Articles Concanavalin A-Induced Posterior Subcapsular Cataract

Articles
Concanavalin A-Induced Posterior Subcapsular Cataract:
A New Model of Cataractogenesis
Arlene Gwon,*~f Christine Mantras,* Lawrence Gruber* and Crystal Cunanan*
Purpose. To evaluate the effect of Concanavalin A (Con A) on cataract formation in New
Zealand Albino rabbits. Uveitis is a chronic inflammatory condition of the eye involving the
anterior and/or posterior segments. It may be acute or chronic and is associated with the
development of posterior subscapular cataract over time. Con A is a nonspecific inflammatory
agent and mitogen for T cells and some B cells. Used extensively in immunogenic studies Con
A has been shown to induce uveitis after intravitreal injection in New Zealand Albino rabbits.
Methods. In two separate studies, Con A was injected intracamerally or intravitreally into one
eye of 12 New Zealand Albino rabbits and an equal volume of balanced salt solution was
injected into the opposite eye as a control. In a third study, the effect of topical steroids after
intravitreal injection of Con A was evaluated. In all studies, anterior and posterior inflammation
and the development of cataract was monitored by slit lamp biomicroscopy and photography.
Cataract formation was also studied histopathologically.
Results. Initially, all eyes treated with Con A demonstrated moderate anterior chamber inflammation
while eyes treated with balanced salt solution showed no inflammation. Three months
after treatment, posterior subcapsular cataracts were present in all rabbit eyes treated with
intravitreal Con A. In the third study, topical steroid treatment of Con A-induced inflammation
significantly reduced anterior chamber inflammation but had no effect on vitreous humor
and posterior subcapsular cataract formation.
Conclusion. This experimental model was the first to demonstrate the development of posterior
subcapsular cataracts after Con-A induced inflammation. The cataract was clinically and
histologically similar to human posterior subscapular cataracts. Invest Ophthalmol Vis Sci.
1993; 34:3483-3488.
IL osterior subcapsular (PSC) cataract is a type of presenile
and senile opacification of the human lens occurring
in 6% of adults between the ages of 43 and 84
years in the Beaver Dam Eye Study. 1 It may be caused
by a variety of conditions and toxins and has been
referred to as "cataracta complicata." It is known to
occur in inflammation, widespread degenerative
states, and when ocular circulation is gravely impaired.
Such cataracts are presumably due to abnormal lens
metabolism and associated with diffusion into the lens
From *Allergan Pharmaceuticals and the ^University of California at Irvine,
Irvine, California.
The results of this paper were presented in part at ARVO, 1992 in a poster titled
"Concanavalin A-Induced Posterior Subcapsular Cataract: A New Model of
Cataractogenesis" (1758-35).
Submitted for publication: December 11, 1992; accepted May 27, 1993.
Proprietary interest category: E.
Reprint requests: Arlene Gwon, Allergan Inc., 2525 Dupont Drive, P.O. Box
19534, Irvine, CA 92713-9534.
of toxins from an inflammatory focus, exogenous
drugs or from products of degeneration caused by disease.
The earliest clinical changes are typically seen in
the central or axial region of the posterior lens and
thus decreases vision early in its course. 23
The lectin Concanavalin A (Con A) is a nonspecific
inflammatory agent and mitogen for T cells and some
B cells. It has been used extensively in immunogenic
studies and shown to induce uveitis after intravitreal
injection in New Zealand albino rabbits. 4 " 6 Because of
the prolonged nature of the inflammatory response
with Con A seen in animal studies, it was a good candidate
for study of the development of complications,
such as cataract formation in uveitis.
In the current study, we investigated the role of
Con A-induced inflammation in the formation of PSC
cataracts. Intravitreal injection of Con A was associated
with anterior and posterior uveitis and cataract
formation, whereas intracameral injection was asso-
Investigative Ophthalmology & Visual Science, December 1993, Vol. 34, No. 13
Copyright © Association for Research in Vision and Ophthalmology 3483

3484 Investigative Ophthalmology & Visual Science, December 1993, Vol. 34, No. 13
ciated with acute mild anterior uveitis and no cataract
development. We also evaluated the role of topical
corticosteroids in decreasing the Con A-induced inflammation
and subsequent PSC cataract formation.
Whereas anterior uveitis was significantly less in the
steroid treated group, no difference was noted in the
severity of posterior uveitis and the" development of
PSC cataract.
