Senescence of the retinal pigment epithelium.

Senescence of human cells has largely been studied as an in vitro phenomenon resulting from replicative exhaustion. The literature contains many studies of retinal pigment epithelium (RPE) cells which document replicative senescence. Several studies by Burke and others illustrate the relationship between donor age and replicative lifespan, the relationship between geographical location of RPE in the posterior pole and replicative lifespan, and the phenomena of altered cellular morphology and decreased culture saturation density for senescent RPE cells. Other studies have focused on the alterations of the expression of specific genes or the alteration of enzymatic activities during the senescence of RPE cells in vitro. Recently, a technique utilizing a histochemical staining procedure for beta galactosidase has been developed which identifies senescent cells. Normal beta galactosidase histochemistry which identifies the lysosomal form of the enzyme is performed at pH 4.0, while senescence-associated beta galactosidase activity is observed at pH 6.0 and is observed in the cytoplasm. We have studied the replicative senescence of human RPE cells in vitro using this procedure and have also measured the length of chromosomal telomeres to identify the aging of cultures in vitro. Our results show that RPE cultures accumulate beta galactosidase positive cells as a function of the number of population doublings and that these data correlate with the shortening of chromosomal telomeres to a functional limit observed for many human cell types at senescence. We have also recently extended this work to the development of a senescence-associated beta galactosidase procedure for observing senescent RPE cells in vivo. Basically, the same histochemical procedure is used with a post-staining bleaching step to clearly visualize staining in the RPE. Our first studies were performed on globes from Rhesus monkeys at a variety of ages from 1 year to 29 years of age. The results show the accumulation of beta galactosidase positive cells in the older monkey eyes. We have also examined several human eyes in an attempt to observe whether any relationship exists between beta galactosidase staining and age, pathology (diabetes, basal laminar deposits), and geographical location (macula vrs. periphery). These studies represent a first effort to determine if senescent RPE are present in vivo. It will be important to extend these studies so that these data might be expressed on a quantitative bases.