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X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts.

Pendergrass W, Zitnik G, Tsai R, Wolf N - Mol. Vis. (2010)

Bottom Line: Using DNA- and ROS-specific vital fluorescent dyes, and laser scanning confocal microscopy we have previously described 4 changes in the aging rodent lenses: 1) a significantly decreased density of surface LECs in lenses from old compared to younger mice and rats; 2) a very large increase in retained cortical nuclei and DNA fragments in the secondary lens fibers of old rodent lenses; 3) increased cortical ROS in old rodent lenses; 4) increased cataract concomitantly with the cortical DNA and ROS increases.In addition to vital staining of fresh lenses, we also examined sections from fixed eyes stained with DAPI or hematoxylin and eosin (H&E) and found the same loss of surface LECs and accumulation of undigested nuclei and debris in secondary lens fibers occur with age or following X-irradiation.X-irradiated lenses develop the same abnormalities in a more accelerated fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195, USA. pendergr@u.washington.edu

ABSTRACT

Purpose: To compare age-related cataractous (ARC) changes in unirradiated mice lenses to those induced by head-only X-irradiation of 3 month-old mice.

Methods: lens epithelial cells (LECs) as well as partially degraded cortical DNA were visualized in fixed sections using 4',6-diamidino-2-phenylindole (DAPI) staining, and in fresh lenses using the vital stain Hoechst 33342. reactive oxygen species (ROS) activity was also visualized directly in fresh lenses using the vital dye Dihydrorhodamine (DHR). In fixed lenses an antibody specific for 8-OH Guanosine (8-OH-G) lesions was used to visualize DNA oxidative adducts from ROS damage. Alpha smooth muscle actin was visualized using specific antibodies to determine if myofibroblasts were present. Fluorescence was quantified using Laser Scanning Confocal Microscopy (LSCM). The degree of lens opacity and cataract formation was determined by slit lamp, or from digitalized images of light reflections taken with a low magnification light microscope.

Results: Using DNA- and ROS-specific vital fluorescent dyes, and laser scanning confocal microscopy we have previously described 4 changes in the aging rodent lenses: 1) a significantly decreased density of surface LECs in lenses from old compared to younger mice and rats; 2) a very large increase in retained cortical nuclei and DNA fragments in the secondary lens fibers of old rodent lenses; 3) increased cortical ROS in old rodent lenses; 4) increased cataract concomitantly with the cortical DNA and ROS increases. In the current study we report that these same 4 changes also occur in an accelerated fashion in mice given head-only X-irradiation at 3 months of age. In addition to vital staining of fresh lenses, we also examined sections from fixed eyes stained with DAPI or hematoxylin and eosin (H&E) and found the same loss of surface LECs and accumulation of undigested nuclei and debris in secondary lens fibers occur with age or following X-irradiation. In addition sections from fixed-eyes were examined for ROS damage to DNA with antibodies specific for 8-OH-G lesions. The frequency of 8-OH-G lesions increased dramatically in lenses from old unirradiated mice over 24 months of age, and similarly in X-irradiated lenses by 9-11 months post irradiation. The accumulation of cortical nuclei was not the result of conversion or invasion by myofibroblasts as tested by antibodies to a marker for such cells, alpha smooth muscle actin.

Conclusions: X-irradiation damage induces a large decrease in surface LECs over a period of 3-11 months post X-irradiation of young mice. These changes are similar in extent to those seen in 24-29 months-old control mouse lenses with age-related cataracts. In 24+ month-old unirradiated mice the secondary lens fibers are not able to degrade nuclei or nuclear DNA efficiently and accumulate large numbers of cortical nuclei and nuclear fragments as well as ROS and 8-OHG lesions. X-irradiated lenses develop the same abnormalities in a more accelerated fashion. The extensive loss of LECS and accumulation of undegraded nuclei, ROS, and ROS damage may play a causal role in cataract generation in both unirradiated old mice and in previously irradiated young adult mice.

