that are termed “fibrous” or “long-spaced” collagen (LSC) [58–62]. The finding of collagen type VI in LSC has been discussed controversially, and whether other collagen types exist in the LSC is unclear. Some authors suggest that there are no collagen derivatives at all [63]. LSCs are the main component of basal laminar deposits, but they are also found in the outer part of Bruch’s membrane located between the basement and cytoplasmic membrane of choroidal capillaries. In the outer collagenous layer of young eyes, LSC was also detected, so the hypothesis arose that LSCs are products of the permanent turnover of collagen [13]. LSCs show only weak staining for typical basement membrane components. Furthermore, LSCs contain carbohydrates that are not observed in laminin or collagen type IV [61]. It was suggested that LSC is built up by direct polymerization of basement membrane material and that the epitopes necessary for the recognition by antibodies are lost in the polymerization process [58, 64, 65].

One of the main components of basal laminar deposits (BLD) is carbohydrate-containing material [66], often as part of glycoproteins whose structure is different from the carbohydrate structure of laminin and collagen type IV [61, 67]. The glycosaminoglycans chondroitin sulfate and heparan sulfate are found in BLD with the detection of heparan sulfate only in BLD of AMD eyes [67].

There are two hypotheses about the origin of the drusen constituents: they originate from RPE cells or from the choroidal blood flow. The lipid characteristics of drusen support a RPE origin. This hypothesis is encouraged by the concept that all metabolic waste products of the RPE cells are released on their basal side and are further transported through Bruch’s membrane to the choroidal blood flow [25]. Under this assumption, degenerative changes in RPE cells occur first, and the altered metabolic waste can lead to the formation of drusen [68–72].

The hypothesis that at least some of the Bruch’s membrane deposits originate from the choroid is supported by histological and biochemical lipid analysis and examinations of the vesicular content of the deposits. In these deposits, cholesterol-like substances similar to those found in atherosclerosis were observed [27]. An experimental study with animals showed that diet-specific lipids can be deposited in Bruch’s membrane [73]. The location and shape of nodular drusen components also indicate they could be derived from the choroid.

Deposits in Bruch’s membrane

•RPE proliferation

•phagocytosis of pigmented remnants

•RPE cell death

disciform scar

Fig. 5.10 Schematic differentiation of the morphology of early and late age-related macular degeneration

Finally, it can be concluded that the observed deposits can be derived from the choroid as well as from a malfunctioning RPE metabolism. The individual composition of the deposits may originate from differing sources depending on age-related changes, genetic background and environmental influences.

With regard to the origin of BLDs located between the RPE cytoplasmic membrane and the basement membrane, the scientific evidence seems to point to its derivation from the RPE [60, 61, 63–65]. Because similar deposits can also be found in other tissues, no RPE specificity is attributed to BLD [74, 75]. Despite this, there is a strong link among basal deposits, choroidal neovascularization, scar formation and loss of vision. Therefore, BLDs are an important indicator for the progression of AMD.

Drusen only slightly affect the vision and mark the preliminary early stage of AMD. However, they are a sign that the development of late-stage AMD may follow [76]. During the development of late AMD different forms can occur. Distinct specifications are geographic atrophy of the RPE, choroidal neovascularization and serous pigment epithelial detachment (Fig. 5.10).

5.3Choroidal Neovascularization

Choroidal neovascularization (CNV) is characterized by vessel growth through Bruch’s membrane beneath the RPE. Inflammatory changes and alterations of diffusion characteristics for growth factors in Bruch’s membrane are believed to be pathogenic. The newly formed capillaries are surrounded by connective tissue and evoke a number of reaction from the overlying RPE. The RPE cells can proliferate and inhibit further

Fig. 5.11 Schematic representation of pathological alterations associated with CNV: growth stimulus for the ingrowth of choroidal capillaries by altered concentration of growth factors and deposit of polar lipids. ICL inner collagenous layer of Bruch’s membrane, EL elastic layer of Bruch’s membrane, OCL outer collagenous layer of Bruch’s membrane, VEGF vascular endothelial growth factor

vessel growth or RPE cells may die. These event will result in the different clinical manifestations of choroidal neovascularization with disciform scar formation as the late stage.

Fibrovascular membranes beneath the RPE and the retina are most frequently to blame for a loss of central vision [51, 53, 77]. This complication happens to about 80–85% of AMD patients resulting in limited reading ability. Histologically one can see that initially only small capillaries grow through Bruch’s membrane beneath the RPE (Fig. 5.11). They continue to grow and differentiate into arterioles and venules. Surrounded by fibrotic tissue, they form a fibrovascular membrane to which the RPE may react by proliferation, subsequently surrounding the newly formed vessels [78, 79]. This process can inactivate the vessels temporarily or permanently. However, RPE cells may also die and the proliferating vessels can spread unrestricted into the subretinal space. The combination of these different events can explain the clinical picture of CNV that is rich in variants and has different individual courses. Some CNVs stay stable over a long period of time and show no fluorescein leakage in fluorescence angiography. Sometimes they are only detectable with histological methods; therefore, they are called occult CNV [80]. Other fibrovascular membranes grow fast and cause characteristic sub-pigment epithelial and subretinal exudations and bleedings. In these cases, there is a high risk for the development of a fibrotic disciform scar which causes loss of central vision [51].

The clinical differentiation of choroidal neovascularization can be detected by fluorescence angiography.

