Histologically, the retina is a multilayered sheet of neuronal, glial, and vascular tissue that lines the inside posterior two-thirds of the eye. It is bounded anteriorly by the vitreous humor and posteriorly by the retinal pigment epithelium (RPE), Bruch’s membrane (BM), and choroid. The macula is a circular area of the retina 5.2–5.5 mm in diameter with a center located 17°, or 4.0–5.0 mm temporal, and 0.53–0.8 mm inferior to the center of the optic disc (Fig. 1.1) [1–5]. Of potential significance in 4AQR, the macula receives more total irradiance than the peripheral retina, and the inferior macula receives more irradiance in the blue part of the spectrum (from the sky) than the superior macula [6]. A useful conversion of arc length on the macula to degrees of eccentricity from the fovea is that 1 degree is equivalent to 280 microns, or three first-order retinal vein widths [5]. Among the distinguishing features of the macula are the high density of cones, two or more layers of ganglion cells, as well as the presence of the xanthophyll carotenoids lutein and zeaxanthin within photoreceptor axons, bipolar cells, and ganglion cells [1, 3, 7, 8].

Xanthophylls are not synthesized by the body, but must come from the diet [7]. Zeaxanthin is the predominant xanthophyll in the center of the macula with lutein rising in relative concentration in the parafovea [7]. The xanthophylls reduce chromatic aberration, absorb damaging blue light, and protect retinal membranes from photooxidation [7, 9]. The extent of their distribution is similar to the size of the bull’s-eye macular lesion in 4AQR and suggests that they may have a role in toxicity. For example, 4AQR in its advanced stages manifests outer retinal atrophy that spares the fovea and is greatest from 2 to 8° eccentric to the fovea [10, 11]. Xanthophylls have their greatest concentrations in the fovea and drop to a lower plateau within 4° of the fovea [9]. The levels are generally nondecreasing once adulthood is reached [7].

The central 1.5 mm circular area of the macula is called the fovea, denoted by a gently curved depression in the retinal surface. Within the fovea is a roughly circular avascular area, the foveal avascular zone (FAZ), approximately 400–500 μm in diameter which contains only cones, present at a density of approximately 125,500–140,000/mm² [1, 12].

A cross section through the retina just outside the area centralis shows ten layers (Figs. 1.3 and 1.4). Proceeding from the vitreous to the choroid are the internal limiting membrane (ILM), nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer (ONL), external limiting membrane (ELM), rod and cone inner and outer segments, and RPE [14]. In histologic sections the retina is thicker around the disc, where it is 0.56 mm thick, and tapers to 0.18 mm at the equator and 0.11 mm at the ora as the density of all neural elements decreases peripherally [1]. The topography of the macula includes a central thinner zone, the foveal depression, and a thicker paracentral annulus around the fovea where the ganglion cell layer, inner nuclear layer, and outer plexiform layer of Henle are thickest (Fig. 1.4) [1].

In vivo measurements of the thickness of retinal layers can be measured by spectral domain optical coherence tomography (SD-OCT). Table 1.1 shows measurements made for central and pericentral regions in normal human subjects [5]. Macular thicknesses and the thicknesses of intraretinal layers measured with different instruments will differ slightly because of differences in location of the outer retinal boundary line and in the segmentation algorithms used. For example, the Stratus time domain optical coherence tomography (TD-OCT) instrument measures from the ILM to the inner segment/outer segment (IS/OS) junction, the Spectralis SD-OCT between the ILM and the Bruch’s membrane–choriocapillaris complex, the Cirrus SD-OCT between the ILM and the photoreceptor outer segment–RPE boundary, and the Optovue SD-OCT between the ILM and the external limit of the RPE [15–17].

The central circular area has a radius of 1.4 mm for the Optovue SD-OCT [5]. For the Cirrus SD-OCT the radius of the central circular zone is 1.0 mm [19]. The pericentral annulus for the Optovue measurements extends from 1.4 to 2.6 mm from the fovea [5]. For the Cirrus measurements the annulus extends from 1.0 to 3.0 mm from the fovea [19].

