Accumulation of DNA, nuclear and mitochondrial debris, and ROS at sites of age-related cortical cataract in mice.
OBJECTIVE
Lenses from young and old mice were analyzed by laser scanning confocal microscopy (LSCM) with vital dyes, to determine whether age-related subcapsular and cortical cataracts were linked to the failure of lens fiber cells to degrade nuclei, DNA, and mitochondria properly and whether they result in the overproduction of reactive oxygen species (ROS) at the same sites.


RESULTS
As opposed to the clear DNA-free subcapsular and cortical areas of young adult mouse lenses, these areas in cataractous old mouse lenses were found to contain accumulations of nuclei, nuclear fragments, aggregated mitochondria, and amorphous DNA as cortical inclusions (P < 0.001 between young and old lenses). These inclusions correlated spatially with age-related cataracts and with the presence of ROS. The source of such undegraded material was a large expansion of transition nuclei in the bow region and also direct involution of surface lens epithelial cells (LECs) into the underlying cortex, frequently leaving bare patches devoid of nuclei on the surface of the anterior epithelium.


METHODS
Live lenses were stained vitally for DNA with Hoechst 33342. ROS and mitochondria were stained and quantified with dihydrorhodamine 123 (DHR). In fixed lenses, DNA was stained with propidium iodide (PI) or 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI). The intensity and position of each probe's fluorescence was determined by LSCM. Cataract localization was ascertained by digitalized microscopy of reflected light.


CONCLUSIONS
In aged mice, most subcapsular and cortical cataracts colocalize with accumulations of nuclei, mitochondria, and DNA, These effects are accompanied at the same sites by the production of ROS. The condition is due to the failure of lens fiber cells in the bow region to differentiate properly into the clear fiber state and to the improper involution of cells from the anterior epithelium directly into the underlying cortex, resulting in cataractous opacities.
Pendergrass
William
W
Department of Pathology, University of Washington School of Medicine, Seattle 98195, USA. pendergr@u.washington.edu
Penn
Philip
P
Possin
Daniel
D
Wolf
Norman
N
D000375:Aging / Q000378:metabolism*; D000818:Animals; D001562:Benzimidazoles; D002386:Cataract / Q000378:metabolism*; D002452:Cell Count; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes; D002479:Inclusion Bodies; D007904:Lens Cortex, Crystalline / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride