The cytoarchitecture of normal mouse lens epithelium.

The Cytoarchitecture of Normal Mouse
Lens Epithelium '
NANCY S . RAFFERTY
Department of Anatomy, Northwestern University Medical and
Dental Schools, Chicago, Illinois 60611
ABSTRACT
The lens epithelium of 30 gm male albino CFl mice was characterized by determining the area dimensions and the mitotic rate of the total
and of the several different regions of whole-mount preparations. A ring of nonpigmented iris cells is found to adhere to the outer surface of the lens cuticle,
which serves to delineate an inner central zone from an outer peripheral zone of
the epithelium. A high number of dividing cells in the peripheral area, especially
immediately adjacent to the meridional rows, but including the area overlain by
the iridial fringe of cells, identifies this wide region as the proliferative zone. The
mitotic rate, furthermore, undergoes marked diurnal variation, rising in the late
evening through the early morning hours and diminishing during the late morning and afternoon hours.
The relative large size of the rabbit,
rat, and frog lens has encouraged their
use in a whole-mount technique, developed by Howard ('52). In this technique,
the entire fixed epithelium covering the
anterior surface of the lens, along with
the overlying acellular cuticle, is removed
€rom the lens fiber mass and flattened
Ionto microscope slides. A variety of cytological, histochemical, biochemical and
autoradiographic studies, appropriate to investigations on cell population kinetics or
cataractogenesis, can thus be performed
on the total cell population comprising
this tissue. Owing perhaps to its small size,
the mouse lens has not been favored as
material for such studies, except by a few
investigators (Hanna, '65; von Sallmann,
'57; von Sallmann et al., '57; Voaden and
Leeson, '70; Weinstock and Stewart, '61).
Mouse lens is reported to be susceptible
to x-ray radiation cataract (von Sallmann,
'57; and von Sallmann, et al., '57) and to
drug-induced temporary opacities (Weinstock and Stewart, '61). Injury-induced
cataract in the mouse is presently under
investigation by the author. In the present
report, the feasibility of whole-mount
preparations of the mouse lens epithelium
is studied and the cytoarchitecture of the
lens epithelium described. The normal
mitotic rate and diurnal variations in the
rate, furthermore, form a standard baseANAT. REC., 173: 225-228.
line for comparison with changes induced
by injury.
It was found that the mouse lens is well
suited to whole-mount studies and, due to
its small size, has the advantage that the
total preparation can be scanned with a
1000-fold magnification (e.g., for mitotic
counts) within a two-hour period.
MATERIALS AND METHODS
This study was done during the summer
months. Carworth Farms (CFl) albino
male mice, weighing between 28 and 30
gm were used. The mice were killed by
cervical dislocation, and the eyes immediately removed and placed into 3 :1 ethanol :
acetic acid for 12 to 24 hours. The eyes
were rinsed and stored in 70% ethanol.
The fixed lens was removed through a
slit in the eye and placed in Delafields
hematoxylin for six to seven seconds to
facilitate visualization and removal of the
epithelium. Removal of the epithelium was
accomplished in tap water under the dissecting microscope ( X 25), as described
previously for the frog lens (Rafferty, '67).
However, the shape of the fixed mouse lens
is so nearly round that the anterior and
posterior surfaces are difficult to distinReceived Sept. 28, '71. Accepted Jan. 24, '72.
1 This investigation was supported by Public Health
Service Research grant 7 R01 EY00698-01 from the
National Eye Institute.
225
226
NANCY S. RAFFERTY
guish. It was observed, however, that the
non-pigmented iris leaves a fringe of cells
at its attachment to the anterior surface
of the lens, and this serves as a marker
of the anterior surface. The detached epithelium was transferred with its cuticle
down to a glass slide and four or five
radial cuts were made to flatten it. After
air drying the preparations were restained
in hematoxylin for 15 minutes, dehydrated, and cover slips mounted.
The diurnal variation in mitotic rate of
the epithelium was determined in mice
killed at four hour intervals, beginning at
2 PM, using three mice per interval. The
number of mitoses and their position with
respect to the adhering iridial fringe, were
counted in the entire lens epithelium. The
total area of each epithelium to the beginning of the meridional rows, the area inside the iridial fringe (central zone), and
the area inside, including the iridial fringe
area, were found from the diameters measured under 25 magnifications with a
B & L stage micrometer (Lines A, B and C
in fig. lb, respectively). The area outside
the iridial fringe (peripheral area), and
the area underlying the iridial fringe were
calculated by appropriate subtractions of
the above measured figures. These calculated areas were compared with areas mzasured directly with a Gelman planimeter
on camera lucida drawings of two mouse
lenses.
