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Further experiments on lens regeneration in eyes of the adult newt Triturus v. viridescens

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Anatomioal Laboratory, Yale University School of Medicine and the Osborn
Zoological Laboratory, Pale University, New Haven, Connecticut
The occurrence of lentoids, cataracts and various types of
abnormalities have often been reported by the author in relation to experiments dealing with lens regeneration in eyes
of the adult newt, I'riturzcs v. viridescerzs. It has been shown
that cataracts (Stone and Steinitz, '53a) or other defects
rarely occur in normal lens regeneration if the original lens
is carefully extracted without severe injury to the dorsal iris.
However, cataracts and other lens abnormalities frequently
occur in eyes associated with iris grafts (Sato, '30 and '33;
Stone, '52 and others) or with regenerating retina (Stone and
Steinitz, '53a) or in eyes of hypophysectomized and thyroidectomized animals (Stone and Steinitz, '53b). Also during the embryonic and early larval life of the newt, abnormalities often arise during regeneration of the lens. This has
been reported and reviewed recently by Reyer ( '48 and '50).
Lentoids develop from cells at various levels in the dorsal
iris around the margins of artificially produced accessory
pupils (Okada, '35 ; Stone and Vultee, '49) or along the margin of regenerating dorsal iris membranes (Stone and Grif'Aided by grants from the James Hudson Brown Memorial Fund of the Yale
University School of Medicine and the Institute of Neurological Diseases and
Blindness, United States Public Health Service (B-23).
fith, '54). However it has been demonstrated in numerous
mid-dorsal iris transplants (Sato, '30; Stone, '52) that a lens
develops only from the original pupillary margin and not
from other borders of the graft which also have potential
lens forming cells. Up t o the present time no experiments
have been reported which test the possibility of forcing lens
regeneration only from the ciliary margin of a graft. An
attempt could be made by rotating 180" a mid-dorsal iris
segment to make the ciliary margin of it a part of the free
pupillary border (see fig. 3). If lenses could be induced in
this way to develop from the ciliary margin it would be of
interest to know whether or not they can develop as normally
as those which regenerate from the original pupillary margin
of iris grafts.
Mikami ('37) found that if the pupillary margin of the
dorsal iris of adult eyes of Triturus pywhogaster was cut
away and a middle segment above the wound margin as far
as the o m serrata was then excised and immediately implanted
in the vitreous chamber of lensless eyes, lenses developed
only from the border of the graft nearest the original pupillary
region. It is not known however, if more perfect lenses would
have developed had the wound margin in the pupillary region
of the implant been allowed to heal before the iris was transplanted (see fig. 2).
The presence of the retina has long been cited as an iniportant factor promoting lens regeneration after the normal
lens is removed from the environment ( Wachs, '14 ; Ikeda,
'34 and '36 ; Uno, '43 and '44 ;Zalokar, '44 ; Stone and Steinitz,
'53a). It has also been claimed that a retinal factor determines the location of the lens fiber pole (Wachs, '14;Ogawa,
'21; Sato, '30; Okada, '39). Stone and Steinitz ( '53a) showed
that the fiber forming pole in regenerating lenses pointed
towards strips of regenerating neural retina which were
undergoing differentiation while other regions of the regenerating retina were quite immature. Since the lens fiber pole
in normal regeneration always points inward towards the
retina at a very early stage, it would be of special interest
to observe the direction of the mediolateral axis of a lens
arising from the mid-dorsal iris turned inside out (see fig. 4).
I n this brief introduction experiments have been suggested
to provide better knowledge of the relationship of lens regeneration to injuries and healing in the mid-dorsal iris and the
conditions under which the mediolateral axis of the lens is
established. The following pages are devoted to a description
of these experiments and the results.
All of the experiments reported in this paper were done on
the eyes of the adult newt, Triturus v. vbidescenns. The newts
were collected from ponds in Connecticut, Massachusetts and
Vermont, There were 4 main groups of experiments on 143
eyes. The heads of the animals were finally fixed in Zenker's
and prepared by the usual methods in serial sections stained
with hematoxylin and erythrosin. The general routine employed in the operations and the post operative treatment of
the animals was similar to those described by the author in
previous publications. The steps followed in the different
types of operations will be found in the illustrations and descriptions of the experiments which follow.
1. Vertical slits iw dorsal i f i s - lens removed
The results of previous experiments (Stone, '52) showed
that injuries such as slits along the pupillary margin of the
dorsal iris do not initiate lens regeneration in eyes with
intact lenses. However, in a recent study (Stone and Griffith,
'54) it was observed that the development of accessory lentoids
and abnormal lenses was associated with defects in wound
healing along the border of regenerating dorsal iris tissue. It
was therefore essential to know what effect injuries, such as
slits along the pupillary margin of the iris without tissue removal, would have upon the early stages of lens regeneration.
