January 2000
Volume 41, Issue 1
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Lens  |   January 2000
Repair in the Rat Lens after Threshold Ultraviolet Radiation Injury
Author Affiliations
  • Ralph Michael
    From the Karolinska Institutet, St. Erik’s Eye Hospital, Stockholm, Sweden;
    The Netherlands Ophthalmic Research Institute, Amsterdam;
  • Gijs F. J. M. Vrensen
    The Netherlands Ophthalmic Research Institute, Amsterdam;
    Department of Ophthalmology, State University of Leiden; and
  • Jan van Marle
    Department of Electron Microscopy, Amsterdam Medical Center, University of Amsterdam, The Netherlands.
  • Stefan Löfgren
    From the Karolinska Institutet, St. Erik’s Eye Hospital, Stockholm, Sweden;
  • Per G. Söderberg
    From the Karolinska Institutet, St. Erik’s Eye Hospital, Stockholm, Sweden;
Investigative Ophthalmology & Visual Science January 2000, Vol.41, 204-212. doi:
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      Ralph Michael, Gijs F. J. M. Vrensen, Jan van Marle, Stefan Löfgren, Per G. Söderberg; Repair in the Rat Lens after Threshold Ultraviolet Radiation Injury. Invest. Ophthalmol. Vis. Sci. 2000;41(1):204-212.

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      © ARVO (1962-2015); The Authors (2016-present)

