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Studies of an injury-induced growth in the frog lens.

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Studies of a n Injury-induced Growth in the Frog Lens'
Department of Anatomy, The johns Hopkins University School of Medicine,
Baltimore, Maryland
A tumor-like growth in the lens of Rana pipiens occurs following
mechanical injury to the lens. The frequency of occurrence and size of the growth
are roughly dependent on the extent of the injury infIicted. The growth fUst appears
four days after wounding, grows actively for three weeks and usually begins to regress
after a month. The cells of the growth appeat to originate by proliferation of the
lens epithelial cells surrounding the wound which grow into and actively invade the
lens fibers. T h e lens growth was not transplantable either homologously into the
anterior eye chamber after dissociation, nor isologously in a subcutaneous site. Protein, LDH, MDH, and G-6-PD patterns obtained after electrophoretic separation in
starch gels were similar for normal and the abnormal lenses. Whether a true tumor
or hyperplasia, the frog lens exhibits an unique reaction to the injury stimulus which
is not found in mammals (man, rabbit, mouse). The controversial tumor immunity
of the mammalian lens appears to be related to an innate resistance of the epithelium
to proliferation which is illustrated in part by the species difference in the reactivity
of the mammalian lens and the amphibian lens to injury.
In an earlier series of experiments (Rafferty, '61), it was observed that the homologous implantation of an embryo into the
anterior chamber of the adult frog eye was
invariably accompanied by a tumor-like
growth in the substance of the host lens.
The growth was characterized by an initially progressive, but subsequently selflimiting invasiveness of the lens fibers.
Mitoses were regularly observed in large
numbers, and intense cytoplasmic basophilia suggested an unusually active protein synthesis in the large, variouslyshaped cells of the growth. The occurrence
of this lens growth was of interest for two
reasons: First, because the lens, in mammals at least, is considered by pathologists
to be one of the few tissues that is refractory to tumors (Mann, '47; Sachs and
Larsen, '48; Hallermann and Meisner,
'54); and secondly, because the association of the lens growth with the embryo
implant suggested that an induction phenomenon may be in operation here. Further study, however, showed that the
growth is stimulated by injury and can
be initiated by merely pricking the lens
with a needle, without the introduction
of an implant.
The uniqueness of this response to injury by the frog lens and the fact that
such a pathology has not been recorded
before for any vertebrate species prompted
further studies to clarify the nature of
the growth, i.e., whether a neoplasm or
hyperplasia; the origin of the cells of the
growth; and its occurrence in other amphibians.
Rana pipiens from Alburg, Vermont
were used for the major part of the work.
In addition, newly metamorphosed Rana
clamitans (Baltimore), Triturus uiridescens (Baltimore and North Carolina), and
Bufo valliceps (Louisiana) were used to
study the response of the lens of other amphibians to injury. The left lens of anesthetized frogs and toads was injured by
pricking it with a 15-gauge needle thrust
through the cornea near the limbus. In
the smaller species used the lens was
pricked with a 27-gauge needle. The corneal wound was covered with sulfathiazole.
In an attempt to characterize the growth
in the lens as either a neoplasm or as a
hyperplasia, transplantation studies and
preliminary biochemical studies using zone
electrophoresis were undertaken. Forqhese
experiments, two to three week-old growths
were used. The whole lens was removed
and either transplanted under the thigh
1This investigation was carried out during t$e
tenure of a traineeship (Graduate Tralning Grant In
Anatomy no. 26-286( CZ)) from the National Instltutes
of Health, U. S. Public Health Service.
skin of the same animal or dissociated for
one hour in 2% versene and transplanted
into the anterior chamber of another host.
For electrophoresis ten injured lenses were
ground in an equal volume of distilled
water in a glass homogenizer. The right
lenses, treated similarly, served as normal
controls. The soluble protein fraction was
obtained in the supernatant after spinning
in an International table centrifuge at
14,000 g for 20 minutes. Horizontal starch
gel electrophoresis was performed using
the materials and methods described by
Smithies (’55) and Markert and Hunter
(’59). Except for the separation of glucose-6-phosphate dehydrogenase (G-SPD),
the best results were obtained when the
samples of lens were electrophoresed in
phosphate buffer at pH 6.4 at a voltage
drop of 5 volts/cm for 17 hours at 6.5 *
.5”C. For G-6-PD, electrophoresis was carried out in Tris-HC1 buffer, pH 7.1 under
the above conditions. The starch strips
were sliced in half longitudinally and a
piece of each stained for protein in Amido
Black for 15 minutes and washed and
stored in Smithies’ solution (Smithies, ’55).
