Current Eye Research
Volume 1 1 riumber 12 1992, 1147- 1160
Selenite nuclear cataractogenesis: a scanning electron microscope study Ruth S.Anderson and Thomas R.Shearer
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Departments of Biochemistry and Ophthalmology, Schools of Dentistry and Medicine, Oregon Health Sciences University, Portland, OR 97201, USA
ABSTRACT The sequential changes during selenite nuclear cataractogenesis were examined with a scanning electron microscope (SEM) and correlated with slit lamp observations. A posterior opacity, visible with the slit lamp 1-2 days after injection of sodium selenite, was found to consist of masses of vacuoles in the superficial posterior cortex by SEM. 2-3 days post injection, a biomicroscopic refractile ring around the nucleus was represented by SEM abnormalities suggesting membrane damage and possible loss of cytosol in the perinuclear region. All normal structure in this region was lost by 5 days after injection when the central nucleus had become opaque. SEM also showed evidence for damage in areas which were still clear by slit lamp examination. Changes, characteristic of aging, were found near selenite induced damage in peripheral (younger) fibers.
formed, slit lamp examination showed that masses of vacuoles had developed around the equator, and later total cortical opacity was observed in a high proportion of the selenite injected rats (4,5). Light microscopy showed gross destruction of the cortex at the time the cortex was opaque. The rats which did not develop cortical opacity showed only minor biomicroscopic changes but rather severe histologic abnormalities (5). The cortices in both groups eventually became clear biomicroscopically and had near normal histology but the nuclear cataract was permanent. Thus, light microscopy has described general histological changes occurring during selenite
INTRODUCTION
In young rats, a single injection of sodium
cataractogenesis, but the technique was limited by relatively low magnification. Transmission electron
selenite causes rapid formation of cataracts, which
microscopy (TEM) provided high magnification of
are useful models for the study of cataractogenesis
selenite nuclear cataract (61, but only small areas
(11. Biomicroscopic observations showed a dense
could be examined. Scanning electron microscopy
posterior subcapsular opacity within 24 hours after
(SEMI allowed the whole lens to be examined in one
injection, a later perinuclear refractile ring, and dense
field for areas of interest which can then be examined
nuclear cataract 3--5 days post injection (2). Light
in detail at higher magnification. Bunce e t al. (7)
microscopy showed the posterior subcapsular opacity
used SEM to examine lenses from rats with mature
to be due to multiple vacuoles in the peripheral cortex
nuclear cataracts, but not during their development.
and the perinuclear refractile ring was associated
The purpose of the present study was to use SEM to
with intense basophilia and distorted fiber outlines.
localize morphologic changes in different regions of
The nuclear opacity consisted of a severely
the lens at various stages of selenite
degenerated peripheral nucleus surrounding a central
cataractogenesis. These were then compared with
granular region (3). After the nuclear opacity had
slit lamp and biochemical observations.
Received on February 26. 1992: accepted on November I I , 1992
G Oxford University Press
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Current Eye Research photographed with Polaroid 107C or 667 film. When
MATERIALS AND METHODS Litters of rats were obtained from B & K International (formerly Bantin and Kingman), Fremont,
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CA. and injected with 3 0 umoles Na,SeO,/kg
on the
available, figures in this report show cross fractures together with longitudinal fibers. We avoided artifact as much as possible by
day the eyes opened(13-14 days after birth).
processing control and experimental
Controls were uninjected littermates. Mother rats
lenses simultaneously so that fixation, dehydration,
were maintained on standard laboratory chow
critical point drying and coating were identical. We
(Wayne F-6) and tap water ad libitum. Eyes were
were also careful to observe comparable regions in
examined daily with the slit lamp after dilation with a
experimental and control lenses. Animals were used
mixture of atropine and phenylephrine and processed
in accordance with the ARVO Resolution on the use
for scanning electron microscopy at appropriate
of Experimental Animals in Research.
stages. Lenses with opaque nuclear cataract were used for SDS-PAGE electrophoresis as previously
RESULTS
described (8).
The B & K strain of Sprague Dawley rats used in this
Preparation of lenses for SEM was based on the
study developed nuclear cataracts slightly more
method used by Kuszak et al. (9). Rats were killed
slowly and cortical cataracts somewhat more rapidly
with carbon dioxide and eyes were removed
than those used in previous studies (2).The
immediately. After the backs of the eyes were
sequence of biomicroscopic changes noted above and
opened, they were immersed in 2.5% glutaraldehyde
their biomicroscopic appearance was, however,
in 0.07 M sodium cacodylate buffer, pH 7.2 for I - - 2
identical. Since slit lamp photographs have been
hours. The lenses were then carefully removed from
published previously (21, they are not included here.
the globes and reimmersed in fresh fixative. Lenses
All control lenses were biomicroscopally clear. For
were fixed for 3 days (with several changes) at room
selenite lenses, the characteristic slit lamp
temperature and then washed in 0.07M cacodylate
appearance together with the time after injection are
buffer for 24 hours. They were post-fixed in 1 %
listed in the text headings below. Additional slit lamp
OsO, at 4°C for 24 hours. Osmium was washed out
information is added in the text if it relates to SEM
in cacodylate buffer (24 hours), and lenses were
findings,
dehydrated in a graded series of acetone
Control lenses observed by SEM showed little
concentrations starting with 30% and ending with
change during the five day period reported here. The
acetone dried over a molecular sieve. Lenses were
locations of the regions studied in selenite
critical point dried in a Pelco 1250 critical point dryer.
cataractogenesis are shown in Figure 1. This
Fractures were initiated on the dried lenses with a
diagram is superimposed on a low magnification SEM
razor blade or fine scalpel to expose an AP plane in
photograph of the lens of an 18 day old rat which is
the center or periphery of the lens. The former were
a control for five days post selenite injection. In later
oriented to show narrow sides and the latter the
figures, SEM details of control lens fiber morphology
broad sides of the fibers. After being glued to
are shown together with selenite induced changes in
aluminum stubbs, the specimens were sputter coated
the same regions. For orientation characteristics of
with 30nm paladium-gold. Observations were made
the regions in control rat lenses are summarized
with a JSM-T330A scanning electron microscope and
below.
1148
Current Eye Research Anterior Pole
The central and peripheral nucleus (Fig. 1) are distinguished from one another because each region reacts very differently to selenite injection. Unlike
AY
\
the central nucleus, the peripheral nucleus has Y sutures and must therefore contain secondary fibers. In conventional terminology, the central and
rrixlUC
-
peripheral nucleus are probably comparable to the
CN-
embryonic and fetal nucleus, respectively. In control
PN-
uator
lenses, the fibers of the central nucleus were Curr Eye Res Downloaded from informahealthcare.com by University of Auckland on 11/30/14 For personal use only.
convoluted and the surface was relatively smooth, but multiple small mounds covered the surface of the peripheral nuclear fibers. The perinuclear region refers t o a narrow band of fibers immediately adjacent to the peripheral nucleus, which appears to be especially susceptible to early damage by selenite. In the perinuclear region of control lenses, SEM showed fibers which were uniform in size and shape. Their surfaces were relatively smooth except for faint ridges and sparse
PY /f Posterior Pole Figure 1. Low magnification of control lens with superimposed diagram for orientation. CN -= central nucleus; PN = peripheral nucleus; Perinuc = perinuclear region; CX = cortex; SF = superficial fibers; AY = anterior Y suture; PY = posterior Y suture.
interlocking devices. With the slit lamp, this region showed no special differences from the rest of the
lens fibers are similar to those reported by other
lens. By SEM, the control cortex consisted of
investigators (9-14) whose studies were concerned
mature, terminally differentiated fibers and
with lenses from adult animals and often involved
superficial immature fibers. The mature fibers had
other species. Our study used lenses from immature
abundant interlocking devices in mid cortex, but
rats, which can account for minor differences.
superficial fibers showed no interlocking devices.
