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Ultrastructure of UVR-B-induced cataract and repair visualized with electron microscopy Linda M. Meyer,1 Alfred R. Wegener,2 Frank G. Holz,2 Martin Kronschl€ager,3 Jan P. Bergmanson4 and Per G. Soderberg3 1
Herzog Carl Theodor Eye Clinic, Munich, Germany University Eye Clinic Bonn, Bonn, Germany 3 Department of Neuroscience, Uppsala University, Uppsala, Sweden 4 University of Houston, College of Optometry, Houston, TX, USA 2
ABSTRACT. Purpose: The aim of the study is to investigate and visualize the ultrastructure of cataract morphology and repair, after in vivo exposure to double threshold dose UVR-B in the C57BL/6 mouse lens. Methods: Twenty-six-week-old C57BL/6 mice received in vivo double threshold dose (6.4 kJ/m2) UVR-B for 15 min. The radiation output of the UVR-source had kMAX at 302.6 nm. After a latency period of 1, 2, 4 and 8 days following UVR-B exposure, the induced cataract was visualized with electron microscopy techniques. Induced, cataract was quantified as forward lens light scattering. Damage to the lens epithelium and the anterior cortex was investigated with light microscopy in toluidine blue-stained semi-thin sections, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dark field illumination photography. Results: UVR-B-exposed lenses developed anterior subcapsular and/or cortical and nuclear cataract after 1 day. Lens light scattering peaked 2 days after exposure. Lens epithelial cell damage was seen in TEM as apoptotic cells, apoptotic bodies, nuclear chromatin condensation, and swollen and disrupted anterior cortex fibres throughout the sections of the whole anterior lens surface. These morphologic changes were also visualized with SEM. Within 8 days, anterior subcapsular cataract was repaired towards the anterior sutures. Conclusion: UVR-B exposure of double cataract threshold dose induces a subtotal loss of epithelial cells across the whole anterior surface of the lens. This damage to the epithelium is repaired by epithelial cell movement from the equator towards the lens sutures, thus in retrograde direction to regular epithelial cell differentiation. Key words: cataract – scanning electron microscopy – transmission electron microscopy – ultraviolet radiation
Acta Ophthalmol. 2014: 92: 635–643 ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
doi: 10.1111/aos.12376
Introduction The transparency of the crystalline lens depends on the regular and ordered
spacing of its cells and proteins. Disruption of this order results in local changes in the refractive index causing
light scattering or cataract. In the lens, the highly organized microarchitecture of lens fibres can be altered by protein aggregation, membrane degeneration, fluctuations of protein density and protein phase separation (Bettelheim 1985). Binding of high-molecularweight aggregates to cellular membranes has also been reported to cause light scattering in all forms of cataract (Tripathi & Tripathi 1983; Vrensen & Willekens 1990). Cataract is still a major health issue with estimated 20 million people blind from the disease (Resnikoff & Pararajasegaram 2001; BrØnsted et al. 2013). Even though cataract surgery cures the disease in industrialized countries, this therapy is by far not available to all patients those are affected worldwide. The national and international impact of the disease on health care budgets is enormous. In the United States alone, 1.3 million cataract procedures are performed annually accounting for 10% of the US annual health care budget or 4.1 billion US dollars (Busbee et al. 2003). This figure is expected to continuously grow in the future due to the demographic development of the world’s population (Taylor 2005). Thus, the need for novel strategies to prevent or slow down cataract progression in vivo becomes evident. An extensive body of evidence links the exposure of the eye to ultraviolet radiation type B (UVR-B) to cataract development in humans and animals (Jose & Pitts 1985; Taylor et al. 1988; Zigman et al. 1991; Klein et al. 1992;
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Schein et al. 1994; West et al. 1998). Proven most deleterious to the lens are wavelengths around 300 nm UVR-B (Pitts et al. 1977; Merriam et al. 2000). Damage to the lens by UVR-B exposure occurs via protein cross-linking, membrane damage and dysregulation of lens enzyme activity resulting in swelling and disruption of lens epithelial cells and cortical lens fibres (Breadsell et al. 1994). From the previous studies, we know that UVR-B-induced cataract at close to threshold exposure doses is characterized by distinct morphological patterns and repair mainly confined to the anterior subcapsular region and superficial cortex of the lens (Meyer et al. 2005). However, these observations were purely macroscopic and allow no definite conclusion on cellular processes in the lens epithelium and superficial lens fibres (Meyer et al. 2009). To further understand UVR-Binduced cataract morphology and epithelial repair mechanisms involved at a cellular level, we investigate here the ultrastructure of the mouse lens epithelium, lens fibres and lens fibre membranes with scanning and transmission electron microscopy techniques following double cataract threshold UVR-B exposure.
Materials and Methods Experimental animal
Six-week-old female C57BL/6 mice were obtained from Charles River Laboratories. The animals were kept and treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and to the Declaration of Helsinki. Ethical approval was obtained for all experiments from the Northern Stockholm Animal Experiments Committee. UVR-B source and exposure
UVR-B in the 300-nm wavelength region (UVR-B-300 nm) was generated with a high-pressure mercury lamp. The emerging radiation was collimated, passed through a water filter and a double monochromator (kMAX = 303 nm with 5 nm [FWHM]), and finally projected in a narrow beam on the cornea of the exposed eye (S€ oderberg et al. 1990; Michael et al. 1996). The
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diameter of the field on the cornea was 6 mm. The contralateral eye was shielded with a black paper shield during exposure. The intensity of UVR was measured with a thermopile that converts UVR-B radiant energy into voltage and allows for stable measurement conditions. The thermopile was calibrated to a National Institute of Standard (NIST) traceable source. The exposure system has been described in detail before (Meyer et al. 2013). Experimental procedure
One eye of each mouse was exposed in vivo to UVR. All animals were checked with a slit lamp prior to UVR exposure to exclude congenital cataract. The unexposed eye was kept as a control. Ten minutes preceding the exposure, the animal was anaesthetized with a mixture of 40 mg/kg ketamine and 5 mg/kg xylazine injected intramuscularly. Five minutes after the injection, 1 % tropicamide was instilled in both eyes to induce mydriasis. The mice were unilaterally exposed for 15 min to double threshold dose (6.4 kJ/m2) UVR-B for 15 min. The radiation output of the UVR-B source had k-MAX at 302.6 nm. After a latency period of 1, 2, 4 and 8 days following UVR-B exposure, the induced cataract was quantified as forward lens light scattering and visualized with electron microscopy techniques and in retro-illumination photography. These observation periods were chosen based on the earlier experimental data (Meyer et al. 2005) to allow for a comparison of both studies. Cataract quantification as lens light scattering
The intensity of forward lens light scattering was measured with a light dissemination meter (S€ oderberg et al. 1990). This instrument uses the principle of dark field illumination. The illuminating light transilluminates a transparent object (e.g. a mouse lens) at 45° against the horizontal plane. At this angle, the light cannot enter the objective aperture. If the object scatters light in the forward direction, a defined fraction of light reaches the objective and is measured by a photodiode. The scattering standard was a lipid emulsion of Diazepam (Stesolid Novum, Dumex-Alphapharma, Den-
mark). Light scattering was therefore expressed in transformed equivalent Diazepam concentration [tEDC] (S€ oderberg et al. 1990). Light microscopy
Damage to the lens epithelium and the anterior cortex was investigated with light microscopy in toluidine bluestained semi-thin sections. Lenses of all time intervals were fixed in 0.08 M cacodylate-buffered glutaraldehyde (1.25%)–paraformaldehyde (1%) solution (ph 7.3) for at least 7 days at 8°C (Vrensen et al. 1995). Each lens was postfixed in a 0.1-M cacodylate-buffered solution of 1% osmium tetroxide supplemented with 1.5% potassium ferricyanide for 1 hr at 4°C and thereafter dehydrated in a graded series of ethanol up to 100% and embedded in epoxy resin. Semi-thin sections of the embedded lenses were stained with 1% toluidine blue for light microscopy. Dark field illumination photography
Cataract morphology was documented with a microscope camera system. Via a dissection microscope (Zeiss), high-resolution photographs were taken at different magnifications from control lenses and exposed lenses in incident illumination and in dark filed illumination. Scanning electron microscopy
Exposed and non-exposed lenses of three lenses at each studied time interval were fixed in 0.08 M cacodylate-buffered glutaraldehyde (1.25%)–paraformaldehyde (1%) solution (ph 7.3) for at least 7 days at 8°C (Vrensen et al. 1995). Lenses were dissected for scanning electron microscopy (SEM) by cutting them into two halves. The capsule was partially removed to expose lens epithelium. The lens cortex and nucleus was prepared to study lens fibres at different depths. The dissected pieces were dehydrated in a graded series of ethanols and dried by immersion for 20 min in hexamethyldisilazane (H 4875; Sigma Chemical, St. Louis, MO), followed by drying for 8 hr on filter paper. The pieces were mounted with carbon glue and sputter-coated with gold and studied in a scanning electron microscope (Zeiss, Ultra 55, Karolinska Instituet, Core Facility).