The experiment illustrated in figure 1A was done on 29
eyes. A long horizontal incision was made in the cornea across
the center of the pupillary space. Then by means of iridectomy scissors 5 slits were produced in the dorsal iris cxtending from the free pupillary margin to about one-half of the
distance towards the corneoscleral junction. The lens was
removed immediately (fig. 1A) leaving a fringe of narromstrips of iris tissue suspended in the aqueous chamber.
2 0 DAYS
2 0 bAVS
31 D A Y S
Fig. 1 Schematic representation of operations and results of lens regeneration
i n a newt in which, a t the time of lens removal, the dorsal iris was slit several
times in the riglit eye ( A ) and remained intact in the left eye ( A l ) . Lens
regeneration m s usually retarded at first in the right eye (B) compared with
the control left eye ( E l ) . Within a month there was usually no difference between the lenses (C and C l ) .
This operation was done on both tlie right and left eyes of
6 animals which were preserved in Zenker’s at the end of
22 days. Serial sections of the 12 eyes were prepared for
histological examination. I n 17 other animals the iris incisions
and lens removal mere done in the right eyes (fig. 1A) while
a t the same time only the lens was removed in the control
eye (fig. 1, A l ) . Groups of these animals were preserved in
formalin, 20, 26, and 31 days after operation. I n 80% alcohol
the anterior half of each eye was removed so that under the
microscope the relations of the regenerating lens and iris
could be examined from the inside and illustrated as in fi,aures
5A, 5B, 6A, and 6B.
I n two cases mid-portions of the dorsal iris adhered to the
corneal scar. At the end of two weeks only small lentoids
had developed. I n all other cases the iris wounds healed duriiig the first week. As late as three weeks after operation
faintly pigmented lines could be seen marking the positions
of the incisions (fig. 1, lB, dotted lines radiating above lens
regenerate). By this time the majority of lens regenerates
in eyes with iris wounds were smaller (fig. 1 B) than those
in control eyes (fig. 1, B1) with the normal iris. Some, however, were equal in size to the normal control lens regenerates.
Those preserved 26 and 31 days after operation showed well
defined spherical lenses and when compared with the normal
controls there was very little difference in size (fig. 1C ancl
C1; 6A and 6B). Only two were slightly smaller than the
normal control lens regenerates.
Irregular healing of incisions in the middle of the lens
forming area was associated with partially double lenses iii
two cases. One is shown very well in a right eye in figme 5A
which can be compared with the normal lens regenerate in
tlie control left eye (fig. 5B) 20 days after operation. Slight
irregularities were also seen in the anterior subcapsular epithelium as in the case shown in figure 7, indicated by arrow.
No cataracts were found in the 12 eyes studied histologically.
TThcther or not they were present in any of the other 17 eyes
examined only in the gross preparations it is not possible to
say, for details essential f o r detecting early cataracts were
Since some of the incisions along the pupillary margin
passed through the lens forming area, it is interesting t o
Fig. 2 Schematic representntiou comparing the stops jn two series of 011cr:itions and the results. 'In one group the pupilhry margin of the iris x i s
reniovod froni donor eye ( A ) . Four days later middle third of the dorsal iris
cxtonding to the ciliary region was excised froin donor eye (E) and implanted
in host eye immediately after lens romoval (C). In the other group the pupillary
inargin of the dorsal iris was removed from donor eye ( A l ) and the rtmainhg
middle third was excised immediately (BI) and transplanted to host eye after
lens removal ( C l ) . Much smaller defective lenses dereloped from the iris grafts
in the latter case ( D l ) than in grafts with hcaled pupillary margins at the
tiinc of trnnsplantntion (D).
note that in most instances the wounds healed quickly enough
so that no gross defects occurred in the lens regenerates. If
the free pupillary margin was uneven greater defects appeared
in the structure of the lens. I n all cases the orientation of the
lenses was normal. The inner fiber forming pole was directed
towards the retina and the outer pole, covered by the subcapsular epithelium, faced the aqueous chamber.
2. Dorsal iris grafts without pupillary margin
I t has been shown that lens regeneration begins very early
from the border of a regenerating iris membrane (Stone and
Griffith, '54) at any level in the middle third of the dorsal
iris where the potency for lens regeneration is the highest.
It has also been shown by several investigators that lenses
readily regenerate from the pupillary margin of dorsal iris
grafts implanted in lensless eyes but little if any iris tissue
regenerated from the transplant. Therefore in the following
experiments a test was made of lens regeneration from iris
grafts from which the pupillary margin had been removed.
