Limits...
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.

Show MeSH

Related in: MedlinePlus

Typical images of DAPI-stained paraformaldehyde-fixed sections from lenses unirradiated control lenses and lenses from mice X-irradiated at 3-month of age. Panel A was from unirradiated 3-month controls, panel B was from 14 month unirradiated controls, panel C was from 14-month X-irradiated lens, and panel D was from a 26-month old unirradiated control. Each image was derived from a panorama taken with a 10× objective (see Methods). The dashed lines show the regions of the lenses used for counting DNA fragments in different regions of cortex (see Figure 9A,B). These include both bow regions, and the cortex below the central zone. The posterior cortex was included if nuclear fragments were present. The non-lens portions of the eye have been deleted for clarity. Original magnification was 200×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2925908&req=5

f8: Typical images of DAPI-stained paraformaldehyde-fixed sections from lenses unirradiated control lenses and lenses from mice X-irradiated at 3-month of age. Panel A was from unirradiated 3-month controls, panel B was from 14 month unirradiated controls, panel C was from 14-month X-irradiated lens, and panel D was from a 26-month old unirradiated control. Each image was derived from a panorama taken with a 10× objective (see Methods). The dashed lines show the regions of the lenses used for counting DNA fragments in different regions of cortex (see Figure 9A,B). These include both bow regions, and the cortex below the central zone. The posterior cortex was included if nuclear fragments were present. The non-lens portions of the eye have been deleted for clarity. Original magnification was 200×.

Mentions: We also compared the number of cortical nuclei present in fixed-eye lens sections using both DAPI and H&E stains. The H&E staining and DAPI staining were very similar with every DAPI-stained-nucleus also being stained by H&E (Figure 7). The DAPI was used for further analysis because it was brighter and permitted more rapid examination of the lens sections. Examples of fixed-eye sections from 3 month, 14 month, and 26-month-old unirradiated control lenses, and a 14-month-old X-irradiated lens (Figure 8C) are compared in Figure 8A-D. These sections were cut through the middle of the whole eye at right angles to the anterior surface to reveal nuclei and nuclear fragments beneath the central zone as well as in the bow region. As in the vital dye study, the old lenses and the 14-month old X-irradiated lenses always contained large numbers of DNA positive material in the cortex beneath the central zone and a greatly expanded bow region. In old animals or cataractous X-rayed animals the debris sometimes filled the whole outer cortex of the lens. For this analysis, the area beneath the “central zone” of the lens was set arbitrarily at the width of the lens nucleus. Figure 9A depicts the total number of nuclear fragments in the entire outer cortex. The total number of DNA fragments increased steadily and significantly with age in unirradiated lenses (Figure 9A), but the 14 month X-irradiated lenses (11 months post-irradiation) did not have more total cortical nuclei than age-matched unirradiated controls although the abnormal cortical nuclei were spread over a larger part of the cortex. This is probably due to the presence of fewer layers of secondary fiber cells in X-irradiated lenses as a result of X-ray-induced damage to LEC replication. In support of this possibility we measured the depth that the nuclei in the bow regions extended below the surface (from the anterior surface to the deepest fragments). As expected, the bows in the 14-month old X-rayed mice did not penetrate as deeply into the cortex as those of the age-matched controls, again probably as a result of X-ray damage to the LECs [46]. The nuclei in the bows of 14-month old X-rayed mice lenses extended only 284±19 microns into the cortex compared to 369±21 microns for age-matched controls (p<0.005) compared to only 138 microns in 3-month-old unirradiated controls and 522±15 microns in the 24-month-old mice lenses.


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 images of DAPI-stained paraformaldehyde-fixed sections from lenses unirradiated control lenses and lenses from mice X-irradiated at 3-month of age. Panel A was from unirradiated 3-month controls, panel B was from 14 month unirradiated controls, panel C was from 14-month X-irradiated lens, and panel D was from a 26-month old unirradiated control. Each image was derived from a panorama taken with a 10× objective (see Methods). The dashed lines show the regions of the lenses used for counting DNA fragments in different regions of cortex (see Figure 9A,B). These include both bow regions, and the cortex below the central zone. The posterior cortex was included if nuclear fragments were present. The non-lens portions of the eye have been deleted for clarity. Original magnification was 200×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Typical images of DAPI-stained paraformaldehyde-fixed sections from lenses unirradiated control lenses and lenses from mice X-irradiated at 3-month of age. Panel A was from unirradiated 3-month controls, panel B was from 14 month unirradiated controls, panel C was from 14-month X-irradiated lens, and panel D was from a 26-month old unirradiated control. Each image was derived from a panorama taken with a 10× objective (see Methods). The dashed lines show the regions of the lenses used for counting DNA fragments in different regions of cortex (see Figure 9A,B). These include both bow regions, and the cortex below the central zone. The posterior cortex was included if nuclear fragments were present. The non-lens portions of the eye have been deleted for clarity. Original magnification was 200×.
Mentions: We also compared the number of cortical nuclei present in fixed-eye lens sections using both DAPI and H&E stains. The H&E staining and DAPI staining were very similar with every DAPI-stained-nucleus also being stained by H&E (Figure 7). The DAPI was used for further analysis because it was brighter and permitted more rapid examination of the lens sections. Examples of fixed-eye sections from 3 month, 14 month, and 26-month-old unirradiated control lenses, and a 14-month-old X-irradiated lens (Figure 8C) are compared in Figure 8A-D. These sections were cut through the middle of the whole eye at right angles to the anterior surface to reveal nuclei and nuclear fragments beneath the central zone as well as in the bow region. As in the vital dye study, the old lenses and the 14-month old X-irradiated lenses always contained large numbers of DNA positive material in the cortex beneath the central zone and a greatly expanded bow region. In old animals or cataractous X-rayed animals the debris sometimes filled the whole outer cortex of the lens. For this analysis, the area beneath the “central zone” of the lens was set arbitrarily at the width of the lens nucleus. Figure 9A depicts the total number of nuclear fragments in the entire outer cortex. The total number of DNA fragments increased steadily and significantly with age in unirradiated lenses (Figure 9A), but the 14 month X-irradiated lenses (11 months post-irradiation) did not have more total cortical nuclei than age-matched unirradiated controls although the abnormal cortical nuclei were spread over a larger part of the cortex. This is probably due to the presence of fewer layers of secondary fiber cells in X-irradiated lenses as a result of X-ray-induced damage to LEC replication. In support of this possibility we measured the depth that the nuclei in the bow regions extended below the surface (from the anterior surface to the deepest fragments). As expected, the bows in the 14-month old X-rayed mice did not penetrate as deeply into the cortex as those of the age-matched controls, again probably as a result of X-ray damage to the LECs [46]. The nuclei in the bows of 14-month old X-rayed mice lenses extended only 284±19 microns into the cortex compared to 369±21 microns for age-matched controls (p<0.005) compared to only 138 microns in 3-month-old unirradiated controls and 522±15 microns in the 24-month-old mice lenses.

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