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Samples of 4 lenses per group were fixed for transmission electron microscopy (TEM) in 0.08 M cacodylate-buffered solution of 1.25% glutaraldehyde and 1% paraformaldehyde (ph 7.3) for 7 days at 4°C. Each sample was postfixed in 0.1-M cacodylate-buffered solution and 1% osmium tetroxide supplemented with 1.5% potassium ferricyanide for 1 hr at 4°C and thereafter dehydrated in a graded series of ethanol and finally embedded in epoxy resin. Ultrathin sections were mounted on copper grids. The section were double stained in 3.5% uranyl acetate for 20 min at room temperature, followed by Reynold’s lead citrate for 10 min at room temperature. The grids then were examined in a Tecnai G2 Bio Twin Spirit (FEI Company, USA) transmission electron microscope.
Lens light scattering (tEDC)
Transmission electron microscopy
EXPO CONTROL
0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0
1
2
4
8
Days after UVR-B exposure
Fig. 1. Mean forward lens light scattering 1, 2, 4 and 8 days following exposure to double cataract threshold dose UVR-B (6.5 kJ/m2). Bar is 95% confidence interval for the mean.
Statistical parameters
The significance levels were set to 0.05 and the confidence coefficients to 0.95 considering the sample size. Light scattering difference between exposed and non-exposed lenses was analysed with 95% confidence intervals for mean lens light scattering.
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Results Cataract quantified as lens light scattering
Exposed lenses scattered light significantly higher at all postexposure intervals than non-exposed lenses. The difference was significant at 2 and 4 days post-UVR-B exposure (Fig. 1). Cataract morphology
Predominantly, anterior subcapsular cataract with characteristic morphological features developed after the double threshold UVR-B exposure (Fig. 2). As soon as 1 day following the exposure, fine granular opacities were visible throughout the whole anterior surface of the lens but not reaching to the equator. At 2 days, these opacities organized in an annulus shaped demarcation line (Fig. 2B). Between days 2– 4, this opaque annulus shrank towards the centre and moved concentrically towards the anterior lens pole. At day 8, a dense triangular-shaped opacity
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Fig. 2. Cataract morphology and repair in vivo after double cataract threshold UVR-300-nm exposure in dark filed illumination photography. Anterior subcapsular opacities with granular appearance resemble epithelial damage. (A) One day after exposure, granular opacities are spread out equally over the anterior surface of the lens but do not reach the equator. At 2 days, the rim of the opaque area organizes in an annulus (B) that continuously shrinks towards the anterior lens pole between day 2 and 4 (C). At day 8, a triangular-shaped subcapsular opacity has formed at the anterior lens sutures (D). Control lenses are clear (E). Viewed from the side, the anterior pole is thickened and slightly elevates the anterior lens capsule (F arrow).
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was left close to the lens sutures leaving the mid-periphery of the lens visibly clear (Fig. 2D). Looking at the anterior lens pole from the side, the central opacity displayed a thickened appearance slightly elevating the (A)
(B)
anterior lens capsule above (Fig. 2F). Non-exposed lenses were clear as seen under the microscope (Fig. 2E). A schematic drawing illustrates the observed movement and repair of central epithelial cells towards the lens sutures following in vivo UVR-B damage (Fig. 3) in the sagittal axis. Three out of 20 animals developed additionally to the anterior subcapsular opacities cortical and cortico-
Fig. 3. Schematic drawing of epithelial cell repair and central movement following in vivo UVR-B exposure in sagittal section. Epithelial cells with regular shape in unexposed lens (A). One day after UVR-B exposure, epithelial cells are damaged severely and are partially missing (B). Two days after exposure, epithelial cells in the equatorial region start to elongate towards the sutures (C). At day 4, epithelial cells migrate towards the lens sutures and phagocytose damaged and apoptotic cells on their way (D). Eight days after UVR-B damage, the mid-periphery of the lens epithelium is repopulated by normal epithelial cells. Remaining cell debris is concentrated to the centre of the anterior epithelium in a multilayered layer (E).
nuclear cataract (Figs 4A and 5A) already 1 day after UVR-B exposure. That type of cataract was not subject to macroscopic visible changes until day 8 after exposure. An overview over the evolution of different cataract morphologies is given in Table 1. Scanning electron microscopy
In sagittal sections, the non-exposed lenses had a normal capsule, epithelium and ordered alignment of fibre cells (Figs 6 and 7A). Superficial cortical lens fibres and deeper cortical as well as nuclear fibres displayed intact interdigitations and ball and socket joints (Fig. 7). Following UVR-B exposure, the damaged epithelial cells showed an irregular shape and a flake-like appearance (Fig. 7B). Adjacent superficial cortical fibres are swollen and fibre-to-fibre interdigitations are damaged (Fig. 7B, arrow). Lenses with anterior subcapsular plus cortical cataract revealed in SEM
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Fig. 4. Cortical cataract 1 day after in vivo exposure to double threshold dose UVR-B 300 nm. Exposed lens in retroillumination photography (A). Cortical fibers of cataractous lens with membrane damage and destroyed microarchitecture of ball and socket joints, and membrane interdigitations in SEM imaging (B–D). Scale bar 10 lm (B) 20 lm and 4 lm (D). Blue rectangle resembles localization of section in sagittal view.
