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Propagation and prolongation of mitotic activity in the formation of injury-induced lentomas in Rana pipiens.

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Propagation and Prolongation of Mitotic Activity
in the Formation of Injury-induced Lentomas
in R a m pipiens '
NANCY S . RAFFERTY
Department of Anatomy, The Johns Hopkins University
School o f Medicine, Baltimore, Maryland
ABSTRACT
Autoradiography following labeling with H-3-thymidine was used to
study the pattern of growth of injury-induced lentomas in Rana pipiens. This reaction
to injury was compared with the more controlled reaction in the rabbit lens, which
has been extensively studied by others.
These studies show that, unlike that of the rabbit, the entire lens epithelium of
the frog reacts within a day to the injury stimulus by disarrangement of its normal
architecture. H-3-thymidine is incorporated between 24 and 40 hours post-injury,
followed subsequently by widespread mitotic activity. Peak thymidine incorporation
occurs at three days and peak mitosis a t five days post-injury. However, the stimulus
to mitotic activity is propagated after the first week largely among the superficial cells
of the enlarging lentoma and adjacent normal epithelium; DNA synthesis and mitosis
i n the deeper cells of the lentoma diminish at a week and cease by two weeks postinjury, and by three weeks in the superficial cells.
It is concluded that the frog lentoma is formed by accretion from the surface as a
result of extensive multiplication of cells throughout the injured epithelium. The
reaction of frog and rabbit lenses to injury differs both in the extensiveness of the
initial reaction and the duration of the stimulus to proliferation.
A penetrating mechanical injury of the
lens of adult Rana pipiens initiates widespread proliferation of the cells of the subcapsular epithelium. The accumulation of
cells in the wound area beginning on the
third and fourth day after injury results in
a dense epithelial growth which, because
of its cytology and behavior in invading the
lens fibers, was designated a lentoma or
lens growth (Rafferty, '63). The cells of
the lentoma are intensely active for a few
weeks following the injury: mitoses are
numerous and cytoplasmic basophilia is
pronounced. This activity then gradually
decreases and degenerative changes in the
cells become apparent. Although an occasional lentoma has been found to persist
for a year after the initiating injury, most
disappear, to be replaced by normal lens
fibers after three months. It is not known
whether the lentoma is a self-limiting benign tumor, a reparative growth, or a peculiar hyperplasia of long duration. Whichever it is, it has not been observed following injury to the rabbit lens (Harding,
Donn and Doblisrinivasan, '59; Harding
and Srinivasan, '61 ; Harding, Feldherr and
Srinivasan, '61 ) , and is perhaps unique to
amphibians.
ANAT. REC..153: 111-128.
Autoradiographic techniques following
injection of H-3-thymidine have been used
to study the growth pattern of the lentoma
and the movement of the cells involved in
its formation. The results reported here
show that the injury stimulus to proliferation is propagated for the first few days
among the cells in all regions of the lens
epithelium (except near the bow where
fiber differentiation is beginning); and for
the next two weeks among both the cells
of the normal epithelium adjacent to the
enlarging growth and the most superficial
cells of the growth, which continue dividing to form the lentoma. The originally
injured cells which in time come to lie
deepest in the lentoma do not continue to
proliferate, or do so very slowly after a
week post-injury. The superficial cells of
the lentoma cease DNA synthesis in most
cases between two and three weeks after
injury, although the lentoma then persists
for several months.
1 This investigation was supported i n part by Public
Health Service Research grant NB-056 35-01 from the
National Institute of Neurological Diseases and Blindness and i n part by a n Institutional Research Grant
f r o 4 The Johns Hopkins University (allocation from
NIH General Research Support grant 1-Sol-FR005378-01).
111
112
NANCY S. RAFFERTY
MATERIALS AND METHODS
The lenses of anesthetized Rana pipiens
(Alburg, Vermont) were injured by sticking a 27-gauge needle into either the midcentral zone or the central zone near the
pupillary margin of the iris. However,
lenses used for preparation of whole mounts
were injured with an insect pin, 0-gauge,
in the mid-central zone, in order to inflict
a smaller wound which facilitates stripping off the epithelium. The corneal wound
was covered with sulfathiazole. The frogs
were kept at room temperature (22-25°C).
