Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

nerve fiber layer close to the internal limiting membrane. The retinal arterial circulation in the human eye is a terminal system with no arteriovenous anastomoses or communication with other arterial systems. Thus, the blood supply to a specific retinal quadrant comes exclusively from the specific retinal artery and vein that supply that quadrant. Any blockage in blood supply therefore results in infarction. As the large arteries extend within the retina toward the periphery, they divide to form arteries with progressively smaller diameters until they reach the ora serrata where they return and are continuous as a venous drainage system. The retinal arteries branch either dichotomously or at right angles to the original vessel. The arteries and venules generated from the retinal arteries and veins form an extensive capillary network in the inner retina as far as the external border of the inner nuclear layer. Arteriovenous crossings occur more often in the upper temporal quadrants with the vein usually lying deeper than the artery at these crossings.

Branches from the central retinal vessels dive deep into the retina forming two distinct capillary beds, one in the ganglion cell layer (superficial capillary plexus) and the other in the inner nuclear layer (deep capillary plexus). Normally, no blood vessels from the central retinal arteries extend into the outer plexiform layer (Figure 3). Thus, the photoreceptor layer of the retina is free of the blood vessels supplied by the central retinal artery. The choriocapillaris provides the blood supply to photoreceptors. Since the fovea contains only photoreceptors, this cone-rich area is free of any branches from the central retinal vessels (Figure 3).

In some individuals (18%), a cilioretinal artery derived from the short posterior ciliary artery (from the choroid vasculature) enters the retina around the termination of Bruch’s membrane, usually on the temporal side of the optic nerve, and courses toward the fovea, where it ends in a capillary bed and contributes to the retinal vasculature. In approximately 15% of eyes with a cilioretinal artery, the branches supply the macula exclusively, whereas in other individuals, they can nourish the macular region and regions of the upper or lower temporal retina.

Physiology

The rate of blood flow through the retinal circulation is approximately 1.6–1.7 ml g–1 of retina with a mean circulation time of approximately 4.7 s. The flow rate through the retinal vessels is significantly slower than that through the choroidal vasculature.

The arteries around the optic nerve are approximately 100 mm in diameter with 18-mm-thick walls. These decrease in diameter in the branched arteries located in the deeper retina to around 15 mm. The walls of the retinal arteries have the characteristics of other small muscular arteries and are composed of a single layer of endothelial cells: a

subendothelial elastica, a media of smooth muscle cells, a poorly demarcated external elastic lamina, and an adventitia comprised of collagen fibrils. Near the optic disk, the arterial wall has five to seven layers of smooth muscle cells, which gradually decrease to two or three layers at the equator and to one or two layers at the periphery. These vessels continue to have the characteristics of arteries, and not arterioles, up to the periphery. The retinal arteries lose their internal elastic lamina soon after they bifurcate at the optic disk. This renders them immune from developing temporal arteritis and distinguishes them from muscular arteries of the same size in other tissues. As a compensatory mechanism, the retinal arteries have a thicker muscularis, which allows increased constriction in response to pressure and or chemical stimuli.

The major branches of the central vein close to the optic disk have a lumen of nearly 200 mm with a thin wall made up of a single layer of endothelial cells having a thin basement membrane (0.1 mm), which is continuous with the adjacent media comprised predominantly of elastic fibers, a few muscle cells, and a thin adventitia. The larger veins in the posterior wall have three to four layers of muscle cells in the media. As the retinal veins move peripherally from the optic disk, they lose the muscle cells, which are replaced by pericytes. The lack of smooth muscle cells in the venular vessel wall results in a loss of a rigid structural framework for the vessels, resulting in shape changes under conditions of sluggish blood flow (e.g., diabetes mellitus) or increased blood viscosity (polycythemia), or with increased venous pressure (papilledema or orbital compressive syndromes).