For enzyme determinations, the staining
methods used are described below.
Histological examination was made of
whole eyes fixed in Bouin’s solution, sectioned serially at 10 on an equatorial
plane, and stained in either Delafield’s
hematoxylin, azure I1 and eosin, or in
Mallory’s triple connective tissue stain.
Table 1 shows the results obtained when
the lens was injured by mechanically disturbing it in a variety of ways. In some
cases (dry renal tumor, dry liver), a small
tear was made in the cornea and a 1 X 1
mm piece of liver or Luck6 renal adenocarcinoma was gently pushed through the
opening without poking an instrument into
the anterior chamber, to avoid touching
the lens. In this group of ten animals, six
small and one large lens growths resulted.
In another group of ten animals, a cell
suspension of liver in saline or saline alone
was injected through a 25-gauge needle
thrust into the anterior chamber in such a
way as to avoid pricking the lens. There
resulted six small and one large lens
growths. When a 15-gauge needle was
poked into the anterior chamber, either
dry or to inject saline, it was not possible
to avoid the lens; a growth occurred in
every lens and eight of the ten were medium to large in size. It appears, therefore, that the greater the mechanical insult the higher the frequency of the lens
growth, and in addition, the more vigorous
the growth, as determined by an increase
in mitotic figures.
Grossly, the lens containing a growth
has an opaque white appearance which
can usually be readily detected with the
naked eye. A large growth is shown in
figure 2.
The histological picture of the growth
in the lens suggests that the cells arise
from the lens epithelium. As seen in figure
3 a fountain of cells arising at the epithelial surface pushes into the core of
the lens, or else a central growth is connected to the lens epithelium by strings
of cells (fig. 4). The cells of the growth,
however, stain more intensely basophilic
than the cells of the lens epithelium, and
their size and shape are more variable.
The “transformation” of normal lens epithelial cells into the more disorganized,
basophilic, and frequently dividing cells
Lens injury experiments: Rana pipiens. Fixation three weeks a f t e r injury
Agent implanted,
injected, or
po,ked into
anterior chamber
No. of
1. Renal tumor (dry)
2. Liver (dry)
3. Liver suspension (25 g needle)
4. Saline (25 g needle)
5. 15 g needle (dry)
6. Saline ( 1 5 g needle)
Five to twelve mitoses observed per section.
of the growth is seen in figure 5. The cells about the fourth week when pycnotic nuof the lens growth have never been ob- clei are present. By the sixth week many
served to contain pigment nor to be sur- nuclei have degenerated in some of the
rounded by connective tissue, which would growths and the cytoplasm has lost its
implicate an origin from the iris. There basophilia. Many of the growths have
is usually no cellular connection between totally degenerated by the third month
the abnormal lens and the iris and almost and have been replaced by normal lens
never with the cornea.
fibers (table 2). A few growths, however,
That the growth invades the lens fibers have been found to persist a year after the
is unquestionable. As seen in figures 6 initiating injury.
and 7 the large mass of the growth has
The growth in the lens was not shown
broken down the lens fibers and has not
to be transpIantable under either of the
merely pushed between the fibers, as the
adjacent intact fibers show little distortion conditions used. When a suspension of
due to compression. Also fragments of 17 lenses containing growths was translens fibers are present within the cellular planted homologously into the anterior
mass of the growth (figs. 4 and 6). Fur- chamber of 20 hosts the material rapidly
thermore, the total size of the lens con- degenerated after the first week and none
taining a growth is not increased. A more could be found in the anterior chambers
advanced stage of an invading growth, between the second and ninth week.
When whole lenses containing growths
which is now compounded by phagocytic
were transplanted isologously under the
invasion, is shown in figure 8.
