The photoreceptor cells form a functional unit with the underlying retinal pigment epithelium, a mononuclear cell layer located between the choroidal vascular network and photoreceptor outer segments. Retinal pigment epithelial cells regulate important functions, such as the delivery of nutrients and metabolites to the photoreceptors, and control retinoid metabolism, outer segment phagocytosis, neuroretinal adhesion, interphotoreceptor metabolism, absorption of light by the melanosomes, and participation in the constitution of the blood–retina barrier. Their alterations are therefore a common feature within the retinal aging process.

The extremely large number of genes described and implicated in hereditary retinal diseases indicates photoreceptor cells as most sensitive to biochemical modifications and led us also to a better understanding of functional and morphological changes that may occur during normal and pathological retinal aging.

MORPHOLOGICAL ALTERATIONS

Morphological changes accompanying the aging process primarily involve the photoreceptor cells and underlying retinal pigment epithelial cells. They are manifested by cell atrophy and cell loss, depigmentation and hyperpigmentation of the retinal pigment epithelium, progressive accumulation of lipofuscin, drusen formation, thickening of Bruch’s membrane, and the appearance of basal deposits [3, 4].

Neural Changes

Neural cell loss is one major characteristic of aging in the human retina, with rod photoreceptor cells more affected than cone photoreceptor cells [5, 6]. Approximately half of all rods in whole retina are lost between the second and the fourth decade, with an annual disappearance of 970 cells/mm2 [6]. Curcio and colleagues showed that the density of rods in the central retina decreases by 30% between the ages of 34 and 90 years, whereas the number of cones remains stable [5]. However, the kinetics of rod loss does not follow a sigmoidal curve and suggests that the neural cell death rate is not related solely to the accumulation of damage [7].

Rod vulnerability is a frequent phenomenon in hereditary retinal diseases. In retinitis pigmentosa, most of the mutated genes known today are expressed specifically in rods. In these cases, the disease develops sequentially, with an initial rod loss followed by a secondary cone loss [8, 9]. Studies of animal models of retinitis pigmentosa have shown that cones are lost some time after rods and suggest that the cone loss is independent from the initial mechanism that causes the death of rods [10]. Apparently, the survival of cones depends on the presence of rods, even if these rods are not functional any longer. A rod-derived viability factor has been described recently and supports the hypothesis that rods secrete a factor mandatory for cone survival [11–14]. A similar mechanism may be present in retinal aging [15].

Mitochondrial alterations may play an important role in the aging process of photoreceptor cells. Mitochondria are mandatory for the synthesis of adenosine triphosphate (ATP), which includes the photoreceptor-specific ATP-binding cassette transporter, a key agent in the retinoid cycle between the retinal pigment epithelium and photoreceptors, through oxidative phosphorylation. As highly metabolically active cells, photoreceptors are the

prime site for acquired mitochondrial DNA (mtDNA) mutations [16]. During its whole life, the retina is exposed to light of variable wavelength, including ultraviolet (UV) light that may contribute to mtDNA damage in retinal cells.

As in other organs, retinal cells encounter a cumulative amount of oxidative and metabolic stress. The accumulation of damaged molecules leads to dysfunction of various metabolic and signaling pathways with subsequent impaired cellular function and cell death. The outer retina is exposed to a relatively high oxygen tension that is close to that found in arterial blood. The photoreceptor membranes are rich in polyunsaturated fatty acids. The combination of these different elements results in a tissue that is especially prone to oxidative damage [17].

Astrocytes display higher levels of glial fibrillary acidic protein and more cytoplasmic organelles [18], indicating an increased cell metabolism in the aging retina. Because of the relatively high antioxidant content, astrocytes are especially resistant to oxidative stress, suggesting that they may be able to protect neurons from free radicals by upregulating enzymatic and nonenzymatic antioxidant defenses [18]. The decreased number of ganglion cells is in line with the rod photoreceptor loss described [6, 19]. About 40% of all ganglion cells are lost by the ninth decade, which implies that their loss contributes to visual function deficits found in aged individuals.

Retinal Pigment Epithelium and Lipofuscin Formation

An apparently universal feature of aging is the accumulation of fluorescent, nondegradable material, termed lipofuscin, which is observed primarily in all postmitotic and long-lived cells in a variety of organisms. It has been hypothesized that this material forms due partly to ageand disease-dependent defects in the proteolytic capacity of cells, resulting in toxic biomolecules that may interact with normal cell function [20, 21].