Tritium-labeled thymidine (New England
Nuclear Corp.; specific activity, 6.7 c/mM)
was injected intraocularly in the earlier
experiments in a dose of 5 to 9 pc, and in
later experiments, intraperitoneally in a
dose of 1 uc/gram body weight. Intraperitoneal injection of the label was found to
be easier since the animals did not have to
be anesthetized; and more accurate since
leakage did not occur as it often did from
the anterior chamber. In addition, the
intraperitoneal route avoided possible second injury to the lens. However, results
obtained using either injection site were
comparable.
Two groups of experiments were performed: (Group 1 ) In order to locate the
cells that contribute to the lentoma (i.e.,
those preparing to proliferate), and to determine the time of maximum DNA synthesis, the H-3-thymidine was injected at
varying intervals up to a month after the
injury, and the lenses fixed four hours
later. Whole mounts and sectioned material were prepared from this group.
(Group 2 ) In order to follow the movement of the proliferating cells, labeled
thymidine was injected 72 hours after injury (the time that was determined by the
first group of experiments to be the peak
of thymidine uptake) and the lenses fixed
at daily and weekly intervals thereafter.
Sectioned material only was prepared from
this second group.
The eyes were enucleated, fixed in
Bouin's solution overnight, embedded in
paraffin, and serially sectioned at 8 ~ton
the equatorial plane. In addition, some
lenses were sectioned sagitally in order to
determine the labeling pattern of cells
away from the wound near the bow.
Autoradiograms were prepared by dipping the slides in NTB2 Kodak emulsion
and developed, following a two week exposure, according to the methods recommended by Kopriwa and Leblond ('62).
The sections were stained in Delafields
hematoxylin for 15 minutes.
Whole mounts of lens epithelium of
group 1 eyes were prepared according to
the method of Harding, Hughes, Bond and
Schork ('60) after overnight fixation of
whole lenses in cold 3: 1 absolute alcohol:
glacial acetic acid. Autoradiograms were
prepared as for the sectioned material.
RESULTS
Group 1. Lenses fixed four hours
after injection o f H-3-thymidine
The overall picture of the response of the
lens epithelium to the injury stimulus is
shown in the whole mount autoradiograms
in figures 2-7. The earliest detectable reaction to the injury is a disarrangement of
the cells as shown in the 24-hour specimen
(fig. 2 ) , but seen as early as 18 hours.
At 18 and 24 hours after injury there is no
increase in incorporation of H-3-thymidine
relative to the control (uninjured) lens
epithelium. (Labeled cells in the control
lens have a typical distribution in the proliferative zone as described by Mikulicich
and Young, '63, in the rat; and Thomson,
Pirie and Overall, '62, and Harding,
Hughes, Bond and Schork, '60, in the rabbit.) There is a lag of more than 24 hours
and less than 40 hours after the injury
before a noticeable uptake of labeled thymidine occurs (fig. 3). At 48 hours a significant increase in H-3-thymidine incorporation has occurred in all regions of. the
epithelium except near the bow where lens
fiber formation takes place (fig. 4 ) . At
72 hours after injury the labeled thymidine
is heavily incorporated into more than half
of the nuclei and mitotic figures have increased in number (figs. 1, 5). DNA synthesis gradually falls off in the next few
days (fig. 6), and on the seventh day most
of the labeled nuclei are confined to the
normal proliferative zone of the epithelium
and to the area adjacent to the injury
where the cells have accumulated to form a
lentoma (fig. 7). Mitoses are seen to reach
a peak in number around the fifth day after
injury. The mitotic figures are unlabeled
MITOTIC ACTIVITY I N LENTOMA FORMATION
since the cells are exposed to the H-3thymidine only for four hours before fixation and the dividing cells which are seen
in the obvious mitotic phases may not
have been in the synthetic stage at the time
of the injection of the labeled thymidine.
The whole mount autoradiograms, therefore, show that peak DNA synthesis occurs
at about three days after injury, that a l l
regions of the epithelium except near the
bow are at first involved, and that at a
week after injury the reactive cells are
beginning to be confined to the wound area.
More precise data were determined from
sectioned material and are shown in table 1.
Counts were made of labeled nuclei appearing in the lens epithelium and lentoma
of each 8 v section and the average of
usually ten sections of each lens calculated.
