1.2 Microanatomy of the Retina

Fig. 1.1 Fundus photograph from a normal eye showing the macula (green circle) and fovea (black circle). The normal central retinal artery (black arrow) is located nasal to the central retinal vein (green arrow) in the optic disc. The normal ratio of diameter of a retinal vein to a retinal artery at a given distance from the center of the optic disc is approximately 1.3:110

nuclear layer, external limiting membrane, rod and cone inner and outer segments, and retinal pigment epithelium (RPE).14 The retina is thicker around the disc, where it measures 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.11 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. 1.3).11

1.2 Microanatomy of the Retina

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells external to the photoreceptors. These cells do not divide after embryogenesis. They are multifunctional, pumping ions and water toward the choroid (Fig. 1.2), participating in the cycling of visual pigments in concert with photoreceptors, and degrading photoreceptor outer segment discs. The lateral cell membranes of the RPE are connected by zonula occludentes

inhibiting the extracellular diffusion of water and ions and constituting the outer bloodÐretina barrier (Fig. 1.4).11

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, which contain ribosomes and mitochondria, at the inner segment/outer segment (IS/OS) junction, a landmark easily seen in normal spectral domain OCT images (Fig. 1.3). 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 mucoploysaccharides. The matrix can be disrupted in situations of serous retinal detachment as seen in RVO.16 The retina contains 110Ð125 million rods and 6.3Ð6.8 million cones.11 The cone outer segments are taller than the rod outer segments accounting for the subfoveal hump in the IS/OS junction (Fig. 1.3). The length of the outer segments and inner segments together is 58Ð67 m in the fovea but 37Ð40 m at the equator and beyond.11

The outer nuclear layer, comprising the photoreceptor cell bodies, is located just internal to the external limiting membrane (ELM), a band of zonulae adherens that connect apposed Muller cells and photoreceptors (Figs. 1.2 and 1.3). 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.11 Rods and cones are connected to adjacent Muller cells by zonulae adherens but are commonly separated from other photoreceptors.11 The outer nuclear layer has gentle topographic variation in thickness, from 45 m nasal to the disc, to 22 m temporal to the disc, to 50 m in the perifovea, to 27 m in the remainder of the peripheral retina.11 In studies of macromolecular diffusion in an experimental pig model, the outer nuclear layer was a bottleneck for molecules applied to the outer retinal surface.17

Fig. 1.2 Diagram of the stratiÞed cellular nature of the retina. The axons of the ganglion cells comprise the nerve Þber layer and optic nerve. BM BruchÕs membrane, RPE retinal pigment epithelium, IS/OS inner segment/outer

segment of photoreceptors, ELM external limiting membrane, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer, NFL nerve Þber layer

Fig. 1.3 Spectral domain OCT image from a normal right eye depicting the layers of the retina. The vitreous is the black empty space at the top above the retina. The foveal depression is seen in the center. The cones are taller than the rods producing greater separation between the inner-segment/outer- segment junction and the apical retinal pigment epithelium at

the fovea. NFL nerve Þber layer, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, ELM external limiting membrane, IS/OS inner-segment/outer-segment junction, RPE retinal pigment epithelium, BM BruchÕs membrane, C choroid, N nasal, T temporal, S superior, I inferior

Fig. 1.4 Electron micrograph of a zonula occludens (smaller arrow) between cells of the ECV304 cell line. Such junctions between endothelial cells and retinal pigment epithelial cells serve as the basis of the bloodÐretina barrier. The larger arrow denotes a maculae adherens intercellular junction (Reproduced with permission from Penfold15)

The outer plexiform layer lies between the inner and outer nuclear layers and describes a zone of synapses between rod and cone inner segments and the dendrites of horizontal cells (Figs. 1.2 and 1.3). It is characteristically thinner than the inner plexiform layer and may partially impede diffusion of molecules from the inner retinal to outer retinal interstitium. HenleÕs 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.11

The inner nuclear layer contains the cell bodies of bipolar, horizontal, and amacrine cells (Figs. 1.2 and 1.3) which mediate the initial processing of signals from rods and cones and have receptive Þelds 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.11 Muller cell processes wrap around the axons and dendrites of the intermediate cells of the retina and around capillaries.11 As with the outer nuclear layer, cystic spaces may develop in this layer when macular edema occurs in the setting of RVO. The inner nuclear layer is a relative bottleneck for the diffusion of macromolecules applied to the vitread side of the retina.17

