studied the impact of ageing on photoreceptor cell density in foveal and temporal equatorial regions in eyes from donors with ages spanning from the second to the ninth decade of life [35]. Equatorial cones decreased at a uniform rate of 16 cells/mm2/year while age-associated decrease was nonuniform for equatorial rods with the greatest loss being between the second and the fourth decades (970 cells/mm2/year). By contrast, while the variability of cone density at the foveal center was large between individuals within each decade, no signiÞcant differences were found in cone densities at the foveal center from the second to ninth decade. The authors concluded that rod photoreceptors are more vulnerable to loss during ageing than cone photoreceptors and that photoreceptor loss accompanying ageing is less pronounced in the fovea than in the peripheral retina [35]. These observations were supported by Curcio and colleagues who reported that the total number of foveal cones was remarkably stable while, in contrast, rod density decreased by 30%, beginning inferior to the fovea in midlife and culminating in an annulus of deepest loss at 0.5Ð3-mm eccentricity by the ninth decade [36]. The space vacated by dying rods was accommodated by larger rod inner segments. Why the rods of central retina, which share a common support system and light exposure with the neighbouring cones, are preferentially vulnerable to ageing remains an area of considerable debate. However, there is now some evidence that rods may secrete cone survival factors [37Ð39].

However, in addition to a signiÞcant loss of retinal ganglion cells and photoreceptors, there is also a signiÞcant age-related reduction in capillary density, synaptic bodies, intercellular connections, and protein content in the neural retina [31], a 21% decline in rod bipolar cells from 35 to 62 years [40] and an increase in retinal remodeling and sprouting of dendrites [28, 41, 42].

It has been proposed that retinal ageing is accompanied by activation of gene-sets which are involved in local inßammatory responses [43]. Complement activation, upregulated chemokine expression, and microglial activation have been observed in aged mice [43]. Deterioration of blood-retinal barrier function in aged rats has been evidenced by leakage of intravascular tracer into the retinal parenchyma and reduced tight junctional integrity and the presence of major histocompatibility complex (MHC) class II-positive resident microglia, activated T cells, and monocyte-like cells

[44]. In the ageing retina, the two inßammatory pathways affected as a result of age-related tissue stress: The complement cascade and retinal microglia activation suggest a low-grade chronic inßammation. This lowgrade chronic inßammation induced by endogenous noxious stress has recently been proposed by Medzhitov to represent a modiÞed form of inßammation, termed para-inßammation. Although the physiological purpose of para-inßammation is to restore tissue functionality and homeostasis, it may become chronic or convert into inßammation if tissue stress or malfunction persists for a sustained period as occurs in the aged retina. Chronic para-inßammation contributes to the initiation and progression of many human diseases including age-related neurodegenerative diseases [45].

3.5Ageing of the RPE

The RPE is a hexanocuboidal monolayer of cells located between the neural retina and the choroid that plays a critical role in ensuring the function and survival of the overlying photoreceptor cells [3, 46]. These functions include: maintenance of a bloodretinal barrier; transport and storage of retinoids; phagocytosis and degradation of photoreceptorÕs outer segments; protection of the outer retina from oxidative damage; neural and vascular protection through the secretion of growth factors and cytokines. However, while we have a classic view of the RPE, it must be emphasized that the structure and function of the RPE varies considerably depending upon both retinal location and ageing [3, 46Ð49]. Macular RPE cells, which cover an area of around 5Ð8 mm diameter, measure about 14 mm across while extramacular RPE cells can measure up to 60 mm with cell size and shape becoming particularly irregular [47, 50, 51]. Macular cells are likely to be the most metabolically active since they are located in a region critical for visual function, and signiÞcant differences in functional activity between central and peripheral cells have been reported [3, 52Ð54]. The RPE is particularly prone to ageing since it is normally a non-divid- ing cell layer throughout life and is exposed to high levels of oxidative stress; these factors together are conducive toward the lifelong accumulation of cellular damage. Cumulative damage will result in structural