The RPE is a monolayer of hexagonal cells external to the photoreceptors (Fig. 1.3). These cells do not divide after embryogenesis. They are multifunctional, pumping ions and water toward the choroid, absorbing photons not involved in phototransduction, protecting the retina from oxidative stress, and participating in the cycling of visual pigments in concert with photoreceptors [6]. The lateral cell membranes of the RPE are connected by zonula occludentes inhibiting the extracellular diffusion of water and ions and constituting the outer blood–retina barrier (BRB) [1].

There are regional variations in RPE biochemistry. Levels of cathepsin D, aryl sulfatase, and acid phosphatase enzyme activity are higher in the macula than in the periphery [6]. Some of these enzymes are inhibited by 4AQs suggesting a potential association with the maculocentric distribution of 4AQR.

The RPE recycles large amounts of photosensitive cell membranes derived from the discs of the photoreceptor outer segments [1]. The apposition of photoreceptor discs to apical RPE plasma membrane triggers a signal for phagocytosis of the discs (Fig. 1.5) [20, 21]. Discs are encompassed by invaginated RPE plasma membrane and the shed discs are internalized into the RPE cell as a phagosome [21–23]. The phagosome moves basally while fusing with lysosomes containing 60 or more hydrolytic enzymes that progressively degrade the discs as well as melanin (Fig. 1.6) [6, 21, 22, 24, 25]. Phagosomes with degraded discs are termed myeloid or myelin bodies. Apposition of RPE to photoreceptor outer segments is essential for disc metabolism. Isolated retinas without apposed RPE do not shed discs, but begin to do so when the retina is reapposed to RPE. Both rods and cones shed discs, but the timing differs by photoreceptor types. Although there are exceptions in certain species, the general pattern is that cones shed their discs with the onset of darkness and rods shed theirs with the onset of light [6]. Melatonin synthesized by the retina primes shedding [24].

In the retina of the rhesus monkey, each RPE cell in the retinal periphery degrades approximately 4,000 rod outer segment discs per day [24]. The phagocytic load per RPE cell increases with age as RPE cells drop out and fewer remaining cells distribute the burden [6]. Disruption of photoreceptor outer segment recycling by RPE damages the photoreceptors and probably has a role in 4AQR. For example, the photoreceptors prematurely die in the Royal College of Surgeons (RCS) rat, which has a mutation impairing RPE phagocytosis [20, 27].

The product of degradation of photoreceptor outer segments by RPE is lipofuscin, which is contained within lysosomes as undigested residues of oxidatively damaged lipids (Fig. 1.7) [6, 24, 28–31]. Fluorophores within the discs resist full degradation in the RPE lysosomes. Their presence gives lipofuscin its autofluorescence [32]. The RCS rat has decreased levels of RPE lipofuscin because of impaired photoreceptor outer segment phagocytosis [27, 33]. Lipofuscin has its highest concentration in the central retina, where it increases in concentration over a lifetime [28, 34]. It occupies 1 % of the cytoplasmic volume of RPE cells in the first decade of life, but 19 % in the decade 81–90 [28]. In 4AQR, parafoveal hyperautofluorescence is a consequence of the disrupted RPE metabolism of photoreceptor outer segments (Fig. 1.8) [35]. Most investigators consider lipofuscin to be detrimental to cell function, as testified by increased levels in 4AQR, Stargardt disease, age-related macular degeneration, and retinitis pigmentosa (Fig. 1.8) [6, 30, 36]. The toxic component of lipofuscin is A2E, which raises lysosomal pH, inhibits lysosomal degradation of proteins, predisposes RPE cells to blue light-induced apoptosis, and has damaging detergent properties for cell membranes [25, 30, 37–40].

By SD-OCT the average thickness of the RPE layer in a central circle of 1 mm diameter in the macula is 18.3 ± SD 2.4 μm. The RPE in the pericentral annulus of RPE from 1 to 2 mm eccentric to the fovea has similar thickness, but more peripherally the RPE thickness increases. In the annulus from 3 to 6 mm eccentric to the fovea, the average thickness of the RPE is 19.6 ± SD 1.8 μm [18]. The RPE thickness in the central macula increases with age due in part to accumulation of lipofuscin within the cells [18]. Average RPE cell diameter increases with age as some RPE cells die and the remainder spread out to fill in gaps [6]. Toxicologic studies in embryonal chick retina suggest that RPE is more sensitive to chloroquine toxicity than retinal neuronal cells, but the reverse seems to be the case in the primate [42, 43].