OBSERVATIONS
Figure l a illustrates a typical whole
mount preparation of mouse lens epithelium. The adhering iridial fringe is seen as
a darkened ring, separating a central from
a peripheral area (but, of course, the iris
cells are separated from the lens cells by
the lens cuticle). The lens cells of both
areas are polygonal and regular. The
density of the cell population appears to
increase from the central to the peripheral
areas. Near the periphery the epithelial
cells align themselves into orderly meridional rows (fig. 2 ) , and as they approach
the lens bow region their elongation and
differentiation into lens fibers takes place.
The equatorial diameter of the intact
fixed lens is approximately 2.3 mm, and
the polar diameter, 2.1 mm. The average
diameter of the 33 flattened epithelia is
2.90 mm, and the average surface area is
6.59 -+. 0.05 mm'. The central zone within
the circular attachment of the iris has an
average diameter of 1.65 mm and a surface area of 2.17 5 0.12 mm'. The iridial
fringe area averages 0.43 mm'. By subtraction, the peripheral area outside of the
iridial fringe is 3.99 mm2. These dimensions are summarized in figure lb.
Fig. l a Whole-mount preparation of mouse lens epithelium showing adhering iridial fringe (IF),
separating a central zone ( C Z ) from a peripheral area which includes the proliferative zone (PZ),
and which gives way to the meridional rows ( M R ) and lens bow (LB). X 2#5.
Fig. l b Line drawing of the epithelium shown in l a with summary figures of area dimensions
(mm2) of different regions.
227
NORMAL MOUSE LENS EPITHELIUM
Fig. 2 Portion of whole-mount preparation of
mouse lens epithelium showing meridional rows
((top) and mitoses (arrows) in the proliferative
zone. x 250.
These calculated areas agree within
11 % with direct planimeter measurements
d camera lucida outlines: for example,
total area, planimeter measurement is
5.89 mm2,compared with 6.59 mm', calculated area; central area, 2.19 mm2, com-
pared with 2.17 mm'; and peripheral area,
3.70 mm2, compared with 3.99 mm'. The
planimeter measurements are the more
accurate because they exclude the wedgeshaped spaces at the periphery which result from the radial cuts necessary for
flattening the preparations. Areas calculated from diameter measurements include
these spaces and the area of the peripheral
region especially is increased by the latter
method.
The average number of mitoses found
in each region of the epithelium at each
time interval is presented in table 1. If the
mitoses are converted into the number per
mm2, the highest number corresponds to
the peripheral area. This area includes the
normal proliferative zone of vertebrate
lenses. Although dividing cells are found
throughout the peripheral area, the greatest concentration appears just anterior to
the meridional rows. Some mitoses are observed a few cells anterior to the rows
(fig. 2).
The total numbers of mitoses counted
in epithelia obtained at four hour intervals (table 1 ) point to a mitotic peak in
the late evening and early morning hours,
and a decline in mitotic rate in the late
morning and afternoon hours.
DISCUSSION
The normal lens epithelium of male
albino CF1 mice weighing 30 gm is shown
to have a high rate of cell division which
fluctuates diurnally, rising in the late
evening and early morning hours and
diminishing in the daytime hours. This
diurnal variation agrees with that de-
TABLE 1
Average number of mitoses in three regions of mouse k n s epithelium
Time of
sacrifice
Number
of eyes
Peripheral
area
Central
area
Area underlying
iridial fringe
Total
epithelium
6
5
5
6
6
5
226.0225.4
238.42 10.2
283.22 16.1
279.02 15.3
358.8 2 31.5
302.0 f36.4
22.0 2 2.6
12.8f3.1
24.4 f6.3
27.8 2 6.5
3 7 . 6 2 5.7
15.223.2
10.5 2 3.1
10.620.8
15.0?2.2
17.024.7
36.0 6.8
12.0-C3.4
258.5 2 30.2
261.8-C 10.6
322.6" 14.2
323.8 C 19.5
432.5-C31.7
329.2 2 39.2
281.8
23.8
17.3
323.0
2 Pm
6 pm
10 pm
2 am
6 am
10 am
Average number
of mitoses
Average area
(33 epithelia)
Average number
mitosis/mmz
3.99 mm2
70.6
2.17 mm2
10.9
*
0.43 mm2
40.2
6.59 mm2
49.0
228
NANCY S. RAFFERTY
scribed by von Sallmann ('52) in the lens
epithelium of chinchilla rabbits; it does
not agree with the findings in Swiss mouse
lens by Voaden and Leeson ('70), who
found high levels of mitotic activity in the
early morning ( 7 AM) and low levels in
the late evening (10 P M ) . The discrepancy
may result from their use of female mice,
of a different strain, or the fact that their
data did not include the intervals between
10 PM and 7 AM, when the mitotic activity
may have shown the upswing described in
the present report. In any case, if the total
number of mitoses from the six intervals
studied in this report are averaged, a value
of 323 mitoses per lens epithelium is obtained. This value coincides with the midday mitotic averages, which is fortuitous
in planning future experiments in which
diurnal variation may be a critical factor.