I n this series there were two groups of experiments involving 20 eyes. A long horizontal incision was made in the
cornea below the center of the pupillary space extending
to the corneoscleral junction. From these points incisions
were extended dorsally so that a corneal flap could be elevated
to expose the dorsal iris. I n one group of eyes a narrow strip
of all the dorsal pupillary margin of the iris was excised
and discarded (fig. 2A). The lens was left in place and the
wound was allowed to heal during the next 4 days. The presence of the normal lens during this period inhibited any beginning stages of lens regeneration after wound healing in
the dorsal iris (Stone and Griffith, '54). At the end of this
period the donor eye was reopened and the middle third of
the dorsal iris, from the previous wound margin to the upper
ciliary region, was excised (fig. 2 B) and transplanted to
a new host eye from which the lens had just been removed
(fig. 2 C) through a slit in the cornea. It was possible later
t o determine from which end of the graft a lens was regen-
erating, for the upper or ciliary border of the implant could
easily be distinguished from the lower pigmented margin
by the presence of the characteristic columnar non-pigmented
cells on the inner surface. The eyes were observed while
living and the heads were preserved in Zenker’s 23 days after
implantation of the iris. Histological studies were made of
all cases.
I n every case a well defined lens regenerate was attached
to the dorsal iris of the host eye, and whenever a lens regenerated from the implanted iris it arose not from the ciliary
region but from the margin which was nearest the former
pupillary border (fig. 2 D). Ten of the 12 iris transplants
were large well organized grafts. One was small due to an
injury, but attached to it was a small lens vesicle. One graft
was completely resorbed. No lens regenerated from 4 transplanted iris segments. I n three of these the margin of the
graft nearest the former pupillary border from which the
lenses usually arise, was fused with the inner surface of the
host dorsal iris. This was sufficient to prevent lens regeneration from that border of the implanted iris. I n each of 7
grafts in which this border was free a lens regenerate was
seen arising from it. I n every case the lens was smaller than
the one developing from the host iris. The largest lens measured 180p in diameter while two much retarded lentoids
measured only 50p. The average was 1 2 0 p compared with
an average of 200 p for the lenses arising from the host iris.
Four of the lens regenerates derived from transplants were
in various stages of cataract formation. One of them with a
degenerating center is shown in figure 8, indicated by an arrow, in an eye 23 days after the 4-day healed iris was transplanted into an eye immediately after lensectomy. The folded
graft can be seen lodged against the pupillary margin of the
host iris from which a lens was regenerating. This compressed
lens has many small vacuoles scattered among degenerating
central lens fibers.
Similar operations were done in another group of experiments, but after the pupillary rim of the dorsal iris was re-
moved and discarded (fig. 2, A l ) the dorsal segment of the
iris was excised immediately (fig. 2, B1) and transplanted t o
another host eye as soon as the normal lens was removed
(fig. 2, Cl). Therefore the wound bordering the formed pupillary region of the graft in these cases was not allowed to
heal before transplantation. One could then determine what
if any effect this might have on lens regeneration.
I n a total of 8 eyes the iris grafts were large and well
organized when they were preserved for histological examination 22 days after operation. One large iris graft was much
folded and fused with the host iris. No lens was regenerating
from this transplant. Lens regenerates were developing from
the remaining 7 grafts. In 6 of them it was obvious that they
were attached to the margin of the graft nearest the former
pupillary border. I n another case it was impossible to determine with certainty the border of the graft to which the lens
was attached because of the complex folding which took place
in the implanted iris membrane. All of the 7 lens regenerates
were lentoids, much retarded in growth. Three were very
small vesicles containing two to 5 lens fiber cells. One vesicle
was a very young regenerating lens, stage 3.
The three largest lentoids ranged from 110 p to 150 ~1 in
diameter and in all of them the fiber cells were degenerating.
A photomicrograph of the largest of these is shown in figure
9 attached to the graft, indicated by the arrow. It is an irregular shaped lens with a large central degeneration area.
The anterior subcapsular epithelium is extremely thin. The
plane of section passing through the lens regenerate from the
host dorsal iris, shown in figure 9, does not pass through the
center of this lens. However it is a normal lens and like 6
others its size was well within the range of normal 22-day
regenerates (Stone and Steinitz, '53a) the average measnrement being 238 p in diameter. Two of the host lenses showed
early stages of cataract formation. They were associated
with pressure exerted by the iris graft. There was one case in
which the lens developing from the host iris was much deformed and retarded because the much folded graft had fused
wit.h most of the lens forming margin of the host dorsal iris.
The results of these two groups of experiments summarized
in figure 2, D and D1, indicate that grafts from the middle
third of the dorsal iris tend to develop lenses from the border
nearest the pupillary region and not from the ciliary portion.
It is also obvious that far better lens regeneration occurs when
the pupillary border of the dorsal iris of the graft is allowed
to heal before transplantation (fig. 2 D) than when it is transplanted immediately after the pupillary margin is removed
(fig. 2, D l ) . It should also be mentioned that the fiber formins
poles of all the lens regenerates in these eyes were directed
normally towards the retina.
rotated 280" dorsouemtrally
on the mediolateral axis
3. Dorsal iris grafts
The scheme of operation for this group of 58 eyes is shown
diagrammatically in figure 3. A corneal flap was prepared and elevated as in the previous operations so that a
mid-dorsal segment of iris tissue could be excised, extending
from the pupillary margin to the upper ciliary region of the
iris (fig. 3 A ) . After the pupillary margin of the iris segment
was excised and discarded (fig. 3 A ) the graft was rotated
180" (fig. 3 B) and fitted into the dorsal iris wound previously
prepared in the new host eye. The ciliary margin of the graft
then formed the mid-dorsal border of the pupillary space
(fig. 3 C). The original lens was temporarily left intact (fig.
