Download A histologic study of lens regeneration in aphakic rabbits.

Investigative Ophthalmology & Visual Science, Vol. 31, No. 3, March 1990
Copyright © Association for Research in Vision and Ophthalmology
A Hisfologic Study of Lens Regeneration
in Aphakic Rabbits
Arlene E. Gwon,*t Lawrence J. Gruber,j- and Karen E. Mundwilerf
Lens regeneration occurs in New Zealand albino rabbits after endocapsular lens extraction, which
leaves the anterior and posterior lens capsules relatively intact. Slit-lamp photography, histologic
studies, and lens protein analysis confirmed the differentiation of lens fibers. In the current study, we
performed a sequential analysis of the regenerating rabbit lens. After endocapsular phacoemulsification and irrigation/aspiration of the lens, rabbits were sacrificed at different time points for histologic
evaluation. Similarities with embryologic development of the lens were evident, although in some
sections, abnormal cellular proliferation occurred. By the 6th day after surgery, a monolayer of lens
epithelial cells lined both the anterior and posterior capsules. At 1 month, the posterior epithelial cells
had elongated, and nuclei had migrated anteriorly. At 2 months, lens cells were differentiating at the
equatorial zone with gradual elongation, anterior migration of nuclei, and eventual loss of nuclei. Invest
Ophthalmol Vis Sci 31:540-547,1990
in a normal lens, ie, epithelial cells and lensfibercells
with larger than normal interfibrillar spaces.
According to Randolph (1899), Gonin noted enlargement and karyokinesis of the epithelial cells on
the anterior capsule as early as 1 day after surgery,
with full proliferation occurring 2 days postoperatively.7 In some cases, Gonin found that anterior
capsule cells became detached and acted as centers of
proliferation on the posterior capsule. In the equatorial region, at the most peripheral parts of the posterior capsule, epithelial cells were seen to extend and
elongate. The lens fibers that were not removed during surgery tended to swell and become granular.
From these observations, Gonin concluded that the
new lens was formed by the hypertrophy of the lens
fibers remaining after surgery and continued to develop at the equatorial zone.
In 1960, Stewart showed that when embryonic tissue was implanted into the capsular bag after lens
evacuation, the new lens fibers were aligned in the
typical concentric pattern of the mature lens.8 He
demonstrated also that lens differentiation occurred
at the equator.
More recently, Gwon et al showed that regenerated
rabbit lenses differentiate normally at the equatorial
zone and also at the anterior capsulotomy site where
anterior and posterior capsules are in close approximation.9 In addition, Gwon and Enomoto verified
the production of a, /3, and 7 crystallins in the regenerated rabbit lens with proportions similar to fetal or
normal lens.10
To better understand the process of lens regeneration or healing after endocapsular lens extraction,
The progressive steps in the process of spontaneous
regeneration of tissue of ectodermal origin have been
described well for the areas of skin epidermis and
corneal epithelium.12 Another ectodermal derivative,
the crystalline lens, was reported in 1827 to regenerate in rabbits.3 However, research in this area has
progressed more slowly. Investigators have found that
the lens regenerative process is dependent on an intact anterior and posterior lens capsule.4 After extraction of the lens capsular contents, regenerating
lens tissue first is noted 2 weeks postoperatively, beginning in the periphery of the capsule and occurring
more rapidly in younger rabbits.5
In 1842, Valentin described for the first time the
regenerated rabbit lens on a microscopic level, demonstrating the presence of characteristic round or
polyhedral-shaped crystalline cells.6 He suggested
that regeneration takes place by effusion into the
capsule of initially liquid cytoblastic masses, which
subsequently develop into lens cells and fibers.
Thirty years later, in 1872, Milliot reported that
lens fibers formed in the equatorial region and were
aligned linearly, similarly to the lenses in embryos or
very young rabbits.5 Furthermore, the regenerated
lens contained the same anatomic elements present
From the *University of California at Irvine and fAllergan
Pharmaceuticals, a division of Allergan, Inc., Irvine, California.
Presented in part at the International Congress of Eye Research,
San Francisco, September, 1988.
Submitted for publication: November 9, 1988; accepted July 14,
1989.