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Changes in the density of surface LECs in fixed-eye sections. The LEC densities in the central zone of unirradiated control and X- irradiated lens sections were determined by counting the DAPI stained nuclei per mm of lens surface (see Methods). X-irradiation was at 3 months. The surface LEC nuclei/mm densities decreased with age (p<0.001 between unirradiated 3-month and 25 month-old mice lenses). The p values shown are for a comparison of the LEC densities in X-irradiated and age-matched controls at 7 months, 12 months, and 14 months using a single tailed Mann–Whitney test. The error bars are for the standard error of the mean. Each bar represents the mean for 6–8 mice.
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f4: Changes in the density of surface LECs in fixed-eye sections. The LEC densities in the central zone of unirradiated control and X- irradiated lens sections were determined by counting the DAPI stained nuclei per mm of lens surface (see Methods). X-irradiation was at 3 months. The surface LEC nuclei/mm densities decreased with age (p<0.001 between unirradiated 3-month and 25 month-old mice lenses). The p values shown are for a comparison of the LEC densities in X-irradiated and age-matched controls at 7 months, 12 months, and 14 months using a single tailed Mann–Whitney test. The error bars are for the standard error of the mean. Each bar represents the mean for 6–8 mice.

Mentions: The LEC density of the Central Zone was significantly reduced relative to unirradiated controls as measured by vital staining with Hoechst 33342. Figure 3 compares the number of LECs per mm2 of the vitally stained lens epithelia. A rapid decrease in the surface LEC density of the Central Zone occurred by 3 months following X-irradiation. Before X-irradiation was administered at 3 months of age, the LEC density was 3,641 nuclei/mm2, and by 3 months post X-irradiation (6 months of age), the cell density had dropped to 2,356 (63% of starting value), and by 11 months post irradiation, the cell density had dropped to 1,856 /mm2, (51% of young controls). This decrease in LEC density in X-irradiated mouse lenses was much more rapid than the gradual decrease in the unirradiated controls to near the same level (2,090 cells/mm2 at 29 months of age). In both X-irradiated and controls this decrease in LEC density preceded cataract formation or other changes reported below. In a separate study of the LEC density, fixed-eye lens sections were also analyzed (Figure 4). The number of nuclei per linear mm of the Central Zone surface were counted using DAPI to stain the sections. This second study on fixed-eye sections produced similar results to those seen in the vital staining of whole lenses, indicating that the LEC densities of X-irradiated mice lenses were reduced significantly by 4 months after X-irradiation (p<0.001). In both studies the decrease in LEC density preceded cataract formation by several months both in X-irradiated and in old unirradiated control mice.


X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts.

Pendergrass W, Zitnik G, Tsai R, Wolf N - Mol. Vis. (2010)

Changes in the density of surface LECs in fixed-eye sections. The LEC densities in the central zone of unirradiated control and X- irradiated lens sections were determined by counting the DAPI stained nuclei per mm of lens surface (see Methods). X-irradiation was at 3 months. The surface LEC nuclei/mm densities decreased with age (p<0.001 between unirradiated 3-month and 25 month-old mice lenses). The p values shown are for a comparison of the LEC densities in X-irradiated and age-matched controls at 7 months, 12 months, and 14 months using a single tailed Mann–Whitney test. The error bars are for the standard error of the mean. Each bar represents the mean for 6–8 mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2925908&req=5

f4: Changes in the density of surface LECs in fixed-eye sections. The LEC densities in the central zone of unirradiated control and X- irradiated lens sections were determined by counting the DAPI stained nuclei per mm of lens surface (see Methods). X-irradiation was at 3 months. The surface LEC nuclei/mm densities decreased with age (p<0.001 between unirradiated 3-month and 25 month-old mice lenses). The p values shown are for a comparison of the LEC densities in X-irradiated and age-matched controls at 7 months, 12 months, and 14 months using a single tailed Mann–Whitney test. The error bars are for the standard error of the mean. Each bar represents the mean for 6–8 mice.
Mentions: The LEC density of the Central Zone was significantly reduced relative to unirradiated controls as measured by vital staining with Hoechst 33342. Figure 3 compares the number of LECs per mm2 of the vitally stained lens epithelia. A rapid decrease in the surface LEC density of the Central Zone occurred by 3 months following X-irradiation. Before X-irradiation was administered at 3 months of age, the LEC density was 3,641 nuclei/mm2, and by 3 months post X-irradiation (6 months of age), the cell density had dropped to 2,356 (63% of starting value), and by 11 months post irradiation, the cell density had dropped to 1,856 /mm2, (51% of young controls). This decrease in LEC density in X-irradiated mouse lenses was much more rapid than the gradual decrease in the unirradiated controls to near the same level (2,090 cells/mm2 at 29 months of age). In both X-irradiated and controls this decrease in LEC density preceded cataract formation or other changes reported below. In a separate study of the LEC density, fixed-eye lens sections were also analyzed (Figure 4). The number of nuclei per linear mm of the Central Zone surface were counted using DAPI to stain the sections. This second study on fixed-eye sections produced similar results to those seen in the vital staining of whole lenses, indicating that the LEC densities of X-irradiated mice lenses were reduced significantly by 4 months after X-irradiation (p<0.001). In both studies the decrease in LEC density preceded cataract formation by several months both in X-irradiated and in old unirradiated control mice.