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(A)
(B)
(C)
(D)
Fig. 5. Corticonuclear cataract 1 day after in vivo exposure to double threshold dose UVR-B 300 nm. Exposed lens in retroillumination photography (A). View on exposed lens from anterior (B). Beneath the capsule are damaged epithelial cells (asterix) and destroyed superficial cortical fibres (arrow). Damaged fibres in higher magnification: deep cortical (C) and superficial cortical fibres (D) with vacuolar structures. Scale bar 20 lm (B) 10 lm (C) and 5 lm (D). Blue rectangle resembles localization of section in sagittal view. Table 1. Morphological development of cataract type during the observation period. Cataract morphology Time
Anterior subcapsular, n
+Cortical, n
+Corticonuclear, n
Day Day Day Day
17 17 17 17
1 1 1 1
2 2 2 2
1 2 4 8
n = Number of animals.
pronounced swelling of superficial and deep cortical fibres (Fig. 4B,C,D) already 1 day following UVR-B exposure. Ball and socket joints are partially destroyed, and large cavities are visible between lens fibres (Fig. 4D). In lenses with nuclear cataract (Fig. 5A), the fibre microarchitecture is severely damaged (Fig. 5B–D). Beneath the epithelial layer (Fig. 5 asterix), superficial cortical fibres are massively swollen and have lost their regular ultrastructure (Fig. 5B, arrow). Approximately up to 300 lm beneath the epithelium giant vacuoles and irregular cell debris
have replaced any normal lens fibre anatomy (Fig. 5D). Deeper down towards nuclear lens fibres cells, swollen fibre cells can be distinguished again but their cell-to-cell contacts and membrane interdigitations are clearly altered (Fig. 5C). The lens capsule appeared regular with no visible damage in all described cataract forms. Transmission electron microscopy
Already 1 day following double cataract threshold UVR-B exposure, the central epithelium had lost epithelial
cells as confirmed in semi-thin sections stained with toluidine blue (Fig. 8A, arrows). Remaining central epithelial cells underwent karyopyknosis (Fig. 8A, triangle). Cells at the nuclear bow appeared unaffected (Fig. 8B). The macroscopically visible annular demarcation line that evolved at the rim of damaged epithelial cells 2 days after exposure was build up as the junction line between regenerating epithelial cells moving in from the equator and apoptotic epithelial cells with swollen cytoplasms and vacuoles in the central epithelium (Fig. 8C, arrow indicates direction of migration). These apoptotic cells were phagocytosed and removed by pushing them towards the lens sutures (Fig. 8D). Healthy epithelial cells in the pre-equatorial region of the lens elongated and stretched out to fill the gap and by this lost their cuboidal appearance (Fig. 8E). Control lenses at days 1, 2, 4 and 8 displayed a regular epithelial pattern in light microscopy as well as in TEM imaging (Fig. 8F,G) at the anterior pole as well as in the nuclear bow. Eight days after UVR-B exposure, the cell debris was pushed towards the lens sutures, thus ending in a triangular pattern at the anterior lens pole (Fig. 9). Here, the epithelial cells were stacked in a multilayered fashion and displayed chromatin condensation, membrane infolds, multiple phagocytosed apoptotic bodies and signs of karyopyknosis (Fig. 9A–D). Cortical fibres just below the multilayered central epithelium were irregular in shape with multiple vacuoles (Fig. 9A,B) but could be clearly demarcated from the regenerating epithelium.
Discussion Lens exposure to double cataract threshold UVR-B dose (Meyer et al. 2008) induces macroscopically mainly anterior subcapsular but also cortical and nuclear cataract in the mouse lens. However, the ultrastructure of these different types of cataract on a cellular level has not been studied before. Here, we present insight into the ultrastructure of the lens epithelium, cortex, nucleus, and into lens repair after UVR-B-induced damage with TEM and SEM techniques.
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(A)
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Fig. 6. Ultrastructure of unexposed mouse lens in SEM imaging. Superficial (A, B) and deep cortical fibres (C, D) display ball and socket joints, and membrane interdigitations. Scale bar is 8 lm (A, B, C) and 2 lm (D). Blue rectangle resembles localization of section in sagittal view.
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Fig. 7. SEM image of anterior polar region of non-exposed lens (A) with regular capsule, epithelium and superficial cortex. One day following UVR-B exposure, the epithelial cells appear flake-like and flattened out. Adjacent fibre cells lying underneath have a swollen appearance (B). Scale bar is 10 lm (A) and 20 lm (B). Blue rectangle resembles localization of section in sagittal view.
Lens epithelium
The macroscopically flake-like opacities that we observed 1 day after
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exposure on the lens surface resemble in our opinion damaged epithelial cells that, due to swelling of their cytoplasm, chromatin condensation and early
apoptosis, give rise to light scattering. Galichanin and co-workers described signs of apoptosis already one hour, visible cataract 7 hr after UVR-B exposure in a study with rat lenses (Galichanin et al. 2010). In vitro studies with cultured lens epithelial cells confirm early apoptosis with TUNEL labelling and up-regulation of p53 and caspase 3 expression on mRNA and protein levels that can last up to 1 week after UVR-B exposure (Michael et al. 1998; Ayala et al. 2007). In our study, the earliest observation point was 24 hr following exposure. Similar to previous findings (Meyer et al. 2005), lens light scattering in the mouse lens peaks at 2 days postUVR-B exposure. Thus, the observed anterior subcapsular cataract is in line with previous findings. However, we can demonstrate that epithelial damage is repaired by cell migration from the lens equator towards the anterior pole and lens sutures. It is believed that epithelial cells of the normal epithelial monolayer are derived from daughter cells in the germinative zone at the lens equator (Rafferty & Rafferty 1981). It was demonstrated already in 1988 that lens epithelial cells contain actin and are capable of undergoing structural modifications for example in a setting of experimental epithelial injury (Liou & Rafferty 1988). In their model, the authors describe flattening of epithelial cells and extension of epithelial cell processes into induced wound spaces. Furthermore, they report amplified membrane folding in the rest of the epithelium following local epithelial damage. Epithelial cells that loose contact to the adjacent cell are stimulated to proliferate into the direction of cellular loss (Yamada et al. 2001). The elongated epithelial cells in the outer periphery as well as the demonstrated central migration of healthy epithelium reported in our study are both findings that support the regenerative capacity of lens epithelial cells in vivo. We confirm that lens epithelial cells are capable of repairing UVR-B-induced damage within 8 days via cell migration from the equator towards the anterior pole and by cleaning damaged cells on the way. The intact lens capsule might function in this context as a guiding structure for cellular repopulation of areas with epithelial damage. The described triangular shape of the remaining cataract at
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Fig. 8. TEM images and semi-thin sections stained with 1% tolouidine blue of anterior subcapsular cataract following in vivo exposure to double cataract threshold dose UVR-B 300 nm at different postexposure intervals. Epithelial cells are lost 1 day postexposure (A, arrows) Clearly visible is the debridement of the central epithelium. Arrowhead indicates apoptotic epithelial cell with karyopyknosis. The nuclear bow and the germinative zone are unaffected (B). Two days after exposure, epithelial cells with swollen cytoplasms are found at the rim of the cataractous area (C). Arrowhead indicates the direction of repopulation. The basal cell membrane is ruptured (C, TEM, arrow) and apoptotic bodies are found (C, asterix). Four days after exposure (D), epithelial damage is removed by cell migration from equator (arrow equals the direction of migration). Epithelial cells in pre-equatorial region are elongated (E). Control lenses at days 1, 2, 4 and 8 show regular epithelial cells in the anterior polar region in light microscopy and TEM (F) as well as in the equatorial zone (G). Scale bar is 20 lm (semi-thin sections) and 2 lm (TEM).
day eight is in our opinion due to the microanatomy of the lens sutures that are Y- and inverted Y-shaped in the mouse lens.