3 B ) in the host eye in order to control later the time at which
lens regeneration might begin. Therefore between 19 and 45
days after operation the normal lens was removed (fig. 3 C)
through a slit in the cornea to release lens regeneration from
the graft of host iris (fig. 3, D and E).
Eight eyes were lost clue to deaths of the hosts between
21 and 44 days after operation. There were 20 eyes in which
the grafts were either resorbed or pushed aside early by the
regenerating dorsal iris of the host. These eyes were discarded between 33 and 76 days after operation since they
failed to fulfill the conditions required by the experiment.
This left 30 eyes which were preserved f o r histological examination between 45 and 75 days after the first operation, i.e.,
transplantation of the iris segment (fig. 3 B).
Fig. 3 Schematic reprcsentatioii of operations and rrsults in which a middorsal iris graft is removed from donor eye (,4) and pupillary margin excised
and discarded. The graft is then rotated 180" on inediolateral axis and implanted
in host eye (B) i n denuded area already prepared. The normal leiis is removed
19 to 45 days later froin host eye (C). The former ciliary inargin of graft iiiay
give rise t o a lrns ( D ) . If it fails n lens regenerates from host iris on either side
of graft (E).
I n many of these 30 cases the rotated grafts healed quite
well in place, There was a tendency for surrounding regenerating iris tissue to push the graft ahead of it, especially
when it had not healed completely along all edges. Sometimes
it formed an elongated cylindrical roll which became lodged
behind the regenerating dorsal iris of the host eye. This is
shown in a case (fig. 10) in whioh the original lens was removed 19 days after the first operation. The eye was preserved 26 days later and the large lens shown in figure 10
was derived from the host iris slightly nasal to the mid-dorsal
line. N o lens regenerated from the graft. I n fact there were
26 of the 30 eyes in which large surviving grafts failed to
give rise to lens regenerates even though they were well
healed to the host iris and stretched across the mid-dorsal
pnpillary margin. A good example is shown in figure 11taken
from a right eye 28 days after lens removal and 47 days after
the iris was transplanted. The graft appears as a thick narrow
membrane attached to the host dorsal iris. The typical tall
non-pigmented cells lining the inner surface of the ciliary
margin of the graft are distinctly shown, indicated by the
arrow. The normal 28 day lens regenerate shown in the figure
11 was not derived from the graft but from the host dorsal
iris on the nasal side of the transplant.
There were only 4 eyes in which lenses were regenerating
from the ciliary margins of well healed grafts. An example
is illustrated in figure 1 2 in which a partly deformed lens is
regenerating from the thick wall of the grafted iris, 2.5 days
after the original normal lens was removed and 37 days after
the iris was transplanted. Another example is shown in fig
13D taken from a left eye with a lens regenerating from the
well defined graft, 60 days after transplantation of the iris
and 25 days after the normal lens was removed. The right
eye from the same animal from which figure 13D is taken
illustrates a condition found in three eyes in this group of
experiments where the well preserved iris graft failed to give
rise to a lens regenerate (fig. 13B), but on the nasal (fig. 13A)
and temporal (fig. 13C) sides of the broad graft a lens developed from the margin of the slowly regenera.ting host iris.
The one on the nasal side (fig. 13A) measures 160 p in diameter and is a retarded stage 10 lens with vacuoles in the
lens fibers. The one on the temporal side (fig.13C) measures
:.. .:<j.
. ,.
Fig. 4 Schematic representation of operation and results in which a middorsal iris segment is removed ( A ) , turned inside out (rotation on dorsoventral
axis) and reiinplanted. The normal lens was removed 8 to 13 days later (B). A
lens often regenerated from reversed pupillary margin ( C and D) with fiber pole
normally oriented. Occasionally a lens regenerated on either side of graft from
host dorsal iris (D).
200 p in diameter and is an early stage 11with no degenerating
fibers. There is a small lentoid developing above it.
It is obvious from the results of these experiments that a
rotated dorsal iris graft can develop a lens from its ciliary
border if it forms part of the pupillary margin (fig. 3 D ) .
However, the graft even though well established in the middorsal region usually acts as a wedge between the lens forming
cells on either side of it so that one or two lenses (fig. 3 E )
regenerate from the host iris. It is also difficult to seal the
graft in a permanent position, for the regenerating dorsal
iris tissue of t,he host invades the wound area and often dislodges the rotated transplant. The fiber forming poles of the
lens regenerates in these eyes were also normally oriented
and facing the retina.