Posterior subcaDsular cataract (1--2 davs Dost
They appeared to be coated with fine particles so
selenite iniectionl
that their margins were not clearly defined. The structure of the Y sutures was complex and in
In the biomicroscopically clear control lenses, SEM showed no vacuoles in posterior superficial cortical
our preparations they were sometimes open,
fibers, but they were covered with masses of
especially near the periphery. This could have been
unidentified small particles (Fig. 2a). In selenite
artifactual. However, it is more likely due to the
lenses, the posterior superficial opacity observed with
immaturity of the rats used in this investigation.
the slit lamp, was associated with SEM appearance
Open Y sutures in comparable rats were also
of patches of vacuoles in the peripheral posterior
observed by light microscopy in a previous study (3).
cortex (Fig. 2b and 2c). The vacuoles appeared to be
The epithelium, present only on the anterior surface
intracellular. Vacuolated fibers were larger than
of the lens, was used for orientation to localize the
adjacent fibers which were similar to those of
anterior and posterior sutures and other areas in the
controls from the same region.
lens. Our observations on SEM appearance of normal
perinuclear and nuclear region showed no
Although the
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Current Eye Research
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biomicroscopic opacity, SEM showed intracellular
Fibers in the perinuclear region of selenite lenses
damage in selenite lenses. In control lenses, the
were distorted (Fig. 3c), lacked interlocking devices
membranes appeared to be intact and the cytosol of
and the surfaces showed pronounced ridges (Fig. 3d).
cross-fractured lens fibers was apparently uniform
Patches of membrane were lost, possibly at the site
(Fig. 2d). Most of the membranes of perinuclear
of the missing interlocking devices.
fibers of selenite lenses also remained intact except
rather sparse clumped cytoplasm within the fibers
for possible loss of interlocking devices.
where the internal structure was visible.
Where
There was
patches of membrane were lost during preparation,
Vacuoles in the posterior superficial cortex had
multiple small empty spaces were interspersed
regressed at this stage, but fibers in this area seemed
between solid structures in the cytosol (Fig. 2e).
to be undergoing further degeneration (Fig. 3e). They
Cross-fractured fibers in the nucleus also had a
were distorted and ridged, and a few small spherical
porous appearance with somewhat less open space
bodies were found in the damaged area. No
(data not shown). Anterior and equatorial cortical
abnormality was found in the other parts of the
fibers showed no difference between control and
cortex.
experimental lenses.
Central nuclear oDacitY (five davs Dost iniectionl iniectionl
Biomicroscopically in selenite lenses, the center of
In selenite lenses, the refractile perinuclear ring
the nucleus was totally opaque and surrounded by a
Refractile Derinuclear ring (2--3 davs
DOSt
visible with the slit lamp corresponded to the
clear or faintly hazy area within the refractile
perinuclear region observed with SEM. The SEM
perinuclear ring
photographs shown in Fig. 3a--3d were from a
refractile rings had appeared in the cortex which was
fracture of the equatorial region peripheral to the
otherwise completely transparent. Some lenses also
nucleus which were oriented to show the broad side
showed large superficial equatorial vacuoles with the
of fibers in the perinuclear region. The broad sides of
slit lamp.
control lens fibers from this region were uniform in
.
By this time, multiple concentric
The most striking SEM feature in selenite lenses
size (Fig. 3a) and had minor ridges (Fig. 3b). Fiber
was the large number of spherical bodies
surfaces and interlocking devices were intact.
(Morgagnian globules
Cytosol in fractured control fibers appeared solid (Fig.
structure in the perinuclear region. The spherical
3b).
bodies were concentrated in the Y sutures (Fig. 4a),
Figure 2. In selenite lenses, biomicroscopy showed posterior subcapsular opacity 1-2 days post injection. a. Control: Posterior superficial cortex. Fibers are covered with small particles, but there are no vacuoles. (500X, Bar = 50 um) b. Selenite: Posterior superficial cortex showing masses of vacuoles. (500X, Bar =50um) c. Higher magnification of 2b, vacuoles appear to be intracellular, note patch of degenerating cytoplasm (arrow). (2000X, Bar = 10 um)
d. Control: Perinuclear region, cross-sections of fibers (arrows) show solid uniform contents. There were no longitudinal fractures. (2000X, Bar = 1Oum) e. Selenite: Perinuclear region, cross-sections of fibers had a perforated appearance (broad arrow), longitudinal fractures showed multiple small empty spaces, a periodic arrangement of remaining solid material near membrane (small arrows) and possible damage to interlocking devices. (2000X, Bar = 10um)
?I and the total loss of normal
-
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Current Eye Research
1152
Current Eye Research but they were also found at the equator (Fig. 4b) and
the remaining contents (Fig. 4i). In the outer cortex
often extended around the nucleus. In contrast the
of selenite lenses, patches of ridged fiber surface and
fibers in the perinuclear region of control lenses had
multiple small mounds were present (Fig. 4j).
relatively uniform elongated fibers (Fig. 4c).
Superficial equatorial vacuoles, visible with the slit
Individual nuclear fibers from control lenses
lamp, were also observed by SEM (Fig. 4k). These
remained intact and appeared to be filled with solid
vacuoles often contained inclusions which appeared
uniform cytoplasm (Fig. 4d and 4e). In selenite
to consist of tangles of threadlike structures (Fig. 41).
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lenses, fibers of the peripheral nucleus were severely damaged. The membranes, apparently more fragile
Loss of cytosolic material in both the cortex and
than those of controls, fractured during preparation
nucleus of lenses with opaque nuclear cataracts 5
revealed large empty spaces, suggesting loss of
days after selenite injection was confirmed by SDS-
cytosol (Fig. 4f). Note that the biomicroscopically
PAGE electrophoresis (Fig. 5). Electrophoresis
dense opacity was in the central nucleus and that the
showed loss of soluble and insoluble
peripheral nucleus was clear or showed only a faint
(cytoskeletal) proteins from both the nucleus and
haziness by slit lamp examination. The membranes in
cortex. Loss of high molecular weight proteins was
the central nucleus also fractured, but, in contrast to
accompanied by increased low molecular weight
relatively empty peripheral nuclear fibers, the
polypeptides.
cytoplasm of fibers in the central nucleus appeared to be filled with particulate material (Fig. 49). Cortical fibers of control lenses had smooth membranes which did not fracture during preparation
DISCUSSION SEM gave new information on the cytologic
and had many interlocking devices (Fig 4h). In
changes in lenses undergoing selenite
selenite lenses, the cortex was clear by slit lamp
cataractogenesis. It also confirmed previous light
examination, but SEM showed extensive localized
microscope studies (3) which demonstrated that early
destruction at all levels of the cortical fibers. In the
posterior superficial opacity was due to extensive
mid cortex of selenite lenses, there was apparent
vacuolization, followed by disturbances in the
localized loss of cytosolic material and clumping of
perinuclear region and later abnormalities in the
Figure 3. In selenite lenses, biomicroscopy showed refractile perinuclear ring 2-3 days post injection. Fig. 3a--3d are oriented to show the broad side of fibers in perinuclear region. a. Control: Size and shape of fibers is uniform except for minor artifactual variation. Surface with typically few interlocking devices is intact. (2000 X, Bar = 10um) b. Control: Higher magnification of part of 3a; ridges on fiber surfaces are barely visible. (5000X, Bar = 5um) c. Selenite: Size and shape of fibers is highly variable
so that some fibers appear to be swollen and others shrunken. Note variation within individual fibers, loss of interlocking devices and localized loss of membrane (arrows). (2000X, Bar = 1Oum) d. Higher magnification of part of Fig. 3c showing localized loss of membrane and clumped fiber contents. Note elaborate system of ridges on fiber surfaces. (5000X, Bar = 5um) e. Selenite: Peripheral posterior cortex showing remnants of vacuoles (straight arrows), distorted fibers with ridged surfaces (curved arrows) and small spherical bodies. (2000X, Bar = 10um)
~
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Current Eye Research
1155
Current Eye Research nucleus which correlated with biomicroscopic
Possible loss of membrane at the site of missing
opacity.
