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1 From the Karolinska Institutet, St. Erik’s Eye Hospital, Stockholm, Sweden; 2 The Netherlands Ophthalmic Research Institute, Amsterdam; 3 Department of Ophthalmology, State University of Leiden; and 4 Department of Electron Microscopy, Amsterdam Medical Center, University of Amsterdam, The Netherlands.
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Abstract |
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METHODS. One eye of young, female Sprague–Dawley rats was exposed to 5
kJ/m2 narrowband ultraviolet radiation (UVR)
(
max = 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.
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Introduction |
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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 fibers7 and lens proteins.8
Clinical studies9 10 and experimental studies with mice,11 rats,1 2 3 4 5 12 rabbits,13 squirrels,14 and trout15 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/m2 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 days2 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
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Methods |
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UVR Exposure
Collimated radiation from a high-pressure mercury lamp (HBO 200 W;
Osram, GmbH, München, Germany), passed through water and
interference filters (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/m2 UVR for 15 minutes,
with a narrow beam that covered only the cornea and the eyelids of the
exposed eye.
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Electron Microscopy
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).
Lens Fiber Membrane Staining
Lenses from five animals, kept for either 7 or 56 days
postexposure were fixed in a phosphate-buffered paraformaldehyde (2%)
solution (pH 7.3) for 3 days at 8°C, dehydrated with ethanol, and
routinely embedded in paraffin. Coronary sections (4 µm) were dewaxed
with xylen and rehydrated. The sections were incubated with
fluorescein-conjugated wheat germ agglutinin (10 µg/ml in PBS, L
4895; Sigma) for 2 hours at room temperature.23
Wheat germ
agglutinin binds to cell-surface glycoproteins of the fiber
membrane.24
The stained slides were mounted with antifade
Vectashield (Vector Laboratories, Burlingame, CA) and examined with a
confocal microscope (Leica Laser Technik GmbH; Heidelberg, Germany)
using a 25x objective with a 0.8 numerical aperture. The acquired
images were stored as TIFF files in 512 x 512 pixel format. Power
spectra of two-dimensional Fourier transformations of the images were
obtained with the Image Processing Toolkit (Reindeer Games,
Gainesville, FL).
Freeze–Fracture
The remaining parts from lenses, fixed for electron microscopy as
described above, were prepared for freeze–fracture. Dissected pieces
were infiltrated with 2.3 M sucrose for cryoprotection and quick-frozen
in liquid ethane. The specimens were fractured in a Balzers BAF 300
(Liechtenstein) at 160 K and a pressure of 10-5
Pa and were replicated with 2 nm platinum at 45° evaporation angle
and with 20 nm carbon at 90°. The replicas were cleaned with
perchloric acid and inspected in a transmission electron microscope (EM
420; Philips Industries).
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Results |
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The average coronary lens radius of a 7-week rat (7 days postexposure) was compared with a 14-week rat (56 days postexposure). The lens radius increased from 2010 ± 20 to 2220 ± 40 µm, as measured from the intact lens in BSS (mean with confidence interval, n = 5). Because of the tissue shrinkage during fixation and embedding, this corresponds to an increase from 1760 ± 130 to 2000 ± 120 µm for the lenses in paraffin (n = 5). During the 49 days between observations at ages 7 and 14 weeks, approximately 60 new fiber layers are formed (counted in Fig. 5D ).
The Fourier transform from the superficial cortex of nonexposed control lenses showed a regular pattern of peaks (Fig. 5E) and revealed an average fiber thickness of 3.9 µm and a fiber width of 7.5 µm in paraffin. At 7 days postexposure, no peaks were visible in the Fourier transform from the superficial cortex (Fig. 5F) . However, at 56 days postexposure, the Fourier transform showed a regular pattern of peaks from the superficial cortex, but not from cortical areas between 200 and 350 µm below the lens capsule (data not shown).
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Discussion |
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The epithelial monolayer was altered at 1 day postexposure by apoptotic bodies26 27 in both the central and equatorial epithelium (Figs. 3B 4F) , and the spacing and arrangement of the epithelial cells was disturbed (Fig. 4B) . An earlier publication from our group using the TdT-dUTP terminal nick-end labeling (TUNEL) assay showed that programmed cell death in the rat lens peaks 24 hours after UVR exposure.20 The apoptotic changes and therefore the loss of metabolic competent cells may disturb the osmoregulation of the epithelium and interfere with the protein and water balance of the underlying fibers. The local derangement of water and ion homeostasis in the epithelium may lead to an extracellular accumulation of calcium.22
The disturbances of the lens epithelium at 1 day postexposure seem to account for the observed anterior superficial opacities. Furthermore, the flakelike appearance of these opacities can be explained by the nonuniform distribution of the apoptotic bodies in the epithelium. By 7 days postexposure, the flakelike opacities disappeared and were replaced by a corrugated opaque lens surface and equatorial opacities (Fig. 2C) . The coincidence of the disappearance of both the flakelike opacities and the apoptotic bodies in the epithelium supports their association.
