title: X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195, USA. pendergr@u.washington.edu
authors.forename: William
authors.initials: W
authors.lastname: Pendergrass
authors.forename: Galynn
authors.initials: G
authors.lastname: Zitnik
authors.forename: Ryan
authors.initials: R
authors.lastname: Tsai
authors.forename: Norman
authors.initials: N
authors.lastname: Wolf
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000375:Aging / Q000473:pathology*; D000818:Animals; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology*; D002452:Cell Count; D004247:DNA / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology*; D005260:Female; D007211:Indoles / Q000378:metabolism; D007904:Lens Cortex, Crystalline / Q000378:metabolism* / Q000473:pathology* / Q000601:surgery; D007907:Lens Nucleus, Crystalline / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013194:Staining and Labeling; D016707:Tissue Fixation; D014965:X-Rays
chemical_list: D007211:Indoles; D017382:Reactive Oxygen Species; C007293:DAPI; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine
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