Fig. 1. An electron micrograph of human retinal pigment epithelium (RPE) from a 52-year- old donor. AM apical microvilli, BI basal interdigitations, BM Bruch’s membrane, JC apicolateral junctional complexes, L lipofuscin granules, M melanosomes located toward the apical portion of the cell, Mt a high density of basically located mitochondria, POS photoreceptor outer segment. Magnification ×13,100 (Reproduced courtesy of John Marshall, St. Thomas’s Hospital, London.)

AGING CHANGES IN THE FUNDUS

The RPE and its inherent pigmentation are partly responsible for the characteristic red color of the fundus. It is noteworthy that with age the fundus becomes much paler, probably reflecting a loss of melanosomes and an increase in lipofuscin granules. By age 60, the appearance of the fundus begins to show a number of changes. These can include loss of fundus reflexes, greater visibility of larger choroidal vessels, areas of hypoand hyperpigmentation, a marked increase in fundus autofluorescence, and the appearance of focal sub-RPE deposits called drusen [7, 8]. Many of these changes represent the hallmark of early age-related macular degeneration (AMD) or age-related maculopathy. These changes can become more apparent in some individuals, in whom RPE atrophy, confluent drusen, hypoand hyperpigmentation, regions of hypofluorescence, RPE detachment, or subretinal neovascularization can be observed (Fig. 2). Such presentation is typically associated with the later stages of AMD and often coincides with visual impairment. It should be emphasized that age-related changes in the neural retina and RPE show tremendous interindividual variation, which presumably reflects the degree of environmental exposure and genetic susceptibility.

AGE-RELATED CHANGES IN RPE MORPHOLOGY

There have been numerous studies to determine changes in RPE cell density with age. It appears that RPE cell density decreases by about 0.36% per year in the peripheral and equatorial retina with increasing age [9]. However, despite the potentially higher

Fig. 2. Examples of two patients in their late 70s who presented to clinic with visual acuity of 20/40 (6/9) in both eyes. On examination, one patient was found to have large confluent soft drusen (A), but the corresponding autofluorescence images showed intact retinal pigment epithelium (RPE) by relative homogeneous autofluorescence (B). The other patient had less drusen, which were smaller (C), but the autofluorescence images showed the RPE was not intact, indicated by multiple areas of increased autofluorescence throughout the macula. Despite the biomicroscopy findings, the second patient is at higher risk for progression to exudative age-related macular degeneration (AMD) because the RPE is not intact. (Images kindly provided by Dr. Erik Van Kuijk, University of Texas Medical Branch, Galveston.)

metabolic load on the macular RPE, there is no significant reduction in RPE cells in the central retina [10], suggesting that the cells are more resistant to attrition than their peripheral counterparts. Interestingly, Dorey et al. reported that RPE cell loss is greater in blacks than whites [11]; however, this has yet to be substantiated by further studies. It is reasonable to postulate that if RPE loss occurs at a faster rate than overlying

Fig. 3. A Confocal image of flat mount human retinal pigment epithelium (RPE) showing the variable distribution of lipofuscin between individual cells. The annulus devoid of lipofuscin surrounds a druse similar to that shown in D. B The variable relative fluorescence intensity (RFI) between individual RPE cells from a 68-year-old. C, D Fluorescent images of retinal cross sections showing the different distributions of lipofuscin associated with drusen. (Images provided by M. Boulton and K. Njoh.)

photoreceptor cells, then the overall functional load on the RPE will significantly increase. However, reports remain contradictory since one study has reported that the ratio of RPE cells to photoreceptor cells decreases with age across the fundus [11], while a subsequent study failed to reveal any significant changes in the central retina [10]. The inability to generate unequivocal data on RPE cell loss with age suggests that this is not a major event during our life span. In the peripheral retina where cell loss is apparent, there is an overall increase in the area of remaining cells brought about by spreading to fill in the gaps. This increase in cell area is associated with an increase in height of RPE cells [11, 12]. It should be emphasized that there is considerable cell–cell variability throughout the monolayer with respect to appearance, pigment content, and protein expression [13], and this variability increases with age (Fig. 3a,b).

However, there is considerable evidence of morphological change to RPE cells at an ultrastructural level [1, 3]. This includes loss of the typical epithelial cobblestone morphology and the appearance of a more pleotrophic cell layer; hyperplasia and regions of multilayered cells; disorganization of apical microvilli; a reduction in basal interdigitations; and an increase in intracellular pigment granules.

Melanosomes

The RPE melanosomes show a regional distribution across the fundus, decreasing from the equator to the posterior pole with a marked peak at the macula [12, 14]. While this differential distribution of melanosomes is maintained throughout life, there is a significant decline in the number of granules in all regions after age 40. Feeney-Burns estimated the decline in melanin granules to be about 35% between the early and late decades of life [12]. This decline will result in decreased light absorption and reduced binding of xenobiotics and ions in the aged RPE [15]. The loss of melanosomes correlates with the association of melanosomes with lysosomes or lipofuscin granules and voiding of material into the sub-RPE space. Furthermore, aged RPE melanin granules have often lost their cigar shape and are less electron dense, possibly due to partial degradation by lysosomal enzymes [12].

RPE melanosomes demonstrate a number of biophysical changes with increasing age. Analysis of the absorption spectra of melanosomes from young and old donors demonstrates an age-dependent increase in the absorption of intact melanosomes between 250 and 450 nm [16]. However, the overall effect of this age-related increase in absorption by melanosomes is negated by the age-related loss of melanosomes. Not surprisingly, melanosomes exhibit a weak fluorescent emission when excited at 364 nm. However, RPE melanin demonstrates an age-dependent decrease in the blue emission of young melanosomes, with a shift toward red in the fluorescence spectrum with increasing age [16].

Lipofuscin

Lipofuscin granules accumulate within the RPE throughout life, eventually occupying up to 19% of cytoplasmic volume by 80 years of age [12, 17]. These granules, which are less electron dense than melanosomes, accumulate in the midto basal cytoplasm of RPE cells and are normally around 1 m in diameter [3]. Their composition is complex, and studies are so far equivocal due to the variable “purities” of the granules used for the analyses [18–20]. However, recent evidence would indicate that lipofuscin granules have minimal protein content [21]. Topographically, maximal accumulation of lipofuscin granules occurs in the posterior pole, albeit with a decrease at the fovea. This correlates with the density distribution of rod photoreceptors, which are thought to be the primary substrate for lipofuscin. A characteristic feature of lipofuscin is its goldenyellow fluorescence when excited by short-wavelength light. It appears that the overall fluorescent intensity of lipofuscin granules increases with age by as much as 40% [16]. However, studies demonstrated considerable heterogeneity in the emission properties of individual granules from the same donor [22, 23].

Pigment Complexes

With increasing age, a variety of pigment complexes is present with the RPE [24]. Predominant are melanolysosomes and melanolipofuscin. The origin of melanolipofuscin is unclear. The common view is that it represents fusion of melanosomes with lipofuscin. However, the observation of predominantly lipofuscin-containing complexes may suggest some melanin synthesis in postmitotic cells. The regional distribution of