The sections used were those through the
maximum area of the lentoma. Although
there is considerable scatter in the data, a
peak of DNA synthesis at around 72 hours
is apparent. Again, as visualized in the
whole mount autoradiograms, the initial
lag in thymidine incorporation is shown,
and the gradual return to the normal or
subnormal activity at 3 to 4 weeks after
injury. Counts of mitotic figures show a
similar increase with time after injury although the data show more variation. As
expected, the increase in mitotic rate is
preceded by an increase in nuclear labeling.
The conspicuous exceptions to the general trend that occurred in some time
categories (table 1) represent either experimental error or possibly individual
variation in reactivity to injury, or both.
The low number of labels counted in some
specimens (table 1, footnote 2 ) , for example, could represent the 15% of R.
pipiens lenses that do not produce lentomas
following injury (Rafferty, '63). On the
other hand, the exceptionally high counts
(table 1, footnote 3) may represent those
lenses which form growths that persist for
periods of time longer than the average.
Such exceptional lens epithelia were also
occasionally seen in the whole mount preparations with respect to the numbers of
labeled nuclei or mitoses or both (cf. figs.
1 and 5 ) , and similar variation has been
reported in the rabbit lens (Harding, Rothstein and Newman, '62). It should also
be pointed out that a source of experi-
113
mental error could be small variations in
the plane of sectioning; since the equatorial region has relatively high mitotic
activity normally, a plane of section which
includes this region would give somewhat
higher counts than a plane which excludes
it.
The temporal distribution of label in the
injured lens epithelium indicates which
cells continue to contribute to the formation of the lentoma. In the first few days
after injury numerous cells in all regions
of the epithelium incorporate H-3-thymidine as do many of the cells throughout the
small lens growth (figs. 3,4,5,9,10). At
five and seven days, however, it is seen
that the deeper cells of the lentoma are
more sparsely labeled, whereas the more
superficial cells of the growth and the cells
in the adjacent epithelium are heavily
labeled (figs. 11,12). With increasing time
after injury fewer of the deeper cells incorporate the labeled thymidine, until at
14 days the lentoma is unlabeled except at
the periphery adjacent to the normal epithelium (fig. 13). At 21 and 28 days after
injury (fig. 14) there is no label found in
the growth and very little in the adjacent
epithelium.
Group 2. Lenses fixed at varying
intervals a f t e r injection of
H-3-thymidine
The movement of the large numbers of
reactive cells labeled on the third day after
injury to the lens was followed by fixing
the lenses at different periods of time after
injection of H-3-thymidine. More than half
of all the cells in the injured epithelium
and the developing lentoma are heavily
labeled at first and the label remains heavy
in these areas for about two weeks. At two
weeks, however, fewer of the cells in the
normal epithelium contain label and the
label is not as dense as earlier. More
heavily labeled cells are seen in the lentoma. At three and four weeks after injection of the H-3-thymidine the thinning of
the label in the normal adjacent epithelium
has progressed, whereas the deeper cells
of the lentoma are still heavily labeled (fig.
15), although not so densely as two weeks
previously.
These observations are interpreted to
support the conclusions derived from the
6
5
93
146
133
10
10
10
I.P.
I.P.
I.P.
6
VA
VB
VB
5 days
19.9
20.3
7.9
0.5
1.1
2.6
214
267
48
10
6
10
1.0.
I.P.
I.P.
4
4
4
IIIB
VB
VA
4 days
0
3.2
0.4
4.1
11.0
5.5
403
285
325
650
473
464
7
10
10
10
10
10
1.0.
I.P.
I.P.
I.P.
I.P.
I.P.
3
1
1
2
2(R)
2(L)
IIIB
VA
VB
VB
VIII
VIII
0
0
0.2
0
14.9
16
0.1
0.3
1.2
0.8
1.2
8wection
Average
no. mitoses/
96
37 2
122
72 hrs
10
5
10
1.0.
1.0.
I.P.
I 2
IIIB 2
VIII 3(R)
13
18
10
10
1.0.
1.0.
- -
Average
c
i,Z$F
8B-sectlon
15
26
12
18
- ___
No.
sections
counted
10
10
10
10
10
1.0.
1.0.
1.0.
I.P.
I.P.
48 hrs
1
1
I 1
I 2
I 3
VIII 1 ( L )
VIII 1 ( R )
of label 1
Site of
injection
IIIA
IIIB
Uninjured
controls
Exp.
No.