Photoreceptors are polarized cells with an outer segment that absorbs quanta of incoming light. The outer segments of photoreceptors are made up of stacks of double membranes derived from a continuously growing and evaginating plasma membrane [24]. Photons of light travel through the translucent inner retina until they strike the photopigment molecules in the stacked discs of the photoreceptor outer segments. The photopigments transduce the light energy in a complex process leading to transmembrane hyperpolarization. The outer segments join the inner segments histologically at the cilium. Outermost in the inner segment is the ellipsoid, a region densely packed with mitochondria for cellular energy production. More vitread to the ellipsoid is the myoid, in which endoplasmic reticulum and golgi apparatus are densely distributed for production of cellular proteins and macromolecules. At the inner terminal of the photoreceptors is the synaptic terminal with many synaptic vesicles filled with neurotransmitters [24]. The IS/OS junction is a landmark easily seen in normal SD-OCT images (Fig. 1.4). Recent evidence suggests that the IS/OS junction actually represents the band of ellipsoids of adjacent photoreceptor outer segments rather than the band formed by the histologic IS/OS junctions, which is just distal to the band of ellipsoids [44]. Between the outer segments and the RPE is the interphotoreceptor matrix, a complex milieu with signaling molecules, glycoproteins, enzymes, and fatty acids in an extracellular matrix of acid mucopolysaccharides. The third highly reflective line seen in SD-OCT images is termed the cone outer segment tips (COST) (Fig. 1.4) and is thought to correspond to the zone of ensheathment of cone outer segments by apical processes of RPE cells [44]. The fourth highly reflective line arises from the RPE and Bruch’s membrane (Fig. 1.4) [44]. All four highly reflective lines in the outer retina can be lost first in the perifovea and later in the central fovea in 4AQR [45, 46].

The retina contains 38.7–125 million rods and 2.2–6.8 million cones [1, 2, 12]. The topographic distribution of rods and cones is illustrated in Fig. 1.9. There is an intuitive relationship of this topographic distribution and the hill of vision as defined by static automated perimetry (Fig.1.10). The density of rods peaks at approximately 150,000/mm [2] in an annular ring around the fovea having a diameter of 3–5 mm, or 10–17° eccentric to the fovea (Fig. 1.9). Although the annular shape of this distribution calls to mind the annular shape of 4AQR, the dimensions do not match. The elliptical annulus of 4AQR has a horizontal and vertical median radius from the fovea of 1.4 and 1.0 mm, respectively (see Chap. 6). The mismatch tends to discount an association of rod density with the pathogenesis of 4AQR. The cone outer segments are taller than the rod outer segments accounting for the subfoveal hump in the IS/OS junction (Fig. 1.4). The length of the outer segments and inner segments together is 58–67 μm in the fovea but 37–40 μm at the equator and beyond [1]. The cones differ from rods in several respects. Their stacked discs are open to the extracellular space. By contrast, in rods the innermost discs are open to the extracellular space, but as the discs move outward with the production of new discs their attachments to the plasma membrane are lost. They become surrounded by plasma membrane and are not bathed in extracellular fluid.

The ONL, comprising the photoreceptor cell bodies, is located just internal to the ELM, a band of zonulae adherens that connect apposed Muller cells and inner segments of photoreceptors (Figs. 1.3 and 1.4) [48, 49]. The ELM is an important landmark seen in SD-OCT images and constitutes a relative diffusion barrier between the interstitium of the inner retina and the subretinal space, as the intercellular space at each zonula adherens narrows to 20 nm [1, 48]. Rods and cones are connected to adjacent Muller cells by zonulae adherens, but are commonly separated from other photoreceptors [1]. The ONL has gentle topographic variation in thickness, from 45 μm nasal to the disc, to 22 μm temporal to the disc, to 50 μm in the perifovea, to 27 μm in the remainder of the peripheral retina [1]. Thinning of the ONL in the perifovea is often seen simultaneously with loss of definition of the IS/OS junction in 4AQR [50, 51].