Of interest is the question how the multilayered epithelial cells that we observed at the anterior lens sutures 8 days after UVR-B exposure will
develop after a longer observation time. We hypothesize that over a time of a few weeks, those cell stacks will undergo apoptosis and phagocytosis
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(A)
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(C1)
(C2)
(C3)
layers as an indirect damage from UVR-B exposure. In most exposed lenses, only the lens epithelium and outer cortical fibres were damaged but in some lenses, severe structural alterations in deeper cortical layers and the nucleus were present additionally to anterior subcapsular cataract (Figs 4 and 5). This observation points to a variation in interindividual UVR-B sensitivity, and we hypothesize that if lens epithelial damage exceeds the individual repair capacity, severe structural alterations occur via the abovedescribed pathways also in deeper lens fibres that then induces cortical and/or nuclear cataract.
Conclusion (D1)
(D3)
(D4)
(D2)
(D5)
(D6)
Fig. 9. TEM images and semi-thin sections stained with 1% toluidine blue of anterior subcapsular cataract following in vivo exposure to double cataract threshold dose UVR-B 300 nm. Eight days after exposure, the epithelium has recovered in the periphery and is multilayered around the anterior pole in an area of visible cataract (A). Small arrowhead indicates the direction of migration, big arrowhead shows the marginal zone of the single-layered and the multilayered epithelium. Fib = fibers. In the outer periphery, the monolayered epithelium is flattened out (B). Arrow indicates the direction of migration. (C1–C3) Around the lens sutures, in a triangular pattern, a multilayered epithelium has formed with phagocytosed cells, cell debris and multiple apoptotic bodies. The origin here is clearly epithelial (arrowheads). In TEM (D1–D6), the multilayered stack of epithelial cells (nuclei marked with black stars, D2, D3, D6) displays multiple membrane infolds, phagosomes and apoptotic bodies (white stars). In D4 and D5, the leading edge of epithelial cells is captured (white arrowheads) with membrane infolds and phagocytosis of apoptotic bodies and karyopyknosis. Scale bar is 2 lm. Cap = capsule, Fib = fibres.
and only a single-layered epithelium will prevail. However, further investigations are necessary to confirm our hypothesis. Lens fibers
Radiant energy from UVR-B 300 nm penetrates approximately 450 lm into the lens and is absorbed completely by the lens epithelium and superficial cortical fibre cells (S€ oderberg et al. 1998). In the epithelium, the result is an impaired metabolic activity and an inactivation of enzymes such as lactate
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dehydrogenase in the outer cortex (Chen et al. 1989; L€ ofgren & S€ oderberg 1995) Due to a disturbed osmoregulation in the lens epithelium, the homeostasis of underlying cortical fibres may also be affected as evidenced by extracellular spaces and vacuoles that were present from 2 days postexposure onwards (Figs 8C,D and 9A,B). Via cell-to-cell communication such as fibre membrane fusions and macromolecular pathways (Shestopalov and Bassnett 2000), the homeostatic imbalance of outer cortical fibres leads to structural alterations in deeper cortical
UVR-B-induced cataract in the mouse lens at double threshold dose is morphologically confined to the anterior third of the lens. Depending on the interindividual sensitivity, damage may also occur in deeper cortical layers and the nucleus. The lens epithelium is equipped with a high capacity of cell repair and migration. We found indications that after a subtotal loss of epithelial cells, this UVR-B-induced damage to the epithelium is repaired by epithelial cell movement from the equator towards the lens sutures, thus in retrograde direction to regular epithelial cell differentiation. However, further research is needed to visualize these dynamic cellular processes in the lens epithelium.
Acknowledgements The authors thank Monika Aronsson for keeping animals and researchers in excellent shape and mood, Margareta Oskarsson and Berit Spangberg for embedding lenses for TEM and SEM, and Margaret Gondo for help with electron microscopy imaging.
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Meyer LM, L€ ofgren S, Ho YS, Lou M, Wegener A, Holz F & S€ oderberg P (2009): Absence of glutaredoxin1 increases lens susceptibility to oxidative stress induced by UVR-B. Exp Eye Res 89: 833–839. Meyer LM, L€ ofgren S, Holz FG, Wegener A & S€ oderberg P (2013): Bilateral cataract induced by unilateral UVR-B exposure – evidence for an inflammatory response. Acta Opthalmol 91: 236–242. Michael R, S€ oderberg PG & Chen E (1996): Long-term development of lens opacities after exposure to ultraviolet radiation at 300 nm. Ophthalmic Res 28: 209–218. Michael R, Vrensen G, van Marle J, Gan L & S€ oderberg PG (1998): Apoptosis in the rat lens after in vivo threshold dose ultraviolet radiation. Invest Ophthalmol Vis Sci 13: 2681–2687. Pitts DG, Cullen AP & Hacker PD (1977): Ocular effects of ultraviolet radiation from 295 to 365 nm. Invest Ophthalmol Vis Sci 16: 932–939. Rafferty NS & Rafferty KI Jr (1981): Cell population kinetics of the mouse lens epithelium. J Cell Physiol 107: 309–315. Resnikoff S & Pararajasegaram R (2001): lindness prevention programmes: past, present, and future. Bull World Health Organ 79: 222–226. Schein OD, West S, Mun˜oz B, Vitale S, Maguire M, Taylor HR & Bressler NM (1994): Cortical lenticular opacification: distribution and location in a longitudinal study. Invest Ophthalmol Vis Sci 35: 363–366. Shestapalov VI & Bassnett S (2000): Threedimensional organization of primary lens fibre cells. Invest Ophthalmol Vis Sci 41: 859–863. S€ oderberg PG, Chen E & Lindstr€ om B (1990): An objective and rapid method for the determination of light dissemination in the lens. Acta Ophthalmol (Copenhagen) 68: 44–52. S€ oderberg PG, L€ ofgren S, Michael R & Gonzalez-Cirre X (1998): New method for measurement of in vivo penetration of UVR into the crystalline lens. SPIE 3246: 43–47. Taylor H (2005): Cataract: how much surgery do we have to do? Br J Ophthalmol 84: 1–2.