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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/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 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  
Methods
Experimental rats were exposed in vivo to a single, threshold dose of UVR. At different time points after UVR exposure, the lenses were extracted and investigated with scanning and transmission electron microscopy (SEM, TEM, respectively). The ultrastructure of lens fiber membranes was studied with freeze–fracture TEM. The cortical lens fiber architecture was mapped with fluorescent membrane staining of coronary lens sections, and their spatial order was analyzed with Fourier transformation. 
UVR Exposure
Collimated radiation from a high-pressure mercury lamp (HBO 200 W; Osram, GmbH, München, Germany), passed through water and interference filters (λmax. = 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. 
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. 
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 25× objective with a 0.8 numerical aperture. The acquired images were stored as TIFF files in 512 × 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). 
Results
Intact Lens
The nonexposed lenses were clear with a smooth surface (Fig. 2A ). One day after UVR exposure, lenses had flaked confluent dots or small granules (approximately 20–50 μm in size) scattered over the anterior surface; the posterior surface looked normal (Fig. 2B) . At 7 days postexposure, the lens surface was rough and appeared corrugated and equatorial opacities were seen (Fig. 2C) . The anterior side of the lens was more opaque than the posterior. Vacuoles (approximately 50–100 μm in size) were visible at the lens equator in the cortex. At 56 days postexposure, the lens surface was clear again (Fig. 2D) ; however, a narrow equatorial cortical light-scattering ring was visible. Because this light-scattering ring was visible in coronary and sagittal views, it was assumed to have a shell shape. The shell-shaped opacity seemed not to disturb the axial view through the lens. The shell-shaped opacity was approximately 50 μm thick and was observed between 280 and 370 μm below the lens surface, which corresponds to 13% to 17% of the total coronary lens radius (2200 μm). 
Lens Epithelium
Sagittal sections of control lenses had a normal alignment of epithelial cells, and the architecture of the nuclear lens bow was regular (Fig. 3A ). One day postexposure, cells with apoptotic morphology were scattered throughout the epithelium (Fig. 3B) . At 7 days postexposure, epithelial cells were missing, and very few apoptotic bodies were found (Fig. 3C) . The epithelium appeared recovered and normal 56 days postexposure (Fig. 3D)
As revealed by electron microscopy, the lens epithelium was disturbed at 1 and 7 days postexposure (Figs. 4B 4C 4F 4G ). At 1 day postexposure the cell interdigitations were disturbed (Fig. 4B) , and numerous apoptotic bodies were seen (Fig. 4F) . At 7 days postexposure, the epithelium contained extracellular spaces approximately 1 to 5 μm in size (Figs. 4C 4G) . At 56 days postexposure, the epithelium appeared normal (Figs. 4D 4H) . No alterations of the lens capsule after UVR exposure were detected by electron microscopy. 
Lens Fibers
No change in the fiber morphology and the nuclear bow architecture was seen at 1 day postexposure (Fig. 3B) . At 7 days postexposure, the nuclear bow was disturbed and the outer cortex had swollen fibers and vacuolar structures (Fig. 5B ). Superficial fibers down to 150 μm or 40 fiber layers were swollen and the fiber order was disturbed. However, the most severe damage did not occur in the most superficial fibers, but in fibers between the 10th and 40th growth shell below the capsule (Figs. 3C 5B) . In these areas, giant vacuolar structures were seen, and the fiber order was severely disturbed. The distance between the area of the most disorganized spatial fiber order and the lens center was approximately 1650 μm at 7 days postexposure (Fig. 5B)
From approximately 150 μm below the capsule, the fibers appeared normal (Figs. 3C 5B) . Fibers with their anterior ends under missing epithelial cells seemed to be more damaged (Fig. 3C) . In addition to the giant vacuolar structures, fibers in the equatorial region in layers 10 to 40 often contained smaller vacuolar structures (1–2 μm in size) at 7 days postexposure (Figs. 6A 6D ). The number of ball and sockets at the endings of damaged fibers was higher than in normal fibers (Fig. 6A)
TEM verified that the smaller vacuolar structures were often located at the base of fiber protrusions (Fig. 7A ). Freeze–fracture showed numerous membranous globules between the fibers in the affected areas at 7 and 56 days postexposure (Figs. 7B 7C) . Membranous globules were dispersed between normal fibers and also were found at the base of ball and sockets (Fig. 7B) . The globules were extracellular protrusions of the fiber membrane, as they were not surrounded by normal granular fiber cytoplasm (Fig. 7C) . The membranes of these globules were free of intramembranous particles and gap junctions. Fiber membranes above and below the damaged growth shells appeared normal, with gap junctions, intramembranous particles, and square array structures (Figs. 7D 7E) . The observations from SEM (Fig. 6D) and freeze–fracture (Figs. 7B 7C) clearly show that the vacuolar structures described above are extracellular in nature. 
At 56 days postexposure, the nuclear bow had regained normal shape (Fig. 3D) . However, disturbed spatial fiber order and swollen fibers were visible predominantly between 200 and 350 μm (60–100 layers) beneath the lens capsule (Figs. 3D 5C) . This was approximately 10% to 18% of the total radius (1975 μm) of the midcoronary paraffin section. The distance between the area of the most disorganized spatial fiber order and the lens center was approximately 1650 μm at 56 days postexposure (Fig. 5C) . The degree of disturbance varied within a lens and between exposed lenses at 56 days postexposure. Fibers in the damaged layers had larger diameters (Fig. 5C) than normal and were growing branches into each other (Fig. 6E) . Large ellipsoidal outgrowths (up to 50 μm) between the fibers were found (Fig. 6B)
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). 
Discussion
Lens Epithelium
The flakelike opacities observed in the superficial lens at 1 day postexposure (Fig. 2B) appear to be similar to the discrete white dots described by Andley and coworkers 25 after in vivo UVR exposure of rabbits. The flakelike opacities in the present experiment were restricted to the anterior surface of the lens, suggesting that they were related to changes in the lens epithelium. 
The epithelial monolayer was altered at 1 day postexposure by apoptotic bodies 26 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 Kojima 28 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 coworkers 29 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 coworkers 33 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 coworkers 22 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 treatment 22 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. 
Conclusions
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. 
 
Figure 1.
 
Relative spectral dose in the corneal plane of the exposed eye. The total dose between 285 and 325 nm was 5 kJ/m2.
Figure 1.
 
Relative spectral dose in the corneal plane of the exposed eye. The total dose between 285 and 325 nm was 5 kJ/m2.
Figure 2.
 
Intact lenses in BSS in equatorial view (top) and anterior view (middle). Bottom: magnification of the anterior view. Nonexposed lens (A), 1 day (B), 7 days (C), and 56 days (D) after UVR exposure. The images in the top, middle, and bottom panels (A) through (D) are from the same lens. At 1 day postexposure (B), lenses showed flaked confluent dots scattered over the anterior surface. At 7 days postexposure (C), equatorial vacuoles (arrowheads) and anterior opacification were visible. The asterisks indicate different camera focus: focus at the lens surface (white asterisk) and focus at the lens equator (black asterisk). At 56 days postexposure (D), a subtle ring-shaped opacity (arrows) was seen in both equatorial and anterior view. Distance between the center of the white gridlines is 790 μm. a, anterior; eq, equator; p, posterior. Scale bars in lower panel, 200 μm.
Figure 2.
 
