age of 80, with the major lipofuscin component A2-E playing the most important role.

The following functional processes of the RPE are influenced by A2-E:

•Activation of complement

•Detergent effect

•Decreased catabolism

•Induction of free radicals after exposition to light

•Enhanced toxicity of light

•Diminished rate of phagocytosis

•More apoptosis

•Inhibition of the proton pump

•Lower lysosomal pH

A progressive accumulation of intracellular debris

during the ageing process promotes apoptosis as a form of cell death and a beginning atrophy of the RPE [1].

5.1.3Deposits in the RPE

The described degenerative changes of the RPE together with age-related changes of the underlying extracellular matrix can lead to a critical shortage of nutrients for photoreceptors and may also lead to malnutrition of the RPE itself. In reaction to this insufficient supply, the clinical manifestations of either neovascular exudative (see Sect. 5.3) or atrophic AMD (see Sect. 5.5) - as the two specifications of the disease – are possible.

5.2Bruch’s Membrane

5.2.1Structure of Bruch’s Membrane

Bruch’s membrane is a tissue layer formed by the choroid and the RPE. Bruch’s membrane is not considered a phospholipid bilayer as in the classical definition of a membrane. Instead, it is a layer comprised of choroid and retinal pigment epithelium and consists of interstitial connective tissue. Hence, Bruch’s membrane may be considered as the inner facet of the choroid and the outer facet of the retina. A pentalaminar structure was revealed by electron microscopy (Fig. 5.3). The five layers are termed, from outside to inside, the choroidal basement membrane, outer collagenous layer, elastic layer, inner collagenous layer and the RPE basement membrane as the innermost stratum. The inner border of Bruch’s membrane is clearly defined by the RPE basement membrane. In

contrast the outer border is restricted by the choroidal basement membrane but is also found between the choroidal capillaries.

The thickness of Bruch’s membrane varies depending on the age and topography, with the thinnest region being 2 mm in the central area of a young eye [3].

5.2.2Age-Related Changes in Bruch’s Membrane

Complex changes appear with increasing age, including thickening of Bruch’s membrane (see Fig. 5.3, compare a and b).

This thickening is part of the normal ageing process [4]. Because of the high interindividual differences for the examined parameters, the authors of this chapter estimated that half of the thickening is due to natural ageing, and the other half is due to other factors such as the genetic disposition or environmental influences. Electron and light microscopic studies have also detected thickening of Bruch’s membrane in AMD patients [5, 6]. Deposits can develop in vesicular and membranous structures [7]. Membrane-shaped debris can appear linear or have similarities to a double-lay- ered phospholipid membrane.

With increasing age, the collagen components in Bruch’s membrane are less soluble. This is especially detectable in the macular region and associated with a significant increase in non-collagenous proteins [8]. The increased amount of different extracellular matrix proteins found in Bruch’s membrane is correlated with increasing age [9]. Furthermore, the production of the extracellular matrix protein vitronectin of RPE cells can be stimulated by activated complement [10]. Enhanced staining for collagen type IV with increasing age was found in immunohistochencal examinations. In contrast, the examination of collagen type III did not reveal any age-dependent changes in relation to the total collagen content [8].

Cross-linked collagen fibrils have an increased resistance against degradation by matrix metalloproteinases [11]. Such cross-linking is characteristic of the normal ageing process of the collagen macromolecules [12] and is also detected in Bruch’s membrane. An early cross-linking of collagen in Bruch’s membrane may be responsible a more rapidly increasing accumulation of debris [13]. Once collagen fibrils have cross-linked a calcification of the elastic layer of Bruch’s membrane is observed [14, 15]. In the latter work, it was extrapolated that the

a

c

b

CIV, CV, Laminin,

Vitronectin, HS

CI, CIII, CV; Fibronectin, KS,

DS

Elastin, CVI, Fibronectin

CI, CIII, CV; Fibronectin, KS,

DS

CIV, CV, CVI, Laminin,

Vitronectin, HS

Marshall et al 1998 (modified)

Fig. 5.3 Structure of Bruch’s membrane. (a) Transmission electron micrograph of Bruch’s membrane of a 3-year-old donor. (b) Transmission electron micrograph of Bruch’s membrane of a 62-year-old donor. (c) Schematic structure of Bruch’s membrane including specification of the thickness of each layer and its composition. BL RPE basal lamina of the retinal pigment

epithelium, ICL inner collagenous layer, EL elastic layer, OCL outer collagenous layer, BL CC basal lamina of the choriocapillaris, CI collagen type I, CIII collagen type III, CIV collagen type IV, CV collagen type V, CVI collagen type VI, KS keratan sulphate, DS dermatan sulphate, HS heparan sulphate

sum of age-related changes slows down the hydraulic conductivity of Bruch’s membrane so that theoretically at the age of 130 no further flow-through will be possible.

In addition to the chemical modifications of the collagen fibers, collagen solubility is also reduced. This

reduction is found in about 50% of the total collagen at the age of 90 or older [8]. The lower solubility results from denaturation in combination with an altered structure and chemical modifications such as glycosylations. The well-known “advanced glycation end products” (AGEs) pentosidine and carboxymethyl

Fig. 5.4 Sketch of age-dependent thickening of Bruch’s membrane (BM) in differently aged specimens: (a) up to 30 years and (b) more than 60 years; PR photoreceptor cells, RPE retinal

pigment epithelium, bm basement membrane, ICL inner collagenous layer, EL elastic layer, OCL outer collagenous layer, CC choroid with capillaries

lysine are found in aged Bruch’s membrane, in basal laminar deposits and in soft drusen [16, 17]. AGEs are strong inducers of cross-links in proteins [18, 19] and may reduce the permeability (pore size) of the extracellular matrix. Moreover, AGEs hamper the enzymatic proteolysis of proteins and disturb the fine-tuned system of synthesis and degradation [20, 21]. The multiple changes in protein structure can make the structure of Bruch’s membrane dense, reduce the aqueous basic matrix between the fibrils and therefore decrease the passage of fluids through Bruch’s membrane (Fig. 5.4).