Taylor HR, West SK, Rosenthal FS, Mun˜oz B, Newland HS, Abbey H & Emmett EA (1988): Effect of ultraviolet radiation on cataract formation. N Engl J Med 319: 1429–1433. Tripathi R & Tripathi B (1983): Morphology of the normal, aging, and cataractous human lens.II. Optical zones of discontinuity and senile cataract. Lens Res 1: 43. Vrensen G & Willekens B (1990): Biomicroscopy and scanning electron microscopy of early opacities in the aging human lens. Invest Ophthalmol Vis Sci 31: 582–587. Vrensen GFJM, Sanderson J, Willekens B & Duncan G (1995): Calcium localization and ultrastructure of clear and pCMPS-treated rat lenses. Invest Ophthalmol Vis Sci 36: 2287–2295. West SK, Duncan DD, Mun˜oz B, Rubin GS, Fried LP, Bandeen-Roche K & Schein OD (1998): Sunlight exposure and risk of lens opacities in a population-based study: the Salisbury Eye Evaluation project. JAMA 280: 714–718. Yamada Y, Kojima M, Vrensen GF, Takahashi N & Sasaki K (2001): Acute ultraviolet B induced lens epithelial cell photo-damage and its repair process. Nihon Ganka Gakkai Zasshi 105: 102–110. Zigman S, Paxhia T, McDaniel T, Lou MF & Yu NT (1991): Effect of chronic near-ultraviolet radiation on the gray squirrel lens in vivo. Invest Ophthalmol Vis Sci 32: 1723– 1732.
Received on October 28th, 2013. Accepted on January 29th, 2014. Correspondence: Linda M. Meyer, MD, PhD Herzog Carl Theodor Eye Clinic Nymphenburger Strasse 43 80335 Munich Germany Tel: +49-89-1270930 Fax: +49-89-1290341 Email: [email protected]
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Ultrastructure of UVR-B-induced cataract and repair visualized with electron microscopy Linda M. Meyer,1 Alfred R. Wegener,2 Frank G. Holz,2 Martin Kronschl€ager,3 Jan P. Bergmanson4 and Per G. Soderberg3 1
Herzog Carl Theodor Eye Clinic, Munich, Germany University Eye Clinic Bonn, Bonn, Germany 3 Department of Neuroscience, Uppsala University, Uppsala, Sweden 4 University of Houston, College of Optometry, Houston, TX, USA 2
ABSTRACT. Purpose: The aim of the study is to investigate and visualize the ultrastructure of cataract morphology and repair, after in vivo exposure to double threshold dose UVR-B in the C57BL/6 mouse lens. Methods: Twenty-six-week-old C57BL/6 mice received in vivo double threshold dose (6.4 kJ/m2) UVR-B for 15 min. The radiation output of the UVR-source had kMAX at 302.6 nm. After a latency period of 1, 2, 4 and 8 days following UVR-B exposure, the induced cataract was visualized with electron microscopy techniques. Induced, cataract was quantified as forward lens light scattering. Damage to the lens epithelium and the anterior cortex was investigated with light microscopy in toluidine blue-stained semi-thin sections, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dark field illumination photography. Results: UVR-B-exposed lenses developed anterior subcapsular and/or cortical and nuclear cataract after 1 day. Lens light scattering peaked 2 days after exposure. Lens epithelial cell damage was seen in TEM as apoptotic cells, apoptotic bodies, nuclear chromatin condensation, and swollen and disrupted anterior cortex fibres throughout the sections of the whole anterior lens surface. These morphologic changes were also visualized with SEM. Within 8 days, anterior subcapsular cataract was repaired towards the anterior sutures. Conclusion: UVR-B exposure of double cataract threshold dose induces a subtotal loss of epithelial cells across the whole anterior surface of the lens. This damage to the epithelium is repaired by epithelial cell movement from the equator towards the lens sutures, thus in retrograde direction to regular epithelial cell differentiation. Key words: cataract – scanning electron microscopy – transmission electron microscopy – ultraviolet radiation
Acta Ophthalmol. 2014: 92: 635–643 ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
doi: 10.1111/aos.12376
Introduction The transparency of the crystalline lens depends on the regular and ordered
spacing of its cells and proteins. Disruption of this order results in local changes in the refractive index causing
light scattering or cataract. In the lens, the highly organized microarchitecture of lens fibres can be altered by protein aggregation, membrane degeneration, fluctuations of protein density and protein phase separation (Bettelheim 1985). Binding of high-molecularweight aggregates to cellular membranes has also been reported to cause light scattering in all forms of cataract (Tripathi & Tripathi 1983; Vrensen & Willekens 1990). Cataract is still a major health issue with estimated 20 million people blind from the disease (Resnikoff & Pararajasegaram 2001; BrØnsted et al. 2013). Even though cataract surgery cures the disease in industrialized countries, this therapy is by far not available to all patients those are affected worldwide. The national and international impact of the disease on health care budgets is enormous. In the United States alone, 1.3 million cataract procedures are performed annually accounting for 10% of the US annual health care budget or 4.1 billion US dollars (Busbee et al. 2003). This figure is expected to continuously grow in the future due to the demographic development of the world’s population (Taylor 2005). Thus, the need for novel strategies to prevent or slow down cataract progression in vivo becomes evident. An extensive body of evidence links the exposure of the eye to ultraviolet radiation type B (UVR-B) to cataract development in humans and animals (Jose & Pitts 1985; Taylor et al. 1988; Zigman et al. 1991; Klein et al. 1992;
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Schein et al. 1994; West et al. 1998). Proven most deleterious to the lens are wavelengths around 300 nm UVR-B (Pitts et al. 1977; Merriam et al. 2000). Damage to the lens by UVR-B exposure occurs via protein cross-linking, membrane damage and dysregulation of lens enzyme activity resulting in swelling and disruption of lens epithelial cells and cortical lens fibres (Breadsell et al. 1994). From the previous studies, we know that UVR-B-induced cataract at close to threshold exposure doses is characterized by distinct morphological patterns and repair mainly confined to the anterior subcapsular region and superficial cortex of the lens (Meyer et al. 2005). However, these observations were purely macroscopic and allow no definite conclusion on cellular processes in the lens epithelium and superficial lens fibres (Meyer et al. 2009). To further understand UVR-Binduced cataract morphology and epithelial repair mechanisms involved at a cellular level, we investigate here the ultrastructure of the mouse lens epithelium, lens fibres and lens fibre membranes with scanning and transmission electron microscopy techniques following double cataract threshold UVR-B exposure.