Intact lenses in BSS in equatorial view (top) and anterior view (middle). Bottom: magnification of the anterior view. Nonexposed lens (A), 1 day (B), 7 days (C), and 56 days (D) after UVR exposure. The images in the top, middle, and bottom panels (A) through (D) are from the same lens. At 1 day postexposure (B), lenses showed flaked confluent dots scattered over the anterior surface. At 7 days postexposure (C), equatorial vacuoles (arrowheads) and anterior opacification were visible. The asterisks indicate different camera focus: focus at the lens surface (white asterisk) and focus at the lens equator (black asterisk). At 56 days postexposure (D), a subtle ring-shaped opacity (arrows) was seen in both equatorial and anterior view. Distance between the center of the white gridlines is 790 μm. a, anterior; eq, equator; p, posterior. Scale bars in lower panel, 200 μm.
Figure 3.
 
Semi-thin sections of the lens nuclear bow region after UVR exposure. The nonexposed lens (A) has a normal epithelium and nuclear bow region. At 1 day postexposure (B), apoptotic bodies were scattered throughout the epithelium (black arrowheads). At 7 days postexposure (C), the nuclear bow was disturbed by large and small vacuolar structures in the outer cortex. Fibers with ends under missing epithelial cells (white arrowhead) seemed to be more involved in the damage. At 56 days postexposure (D), large, irregular fibers were visible in the deeper cortex (black bold arrows). Scale bars, 100 μm.
Figure 3.
 
Semi-thin sections of the lens nuclear bow region after UVR exposure. The nonexposed lens (A) has a normal epithelium and nuclear bow region. At 1 day postexposure (B), apoptotic bodies were scattered throughout the epithelium (black arrowheads). At 7 days postexposure (C), the nuclear bow was disturbed by large and small vacuolar structures in the outer cortex. Fibers with ends under missing epithelial cells (white arrowhead) seemed to be more involved in the damage. At 56 days postexposure (D), large, irregular fibers were visible in the deeper cortex (black bold arrows). Scale bars, 100 μm.
Figure 4.
 
The apical side of lens epithelium in scanning electron micrographs (SEM) (top) and lens epithelial section in transmission electron micrographs (TEM) (bottom) after UVR exposure. Nonexposed lens (A, E), 1 day (B, F), 7 days (C, G), and 56 days postexposure (D, H). The epithelium showing an apoptotic nucleus (asterisk), apoptotic bodies (black arrows), vacuolar structures (white arrows), and a covered basal membrane (arrowheads). c, lens capsule; e, epithelial cell; f, lens fiber. Scale bars, (A, B, C, D) 20 μm; (E, F, G, H) 2 μm.
Figure 4.
 
The apical side of lens epithelium in scanning electron micrographs (SEM) (top) and lens epithelial section in transmission electron micrographs (TEM) (bottom) after UVR exposure. Nonexposed lens (A, E), 1 day (B, F), 7 days (C, G), and 56 days postexposure (D, H). The epithelium showing an apoptotic nucleus (asterisk), apoptotic bodies (black arrows), vacuolar structures (white arrows), and a covered basal membrane (arrowheads). c, lens capsule; e, epithelial cell; f, lens fiber. Scale bars, (A, B, C, D) 20 μm; (E, F, G, H) 2 μm.
Figure 5.
 
Midcoronary paraffin lens sections stained with wheat germ agglutinin. Nonexposed lenses (A, D), 7 days (B), and 56 days (C) after UVR exposure. Nonexposed lenses (A, D) are from contralateral eyes of the exposed lenses (B, C). (B) Section from the lens shown in Figure 2C ; (C) section from the lens in Figure 2D . The insets (E) and (F) are the power spectra of two-dimensional Fourier transformations of the marked subcapsular areas (100 × 100 μm) in (A) and (B), contrast enhanced to show detail. The regular order and equal size of the fibers in the control lens (A) gave a regular pattern of peaks in the Fourier transform (E); fibers of different size (B) gave no peaks in the Fourier transform (F). The outermost left and right rulers show the distance from below the lens capsule and the middle rulers show the distance from the lens center (radius). The increased lens radius of (C) and (D) compared with (A) and (B) reveals the lens growth between 7 and 56 days postexposure.
Figure 5.
 