As reported earlier (Rafferty, '61 ) , the thigh skin of the same animal (16 cases)
lens growth is self-limiting. It reaches its the transplant became ensheathed in a
maximum size between the second and capsule of connective tissue within the
third week after its induction, and at its first week. Within this time aIso, the
maximum rarely completely invades the lens cuticle and epithelium had usually
whole lens and has never been observed to degenerated, although a small nodule of
invade other tissues of the eye, nor to epithelial cells occasionally remained outmetastasize. Signs of degeneration in the side of the connective tissue sheath. Some
cells of the growth can be detected at of these epithelial cells have the appearance of an early stage of lens fiber cells,
with eccentric nuclei displaced to one
Onset and decline of the lens growth
side of an elongated, open cell (fig. 9 ) .
in R a m pipiens'
Such nodules of epithelial cells were recovered from one week to five week old
No. lenses No. lenses
No. of
with no
transplants, but not from four month old
transplants. The transplants recovered
1 day
four months consisted of dense lens
2 days
encapsulated in connective tissue.
3 days
The lens epithelium was missing in each,
4 days
5 days
and there was no unusual growth within
7 days
transplant nor in the surrounding host
10 days
14 days
Samples of normal and abnormal lens
21 days
1 month
were compared for their protein, lactic de1.5 months
hydrogenase, malic dehydrogenase, and
2 months
glucose-6-phosphate dehydrogenase activ3 months
ity after separation by zone electrophoresis
5 months
6.5 months
in starch gels. These enzymes were chosen
9-10 months
for preliminary study because each is rep12 months
1 72
resentative of the three phases in the me1Combination of data from four experiments in
tabolism of carbohydrate in the lens; Viz.,
which material was injected into the anterior chamber
through a 15-gauge needle.
glycolysis, the citric acid cycle, and the
2 Occasionally a lens was found to be absent in the
hexos monophosphate shunt, respectively.
sectioned eye.
The latter shunt appears to be the major
aerobic pathway in carbohydrate metabolism in the lens; the citric acid cycle is of
minor importance (Kinoshita, ’55; and
Pirie and Van Heynigen, ’56). In addition,
the hexose monophosphate shunt has been
shown to play an increased role in various
neoplastic tissues in relation to the increased synthesis of nucleic acids (Angeletti, Suntzeff and Moore, ’60; Weber and
Cantero, ’57).
Only minor differences in the rate of
migration of proteins in the two lens samples were observed. When samples of each,
inserted side by side in the same starch
strip, were electrophoresed in Tris-HC1
buffer, pH 7.1,the normal lens proteins
lagged slightly behind the proteins of
lenses containing growths, whereas in
phosphate buffer, pH 6.4,the migrations
were usually at the same rate (fig. l a )
or occasionally that of the normal lens,
faster. Three to four bands migrated towards the anode and 6-7 bands towards
the cathode under the conditions described
Three to four components of LDH were
revealed and these migrated and stained
the same in both samples (fig. lb). MDH
was resolved into two intensely stained
and two faintly stained anodal, and one
deeply stained cathodal bands. The pattern was similar for both samples (fig. lc).
The results obtained after staining for
G-6-PD were consistenly poor despite numerous attempts to alter the many variables such as the pH, temperature, and
buffer system during electrophoresis, and
the proportion of substrate and coenzyme
in the staining solution. The staining
method used is a variation of that used
by Boyer et al., ’62. Very faint bands
stained after electrophoresis in Tris-HC1,
pH 7.1, and incubation for two-five hours
in the staining solution at 37°C. The
bands were similar for both lens samples,
although the migration of normal lenses
was faster than that of lenses containing
growths (fig. la). The enzyme did not
migrate at all from the origin in phosphate buffer at pH 6.4,and there was no
staining after electrophoresis in Tris-HC1
buffer, pH 8.4or in borate buffer, pH 9.5.
Occurrence in other amphibians
A large growth in the lens comparable
to that studied in Rana pipiens was injuryinduced in six young Rana clamitans. It
is interesting, however, that the toad, Bufo
valliceps, and salamander, Triturus viridescens, responded differently to the injury stimulus. In Bufo, of the ten lenses
injured, only one showed an invading
growth and four a slight thickening of the
epithelium after two weeks. The other
five lenses were in advanced stages of degeneration and phagocytosis.