Materials and Methods Experimental animal
Six-week-old female C57BL/6 mice were obtained from Charles River Laboratories. The animals were kept and treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and to the Declaration of Helsinki. Ethical approval was obtained for all experiments from the Northern Stockholm Animal Experiments Committee. UVR-B source and exposure
UVR-B in the 300-nm wavelength region (UVR-B-300 nm) was generated with a high-pressure mercury lamp. The emerging radiation was collimated, passed through a water filter and a double monochromator (kMAX = 303 nm with 5 nm [FWHM]), and finally projected in a narrow beam on the cornea of the exposed eye (S€ oderberg et al. 1990; Michael et al. 1996). The
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diameter of the field on the cornea was 6 mm. The contralateral eye was shielded with a black paper shield during exposure. The intensity of UVR was measured with a thermopile that converts UVR-B radiant energy into voltage and allows for stable measurement conditions. The thermopile was calibrated to a National Institute of Standard (NIST) traceable source. The exposure system has been described in detail before (Meyer et al. 2013). Experimental procedure
One eye of each mouse was exposed in vivo to UVR. All animals were checked with a slit lamp prior to UVR exposure to exclude congenital cataract. The unexposed eye was kept as a control. Ten minutes preceding the exposure, the animal was anaesthetized with a mixture of 40 mg/kg ketamine and 5 mg/kg xylazine injected intramuscularly. Five minutes after the injection, 1 % tropicamide was instilled in both eyes to induce mydriasis. The mice were unilaterally exposed for 15 min to double threshold dose (6.4 kJ/m2) UVR-B for 15 min. The radiation output of the UVR-B source had k-MAX at 302.6 nm. After a latency period of 1, 2, 4 and 8 days following UVR-B exposure, the induced cataract was quantified as forward lens light scattering and visualized with electron microscopy techniques and in retro-illumination photography. These observation periods were chosen based on the earlier experimental data (Meyer et al. 2005) to allow for a comparison of both studies. Cataract quantification as lens light scattering
The intensity of forward lens light scattering was measured with a light dissemination meter (S€ oderberg et al. 1990). This instrument uses the principle of dark field illumination. The illuminating light transilluminates a transparent object (e.g. a mouse lens) at 45° against the horizontal plane. At this angle, the light cannot enter the objective aperture. If the object scatters light in the forward direction, a defined fraction of light reaches the objective and is measured by a photodiode. The scattering standard was a lipid emulsion of Diazepam (Stesolid Novum, Dumex-Alphapharma, Den-
mark). Light scattering was therefore expressed in transformed equivalent Diazepam concentration [tEDC] (S€ oderberg et al. 1990). Light microscopy
Damage to the lens epithelium and the anterior cortex was investigated with light microscopy in toluidine bluestained semi-thin sections. Lenses of all time intervals were fixed in 0.08 M cacodylate-buffered glutaraldehyde (1.25%)–paraformaldehyde (1%) solution (ph 7.3) for at least 7 days at 8°C (Vrensen et al. 1995). Each lens was postfixed in a 0.1-M cacodylate-buffered solution of 1% osmium tetroxide supplemented with 1.5% potassium ferricyanide for 1 hr at 4°C and thereafter dehydrated in a graded series of ethanol up to 100% and embedded in epoxy resin. Semi-thin sections of the embedded lenses were stained with 1% toluidine blue for light microscopy. Dark field illumination photography
Cataract morphology was documented with a microscope camera system. Via a dissection microscope (Zeiss), high-resolution photographs were taken at different magnifications from control lenses and exposed lenses in incident illumination and in dark filed illumination. Scanning electron microscopy
Exposed and non-exposed lenses of three lenses at each studied time interval were fixed in 0.08 M cacodylate-buffered glutaraldehyde (1.25%)–paraformaldehyde (1%) solution (ph 7.3) for at least 7 days at 8°C (Vrensen et al. 1995). Lenses were dissected for scanning electron microscopy (SEM) by cutting them into two halves. The capsule was partially removed to expose lens epithelium. The lens cortex and nucleus was prepared to study lens fibres at different depths. The dissected pieces were dehydrated in a graded series of ethanols and dried by immersion for 20 min in hexamethyldisilazane (H 4875; Sigma Chemical, St. Louis, MO), followed by drying for 8 hr on filter paper. The pieces were mounted with carbon glue and sputter-coated with gold and studied in a scanning electron microscope (Zeiss, Ultra 55, Karolinska Instituet, Core Facility).
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Samples of 4 lenses per group were fixed for transmission electron microscopy (TEM) in 0.08 M cacodylate-buffered solution of 1.25% glutaraldehyde and 1% paraformaldehyde (ph 7.3) for 7 days at 4°C. Each sample was postfixed in 0.1-M cacodylate-buffered solution and 1% osmium tetroxide supplemented with 1.5% potassium ferricyanide for 1 hr at 4°C and thereafter dehydrated in a graded series of ethanol and finally embedded in epoxy resin. Ultrathin sections were mounted on copper grids. The section were double stained in 3.5% uranyl acetate for 20 min at room temperature, followed by Reynold’s lead citrate for 10 min at room temperature. The grids then were examined in a Tecnai G2 Bio Twin Spirit (FEI Company, USA) transmission electron microscope.
Lens light scattering (tEDC)
Transmission electron microscopy
EXPO CONTROL
0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0
1
2
4
8
Days after UVR-B exposure
Fig. 1. Mean forward lens light scattering 1, 2, 4 and 8 days following exposure to double cataract threshold dose UVR-B (6.5 kJ/m2). Bar is 95% confidence interval for the mean.
Statistical parameters
The significance levels were set to 0.05 and the confidence coefficients to 0.95 considering the sample size. Light scattering difference between exposed and non-exposed lenses was analysed with 95% confidence intervals for mean lens light scattering.
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Results Cataract quantified as lens light scattering
Exposed lenses scattered light significantly higher at all postexposure intervals than non-exposed lenses. The difference was significant at 2 and 4 days post-UVR-B exposure (Fig. 1). Cataract morphology
Predominantly, anterior subcapsular cataract with characteristic morphological features developed after the double threshold UVR-B exposure (Fig. 2). As soon as 1 day following the exposure, fine granular opacities were visible throughout the whole anterior surface of the lens but not reaching to the equator. At 2 days, these opacities organized in an annulus shaped demarcation line (Fig. 2B). Between days 2– 4, this opaque annulus shrank towards the centre and moved concentrically towards the anterior lens pole. At day 8, a dense triangular-shaped opacity
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Fig. 2. Cataract morphology and repair in vivo after double cataract threshold UVR-300-nm exposure in dark filed illumination photography. Anterior subcapsular opacities with granular appearance resemble epithelial damage. (A) One day after exposure, granular opacities are spread out equally over the anterior surface of the lens but do not reach the equator. At 2 days, the rim of the opaque area organizes in an annulus (B) that continuously shrinks towards the anterior lens pole between day 2 and 4 (C). At day 8, a triangular-shaped subcapsular opacity has formed at the anterior lens sutures (D). Control lenses are clear (E). Viewed from the side, the anterior pole is thickened and slightly elevates the anterior lens capsule (F arrow).