Midcoronary paraffin lens sections stained with wheat germ agglutinin. Nonexposed lenses (A, D), 7 days (B), and 56 days (C) after UVR exposure. Nonexposed lenses (A, D) are from contralateral eyes of the exposed lenses (B, C). (B) Section from the lens shown in Figure 2C ; (C) section from the lens in Figure 2D . The insets (E) and (F) are the power spectra of two-dimensional Fourier transformations of the marked subcapsular areas (100 × 100 μm) in (A) and (B), contrast enhanced to show detail. The regular order and equal size of the fibers in the control lens (A) gave a regular pattern of peaks in the Fourier transform (E); fibers of different size (B) gave no peaks in the Fourier transform (F). The outermost left and right rulers show the distance from below the lens capsule and the middle rulers show the distance from the lens center (radius). The increased lens radius of (C) and (D) compared with (A) and (B) reveals the lens growth between 7 and 56 days postexposure.
Figure 6.
 
Scanning electron micrographs at 7 days (A, D) and 56 days (B, E) after UVR exposure and from a nonexposed control (C). At 7 days postexposure (A, D), fibers in the equatorial region contained extracellular spaces of varying size. At 56 days postexposure, fibers in the damaged layers were larger than normal and were growing branches into each other (E). Large ellipsoidal areas between the fibers were filled with fiber material (B). c, lens capsule; e, epithelial cell; f, lens fiber. Scale bars, (A) 50 μm; (B, C, D, E) 10 μm.
Figure 6.
 
Scanning electron micrographs at 7 days (A, D) and 56 days (B, E) after UVR exposure and from a nonexposed control (C). At 7 days postexposure (A, D), fibers in the equatorial region contained extracellular spaces of varying size. At 56 days postexposure, fibers in the damaged layers were larger than normal and were growing branches into each other (E). Large ellipsoidal areas between the fibers were filled with fiber material (B). c, lens capsule; e, epithelial cell; f, lens fiber. Scale bars, (A) 50 μm; (B, C, D, E) 10 μm.
Figure 7.
 
Transmission electron micrograph (A) and freeze–fracture transmission electron micrographs (B, C, D, E) of superficial fibers at 7 days (A, B, C) and 56 days (D, E) after UVR exposure. At 7 days postexposure, fiber protrusions with adjacent extracellular spaces (A, black arrows) and normal protrusions (A, white arrows), ball-and-socket connection with membranous globules (B, black bold arrows) and normal membrane part (B, white bold arrow). Membranous globules (C, black bold arrows) starting from normal fiber membrane (C, white bold arrow) and intercellular space with abnormal cytoplasm (C, black asterisk). At 56 days postexposure, superficial fibers had normal gap junctions (D, white asterisks) and normal square array pattern of intramembranous particles (E, white arrow heads). BS, ball-and-socket; fm, fiber membrane; cyt, cytoplasm. Scale bars, (A, B, C, D) 2 μm; (E) 0.2 μm.
Figure 7.
 
