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Radiation cataracts: mechanisms involved in their long delayed occurrence but then rapid progression.

Wolf N, Pendergrass W, Singh N, Swisshelm K, Schwartz J - Mol. Vis. (2008)

Bottom Line: This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period.As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins.The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington, Seattle, WA 98195-7475, USA. normwolf@u.washington.edu

ABSTRACT

Purpose: This study was directed to assess the DNA damage and DNA repair response to X-ray inflicted lens oxidative damage and to investigate the subsequent changes in lens epithelial cell (LEC) behavior in vivo that led to long delayed but then rapidly developing cataracts.

Methods: Two-month-old C57Bl/6 female mice received 11 Grays (Gy) of soft x-irradiation to the head only. The animals' eyes were examined for cataract status in 30 day intervals by slit lamp over an 11 month period post-irradiation. LEC migration, DNA fragment, free DNA retention, and reactive oxygen species (ROS) presence were established in the living lenses with fluorescent dyes using laser scanning confocal microscopy (LSCM). The extent and removal of initial LEC DNA damage were determined by comet assay. Immunohistochemistry was used to determine the presence of oxidized DNA and the response of a DNA repair protein in the lenses.

Results: This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period. The initially incurred DNA strand breaks were repaired within 30 min, but DNA damage remained as shown 72 h post-irradiation by the presence of the DNA adduct, 8-hydroxyguanosine (8-OHG), and a DNA repair protein, XRCC1. This was followed months later by abnormal behavior by LEC descendant cells with abnormal differentiation and migration patterns as seen with LSCM and fluorescent dyes.

Conclusions: The sudden development of cortical cataracts several months post-irradiation coupled with the above findings suggests an accumulation of damaged descendants from the initially x-irradiated LECs. As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins. The events seen in this study indicate the retention and transmission of progenitor cell DNA damage in descendant LEC. The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

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Loss of surface LEC by death and by abnormal migration in the x-ray cataract. This confocal view shows the loss of surface LEC and their descent into the lens interior at a site well anterior to the lens equator. The irradiated animal is five months post-irradiation. This picture is made from “stacked” multiple images extending from the lens surface to 120 μm into the interior.
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f7: Loss of surface LEC by death and by abnormal migration in the x-ray cataract. This confocal view shows the loss of surface LEC and their descent into the lens interior at a site well anterior to the lens equator. The irradiated animal is five months post-irradiation. This picture is made from “stacked” multiple images extending from the lens surface to 120 μm into the interior.

Mentions: In spite of the initial recovery of DNA strand breaks, a dysfunctional lens status increased over time as confirmed by confocal viewing of living lenses using fluorescent dyes for nuclear fragments, free DNA presence, and localized ROS presence as well as noting aberrant LEC migration within the lens. These changes increased progressively in the irradiated mice when viewed in this manner. They were not present in the first month but developed minimally by the third month and were present as marked changes in cataractous, irradiated lenses at five and seven months post-irradiation (Figure 6) with aberrant LEC migration (Figure 7). Thus, the slit lamp and confocal microscopic sequential examinations were usually in agreement, although the confocal examinations at three months showed some minimal early loss of surface LEC while the slit lamp examinations did not yet reveal any evidence of cataract. We selected only those pairs of irradiated mice with obvious opacities when observed by slit lamp examination and pairs of control mice for the confocal studies at five and seven months post-irradiation to illustrate and measure by degree of respective dye fluorescence the internal and surface LEC lens changes. Thus, the most likely candidates were chosen from the slit lamp results for use in the confocal examinations. The advanced changes in a cataractous mouse lens at five months post-irradiation (shown in the confocal view in Figure 6) displayed Hoechst 33343 fluorescence for displaced DNA presence, dihydrorhodamine conversion to rhodamine for ROS presence, and light refraction at the affected sites (note the spatial overlap for the each of these). Both the free DNA and the ROS computerized fluorescence measurements differed from those of control mice by p<0.01 at five months post-irradiation and p<0.001 at seven months using the Mann–Whitney non-parametric test. These measurements included all regions of the interior lens as “stacked” confocal readings of 6 μm each to a 120 μm depth in the cortex. The overall cortex readings contained an accumulation of both nuclear fragments and free DNA. In general, the ROS intensity was greatest at internal lens regions where DNA intensity was also highest. Sequential depth confocal views also showed the migration of LEC strands to the lens interior from inappropriate surface sites that were not at the normal subequatorial bow region. This misdirected migration was a prominent feature in the cataractous lens, and the internalized strands of cells present there did not undergo resolution of their nuclei as shown in the stacked depths integrated LSCM readings in Figure 7. Each of the above noted events are quite similar to those seen in age-related cataract in non-irradiated old mice of the same strain and in old rats as we have previously reported [28,29].