interlocking devices suggested that these devices are
In selenite injected rats, the earliest lens change observed with the slit lamp was a posterior
Ridges on the fiber cell surface were seen in
subcapsular opacity which appeared within the first
control fibers (Fig. 3b), but they seemed t o be
day after injection and later extended over the whole
exaggerated in selenite lenses (Fig. 3d). Patches of
back of the lens except the posterior Y suture. By
ridged membranes also were found in the cortex at
SEM, this opacity was correlated with vacuoles
the site of the regressing posterior superficial opacity
which appeared to be intracellular within swollen
(Fig. 3e).
superficial fibers (Fig. 2b and 2c). However TEM Curr Eye Res Downloaded from informahealthcare.com by University of Auckland on 11/30/14 For personal use only.
especially susceptible to selenite induced damage.
Ridges have been associated with maturation and
studies would be necessary to confirm this. In
aging of fibers by others (9-13). In selenite
previous studies, lens epithelium showed damage
cataracts, ridges were observed near cataractous
within a few hours after selenite injection (4).
damage in peripheral young fibers (Fig.4j).
Possibly selenite induced damage to the epithelial
they were always found in a damaged area and
pump mechanism led to the accumulation of water at
included young fibers, ridges probably represented
the back of the lens where no epithelium is present.
part of the degenerative process.
During the second day after selenite injection, a
Since
In preliminary studies of early secondary cortical
decrease in the extent of the posterior subcapsular
cataract (which develops after the nuclear cataract),
cataract and a refractile ring around the lens nucleus
we have observed increased amounts of similar
were observed with the slit lamp.
structures and also microvilli, another characteristic
This biomicroscopic refractile ring was correlated to
of aging (12).near cataractous damage in relatively
damage in the perinuclear region shown by SEM (Fig.
young fibers. Thus, selenite cataractous changes
3c and 3d).
appear to mimic aging changes in lens fibers. Similar
Figure 4. In selenite lenses 5 days post injection, biomicroscopy showed an opaque central nucleus, clear peripheral nucleus and clear cortex except for multiple concentric refractile rings. a. Selenite: Perinuclear level anterior Y suture containing masses of spherical bodies (2000X, Bar = 1Oum) b. Selenite: Perinuclear level, equator. No normal fiber structure, apparent fusion of membranes and numerous spherical bodies. (2000X, Bar = 10um) c. Control: Perinuclear fibers uniform in size and shape except for minor artifactual changes (2000X, Bar = 1Oum) d. Control: Peripheral nucleus near posterior Y suture. Fractures show solid fiber contents (arrows). Textured appearance of fibers is due to masses of small mounds which cover the surface of these older fibers. (2000X, Bar = 1Oum) e. Control: Central nucleus showing uniform solid fiber contents (arrows) and intact surfaces. Irregular conformation is associated with normal aging changes. (2000X, Bar = 1Oum)
f. Selenite: Peripheral nucleus near posterior Y suture. Note apparent loss of cytosol (arrows). (2000X, Bar = IOum) g. Selenite: Central nucleus showing some loss of membrane and particulate appearance of fiber contents. (2000X, Bar = 1Oum) h. Control: Mid-cortical fibers, unfractured fiber membrane is smooth except for characteristic numerous interlocking devices. (2000X, Bar = 10um) i. Selenite: Localized damage in mid-cortical fibers. Note fractured membranes with loss of cytosol and clumping of contents. (2000X, Bar = 1Oum) j. Selenite: Outer cortical fibers. Note ridges (arrowheads) and numerous small mounds (arrows) on surface of fibers. (5000X, Bar = 5um) k. Selenite: Superficial cortical vacuoles near equator containing clumps (arrows). Wide arrow points to edge of lens near equator (capsule lost), crack in vacuole is artifact. (IOOOX, Bar= 10um) I. Higher magnification of structure at upper arrow in Fig. 3k. Note threadlike appearance. (10,000 X, Bar = 1um)
1156
Current Eye Research selenite lenses (Fig. 2b, 2c, 2e, 3c, 3d, 4f, 49, 4i, 4j). This indicates that the membranes were more '
Sol
Insol
Sol
Insol
fragile in selenite lenses. TEM might reveal structural membrane damage in the sites indicated by SEM.
97 66
Loss of main intrinsic membrane protein (MIP 26) has been shown in selenite nuclear cataract (17). It is
45
interesting that early membrane damage, as seen by
32
SEM, occurred in the perinuclear region which was separated from the site of future opacity.
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20
When the perinuclear refractile ring became visible
14
5
S
C Se C Se
C Se C Se
with the slit lamp (2-3 days post injection), perinuclear fibers were irregular in shape and size,
Figure 5. SDS-PAGE Electrophoresis by SDS-PAGE of proteins from lens cortex and nucleus of control (C) and cataractous selenite (Se) lenses from rats at 5 days after injection. S = molecular weight standards (kilodaltons). "Sol" = water soluble fraction, and "Insol" = water insoluble fraction. Bands at arrow and below are crystallins and asterisk indicates cytoskeletal region in controls. Note loss of bands from both regions together with new low molecular weight bands in selenite lenses.
possibly due to uneven uptake of water. Rapid changes in dry mass concentration, probably caused by rapid shifts in water distribution within the lens have been described in selenite cataract (18). By the time the nuclear opacity had developed (5 days post injection), extensive fracture of membranes together with apparent loss of cytosol was found in the peripheral part of the nucleus (Fig. 4f) and in the cortex (Fig. 4i) although these regions were
ridges were observed in the microphthalmic
essentially clear biomicroscopically. Fracture of
(Browman strain) rat (13) and the Emory mouse
membranes during preparation suggested increased
cataract (14).
fragility, probably due to loss of protein. Fracture of
In human lens fibers, square arrays (non-
membranes revealed fiber contents and indicated that
communicating junctions) were found in ridges (15)
there was loss of cytosol within affected fibers
and in fine globular elements ( 1 6) similar to the small
where the remaining contents were clumped and
mounds found near selenite cataract (Fig. 4j).
irregular. This contrasted sharply with fibers of
Vrensen et al. ( 1 6) suggested that square arrays
control lenses in which the membranes remained
associated with the fine globular elements were part
intact and cross fractures showed uniform solidly
of a protective mechanism which separated diseased
packed cytosol.
and healthy fibers, thus conserving transparency.
Loss of cytosol in selenite lenses was also
Ridges and small mounds adjacent to damaged areas
suggested by electrophoresis. Electrophoresis of
in selenite cataract should be investigated with
nuclear and cortical proteins by SDS-PAGE showed
freeze-fracture techniques to determine whether
breakdown or loss of crystallins and cytoskeletal
square arrays are present.
proteins with accumulation of lower molecular weight
Membranes of control lenses remained intact after
polypeptides (Fig.5). Similar results were obtained in
preparation for SEM (Fig. 2d., 3a, 3d, 4c, 4d, 4e and
earlier studies which showed that the above
4h), but localized membrane losses occurred in
proteolytic changes were progressive during selenite
1157
Current Eye Research nuclear cataractogenesis. Increased proteolysis in
Calpain proteolysis is important in selenite
the cortex was a new finding and probably reflects
cataractogenesis (8) and may be part of the
the beginning of the secondary cortical cataract in
mechanism which produces spherical bodies in
which cortical opacity is first observed with the slit
cataractous lenses.
lamp 15--20 days after selenite injection in this strain of rats (unpublished data).