The retinal capillary network is spread throughout the retina, diffusely distributed between the arterial and venous systems. There are three specific areas of the retina that are devoid of capillaries. The capillary network extends as far peripherally as the retinal arteries and veins up to 1.5 mm from the posterior edge of the troughs of the ora serrata (ora bays), leaving the ora serrata ridges (ora teeth) without any retinal circulation. The 400-mm- wide capillary-free region centered around the fovea is another area lacking retinal capillaries. Finally, the retina adjacent to the major arteries and some veins lacks a capillary bed.

The retinal capillary wall is comprised of the endothelial cells, basement membrane, and intramural pericytes. The retinal capillary lumen is extremely small (3.5–6 mm in diameter), requiring the circulating red blood cells to undergo contortions to pass through. Unlike the choriocapillaris, the endothelial cells of retinal capillaries are not fenestrated. The edges of endothelial cells show interdigitation and are joined by the zona occludens at their lumenal surface. The endothelial cells of the retinal arteries are linked by tight junctions, which prevent the movement of large molecules in or out of the retinal vessels. These tight junctions, in concert with the Muller

cells and astrocytes, establish the blood–retinal barrier that prevents the passage of plasma proteins and other macromolecules in or out of the capillary system. The pericytes (also called mural cells) are embedded within the basal lamina of the retinal capillary endothelium. The pericytes are believed to play an important role in the stabilization of the retinal capillary vasculature.

Pathology

Diabetic retinopathy (maculopathy) is the leading cause of vision loss in patients with type 2 diabetes and is characterized by the hyperpermeability of retinal blood vessels, with subsequent formation of macular edema and hard exudates. Early changes in the retinal vasculature include thickening of the basement membrane, loss of pericytes, formation of microaneurysms, and increased permeability. It is generally hypothesized that these changes lead to the dysfunction of the retinal vessels, loss of vessel perfusion, hypoxia, induction of retinal VEGF expression, and pathological neovascularization as seen in proliferative diabetic retinopathy. In severe cases, retinal detachment can occur as a result of traction caused by fibrous membrane formation. Laser photocoagulation and VEGF-blocking agents are currently being used as therapeutic approaches for this disease.

Retinopathy of prematurity is a potentially blinding disorder affecting premature infants weighing 1250 g or less and with a gestational age of less than 31 weeks. This

disease is characterized by abnormal retinal vascularization and can be classified into several stages ranging from mild (stage I), with mildly abnormal vessel growth, to severe (stage V), with severely abnormal vessel growth and a completely detached retina. The pathophysiology of this disease has been extensively studied and several factors have been implicated. The premature birth of an infant before the retinal vasculature has extended to the periphery results in stoppage of the normal blood vessel growth that is driven by the hypoxia-mediated expression of VEGF. This is compounded by the oxygen therapy given to alleviate the respiratory distress, which reduces VEGF expression leading to vaso-obliteration. Once the infant is taken off oxygen, there is a relative hypoxia, upregulation of VEGF, and florid abnormal neovascularization.

See also: Blood–Retinal Barrier; Development of the Retinal Vasculature.

Further Reading

Hogan, M. J., Alvarado, J. A., and Weddell, J. (1971). Histology of the human eye. Philadelphia, PA: W.B. Saunders.

Jakobiec, F. A. (1982). Ocular anatomy, embryology and teratology.

Philadelphia, PA: Harper and Row.

Saint-Geniez, M. and D’Amore, P. A. (2004). Development and pathology of the hyaloid, choroidal and retinal vasculature.

International Journal of Developmental Biology 48: 1045–1058. Wolff, E. (1933). The anatomy of the eye and orbit. Philadelphia, PA:

P. Blakiston’s and Co.

Glossary

Angiogenesis – The growth of new blood vessels through the process of budding from existing vessels. Angiogenesis occurs in response to a stimulus such as hypoxia.

Inner plexus – The network of superficial blood vessels that lies on the inner surface of the retina. Mural cells – Includes pericytes and smooth muscle cells. These cells surround the endothelial cell tubes and contribute to the formation of stable vessels. Outer plexus – The deeper network of blood vessels that lies at the junction of the inner nuclear layer and the outer plexiform layer of the retina. This layer of vessels forms by budding from the inner plexus, and consists entirely of capillaries.