Radiation cataracts: mechanisms involved in their long delayed occurrence but then rapid progression.

Wolf N, Pendergrass W, Singh N, Swisshelm K, Schwartz J - Mol. Vis. (2008)

Loss of surface LEC by death and by abnormal migration in the x-ray cataract. This confocal view shows the loss of surface LEC and their descent into the lens interior at a site well anterior to the lens equator. The irradiated animal is five months post-irradiation. This picture is made from “stacked” multiple images extending from the lens surface to 120 μm into the interior.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254966&req=5

f7: Loss of surface LEC by death and by abnormal migration in the x-ray cataract. This confocal view shows the loss of surface LEC and their descent into the lens interior at a site well anterior to the lens equator. The irradiated animal is five months post-irradiation. This picture is made from “stacked” multiple images extending from the lens surface to 120 μm into the interior.
Mentions: In spite of the initial recovery of DNA strand breaks, a dysfunctional lens status increased over time as confirmed by confocal viewing of living lenses using fluorescent dyes for nuclear fragments, free DNA presence, and localized ROS presence as well as noting aberrant LEC migration within the lens. These changes increased progressively in the irradiated mice when viewed in this manner. They were not present in the first month but developed minimally by the third month and were present as marked changes in cataractous, irradiated lenses at five and seven months post-irradiation (Figure 6) with aberrant LEC migration (Figure 7). Thus, the slit lamp and confocal microscopic sequential examinations were usually in agreement, although the confocal examinations at three months showed some minimal early loss of surface LEC while the slit lamp examinations did not yet reveal any evidence of cataract. We selected only those pairs of irradiated mice with obvious opacities when observed by slit lamp examination and pairs of control mice for the confocal studies at five and seven months post-irradiation to illustrate and measure by degree of respective dye fluorescence the internal and surface LEC lens changes. Thus, the most likely candidates were chosen from the slit lamp results for use in the confocal examinations. The advanced changes in a cataractous mouse lens at five months post-irradiation (shown in the confocal view in Figure 6) displayed Hoechst 33343 fluorescence for displaced DNA presence, dihydrorhodamine conversion to rhodamine for ROS presence, and light refraction at the affected sites (note the spatial overlap for the each of these). Both the free DNA and the ROS computerized fluorescence measurements differed from those of control mice by p<0.01 at five months post-irradiation and p<0.001 at seven months using the Mann–Whitney non-parametric test. These measurements included all regions of the interior lens as “stacked” confocal readings of 6 μm each to a 120 μm depth in the cortex. The overall cortex readings contained an accumulation of both nuclear fragments and free DNA. In general, the ROS intensity was greatest at internal lens regions where DNA intensity was also highest. Sequential depth confocal views also showed the migration of LEC strands to the lens interior from inappropriate surface sites that were not at the normal subequatorial bow region. This misdirected migration was a prominent feature in the cataractous lens, and the internalized strands of cells present there did not undergo resolution of their nuclei as shown in the stacked depths integrated LSCM readings in Figure 7. Each of the above noted events are quite similar to those seen in age-related cataract in non-irradiated old mice of the same strain and in old rats as we have previously reported [28,29].

Bottom Line: This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period.As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins.The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington, Seattle, WA 98195-7475, USA. normwolf@u.washington.edu

ABSTRACT

Purpose: This study was directed to assess the DNA damage and DNA repair response to X-ray inflicted lens oxidative damage and to investigate the subsequent changes in lens epithelial cell (LEC) behavior in vivo that led to long delayed but then rapidly developing cataracts.

Methods: Two-month-old C57Bl/6 female mice received 11 Grays (Gy) of soft x-irradiation to the head only. The animals' eyes were examined for cataract status in 30 day intervals by slit lamp over an 11 month period post-irradiation. LEC migration, DNA fragment, free DNA retention, and reactive oxygen species (ROS) presence were established in the living lenses with fluorescent dyes using laser scanning confocal microscopy (LSCM). The extent and removal of initial LEC DNA damage were determined by comet assay. Immunohistochemistry was used to determine the presence of oxidized DNA and the response of a DNA repair protein in the lenses.

Results: This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period. The initially incurred DNA strand breaks were repaired within 30 min, but DNA damage remained as shown 72 h post-irradiation by the presence of the DNA adduct, 8-hydroxyguanosine (8-OHG), and a DNA repair protein, XRCC1. This was followed months later by abnormal behavior by LEC descendant cells with abnormal differentiation and migration patterns as seen with LSCM and fluorescent dyes.

Conclusions: The sudden development of cortical cataracts several months post-irradiation coupled with the above findings suggests an accumulation of damaged descendants from the initially x-irradiated LECs. As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins. The events seen in this study indicate the retention and transmission of progenitor cell DNA damage in descendant LEC. The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

Show MeSH
Related in: MedlinePlus