In Triturus the injured lens responded
in either of two ways: it formed a slight
localized thickening of the lens epithelium
(13 of 31 cases); or degenerated (18 of
31 cases). In 15 of the 18 cases in which
the original lens degenerated ( a small
dense nodule remaining behind the iris
in the vitreous body), a new lens was regenerated from the dorsal iris. In figure
10 an early lens regenerate in the form
of a depigmented vesicle is seen arising
from the iris. The original lens, much
reduced in size and surrounded by phagocytes, is seen in the vitreous. In those
eyes fixed one month after the injury to
the lens, the latter is absent and a complete, smaller than normal, regenerate lens
is present, still attached by a few cells
to the dorsal iris.
In the 13 injured Triturus lenses which
persisted the lens epithelium showed a
thickening and a small amount of invasion
of the lens fibers. However, mitoses were
not observed even during the first week
and after two weeks the cells of the growth
lost their basophilia and the nuclei had
a vacuolated appearance. One-month old
injured lenses had the same appearance.
Until the experimental induction of
epithelial tumors in transplanted mouse
lenses by Mann (’47) the mammalian lens
had been attributed with an innate “tumor
immunity” inherent in some vague intrinsic resistance to neoplasia. Mann’s experiments indicated, however, that the observed tumor immunity probably resides
in the anatomical isolation of the lens
from the blood circulation, for when 38
mouse lenses were brought into contact
with the circulation after transplantation
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* I
a .
6.6- ‘D
Fig. 1 Protein and enzyme patterns obtained from normal lenses (NL) and lenses containing
two- to three-week old growths (LG) after electrophoretic separation in starch gels. ( a ) protein
( P I . ( b ) lactic dehydrogenase (LDH) stained by immersing the starch strips for one hour at room
temperature in the dark in the following mixture: 120 m l Tris-HC1 buffer, 0.3M,pH 8.5; 30 ml
Na-lactate, 0.5 M; 30 mg DPN; 30 mg Nitro-B.T.; 8 mg phenazine methosulfate. (c) malic dehydrogenase (MDH) stained under the same conditions as LDH but substituting 30 ml Na-malate,
0.5 M, for the lactate. ( d ) glucose-6-phosphate dehydrogenase (G-6-PD) stained by incubating the
starch strips for two to five hours a t 37OC in the dark in the following mixture: 120 ml Tris-HC1
buffer, 0.5 M, pH 8.5; 120 mg glucose-6-phosphate (disodium salt); 10 m l MgCL, 0.03 M; 30 mg
TPN; 30 mg Nitro-B.T.; 8 mg phenazine methosulfate.
along with methylcholanthrene under the
flank skin, three tumors of the lens epithelium resulted. Conversely, the injection of methylcholanthrene into rabbit
lenses left in situ in the eye resulted in no
tumors (number of cases not cited). Thus,
unless there is a species difference in the
reaction of the rabbit and mouse lens to
the carcinogen (which is possible), the
isolation of the intact lens is implicated
in its tumor immunity.
There is only one case reported in the
literature of a human lens tumor: a metastasis to the lens of a melanosarcoma originating in the ciliary body (Hallermann
and Meisner, ’54). There was no vascular
or direct contact between the primary
melanosarcoma and the lens, but metastasis was thought to be accomplished
through the fragmentation of the primary
tumor into the aqueous humor and the
subsequent attachment of a fragment to
the lens capsule. The usual vascular route
for metastatic dissemination was circumvented in this case.
Sachs and Larsen (’48) in searching for
an explanation of the observed immunity
of the mammalian lens to tumors pointed
out the barriers imposed on an oncogenic
agent in passing from the blood to the
lens: the ciliary process, which prevents
the passage of larger molecules; and the
lens cuticle which acts as a semipermeable membrane. Although these anatomical barriers may prevent the passage of
metastasizing cells and large carcinogenic
molecules from the blood stream to the
lens, there is still to be explained the lack
of neoplastic transformation of the continually dividing cells of the mammalian
lens epithelium. It is well known that the
lens epithelium undergoes continual mitotic activity in the germinative zone
throughout life. Mitotic counts made in
the epithelium of mature rabbits by von
Sallmann (’52) were found to be 60:
100,000 cells in the germinative zone and
0.11:100,000in the central zone. High
mitotic activity in the germinative zone
has further been illustrated by labeling
with tritiated thymidine (Hanna and
O’Brien, ’61; and Harding, Hughes, Bond
and Schork, ’60). In view of this respectable mitotic rate a certain number of
mutational changes could be expected to
occur which might rarely, at least, result
in a neoplastic condition. Yet, a spontaneous intrinsic tumor of the lens epithelium
has never been reported for any mammal. Since it is difficult to understand
how the anatomical isolation of the lens
by itseIf may affect a reduction in mutations, it seems more likely that the cells
of the mammalian lens epithelium do indeed possess an inherent resistance to
neoplastic change.