Reprint requests: Arlene Gwon, MD, Allergan Pharmaceuticals,
2525 Dupont Drive, Irvine, CA 92715.
540
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RABDIT LENS REGENERATION / Gwon er ol
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and to verify that the process develops from residual
lens epithelial cells (rather than retained lens fibers),
we examined the histologic findings in the early postoperative period during the phase of early lens regrowth in rabbits. Previous histologic work has focused on the developed regenerated lens when lens
fiber differentiation is restricted to the lens equatorial
zone. 578
Materials and Methods
Surgical Procedure
Six young New Zealand albino rabbits, weighing
approximately 2.5-3 kg, were anesthetized with approximately 5 mg/kg Xylazine (Fermenta Animal
Health) and 50 mg/kg ketamine (Aveco, Fort Dodge,
IA), intramuscularly. The surgical eye was dilated
with 1% cyclopentolate (Alcon, Fort Worth, TX) and
2.5% phenylephrine (Winthrop, New York, NY);
eyelashes were trimmed; and the ocular area was dis-
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infected with povidone iodine. A wire lid speculum
was inserted to retract the lids, and a limbal incision
was made at 11 o'clock with a 2.85-mm keratome. A
21-gauge phacoemulsification tip was inserted
through the corneal wound and used to perform the
anterior capsulotomy and removal of the lens nucleus
with balanced salt solution as the irrigant (Fig. IA).
Considerable care was taken to remove all lens cortical material by diligent irrigation and aspiration. At
the completion of the procedure, the corneal incision
was closed with three interrupted nylon sutures
(10-0).
At the end of surgery, 0.25 ml (20 mg) of gentamicin (Solo Pak, Franklin Park, IL) was injected subconjunctivally, and Polymixin B-bacitracin-neomycin ointment (Pharmaderm, Melville NY) was applied topically. Postoperatively, all surgical eyes were
treated topically with 1% tropicamide (Alcon, Humacao, PR) and 0.3% Gentamicin (Allergan, Irivine,
CA), four times daily for 7 days. All experimental
Fig. 1. (A) Schematic diagram of endocapsular phacoemulsification technique. (B-D) Light micrographs of epithelial cells in capsular bag, 1
day postoperatively. (Anterior lens surface, right; posterior, left.) (B) Anterior epithelial cells are multilayered in some areas (X60). (C) Single
layer of flat cells with elongated nuclei migrate along the posterior capsule (X60). (D) Cellular proliferation in equatorial region (X10).
(Hematoxylin and eosin)
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / March 1990
Vol. 31
animals were treated in accordance with USDA
guidelines and the ARVO Resolution on the Use of
Animals in Research.
Histologic Procedure
Slit-lamp biomicroscopy and photography were
performed periodically and rabbits were sacrificed at
various time periods for histologic examination. For
each rabbit, both eyes and their associated structures
were removed and preserved in 10% neutral buffered
formalin. After fixation, the globe was sectioned
through the cornea, pupil, and optic nerve with the
lens in situ. Tissues were processed in an automatic
tissue processor in a 14-h processing cycle which included dehydration in reagent grade alcohol, clearing
in xylenes, and infiltration with paraffin. Paraffinembedded tissues were sectioned at 5 nm and stained
with hematoxylin and eosin.
Results
Day 1
Postoperatively, the corneas of the rabbits were relatively clear, with little or no edema around the corneal incision site. The anterior chamber contained
moderate to heavy amounts of fibrin.
Histologically, a single monolayer of epithelial cells
lined the anterior capsule, with some multilayering
and some gaps where cells had most likely been lost at
the time of surgery (Fig. IB). Migrating cells, as indicated by their long flat appearance, began to appear
along the posterior capsule (Fig. 1C). Cells had begun
proliferating in the equatorial zone, and some eosinophilic debris was seen (Fig. ID).
Day 4
The corneas were clear and nonedematous, and the
fibrin reaction in the anterior chamber was resolving
by the 4th day after surgery.
Epithelial cells had proliferated along the posterior
capsule and were in contact with a single layer of
anterior epithelial cells (Fig. 2A). Multiple vacuoles
were seen in the epithelial cell layers (Fig. 2A). At the
equator, among some debris and scattered neutrophils, lensfibershad begun differentiating, with anterior displacement of cell nuclei (Figs. 2B, C). Some of
the differentiated fibers, however, may have been material left in the capsule bag at the time of surgery.