MATERIALS AND METHODS
Intracameral Injections
Topical anesthesia with 0.5% proparacaine solution
(Allergan, Irvine, CA) was applied to both eyes of six
juvenile female New Zealand Albino rabbits weighing
2.5 to 3 kg. Approximately 1 ml Con A at 1 mg/ml
(total dose of 100 ug) was injected into the anterior
chamber of one eye of each rabbit and the contralateral
eye received an equal volume of balanced salt solution
(without prior paracentesis). After the injections,
the animals received one drop each of 1 % tropicamide
(Alcon, Humacao, Puerto Rico) and 0.3%
gentamicin (Solo Pak, Franklin Park, IL), four times
daily for 7 days.
Intravitreal Injections
Eighteen juvenile female New Zealand albino rabbits
weighing approximately 2.5 to 3 kg were anesthetized
with a 2 to 3 ml intramuscular injection of a 1:5 mixture
of 100 mg/ml xylazine base (Haver, Shawnee, KS)
and 50 mg/ml ketamine HC1 (Aveco, Fort Dodge, IA)
combined with sterile water. The eyelashes were
trimmed and the fur surrounding the eye was prepped
with povidone iodine (Professional Disposables, Inc.,
Orangeburg, NY). A lid speculum was inserted and
intravitreal injections were placed at approximately 2
to 3 mm posterior to the corneoscleral limbus in the
superotemporal quadrant, using a 30-gauge needle.
Group 1. Six rabbits received a 1 ml injection of
Con A at 1 mg/ml (Sigma Chemical Co., St. Louis, MO
and ICN Biochemicals, Cleveland, Ohio), yielding a
total dose of 100 /tg in one eye. The fellow eye received
an injection of equal volume of balanced salt solution
(Allergan Medical Optics, Irvine, CA).
Group 2. Twelve rabbits received intravitreal Con
A. Postoperatively, six of these rabbits received 1%
Pred Forte (Allergan, Irvine, CA) four times daily in
the test eye for 3 weeks.
Postoperatively, all test eyes received 1% tropicamide
(Alcon, Humacao, Puerto Rico) and 10% phenylepherine
(Winthrop, New York, NY) four times daily
to each eye for 2 weeks to maintain dilation.
Slit Lamp Biomicroscopy/Photography
All eyes were examined with slit lamp biomicroscopy at
least biweekly for 1 month, weekly for 2 months, and
monthly thereafter. Slit lamp photography was performed
at months 2, 3, and 4. Biomicroscopy findings
were graded on a scale from 0 to 4 with 0 = none, 1 =
trace, 2 = mild, 3 = moderate, and 4 = severe (Table
1). Intergroup comparisons used the exact P value derived
from the Wilcoxon Rank Test.
Histopathology
Rabbit eyes were fixed in 10% neutral buffered formalin.
After washing the eyes in tap water, the globe was
sectioned from the central cornea through the pupil to
the optic nerve with the lens in situ. Tissue was processed
in an automatic tissue processor overnight. The
tissue was then dehydrated in reagent grade alcohol,
cleared with xylene and infiltrated with paraffin. Paraffin-embedded
tissues were sectioned at 5 nm and
stained with hematoxylin and eosin.
All animals were handled in accordance with
USDA guidelines and the ARVO Resolution on the
Use of Animals in Research.
RESULTS
Biomicroscopy
Intracameral Injections. Four eyes that received balanced
salt solution intracamerally had no evidence of
irritation or inflammation at any time during the 6week
observation period. Two eyes had minor irritation,
which resolved by day 4 and remained clear
through the 6-week observation period: one eye
showed mild anterior chamber cells, which resolved by
TABLE l. Anterior Chamber Inflammation
Grading Scale
Cells
None
Trace
Mild
Moderate
Severe
Flare
None
Trace
Mild
Moderate
Severe
0
+ 1
+2
+3
+4
0
+ 1
+2
+3
+4
No cells seen per high power field
1-9 cells seen per high power field
10-25 cells seen per high power field
26-50 cells seen per high power field
Too many cells to count per high
power field
No Tyndall effect
Tyndall beam in the anterior chamber
has mild intensity
Tyndall beam in the anterior chamber
has strong intensity
Tyndall beam is very intense, aqueous
has white, milky appearance
Tyndall beam has marked intensity,
fibrin fills anterior chamber and
obscures view of the pupil

Posterior Subcapsular Cataract Model 3485
day 4; one eye that had a slight iris nick during injection
had some fibrin on the iris at 24 hours, which
resolved by day 4. All eyes receiving intracameral injection
of Con A had slight irritation and pupil miosis
immediately after injection. At 24 hours, there was
mild to moderate cells and fibrin in the anterior
chamber. Two eyes also had moderate corneal edema
and haze. These eyes were treated with 1% prednisolone
acetate (Allergan, Irvine, CA), one drop four
times a day for 5 days. By 1 week postinjection, all
inflammatory signs had resolved and all eyes remained
normal until the animals were killed at 6 weeks.
