Ultrastructural Study of Selenife-lnduced
Nuclear Cataracts
Nancy J. Russell,* Joyce E. Royland,* Elron L. McCowley,* and Thomas R. ShearerjThe formation of the selenite-induced cataract was investigated by examining the ultrastructure of the
cataract with transmission electron microscopy (TEM). A lacy, or honeycomb, appearance of the nuclear
cataract seen by light microscopy was resolved by TEM to be due to the aggregation or precipitation
of cytoplasmic material. Despite severe intracellular changes the fibers retained their close apposition
to one another. These results are consistent with the hypothesis that a major lesion in selenite-induced
nuclear cataracts is the formation of insoluble cytoplasmic aggregates. Invest Ophthalmol Vis Sci
25:751-757, 1984
Elevated doses of the essential trace mineral selenium
cause nuclear cataracts in suckling rats.1"4 These cataracts develop within 4 days after oral or a single subcutaneous injection of sodium selenite, Na2SeO3.2"4
Selenium is a strong sulfhydryl oxidant in vitro,5 and,
thus, the selenite-induced cataract may serve as a useful
model for studies on the role of oxidant stressors in
cataractogenesis.
Selenium-induced cataracts are predominantly nuclear, but slit-lamp studies have shown that swollen
fibers and refractive changes appear in the lens cortex
prior to the formation of the nuclear cataract.3 Very
little is known about microscopic changes that occur
during the formation of these lesions, although a previous light microscopic study6 showed the width of the
nuclear lens fibers to be increased, whereas the width
of the cortical fibers was decreased. Thus, the purpose
of the present study was to investigate the mechanism
of selenite-induced cataracts by examining the subcellular features of the cataract with transmission electron microscopy.
from days 2-10 postpartum and control pups received
daily injections of saline alone.
The pups were killed on day 11 postpartum by ether
and the eyes immediately removed and placed in 2.5%
glutaraldehyde buffered with sodium cacodylate (pH
7.2). Surgical and fixation procedures were carried out
at room temperature. Microscopic changes due to the
development of cold cataracts were distinguished easily
from those produced by selenite. After a few minutes
of submersion in the fixative, two incisions were made
in the eye to facilitate fixation of the lens. The lenses
were fixed intact for 3 days, and one of each pair of
lenses was dissected into 10 specific pieces, which were
washed in buffer and postfixed in 2% osmic acid. The
second lens of each pair was left intact but otherwise
processed in the same manner as the dissected lens.
After postfixation, the tissues were washed in maleate
buffer (pH 5.2) and stained en bloc with uranyl acetate.
Subsequently, the tissues were dehydrated in ethanol
and embedded in a low viscosity resin.7 One-micronthick sections were cut from the resin blocks, placed
on glass slides, and stained with toluidine blue for light
microscopy observation.8 Thin sections were prepared
with a diamond knife, picked up on formvar coated
grids, and stained with uranyl acetate9 and lead citrate.10 Sections were viewed with a Philips 300 and
Siemens 101 electron microscope.
Materials and Methods
Pregnant Sprague-Dawley rats (DOBS, Charles
River; Wilmington, MA) were housed individually and
provided distilled water and Wayne F-6 Lab Blox ad
libitum. After birth, litters were culled to 8 pups per
litter. Pups in the experimental group received daily
injections of sodium selenite (0.5 mg Se/kg) in saline
Results
Light Microscopy
Light microscopic observations of lenses from pups
in the control group showed that the tissue was composed of elongated cells (fibers) that were hexagonal
in cross section. The lens fibers in the outer third of
the lens stained a pale blue with toluidine blue. Progressively darker staining was seen in the more centrally
From the Department of Pharmacology, School of Medicine,*
and the Department of Biochemistry, School of Medicine and Dentistry,! the Oregon Health Sciences University, Portland, Oregon.
Submitted for publication: March 18, 1983.
Reprint requests: Dr. Nancy J. Russell, Department of Pharmacology, School of Medicine, Oregon Health Sciences University, 3181
S.W. Sam Jackson Park Road, Portland, OR 97201.