Transmission electron micrograph (A) and freeze–fracture transmission electron micrographs (B, C, D, E) of superficial fibers at 7 days (A, B, C) and 56 days (D, E) after UVR exposure. At 7 days postexposure, fiber protrusions with adjacent extracellular spaces (A, black arrows) and normal protrusions (A, white arrows), ball-and-socket connection with membranous globules (B, black bold arrows) and normal membrane part (B, white bold arrow). Membranous globules (C, black bold arrows) starting from normal fiber membrane (C, white bold arrow) and intercellular space with abnormal cytoplasm (C, black asterisk). At 56 days postexposure, superficial fibers had normal gap junctions (D, white asterisks) and normal square array pattern of intramembranous particles (E, white arrow heads). BS, ball-and-socket; fm, fiber membrane; cyt, cytoplasm. Scale bars, (A, B, C, D) 2 μm; (E) 0.2 μm.
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. 
Söderberg PG. Experimental cataract induced by ultraviolet radiation. Acta Ophthalmol (Copenh). 1990;68:1–77.
Söderberg PG. Development of light dissemination in the rat lens after exposure to radiation in the 300 nm wavelength region. Ophthalmic Res. 1990;22:271–279. [CrossRef] [PubMed]
Michael R, Söderberg PG, Chen E. Long-term development of lens opacities after exposure to ultraviolet radiation at 300 nm. Ophthalmic Res. 1996;28:209–218. [CrossRef] [PubMed]
Michael R. Threshold Dose Estimation for Ultraviolet Radiation Induced Cataract. Thesis. 1997;1–36. Karolinska Institutet Stockholm.
Michael R, Söderberg PG, Chen E. Dose-response function for lens forward light scattering after in vivo exposure to ultraviolet radiation. Graefes Arch Clin Exp Ophthalmol. 1998;236:625–629. [CrossRef] [PubMed]
Bettelheim FA. Physical basis of lens transparency. Maisel H eds. The Ocular Lens: Structure, Function and Pathology. 1985;265–295. Marcel Dekker New York.
Hemenger RP. Small-angle intraocular light scatter: a hypothesis concerning its source. J Opt Soc Am [A]. 1988;5:577–582. [CrossRef] [PubMed]
Tardieu A, Delaye M. Eye lens protein and transparency: from light transmission theory to solution X-ray structural analysis. Annu Rev Biophys Biophys Chem. 1988;17:47–70. [CrossRef] [PubMed]
Taylor HR. Ultraviolet radiation and the eye: an epidemiologic study. Trans Am Ophthalmol Soc. 1990;87:802–853.
Cruickshanks KJ, Klein BE, Klein R. Ultraviolet light exposure and lens opacities: the Beaver Dam Eye Study. Am J Public Health. 1992;82:1658–1662. [CrossRef] [PubMed]
Jose JG, Pitts DG. Wavelength dependency of cataracts in albino mice following chronic exposure. Exp Eye Res. 1985;41:545–563. [CrossRef] [PubMed]
Breadsell RO, Wegener A, Breipohl W. UV-B radiation-induced cataract in the royal collage of surgeons rat. Ophthalmic Res. 1994;26:848–849.
Pitts DG, Cullen AP, Hacker PD. Ocular effects of ultraviolet radiation from 295 to 365 nm. Invest Ophthalmol Vis Sci. 1977;16:932–939. [PubMed]
Zigman S, Paxhia T, McDaniel T, Lou MF, Yu NT. Effect of chronic near-ultraviolet radiation on the gray squirrel lens in vivo. Invest Ophthalmol Vis Sci. 1991;32:1723–1732. [PubMed]
Cullen AP, Monteith-McMaster CA. Damage to the rainbow trout (Oncorhyncus mykiss) lens following an acute dose of UVB. Curr Eye Res. 1993;12:97–106. [CrossRef] [PubMed]
Lerman S. Human ultraviolet radiation cataracts. Ophthalmic Res. 1980;12:303–314. [CrossRef]
Müller–Breitenkamp U, Hockwin O, Siekmann H, Dragomirescu V. Ultraviolet radiation as cataract risk factor—a case report. Dev Ophthalmol. 1997;27:76–80. [PubMed]