Intravitreal Injections. Inflammation. At day
one, all eyes receiving balanced salt solution were normal
without evidence of inflammation in the anterior
or posterior segment and remained normal throughout
the 6-month observation period. All eyes that received
Con A intravitreally had fibrin in the vitreous
humor and a few had a preretinal hemorrhage on indirect
ophthalmoscopy at day 1. The anterior segments
of these eyes were normal (Table 2).
By day 3, there was evidence of inflammation in all
Con A-treated eyes with moderate cells and fibrin in
the anterior chamber, on the anterior lens capsule and
in the vitreous humor. Anterior segment inflammation
gradually subsided by 4 to 7 days while posterior segment
inflammation increased with moderate cells and
fibrin noted in the vitreous and on the posterior surface
of the posterior lens capsule.
Inflammation persisted through day 9 in the steroid
group and day 16 in the nonsteroid group. Anterior
chamber cells were significantly less in the steroidtreated
group at all times except days 3, 7, and 9 (Table
2). Anterior chamber flare and fibrin was minimal
in both groups throughout the evaluation period, and
significantly less in the steroid group at day 9 only
(Table 3). Vitreous cells were moderate in both groups
TABLE 2.
Anterior
Days
0
1
2
3
5
7
9
13
16
23
Intravitreal Concanavalin A:
Chamber Cells
Steroids
(n = 6)
0.0
0.0
0.16 ±0.15
0.8 ±0.15
0.66 ±0.19
0.3 ±0.19
0.33 ± 0.3
0.0
0.0
0.0
No Steroids
(n = 12)
0.0
0.0
1.41 ±0.31
1.5 ±0.15
1.58 ±0.14
1.0 ±0.24
1.16 ±0.24
1.25 ±0.21
1.41 ±0.14
0.0
P Value
0.026
0.317
0.045
0.705
0.193
0.045
0.023
Values are mean ± SE. Biomicroscopy findings are graded on a
scale from 0 to 4; 0 = none, 1 = trace, 2 = mild, 3 = moderate,
and 4 = severe.
TABLE 3. Intravitreal Concanavalin A:
Anterior Chamber Flare/Fibrin
Days
0 1
2 3
5 7
9
13
16
23
Steroids
(n = 6)
0.0
0.5 ±0.2
0.0
0.16 ±0.15
0.0
0.0
0.0
0.0
0.0
0.0
No Steroids
(n = 12)
0.0
0.0
0.16 ±0.16
0.33 ±0.14
0.25 ±0.13
0.16 ±0.11
1.0 ±0.3
0.0
0.16 ±0.11
0.0
P Value
0.317
0.157
0.317
0.317
0.014
0.157
Values are mean ± SE. Biomicroscopy findings are graded on a
scale from 0 to 4; 0 = none, 1 = trace, 2 = mild, 3 = moderate,
and 4 = severe.
and not significantly different (Table 4). After the seventh
week it was difficult to evaluate vitreous inflammation
because of the development of cataracts.
Cataract development. The lenses of steroid and
nonsteroid Con A groups remained clear until 2 weeks
when a grainy, lacy pattern of cells and fibrin were
noted on the posterior capsule surface (Fig. 1). As
early as 5 weeks, vacuoles were noted in the posterior
subcapsular lens area in 3 of 6 steroid treated eyes and
11 of 12 nontreated eyes. By 7 weeks, a posterior subcapsular
cataract was noted in 3 of 6 steroid eyes and
all of the nontreated eyes.