The number of mitoses reported in the
total lens epithelium is considerably higher
than that found by von Sallmann et al.
('57) in Swiss strain mice: 323 compared
with 42 mitoses, respectively. This differ'ence probably results from the exclusion
by the latter authors of both early prophases and late telophases, which in this
experiment are included, and from other
differences in their counting techniques
(e.g., their counts were made under lower
magnification). Miki ('61) pointed out that
both body weight and the season of the
year, among other factors, influence the
mitotic rate in the lens epithelium of rats:
the rate increases as the body weight increases, and peaks during the summer
season.
These factors were appreciated by
Hanna ('65) who carefully studied the
patterns of DNA, RNA, and protein synthesis in the developing lens of Swiss mice.
He describes the 180-day or older mouse
lens as having a proliferative zone confined to a narrow band just anterior to the
lens equator, opposite the ciliary processes.
This mature condition evolves from a
broader proliferative zone involving most
of the epithelium of very young mice, and
which with aging contracts to a restricted
band. In the epithelia of 30 gm mice (approximately eight weeks of age) studied
in the present report, the proliferative
zone appears wide with a somewhat higher
concentration of mitoses close to the meridional rows. The area underlying the iridial
fringe should perhaps be included in the
proliferative zone since it has a higher
concentration of mitoses than the adjacent
central area.
The adhering iridial fringe provides a
convenient marker between adjacent areas
of the lens epithelium which otherwise has
a remarkably uniform appearance, though
a different physiological activity. The
mitotic rate in the whole epithelium and
in the different areas delineated by the
iris cells, and the diurnal rhythmicity in
mitoses described in this report form a
normal baseline against which changes induced by injury may be compared.
LITERATURE CITED
Hanna, C. 1965 Changes in DNA, RNA, and
protein synthesis in the developing lens. Invest. Ophthal., 4: 480-491.
Howard, A. 1952 Whole mounts of rabbit lens
epithelium for cytological study. Stain Tech.,
27: 313-315.
Miki, T. 1961 Fluctuation in mitosis count of
lens epithelium; influence of age, season, and
adrenal hormones. Acta SOC.Ophthal. Japan,
65: 2207-2224.
Rafferty, N. S. 1967 Proliferative response in
experimentally injured frog lens epithelium:
autoradiographic evidence for movement of
DNA synthesis towards the injury. J. Morph.,
121: 295-310.
von Sallmann, L. 1952 Experimental studies
on early lens changes after roentgen irridiation.
111. Effect of x-radiation on mitotic activity and
nuclear fragmentation of lens epithelium in
normal and cysteine-treated rabbits. Arch.
Ophthal., 47: 305-320.
1957 The lens epithelium in the pathogenesis of cataract. Am. J. Ophthal., 44:
159-1 70.
von Sallmann, L., L. Caravaggio, C. M. Munoz
and A. Drungis 1957 Species differences in
the radiosensitivity of the lens. Am. J. Ophthal.,
4 3 : 693-704.
Voaden, M. J., and S. J. Leeson 1970 A
chalone in the mammalian lens. 1. Effect of
bilateral adrenalectomy on the mitotic activity
of the adult mouse lens. Exp. Eye Res., 9:
57-66.
Weinstock, M., and H. C. Stewart 1961 Occurrence in rodents of reversible drug-induced
oaacities of the lens. Brit. J. Ophthal., 45:
468214.