The outer plexiform layer lies between the inner nuclear layer and ONL and describes a zone of synapses between rod and cone inner segments and the dendrites of horizontal cells (Figs. 1.3 and 1.4). It is characteristically thinner than the inner plexiform layer and may partially impede diffusion of molecules from the inner retinal to outer retinal interstitium. Henle’s layer designates the outer plexiform layer adjacent to the fovea where the axons of the rods and the cones turn and travel more parallel with the plane of the retina and away from the fovea [1]. The lengths of individual fibers of Henle are not known for humans, but are longer for the fibers belonging to more centrally located photoreceptors because there is greater distance between these more central photoreceptors and their connecting bipolar cells displaced laterally from the center of the macula [2].

The inner nuclear layer contains the cell bodies of bipolar, horizontal, and amacrine cells (Figs. 1.3 and 1.4) which mediate the initial processing of signals from rods and cones and have receptive fields of varying diameter. The bipolar cells are the most numerous. The cell bodies of the Muller cells are also contained within this layer. Muller cells span the thickness of the retina and are involved in glucose metabolism and ionic and water transport within the retina [1]. Muller cell processes wrap around the axons and dendrites of the intermediate cells of the retina and around capillaries [1]. The inner nuclear layer is a relative bottleneck for the diffusion of macromolecules applied to the vitreal side of the retina [52].

The inner plexiform layer is located between the ganglion and inner nuclear cell layers and ranges in thickness from 18 to 36 μm (Figs. 1.3 and 1.4) [1]. In addition to Muller cell branches and retinal blood vessels, the inner plexiform layer contains synaptic processes of the bipolar, ganglion, and amacrine cells. The bipolar cell axons bring signals from the outer retina to the processing amacrine cells and to the dendrites of the more superficially located ganglion cells. There are at least 25 types of amacrine cells in the human retina, and the lateral span of their dendrites increases with eccentricity from the fovea.

The ganglion cell layer lies between the inner plexiform layer and the nerve fiber layer. In histological sections it varies in thickness from 10 to 20 μm in the nasal retina to 60 to 80 μm in the perifovea (Figs. 1.3 and 1.4) [1]. Measured by SD-OCT, the average thickness of an annulus from 1 to 3 mm from the fovea is 50.6 ± SD 5.6 μm (Table 1.1). The average thickness of an annulus from 3 to 6 mm from the fovea is 28.5 ± SD 3.0 μm [18]. This variability in thickness corresponds to the presence of a single lamina of ganglion cells in most of the retina, but 8–10 laminae as the fovea is approached from the optic disc [1].

The average number of ganglion cells in the retina is 1.07 ± 0.4 million [2]. Ganglion cell densities are highest in a horizontally oriented elliptical ring extending from 0.4 to 2.0 mm from the fovea [2]. The size and shape of this area resemble the area of funduscopic damage in 4AQR, which is known to involve ganglion cells at an early stage [43]. The pericentral ganglion cell layer thickness decreases with age, presumably due to attrition of ganglion cells [18]. The ratios of ganglion cells to photoreceptors are 1:100 rods and 1:4 cones, respectively, except in the macula where the ratio of ganglion cells to cones may be as large as 1:2 [2]. This translates physiologically into a smaller receptor field for each ganglion cell in the macula and, therefore, greater acuity. Macular visual field sensitivity correlates with ganglion cell/inner plexiform layer thickness as measured by SD-OCT [53]. The ganglion cells’ dendrites extend toward the outer retina and synapse with retinal bipolar and amacrine cells in the inner plexiform layer. The ganglion cell axons are long, making up the retinal nerve fiber layer. They travel within the optic nerve (Fig. 1.3), through the optic chiasm and eventually synapse with cells in the lateral geniculate body. In a primate model of chloroquine toxicity, ganglion cells showed histologic damage earliest [43].