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Calcium concentration was elevated in both the
Threadlike structures observed in vacuoles of newly damaged fibers (Fig. 41) may have been cytoskeleton detached from the membrane, but not yet destroyed.
cortex and nucleus (data not shown) of selenite
Possibly, breakdown and loss of soluble lens protein
lenses from the strain of rats used in this study. High
(8) during selenite cataractogenesis exposed these
calcium concentration in the nucleus, but not the
structures and made them visible with SEM. Both
cortex in other strains also was shown previously (7,
ankyrin and spectrin, involved in attachment of other
8, 19) and is compatible with the hypothesis that
cytoskeletal elements to the plasma membrane (361,
calpain II, a calcium activated protease, plays an
have been found in lens (37,381. Both are substrates
important role in the observed degenerative changes.
for calpain (39, 40) and loss of cytoskeletal elements
It was surprising that the central nucleus, which was
has been described in human cataracts (41, 42).
totally opaque by slit lamp examination, showed
Further study of the cytoskeleton in the selenite
relatively high amounts of cytoplasmic content (Fig.
cataract model seems warranted. Studies should
49). This may have been related to the high
include light microscope and SEM observations on
concentration of g crystallins in the nucleus which
lenses which have been treated to remove soluble
are not degraded in selenite cataract (20).
proteins. TEM studies with antibody labelled
Spherical bodies similar to those found in the perinuclear region have been reported in several human cataracts (21, 22) as well as experimental
cytoskeletal elements at different stages and locations also would be useful. The present SEM investigation has shown that
sugar (231, radiation (24, 25) and genetic (14, 26)
biomicroscopically clear areas in the lens had already
cataracts. Mousa e t al. (27) induced formation of
sustained severe damage including loss of cytosol
similar spherical bodies by incubation of rat lenses
and membrane defects. SEM observations were
with agents which caused microfilament and tubulin
supported by biochemical data in this and earlier
disorganization. Spherical bodies resembling those in
studies. These findings are compatible with the
lenses also have been observed in other types of
hypothesis that early selenite-induced damage to the
cells. These "blebs" could be induced to form by
lens epithelium allows a net influx of calcium causing
high calcium levels (28-30) and were associated with
activation of calpain resulting in partial proteolysis
increased membrane permeability (31) and
and opacity.
disturbances of the cytoskeleton (32-34). Nicotera et
al. (35) reported that leupeptin, a calpain inhibitor, protected hepatocytes from blebbing (and loss of viability) and the authors suggested that calcium
ACKNOWLEDGEMENTS Partially supported by NIH Grant #EY03600 to
activated protease attack on cytoskeleton was part
T.R.S. The authors thank Mr. Mitsuoshi Azuma and
of the mechanism which caused certain blebs.
Mr. Jay Wright for expert technical assistance.
1158
Current Eye Research CORRESPONDING AUTHOR
14.
R.S. Anderson, Ph.D., Department of Biochemistry,
m,
Oregon Health Sciences University, 61 1 S.W. Campus Dr., Portland, OR 97201
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2.
3.
15.
228-245.
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Shearer, T.R., David, L.L., Anderson, R.S. and Azuma, M. ( 1 992) Review of selenite cataract. Curr. Eye Res. 11,357-369. Shearer, T.R., Anderson, R.S., Britton, J.L. and Palmer, E.A. ( I 983) Early development of selenium-induced cataract: Slit lamp evaluation. Exp. Eye Res. 36, 781-788. Shearer, T.R. and Anderson, R.S. (1985) Histologic changes during selenite cataractogenesis: A light microscopy study. 557-565. Exp. Eye Res. Anderson, R.S., Shearer, T.R. and Claycomb, C.K. ( 1 986) Selenite-induced epithelial damage and cortical cataract. Curr. Eye Res. 5,53-61. Anderson, R.S.,Trune, D.R. and Shearer, T.R. ( 1 988) Histologic changes in selenite cortical cataract. Invest. Ophthalmol. Vis. Sci.29.
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Russell, N.J., Royland, J.E., Macawley, E.L. and Shearer, T.R. (1984) Ultrastructural study of selenite-induced nuclear cataracts. Invest. Ophthalmol. Vis. Sci. 25, 751-757. Bunce, G.E., Hess, J.L. and Batra, R. (1984) Lens calcium and selenite induced cataract. Curr. Eye Res. & 31 5-320. David, L.L. and Shearer, T.R. (1984) Calciumactivated proteolysis in the lens nucleus during selenite cataractogenesis. Invest. Ophthalmol. Vis. Sci. 25, 275-1283. Kuszak, J.R., Macsai, M.S. and Rae, J.L. ( 1 983) Stereo scanning electron microscopy of the crystalline lens. Scan. Electron Microsc. 983/111, 141 5-1426. Kuwabara, T. (1975) The maturation of the lens cell: A morphologic study. Exp. Eye 427-443. Res. Sakuragawa, M., Kuwabara, T., Kinoshita, J.H. and Fukui, H.N.(1975)Swelling of the lens fibers. Exp. Eye Res. 21, 381-394. Kuszak, J.R., Ennesser, C.A., Umlas, J., Macsai-Kaplan, M.S. and Weinstein, R.S. (1988) The ultrastructure of fiber cells in primate lenses: A model for membrane senescence. J. Ultrastruct. Res. JQQ, 60-74. Lo, W-K. and Kuck, J.F.R. (1987) Alterations in fiber cell membranes of Emory mouse cataract: a morphologic study. Curr. Eye Res.
a,
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12.
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6, 433-444.
Vrensen, G., Van Marle, J., Willekins, B. and Van Veen, H. (1990) Square arrays in early cortical lens opacities. Invest. Ophthalmol. Vis. Sci. 31, 2476-2481. David, L.L., Takemoto, L.J., Anderson, R.S. and Shearer, T.R. ( 1 988) Proteolytic changes in main intrinsic polypeptide (MIP26) from membranes in selenite cataract. Curr. Eye Res.
7, 41 1-417. 18.
a,
4.
Hollenberg, M.J., Wyse, J.P.H. and Lewis, B.J. (1976) Surface morphology of lens fibers from eyes of normal and microphthalmic (Browman) 425-438. rats. Cell Tiss. Res. Lo, W-K. and Harding, C.V. (1984) Square arrays and their role in ridge formation in human lens fibers. J. Ultrastruct. Res. 86,
19.
20.
Palmquist, B-M., Fagerholm, P. and Landau, I. ( 1986) Selenium-induced cataract--A correlation of dry mass and light scattering. Exp. Eye Res. 42,35-42. Hightower, K.R., David, L.L. and Shearer, T.R. ( 1 987) Regional distribution of free calcium in selenite cataract: Relation to calpain II. Invest. Ophthamol Vis. Sci. 28, 1 702- 1 706. David, L.L., Dickey, B.M. and Shearer, T.R. (1987) Origin of urea-soluble protein in selenite cataract. Invest. Ophthalmol. Vis. Sci.
28, 1148-1 156. 21.
22.
23.
24.
25.
26.