One distinguishes between classical (Fig. 5.12) and occult neovascularization (Fig. 5.13). A CNV is termed classic if a well-limited area of hyperfluorescence develops in early fluorescence angiography which diffusely leaks dye during the further course of the angiographic examination. The histological correlation is fibrotic vascularized tissue penetrating the RPE and growing subretinally [81–83] (Fig. 5.12a, b). When in fluorescence angiography only diffuse unspecific hyperfluorescent areas composed of multiple spots or in late angiography pictures of just diffuse leakage of unsure origin are detectable, these membranes are termed occult choroidal neovascularizations [84, 85]. Histologically, this subtype is characterized by a subpigment epithelial fibrovascular membrane growing through the outer layers of Bruch’s membrane and spreading under the RPE layer [82, 83] (Fig. 5.13a, b). Optical coherence tomography (OCT) is a noninvasive method simplifying the differentiation between classic (Fig. 5.12c, d) and occult CNV (Fig. 5.13c, d) by providing high quality illustrations of both.

Only in a few cases did the fibrovascular membrane grow exclusively subretinally, as revealed by histological examinations in post-mortem eyes with exudative AMD and excised neovascular membranes. Much more frequently the fibrovascular vessel membrane is composed of subpigment epithelial and subretinal parts [81–83]. These findings indicate that CNV in AMD begins with the ingrowth of the membrane in the outer layers of Bruch’s membrane. As disease progresses the RPE is penetrated and the choroidal neovascularization develops subretinally [86]. Composition of choroidal neovascularization always includes fibrovascular tissue containing fibroblasts and sometimes bleeding, with the vascular endothelium and RPE being the two most frequent cellular components. In nearly all cases of BLD non-cellular elements are detectable [82]. The strong association of these diffuse subpigment epithelial deposits with the development of the different subtypes may point to an important role of BLD in the development of both types of choroidal neovascularization [69, 76, 87– 89]. The accompanying deposition of lipids and drusen characterizes the water permeability of Bruch’s membrane and may further be responsible for the individual characterization of the late exudative AMD leading to a CNV with or without serous pigment epithelial detachment (“dual pathogenic pathway”) [89].

Etiology. Different mechanisms are under suspicion of causing choroidal neovascularization. Cells of the

Fig. 5.12 Classical subretinal neovascularization (CNV) (a) in fluorescence angiography characterized by a well-defined area of hyperfluorescence, (b) histological section of the same lesion (periodic acid Schiff’s staining, magnification 100-fold) with RPE cells surrounding the subretinal neovascular membrane

rich in fibroblasts; star: neovascularized tissue rich in fibroblasts, arrow: new sprouting vessel, open arrow: retinal pigment epithelium. (c) Classic CNV in optical coherence tomography in top view and (d) illustration of a sectional plane (corresponding to the green arrow in c)

immune system such as leukocytes and macrophages in the damaged Bruch’s membrane indicate a role for an immune reaction and underlying inflammatory processes [90–95]. The presence of macrophages [94, 95] and of complement system components as part of innate immunity in the drusen underlines a possible involvement of the immune system [74]. Retinal microglia cells can also function as antigen-presenting cells, and in early AMD changes in this cell type are detectable. The discovery of cytokines as soluble immune mediators with a modulating function on the expression of growth factors and

other cytokines supports the role of the immune system in AMD [96, 97]. Further it is discussed whether hypoxia may stimulate microglia in the retina. The so-called “advanced glycation end products” (AGE) are also possible activators of an immune response in Bruch’s membrane. By reducing oxidation processes caused by glycosylation and oxidation of proteins and lipids in tissues, they are particularly important when a slow metabolic turn-over is present. After the formation of choroidal capillaries first under and later penetrating through the RPE, a form of granulated tissue differentiates.

Fig. 5.13 Occult CNV (a) with large bleeding seen in fluorescence angiography as cloudy hyperfluorescent area at the lower border, (b) histological section of the same lesion (periodic acid Schiff’s staining, magnification 100-fold) with a widespread subpigment epithelial part including tissue rich in fibroblasts

(star) and newly sprouted vessels (arrow). The RPE is accompanied by plenty of diffuse deposits (open arrow). (c) Occult CNV in optical coherence tomography angiography in top view and (d) as sectional plane of the green arrow shown in (c)

This process ends with the of a disciform scar and destruction of the overlying retina [6, 50, 53, 77].

Phospholipids themselves and other associated chemical substances may trigger a low-grade chronic reaction with inflammatory characteristics [23, 42]. Above all eicosanoides as degradation products of arachidonic acid are thought to be the cause. Prostaglandins are well-known inflammation-inducing factors and are derived from long chain unsaturated arachidonic acid. In addition, the ultrastructural destruction and alteration of Bruch’s membrane itself may provoke an inflammatory process [7, 58, 94]. With increasing age there is progressive generation and

deposition of peroxidized lipids and other oxidatively modified substances. A direct induction of capillary growth by peroxidized lipids and other oxidatively damaged products of the fatty acid metabolism is postulated [44] as seen in an animal model with lightinduced retinal damage [98].

Another cause for capillary ingrowth may be the altered permeability of Bruch’s membrane modified by deposited lipids (Fig. 5.14) [23, 24, 38, 45, 54, 99– 101]. This again causes an alteration in the diffusion of growth factors secreted by RPE cells [38]. These are released basally and are necessary for the maintenance of the normal choroidal architecture. A diminished