24 hrs
~
Time after
injury when
Iabel was
injected
~
Two small lens growths.
Large lens growth. Figure 11.
Medium lens growth.
Small lens growth.
Epithelium thickened.
Epithelium normal, lens growth not present.
Small lens growth.
Epithelium thickened.
Small lens growth. Figure 10.
Epithelium thickened. Large lens growth.
Medium lens growth.
Large lens growth.
Epithelium thickened.
Epithelium normal.
Small lens growth at wound. Figure 9.
Epithelium normal.
Epithelium normal. Figure 8.
-
Remarks
-
TABLE 1
Temporal uptake of H-3-thymidine and mitotic counts in control and injured lens epithelium. Lenses fixed four hours after injection of label
3
IIIA
7 days
10
10
10
I.P.
I.P.
I.P.
VA 16
VB 15
VB 16
I.O.,intraocular injection; I.P., intraperitoneal injection.
Lenses showing exceptionally few labeled nuclei.
3 Lenses showing exceptionally numerous labeled nuclei.
2
1
10
I.P.
VA 15
28 days
~
10
10
10
10
I.P.
I.P.
I.P.
I.P.
11
12
11
12
VA
VA
VB
VB
21 days
10
10
10
10
1.0.
1.0.
I.P.
I.P.
14
10
6
6
I
IIIB
VB
VB
3
10
10
10
10
10
10
10
10
10
1.0.
I.P.
I.P.
I.P.
I.P.
1.0.
I.P.
I.P.
I.P.
I.P.
14 days
IIIB 5
VA 9
VA 13
VB 9
VB 13
7
8
7
8
VA
VA
VB
VB
6 days
2
6
4
34
14
12
6
9
298
45
44
22
58
93
86
86
61
395
61 2
119
101
133
0.2
0.3
0.4
0.7
0.4
1.0
0.3
0.5
2.9
0.5
2.3
2.7
5.6
10.0
8.5
12.5
7.5
8.6
9.2
13.5
4.5
13.0
Medium lens growth labeled at surface only, degenerating centrally.
Scattered degenerating cells of lens growth, not
labeled.
Large lens growth not labeled, some cells degenerating.
Scattered degenerated cells of lens growth.
Small lens growth, not labeled.
Small lens growth, not labeled.
Small lens growth, not labeled.
Large lens growth, not labeled. Figure 14.
Large lens growth. Epithelium thickened.
Medium lens growth.
Medium lens growth.
Large lens growth. Figure 13;not labeled.
Epithelium very thickened.
Medium lens growth.
Small lens growth.
Large lens growth. Figure 12.
Large lens growth.
Large lens growth.
Large lens growth.
Medium lens growth.
Large lens growth.
Medium lens growth.
Epithelium thickened. Large lens growth.
5
4
F=I
29
E
0
H
8
r
5
116
NANCY S. RAFFERTY
Fig. 1 Autoradiogram of a whole mount of injured lens epithelium from a frog injected intraperitoneally with H-3-thymidine 72 hours after injury, and fixed four hours later. Focus was made
on the mitoses which are unusually numerous in this preparation; grains over nuclei which incorporated H-3-thymidine are out of focus. X 480.
Group 1 experiments that the deeper cells
of the lentoma represent the originally injured cells, and that these do not proliferate as continuously or rapidly as the more
superficial cells and adjacent epithelial
cells. If there is little or no turnover of
DNA in non-dividing cells, then the heavier
label in the deeper cells indicates that these
are dividing very slowly if at all after
attaining a position away from the surface;
and the diluted label in the adjacent epi-
thelium suggests that these cells are dividing more rapidly or constantly, and are the
continuing source of cells to the enlarging
lentoma. In addition to adding new cells
to the lentoma, cells of the rapidly dividing
epithelium move from the central zone
toward the proliferative zone where further
division would dilute the label. Nuclei containing dilute label were seen in the bow
region three and four weeks after injection
of the thymidine.