751
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / June 1984
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Fig. 1. Light micrograph of the nuclear region in a lens from a
control animal. The cytoplasm of the nuclear lens fibers is dark and
featureless (X750).
located fibers of the deep cortical region. Many darkly
stained round spots were observed in each fiber. Similar
spots are known to appear in the lenses of young rats
and mice when the eye is cooled below body temperature, such as after enucleation or submersion of the
eye tissues in cool solutions." The fibers of the lenticular nucleus were featureless and stained a homogenous dark purple or maroon (Fig. 1).
In the selenite-treated animals, the lens fibers of the
cortical and deep cortical regions were similar to those
seen in the control lenses. In contrast, the center of
mm
}..
Fig. 2. Light micrograph of cataractous nuclear region. The cytoplasm has lost its dense, featureless appearance. The dark cell
linings give the tissue an overall honeycomb pattern. Networks of
dense material extending across the lens fibers (arrows) connect with
peripheral material to produce a lacy pattern (X750).
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Fig. 3. Electron micrograph of cortical cells from the lens of a
control animal. Lens fibers are hexagonal in cross-section and contain
electron opaque spots within the cytoplasm, especially along the
plasma membrane. The spots are associated with the formation of
cold cataracts (X8000).
the lens showed pronounced changes after selenite.
The whole nuclear region had a lacy or honeycomblike pattern (Fig. 2) consisting of a network of darkly
stained material surrounded by thick, darkly stained
"membranes" separated by clear areas.
Transmission Electron Microscopy
The ultrastructural appearance of cells of the cortical
regions of the control and cataractous lenses of the
11 -day-old rat pups were similar. The fibers contained
a granular cytoplasm of medium electron opacity and
accumulations of more electron opaque material (Fig.
3). The accumulations consisted of a few round masses
scattered throughout the cytoplasm with larger masses
present in the peripheral cytoplasm. The larger masses
were found along the bends in the cell membrane which
give the lens fibers their characteristic hexagonal crosssection. These opacities were identical in size, shape,
and position to the darkly staining cytoplasmic inclusions seen by light microscopy, and they are the cytoplasmic "cold spots" associated with the cooling of
the lens.
In contrast to that seen in the cortical regions, the
ultrastructural appearance of the nuclear region of the
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ULTRASTRUCTURE OF SELENITE-INDUCED CATARACTS / Russell er ol.
control and cataractous lenses were strikingly different
from each other. In the control lenses the nuclear region
contained cells that were apposed closely and were
filled with an electron opaque, featureless cytoplasm
(Fig. 4), whereas the nuclear region of the cataractous
lenses (Fig. 5) contained enlarged, irregularly shaped
fibers, each of which had a lacy appearance. The honeycomb pattern formed by "darkly stained membranes," as seen by light microscopy, was resolved by
electron microscopy into a layer of very electron
opaque cytoplasm lying along the cell membrane (Fig.
6). Although the swollen lens fibers in most areas of
the cataractous region appeared to have remained intact, the boundaries of some could not be seen clearly
(Fig, 5). Low magnification electron micrographs taken
of these regions suggested that some cell rupture and/
or cell fusions had occurred. However, at higher magnification it was found that some of the observed
"breaks" were actually the sites of gap junctions. These
penta-laminate junctions had little electron opaque
material on either side (Fig. 7).
The electron opaque cytoplasmic material also extended in an irregular network across the nuclear lens
fibers. Generally this material was surrounded by a
lighter, flocculent material (Fig. 8). However, in the
central regions of the cataract (Fig. 6), and occasionally
in more peripheral areas, lens fibers lacked the light
flocculent material.
In the transition zone between the cataract and the
noncataractous areas, three types of lens fibers were
found: deep cortical fibers; transitional, possibly precataractous fibers; and cataractous lens fibers (Fig. 8).