Söderberg PG. Acute cataract in the rat after exposure to radiation in the 300 nm wavelength region. A study of the macro-, micro- and ultrastructure. Acta Ophthalmol (Copenh). 1988;66:141–152. [PubMed]
Wegener AR. In vivo studies on the effect of UV-radiation on the eye lens in animals. Doc Ophthalmol. 1994.3–4:221–232.
Michael R, Vrensen G, van Marle J, Gan L, Söderberg PG. Apoptosis in the rat lens after in vivo threshold dose ultraviolet irradiation. Invest Ophthalmol Vis Sci. 1998;13:2681–2687.
Wester U. Solar ultraviolet radiation on the canary islands and in Sweden—a comparison of irradiance levels. Paschier W eds. Human Exposure to UVR. 1987;275–279. Elsevier Science Publishers Amsterdam.
Vrensen GFJM, Sanderson J, Willekens B, Duncan G. Calcium localization and ultrastructure of clear and pCMPS-treated rat lenses. Invest Ophthalmol Vis Sci. 1995;36:2287–2295. [PubMed]
Bond J, Green C, Donaldson P, Kistler J. Liquefaction of cortical tissue in diabetic and galactosemic rat lenses defined by confocal laser scanning microscopy. Invest Ophthalmol Vis Sci. 1996;37:1557–1565. [PubMed]
Kistler J, Gilbert K, Brooks HV, Jolly RD, Hopcroft DH, Bullivant S. Membrane interlocking domains in the lens. Invest Ophthalmol Vis Sci. 1986;27:1527–1534. [PubMed]
Andley UP, Fritz C, Morrison AR, Becker B. The role of prostaglandins E2 and F2 alpha in ultraviolet radiation-induced cortical cataracts in vivo. Invest Ophthalmol Vis Sci. 1996;37:1539–1548. [PubMed]
Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–257. [CrossRef] [PubMed]
Wyllie AH. Apoptosis: an overview. Br Med Bull. 1997;53:451–465. [CrossRef] [PubMed]
Yamada Y, Kojima M. Repairing process of rabbit lens epithelial cells damaged by ultraviolet B. 1998;23:225–236. J Kanazawa Medical University Thesis
Andley UP, Song Z, Mitchell DL. DNA repair and survival in human lens epithelial cells with extended lifespan. Curr Eye Res. 1999;18:224–230. [CrossRef] [PubMed]
Söderberg PG, Löfgren S, Michael R, Gonzalez–Cirre X. New method for measurement of in vivo penetration of UVR into the crystalline lens. SPIE. 1998;3246:43–47.
Chen E, Söderberg PG, MacKerell AD, Lindström B, Tengroth B. Inactivation of lactate dehydrogenase by UV radiation in the 300 nm wavelength region. Radiat Environ Biophys. 1989;28:185–191. [CrossRef] [PubMed]
Löfgren S, Söderberg PG. Rat lens glycolysis after in vivo exposure to narrow band UV or blue light radiation. J Photochem Photobiol B. 1995;30:145–151. [CrossRef] [PubMed]
Reddy GB, Bhat KS. UVB irradiation alters the activities and kinetic properties of the enzymes of energy metabolism in rat lens during aging. J Photochem Photobiol B. 1998;42:40–46. [CrossRef] [PubMed]
Li LK, So L, Spector A. Membrane cholesterol and phospholipid in consecutive concentric sections of human lenses. J Lipid Res. 1985;26:600–609. [PubMed]
Halliwell B, Gutteridge J. Free Radicals in Biology and Medicine. 1985;139–189. Claredon Press Oxford.
Cenedella RJ. Cholesterol and cataracts. Surv Ophthalmol. 1996;40:320–337. [CrossRef] [PubMed]
Cenedella RJ, Shi H. Spatial distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger RNA in the ocular lens: relationship to cholesterologenesis. J Lipid Res. 1994;35:2232–2240. [PubMed]
Chandy G, Zampighi GA, Kreman M, Hall JE. Comparison of the water transporting properties of MIP and AQP1. J Membr Biol. 1997;159:29–39. [CrossRef] [PubMed]
Verbavatz JM, Ma T, Gobin R, Verkman AS. Absence of orthogonal arrays in kidney, brain and muscle from transgenic knockout mice lacking water channel aquaporin-4. J Cell Sci. 1997;110:2855–2860. [PubMed]
Figure 1.
 