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was left close to the lens sutures leaving the mid-periphery of the lens visibly clear (Fig. 2D). Looking at the anterior lens pole from the side, the central opacity displayed a thickened appearance slightly elevating the (A)
(B)
anterior lens capsule above (Fig. 2F). Non-exposed lenses were clear as seen under the microscope (Fig. 2E). A schematic drawing illustrates the observed movement and repair of central epithelial cells towards the lens sutures following in vivo UVR-B damage (Fig. 3) in the sagittal axis. Three out of 20 animals developed additionally to the anterior subcapsular opacities cortical and cortico-
Fig. 3. Schematic drawing of epithelial cell repair and central movement following in vivo UVR-B exposure in sagittal section. Epithelial cells with regular shape in unexposed lens (A). One day after UVR-B exposure, epithelial cells are damaged severely and are partially missing (B). Two days after exposure, epithelial cells in the equatorial region start to elongate towards the sutures (C). At day 4, epithelial cells migrate towards the lens sutures and phagocytose damaged and apoptotic cells on their way (D). Eight days after UVR-B damage, the mid-periphery of the lens epithelium is repopulated by normal epithelial cells. Remaining cell debris is concentrated to the centre of the anterior epithelium in a multilayered layer (E).
nuclear cataract (Figs 4A and 5A) already 1 day after UVR-B exposure. That type of cataract was not subject to macroscopic visible changes until day 8 after exposure. An overview over the evolution of different cataract morphologies is given in Table 1. Scanning electron microscopy
In sagittal sections, the non-exposed lenses had a normal capsule, epithelium and ordered alignment of fibre cells (Figs 6 and 7A). Superficial cortical lens fibres and deeper cortical as well as nuclear fibres displayed intact interdigitations and ball and socket joints (Fig. 7). Following UVR-B exposure, the damaged epithelial cells showed an irregular shape and a flake-like appearance (Fig. 7B). Adjacent superficial cortical fibres are swollen and fibre-to-fibre interdigitations are damaged (Fig. 7B, arrow). Lenses with anterior subcapsular plus cortical cataract revealed in SEM
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(D)
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Fig. 4. Cortical cataract 1 day after in vivo exposure to double threshold dose UVR-B 300 nm. Exposed lens in retroillumination photography (A). Cortical fibers of cataractous lens with membrane damage and destroyed microarchitecture of ball and socket joints, and membrane interdigitations in SEM imaging (B–D). Scale bar 10 lm (B) 20 lm and 4 lm (D). Blue rectangle resembles localization of section in sagittal view.
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(A)
(B)
(C)
(D)
Fig. 5. Corticonuclear cataract 1 day after in vivo exposure to double threshold dose UVR-B 300 nm. Exposed lens in retroillumination photography (A). View on exposed lens from anterior (B). Beneath the capsule are damaged epithelial cells (asterix) and destroyed superficial cortical fibres (arrow). Damaged fibres in higher magnification: deep cortical (C) and superficial cortical fibres (D) with vacuolar structures. Scale bar 20 lm (B) 10 lm (C) and 5 lm (D). Blue rectangle resembles localization of section in sagittal view. Table 1. Morphological development of cataract type during the observation period. Cataract morphology Time
Anterior subcapsular, n
+Cortical, n
+Corticonuclear, n
Day Day Day Day
17 17 17 17
1 1 1 1
2 2 2 2
1 2 4 8
n = Number of animals.
pronounced swelling of superficial and deep cortical fibres (Fig. 4B,C,D) already 1 day following UVR-B exposure. Ball and socket joints are partially destroyed, and large cavities are visible between lens fibres (Fig. 4D). In lenses with nuclear cataract (Fig. 5A), the fibre microarchitecture is severely damaged (Fig. 5B–D). Beneath the epithelial layer (Fig. 5 asterix), superficial cortical fibres are massively swollen and have lost their regular ultrastructure (Fig. 5B, arrow). Approximately up to 300 lm beneath the epithelium giant vacuoles and irregular cell debris
have replaced any normal lens fibre anatomy (Fig. 5D). Deeper down towards nuclear lens fibres cells, swollen fibre cells can be distinguished again but their cell-to-cell contacts and membrane interdigitations are clearly altered (Fig. 5C). The lens capsule appeared regular with no visible damage in all described cataract forms. Transmission electron microscopy
Already 1 day following double cataract threshold UVR-B exposure, the central epithelium had lost epithelial
cells as confirmed in semi-thin sections stained with toluidine blue (Fig. 8A, arrows). Remaining central epithelial cells underwent karyopyknosis (Fig. 8A, triangle). Cells at the nuclear bow appeared unaffected (Fig. 8B). The macroscopically visible annular demarcation line that evolved at the rim of damaged epithelial cells 2 days after exposure was build up as the junction line between regenerating epithelial cells moving in from the equator and apoptotic epithelial cells with swollen cytoplasms and vacuoles in the central epithelium (Fig. 8C, arrow indicates direction of migration). These apoptotic cells were phagocytosed and removed by pushing them towards the lens sutures (Fig. 8D). Healthy epithelial cells in the pre-equatorial region of the lens elongated and stretched out to fill the gap and by this lost their cuboidal appearance (Fig. 8E). Control lenses at days 1, 2, 4 and 8 displayed a regular epithelial pattern in light microscopy as well as in TEM imaging (Fig. 8F,G) at the anterior pole as well as in the nuclear bow. Eight days after UVR-B exposure, the cell debris was pushed towards the lens sutures, thus ending in a triangular pattern at the anterior lens pole (Fig. 9). Here, the epithelial cells were stacked in a multilayered fashion and displayed chromatin condensation, membrane infolds, multiple phagocytosed apoptotic bodies and signs of karyopyknosis (Fig. 9A–D). Cortical fibres just below the multilayered central epithelium were irregular in shape with multiple vacuoles (Fig. 9A,B) but could be clearly demarcated from the regenerating epithelium.
Discussion Lens exposure to double cataract threshold UVR-B dose (Meyer et al. 2008) induces macroscopically mainly anterior subcapsular but also cortical and nuclear cataract in the mouse lens. However, the ultrastructure of these different types of cataract on a cellular level has not been studied before. Here, we present insight into the ultrastructure of the lens epithelium, cortex, nucleus, and into lens repair after UVR-B-induced damage with TEM and SEM techniques.
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Fig. 6. Ultrastructure of unexposed mouse lens in SEM imaging. Superficial (A, B) and deep cortical fibres (C, D) display ball and socket joints, and membrane interdigitations. Scale bar is 8 lm (A, B, C) and 2 lm (D). Blue rectangle resembles localization of section in sagittal view.
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Fig. 7. SEM image of anterior polar region of non-exposed lens (A) with regular capsule, epithelium and superficial cortex. One day following UVR-B exposure, the epithelial cells appear flake-like and flattened out. Adjacent fibre cells lying underneath have a swollen appearance (B). Scale bar is 10 lm (A) and 20 lm (B). Blue rectangle resembles localization of section in sagittal view.