4. Dorsal
ivis g r a f t s rotated 180" ow the dorsoventral
axis - turned inside out
There were 20 eyes in this group of experiments. The steps
in the operations and a summary of the results are shown
in the schematic figure 4 above. 9 corneal flap was elevated
in a manner already described in the other experiments. A
segment of the mid-dorsal iris including the pupillary margin
was excised extending dorsally about two-thirds of the distance towards the ora serrata (fig. 4 A ) . The iris membrane
was then turned inside out, i.e., rotated 180" on its dorsoventral axis, and returned to the denuded area as indicated in
figure 4 A . The cut edges were brought into apposition and
pressed together to aid healing of the graft. The lens was
left intact and the corneal flap replaced. Eight t o 13 days later
the normal lens was removed (fig. 4 B) in all but one specimen
to release lens regeneration in the eye (fig. 4, C and D). All
hosts were preserved in Zenker's 19 days later when all the
lens regenerates present would still be attached to their source
of origin. The time of preservation varied from 27 to 31 days
after the reversal of the seapent of the dorsal iris.
The main objective of this experiment was not only to test
the ability of the reversed pupillary margin of the dorsal iris to
remain in position and give rise to a lens but to observe in
which direction the lens fiber pole became oriented during
development. Normally the fiber forming pole of the lens
regenerate points inward towards the retina while the outer
pole covered by the subcapsular epithelium faces the aqueous
chamber. Two eyes were lost due to the death of the host
8 days after operation. Eighteen eyes which were observed
while living became available for histological examination.
The grafts usually healed well in place. There was a tendency, as in the previous experiments, for the surrounding
host iris to regenerate slowly and push the graft ahead of
it towards the nasal or temporal sides. I n one the normal
lens was not removed. Therefore no lens could regenerate
from the reversed iris segment. I n one case the graft degenerated rather rapidly due to severe injury at the time of
operation. I n 8 eyes no lenses regenerated from grafts, which
were reduced in size and remained fused with the regenerating
margin of the host iris. Large lens regenerates developed
from the regenerating host iris. I n one case two lenses were
regenerating, one on either side of the small mid-dorsally located graft.
I n 7 eyes the grafts gave rise to lens regenerates. Figure
14 shows in a left eye, 47 days after the iris was reversed and
19 days after the normal lens was removed, a large lens regenerate 280 p in diameter, attached to the thick reversed
iris segment. One can see that the poles of the mediolateral
axis of the lens are normally oriented, the fiber pole directed
towards the retina and the outer one, covered by the subcapsular epithelium, facing the aqueous chamber. There are
only slight irregularities in the arrangement of central fiber
cells but no areas of degeneration are present.
Figure 15 shows another iris graft fused with the ciliary
margin of the host iris. It is thick and folded with a lens
regenerate measuring 310 p in diameter attached to it. This
lens has two primary lens fiber centers and is slightly irregular
in shape. Like the one shown in figure 14 the lens fiber pole
is normally directed inward towards the center of the retina.
I n figure 16A a folded graft in a right eye is shown fused
with the ciliary margin of the host iris. The large lens regenerate with its two primary lens centers measures 260 p in diameter and is attached to the graft. It is also directed
normally towards the center of the retina.
I n the left eye of the same case from which figure 16A is
taken there were three lens regenerates, one on either side
of the graft derived from host dorsal iris tissue and one re-
generating from the graft (schematically shown in fig. 4 D).
One of the lenses developing from the host iris on the nasal
side of the reversed iris segment is shown in figure 16B. It
is a small stage 10 regenerate measuring 1 7 0 in
~ diameter.
Its development was retarded but there were no degenerating
areas in it. The graft was thick and attached along the middorsal border of the host iris. It formed a C-shaped fold extending inward and attached t o its uneven free margin can
be seen in figure 16C a small stunted stage 8 lentoid, indicated
by arrow. The fiber pole of the lentoid is directed towards
the retina. Also appearing below the lentoid is a portion
of the host lens, which was shown in figure 16B to be rising
from the host iris. On the temporal side of this graft another
lens regenerate, a stage 10, was also attached to the host
dorsal iris. I t measured 200 p in diameter and possessed numerous small vacuoles in the central fibers.
I t is obvious from these results that reversing the dorsal
iris appeared to have no visible effect on the polarization of
the lenses which regenerated from the grafts. Although there
were two small lentoids measuring 40 p and 80 v in diameter,
there were other large ones which ranged from 190 p to 310 p.
the average of them being 2 6 0 ~ .These were developing at
about the same rate as most of the regenerates arising from
the host dorsal iris. Although none were cataracts there were
deformities in the arrangement of lens fibers. With the exception of the two pairs of smaller lenses which developed
on either side of the grafts the average diameter of the host
lens regenerates was 2 ' 7 7 ~ . Five of them were developing
cataracts. The fiber poles of these regenerates as in all others
were normally directed towards the retina.
