Senescence-associated beta-galactosidase histochemistry for the primate eye.
OBJECTIVE
To develop a senescence-associated beta-galactosidase histochemistry and bleaching protocol for the primate posterior pole.


METHODS
Rhesus monkey eyes of different ages were enucleated after death, fixed in 4% paraformaldehyde for up to 16 hours, and cryoprotected using a graded sucrose infiltration technique. Ten-micrometer tissue sections were treated with beta-galactosidase, pH 4 (lysosomal) or pH 6 (senescence-associated) activity, for various times. Bleaching of retinal pigment epithelial (RPE) cell and choroidal melanocyte pigment was performed after beta-galactosidase histochemistry using 0.1% to 1% potassium permanganate incubation for 1 minute to 2 hours followed by 0.5% oxalic acid immersion.


RESULTS
A 6-hour incubation with beta-galactosidase, pH 4 or 6, demonstrated optimal staining of the RPE. Uniform staining of the RPE for pH 4 beta-galactosidase was seen in both young and old eyes. In contrast, senescence-associated beta-galactosidase (pH 6) staining was seen in the RPE of 16 and 29-year-old, but not 1- and 2-year-old eyes. Senescence-associated beta-galactosidase staining was evident in RPE cells adjacent to cuticular drusen. Optimal bleaching without loss of beta-galactosidase staining was obtained using a 25-minute incubation with 0.05% permanganate.


CONCLUSIONS
The senescence-associated beta-galactosidase histochemistry assay, adapted for use in the primate posterior pole, showed staining of RPE cells in older eyes. Visualization of beta-galactosidase activity in the RPE was enhanced by permanganate bleaching of melanin pigment. This technique could be valuable for identifying senescent RPE cells in human eyes.
Mishima
K
K
Department of Ophthalmology, University of California, Davis 95616-8794, USA.
Handa
J T
JT
Aotaki-Keen
A
A
Lutty
G A
GA
Morse
L S
LS
Hjelmeland
L M
LM
D000375:Aging / Q000502:physiology*; D000818:Animals; D016922:Cellular Senescence / Q000502:physiology; D002829:Choroid / Q000201:enzymology*; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D008253:Macaca mulatta; D008867:Microtomy; D010857:Pigment Epithelium of Eye / Q000201:enzymology*; D016610:Tissue Embedding; D001616:beta-Galactosidase / Q000378:metabolism*
D001616:beta-Galactosidase