Day 6
Corneas, anterior chambers, and lens capsules were
clear by the 6th day.
Fig. 2. Light micrographs, 4 days postoperatively. (Anterior lens
surface, right; posterior, left.) (A) Anterior cuboidal and posterior
columnar cells line the lens capsule (X60). (B) Cells proliferate
along the posterior capsule (X10). (C) Differentiating lens fibers
(XlO). (Hematoxylin and eosin)
Histologically, a single layer of epithelial cells,
elongating in an anterior-posterior direction with anterior displacement of their nuclei, lined the posterior
capsule (Fig. 3A). In general, thecapsules were linear
and regular (Figs. 3A, B). Toward the central region
of the lenses, the capsules were wrinkled and undu-
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RADBIT LENS REGENERATION / Gwon er ol
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h
Fig. 3. Light micrographs, 6 days postoperatively. (Anterior lens surface, right; posterior, left.) (A) Posterior capsule cells elongated with
anterior displacement of nuclei (X10). (B) Posterior capsule cells elongated with anterior displacement of nuclei (X60). (C) Irregular cellular
proliferation associated with capsular wrinkling (XlO). (D) Anterior capsule rolled outward at site of surgical incision (X10). (Hematoxylin
and eosin)
lating. Here, in the central region, multiple stellate
cells with large vacuoles were seen in a hyperproliferative state (Fig. 3C). Similarly, a proliferation of cells
with less definitive orientation was seen at the site of
the anterior capsulotomy, where the capsules were
less taut (Fig. 3D).
Day 7
Under slit-lamp examination, new lens growth was
seen in the periphery of the capsular bag, and the
anterior capsulotomy appeared to be sealed by fibrin
in some instances (Fig. 4A).
Posterior lens cells continued to elongate in an anterior-posterior direction, and nuclei were displaced
anteriorly (Fig. 4B). Cells in the equatorial zone had
begun to differentiate, and nuclei had begun to migrate anteriorly. Eosinophilic, partially disorganized,
lens material filled the space between anterior and
posterior epithelial cells (Fig. 4C). In some cases de-
velopment progressed at different rates, as in the case
pictured (Figs. 4C, D), where development in the
equatorial region (Fig. 4C) was more advanced than
in the central lens region (Fig. 4D).
Month 1
By 1 month, slit-lamp examination revealed that
approximately 50% of the capsular bag wasfilledwith
lens regrowth (Fig. 5A).
Histologically, the differentiating fibers from the
posterior cells had elongated considerably and the
nuclei had migrated anteriorly, forming a horizontal
band across the posterior cortical region (Fig. 5B). In
the equatorial region, normal fiber differentiation
continued, with elongation of cells and anterior
displacement of nuclei along the posterior capsule
(Fig. 5C).
A single layer of cuboidal epithelial cells and vacuoles lined the anterior capsule; the lens cortex be-
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / March 1990
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Hg. 4. Light micrographs, 7 days postoperatively. (Anterior
lens surface, right: posterior. left) (A) Slit lamp photograph of
regenerated lens material at I week. Arrow points to new lens
growth. (B) Elongated cells with anteriorly displaced nuclei line
posterior capsule (X60). (C) Lensfiberdifferentiation in the posterior equatorial region (X10). (D) Cuboidal cells lines anterior
and posterior capsule (X10). (Hematoxylin and eosin)
neath was vacuolated and in some areas had separated from the anterior epithelial cells (Fig. 5B).
ranged (Fig. 6C). In other areas, with larger vacuoles,
the anterior lens cortex had separated from the epithelium (Fig. 6D).
Month 2
Between the 2nd and 3rd months, lens regrowth
filled the capsular bag, except in the area of the original anterior capsulotomy (Fig. 6A).
