Senescence of the retinal pigment epithelium

Senescence of human cells has largely been studied as an in vitro phenomeno n resulting from replicative exhaustion. The literature contains many studi es of retinal pigment epithelium (RPE) cells which document replicative sen escence. Several studies by Burke and others illustrate the relationship be tween donor age and replicative lifespan, the relationship between geograph ical location of RPE in the posterior pole and replicative lifespan, and th e phenomena of altered cellular morphology and decreased culture saturation density for senescent RPE cells. Other studies have focused on the alterat ions of the expression of specific genes or the alteration of enzymatic act ivities during the senescence of RPE cells in vitro. Recently, a technique utilizing a histochemical staining procedure for beta galactosidase has bee n developed which identifies senescent cells. Normal beta galactosidase his tochemistry which identifies the lysosomal form of the enzyme is performed at pH 4.0, while senescence-associated beta galactosidase activity is obser ved at pH 6.0 and is observed in the cytoplasm. We have studied the replica tive senescence of human RPE cells in vitro using this procedure and have a lso measured the length of chromosomal telomeres to identify the aging of c ultures in vitro. Our results show that RPE cultures accumulate beta galact osidase 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-as sociated beta galactosidase procedure for observing senescent RPE cells in vivo. Basically, the same histochemical procedure is used with a post-stain ing bleaching step to clearly visualize staining in the RPE. Our first stud ies 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 galact osidase positive cells in the older monkey eyes. We have also examined seve ral human eyes in an attempt to observe whether any relationship exists bet ween beta galactosidase staining and age, pathology (diabetes, basal lamina r deposits), and geographical location (macula vrs. periphery). These studi es represent a first effort to determine if senescent RPE are present in vi vo. It will be important to extend these studies so that these data might b e expressed on a quantitative bases.