The present report by the author is part of an extensive
investigation on the eye of the adult newt to determine as far
as possible all the factors which not only release and inhibit
lens regeneration but which also influence in any way its
normal and abnormal development. The lens in the eye of
-A D U L T
the adult Triturus v. viridescens can be carefully removed
without visible injury to the dorsal iris. Although injuries
or wounds do not, perse, (Stone, '52) favor or prevent lens
regeneration, this does not imply that a lens arising from the
wound region in the iris will always be normal.
Wachs ('14) working on several species of salamander
larvae stated that he saw no difference in lens regeneration
whether the lens was removed with care or associated with
injuries to the iris. It is true that lens regeneration occurs
readily in certain salamander eyes even following severe injuries to the iris, such as the removal of large amounts of the
dorsal iris (Stone and Griffith, '54). Some lens regenerates
may be normal and some may show various defects or develop
into cataracts. Some of these defects are associated with
pressure from surrounding structures with which the lens
regenerates come into contact (Stone, '52). Some on the other
hand are the result of uneven healing in an iris wound from
which a new iris membrane is regenerating (Stone and Griffith, '54).
From the results of experiments reported in this paper it
is quite obvious that the healing of wounds, such as slits
in the dorsal iris (fig. 1 A ) can be followed by normal lens
regeneration (figs. 6 8 and 6B), even though the rate of
development and size of the regenerate may be temporarily
delayed (fig. 1,B and C). On the other hand if wound healing
is uneven along the lens forming margin of the iris various
defects later appear in the lenses even resulting in two partially fused lens bodies (compare figs. 5A and 5 s ) .
Large normal appearing lenses will develop from iris grafts
placed into lensless eyes of the adult newt, provided the lens
regenerates are not subjected to mechanical pressure from
surrounding eye tissue (Stone, '52). However, it has been
the common experience of various investigators, as already
mentioned, that the lens regenerate always arises from the
former pupillary region of the graft and not from other
borders. This is still maintained by a graft even though the
original pupillary margin is excised before transplantation
takes place. The foregoing experiments have shown that the
removal of the pupillary margin of the graft has a marked
effect upon the lens regenerates. All the lenses were retarded
in development and many cataracts appeared among them.
Lens regeneration was slightly favored if the pupillary margin
of the graft was allowed to heal for 4 days previous to transplantation (fig. 2, D and D l ) . I n any event after the middorsal iris segment was detached from the rest of the iris
and transplanted the potency for the lens formation was still
the highest in the pupillary region.
However, if this same region, from which the pupillary
margin was removed is sealed to dorsal iris tissue in a 180"
rotated graft (fig. 3 C ) the ciliary margin of this rotated iris
can then be forced to regenerate a lens (fig. 3 D). Therefore
the less potent lens forming region of the rotated iris, i.e.,
the ciliary border, now takes over the role of lens regeneration
just as it does when it forms a free border of an experimentally produced accessory pupil in the upper iris (Stone,
'53). although the potcncy for lens formation in this ciliary
region is present, it is normally low, being called forth only
when it is a free border of a segment of iris united with the
surrounding iris of the eye.
It was stated in the beginning of this paper that earlier
investigators have cited the presence of the retina as an
important factor promoting lens regeneration. It has recently
been shown that the rate of lens regeneration and the size
of the lens regenerate is definitely affected by some retinal
factor (Stone and Steinitz, '53b). It was also mentioned that
the location of the lens fiber pole might be influenced by the
presence of the retina.
The results of the present experiments give strong evidence
to support this point of view. The lens fiber pole in all regenerates was directed, as in normal lens regeneration, towards the retina. Particularly significant were the lenses
developing from mid-dorsal iris se,gments turned inside out,
i.e., rotated 180" on the dorsoventral axis (fig. 4, C and D).
The polarity of the lens regenerates from the reversed iris
was not reversed. I n every case the fiber pole faced inward
towards the retina as it does in normal lens regeneration.
These are considered strong proof that the retina is playing
an important role in determining the mediolateral axis of the
lens. The results of other experiments t o be published later
will deal exclusively with this problem and give further evidence to support this view.
The experiments described here have been concerned with
the most potent lens forming region of the dorsal iris, the
middle third. On either side, along the dorsal pupillary margin, the potency for lens formation rapidly diminishes in
the nasal and temporal directions (Sato, '30; Stone, '52). I f
the middle portion of this region is replaced by a non-lens
forming ventral iris graft, two lenses then develop, one on
either side (Stone, '53, fig. 5). If the same areas of the
dorsal iris are separated by an intervening segment of lens
forming dorsal iris tissue, as in the present experiments (figs.
3 C and 4 B), a lens will still develop on either side of the
graft (fig. 3 E ) even though the graft may also give rise to
a lens (fig. 4 D). Multiple lens regeneration in a lensless eye
therefore depends upon an exposed free margin of dorsal
iris tissue which is separated far enough from other similar
regions in order to organize a separate lens forming unit.
