± S
Age
6 vvk.
6 mo.
1 yr.
6 A.M
7.6 ± 0.2 (12)°
4.3 ± 0.3 (8)
4.0 ± 0.3 (8)
12 Noon
8.6 ± 0.2 (18)
5.9 ± 0.6 (8)
3.8 ± 0.2 (9)
6 P.M.
11.6 + 0.7 (7)
10.5 + 0.8 (10)
9.2 + 0.4 (10)
12 Midnight
8.0 ± 0.4 (10)
8.4 ± 0.6 (12)
6.2 ± 0.3 (8)
Average
8.9
7.3
5.8
"The number of preparations in each group is listed in parentheses.
ratio of labeled cells to mitoses (Table
VI). Periods of low mitotic activity are
characterized by high H/M values and
periods of high mitotic activity by low
H/M values. The low ratios recorded in
the morning hours for the older animals
were offset by higher figures during evening hours. When the average daily ratios
were calculated for each age group the decrease in the number of labeled cells relative to mitoses, though still apparent, was
less marked than in the series of animals
killed in the forenoon.
Discussion
Sippel7 has presented correlations between the weight of the rat lens and age,
and between lens weight and surface area
from which a growth curve for the surface
area of this lens can be constructed. The
expansion of epithelial area with age,
directly measured in flat mounts, closely
parallels that of the total surface area as
calculated by Sippel. It is, thus, related
to lens growth. Despite the rapid enlargement of the lens during the first year of
life, the size of the epithelial population
is remarkably stable. The small increase
in number of epithelial cells between 2
and 8 weeks of age indicates that some of
the proliferative activity during this interval contributes to an increase in the
epithelial population. After 8 weeks of age
the number of epithelial cells remains unchanged and signs of cell death are infrequent. Disregarding replacement of the
apparently few cells lost through death,
each mitosis corresponds to migration of a
cell from the epithelium in the course of
differentiation to a lens fiber. In this age
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Volume 5
Number 6
Effect of age on cell division of lens epithelium 565
span, then, the number of mitoses in the
normal rat lens is directly related to the
rate of fiber formation. The high incidence
of cell degeneration and death observed in
very old rats1 may alter the "steady-state."
Messier and Leblond8 classified the lens
epithelium as a slowly expanding population. By their own criteria such a tissue is
characterized by a low labeling index and
indefinite persistence of radioactive cells.
New cells are permanently added to the
population. They noted that the lens epithelium was an unusual type of expanding
population in that 3H-labeled cells migrated to become lens fibers. It appears
more reasonable to call the lens epithelium,
in adult life, a steady-state renewal system,
as suggested by Srinivasan and Harding0
with cell birth balanced by loss of cells
through differentiation.
The observed decrease in both mitotic
activity and 3H-thymidine incorporation
during the first year of life confirms the
reports of previous workers. The 48 per
cent reduction in number of mitoses between the ages of 6 weeks and 6 months
is comparable to the 33 per cent decrease
reported by us1 between the ages of 6 to
7 weeks and 20 weeks, and to the 33 per
cent reduction noted by Cotlier2 between
7 to 8 weeks and 16 weeks. Although we
found no further reduction by 1 year,
Cotlier described an additional 40 per cent
decrease. Mikulicich and Young3 determined both mitotic index and labeling
index in a group of very young rats. Their
results were based upon counts from
sagittal sections of the lens. The mitotic
index fell approximately 83 per cent between birth and 5 weeks of age with a
decrease of 63 per cent in the labeled cell
index. These values correspond to the rapid
reduction of proliferative activity that we
recorded during the same age interval. At
ages where data from the two studies can
be directly compared, the mitotic indices
of Mikulicich and Young were 4 to 6 times
greater and the 3H-labeled cell indices
were 3 times larger than those obtained
in our series of animals. Hanna and
O'Brien10 reported a reduction in the number of labeled cells in sections of the rat
lens from birth to 10 months af age. Since
they did not state the total number of cells
counted per section, no basis for comparison was provided, and the significance of
the reduction cannot be judged. Discrepancies in data from different laboratories may result from real differences in
proliferative activity in rats of different
strains or may be caused by fluctuation of
activity at varying times of day. They may
also arise from differences in experimental
procedure such as, counting small portions
of the population in sections as opposed to
total counts of flat mounts.
The change in the ratio of labeled cells
to mitoses with increasing age suggests
some alteration in timing of the division
cycle. The number of mitoses and 3Hlabeled cells in a population is assumed to
be proportional to the respective durations
of the M and S phases. The assumption,
however, requires that the cells divide
asynchronously. Some degree of synchronization of division was known to exist in
the lens epithelium of young rats since the
rate at which cells entered mitosis changed
during the day.'1 Scullica and Zoldan11 were
unable to detect a statistical difference between mitotic counts at noon and midnight
in a small number of Wistar rats, but the
existence of a daily periodicity of mitotic
activity was confirmed in the present study.