Fig. 3.3 Diagrammatic representation of age-related changes in the RPE. A young cell is represented in (a) as a comparison with an aged RPE cell (b) which shows increased diameter, reduced numbers of microvilli, loss of orientation of melanosomes and their partial degradation, the appearance of lipofuscin and pigment complexes, reduced numbers of mitochondria and basal interdigitations, formation of basal laminar deposits and drusen, and reduced choriocapillaris density

a

Photoreceptor

Outer Segment

Melanosomes

Tight Junction

Nucleus

Mitochondria

Bruch’s Membrane

Choriocapillaris

b

Reduced number

of microvilli

Melanosomes partially degraded with loss of

orientation Apperance of

pigment complexes

Accumulation of lipofuscin granules

and functional changes in the RPE (Fig. 3.3) and ensuing RPE dysfunction can lead to many of the pathological changes associated with atrophic AMD.

3.5.1Changes in RPE Cell Density

Although numerous studies have been undertaken to determine the change in RPE cell density with increasing age, outcomes vary and are highly dependent on the region of the fundus assessed. Panda-Jonas and colleagues assessed RPE cell number in 53 normal human donor eyes from individuals with donor age ranging from 40 to 77 years [55]. They reported that the total number of RPE cells ranged from 2,130,500

to 4,653,200 and was positively correlated with the number of rods and cones and the total area of the retina. The RPE cell density decreased signiÞcantly from the fovea to the midperiphery and was lowest in the outer peripheral fundus regions. Interestingly, RPE cell density was highest in the nasal fundus region compared with any other fundus quadrant. Overall, the retinal pigment epithelial cell density decreased by about 0.3%/year with increasing age [55]. Gao and HollyÞeld observed similar cell loss of equatorial RPE at a uniform rate of 14 cells/mm2/year from the second to the ninth decade (Table 3.1) [35]. However, no signiÞcant differences were found in RPE cell densities at the foveal center from the second to ninth decade, suggesting that the densities of RPE cells

remain stable throughout life in the normal retina and that RPE cells in this region are more resistant to attrition than their peripheral counterparts. By contrast, Del Priore and colleagues reported signiÞcant number of apoptotic cells in the macular region with the highest density in the fovea in aged eyes [56]. It has been suggested that RPE cell loss is greater in blacks than whites [57]. RPE cell loss is associated with an increase in the area of remaining cells which are required to Þll in the gaps left by the dead cells and is accompanied by an increase in height of RPE cells [57, 58]. It is reasonable to hypothesize that if RPE loss, especially in the peripheral and equatorial retina, occurs at a faster rate than overlying photoreceptor cells, then the overall functional load on the RPE will signiÞcantly increase. However, the ratio of RPE cells to photoreceptors requires clariÞcation since one study has reported the ratio of RPE cells to photoreceptor cells to decrease with age across the fundus [57] while a second failed to Þnd any association [35]. The inability to generate unequivocal data on RPE cell loss with age suggests that this is not a major event during our lifespan.

3.5.2Subcellular Changes in the RPE

Ultrastructural studies show a loss of the typical epithelial cobblestone morphology and the appearance of a more pleotropic cell layer; hyperplasia and regions of multilayered cells; disorganization of apical microvilli; a reduction in basal interdigitations and an increase in intracellular pigment granules [3, 47]. However, there is considerable cell-to-cell variability throughout the monolayer with respect to appearance, pigment content, and protein expression [59], and this variability increases with age.