This unit must have support from the near-by retina to produce a lens.
1. Four groups of experiments on 143 eyes of the adult
newt, Tritzcrus v.uiridescens, were made to study the relations
of lens regeneration to injuries and healing in the mid-dorsal
iris and to examine some of the conditions under which polarization of the lens is established.
2. Wound healing in vertical slits made along the pupillary
margin of the mid-dorsal iris in lensectomized eyes was rapid
but imperfections in it were associated with partially double
lenses and other morphological irregularities. By the end
of a month some lens regenerates were equal in size to
normal controls although earlier they were smaller.
L. 13. STOKE
3. Mid-dorsal iris grafts lacking the original pnpillav
margin developed lenses only at the border from which the
pnpillai-y margin had been excised. Better lens regeneration
followed if the border of the graft was allowed to heal for 4
days before transplantation. However, all the lens regenerates were retarded in development and many cataracts
appeared among them.
4. The middle segment of the dorsal iris was rotated 180"
making the ciliary margin of the graft a part of the free
pupillary border. When tho graft. was not dislodged by regenerating host iris, lens regeneration was forced from the
ciliary margin in a few cases. The grafts usually aeted as a
wedge between two lens forming areas in the host dorsal iris.
T,enses arose from one or both of these areas.
5. The mid-dorsal segment of the iris was turned inside
out, i.e., rotated 180" on its dorsoventral axis. Lenses developed from the reversed pupillary margin in some cases
but the h e r and outer poles were not reversed. The fiber
pole was normally directed towards the retina. which appsars
to influence the polarization of the regenerating lens.
6. Lenses also regenerated from host dorsal iris 011 either
side of a reversed dorsal ins graft even though the latter
gavo rise to a lens.
7. Many lentoids and cataracts were found in these experimental eyes.
IKEDA, Y. 1934 Heitrag zur Aualyse der Wolffechen Tdusunregcnsration durrh
xenoplnetisch ImphstLLtion der Ins in das eutlinste Auge bei TrMm und
ITynabCti. Arb. mat. Inst. Bcndui, 16: 6WX.
-.--1936 Beitriige cur Fragc. der Fahigkeit zur Linseiiregeneration hei
eincn A r t von Hynobins (ICg(n0biw %nna?bgsoTago). Ibid., 18: 17-50.
MIKAUI, Y. 1937 Reitriigo zur Analyae der Linsenregeneration (I.). ifber die
Linsenpotcnzgefiille an der Iris. Reprinted from Botany und Zoolom,
6: 75-80.
OKADA,Y o R. 1955 "he lens potc?ncy posterior to the iris. Proc. Imp. Bead.
Japan, 11: 11.5-118.
I939 Studies in lens regeueration in Anuran Amphihia. PW
liminnry observations and experiments. ?&ern. Coll. &i. Kyoto Imp.
Univ., B, 16: 159-188.
O a n l ~ a ,C. 1921 Experiments on the regeneration of lens i n Diemyctylus. J.
Exp. ZOO^., 33: 395-4137.
G. 1929 Uber den Einfluss der Rotgenstahlen auf die Regeneration
dcr Linse. Arch. f. Entw. merh., 121: 39-71.
RIUYER,R. W. 1948 An experimental study of lens regeneration in TTriturus
viridescens viridcscens. I. Regeneration of a lens after extirpation in
embryos and larvae of different ages. J. Exp. Zool., 107: 217-268.
1950 An expcrimentirl study of lens regeneration in T r i t u r u s virideacens airdescens. 11. Lens development from the dorsal iris in the
absence of the embryonic lens. J. Exp. Zool., 113: 317-354.
SATO,T. 1930 Beitrage zur Analyse der Wolff 'schen Ihsenregeneration. I.
Arch. f . Entw. mech., 12.2: 451-493.
1933 Beitrage zur Analyse der Wolff 'schen Linscnregeneration. 11.
Arch. f. Entw. mech., 130: 19-78.
L. S. 1952 An experimental study of the inhibition and release of lens
regeneration in adult eyes of Triturus v i d e s o e n s viridescens. J. Exp.
ZOO^., 121: 181-224.
1953 A n experimental ana1;rsis of lens regeneration. Am. J. Ophthal.,
36, Pt. I l : 31-39.
RTOXE, L. S.,AND R. H. G R I F ~ T H1954 Regeneration of the iris and lens in eyes
of adult T r h r u s ?I. airidexens. J. Exp. Zool., 127: 153-180.
~ T O X E ,L. S., AND H. STEIXITZ1953a The regeneration of lenses i n eyes with
intact and regenerating retina in adult Triturtis w. viridescens. J. Exp.
ZOO]., 164 : 435-468.
39.5313 Effects of hypophysectomy and thyroidectomy on lens and
retina regeneration in tlir adult newt, l'rtturus v. viridescrns. J. Exp.