The accumulation of lipofuscin in retinal pigment epithelial cells appears to be an important marker of retinal aging. Oxidative damage seems to play an important role in age-related retinal pigment epithelial cell damage, but the mechanisms are not completely understood. Lipofuscin is a by-product of photoreceptor outer segment turnover

[22]and is a primary source for reactive oxygen species, responsible for cellular and extracellular matrix alterations. Lipofuscin accumulation in postmitotic retinal pigment epithelial cells serves as a clear example of an aging cell. It accumulates in an agedependent manner in the lysosomal compartment of the retinal pigment epithelial cells

[23]and is most likely harmful when present in sufficient amount [24]. Lipofuscin becomes apparent within the retinal pigment epithelium by the age of 10 years. By the age of 40 years, already 8% of the cytoplasmic volume is occupied, and by 80 years of age this figure has risen to more than 20%.

Lipofuscin may influence dramatically the physiology of the retinal pigment epithelium due to its potential toxicity. Lipofuscin is a heterogeneous material composed of a mixture of lipids, particularly lipid peroxides; proteins; and different fluorescent compounds, derived mainly from vitamin A, as a by-product of the visual cycle. N-Retinylidene- N-retinylethanolamine (A2E) is the major autofluorescent component of lipofuscin. It is formed after hydrolysis of its precursor, A2E-phosphatidyletholamine (A2-PE), and may alter the process of lysosomal degradation in retinal pigment epithelial cells by inhibition of the ATP-dependent lysosomal proton pump [25] as well as by its detergent

and phototoxic properties [21, 26]. Results obtained in cultured retinal pigment epithelial cells indicated further that A2E is able to induce apoptosis via a mitochondria-related and wavelength-dependent mechanism [27]. More superoxide anions are generated in granules exposed to blue light (400–520nm) than in granules exposed to red light (660–730 nm) or full white light [28]. Further analysis of isolated lipofuscin granules allowed the observation of other toxic molecules, including malondialdehyde (MDA), 4-hydrox- ynonenale (HNE), advanced glycation end products (ÂGE) [29], and A2E) [30]. These proteins have shown posttranslational modifications, underscoring the potential contribution of oxidative damage in lipofuscin biogenesis with subsequent impaired cellular function and cell death [31, 32].

An additional retinal pigment epithelial lipofuscin fluorophore that originates as a condensation product of two molecules of all-trans retinal (ATR) dimer and forms a protonated Schiff base conjugate with phosphatidylethanolamine (ATR dimer–PE) has been identified in isolated bovine photoreceptor outer segments, although in much smaller quantities than A2E or its precursor A2-PE. This ATR dimer may play an important role in the photoreactivity of retinal pigment epithelial lipofuscin as it undergoes a photooxidation process and has UV-visible absorbance maxima at 285 and 506 nm [33].

Bruch’s Membrane and Choroid

A number of age-related changes have been described in Bruch’s membrane, which is situated between the retinal pigment epithelium and the choriocapillaris [34–37]. Its most prominent alterations are the formation of drusen and basal deposits. These deposits contain a variety of inflammation-related proteins, including C-reactive protein, vitronectin, α-antichymotrypsin, amyloid P component, and fibrinogen [38–41]. They are observed also in atherosclerosis, dermal elastosis, membranoproliferative glomerulonephritis type II, and Alzheimer’s disease [40, 41], strengthening the hypothesis for local inflammation with complement activation and immune complex deposition in the formation of drusen. The identification and localization of multiple complement activators (nuclear fragments, membrane-bound vesicles, lipofuscin, cholesterol, and microfibrillar debris) [42, 43] as well as terminal complement compounds support the conclusion that they act as a trigger for the activation of the complement cascade, a basic physiological reaction to foreign cells, dead cells, or cell fragments. Cellular debris that derives from compromised retinal pigment epithelial cells may act as an additional chronic inflammatory stimulus for drusen formation. Failure to eliminate the entrapped material generates an additional local proinflammatory signal, sufficient to trigger subsequent events, including local upregulation of cytokines, acute phase reactants, and other proinflammatory mediators as well as the invasion of incipient drusen by processes of dendritic cells from the choroid.

Bruch’s membrane functions as a physical barrier to cell movement, restricting the passage of cells between the choroid and the retina. Virtually all of the nutrition for the central retina derives from the choroid. In young human eyes, this layer is only 2 µ thin, but gradually thickens with increasing age, reaching about 6 µ late in life. Massive accumulation of esterified cholesterol renders the Bruch’s membrane increasingly hydrophobic with age [44]. A growing number of fibers with a higher incidence of fibers displaying an atypical banding periodicity as well as increased calcification [36] leads