MITOTIC ACTIVITY IN LENTOMA FORMATION
117
DISCUSSION
the nuclei in all regions of the epithelium
Previous findings (Rothstein, Weinsieder are labeled and are presumably preparing
and Blaiklock, '64; Rafferty, '63) and those for division. Peak mitotic activity in the
reported here with the injured frog lens are frog lens epithelium is seen five days after
strikingly different from those obtained by injury. It should be pointed out that the
Harding and his cc-workers ('59, '61) in two-day interval between peak uptake of
the rabbit. The greatest difference is ob- H-3-thymidine and peak mitosis does not
served in the mitotic kinetics of the injured necessarily indicate an extraordinarily long
epithelium. In the rabbit lens epithelium Gz period (post-synthetic phase) in the cell
the injury stimulus, as manifested by the cycle of injured frog lens epithelium. From
appearance of radioactive nuclei followed the observation of the sharp rise in mitosis
by cells in mitosis, moves outward from the on the fifth day post-injury from near a
wound as a band at the rate of 17 v/hour base value at four days, it appears unlikely
(Harding and Srinivasan, '61); but in the that the dividing cells seen on the fifth day
frog, the entire lens epithelium appears to represent a second mitosis. However, the
react simultaneously. The first observable true peaks of DNA synthesis and mitosis
reaction is a disarrangement of the normal may not have been determined, since the
architecture of the epithelium within a day points were taken at 24-hour intervals. If,
post-injury. This is followed a day later by for example, the true peak of DNA synwidespread DNA synthesis (also Rothstein, thesis were closer to the fourth day rather
Weinsieder and Blaiklock, '64), and sub- than the third day after injury, and the
sequently by mitosis. Thus the mitotic mitotic peak were closer to the fourth
stimulus in the frog lens does not move rather than the fifth day, the interval beoutward from the wound as a slowly prog- tween the S (synthetic) and M (mitotic)
ressing wave but affects cells in all regions phases, which is the G2 period, may be conof the epithelium at about the same time. siderably shorter than that implied by the
From the observation that more than half data reported here. The Gz period of the
of the nuclei are labeled at 72 hours post- cell cycle of numerous mammalian tissues
injury, it is strongly suggested that the in vivo is in the range of 1 to 4 hours, but
initial mitotic activity tends to be syn- may be as long as a day in the regenerating
limb blastema of Triturus viridescens (Hay
chronous.
However, the actual onset of cellular and Fischman, '61 ).
In addition to these differences in the
proliferation is initiated earlier after injury
in the rabbit lens. Whereas the rabbit lens early response to injury, the stimulus for
has been shown to begin incorporating H-3- multiplication persists longer in the cells
thymidine at 16 hours after injury (Har- of the frog epithelium. The lens epithelium
ding and Srinivasan, '61), the injured frog of the rabbit ceases proliferation when the
lens has a preincorporation period of be- wound area is healed over, whereas the
tween 24 and 40 hours (or 24 to 48 hours: frog lens epithelium continues to prolifsee Rothstein, Weinsieder and Blaiklock, erate for at least two weeks. This does not
'64). For the sake of comparison with represent merely a slower response, since
another amphibian, in the regenerating the accumulating cells in the wound area
limb blastema of Triturus viridescens, Hay invade the lens fibers to form a prominent
and Fischman ('61) found that uptake of lentoma which is added to by division of
H-3-thymidine began 4 to 5 days after am- the cells at the surface of the lens growth
putation in the dedifferentiating inner and in the adjacent normal epithelium.
tissues of the stump. In that system, how- The deeper cells of the frog lentoma are
ever, the delay between the time of injury the first to cease mitosis, as shown by
and the time of DNA synthesis may result cessation of thymidine uptake between one
from the necessity of the inner tissues to and two weeks after injury. The last cells
to cease division appear to be the surface
dedifferentiate before they proliferate.
Peak DNA synthesis occurs one day after cells of the lentoma.
In summary, therefore, it is suggested
injury in the rabbit (Harding, Donn and
Doblisrinivasan, '59) and three days post- that the formation of lentomas in the frog
injury in the frog, when more than half of results either from the rapidity with which
118
NANCY S. RAFFERTY
the mitotic stimulus is propagated throughout the whole epithelium, or from the prolonged duration of the mitotic stimulus, or
both. In connection with the first possibility it should be emphasized that although
the disarrangement of the epithelium is
noted soon after injury, the actual onset of
DNA synthesis does not begin until almost
two days after injury; but when it begins,
it begins everywhere in the epithelium, and
not only in the region immediately adjacent to the wound as in the rabbit. The
dividing cells are accumulated in the
wound area as a result of the mitotic pressure, invaginating, in most cases, as more
cells are added at the surface. As regards
the duration of mitotic activity, it is clearly
prolonged in the injured frog lens epithelium compared with that of the rabbit, and
most likely is a contributing factor in the
formation of lentomas in the frog.