The inner plexiform layer is located between the ganglion and inner nuclear cell layers and ranges in thickness from 18 to 36 m (Figs. 1.2 and 1.3).11 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 superÞcially 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 Þber layer varying in thickness from 10 to 20 m in the nasal retina to 60Ð80 m in the perifovea (Figs. 1.2 and 1.3).11 This variability in thickness corresponds to the presence of a single lamina of ganglion cells in most of the retina, but increasing to 8Ð10 laminae as the fovea is approached from the optic disc.11 The ratios of ganglion cells to rods and cones are 1:100 rods and 1:4 cones, except in the macula where the ratio is greater. This translates physiologically into a smaller receptor Þeld for each ganglion cell in the macula and, therefore, greater acuity. The ganglion cellsÕ 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 Þber layer. They travel within the optic nerve (Fig. 1.2), through the optic chiasm and eventually synapse with cells in the lateral geniculate body.

The nerve Þber layer is thickest adjacent to the optic disc, where it is 20Ð30 m (Figs. 1.2 and 1.3).11 The nerve Þbers 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 deÞnes the orientation of the horizontal raphe. Since the axons of the papillomacular bundle are the Þrst 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 Þber layer thickens. It is absent within the fovea and very thin in the far periphery. Ischemia disrupts physiologic axoplasmic ßow and produces both proximal and distal axonal degeneration.18 The funduscopic correlates of these processes are cotton wool spots and optic disc edema in acute ischemia and the featureless retina lacking nerve Þber layer striations in chronic retinal ischemia.18,19

The normal bloodÐretina barrier 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. 1.4). These prevent the easy passage of paracellular small molecules between the neurons of the retina and the vascular system. Several transcellular transport systems allow the transcellular passage of metabolites.15 The space between cells of the retina is similar to that of the brain and is passable by peroxidase (approximately 2.5Ð5-nm diameter).20-24 The maximum size of a molecule that can diffuse through the intercellular spaces of the retina is estimated to be 6.2 ± 0.5 nm in the normal eye. In eyes with RVO and breakdown of the bloodÐ retina barrier, larger molecules can diffuse through the retina.17 The bloodÐaqueous barrier functions in a manner similar to the bloodÐretina barrier, and all forms of RVO cause disruption of both barriers, presumably mediated by VEGF released from the ischemic retina.25,26 The intercellular space between the photoreceptors and the RPE and between adjacent photoreceptors external to

the ELM is termed the subretinal space. The subretinal space can expand dramatically with pooled ßuid in RVO.

The internal limiting membrane (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, Þbronectin, and collagen.17 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.18 Muller cell processes form a continuous but uneven border of attachment with the ILM. The internal limiting membrane constitutes a barrier for vitreous molecules diffusing toward the retina. Thus, for example, the retinal ILM is impermeable to tissue plasminogen activator in the rabbit.27 Ranibizumab (MW 49 kD) was developed in part because of concerns that bevacizumab (MW 149 kD) might not diffuse through human ILM. The ILM overlying the optic disc is a thinner layer known as the limiting membrane of Elschnig. The central part is even thinner and is known as the central meniscus of Kuhnt. The ILM overlying the optic disc may therefore be permeable to molecules that cannot penetrate the retinal ILM.28

The anatomic relationship of the vitreous and the retina is important for certain complications of BRVO and CRVO. Attachment of the vitreous to the retina in a BRVO with extensive capillary nonperfusion is associated with a higher rate of secondary retinal neovascularization. It is thought that the vitreous provides a scaffold for growth of the new vessels.29,30 Similarly, posterior vitreous detachment (PVD) protects against development of retinal and disc neovascularization in ischemic CRVO, but not anterior segment neovascularization.18 In nonischemic CRVO, but not in ischemic CRVO, posterior vitreous detachment protects against development of macular edema.18 The attached posterior vitreous is thought to retard diffusion of chemical mediators of hyperpermeability away from the site of action in the retinal microvasculature.18 Collagen Þbers of the posterior cortical vitreous are bound to the internal limiting membrane of the retina through the adhesion molecules laminin and Þbronectin.31