Data obtained from the comparison of
diurnal fluctuation in mitosis and 3Hthymidine incorporation in rats of different
ages demonstrated that the H/M ratio
varied over the day because of time dependent changes in mitotic counts. No consistent pattern of diurnal variations in 3Hthymidine incorporation was seen. A 6 hour
shift of peak mitotic activity between the
ages of 6 weeks and 3 to 12 months accounted to some extent for the reduction
of 3H-cells relative to mitosis during the
morning hours in older animals.
Alteration of the diurnal rhythm of
mitotic activity with age has also been
observed in the corneal endothelium of
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Investigative Ophthalmology
December 1966
566 von Sallmann and Grimes
young rabbits.12 When early morning and
late afternoon counts were compared in
this tissue, the fluctuation was twice as
great in 10-week-old rabbits than in 2week-old rabbits. Since only two time
periods of the day were studied in these
experiments, what appeared to be an
exaggeration of the daily variation in
mitotic counts with age have been due to
a time shift in maximum and minimum
activity such as occurs in the rat lens.
When all H/M values obtained at 6 hour
intervals during the day are averaged for
rats of different ages, older animals still
show a lower number of labeled cells relative to mitoses. While it is possible that
the duration of the M and S phases do
change with age, Cotlier- found there was
no difference in the duration of mitosis
between 7- to 8-week-old and 1-year-old
rats. The remaining explanation of the discrepancy on this basis is a more rapid rate
of DNA synthesis in older animals which
is unlikely. Although no consistent pattern
of diurnal variations in 3H-thymidine incorporation was seen in the rat lens epithelium in this experiment, examinations at
shorter intervals during the day may reveal
that rhythmic fluctuations do exist. Undetected periodicity of DNA synthesis could
contribute to a fuller explanation of the
differences in H/M values in young and
old animals.
Daily fluctuations in the number of
nuclei in DNA synthesis have recently
been observed in several tissues of the
mouse.8- J3> 14 A rhythmic uptake of 3Hthymidine by urodele larval epidermis
over the 24 hour period has also been reported.15 Pilgrim, Erb, and Maurer13
demonstrated that a peak in 3H-labeled
cell index preceded the peak mitotic index
by approximately 12 hours in various
epithelia of the digestive tract. The time
interval between the two maxima corresponded to that expected from available
knowledge of the durations of the S and
Ga phases in these tissues. The authors
concluded that the causes of fluctuation in
the mitotic index are complex, but are due,
partially at least, to synchronization of Sphase cells. In the case of mouse corneal
epithelium11 the peak of 3H-labeled cell
index coincided with the peak mitotic
activity and the periodicity of DNA synthesis could not be related to that of mitosis
in terms of the cell cycle.
The complexities inherent in studies of
cell proliferation in the living animal receive emphasis from the results of this
investigation of diurnal variations and age
effects in the lens epithelium. The influence
of such factors should be considered carefully in evaluating normal proliferative
capacity of cell populations and in interpreting the changes produced by experimental interference with the system.
REFERENCES
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O., Welch, C , Kimura, S. F., Munoz, C M . ,
and Drungis, A.: Effects of high energy
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2. Cotlier, E.: The mitotic cycle of the lens
epithelium. Effect of age and galactose, Arch.
Ophth. 68: 801, 1962,
3. Mikulicich, A. C , and Young, R. W.: Cell
proliferation and displacement in the lens
epithelium of young rats injected with
tritiated thymidine, INVEST. OPHTH. 2: 344,
1963.
4. von Sallmann, L., Grimes, P., and McElvain,
N.: Aspects of mitotic activity in relation to
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5. Scullica, L., Crimes, P., and McElvain, N.:
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6. von Sallmann, L., Caravaggio, L., Munoz, C ,
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radiosensitivity of the lens, Am. J. Ophth.
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7. Sippel, T. O.: Energy metabolism in the
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4: 452, 1965.
10. Hanna, C , and O'Brien, J.: Cell production
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Volume 5
Number 6
Effect of age on cell division of lens epithelium 567
and migration in the epithelial layer of the
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11. Scullica, L., and Zoldan, T.: Modificazioni
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del ratto in different! condizione ambientali,
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12. von Sallmann, L., Caravaggio, L., and Grimes,
P.: Studies on the corneal endothelium of
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13. Pilgrim, C , Erb, W., and Maurer, W.: Diurnal
fluctuations in the number of DNA svn-
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14. Chumak, M. C : A study of the mitotic cycle
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15. Scheving, L. E., and Chiakulas, J. J.: Twentyfour hour periodicity in the uptake of tritiated
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