Relative spectral dose in the corneal plane of the exposed eye. The total dose between 285 and 325 nm was 5 kJ/m2.
Figure 1.
 
Relative spectral dose in the corneal plane of the exposed eye. The total dose between 285 and 325 nm was 5 kJ/m2.
Figure 2.
 
Intact lenses in BSS in equatorial view (top) and anterior view (middle). Bottom: magnification of the anterior view. Nonexposed lens (A), 1 day (B), 7 days (C), and 56 days (D) after UVR exposure. The images in the top, middle, and bottom panels (A) through (D) are from the same lens. At 1 day postexposure (B), lenses showed flaked confluent dots scattered over the anterior surface. At 7 days postexposure (C), equatorial vacuoles (arrowheads) and anterior opacification were visible. The asterisks indicate different camera focus: focus at the lens surface (white asterisk) and focus at the lens equator (black asterisk). At 56 days postexposure (D), a subtle ring-shaped opacity (arrows) was seen in both equatorial and anterior view. Distance between the center of the white gridlines is 790 μm. a, anterior; eq, equator; p, posterior. Scale bars in lower panel, 200 μm.
Figure 2.
 
Intact lenses in BSS in equatorial view (top) and anterior view (middle). Bottom: magnification of the anterior view. Nonexposed lens (A), 1 day (B), 7 days (C), and 56 days (D) after UVR exposure. The images in the top, middle, and bottom panels (A) through (D) are from the same lens. At 1 day postexposure (B), lenses showed flaked confluent dots scattered over the anterior surface. At 7 days postexposure (C), equatorial vacuoles (arrowheads) and anterior opacification were visible. The asterisks indicate different camera focus: focus at the lens surface (white asterisk) and focus at the lens equator (black asterisk). At 56 days postexposure (D), a subtle ring-shaped opacity (arrows) was seen in both equatorial and anterior view. Distance between the center of the white gridlines is 790 μm. a, anterior; eq, equator; p, posterior. Scale bars in lower panel, 200 μm.
Figure 3.
 
Semi-thin sections of the lens nuclear bow region after UVR exposure. The nonexposed lens (A) has a normal epithelium and nuclear bow region. At 1 day postexposure (B), apoptotic bodies were scattered throughout the epithelium (black arrowheads). At 7 days postexposure (C), the nuclear bow was disturbed by large and small vacuolar structures in the outer cortex. Fibers with ends under missing epithelial cells (white arrowhead) seemed to be more involved in the damage. At 56 days postexposure (D), large, irregular fibers were visible in the deeper cortex (black bold arrows). Scale bars, 100 μm.
Figure 3.
 
Semi-thin sections of the lens nuclear bow region after UVR exposure. The nonexposed lens (A) has a normal epithelium and nuclear bow region. At 1 day postexposure (B), apoptotic bodies were scattered throughout the epithelium (black arrowheads). At 7 days postexposure (C), the nuclear bow was disturbed by large and small vacuolar structures in the outer cortex. Fibers with ends under missing epithelial cells (white arrowhead) seemed to be more involved in the damage. At 56 days postexposure (D), large, irregular fibers were visible in the deeper cortex (black bold arrows). Scale bars, 100 μm.
Figure 4.
 
The apical side of lens epithelium in scanning electron micrographs (SEM) (top) and lens epithelial section in transmission electron micrographs (TEM) (bottom) after UVR exposure. Nonexposed lens (A, E), 1 day (B, F), 7 days (C, G), and 56 days postexposure (D, H). The epithelium showing an apoptotic nucleus (asterisk), apoptotic bodies (black arrows), vacuolar structures (white arrows), and a covered basal membrane (arrowheads). c, lens capsule; e, epithelial cell; f, lens fiber. Scale bars, (A, B, C, D) 20 μm; (E, F, G, H) 2 μm.
Figure 4.
 
The apical side of lens epithelium in scanning electron micrographs (SEM) (top) and lens epithelial section in transmission electron micrographs (TEM) (bottom) after UVR exposure. Nonexposed lens (A, E), 1 day (B, F), 7 days (C, G), and 56 days postexposure (D, H). The epithelium showing an apoptotic nucleus (asterisk), apoptotic bodies (black arrows), vacuolar structures (white arrows), and a covered basal membrane (arrowheads). c, lens capsule; e, epithelial cell; f, lens fiber. Scale bars, (A, B, C, D) 20 μm; (E, F, G, H) 2 μm.
Figure 5.
 
Midcoronary paraffin lens sections stained with wheat germ agglutinin. Nonexposed lenses (A, D), 7 days (B), and 56 days (C) after UVR exposure. Nonexposed lenses (A, D) are from contralateral eyes of the exposed lenses (B, C). (B) Section from the lens shown in Figure 2C ; (C) section from the lens in Figure 2D . The insets (E) and (F) are the power spectra of two-dimensional Fourier transformations of the marked subcapsular areas (100 × 100 μm) in (A) and (B), contrast enhanced to show detail. The regular order and equal size of the fibers in the control lens (A) gave a regular pattern of peaks in the Fourier transform (E); fibers of different size (B) gave no peaks in the Fourier transform (F). The outermost left and right rulers show the distance from below the lens capsule and the middle rulers show the distance from the lens center (radius). The increased lens radius of (C) and (D) compared with (A) and (B) reveals the lens growth between 7 and 56 days postexposure.
Figure 5.
 
