Abstract
purpose. To investigate the development and recovery of lens damage after in
vivo close-to-threshold exposure to ultraviolet B radiation.
methods. One eye of young, female Sprague–Dawley rats was exposed to 5
kJ/m2 narrowband ultraviolet radiation (UVR)
(λmax = 302 nm) for 15 minutes. Groups of rats were
killed 1, 7, and 56 days after exposure. The structure of the exposed
and nonexposed lenses was examined with light microscopy, scanning
electron microscopy, transmission electron microscopy,
freeze–fracture, fluorescent membrane staining, and Fourier transform
analysis.
results. One day after UVR exposure the lens surface had flakelike opacities.
Seven days after exposure, the lens surface appeared opaque and
corrugated, and the equatorial cortex had small opacities. At 56 days
postexposure, the surface and equator appeared clear, but the cortex
had a subtle shell-shaped opacity. At 1 day postexposure, apoptotic
cell death occurred in the lens epithelium, but the cortical fibers
were normal. At 7 days postexposure, the epithelium and the fibers
between the 10th and 40th growth shell below the capsule contained
extracellular spaces of different sizes. After 56 days, the epithelial
layer appeared normal, and the extracellular spaces had disappeared;
but abnormal fibers were found between the 60th and 100th growth shell
below the capsule. Fibers above and below the damaged growth shells
appeared fully normal.
conclusions. A close-to-threshold dose of UVR causes cataract, which is largely
reversible. The UVR exposure leads to apoptosis in the lens epithelium,
and after a latency period of several days, lens fibers are abnormal.
Extracellular spaces develop in the epithelium and fibers. Within
several weeks after exposure, the epithelium fully recovers and new
fibers develop normally. The originally affected fibers are repaired.
However, this repair is incomplete, leaving a small zone of enhanced
light scattering in the equatorial
cortex.
Previous studies from this laboratory have documented that
ultraviolet radiation (UVR) exposure of the rat lens leads to increased
light scattering.
1 2 3 4 5 To understand this effect, we
studied the morphology of the lens epithelium and the lens fibers, the
ultrastructure of the fiber membranes and the spatial order of the
fibers.
The transparency of the crystalline lens depends on the regular or
ordered spacing of its cells and proteins. Disturbance of this
order—such as protein aggregation, membrane degeneration, fluctuations
in protein density and phase separation—results in local changes of
refractive index, which cause light scattering.
6 The
understanding of cataract formation can be improved by study of the
spatial organization of lens fibers
7 and lens
proteins.
8
Clinical studies
9 10 and experimental studies with
mice,
11 rats,
1 2 3 4 5 12 rabbits,
13 squirrels,
14 and trout
15 document a
dose–response relationship between UVR exposure and subsequent lens
opacities. Rare cases of human cataract have been correlated with
accidental UVR exposure.
16 17
UVR may damage the lens by several mechanisms: protein cross-linking,
DNA damage, dysfunction of enzymes, and membrane damage. UVR injury
leads to swelling and disruption of lens epithelial cells and cortical
lens fibers.
12 18 Swollen mitochondria, subcapsular
vacuoles and chromatin condensation, and nuclear fragmentation are
found in the epithelium.
18 Long-term, repeated,
subthreshold UVR leads to epithelial hyperplasia.
19 Threshold exposure to UVR induces programmed cell death (apoptosis) in
the lens epithelium 24 hours after exposure.
20
The UVR dose in the current experiment (5 kJ/m
2 at 300 nm)
is close to threshold for cataract in rabbits and
rats.
3 13 Earlier experiments have shown that light
scattering after UVR exposure develops within 7 days
2 and
increases exponentially, depending on the dose.
5 After a
threshold dose of UVR, the rat lens develops opacities that may be
repaired. In contrast, after suprathreshold UVR the rat lens is unable
to repair the injury.
3
A wavelength centered at 300 nm was chosen because of its biological
and environmental importance. The cornea begins to transmit UVR above
290 nm, and the lens begins to transmit above 340 nm. The lens absorbs
nearly all energy between these wavelengths, and only radiation energy
that is absorbed by a tissue can have a damaging effect. The intensity
of UVR on the earth surface depends on the path length of solar
radiation through the atmosphere and is a complex function of altitude,
latitude, time of day, and stratospheric ozone. The intensity of UV B
radiation (280–315 nm) varies more with the above factors than longer
wavelength UVR. For example, the annual maximum value at 300 nm at the
Canary Islands (28°N) is about seven times as high as the maximum
reached in Stockholm (59°N).
4 21
Collimated radiation from a high-pressure mercury lamp (HBO 200 W;
Osram, GmbH, München, Germany), passed through water and
interference filters (λ
max. = 300 nm,
half-bandwidth, 10 nm) was projected on the cornea of one
eye.