The studies presented in this report indicate that there is a species difference in
the behavior of the lens epithelium to
injury. Whether the injury-induced growth
in the frog lens is a tumor or a peculiar
hyperplasia is relatively unimportant; what
is important is the fact that the stimulus
of mechanical injury results in a rapid
increase in mitoses in the cells around
the wound which gives rise to an actively
invading growth. The injured mammalian
lens epithelial cells also respond by initially dividing until the wound is healed
over, but excessive proliferation does not
occur. Thus, Harding, Donn, and Doblisrinivasan (’59), working with rabbits,
found that the epithelial cells in the region around the wound showed a high
rate of incorporation of tritiated thymidine, the maximum occurring between 24
and 48 hours after the wounding. They
make the statement:
“If all the radioactive cells in the 24 and
48 hour preparations would have ultimately divided, it would appear that injury stimulates a very high rate of cellular
proliferation in the surrounding epithelium.”
In the rabbit all the cells around the
wound do not divide; in the frog it appears that they do, resulting in an invading growth.
The lenses of the toad and salamander
studied are also labile, but their response
to injury may be expressed in a different
manner: phagocytic breakdown, and, in
the case of Triturus, replacement of the
injured lens by a new lens regenerated
from the dorsal iris. A difference in behavior of the lens to injury was noted
between Triturus viridescens and Amblystoma punctatum by Stone (‘43) and Stone
and Cole (’43). Thus, they found that
the Triturus lens is less resistant to injury
than the lens of Amblystoma, whose high
lens survival they correlated with the inability of the eye to regenerate a new lens.
Stone et al. (‘34), Stone and Sapir (‘40),
Stone and Farthing (’42), Stone (’52, ’54),
and Stone and Steinitz (’53) have frequently remarked on the lability of the
adult Tritirus lens when the eye is transplanted from one animal to another or
otherwise injured. They found that the
injured lens either rapidly degenerates or
becomes cataractous (vacuolated or cystic).
Stone (’52) and Reyer (’56) noted, however, that the subcapsular epithelium becomes quite flat in these cataractous
lenses, and they considered the vacuolation of the fibers as a slow degenerative
In a study of lens regeneration in a
variety of larval and adult anurans, urodeles, and fishes, Stone and Sapir (’40)
described a few cases in reimplanted lens
fragments of larval Amblystoma tigrinum
and of larval Rana pipiens of a thickened lens epithelium having an “embryonic appearance.” Their figures 6, 10 and
11 show proliferation of the lens epithelium which somewhat resembles the early
stages in the development of the lens
growth described in this report in adult
Rana pipiens. They did not observe, however, any unusual behavior of the epithelium in lens fragments remaining after
incomplete lentectomy or in reimplanted
lens fragments in eyes of larval Rana
clamitans, Rana sylvatica, Amblystoma
punctatum, and of adult Triturus viridescens and Fundulus heteroclitus.
At the present time identification of the
frog lens growth as a tumor or reparative
hyperplasia is not possible. In support of
its being a benign epithelioma are its invasiveness, progressive growth for three
to four weeks during which it exhibits a
high mitotic rate, and its occasional persistence for a year, or perhaps longer.
The properties of self-limiting growth and
of eventual total regression are characteristics of many benign epithelial tumors
such as the Shope rabbit fibroma, squirrel
fibroma, and myxoma and papilloma in
wild rabbits (see Gross, ’61, for a review).
The finding that the frog lens growth
was not transplantable under the conditions reported here does not rule it out
as a benign tumor, as transplantability is
more characteristic of malignant growths
than of benign ones. The conditions required for the successful transplantation
of a benign tumor are often stringent, and
no attempt was made here to test all of the
conditions. Some tumors have been found
not to be transplantable at all (see Stewart et al., ’59, for a review).
It is hoped that further biochemical
analysis of the lens growth may give a
better indication of its nature. There is a
sizable body of data in the literature which
indicates that neoplasms may exhibit qualitative and quantitative changes in the composition of their proteins and enzymes
compared with comparable normal tissues.