ZOO^., 124 : 469-504.
L. S., AND J. H. VULTEE 1949 Inhibition and release of lens regeneration
i n the dorsal iris of Triturus v. viridescens. Anat. Rec., 109: 144-145.
['NO, &I. 1943-44 Zur Frage des Nerhanismus der Wolff 'sclien Linsenregeneration. Jap. J . Med. Sc. I (Anat.), 11: 75-100.
WACHS, H. 1914 N e w Versuche zur Wolff 'schen Linsenregeneration. Arch. f .
Entw. mech., 33: 3-51.
ZALOKAR,M. 1944 Coiitribution a 1'Ctude de la r k g e n h t i o n du cristallin chez
le Triton. Revue Suisse de Zoologie, lil, no. 26 : 443-521.
5 8 and 5R Concern, respectively, drawings of the inner surfaces of dissected
anterior halves of right and left eyes of same newt 20 days after lens
remoral. Figure BA shows an early double lens regenerate 20 days after
vertical slits were made in the dorsal iris (fig. 1A ) . Figure 5B shows
lens regeneration from normal dorsal iris following lens removal (fig. 1,
A l ) . x 35.
BA ;tnd BB Concern, respectively, right and left q e s of same animal, 31 days
after operations shown in figure 1, A and A l . There are no residual effects
of the slits in the dorsal iris upon the lens regenerate in the right eye (fig.
6A). Figure 6B shows lens regeneration from normal iris following lens
removal (fig. 1, A l ) . X 35.
Showing in a left eye a lens regenerate attached t o dorsal iris, 22 daps after
operation illustrated i n figure 1 A . Plane of section passes through scar
of a slit in the iris. Anterior subcapsular epithelium is abnormal, indicated
by arrow. X 70.
Showing in a right eye a small cataract, indicated by arrow, attached t o
former pupillarp region of iris graft, 23 days after removal of normal lens
and transplantation of iris which was allowed t o heal 4 days before grafting.
Operation is same as shown in figure 2, A, B, and G. Retarded lens regenerate
is also sho\vn attached to host dorsal iris. X 70.
Showing in a left eye a small cataract, indicated bp arrow, attached t o former
pupillarp region of iris graft, 22 d:tgs after removal of normal lens and
transplantation of iris. Operation is same as shoini in figure 2, A l , B1, and
CI. Lens developing from host dorsal iris is much larger. X 70.
10 Showing in n left eye :I cylindrical &aped roll of iris graft lodged hehind
host iris, 45 days after iris transplantation and 26 days after remoral of
normal lens. No lens regenerated from iris graft. The large lens regenerated
from the host dorsal iris. X 70.
11 Bliowing in a right eye :i thick Iiarrow iris graft fused with the host iris 47 days
after oporation. The large lens regenerated from host dorsal iris 28 d a y
after removal of normal lens. X 70.
Showing in ii right eye a deformed lens regenerating from cilary margin
of rotated iris graft (fig. 3, A, B, and C), 25 days after normal lens was
rcinoved and 3 7 days after iris was tranpplanted. X 70.
138, 1:<l3. 13C, and 131) Concern the same animal 25 days after lene renioval
and 60 days a f t e r rotatioil of i r k (see fig. 3 , A , B. and C). Figures 13A and
13C b110\v in iiytrt e j c lens iegcncr:ites from host i l i a 011 nasal and trmporal
?;ides of t h e well preserved graftcd iris (fig. 138). The g r a f t failed t o form
a lens. Figure 13 D sliows iii the left epe a lens regenerating froin the
ciliary niargiii of the rotxtecl iris graft. x 70.
14 Showing i n it left eye (47 days a f t e r turning dorsal iris segment inside out and
d:i) s after rerno\ m g norni:il lens) LI lrns regerwrnting from the rcverserl
iris b u t with inner fiber pole directed normally tonards the retina. X 70.
15 Sliuwing in a right eye ( 4 7 d:tys aftor reversing dorsal iris segment and
19 (lais a f t e r lens renioval) a lrns regenerating froin thick folded graft.
Tnncr fiber pole i s directed normally towards re tin:^. X i 0 .
16A, 18H, and 16C Coneern the s:iine animal 51 da>s a f t e r iris wits reversed!
and 19 days a f t r r normal lens was remored. Figure 16A sliows large lens
r e g c n c ~ ~ a tattached
t o graft. Iiiner fiber pole is directrd iiorinallg tovardP
retina. Bigure l 6 U shows a small stage 10 lens attached to host iris on nasal
side of iris graft. Fignre 16C shows a retarded lentoid, indicated by arrow,
derived froin iris graft. I t s fiber polrr point upward towards rotin:i. Portion
of the lens shown in figure 1613 also appears below graft. x 70.
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lens, adults, experimentov, newt, viridescens, regenerative, triturus, eyes
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