The first of these, the normal deep corticalfibers,contained a large number of electron opaque spots. These
spots had a dark central core, and they were surrounded
by only faint cytoplasmic material. Abutting these cells
was the second type offiber,the transitional lens fiber,
which contained irregularly shaped aggregates of material of medium electron opacity. These aggregates
were surrounded by a light flocculent material. The
transitional fibers, in turn, abut the third fiber type,
the cataractous lens fiber. The cataractous fibers contained large amounts of very electron-opaque material
interspersed with small irregular patches of a much
lighter material. Those cataractous fibers, located peripherally, contained only small areas of the pale material against a dark background, whereas those more
centrally located contained afineflocculentcytoplasm
in which fine networks of very opaque cytoplasm appeared against a light background. In the most central
regions, only opaque material remained in each lens
fiber.
Although the cataracts in these 11 -day-old rat pups
generally were limited to the nuclear region, spurs of
cataractous fibers 2-3-cells thick extended into the
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753
Fig. 4. Electron micrograph of the nuclear region from the lens
of a control animal. As in the light micrographs, the cytoplasm is
featureless and cells are closely apposed (X8000),
cortical regions (Fig. 9). These radiated from the periphery of the cataract along suture lines towards the
lens surface.
In the preparation of the nuclear and perinuclear
regions for electron microscopic observation, it was
observed that the cataractous tissue was invariably better infiltrated with plastic resin and the lens tissue less
brittle than similarly prepared regions of noncataractous lenses. As a consequence, it was much easier to
obtain useable thin sections of cataractous than of normal nuclear lens tissue. These differences indicated
that in the selenite-induced cataract the nuclear region
had become more permeable and less dense than that
of the control lenses.
Discussion
The major contribution of the present study is the
ultrastructural description of selenite-induced nuclear
cataracts. To our knowledge, this is the first ultrastructural description of a mineral-induced cataract. The
description was confined to the deep cortical and nuclear regions of early, but pronounced, selenite cataracts. A previous light microscopic study5 of mature
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1984
Vol. 25
Fig. 5. Low magnification electron micrograph of deep cortical, perinuclear region (c), transition zone (t), and cataractous nuclear region
(n) (X2000).
selenite-induced cataract revealed that the cataract had
the following characteristics: atypical lens fibers, which
were enlarged in the nuclear area, but decreased in
the cortical region of the lens; microglobular formations
and possible calcification in the nuclear area; increased
vacuolization in the equatorial region under the anterior epithelium; focal hyperplasia of the anterior epithelium; cellular damage in the cortex; decreased lens
diameter; and decreased anterior-posterior lens dimension. A light microscopic study of the seleniteinduced cataract in rabbits showed proliferation of epithelial cells and some vacuolization.12 From the light
microscopy of the present study, we can now add that
the nuclear region of lenses showing selenite-induced
cataract had a lacy or honeycombed appearance, which
was due to aggregation of the cytoplasmic material.
Transmission electron microscopy further revealed
the nature of the lacy or honeycomb appearance of
the nuclear fibers of the cataractous lens. Unlike the
dense, featureless cytoplasm normally seen in the nuclear region of control animals, the cytoplasm of nu-
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clear fibers from rats with selenite-induced cataracts
appeared to have precipitated. This precipitation of
the cytoplasm may have caused the observed distortion
in cell shape.
An interesting observation was the presence of a
band of precipitated cytoplasm along the cell membrane except at regions of cell junctions, which made
many of the cells appear continuous with one another
when examined at low magnifications. Although these
junctions are commonly referred to as gap junctions,13"15 Zampighi and co-workers16 recently have
shown in the bovine lens that such junctions are both
structurally and chemically different from gap junctions
found in the liver. Although the function of the
"gap" 13 " 15 or "penta-laminar" 16 junctions in the lens
has yet to be determined, the absence of precipitated
cytoplasm along the junction suggests that the cytoplasmic matrix simply may be attached to the cell
membrane except at the junctions, or that the material
may be pushed to the side of each junction by the
movement of water from one cell to another.
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ULTRASTRUCTURE OF SELENITE-INDUCED CATARACTS / Russell er ol.