1 The spectrum of the radiation is given in
Figure 1 . Altogether, 31 female Sprague–Dawley rats were exposed unilaterally
at the age of 6 weeks. Ten minutes before exposure, each animal was
anesthetized by an intraperitoneal injection of a mixture of 94 mg/kg
ketamine and 14 mg/kg xylazine. Five minutes after injection,
mydriaticum tropicamide was instilled in both eyes. After another 5
minutes, the eye was exposed to 5 kJ/m
2 UVR for 15 minutes,
with a narrow beam that covered only the cornea and the eyelids of the
exposed eye.
The rats were killed with carbon dioxide 1, 7, and 56 days after UVR
exposure. All animals were kept and treated according to the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
After removing the lens by a posterior scleral incision, it was placed
in balanced salt solution (BSS), cleared of adherent ciliary body and
photographed with a stereomicroscope (MZ 6; Leica AG, Heerbrugg,
Germany) against a black background with a white grid.
Both the exposed and nonexposed lenses from seven animals at each
time interval were fixed in a 0.08 M cacodylate-buffered glutaraldehyde
(1.25%)–paraformaldehyde (1%) solution (pH 7.3)
22 for
at least 7 days at 8°C.
Lens parts were dissected for SEM. The lens capsule was stripped off to
view the basal side of the lens epithelium. Fibers were removed from
the epithelium to expose its apical side. The lens cortex was prepared
to view lens fibers at different depth. The dissected pieces were
dehydrated in a graded series of ethanols and dried by immersion for 20
minutes in hexamethyldisilazane (H 4875; Sigma Chemical, St. Louis,
MO), followed by drying for 8 hours on filter paper. The pieces were
mounted with carbon glue and sputter-coated with gold and studied in a
scanning electron microscope (SEM 505; Philips Industries, Eindhoven,
The Netherlands).
Other lens parts were dissected and postfixed for TEM in a buffered 1%
osmium tetroxide solution (24505; Merck, Rahway, NJ) supplemented with
1.5% potassium ferricyanide (4973; Merck), dehydrated in a graded
series of ethanols, and embedded in epoxy resin. Sagittal sections of
80 to 100 nm contrasted with uranyl acetate and lead citrate were
studied in a transmission electron microscope (EM 201; Philips
Industries).
Semi-thin lens sections in epoxy resin were stained with toluidine blue
for photomicroscopy (DM RB; Leica, AG).
In vivo threshold dose UVR leads within 1 day to apoptosis and
disintegration of the lens epithelium, associated with flakelike
opacities at the lens surface. After 1 week the epithelium and the
equatorial parts of superficial lens fibers contain extracellular
spaces. The extracellular spaces together with locally disarranged
fibers produce a corrugated opaque lens surface and equatorial
opacities. Within several weeks after exposure, the lens epithelium
recovers, and new fibers develop normally. The lens fibers regain
normal osmotic properties and fill up the extracellular spaces. Repair,
however, is incomplete, and disarranged fibers remain in the cortex,
producing a subtle shell-shaped opacity. In this way, subtle damage to
the lens fibers induced by UVR may accumulate during lifetime and
contribute to the formation of cortical cataract.
UVR exposure initially causes DNA damage in the lens epithelium, which
is repaired within a few days keeping the cells either functioning
properly or removing them by programmed cell death. New epithelial
cells proliferate and enable the epithelium to be repaired completely.
Damage to the lens fibers is delayed compared with the epithelium and
is restricted to the fibers differentiating at the moment of exposure.
How both events, in epithelium and fibers, are linked remains a
challenging question. UVR may reach the lens equator by multiple
scattering and might damage differentiating fibers directly, there may
be signals from the injured and repairing epithelium that causes the
damage of the lens fibers, or the disarranged epithelium is not able to
optimally regulate water and ion homeostasis.
Supported by St. Eriks Ögonforskningsstiftelse, Anders Otto Swärds Stiftelse, Clas Groschinskys Minnesfond, ELFAs
Forskningsstiftelse, Swedish Society for Medical Research, Carmen och Bertil Regnérs Stiftelse, and Erik och Edith Fernströms Stiftelse.
Submitted for publication March 31, 1999; revised August 3, 1999; accepted September 3, 1999.
Commercial relationships policy: N.
Corresponding author: Ralph Michael, Research Laboratory, St. Erik’s Eye Hospital, S-112 82 Stockholm, Sweden.
[email protected]
The authors thank Ben Willekens for preparing the SEM specimens and
taking the SEM photographs; Anneke de Wolf for cutting ultrathin
sections for TEM; and Agneta Bonnevier, Margareta Oskarsson, and Berit
Spångberg for making paraffin sections. They also thank Marina
Danzman, Niko Bakker, Ton Put, and Maud Leindahl for photographic
assistance and John Merriam for English language check.
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