The extracellular spaces in the epithelium together with abnormal superficial fibers seem to account for the corrugated opaque lens surface at 7 days postexposure. The extracellular spaces in the epithelium (Figs. 4C 4G) were probably caused by disturbed osmoregulation and programmed cell death, which cleared damaged cells from the epithelium. The epithelial alignment was still incomplete, but seemed to start reestablishing probably by cell movement and proliferation. The extracellular spaces were filled by adjacent cells covering also the basal membrane (Fig. 4G) . By 56 days after exposure, the arrangement and spacing of the epithelium returned to normal (Figs. 3D 4D 4H) . The disappearance of the extracellular spaces indicates a recovery of the osmoregulation. Whether the epithelium recovers by cell movement or cell proliferation, or both, is still under discussion. Recent results from Yamada and Kojima28 support the idea of cell proliferation in the central lens epithelium after UVR injury. They observed proliferating cell nuclear antigen positivity, indicative of start-up of proliferation and mitosis in the central epithelium at 2 days after in vivo UVR exposure in the 20-week-old rabbit (0.5–3 kJ/m2 corneal dose at 310 nm).
The epithelium at 56 days after UVR appeared normal, as did the lens fibers differentiated from these cells. The recovered morphology of the epithelium and of the superficial fibers account for the regained clear lens surface at 56 days postexposure. With the recovery of the epithelium and the superficial cortex, the corrugated surface seen at 7 days postexposure disappeared (Figs. 2C 2D) .
Although short-term threshold UVR leads to programmed cell death, long-term repeated UVR exposure at subthreshold levels leads to epithelial hyperplasia.19 With subthreshold exposure, epithelial cells may not die but might be stimulated to create a multilayered shield to protect against UVR. DNA damage and repair play certainly an important role in the recovery of the lens epithelium after UVR injury. Andley and coworkers29 showed that photoproducts [cyclobutane pyrimidine dimers and (6-4) photoproducts] are repaired within 1 to 2 days after UVR exposure, depending on the UVR dose and the type of photoproduct. This work was done in cultured human lens epithelial cells using the same waveband and UVR dose at the level of the epithelial cells (0.4 and 0.8 kJ/m2 epithelial dose) as in our present experiment (5 kJ/m2 corneal dose = 0.5 kJ/m2 epithelial dose with 10% corneal transmission assumed). They concluded that cells either repair DNA damage and proceed in the cell cycle or do not repair and are eliminated by cell death.
Lens Fibers
Except for the superficial flakelike opacities, no lens opacities
or increased light scattering was observed at 1 day postexposure (Fig. 2B)
. No histologic and ultrastructural changes were seen in the lens
fibers at this time point, suggesting that the morphology of the fibers
is not altered immediately after UVR exposure.
UVR at 300 nm is absorbed by the lens epithelium and the superficial cortex because this radiation penetrates approximately 450 µm into the lens.30 A few hours after UVR exposure, glycolysis is inhibited and lactate dehydrogenase is inactivated in the lens cortex.31 32 Reddy and coworkers33 also showed a decrease of enzyme activity in the rat lens immediately after UVR exposure. Furthermore, the disturbed osmoregulation of the lens epithelium may influence the homeostasis of the underlying fibers. However, these metabolic changes in the lens fibers at 1 day postexposure do not result in visible morphologic changes or light scattering.
Light and electron microscopy suggest that the equatorial opacities detected at 7 days after UVR were caused by vacuolar structures and extracellular spaces (Figs. 2C 3C 5B 6A 6D) . Inasmuch as these extracellular spaces were restricted to the lens equator, they probably account for the observed equatorial opacities. After UVR exposure, the equatorial section of a cortical fiber was more damaged than either the anterior or posterior ends. The equatorial section of the lens is the most metabolically active region, as indicated by the presence of endoplasmatic reticulum, mitochondria, nucleus, and Golgi apparatus. The membranes of fiber ends contain more cholesterol than phospholipids, compared with the lens equator.34 Because high concentrations of cholesterol depress peroxidation,35 fiber ends may be less sensitive to UVR insults.
The extracellular spaces between the equatorial parts of cortical fibers appeared probably between 1 and 7 days postexposure as a result of disturbed water homeostasis. These layers contained areas with extracellular membranous globules devoid of intramembranous particles (Figs. 7B 7C) . Such membranous globules impair the pumping ability of the membranes, leading to osmotic swelling. Vrensen and coworkers22 previously found cortical fibers with similar extracellular spaces in p-chloromercuri-phenylsulfonate (pCMPS) treated rat lenses. In the pCMPS cataracts, water follows the accumulation of calcium. It seems most likely that the membranous changes found in the present study lead to accumulation of calcium in the extracellular space, followed by water accumulation.