Harding, C,V., Chylack, L.T., Susan, S.R., Decker, J.G. and Lo, W.K. (1980) Morphological changes in cataract: The ultrastructure of human lens opacities, localized by Cooperative Cataract Research Group procedures. In "Red Blood Cell and Lens Metabolism" (ed. Srivastava, S . ) . pp. 27-39. Elsevier, New York. Harding, C.V., Maisel, H., Chylack, L.T., Susan, S.R., Lo, W.K. and Bobrowski, W.F. ( 1 985) The structure of the human cataractous lens. In "The Ocular Lens" (ed. Maisel, H.). pp. 367-404. Marcel Dekker, Inc., New York. Creighton, M.O., Ross, W.M., StewartDeHaan, P.J., Sanwal, M. and Trevithick, J.R. (1 985) Modeling-cortical cataractogenesis VI1:Effects of vitamin E treatment on galactoseinduced cataracts. Exp. Eye Res.40. 213-222. Rini, F.J., Worgul, B. and Merriam, G. R. (1983) Scanning electron microscopic analysis of radiation cataracts in rat lenses. Ophthalmic Res. B,146-159. Ross, W.M., Creighton, M.O., Inch, W.R. and Trevithick, J. R. (1983) Radiation cataract formation diminished b y vitamin E in rat lenses in vitro. Exp. Eye Res. 36,645-653. Brooks. H.V., Jolly, R.D. and Paterson, C.A.
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27.
28.
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29.
(1983)The pathology of inherited cataract of sheep. Curr. Eye Res. 2,625-632. Mousa, G.Y., Creighton, M.O. and Trevithick, J.R. (1979)Eye lens opacity in cortical cataracts associated with actin-related globular degeneration. Exp. Eye R e s . 2 , 379-391. Jewel, S.A., Bellomo, G., Thor, H., Orrenius, S. and Smith, M.T. (1982)Bleb formation in hepatocytes during drug metabolism is caused by disturbances in thiol and calcium ion homeostasis. Science, 217, 1257-1259. Phelps, P.C., Smith, M.W. and Trump, B.F. (1989)Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells. Lab. Invest. 60,630642.
30. Tsai, W.J., Chen, J.C. and Wang, C.T. (1988).Changes in both calcium pool size and morphology of human platelets incubated in various concentrations of calcium ion. Calcium-specific bleb formation on platelet membrane surface. Biochim. Biophys. Acta,
940. 105-120. 31. Nieminen, A-L., Gores, G.J., Wray, B.E., Tanaka, Y., Herman, B. and LeMasters, J.J. (1988)Calcium dependence of bleb formation and cell death in hepatocytes. Cell Calcium,
9,237-246. 32. Eriksson, J.E., Paatero, G.1.L., Meriluoto, J.A.O., Codd, G.A., Kass, G.E.N., Nicotera, P. and Orrenius, S. (19891 Rapid microfilament reorganization induced in isolated rat hepatocytes by microcystin-LR, a cyclic peptide toxin. Exp. Cell Res. 86-
m,
100. 33. Mirabelli, F., Salis, A., Marinoni, V., Finardi, G., Bellomo, G., Thor, H. and Orrenius, S. (1988)Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin. Arch. Biochem. Biophys.
261-269. 34. Sage, M.D., and Jennings, R.B. (1988).
35.
Cytoskeletal injury and subsarcolemmal bleb formation in dog heart during in vitro total 327-337. ischemia. Am. J. Pathol. Nicotera, P., Hartzell, P. Davis, G. and The formation of plasma Orrenius, S. (1986). membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2 is mediated by activation of a non-lysosomal proteolytic system. FEBS Lett. 264, 261-269. Bennett, V. (1 990)Spectrin-based membrane skeleton: A rnultipotential adaptor between plasma membrane and cytoskeleton. Physiol. Rev. 170,1029-1065. Allen, D.P., Low, P.S., Dola, A. and Maisel, H. (1 987)Band 3 and ankyrin homologues are present in eye lens: Evidence for all major
+
36.
37.
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erythrocyte membrane components in same non-erythroid cell. Biochem. Biophys. Res. Comm. 149,266-275. Truscott, R.J.W., Marcantonio, J.M., Tomlinson, J. and Duncan, G. (1989) Calcium-induced cleavage and breakdown of spectrin in the rat lens. Biochem. Biophys. Res. Comm. 1472-1477. Boivin, P., Galand, C. and Dhermy, D. (1990). In vitro digestion of spectrin, protein 4.1 and ankyrin by erythrocyte calcium dependent neutral protease (calpain 1). Int. J. Biochern.
m,
39.
22, 1479-1489. 40. Hall, T.G. and Bennett, V. (1987) Regulatory
domains of erythrocyte ankyrin. J. Biol.Chem.
262, 10537-10545. 41. Maisel, H. and Ellis, M. (1984)Cytoskeletal 42.
proteins of the aging human lens. Curr. Eye Res. 3,369-381. Tagliavini, J., Gandolfi, S.A. and Mariani, G. (1 986)Cytoskeletal abnormalities in human senile cataract. Curr. Eye Res. 5,903-910.
Volume 1 1 riumber 12 1992, 1147- 1160
Selenite nuclear cataractogenesis: a scanning electron microscope study Ruth S.Anderson and Thomas R.Shearer
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Departments of Biochemistry and Ophthalmology, Schools of Dentistry and Medicine, Oregon Health Sciences University, Portland, OR 97201, USA
ABSTRACT The sequential changes during selenite nuclear cataractogenesis were examined with a scanning electron microscope (SEM) and correlated with slit lamp observations. A posterior opacity, visible with the slit lamp 1-2 days after injection of sodium selenite, was found to consist of masses of vacuoles in the superficial posterior cortex by SEM. 2-3 days post injection, a biomicroscopic refractile ring around the nucleus was represented by SEM abnormalities suggesting membrane damage and possible loss of cytosol in the perinuclear region. All normal structure in this region was lost by 5 days after injection when the central nucleus had become opaque. SEM also showed evidence for damage in areas which were still clear by slit lamp examination. Changes, characteristic of aging, were found near selenite induced damage in peripheral (younger) fibers.
formed, slit lamp examination showed that masses of vacuoles had developed around the equator, and later total cortical opacity was observed in a high proportion of the selenite injected rats (4,5). Light microscopy showed gross destruction of the cortex at the time the cortex was opaque. The rats which did not develop cortical opacity showed only minor biomicroscopic changes but rather severe histologic abnormalities (5). The cortices in both groups eventually became clear biomicroscopically and had near normal histology but the nuclear cataract was permanent. Thus, light microscopy has described general histological changes occurring during selenite
INTRODUCTION
In young rats, a single injection of sodium
cataractogenesis, but the technique was limited by relatively low magnification. Transmission electron
selenite causes rapid formation of cataracts, which
microscopy (TEM) provided high magnification of
are useful models for the study of cataractogenesis
selenite nuclear cataract (61, but only small areas
(11. Biomicroscopic observations showed a dense
could be examined. Scanning electron microscopy
posterior subcapsular opacity within 24 hours after
(SEMI allowed the whole lens to be examined in one
injection, a later perinuclear refractile ring, and dense
field for areas of interest which can then be examined
nuclear cataract 3--5 days post injection (2). Light
in detail at higher magnification. Bunce e t al. (7)
microscopy showed the posterior subcapsular opacity
used SEM to examine lenses from rats with mature
to be due to multiple vacuoles in the peripheral cortex
nuclear cataracts, but not during their development.
and the perinuclear refractile ring was associated
The purpose of the present study was to use SEM to
with intense basophilia and distorted fiber outlines.
localize morphologic changes in different regions of
The nuclear opacity consisted of a severely
the lens at various stages of selenite
degenerated peripheral nucleus surrounding a central
cataractogenesis. These were then compared with
granular region (3). After the nuclear opacity had
slit lamp and biochemical observations.