Bottom Line: Using DNA- and ROS-specific vital fluorescent dyes, and laser scanning confocal microscopy we have previously described 4 changes in the aging rodent lenses: 1) a significantly decreased density of surface LECs in lenses from old compared to younger mice and rats; 2) a very large increase in retained cortical nuclei and DNA fragments in the secondary lens fibers of old rodent lenses; 3) increased cortical ROS in old rodent lenses; 4) increased cataract concomitantly with the cortical DNA and ROS increases.In addition to vital staining of fresh lenses, we also examined sections from fixed eyes stained with DAPI or hematoxylin and eosin (H&E) and found the same loss of surface LECs and accumulation of undigested nuclei and debris in secondary lens fibers occur with age or following X-irradiation.X-irradiated lenses develop the same abnormalities in a more accelerated fashion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195, USA. pendergr@u.washington.edu

ABSTRACT

Purpose: To compare age-related cataractous (ARC) changes in unirradiated mice lenses to those induced by head-only X-irradiation of 3 month-old mice.

Methods: lens epithelial cells (LECs) as well as partially degraded cortical DNA were visualized in fixed sections using 4',6-diamidino-2-phenylindole (DAPI) staining, and in fresh lenses using the vital stain Hoechst 33342. reactive oxygen species (ROS) activity was also visualized directly in fresh lenses using the vital dye Dihydrorhodamine (DHR). In fixed lenses an antibody specific for 8-OH Guanosine (8-OH-G) lesions was used to visualize DNA oxidative adducts from ROS damage. Alpha smooth muscle actin was visualized using specific antibodies to determine if myofibroblasts were present. Fluorescence was quantified using Laser Scanning Confocal Microscopy (LSCM). The degree of lens opacity and cataract formation was determined by slit lamp, or from digitalized images of light reflections taken with a low magnification light microscope.

Results: Using DNA- and ROS-specific vital fluorescent dyes, and laser scanning confocal microscopy we have previously described 4 changes in the aging rodent lenses: 1) a significantly decreased density of surface LECs in lenses from old compared to younger mice and rats; 2) a very large increase in retained cortical nuclei and DNA fragments in the secondary lens fibers of old rodent lenses; 3) increased cortical ROS in old rodent lenses; 4) increased cataract concomitantly with the cortical DNA and ROS increases. In the current study we report that these same 4 changes also occur in an accelerated fashion in mice given head-only X-irradiation at 3 months of age. In addition to vital staining of fresh lenses, we also examined sections from fixed eyes stained with DAPI or hematoxylin and eosin (H&E) and found the same loss of surface LECs and accumulation of undigested nuclei and debris in secondary lens fibers occur with age or following X-irradiation. In addition sections from fixed-eyes were examined for ROS damage to DNA with antibodies specific for 8-OH-G lesions. The frequency of 8-OH-G lesions increased dramatically in lenses from old unirradiated mice over 24 months of age, and similarly in X-irradiated lenses by 9-11 months post irradiation. The accumulation of cortical nuclei was not the result of conversion or invasion by myofibroblasts as tested by antibodies to a marker for such cells, alpha smooth muscle actin.

Conclusions: X-irradiation damage induces a large decrease in surface LECs over a period of 3-11 months post X-irradiation of young mice. These changes are similar in extent to those seen in 24-29 months-old control mouse lenses with age-related cataracts. In 24+ month-old unirradiated mice the secondary lens fibers are not able to degrade nuclei or nuclear DNA efficiently and accumulate large numbers of cortical nuclei and nuclear fragments as well as ROS and 8-OHG lesions. X-irradiated lenses develop the same abnormalities in a more accelerated fashion. The extensive loss of LECS and accumulation of undegraded nuclei, ROS, and ROS damage may play a causal role in cataract generation in both unirradiated old mice and in previously irradiated young adult mice.

Show MeSH
Related in: MedlinePlus