Thiol groups are involved in the in interand intramolecular protein cross-linking. With increasing age there is a linear decrease of free thiol groups in amino acids which is interpreted as an augmented occurrence of disulfide bonds [3]. This cross-link age may occur between structural fibres, between structural proteins as well as between diffusing proteins. The accumulation of chemical changes in structural proteins and the matrix hampers the extracellular matrix renewal of Bruch’s membrane.

In addition to the changes in the collagenous layers of Bruch’s membrane, the composition of the extracellular matrix proteins in the basement membranes can be modified especially in the RPE. With increasing age decreased immunohistochemical staining for collagen IV and laminin−particularly in the basement membrane of the RPE when compared to the basement membrane of the choroidal endothelium−is observed [22]. Because of the intact ultrastructure of the basement membrane, an enhanced cross-linking that results in antigen masking of the recognized epitopes is suspected.

Furthermore, with increasing age an increased deposition of lipids alters the biochemical characteristics of

Bruch’s membrane (Fig. 5.5a, b) [23]. This correlation was found true for neutral fatty acids detected by oil red O staining and for phospholipids detected by Sudan black B staining, although the ratio of both lipid species and the total amount of extracted lipids varied between the different specimens. In eyes of individuals aged 30 years or less, no lipid staining was detected, while different staining patterns occurred in specimens of patients aged 31–60 years. Donor eyes of individuals aged 61 years or more always showed a more or less stronger positive staining for both types of lipids [23]. Further thin layer and gas chromatographic analyses of Bruch’s membrane lipids confirmed the age-related increase of fatty acids and a great interindividual difference with regard to their composition and increment [25]; see review [26]. The results concerning the composition of phospholipids point to a cellular rather than a plasmatic origin. The authors of this chapter suggest the RPE as a likely source.

The age-dependent increase of primarily neutral fatty acids, esterified and non-esterified cholesterol in Bruch’s membranes of human donor eyes was confirmed by several studies [22, 27, 28]. In lipoprotein-like particles of Bruch’s membrane esterified cholesterol was the predominantly identified neutral lipid [29] with weaker staining in the peripheral Bruch’s membrane [28]. Additional biochemical studies revealed a stronger deposition of lipids in macular than in peripheral areas of Bruch’s membrane (Fig. 5.5c, d) [24].

Early ultrastructural examinations already showed many round, small (mostly less than 100 mM in diameter) electron-permeable membranous or vesicular open areas in Bruch’s membrane in older eyes [30, 31]. Fatty acids are washed out during the preparation of the specimen in

RPE

BM

CC

10.0

8.0

6.0

4.0

2.0

Age, y

Fig. 5.5 (a, b) Staining of lipids with oil red O of a middle-aged (30–60 years, a) and elderly (>60 years, b) specimen with increasing staining of retinal pigment epithelium (RPE), Bruch’s membrane (BM) and choroid (CC); brown = melanin of RPE. ([23] with friendly permission of Elsevier) (c, d) Positive cor-

relation of the amount of extracted lipids from Bruch’s membrane with increasing donor age. In macular areas a stronger increase of lipid content (c) is detected than in the retinal periphery (d) [24]

conventional electron microscopy. Therefore, only investigations with a fixation technique that preserved neutral fatty acids could demonstrate that these vesicles in Bruch’s membrane were filled with electron-dense material [27, 32]. The vesicular structures in Bruch’s membrane were filled with esterified and non-esterified cholesterol. These lipids were also detectable with oil red O and Sudan black staining. Moreover, the distribution and size of the vesicles were consistent with structures that were rich in lipids and were detected in light microscopic studies. The quick-freeze/deep-etch technique as another method of preparation disclosed different surface and core structures of the lipid particles and provided evidence of the fusion of particles [33]. In peripheral Bruch’s membrane the deposition of lipoprotein particles was also found. However, there was less material aggregation and it took place at a slower rate compared to the macular regions [34].

During the normal ageing process an increased amount of TIMP-3 (tissue inhibitor of matrix metalloproteinases) is detected in Bruch’s membrane. In eyes of AMD patients this increase in TIMP-3 is more pronounced when compared to age-matched controls [35].

TIMP-3 is an inhibitor of matrix metalloproteinases (MMP), enzymes that are important in tissue turn over and whose active forms facilitate permeability of Bruch’s membrane [36]. The inactive proforms of MMP-2 and -9 are found in enhanced amounts in aged Bruch’s membrane, but not the active forms [37]. Therefore, an imbalanced ratio of TIMP and MMP may additionally hamper the dynamic modulation in Bruch’s membrane beside the age-related enhanced brittleness caused by collagen modifications.

All these structural changes lead to sinking hydraulic conductivity, which means the ability to let fluids pass [3, 38, 39]. The main resistance is located in the inner collagenous layer. For the first decades of life, this finding is explained by the reduction in pore number and size built up by the meshed structure of the collagenous fibers [3, 39, 40]. Hence, larger proteins and lipids are slowed down while passing Bruch’s membrane and can accumulate in the different layers. From the fifth decade of life the permeability to water in Bruch’s membrane decreases exponentially, which is explained by the exponential increase in the amount of deposited lipids [3, 22–24, 41, 42].

These lipids were identified as phospholipids and neutral lipids whose accumulation begins in the fourth