The nerve fiber layer is thickest adjacent to the optic disc where it is 20–30 μm (Figs. 1.3 and 1.4) [1]. The nerve fibers remain unmyelinated until they reach the lamina cribrosa. Muller cell processes interdigitate around the ganglion cell axons which sometimes directly contact their neighbors. The axons assume a generally radial course toward the optic nerve except for those immediately temporal to the macula, which arc above and below the papillomacular bundle that defines the orientation of the horizontal raphe. Since the axons of the papillomacular bundle are the first to develop, they form the center of the optic nerve with axons from the more peripheral retina found more peripherally in the optic nerve. As ganglion cell axons converge toward the optic nerve, the nerve fiber layer thickens. It is absent within the fovea and very thin in the far periphery. Ischemia disrupts physiologic axoplasmic flow and produces both proximal and distal axonal degeneration [54]. The funduscopic correlates of these processes are cotton wool spots and optic disc edema in acute ischemia and the featureless retina lacking nerve fiber layer striations in chronic retinal ischemia [54, 55]. The perifoveal thickness of the combined inner nerve fiber layer–ganglion cell layer as measured by SD-OCT is thinned in patients with hydroxychloroquine retinopathy [5].

The normal BRB is based on tight intercellular junctions between vascular endothelial cells and between retinal pigment epithelial cells. In both sites, the barrier is subsumed by the zonulae occludens (Fig. 1.11). These prevent the easy passage of paracellular ions and hydrophilic small molecules between the neurons of the retina and the vascular system. Amphiphilic substances such as the 4AQs pass through cellular membranes by diffusion and are not impeded by the BRB. The 4AQs in toxic concentrations may degrade the BRB. Vitreous fluorophotometry shows that the BRB is intact in patients taking chloroquine without retinopathy [56]. On the other hand, patients with chloroquine retinopathy have increased permeability of the BRB [56].

The ILM, the sole true basement membrane within the retina, separates the retina from the vitreous. The inner stratum of the ILM is laminated with the basement membrane of the Muller cells. The outer stratum is composed of laminin, proteoglycans, fibronectin, and collagen [16]. The ILM varies in thickness from 2,000 nm over the parafovea to 20 nm over the fovea, since the density of Muller cells decreases in the fovea [17]. Muller cell processes form a continuous but uneven border of attachment with the ILM. The ILM constitutes a barrier for vitreous molecules diffusing toward the retina.

The central retinal artery travels through the center of the lamina cribrosa to the optic disc, where it divides into superior and inferior branches that supply the retinal hemispheres. Further equal bifurcations occur downstream as do sidearm branchings of smaller arterioles [58]. Branch retinal arteries lie in the nerve fiber layer or ganglion cell layer, with only the smaller arterioles descending into the inner plexiform layer to supply capillaries [22].

Retinal capillaries reside within various laminae of the inner retina. During normal development, astrocytes in the retina produce vascular endothelial growth factor (VEGF) that induces development of the superficial capillary bed. Later, photoreceptor development in the outer retina causes hypoxia of the inner retina with upregulation of VEGF from the inner nuclear layer and development of the deeper capillary bed within the inner retina [61]. Astrocytes and retinal capillaries colocalize within the retina. Astrocytes and capillaries are absent from the FAZ and in the immediately postoral retina [62].

A superficial network of capillaries called the radial peripapillary capillaries (RPCs) surrounds the optic nerve (Fig. 1.11) [59]. These capillaries lie in the superficial nerve fiber layer and preferentially nourish that layer, but derive from arterioles located deeper at the levels of the outer nerve fiber layer and ganglion cell layer [1]. The RPCs are arranged in parallel rows rather than in the anastomotic net typical of the deeper retinal capillaries. RPCs connect rarely with each other or with deeper retinal capillaries and run parallel to major retinal arteries, rarely crossing them [59].

Besides the RPCs, capillaries of the inner retina assume locations at four depths depending on the thickness of the ganglion cell layer [60]. One lamina of capillaries is present in the nerve fiber layer and ganglion cell layer. Capillaries are regularly found at the outer and inner borders of the inner nuclear layer (which is approximately 40 μm thick); are missing in the inner plexiform layer (approximately 30 μm thick); and are found at the inner or outer boundaries of the ganglion cell layer for the parts where it is approximately 30 μm thick as well as within the ganglion cell layer where it is thicker (50–60 μm in an annulus 0.7–1.8 mm from the fovea) (Fig. 1.12) [60]. The deepest lamina vanishes more proximally in the retinal mid-periphery, the middle lamina vanishes more peripherally, and the most superficial lamina extends almost to the ora serrata which, like the fovea, is bordered by an avascular zone (Fig. 1.13). A capillary-free zone is also found adjacent to retinal arterioles (Fig. 1.14) [1, 62].