Midcoronary paraffin lens sections stained with wheat germ agglutinin. Nonexposed lenses (A, D), 7 days (B), and 56 days (C) after UVR exposure. Nonexposed lenses (A, D) are from contralateral eyes of the exposed lenses (B, C). (B) Section from the lens shown in Figure 2C ; (C) section from the lens in Figure 2D . The insets (E) and (F) are the power spectra of two-dimensional Fourier transformations of the marked subcapsular areas (100 × 100 μm) in (A) and (B), contrast enhanced to show detail. The regular order and equal size of the fibers in the control lens (A) gave a regular pattern of peaks in the Fourier transform (E); fibers of different size (B) gave no peaks in the Fourier transform (F). The outermost left and right rulers show the distance from below the lens capsule and the middle rulers show the distance from the lens center (radius). The increased lens radius of (C) and (D) compared with (A) and (B) reveals the lens growth between 7 and 56 days postexposure.
Figure 6.
 
Scanning electron micrographs at 7 days (A, D) and 56 days (B, E) after UVR exposure and from a nonexposed control (C). At 7 days postexposure (A, D), fibers in the equatorial region contained extracellular spaces of varying size. At 56 days postexposure, fibers in the damaged layers were larger than normal and were growing branches into each other (E). Large ellipsoidal areas between the fibers were filled with fiber material (B). c, lens capsule; e, epithelial cell; f, lens fiber. Scale bars, (A) 50 μm; (B, C, D, E) 10 μm.
Figure 6.
 
Scanning electron micrographs at 7 days (A, D) and 56 days (B, E) after UVR exposure and from a nonexposed control (C). At 7 days postexposure (A, D), fibers in the equatorial region contained extracellular spaces of varying size. At 56 days postexposure, fibers in the damaged layers were larger than normal and were growing branches into each other (E). Large ellipsoidal areas between the fibers were filled with fiber material (B). c, lens capsule; e, epithelial cell; f, lens fiber. Scale bars, (A) 50 μm; (B, C, D, E) 10 μm.
Figure 7.
 
Transmission electron micrograph (A) and freeze–fracture transmission electron micrographs (B, C, D, E) of superficial fibers at 7 days (A, B, C) and 56 days (D, E) after UVR exposure. At 7 days postexposure, fiber protrusions with adjacent extracellular spaces (A, black arrows) and normal protrusions (A, white arrows), ball-and-socket connection with membranous globules (B, black bold arrows) and normal membrane part (B, white bold arrow). Membranous globules (C, black bold arrows) starting from normal fiber membrane (C, white bold arrow) and intercellular space with abnormal cytoplasm (C, black asterisk). At 56 days postexposure, superficial fibers had normal gap junctions (D, white asterisks) and normal square array pattern of intramembranous particles (E, white arrow heads). BS, ball-and-socket; fm, fiber membrane; cyt, cytoplasm. Scale bars, (A, B, C, D) 2 μm; (E) 0.2 μm.
Figure 7.
 
Transmission electron micrograph (A) and freeze–fracture transmission electron micrographs (B, C, D, E) of superficial fibers at 7 days (A, B, C) and 56 days (D, E) after UVR exposure. At 7 days postexposure, fiber protrusions with adjacent extracellular spaces (A, black arrows) and normal protrusions (A, white arrows), ball-and-socket connection with membranous globules (B, black bold arrows) and normal membrane part (B, white bold arrow). Membranous globules (C, black bold arrows) starting from normal fiber membrane (C, white bold arrow) and intercellular space with abnormal cytoplasm (C, black asterisk). At 56 days postexposure, superficial fibers had normal gap junctions (D, white asterisks) and normal square array pattern of intramembranous particles (E, white arrow heads). BS, ball-and-socket; fm, fiber membrane; cyt, cytoplasm. Scale bars, (A, B, C, D) 2 μm; (E) 0.2 μm.
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