755
The absence of other ultrastructural descriptions of
a mineral-induced cataract makes direct comparisons
with other studies impossible. In addition, although
ultrastructural changes in the epithelial and cortical
cells have been studied extensively in many types of
cataracts, cells in the nuclear region have not been
studied well. The reason for this may lie, at least in
part, in the difficulty involved in preparing suitable
thin sections. There are, however, several studies involving hereditary cataracts in mice,17"19 x-ray-induced
cataracts,20 and advanced senile nuclear cataracts,21
all of which have shown evidence of a precipitation
of the cytoplasmic proteins in nuclear fibers resulting
in the formation of some large aggregates of material
not unlike that we have observed in the selenite-induced cataract.
Biochemical studies of selenite-induced cataracts2'4
support the hypothesis that the electron opaque aggregates seen in the cell matrix by electron microscopy
are the result of precipitation of cytoplasmic proteins.
Fig. 1. High magnification electron micrograph of gap junction
and adjacent cell membranes. The zipper-like appearance of the
junction is shown, Only a small amount of dense cytoplasm is seen
on either side of the junction (X95,000).
Fig. 6. Electron micrograph of cataractous area with the fibers cut
in cross-section. Honeycomb pattern persists in most areas. A slight
increase in extracellular space is observed. In each fiber the cytoplasmic material forms a thin band lining the cell and an irregular
network that extends across the fiber. In this example, the clear
spaces between the precipitated cytoplasm are devoid of any flocculent
material. Cells are joined by gap junctions (X8500).
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For example, one of the most outstanding features of
these cataracts is the rapid and significant increase in
the amount of insoluble protein in the lens. Thus, it
is likely that the lacy networks observed in the nuclear
fibers and the cytoplasmic aggregations seen just inside
the membrane are insoluble protein. A similar increase
in insoluble material accompanied by an increase in
the granularity of the cytoplasm has been reported in
x-ray cataracts in rabbits.20
Studies also have shown that the increase in insoluble
protein seen in the selenite-induced cataract is accompanied by a dramatic loss of glutathione and NADPH
before formation of the nuclear cataract.4 Selenite,
which oxidizes the sulfhydryls of glutathione and proteins in vitro,5 has been postulated to cause proteins
to precipitate by disulfide formation in vivo. However,
if this does occur in the selenite-induced cataract, it
must be confined either to a specific class of proteins
or to a limited anatomical spot, since the total lens
concentrations of disulfides, diglutathione, and mixed
disulfides do not increase.22 Clear areas were observed
around the aggregates of precipitated cytoplasm. In
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1984
Vol. 25
jfi*
%*
Fig. 8. Electron micrograph of cells in the transitional region. Cells of the deep cortical region (c), located to the upper left, contain round
opacities in their cytoplasm. A "transitional cell" (t) contains irregular densities filled with a fine flocculent material in which irregularly
shaped aggregates of cytoplasm were present. Many of these aggregates lie against the plasma membrane. To the right are cataractous nuclear
fibers (n). In this transitional region the cataractous fibers contain scattered areas of light llocculent material against the dark background
characteristic of nuclear cells (X9000).
hereditary nuclear cataracts of the Nakano mouse17
the cytoplasm also clumps, forming large aggregates.
This occurs only after there has been a significant in-
crease in lens sodium concentration. The sodium increase has been shown to be the result of a Na-K
ATPase deficiency. Consequently, in the selenium-in-
Fig. 9. Electron micrograph of a spur of abnormal fibers radiating along a suture line. This spur originated near the edge of the cataractous
region at the periphery of the lens nucleus. Normal appearing cortical cells lie to each side of the spur (X34,000).
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ULTRA5TRUCTURE OF SELENITE-INDUCED CATARACTS / Russell er ol.
duced cataract water uptake could be involved since
an increase in total lens water has been observed when
these cataracts are hypermature.23 However, no changes
were observed in the concentrations of sodium, potassium, or water in well-formed nuclear cataracts examined up to 12 days after a single injection of selenite.4'24 These results would suggest that hydration is
a result rather than a cause of the selenite-induced
cataract. However, it will be necessary to measure the
water and ion levels present in cataracts produced by
multiple injections of selenite before it can be determined whether or not the changes we observed were
indeed due to hydration.
Key words: nuclear cataract, selenite, transmission electron
microscopy, light microscopy, lens
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