The observation that fibers with a growth cone interfacing with disturbed epithelium are more damaged suggests that incomplete coverage of the anterior lens capsule with epithelial cells might contribute to the pathologic changes in the cortical lens fibers (Fig. 3C) . Further experiments are necessary to confirm that a local disturbance of the epithelium leads to changes in the underlying fibers.
Small extracellular spaces, similar in appearance to enlarged sockets seen with SEM (Fig. 6D) , were often located by fiber protrusions and ball and sockets (Fig. 7A) . Membranous globules also were found at the base of ball and sockets (Fig. 7B) . Others have detected abnormal fiber protrusions after UVR.12 These observations suggest a correlation between the appearance of extracellular spaces with fiber protrusions and ball and sockets.
It is interesting that after 1 week the most superficial fibers are not damaged as severely as the fibers in layers 10 to 40 below the capsule (Fig. 5B) . Fluorescent membrane staining revealed that approximately 1.2 growth shells of fibers are formed per day in young rats (Fig. 5) . Approximately eight new fiber layers must have formed between the UVR insult and the observation at day 7 postexposure. Hence, the fibers found in layer 10 at 7 days postexposure were the most superficial fibers at the time of the UVR exposure, which explains their severe damage. Similar cataract morphology was found earlier with, e.g., pCMPS at 4 hours after treatment22 and in galactosemic and diabetic cataracts at 1 and 4 weeks, respectively, after treatment.23 It was speculated that the damaged zone reveals a critical stage in fiber development.22
Between 7 and 56 days postexposure, light scattering in the exposed lens decreased substantially (Figs. 2C 2D) . The superficial and equatorial opacities seen at 7 days postexposure disappeared. Instead a subtle shell-shaped opacity was visible deeper in the equatorial lens cortex. The normal morphology and normal spatial order of the lens epithelium and the superficial lens fibers down to 200 µm below the capsule (Figs. 3D 5C) explains the disappearance of the superficial and equatorial opacities.
Lens fiber order is crucial for lens transparency.6 7 A regular pattern of peaks in the Fourier transform is consistent with regular fiber order. The Fourier transform confirmed no spatial fiber order in the equatorial cortex between 0 and 150 µm below the lens capsule at 7 days postexposure (Fig. 5F) and between 200 and 350 µm below the capsule at 56 days postexposure. These locations correspond well with the areas of opacity found at 7 and 56 days postexposure (Figs. 2C 2D) . Because the zone of disturbed spatial fiber order had about the same distance from the lens center at 7 and 56 days postexposure, it seems most likely that the fibers damaged at 7 days postexposure remained in their original growth shell at between 1600 and 1700 µm of the lens radius (Figs. 5B 5C) . The hexagonal pattern of peaks in the Fourier transform (Fig. 5E) is due to the hexagonal shape of the lens fibers.
At 56 days postexposure, the location of the disturbed fiber order found with the fluorescent membrane staining (at 10%–18% of the lens radius, Fig. 5C ) corresponded well to the location of the shell-shaped opacity in the intact lens (at 13%–17% of the lens radius, Fig. 2D ). Furthermore, the disappearance of the extracellular spaces and the reoccupation of these spaces by adjacent fibers in the affected fiber layers explains the decrease of light scattering compared with the equatorial opacities at 7 days postexposure.
At 56 days postexposure, no extracellular spaces were found in the originally damaged fiber layers. Rather these fiber layers contained ellipsoidal outgrowths and branched into each other (Figs. 6B 6E) , suggesting that the extracellular spaces had been reoccupied by fiber material from adjacent fibers which filled in these spaces. Neighboring fibers must have regained osmotic function and have repaired their membranes. Membrane repair and growth requires cholesterol synthesis.36 Both the superficial and deep cortical fibers in the rat lens are capable of cholesterol synthesis.37 However, repair was incomplete, inasmuch as abnormal membranes with membranous globules were still found in the affected fiber layers.
Equatorial epithelial cells formed after UVR exposure appeared normal and fibers developing from these cells were histologically normal. The spatial order of the new fibers was normal, and their membranes were normal with gap junctions, intramembranous particles, and square arrays (Figs. 7D 7E) . Square arrays are thought to be formed by passive water channels (aquaporines),38 39 showing that the new fibers regained normal osmotic function.
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Conclusions |
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TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
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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.
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Acknowledgements |
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Footnotes |
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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. ralph.michael{at}altavista.net
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