Lens epithelium
The macroscopically flake-like opacities that we observed 1 day after
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exposure on the lens surface resemble in our opinion damaged epithelial cells that, due to swelling of their cytoplasm, chromatin condensation and early
apoptosis, give rise to light scattering. Galichanin and co-workers described signs of apoptosis already one hour, visible cataract 7 hr after UVR-B exposure in a study with rat lenses (Galichanin et al. 2010). In vitro studies with cultured lens epithelial cells confirm early apoptosis with TUNEL labelling and up-regulation of p53 and caspase 3 expression on mRNA and protein levels that can last up to 1 week after UVR-B exposure (Michael et al. 1998; Ayala et al. 2007). In our study, the earliest observation point was 24 hr following exposure. Similar to previous findings (Meyer et al. 2005), lens light scattering in the mouse lens peaks at 2 days postUVR-B exposure. Thus, the observed anterior subcapsular cataract is in line with previous findings. However, we can demonstrate that epithelial damage is repaired by cell migration from the lens equator towards the anterior pole and lens sutures. It is believed that epithelial cells of the normal epithelial monolayer are derived from daughter cells in the germinative zone at the lens equator (Rafferty & Rafferty 1981). It was demonstrated already in 1988 that lens epithelial cells contain actin and are capable of undergoing structural modifications for example in a setting of experimental epithelial injury (Liou & Rafferty 1988). In their model, the authors describe flattening of epithelial cells and extension of epithelial cell processes into induced wound spaces. Furthermore, they report amplified membrane folding in the rest of the epithelium following local epithelial damage. Epithelial cells that loose contact to the adjacent cell are stimulated to proliferate into the direction of cellular loss (Yamada et al. 2001). The elongated epithelial cells in the outer periphery as well as the demonstrated central migration of healthy epithelium reported in our study are both findings that support the regenerative capacity of lens epithelial cells in vivo. We confirm that lens epithelial cells are capable of repairing UVR-B-induced damage within 8 days via cell migration from the equator towards the anterior pole and by cleaning damaged cells on the way. The intact lens capsule might function in this context as a guiding structure for cellular repopulation of areas with epithelial damage. The described triangular shape of the remaining cataract at
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Fig. 8. TEM images and semi-thin sections stained with 1% tolouidine blue of anterior subcapsular cataract following in vivo exposure to double cataract threshold dose UVR-B 300 nm at different postexposure intervals. Epithelial cells are lost 1 day postexposure (A, arrows) Clearly visible is the debridement of the central epithelium. Arrowhead indicates apoptotic epithelial cell with karyopyknosis. The nuclear bow and the germinative zone are unaffected (B). Two days after exposure, epithelial cells with swollen cytoplasms are found at the rim of the cataractous area (C). Arrowhead indicates the direction of repopulation. The basal cell membrane is ruptured (C, TEM, arrow) and apoptotic bodies are found (C, asterix). Four days after exposure (D), epithelial damage is removed by cell migration from equator (arrow equals the direction of migration). Epithelial cells in pre-equatorial region are elongated (E). Control lenses at days 1, 2, 4 and 8 show regular epithelial cells in the anterior polar region in light microscopy and TEM (F) as well as in the equatorial zone (G). Scale bar is 20 lm (semi-thin sections) and 2 lm (TEM).
day eight is in our opinion due to the microanatomy of the lens sutures that are Y- and inverted Y-shaped in the mouse lens.
Of interest is the question how the multilayered epithelial cells that we observed at the anterior lens sutures 8 days after UVR-B exposure will
develop after a longer observation time. We hypothesize that over a time of a few weeks, those cell stacks will undergo apoptosis and phagocytosis
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(A)
(B)
(C1)
(C2)
(C3)
layers as an indirect damage from UVR-B exposure. In most exposed lenses, only the lens epithelium and outer cortical fibres were damaged but in some lenses, severe structural alterations in deeper cortical layers and the nucleus were present additionally to anterior subcapsular cataract (Figs 4 and 5). This observation points to a variation in interindividual UVR-B sensitivity, and we hypothesize that if lens epithelial damage exceeds the individual repair capacity, severe structural alterations occur via the abovedescribed pathways also in deeper lens fibres that then induces cortical and/or nuclear cataract.
Conclusion (D1)
(D3)
(D4)
(D2)
(D5)
(D6)
Fig. 9. TEM images and semi-thin sections stained with 1% toluidine blue of anterior subcapsular cataract following in vivo exposure to double cataract threshold dose UVR-B 300 nm. Eight days after exposure, the epithelium has recovered in the periphery and is multilayered around the anterior pole in an area of visible cataract (A). Small arrowhead indicates the direction of migration, big arrowhead shows the marginal zone of the single-layered and the multilayered epithelium. Fib = fibers. In the outer periphery, the monolayered epithelium is flattened out (B). Arrow indicates the direction of migration. (C1–C3) Around the lens sutures, in a triangular pattern, a multilayered epithelium has formed with phagocytosed cells, cell debris and multiple apoptotic bodies. The origin here is clearly epithelial (arrowheads). In TEM (D1–D6), the multilayered stack of epithelial cells (nuclei marked with black stars, D2, D3, D6) displays multiple membrane infolds, phagosomes and apoptotic bodies (white stars). In D4 and D5, the leading edge of epithelial cells is captured (white arrowheads) with membrane infolds and phagocytosis of apoptotic bodies and karyopyknosis. Scale bar is 2 lm. Cap = capsule, Fib = fibres.
and only a single-layered epithelium will prevail. However, further investigations are necessary to confirm our hypothesis. Lens fibers
Radiant energy from UVR-B 300 nm penetrates approximately 450 lm into the lens and is absorbed completely by the lens epithelium and superficial cortical fibre cells (S€ oderberg et al. 1998). In the epithelium, the result is an impaired metabolic activity and an inactivation of enzymes such as lactate
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dehydrogenase in the outer cortex (Chen et al. 1989; L€ ofgren & S€ oderberg 1995) Due to a disturbed osmoregulation in the lens epithelium, the homeostasis of underlying cortical fibres may also be affected as evidenced by extracellular spaces and vacuoles that were present from 2 days postexposure onwards (Figs 8C,D and 9A,B). Via cell-to-cell communication such as fibre membrane fusions and macromolecular pathways (Shestopalov and Bassnett 2000), the homeostatic imbalance of outer cortical fibres leads to structural alterations in deeper cortical
UVR-B-induced cataract in the mouse lens at double threshold dose is morphologically confined to the anterior third of the lens. Depending on the interindividual sensitivity, damage may also occur in deeper cortical layers and the nucleus. The lens epithelium is equipped with a high capacity of cell repair and migration. We found indications that after a subtotal loss of epithelial cells, this UVR-B-induced damage to the epithelium is repaired by epithelial cell movement from the equator towards the lens sutures, thus in retrograde direction to regular epithelial cell differentiation. However, further research is needed to visualize these dynamic cellular processes in the lens epithelium.
Acknowledgements The authors thank Monika Aronsson for keeping animals and researchers in excellent shape and mood, Margareta Oskarsson and Berit Spangberg for embedding lenses for TEM and SEM, and Margaret Gondo for help with electron microscopy imaging.
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Received on October 28th, 2013. Accepted on January 29th, 2014. Correspondence: Linda M. Meyer, MD, PhD Herzog Carl Theodor Eye Clinic Nymphenburger Strasse 43 80335 Munich Germany Tel: +49-89-1270930 Fax: +49-89-1290341 Email: [email protected]
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