Received on February 26. 1992: accepted on November I I , 1992
G Oxford University Press
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Current Eye Research photographed with Polaroid 107C or 667 film. When
MATERIALS AND METHODS Litters of rats were obtained from B & K International (formerly Bantin and Kingman), Fremont,
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CA. and injected with 3 0 umoles Na,SeO,/kg
on the
available, figures in this report show cross fractures together with longitudinal fibers. We avoided artifact as much as possible by
day the eyes opened(13-14 days after birth).
processing control and experimental
Controls were uninjected littermates. Mother rats
lenses simultaneously so that fixation, dehydration,
were maintained on standard laboratory chow
critical point drying and coating were identical. We
(Wayne F-6) and tap water ad libitum. Eyes were
were also careful to observe comparable regions in
examined daily with the slit lamp after dilation with a
experimental and control lenses. Animals were used
mixture of atropine and phenylephrine and processed
in accordance with the ARVO Resolution on the use
for scanning electron microscopy at appropriate
of Experimental Animals in Research.
stages. Lenses with opaque nuclear cataract were used for SDS-PAGE electrophoresis as previously
RESULTS
described (8).
The B & K strain of Sprague Dawley rats used in this
Preparation of lenses for SEM was based on the
study developed nuclear cataracts slightly more
method used by Kuszak et al. (9). Rats were killed
slowly and cortical cataracts somewhat more rapidly
with carbon dioxide and eyes were removed
than those used in previous studies (2).The
immediately. After the backs of the eyes were
sequence of biomicroscopic changes noted above and
opened, they were immersed in 2.5% glutaraldehyde
their biomicroscopic appearance was, however,
in 0.07 M sodium cacodylate buffer, pH 7.2 for I - - 2
identical. Since slit lamp photographs have been
hours. The lenses were then carefully removed from
published previously (21, they are not included here.
the globes and reimmersed in fresh fixative. Lenses
All control lenses were biomicroscopally clear. For
were fixed for 3 days (with several changes) at room
selenite lenses, the characteristic slit lamp
temperature and then washed in 0.07M cacodylate
appearance together with the time after injection are
buffer for 24 hours. They were post-fixed in 1 %
listed in the text headings below. Additional slit lamp
OsO, at 4°C for 24 hours. Osmium was washed out
information is added in the text if it relates to SEM
in cacodylate buffer (24 hours), and lenses were
findings,
dehydrated in a graded series of acetone
Control lenses observed by SEM showed little
concentrations starting with 30% and ending with
change during the five day period reported here. The
acetone dried over a molecular sieve. Lenses were
locations of the regions studied in selenite
critical point dried in a Pelco 1250 critical point dryer.
cataractogenesis are shown in Figure 1. This
Fractures were initiated on the dried lenses with a
diagram is superimposed on a low magnification SEM
razor blade or fine scalpel to expose an AP plane in
photograph of the lens of an 18 day old rat which is
the center or periphery of the lens. The former were
a control for five days post selenite injection. In later
oriented to show narrow sides and the latter the
figures, SEM details of control lens fiber morphology
broad sides of the fibers. After being glued to
are shown together with selenite induced changes in
aluminum stubbs, the specimens were sputter coated
the same regions. For orientation characteristics of
with 30nm paladium-gold. Observations were made
the regions in control rat lenses are summarized
with a JSM-T330A scanning electron microscope and
below.
1148
Current Eye Research Anterior Pole
The central and peripheral nucleus (Fig. 1) are distinguished from one another because each region reacts very differently to selenite injection. Unlike
AY
\
the central nucleus, the peripheral nucleus has Y sutures and must therefore contain secondary fibers. In conventional terminology, the central and
rrixlUC
-
peripheral nucleus are probably comparable to the
CN-
embryonic and fetal nucleus, respectively. In control
PN-
uator
lenses, the fibers of the central nucleus were Curr Eye Res Downloaded from informahealthcare.com by University of Auckland on 11/30/14 For personal use only.
convoluted and the surface was relatively smooth, but multiple small mounds covered the surface of the peripheral nuclear fibers. The perinuclear region refers t o a narrow band of fibers immediately adjacent to the peripheral nucleus, which appears to be especially susceptible to early damage by selenite. In the perinuclear region of control lenses, SEM showed fibers which were uniform in size and shape. Their surfaces were relatively smooth except for faint ridges and sparse
PY /f Posterior Pole Figure 1. Low magnification of control lens with superimposed diagram for orientation. CN -= central nucleus; PN = peripheral nucleus; Perinuc = perinuclear region; CX = cortex; SF = superficial fibers; AY = anterior Y suture; PY = posterior Y suture.
interlocking devices. With the slit lamp, this region showed no special differences from the rest of the
lens fibers are similar to those reported by other
lens. By SEM, the control cortex consisted of
investigators (9-14) whose studies were concerned
mature, terminally differentiated fibers and
with lenses from adult animals and often involved
superficial immature fibers. The mature fibers had
other species. Our study used lenses from immature
abundant interlocking devices in mid cortex, but
rats, which can account for minor differences.
superficial fibers showed no interlocking devices.
Posterior subcaDsular cataract (1--2 davs Dost
They appeared to be coated with fine particles so
selenite iniectionl
that their margins were not clearly defined. The structure of the Y sutures was complex and in
In the biomicroscopically clear control lenses, SEM showed no vacuoles in posterior superficial cortical
our preparations they were sometimes open,
fibers, but they were covered with masses of
especially near the periphery. This could have been
unidentified small particles (Fig. 2a). In selenite
artifactual. However, it is more likely due to the
lenses, the posterior superficial opacity observed with
immaturity of the rats used in this investigation.
the slit lamp, was associated with SEM appearance
Open Y sutures in comparable rats were also
of patches of vacuoles in the peripheral posterior
observed by light microscopy in a previous study (3).
cortex (Fig. 2b and 2c). The vacuoles appeared to be
The epithelium, present only on the anterior surface
intracellular. Vacuolated fibers were larger than
of the lens, was used for orientation to localize the
adjacent fibers which were similar to those of
anterior and posterior sutures and other areas in the
controls from the same region.
lens. Our observations on SEM appearance of normal
perinuclear and nuclear region showed no
Although the
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biomicroscopic opacity, SEM showed intracellular
Fibers in the perinuclear region of selenite lenses
damage in selenite lenses. In control lenses, the
were distorted (Fig. 3c), lacked interlocking devices
membranes appeared to be intact and the cytosol of
and the surfaces showed pronounced ridges (Fig. 3d).
cross-fractured lens fibers was apparently uniform
Patches of membrane were lost, possibly at the site
(Fig. 2d). Most of the membranes of perinuclear
of the missing interlocking devices.
fibers of selenite lenses also remained intact except
rather sparse clumped cytoplasm within the fibers
for possible loss of interlocking devices.
where the internal structure was visible.
Where
There was
patches of membrane were lost during preparation,
Vacuoles in the posterior superficial cortex had
multiple small empty spaces were interspersed
regressed at this stage, but fibers in this area seemed
between solid structures in the cytosol (Fig. 2e).
to be undergoing further degeneration (Fig. 3e). They
Cross-fractured fibers in the nucleus also had a
were distorted and ridged, and a few small spherical
porous appearance with somewhat less open space
bodies were found in the damaged area. No
(data not shown). Anterior and equatorial cortical
abnormality was found in the other parts of the
fibers showed no difference between control and
cortex.
experimental lenses.
Central nuclear oDacitY (five davs Dost iniectionl iniectionl
Biomicroscopically in selenite lenses, the center of
In selenite lenses, the refractile perinuclear ring
the nucleus was totally opaque and surrounded by a
Refractile Derinuclear ring (2--3 davs
DOSt
visible with the slit lamp corresponded to the
clear or faintly hazy area within the refractile
perinuclear region observed with SEM. The SEM
perinuclear ring
photographs shown in Fig. 3a--3d were from a
refractile rings had appeared in the cortex which was
fracture of the equatorial region peripheral to the
otherwise completely transparent. Some lenses also
nucleus which were oriented to show the broad side
showed large superficial equatorial vacuoles with the
of fibers in the perinuclear region. The broad sides of
slit lamp.
control lens fibers from this region were uniform in
.