By 3 months postintravitreal Con A, PSC cataracts
were present in all eyes and were granular/vacuolar in
appearance (Fig. 2). In the six steroid-treated eyes, the
PSC opacities were localized in the central optical axis
TABLE 4. Intravitreal Concanavalin A:
Vitreous Cells
Days
1
2 Q
O
5
7
9
13
16
23
28
W5
W6
W7
Steroids
(n = 6)
0.0
0.0
0 16 + 015
0.16 ±0.15
2.0 ±0.33
2.0 ±0.33
2.33 ±0.19
2.33 ±0.19
2.5 ±0.2
2.5 ±0.2
2.16 ±0.15
2.16 ±0.15
2.0 ±0.24
No Steroids
(n = 12)
0.0
0.42 ±0.14
o F.Q + n 14
U.JO T" U.I i
0.83 ± 0.24
2.75 ±0.3
3.0 ±0.12
3.16 ±0.16
2.66 ±0.14
2.75 ±0.13
2.66 ±0.21
2.5 ±0.34
2.33 ± 0.33
2.0 ±0.25
P Value
0.083
0.083
0.102
0.058
0.033
0.31
>0.999
0.0317
>0.999
0.563
>0.999
Values are mean ± SE. Biomicroscopy findings are graded on a
scale from 0 to 4: 0 = none, 1 = trace, 2 = mild, 3 = moderate,
and 4 = severe.

3486 Investigative Ophthalmology 8c Visual Science, December 1993, Vol. 34, No. 13
FIGURE 1. Slit lamp photograph. A lacy pattern of cells and
fibrin is noted on the posterior surface of the lens in intravitreal
Con A-treated eye at 2 weeks.
or diffuse involving 20% to 40% (n = 3) or 80% to
100% (n = 3) of the posterior circumference, respectively.
In the 12 nonsteroid eyes, PSC opacity was localized
in 5, diffuse in 3, and mixed with anterior and
posterior cortical opacity in 4.
By 16 weeks, four eyes, two in each group, had
developed mature cortical and nuclear cataracts. In
the remaining eyes, the PSC cataracts appeared stable
and nonprogressive.
No cataracts were noted in any of the balanced salt
solution-treated eyes at any time.
Histopathology
At 2 weeks, the lens capsule and anterior epithelium
appeared intact. Small vacuoles were seen in the epithelial
layer that were probably related to a fixation
artifact. Lens fibers appeared normal for the most
FIGURE 2. Slit lamp photograph. A globular, edematous posterior
subcapsular opacity is noted in intravitreal Con Atreated
eye at 11 weeks.
FIGURE 3. Photomicrograph of intravitreal Con A-treated
eye at 14 months. A monolayer of epithelial cells lines the
anterior capsule. Incomplete cell differentiation is noted in
the equator with nuclei displaced toward the posterior lens.
(Bar = 5 //.)
part, staining more deeply in the nuclear region and
paler in the cortical area. There was an occasional separation
of the cortical fibers from the posterior capsule.
Adjacent cortical fibers appeared foamy or swollen
and separation between fibers was somewhat prominent.
Occasional vacuoles were seen. The lens nucleus
appeared normal and cell differentiation in the equatorial
region appeared unremarkable. The vitreous humor
contained numerous inflammatory cells and fibrin
strands.
At 3 months, the lens capsule appeared intact. A
semicontinuous monolayer of epithelium extended
FIGURE 4. Photomicrograph of intravitreal Con A-treated
eye at 3 months. A multilayer of large, rounded bladder-type
cells of Wedl is seen on the posterior capsule. Adjacent cortical
fibers are swollen or globular and there is loss of normal
architecture in the posterior cortex. Multiple inflammatory
cells and a fibrovascular membrane are noted in the vitreous
humor. (Bar = 5 M-)

Posterior Subcapsular Cataract Model 3487
along both the anterior and posterior capsule with loss
of the equatorial bow region. Lens epithelial cell differentiation
was incomplete with nuclei displaced toward
the posterior lens (Fig. 3). The anterior epithelial
layer contained vacuoles and there were some areas of
cell loss. Cells along the posterior capsule were larger
and more rounded in appearance, the so-called "bladder"
cells of Wedl. Adjacent cortical fibers were swollen
or globular (Fig. 4). In other areas, there was loss
of architecture and the cortex was amorphous with an
occasional cell nucleus noted in the more central cortical
regions. The vitreous humor contained numerous
inflammatory cells and a fibrovascular membrane. Similar
changes were noted in the micrographs examined
at 4, 5, and 14 months.