Angeletti, Suntzeff and Moore (’60) found
variations in the chromatographic patterns of several enzymes from a mouse
rhabdomyosarcoma compared with those
of normal muscle. Alkaline phosphatase
was found only in the tumor tissue whereas a-glycerol phosphate dehydrogenase was
localized only in normal muscle, not in
the tumor. Differences between the two
tissues were found in the number of peaks
of glutamic oxaloacetic transaminase, acid
phosphatase, and glucose-6-phosphate dehydrogenase. The latter enzyme showed a
considerable increase in activity in the
rhabdomyosarcoma, in mouse squamous
cell carcinoma (Angeletti, Moore, Solaric
and Suntzeff, ’60), and in rat hepatoma
(Weber and Cantero, ’57). Weber and
Cantero found that the increase in glucose6-phosphate dehydrogenase was specific for
the tumor and was not characteristic of
regenerating liver nor of embryonic or
new-born rat liver.
The preliminary results obtained in
these experiments indicate that G-6-PD in
lenses containing growths and in normal
lenses has similar electrophoretic mobilities and activity. Conclusions should not
be drawn from these results, however,
until a better method of visualizing the
G-6-PD activity has been worked out.
Enzymes such as lactic, malic, and isocitric dehydrogenase were found by Angeletti, Suntzeff and Moore (’60) to have
similar chromatographic patterns and activities in normal and tumor tissues, and
the overall protein patterns of the two were
also generally similar. This was also found
to be true for the two types of lenses studied in this report.
A study of the fine structure of normal
and abnormal lens epithelial cells, already
reported for a number of normal mammalian lenses (Wanko and Gavin, '58),
which is being undertaken presently, may
reveal differences which will better allow
characterization of the lens growth. In
any case, the labile frog lens affords an
excellent system in which to study growth
on the subcellular level. Until the growth
is proved to be otherwise, it is suggested
for convenience to describe it as a lentoma.
Rafferty. N. S. 1961 Fate of implanted embryos in frog eyes. J. Exp. Zool., 147: 3 3 4 2 .
Reyer, R. W. 1956 Lens regeneration from
homoplastic and heteroplastic implants of dorsal iris into the eye chamber of Triturus virdescens and Amblystoma punctatum. Ibid., 133:
Sachs, E., and R. L. Larsen 1948 Cancer and
the lens. Am. J. Ophthal., 31: 561-564.
Smithies, 0. 1955 Zone electrophoresis in starch
gels: group variations in the serum proteins
of normal human adults. Biochem. J., 61:
Stewart, H. L., K. C. Snell, L. J. Dunham and
S. M. Schlyen 1959 Transplantable and
transmissible tumors of animals. Atlas of
Tumor Pathology, Sec. 12, Fasc. 40, pp. 378,
Am. Reg. Path., Armed Forces Inst. of Path.,
Washington, D. C.
Stone, L. S., I. S. Zaur and T. E. Farthing 1934
Grafted eyes of adult Triturus viridescens with
Angeletti, P. U., V. Suntzeff and B. W. Moore
special reference to repeated return of vision.
1960 Chromatographic patterns of protein and
Proc. SOC.Exptl. Biol. Med., 31: 1082-1084.
enzymes in extracts of rhabdomyosarcoma and
Stone, L. S., and P. Sapir 1940 Experimental
muscle in mice. Cancer Res., 20: 1229-1234.
studies on the regeneration of the lens in the
Angeletti, P. U., B. W. Moore, S. Solaric and
eye of anurans, urodeles, and fishes. J. Exp.
V. Suntzeff 1960 Chromatography of proZOol., 85: 71-101.
teins of squamous cell carcinomas and normal
epithelium of mice. hoc. SOC. Exptl. Biol. Stone, L. S., and T. E. Farthing 1942 Return
of vision four times in the same adult salamanMed., 103: 329-331.
der eye (Triturus viridescens) repeatedly
Boyer, S. H., I. H. Porter and R. G. Weilbacher
transplanted. Ibid., 91: 265-285.
1962 Electrophoretic heterogeneity of glucose6-phosphate dehydrogenase and its relation- Stone, L. S., and C. H. Cole 1943 Grafted
eyes of young and old adult salamanders
ship to enzyme deficiency in man. Proc. SOC.