By this time, multiple concentric
The most striking SEM feature in selenite lenses
size (Fig. 3a) and had minor ridges (Fig. 3b). Fiber
was the large number of spherical bodies
surfaces and interlocking devices were intact.
(Morgagnian globules
Cytosol in fractured control fibers appeared solid (Fig.
structure in the perinuclear region. The spherical
3b).
bodies were concentrated in the Y sutures (Fig. 4a),
Figure 2. In selenite lenses, biomicroscopy showed posterior subcapsular opacity 1-2 days post injection. a. Control: Posterior superficial cortex. Fibers are covered with small particles, but there are no vacuoles. (500X, Bar = 50 um) b. Selenite: Posterior superficial cortex showing masses of vacuoles. (500X, Bar =50um) c. Higher magnification of 2b, vacuoles appear to be intracellular, note patch of degenerating cytoplasm (arrow). (2000X, Bar = 10 um)
d. Control: Perinuclear region, cross-sections of fibers (arrows) show solid uniform contents. There were no longitudinal fractures. (2000X, Bar = 1Oum) e. Selenite: Perinuclear region, cross-sections of fibers had a perforated appearance (broad arrow), longitudinal fractures showed multiple small empty spaces, a periodic arrangement of remaining solid material near membrane (small arrows) and possible damage to interlocking devices. (2000X, Bar = 10um)
?I and the total loss of normal
-
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Current Eye Research
1152
Current Eye Research but they were also found at the equator (Fig. 4b) and
the remaining contents (Fig. 4i). In the outer cortex
often extended around the nucleus. In contrast the
of selenite lenses, patches of ridged fiber surface and
fibers in the perinuclear region of control lenses had
multiple small mounds were present (Fig. 4j).
relatively uniform elongated fibers (Fig. 4c).
Superficial equatorial vacuoles, visible with the slit
Individual nuclear fibers from control lenses
lamp, were also observed by SEM (Fig. 4k). These
remained intact and appeared to be filled with solid
vacuoles often contained inclusions which appeared
uniform cytoplasm (Fig. 4d and 4e). In selenite
to consist of tangles of threadlike structures (Fig. 41).
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lenses, fibers of the peripheral nucleus were severely damaged. The membranes, apparently more fragile
Loss of cytosolic material in both the cortex and
than those of controls, fractured during preparation
nucleus of lenses with opaque nuclear cataracts 5
revealed large empty spaces, suggesting loss of
days after selenite injection was confirmed by SDS-
cytosol (Fig. 4f). Note that the biomicroscopically
PAGE electrophoresis (Fig. 5). Electrophoresis
dense opacity was in the central nucleus and that the
showed loss of soluble and insoluble
peripheral nucleus was clear or showed only a faint
(cytoskeletal) proteins from both the nucleus and
haziness by slit lamp examination. The membranes in
cortex. Loss of high molecular weight proteins was
the central nucleus also fractured, but, in contrast to
accompanied by increased low molecular weight
relatively empty peripheral nuclear fibers, the
polypeptides.
cytoplasm of fibers in the central nucleus appeared to be filled with particulate material (Fig. 49). Cortical fibers of control lenses had smooth membranes which did not fracture during preparation
DISCUSSION SEM gave new information on the cytologic
and had many interlocking devices (Fig 4h). In
changes in lenses undergoing selenite
selenite lenses, the cortex was clear by slit lamp
cataractogenesis. It also confirmed previous light
examination, but SEM showed extensive localized
microscope studies (3) which demonstrated that early
destruction at all levels of the cortical fibers. In the
posterior superficial opacity was due to extensive
mid cortex of selenite lenses, there was apparent
vacuolization, followed by disturbances in the
localized loss of cytosolic material and clumping of
perinuclear region and later abnormalities in the
Figure 3. In selenite lenses, biomicroscopy showed refractile perinuclear ring 2-3 days post injection. Fig. 3a--3d are oriented to show the broad side of fibers in perinuclear region. a. Control: Size and shape of fibers is uniform except for minor artifactual variation. Surface with typically few interlocking devices is intact. (2000 X, Bar = 10um) b. Control: Higher magnification of part of 3a; ridges on fiber surfaces are barely visible. (5000X, Bar = 5um) c. Selenite: Size and shape of fibers is highly variable
so that some fibers appear to be swollen and others shrunken. Note variation within individual fibers, loss of interlocking devices and localized loss of membrane (arrows). (2000X, Bar = 1Oum) d. Higher magnification of part of Fig. 3c showing localized loss of membrane and clumped fiber contents. Note elaborate system of ridges on fiber surfaces. (5000X, Bar = 5um) e. Selenite: Peripheral posterior cortex showing remnants of vacuoles (straight arrows), distorted fibers with ridged surfaces (curved arrows) and small spherical bodies. (2000X, Bar = 10um)
~
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Current Eye Research
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Current Eye Research nucleus which correlated with biomicroscopic
Possible loss of membrane at the site of missing
opacity.
interlocking devices suggested that these devices are
In selenite injected rats, the earliest lens change observed with the slit lamp was a posterior
Ridges on the fiber cell surface were seen in
subcapsular opacity which appeared within the first
control fibers (Fig. 3b), but they seemed t o be
day after injection and later extended over the whole
exaggerated in selenite lenses (Fig. 3d). Patches of
back of the lens except the posterior Y suture. By
ridged membranes also were found in the cortex at
SEM, this opacity was correlated with vacuoles
the site of the regressing posterior superficial opacity
which appeared to be intracellular within swollen
(Fig. 3e).
superficial fibers (Fig. 2b and 2c). However TEM Curr Eye Res Downloaded from informahealthcare.com by University of Auckland on 11/30/14 For personal use only.
especially susceptible to selenite induced damage.
Ridges have been associated with maturation and
studies would be necessary to confirm this. In
aging of fibers by others (9-13). In selenite
previous studies, lens epithelium showed damage
cataracts, ridges were observed near cataractous
within a few hours after selenite injection (4).
damage in peripheral young fibers (Fig.4j).
Possibly selenite induced damage to the epithelial
they were always found in a damaged area and
pump mechanism led to the accumulation of water at
included young fibers, ridges probably represented
the back of the lens where no epithelium is present.
part of the degenerative process.
During the second day after selenite injection, a
Since
In preliminary studies of early secondary cortical
decrease in the extent of the posterior subcapsular
cataract (which develops after the nuclear cataract),
cataract and a refractile ring around the lens nucleus
we have observed increased amounts of similar
were observed with the slit lamp.
structures and also microvilli, another characteristic
This biomicroscopic refractile ring was correlated to
of aging (12).near cataractous damage in relatively
damage in the perinuclear region shown by SEM (Fig.
young fibers. Thus, selenite cataractous changes
3c and 3d).
