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J. Jim, J. Cui, K. Chan, J.A. Matsubara; A Histochemical Method to Study Senescence-associated B-Galactosidase Staining in Human Donor Eyes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1722.
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Purpose. To correlate levels of beta-galactosidase associated cellular senescence in the human postmortem eye as a function of age. Methods. Seventy-two donor eyes (7 to 75 years at time of death) were obtained within 24 hrs postmorten and fixed with 4% paraformaldehyde in phosphate buffer saline. Retinal tissue was placed in OCT and frozen in liquid nitrogen. Cryostat sections of 7µm thickness were cut and mounted onto glass slides. Following the protocol of Dimri et al, '95, sections were immersed for 10-14 hours in senescence associated b-gal staining solution, consisting of 1mg of X-gal per ml of 40 mM citric acid and sodium phosphate at pH 6.0, 5mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl2. Next, sections were rinsed and the retinal pigment epithelial layer was bleached for 10-20 minutes using a solution of 0.1% potassium permanganate and 5-25 minutes in a solution of 0.5% oxalic acid. Sections were then rinsed again, counterstained and coverslipped. Slides were viewed under brightfield at 100-400x. Results. Optimal incubation time for senescence-associated b-gal staining was determined to be 10 hours. Optimal time for bleaching of endogenous pigment was 10 minutes with potassium permanganate and 5 minutes with oxalic acid. The tissue sections revealed numerous b-gal positive cellular profiles including RPE, fibroblasts, retinal ganglion and Müller cells. The RPE cells positive for b-gal were quantified by age of donor eye. 12.5% (N=8) of the donor eyes less than 54 yrs demonstrated significant b-gal staining in the RPE layer. The percentage of eyes with significant b-gal staining in the RPE layer rose to 60% (N=5) of the donor eyes between 60-69 yrs, and 75% (N=12) of the donor eyes over 70 yrs of age. Conclusions. Optimization of protocols originally developed in monkey reveals senescence associated b-gal staining of RPE cells increases with age of donor eye. The results reveal that RPE cells of the human eye, like those of rhesus monkeys, undergo cellular senescence with age. Combined with our earlier studies, which showed differential gene expression between senescent and non-senescent RPE cells in vitro, these results suggest that the RPE may play a role in the pathogenesis of age-related degenerative retinal disorders such as AMD.
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