That the differences in behavior of rabbit and frog lens following injury are both
real and species-related seems probable
because of the general consistency in observations of different workers. However,
a discrepancy in reports dealing with the
frog is apparent in the recent report of
Rothstein, Weinsieder and Blaiklock ('64)
who failed to observe mitoses as a sequel
to thymidine uptake following injury. This
result may be explained, however, by the
authors' incubation of lenses in a defined
medium for two and one-half hours before
fixation. Moreover, a previous report (Harding, Rothstein and Newman, '62) indicates
that serum supplementation supports significant mitotic activity of rabbit lenses
cultured for prolonged periods. The in vivo
experiments reported here on Rana pipiens
clearly show that a great increase in
mitoses occurs three to seven days following injury, and although there is considerable variation in the number of dividing
cells between different lenses, a 10- to 20fold increase is not unusual.
It is of some interest that lentoma formation was not noted in the bullfrog experiments, which were terminated after five
days (Rothstein,Weinseider and Blaiklock,
'64), the period at which beginning lentoma formation is seen in R. pipiens. However, the scar formation observed at that
time (fig. 5, Rothstein, Weinsieder and
Blaiklock, '64) is similar in appearance to
the five-day lens growth reported here for
R. pipiens (fig. 6 ) , suggesting strongly that
the injury response in both species may be
the same.
Although this study of the growth pattern of the frog lentoma adds little to our
understanding of its nature, the fact that
the lentoma is enlarged by accretion of
superficial and adjacent cells of the normal epithelium rather than by continual
division of the deeper and presumably
originally injured cells is curious and probably significant. It might be argued that
diffusion of the labeled nucleotide to the
deeper cells of the growth in the temporal
experiments may be inadequate resulting
in apparent cessation of DNA synthesis.
But, in fact, in these experiments the number of mitoses among the deeper cells also
is seen to decrease until none are observed
at three weeks post-injury. Moreover, the
injured cells which were labeled at the
peak of DNA synthesis and allowed to migrate deep within the growth show little
dilution of the label which would be expected if repeated mitosis were occurring.
Cessation of proliferation among the
cells lying deep within the lentoma may,
however, result from inadequate diffusion
of metabolites inwards and outwards, since
diffusion or active transport between the
aqueous and lens are the mechanisms normally involved in the nourishment of the
avascular lens.
LITERATURE CITED
Harding, C. V., A. Donn and B. Doblisrinivasan
1959 Incorporation of thymidine by injured
lens epithelium. Exp. Cell Res., 18: 582-585.
Harding, C. V., W. L. Hughes, V. P. Bond and
P. Schork 1960 Autoradiographic localization
of tritiated thymidine in whole-mount preparations of lens epithelium. A.M.A. Arch. Ophthal.,
63: 58-65.
Harding, C. V., and B. D. Srinivasan 1961 A
propagated stimulation of DNA synthesis and
cell division. Exp. Cell Res., 25: 326340.
Harding, C. V., C. Feldherr and B. D. Srinivasan
1961 The distribution of DNA-synthesizing
cells in lens epithelium following injury. In:
The Structure of the Eye. G . Smelser, ed. Academic Press, N. Y., pp. 273-282.
Harding, C. V., H. Rothstein and M. B. Newman
1962 The activation of DNA synthesis and cell
division in rabbit lens in uitro. Exp. Eye Res.,
I : 457-465.
Hay, E. D., and D. A. Fischman 1961 Origin
of the blastema in regenerating limbs of the
MITOTIC ACTIVITY IN LENTOMA FORMATION
newt Triturus viridescens. Devel. Biol., 3: 2659.
Kopriwa, B. M., and C. P. Leblond 1962 Improvements in the coating technique of radioautography. J. Histochem, Cytochem.,
269284.
Mikulicich, A. G., and R. W. Young 1963 Cell
proliferation and displacement in the lens epithelium of young rats injected with tritiated
thymidine. Invest. Ophthal., 2: 344-354.
119
Rafferty, N. S.
1963 Studies of an injuryinduced growth i n the frog lens. Anat. Rec.,
:'41
299-312*
Rothstein, H., A. Weinsieder and R. G. Blaiklock
1964 Response to injury in the lens epithelium
of the bullfrog (Rana catesbeiana). Exp. Cell
Res., 35: 548-556.
Thomson, D. S., A. Pirie and M. Overall 1962
Autoradiography of lens epithelium after parenteral injection of tritiated thymidine. A.M.A.
Arch. Ophthal., 67: 464-469.
PLATE 1
EXPLANATION OF FIGURES
Autoradiograms of radial segments of whole mounts of lens epithelium
showing the distribution of labeled nuclei a t various times after injury.
X 150.
120
2
Twenty-four hour injured lens epithelium showing incorporation of
H-3-thymidine restricted to proliferative zone in a pattern typical of
control lens. Note, however, disarrangement of cell pattern which i s
noted also 18 hours after injury. (PZ, proliferative zone; CZ, central
zone; Wy, wound out of field.)
3
Forty-hour injured lens epithelium showing incorporation of label by
cells throughout all regions of the epithelium. Two mitoses shown
(arrows).
4
Forty-eight-hour injured lens epithelium showing a n increase in number of radioactive nuclei in all regions of the epithelium. Four mitoses
present (arows).
MITOTIC ACTIVITY I N LENTOMA FORMATION
N a n c y S. Rafferty
PLATE 1
121
PLATE 2
EXPLANATION O F FIGURES
Autoradiograms of radial segments of whole mounts of lens epithelium
showing the distribution of labeled nuclei at various times after injury.
x 150.
5
Seventy-two-hour injured lens epithelium showing peak uptake of
H-3-thymidine throughout all regions, and accumulation of cells adjacent to wound (top). Mitoses present (arrows).
6 Five-day injured lens epithelium showing decreased number of labeled
nuclei, numerous mitoses, and thickening lentoma (top).
7
122
Seven-day injured lens epithelium showing restriction of labeled nuclei in the normal proliferative zone, as i n figure 2, and i n the area
adjacent to the lentoma (out of the field at the top).
MITOTIC ACTIVITY I N LENTOMA FORMATION
Nancy S. Rafferty
PLATE 2
123
PLATE 3
EXPLANATION O F FIGURES
Camera lucida drawings of autoradiograms of sectioned lenses labeled
with H-3-thymidine a t varying times after injury showing spatial distribution with time of label (dots) and mitoses ( x ) in the lens growth and
epithelium.
8
Twenty-four hour injured lens. Wound a t left. (15 labeled nuclei;
no mitoses counted.)
9
Forty-eight-hour injured lens showing injury with accumulating cells
a t upper right. (129 labeled nuclei; 16 mitoses counted.)
10
Seventy-two-hour injured lens showing peak uptake of H-3-thymidine
throughout epithelium and developing lentoma ( a t two points in this
lens). (358 labeled nuclei; no mitoses counted.)
11 Five-day injured lens showing decrease in number of labeled nuclei
compared with 72-hour injured lens, and large number of mitoses in
lens growth (LG). (148 labeled nuclei; 30 mitoses counted.)
124
MITOTIC ACTIVITY IN LENTOMA FORMATION
Nancy S. Rafferty
PLATE 3
125
PLATE 4
EXPLANATION OF FIGURES
Camera lucida drawings of autoradiograms of sectioned lenses labeled
with H-3-thymidine a t varying times after injury showing spatial distribution with time of label (dots) and mitoses ( x ) in the lens growth and
epithelium.
12
Seven-day injured lens showing further thinning of labeled nuclei in
deeper area of lentoma, but heavy label still present i n superficial
cells. (174 labeled nuclei; 14 mitoses counted.)
13 Fourteen-day injured lens showing labeled nuclei restricted to superficial cells of lentoma and epithelium (20 labeled nuclei; 3 mitoses
counted. )
14 Twenty-one-day injured lens showing unlabeled lentoma. ( 12 labels;
1 mitosis counted.)
15 Twenty-four-day injured lens of Group 2 experiments in which cells
labeled on third day post-injury were allowed to migrate for 21 days
before fixation. Note heavy labeling of lentoma and thinner labeling
of lens epithelium. Mitoses not present.
126
MITOTIC ACTIVITY I N LENTOMA FORMATION
Nancy S. Rafferty
PLATE 4
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prolongation, induced, mitotic, formation, propagation, lentomas, activity, pipiens, injury, rana
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