appear to mimic aging changes in lens fibers. Similar
Figure 4. In selenite lenses 5 days post injection, biomicroscopy showed an opaque central nucleus, clear peripheral nucleus and clear cortex except for multiple concentric refractile rings. a. Selenite: Perinuclear level anterior Y suture containing masses of spherical bodies (2000X, Bar = 1Oum) b. Selenite: Perinuclear level, equator. No normal fiber structure, apparent fusion of membranes and numerous spherical bodies. (2000X, Bar = 10um) c. Control: Perinuclear fibers uniform in size and shape except for minor artifactual changes (2000X, Bar = 1Oum) d. Control: Peripheral nucleus near posterior Y suture. Fractures show solid fiber contents (arrows). Textured appearance of fibers is due to masses of small mounds which cover the surface of these older fibers. (2000X, Bar = 1Oum) e. Control: Central nucleus showing uniform solid fiber contents (arrows) and intact surfaces. Irregular conformation is associated with normal aging changes. (2000X, Bar = 1Oum)
f. Selenite: Peripheral nucleus near posterior Y suture. Note apparent loss of cytosol (arrows). (2000X, Bar = IOum) g. Selenite: Central nucleus showing some loss of membrane and particulate appearance of fiber contents. (2000X, Bar = 1Oum) h. Control: Mid-cortical fibers, unfractured fiber membrane is smooth except for characteristic numerous interlocking devices. (2000X, Bar = 10um) i. Selenite: Localized damage in mid-cortical fibers. Note fractured membranes with loss of cytosol and clumping of contents. (2000X, Bar = 1Oum) j. Selenite: Outer cortical fibers. Note ridges (arrowheads) and numerous small mounds (arrows) on surface of fibers. (5000X, Bar = 5um) k. Selenite: Superficial cortical vacuoles near equator containing clumps (arrows). Wide arrow points to edge of lens near equator (capsule lost), crack in vacuole is artifact. (IOOOX, Bar= 10um) I. Higher magnification of structure at upper arrow in Fig. 3k. Note threadlike appearance. (10,000 X, Bar = 1um)
1156
Current Eye Research selenite lenses (Fig. 2b, 2c, 2e, 3c, 3d, 4f, 49, 4i, 4j). This indicates that the membranes were more '
Sol
Insol
Sol
Insol
fragile in selenite lenses. TEM might reveal structural membrane damage in the sites indicated by SEM.
97 66
Loss of main intrinsic membrane protein (MIP 26) has been shown in selenite nuclear cataract (17). It is
45
interesting that early membrane damage, as seen by
32
SEM, occurred in the perinuclear region which was separated from the site of future opacity.
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20
When the perinuclear refractile ring became visible
14
5
S
C Se C Se
C Se C Se
with the slit lamp (2-3 days post injection), perinuclear fibers were irregular in shape and size,
Figure 5. SDS-PAGE Electrophoresis by SDS-PAGE of proteins from lens cortex and nucleus of control (C) and cataractous selenite (Se) lenses from rats at 5 days after injection. S = molecular weight standards (kilodaltons). "Sol" = water soluble fraction, and "Insol" = water insoluble fraction. Bands at arrow and below are crystallins and asterisk indicates cytoskeletal region in controls. Note loss of bands from both regions together with new low molecular weight bands in selenite lenses.
possibly due to uneven uptake of water. Rapid changes in dry mass concentration, probably caused by rapid shifts in water distribution within the lens have been described in selenite cataract (18). By the time the nuclear opacity had developed (5 days post injection), extensive fracture of membranes together with apparent loss of cytosol was found in the peripheral part of the nucleus (Fig. 4f) and in the cortex (Fig. 4i) although these regions were
ridges were observed in the microphthalmic
essentially clear biomicroscopically. Fracture of
(Browman strain) rat (13) and the Emory mouse
membranes during preparation suggested increased
cataract (14).
fragility, probably due to loss of protein. Fracture of
In human lens fibers, square arrays (non-
membranes revealed fiber contents and indicated that
communicating junctions) were found in ridges (15)
there was loss of cytosol within affected fibers
and in fine globular elements ( 1 6) similar to the small
where the remaining contents were clumped and
mounds found near selenite cataract (Fig. 4j).
irregular. This contrasted sharply with fibers of
Vrensen et al. ( 1 6) suggested that square arrays
control lenses in which the membranes remained
associated with the fine globular elements were part
intact and cross fractures showed uniform solidly
of a protective mechanism which separated diseased
packed cytosol.
and healthy fibers, thus conserving transparency.
Loss of cytosol in selenite lenses was also
Ridges and small mounds adjacent to damaged areas
suggested by electrophoresis. Electrophoresis of
in selenite cataract should be investigated with
nuclear and cortical proteins by SDS-PAGE showed
freeze-fracture techniques to determine whether
breakdown or loss of crystallins and cytoskeletal
square arrays are present.
proteins with accumulation of lower molecular weight
Membranes of control lenses remained intact after
polypeptides (Fig.5). Similar results were obtained in
preparation for SEM (Fig. 2d., 3a, 3d, 4c, 4d, 4e and
earlier studies which showed that the above
4h), but localized membrane losses occurred in
proteolytic changes were progressive during selenite
1157
Current Eye Research nuclear cataractogenesis. Increased proteolysis in
Calpain proteolysis is important in selenite
the cortex was a new finding and probably reflects
cataractogenesis (8) and may be part of the
the beginning of the secondary cortical cataract in
mechanism which produces spherical bodies in
which cortical opacity is first observed with the slit
cataractous lenses.
lamp 15--20 days after selenite injection in this strain of rats (unpublished data).
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Calcium concentration was elevated in both the
Threadlike structures observed in vacuoles of newly damaged fibers (Fig. 41) may have been cytoskeleton detached from the membrane, but not yet destroyed.
cortex and nucleus (data not shown) of selenite
Possibly, breakdown and loss of soluble lens protein
lenses from the strain of rats used in this study. High
(8) during selenite cataractogenesis exposed these
calcium concentration in the nucleus, but not the
structures and made them visible with SEM. Both
cortex in other strains also was shown previously (7,
ankyrin and spectrin, involved in attachment of other
8, 19) and is compatible with the hypothesis that
cytoskeletal elements to the plasma membrane (361,
calpain II, a calcium activated protease, plays an
have been found in lens (37,381. Both are substrates
important role in the observed degenerative changes.
for calpain (39, 40) and loss of cytoskeletal elements
It was surprising that the central nucleus, which was
has been described in human cataracts (41, 42).
totally opaque by slit lamp examination, showed
Further study of the cytoskeleton in the selenite
relatively high amounts of cytoplasmic content (Fig.
cataract model seems warranted. Studies should
49). This may have been related to the high
include light microscope and SEM observations on
concentration of g crystallins in the nucleus which
lenses which have been treated to remove soluble
are not degraded in selenite cataract (20).
proteins. TEM studies with antibody labelled
Spherical bodies similar to those found in the perinuclear region have been reported in several human cataracts (21, 22) as well as experimental
cytoskeletal elements at different stages and locations also would be useful. The present SEM investigation has shown that
sugar (231, radiation (24, 25) and genetic (14, 26)
biomicroscopically clear areas in the lens had already
cataracts. Mousa e t al. (27) induced formation of
sustained severe damage including loss of cytosol
similar spherical bodies by incubation of rat lenses
and membrane defects. SEM observations were
with agents which caused microfilament and tubulin
supported by biochemical data in this and earlier
disorganization. Spherical bodies resembling those in
studies. These findings are compatible with the
lenses also have been observed in other types of
hypothesis that early selenite-induced damage to the
cells. These "blebs" could be induced to form by
lens epithelium allows a net influx of calcium causing
high calcium levels (28-30) and were associated with
activation of calpain resulting in partial proteolysis
increased membrane permeability (31) and
and opacity.
disturbances of the cytoskeleton (32-34). Nicotera et
al. (35) reported that leupeptin, a calpain inhibitor, protected hepatocytes from blebbing (and loss of viability) and the authors suggested that calcium
ACKNOWLEDGEMENTS Partially supported by NIH Grant #EY03600 to
activated protease attack on cytoskeleton was part
T.R.S. The authors thank Mr. Mitsuoshi Azuma and
of the mechanism which caused certain blebs.
Mr. Jay Wright for expert technical assistance.
1158
Current Eye Research CORRESPONDING AUTHOR
14.
R.S. Anderson, Ph.D., Department of Biochemistry,
m,
Oregon Health Sciences University, 61 1 S.W. Campus Dr., Portland, OR 97201
Curr Eye Res Downloaded from informahealthcare.com by University of Auckland on 11/30/14 For personal use only.
2.
3.
15.
228-245.
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