(Amblystoma punctatum) showing return of
Nat. Acad. Sci., 48: 1868-1876.
vision. Yale J. Biol. Med., 15: 735-754.
Gross, L. 1961 Oncogenic Viruses. Pergamon
Stone, L. S. 1943 Factors controlling lens rePress, New York, Chap. 11, 1 7 4 6 .
generation from the dorsal iris in the adult
Hallermann, W., and G. Meisner 1954 Die
Triturus viridescens eye. Proc. SOC. Exptl. Biol.
umstrittene Tumorimmunitat der Linse. Klin.
Med., 54: 102-103.
Monatsbl. Augenh., 124: 159-164.
1952 An experimental study of the inHanna, C., and J. E. O'Brien 1961 Cell prohibition and release of lens regeneration in
duction and migration in the epithelial layer
adult eyes of T r i t u c s viridescens viridescens.
of the lens. A.M.A. Arch. Ophthal., 66: 103J+ EXP. 2001.. 121: 181-223.
IU I .
Stone, L. S., and H. Steinitz 1953 The regenHarding,
1959 Incorporation
C. V., A. Donn
of and
B. Doblisrinivasan
by injured
eration of lenses in eyes with intact and regenerating retina in adult Triturus v. virideslens epithelium. Exptl. Cell Res., 18: 582-585.
cens. Ibid., 124: 435-467.
L* Hughes*
p' Bond and Stone, L. S. 1954 Further experiments on lens
P. SchOrk 1960 Autoradiowphic localharegeneration in eyes of the
v. viridescens. Anat. Rec., 120: 599-623.
tion of tritiated thymidine in whole-mount
preparations of lens epithelium. A.M.A. Arch.
L. 1952
Ophthal., 63: 58-65.
on early lens changes after roentgen irradignoshita, J. Ha 1955 Carbohydrate metahation, 111. A. M. A. Arch. Ophthal., 47: 305lism of lens. Ibid., 54: 360-368.
~ T., and
M. ~A. Gavin
The fine
Mann, I. 1947 Induction of an experimental ~
tumour af the lens. Brit. J. Cancer., 1 : 63-67.
structure of the lens epithelium. Ibid., 60:
Markert, C. L., and R. L. Hunter 1959 The dis868479.
tribution of esterases in mouse tissues. J. Weber, G., and A. Canter0 1957 Glucose-6Histochem. Cytochem., 7: 42-49.
phosphate utilization in hepatoma, regenerating and newborn rat liver, and in the liver
P e e , A., and R. van Hep.ningen 1956 Biochemistry of the Eye. Blackwell Scientific
of fed and fasted normal rats. Cancer Res.,
17: 995-1005
Publications, Oxford, Chap. 11, 36-67.
Gross appearance of a one-month old growth in the lens of R a n a
pipiens. x 10.
3 A lens growth in R a n a p i p i e n s initiated by sticking the lens with a
15-gauge needle three weeks previously, showing ingrowth from one
point of the epithelium. Mitoses at arrows. x 200.
A three-week old lens growth in R a n a pipiens showing ingrowth from
the epithelium a t several loci. Note fragments of degenerating lens
fibers within the cellular mass. X 100.
A one-month old lens growth in R a n a p i p i e n s showing the transition
between normal lens epithelial cells (lower left and upper center)
and the denser, more basophilic, disorganized cells of the growth.
Four mitoses a t arrows. X 300.
Nancy S. Rafferty
A three-week old lens growth i n Rana p i p i e n s . The large central
growth has caused relatively little distortion to the surrounding lens
fibers. Mallory’s stain. x 100.
A higher magnification of the advancing edge of the lens growth
shown in figure 6. X 600.
A three-week old lens growth i n Rana pipiens showing a n unusually
widespread invasiveness. Lens cuticle anteriorly may have been
broken during preparation. Centrally the light area contains numerous lymphocytes. X 50.
A n isologous lens transplant to the thigh after five weeks. Host muscle (M); host skin ( S ) ; transplanted lens fibers (LF) ensheathed in
connective tissue to which is attached a clump of young lens fiber
cells. X 100.
10 Eye of Triturus 15 days after injury to the lens, showing the small
dense original lens behind the iris (bottom) and a new lens regenerating from the dorsal iris (top). Mallory’s stain. X 100.
Nancy S. Rafferty
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lens, induced, growth, injury, studies, frog
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