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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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Typical vital staining of the anterior and posterior cortices of 10-month old mice lenses for DNA, ROS, and cataract. DNA (Hoechst 33342) in the lenses is shown in red, ROS (oxidized DHR) is shown in green, and cataract reflected light in white. The LSCM (10× objective) was focused 120 microns below the anterior poles of unirradiated control lenses (upper panel) and X-irradiated lenses (lower panel) at the same age. The donors of the lenses were 3 months old when irradiated and 10 months old when analyzed. The first column depicts the DNA fluorescence in red (Hoechst 33342 fluorescence), middle Column shows ROS in green (oxidized DHR). The right column shows light reflected from cataracts. Original magnification was 200×.
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f5: Typical vital staining of the anterior and posterior cortices of 10-month old mice lenses for DNA, ROS, and cataract. DNA (Hoechst 33342) in the lenses is shown in red, ROS (oxidized DHR) is shown in green, and cataract reflected light in white. The LSCM (10× objective) was focused 120 microns below the anterior poles of unirradiated control lenses (upper panel) and X-irradiated lenses (lower panel) at the same age. The donors of the lenses were 3 months old when irradiated and 10 months old when analyzed. The first column depicts the DNA fluorescence in red (Hoechst 33342 fluorescence), middle Column shows ROS in green (oxidized DHR). The right column shows light reflected from cataracts. Original magnification was 200×.

Mentions: We previously reported that vitally stained lenses from old cataractous mice and rats contained large numbers of undegraded or partially degraded nuclei, and high levels of ROS in the lens cortex [19,20]. In Figure 5 typical examples of vital staining for cortical DNA (Hoechst 33342) and ROS (as oxidized DHR) in X-irradiated mouse fresh lenses and in age-matched unirradiated controls are compared. Figure 6 depicts the relative quantitative intensities of cortical DNA (DNA fragments and cytoplasmic DNA) using the vital dye method in X-irradiated mice and in age-matched unirradiated controls. Both the number of abnormal cortical nuclear fragments, as well as, the intensity of cortical ROS increased significantly in the X-irradiated lenses by 6 months post irradiation using the vital dye method.


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)

Typical vital staining of the anterior and posterior cortices of 10-month old mice lenses for DNA, ROS, and cataract. DNA (Hoechst 33342) in the lenses is shown in red, ROS (oxidized DHR) is shown in green, and cataract reflected light in white. The LSCM (10× objective) was focused 120 microns below the anterior poles of unirradiated control lenses (upper panel) and X-irradiated lenses (lower panel) at the same age. The donors of the lenses were 3 months old when irradiated and 10 months old when analyzed. The first column depicts the DNA fluorescence in red (Hoechst 33342 fluorescence), middle Column shows ROS in green (oxidized DHR). The right column shows light reflected from cataracts. Original magnification was 200×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Typical vital staining of the anterior and posterior cortices of 10-month old mice lenses for DNA, ROS, and cataract. DNA (Hoechst 33342) in the lenses is shown in red, ROS (oxidized DHR) is shown in green, and cataract reflected light in white. The LSCM (10× objective) was focused 120 microns below the anterior poles of unirradiated control lenses (upper panel) and X-irradiated lenses (lower panel) at the same age. The donors of the lenses were 3 months old when irradiated and 10 months old when analyzed. The first column depicts the DNA fluorescence in red (Hoechst 33342 fluorescence), middle Column shows ROS in green (oxidized DHR). The right column shows light reflected from cataracts. Original magnification was 200×.
Mentions: We previously reported that vitally stained lenses from old cataractous mice and rats contained large numbers of undegraded or partially degraded nuclei, and high levels of ROS in the lens cortex [19,20]. In Figure 5 typical examples of vital staining for cortical DNA (Hoechst 33342) and ROS (as oxidized DHR) in X-irradiated mouse fresh lenses and in age-matched unirradiated controls are compared. Figure 6 depicts the relative quantitative intensities of cortical DNA (DNA fragments and cytoplasmic DNA) using the vital dye method in X-irradiated mice and in age-matched unirradiated controls. Both the number of abnormal cortical nuclear fragments, as well as, the intensity of cortical ROS increased significantly in the X-irradiated lenses by 6 months post irradiation using the vital dye method.

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