At this time, differentiation was occurring primarily in the equatorial region (Fig. 6B). The bulk of the
lens material consisted of an eosinophilic amorphous
substance with multiple vacuoles. In some areas, with
fewer vacuoles, the lens material was uniformly ar-
Discussion
Lens fiber differentiation in the embryo has been
shown to involve loss of mitotic activity, marked cellular elongation, intensive synthesis of lens specific
proteins called crystallins, and loss of the cell nucleus. '' In this microscopic study of the regenerating
rabbit lens, we have demonstrated the processes lead-
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RABDIT LENS REGENERATION / Gwon er ol
Fig. S. Light micrographs of lens fiber differentiation, 1 month
postoperatively. (Anterior lens surface, right; posterior, left) (A)
Regenerated lens material at 1 month. Approximately 50% of the
capsular bag isfilledwith new lens growth. (B) Multiple vacuoles in
anterior cortex. Lens fiber differentiation proceeding from posterior capsule (X10). (C) Lens fiber differentiation in equatorial region (X10). (Hematoxylin and eosin)
ing up to the morphologic changes noted in lens fiber
differentiation. As in embryonic development, lens
regeneration proceeds by cellular proliferation along
the anterior and posterior capsule (days 1-7) and is
545
followed by elongation of the posterior epithelial cells
and migration of the nuclei anteriorly (1 month).
During the 2nd month, lens differentiation occurs at
the equatorial zone, with gradual elongation of cells,
anterior migration of nuclei, and eventual loss of the
nuclei. A description of the crystalline changes during
lens regeneration is presented in a previous publication.9
The ability to study the differentiation of lens fiber
cells in vitro has greatly enhanced knowledge of the
biochemical influences on cellular differentiation.
Following the suggestion of Coulombre and Coulombre that a substance from the posterior segment
induces lens fiber differentiation,12 various investigators have isolated this type of factor in the retina and
vitreous. Chamberlain and McAvoy have identified a
factor from the neural retina that appears to be fibroblast growth factor, capable of inducing lens fiber differentiation.13 Similarly, Beebe and associates have
isolated a factor, lentropin, from the vitreous; it is
related to insulinlike growth factors and is capable of
inducing lens epithelial cells to differentiate into
fibers.14
While in vitro studies enable us to isolate biochemical factors, in vivo studies may increase understanding of the morphologic factors involved in lens regrowth. In our study, after evacuation of the lens, the
empty capsular bag became folded as a result of loss
of elasticity. The area of undulating or wrinkled capsule was associated with abnormal cellular proliferation. This finding suggests that the mechanical forces
exerted on the capsule bag may play an important
role in lens fiber differentiation. This is consistent
with the work of Coulombre and Coulombre, who
showed that lens fiber differentiation occurred when
lens epithelial cells attached to capsules were implanted in chick embryos.'5 The new lens fiber "size,
shape, and orientation is remarkably better when the
implant is flat."*5 This also supports the hypothesis
proposed by Bito et al, that "physical forces exerted
on the lens (such as those due to the varying tension
of the suspensory ligaments) and the resulting wear
and tear on the epithelium may be casually related to
the rate of cell proliferation and the distribution of
dividing cells in this tissue."16
In summary, the regenerative process in the New
Zealand albino rabbit after endocapsular lens extraction appears to follow the stages seen in the embryonic development of the lens. In the future, in the
immediate postoperative period, it is possible that
capsule tension may be maintained with pharmacologic agents, and that growth factors may be added to
improve the quality of the regenerating lens.
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / March 1990
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Fig. 6. Light micrographs of regenerated lens, 2 months postoperatively. (Anterior lens surface, right; posterior, left) (A) Regenerated lens
material at 2 months. New lens growthfillscapsular bag except at the anterior capsulotomy site. Arrow points to the anterior capsulotomy site.
(B) Lens fiber differentiation in the equatorial region (X10). (C) Single layer of more normal anterior epithelial cells is associated with more
unifonn arrangement of lens cortex (X10). (D) Anterior cortex consists of amorphous eosinophilic material with vacuoles (X10). (Hematoxylin and eosin)
Key words: lens regeneration, lens fiber differentiation, lens
histology, endocapsular lens extraction, lens capsule
Acknowledgments
The authors wish to acknowledge the scientific contributions and advice of Irving H. Leopold, MD and Daniel M.
Albert, MD, and the editorial assistance of Mary-Jane
Branin, MA. Without the translations by David Harper,
MD, Val Portnoy, and Heidi Wierzba, work done by the
early investigators in this area would have gone undiscovered.
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