3.5.3Accumulation of Lipofuscin

Perhaps the most obvious age-related change in the RPE is the appearance of lipofuscin granules. Lipofuscin granules are normally around 1 mm in diameter and accumulate in the mid to basal cytoplasm of RPE cells [47] throughout life where they may eventually occupy up to 19% of cytoplasmic volume by 80 years of age (Fig. 3.4) [58, 60]. Lipofuscin is contained within secondary lysosomes. The composition

of these granules is complex, and they consist of numerous retinoid derivatives, lipid adducts, and oxidatively modiÞed components [32, 61]. Despite extensive analyses, the precise origin and composition of lipofuscin remains elusive, but there is strong evidence that RPE lipofuscin has minimal protein content [62]. However, the retinoid component has been well studied and A2E appears to be one of the dominant ßuorophores in lipofuscin [63]. Accumulation of lipofuscin correlates with the density distribution of rod photoreceptors which are thought to be the primary substrate, and thus, the highest accumulation of lipofuscin occurs in the posterior pole where rod photoreceptor density is highest. Interestingly, it has been suggested that A2E may show an atypical distribution in the RPE with levels being lower at the posterior pole and increasing toward the periphery [64]. However, this contradiction has yet to be corroborated. A characteristic feature of lipofuscin is its golden-yellow ßuorescence, largely due to the ßuorescence of retinoid components, when excited by short wavelength light. It appears that the overall ßuorescent intensity of lipofuscin granules increases with age by as much as 40% [65]. However, studies demonstrate considerable heterogeneity in the emission properties of individual granules from the same donor [66, 67].

It is now well recognized that lipofuscin is a potent photoinducible generator of a range of reactive oxygen species (ROS) including superoxide anion, singlet oxygen, hydrogen peroxide, and lipid peroxides [68Ð 70]. ROS production is strongly wavelength dependent with, for example, efÞciency increasing with decreasing wavelength by a factor of 10 when excitations of 420 and 520 nm are compared. Furthermore, the photoreactivity of individual lipofuscin granules increases with age [71]. It is thus not surprising that exposure of RPE cells containing lipofuscin to blue light (390Ð550 nm) results in lipofuscin-dependent lipid peroxidation (malondialdehyde and 4-hydroxy- nonenal), protein oxidation (protein carbonyl formation), loss of lysosomal integrity, mitochondrial DNA damage, and RPE cell death (Fig. 3.4) [33, 72, 73]. The photosensitizers principally responsible for ROS generation remain unclear. The most studied of the potential photosensitizers is A2E and its related compounds which can, when exposed to blue light, induce RPE apoptosis [74Ð76]. However, the potency of A2E is at least an order of magnitude less than RPE lipofuscin granules containing equivalent A2E concentrations

Viability as a % Control

100

75

50

**

25

0

Time (Hours)

Fig. 3.4 Age-related accumulation and photoreactivity of lipofuscin in RPE cells. (a) A transmission electron micrograph of human RPE from a 52-year-old donor (Reproduced courtesy of John Marshall, St ThomasÕs Hospital, London). Note, photoreceptor outer segment (POS), melanosomes (M) located toward the apical portion of the cell, lipofuscin granules (L) in the central to basal region, a high density of basically located mitochondria (Mt), BruchÕs membrane (BM). (b) Fluorescence microscopy of tissue sections from 9-, 43-, and 97-year-old donors showing an increase in ßuorescent lipofuscin granules with increasing age. (c) A confocal image of ßat-mount human RPE showing

the variable distribution of lipofuscin between individual cells. The annulus devoid of lipofuscin surrounds a Drusen (Image provided by Boulton and Njoh). (d) The photoreactivity of RPE lipofuscin. Lipofuscin-fed RPE cells (□,■) and cells lacking lipofuscin (○,●) were exposed to 2.8 mW cm2 light (390Ð550 nm [□,○]) or maintained in the dark (■,●) for up to 48 h. The ability of the RPE cells to reduce MTT to a blue formazan product (absorbance measured at 590/630 nm) was utilized as a measure of cell viability. The insets show light micrographs of lipofuscinfed RPE cells exposed to dark (left) and light (right) (Reproduced in part from Boulton [32] and Davies et al. [33])

suggesting that (a) there are more potent metabolites of A2E, (b) retinoids other than A2E are involved, or

(c) the presence of other more reactive chromophores which may be non-retinoid in origin [77, 78]. This is

supported by the work of Rozanowska and colleagues who observed a substantial age-related increase in ROS generation in the chloroform-insoluble fraction of lipofuscin granules but not the chloroform-soluble