DISCUSSION
Posterior subcapsular cataracts can occur spontaneously
in the aging Wistar rat, 7 can occur after the intravitreal
injection of docosahexenoic acid 8 and bacterial
endotoxin 9 and can occur after microwave and
ionizing radiation exposure. 911 In the current studies,
we describe the occurrence of PSC cataracts after Con
A-induced inflammation.
We have shown that intravitreal injection of the
lectin Con A will induce chronic inflammation followed
by development of posterior subcapsular cataract
in New Zealand Albino rabbits. The prolonged
Con A-induced inflammation does not occur when the
compound is injected intracamerally. Although slight
inflammation did occur for a few days after intracameral
injection, chronic inflammation and cataract
formation was not noted. It is possible that the injected
Con A was washed out through the trabecular
meshwork too rapidly to induce a chronic inflammation
and cataract. In contrast, intravitreal injection of
Con A induced a chronic anterior and posterior uveitis
with exacerbations and remissions for up to 2 months.
The inflammation is similar to some types of human
uveitis in its clinical course of exacerbations and remissions,
inflammatory signs of keratoprecipitates, anterior
chamber cells, posterior synechiae formation,
vitreous cells, and cataract formation.
Treatment with topical corticosteroids resulted in
less severe anterior uveitis but had minimal effect on
the posterior uveitis. PSC cataract formation appeared
to be less severe in the steroid group but differences
were small and further studies are needed for
verification. In humans, both uveitis and steroid therapy
are known to induce PSC cataract independently,
so it would be of interest to learn if higher doses of
steroid had an inhibitory effect in this model.
The clinical and morphologic changes in PSC cataract
in humans and radiation-induced PSC in mice,
rats, and the bullfrog have been well described."" 17
Clinically, the posterior subcapsular cataract seen at 3
months after intravitreal injection was similar in clinical
appearance to the vacuolar or lacy type of cataract
described by Eshagian. 2 These cataracts are granular
and appear to be made of multiple watery cysts. They
are seen in senile or age-related, diabetic, retinitis pigmentosa,
and corticosteroid cataracts. 2
The Con A-inflammatory PSC cataract is histologically
similar to that reported in humans 212 " 14 and in
radiation-induced PSC cataract in animals. 1115 " 17 Cell
differentiation in the equatorial region is incomplete
with displacement of the lens bow nuclei toward the
posterior lens pole. Cortical fibers appear irregular in
size and shape. There is migration of cells along the
posterior capsule. These posterior cells are swollen,
"bladder" type cells of Wedl and there is swelling in
the adjacent cortical fibers, with areas of liquefaction
and loss of fiber architecture.
It is also noteworthy the Con A-induced inflammation
remained relatively inactive after 2 months and
the posterior subcapsular cataract progressed very little
from 3 to 14 months in most cases. These results
can be interpreted several ways: the cataract progression
may be dependent on an active inflammatory stimulus;
cataract progression may require the continued
presence of Con A; or the Con A is somehow removed
or inactivated over time. In addition to being a nonspecific
inflammatory agent and a mitogen for T cells,
Con A is widely used as a probe for studying cell-surface
oligosaccharides. 1819 Suzuki et al demonstrated
the binding of Con A to the lens epithelial cells and
lens fibers with nuclei. It is possible that one cause of
the cataract formation in this model is disruption of
cell-cell contacts by the binding of the Con A to the
N-acetyl-glucosamine residues in the cell membranes.
This aberrant binding could lead to cataract formation
early on, but with time the cells may internalize the
Con A, effectively returning the cell surface to its original
state, and thus halting the progression of the cataract.
Whether Con A is binding to the N-acetyl-glucosamine
residues in this model has not been determined.
Nor does there exist any literature describing the use
of lectins such as Con A to alter cell-cell contacts in
any animal model. Finally, there have been no studies
of cell membrane cycling of bound lectin molecules,
although such a precedence exists for growth factor
receptors. Further studies are needed to discern
among these possible mechanisms of action in the formation
of Con A-induced cataractogenesis.
In summary, we have developed a model of Con
A-induced inflammatory posterior subcapsular cataract
that has many characteristics desirable in a model,
including a cataract development rate that would al-

3488 Investigative Ophthalmology & Visual Science, December 1993, Vol. 34, No. 13
low this to be a good assay system for drugs and other
factors affecting cataractogenesis.
Key Words
lens opacity/cataract, Concanavalin A, posterior subcapsular
cataract, steroids
Acknowledgments
The authors thank John Conlon, PhD, for statistical assistance.
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