Pericytes – Mesenchymal cells that are associated with small vessels. These cells are critical for vessel stability and for the formation of the blood–retinal barrier.

Vasculogenesis – The process of vessel formation through the organization of vascular precursor cells into chords and vessels. Vasculogenesis proceeds in the absence of stimuli such as VEGF.

Introduction

The development of the vascular network of the human retina follows a very specific topography and series of events, producing a network of vessels that precisely meets the metabolic demands of the healthy adult retina. Disruption of this process can lead to the underor overproduction of vessels and/or the formation of vessels with pathological characteristics, including a breakdown of the blood–retina barrier (BRB) and inappropriate pericyte ensheathment leading to vessel instability. In this article, we describe the process through which the vasculature develops, and the intrinsic and extrinsic signals that control its formation.

An Overview of Human Adult Retinal

Vasculature

The retina has the highest metabolic demand of any tissue in the body. The conflicting requirements of sufficient blood supply and minimal interference with the light path

to the photoreceptors are met by two vascular supplies: inherent intraretinal vessels supply the inner two-thirds of the retina, and the choroidal vasculature supplies the outer third of the retina.

The vasculature of the adult retina enters and exits through the optic disk (Figure 1(a)). As shown in the fundus photo (Figure 1(b)), four main artery/vein pairs supply the human retina. These vessels are termed the superior nasal, inferior nasal, superior temporal, and inferior temporal branches of the central retinal artery and vein. Smaller arterioles branch off these four main arteries, whereas terminal branches bifurcate toward the peripheral retina. The fovea, a region of the retina that contains the highest density of photoreceptors, remains avascular throughout development and in the adult retina. The inner plexus ramifies in the ganglion cell and nerve fiber layers. Some of these vessels dive down through the inner plexiform and inner nuclear layers to form the outer, or deep, plexus at the junction of the inner nuclear and outer plexiform layers of the retina (Figure 1(c)). Vessels in the inner plexus cover the spectrum from arterioles and venules to capillaries and postcapillary venules, whereas vessels in the outer plexus are mainly capillary in size. Both vascular layers reach almost to the edge of the adult retina, leaving an avascular rim that is thin enough to be adequately oxygenated through diffusion from the choroid. A third intraretinal vascular network, the radial peri-papillary capillaries (RPCs), forms a limited plexus in the nerve fiber layer around the optic disk. The RPCs radiate out from the optic nerve head (ONH) and supply the thick nerve fiber bundles where they exit the retina.

Figure 1(d) is a schematic representation of central nervous system (CNS)/microvascular interface, as would be found typically in the human retina. CNS vessels are characterized by tight junctions between adjacent vascular endothelial cells; the tight junctions are responsible for creating the BRB. Outside the endothelial cell layer lays the basal lamina, which is laid down by endothelial cells and pericytes. This layer is composed predominantly of collagen IV, fibronectin, and laminin. Pericytes are located within the basal lamina, between the vascular endothelial cells and the astrocytic endfeet that form the glia limitans, or glial limiting membrane. In addition to astrocytes, ultrastructural evidence suggests that the glia limitans also includes perivascular cells and microglia.

Vasculature of the Primordial Retina

During embryonic development, the retina forms as an extension of the diencephalon. The rudimentary structures

of the eye are distinguishable by 4 –5 weeks gestation (WG). At the earliest stages in development, the primordial lens and retinal tissues are oxygenated through the hyaloid vasculature, a vascular network that begins as an artery entering the eye through the optic nerve (the central hyaloid artery), splits into hyaloid vessels as it continues forward through the vitreous and around the developing lens, and exits at the front of the eye. This vascular network is present early in human embryonic development and regresses as the retinal vasculature forms and is able to meet the increasing metabolic demands of the eye. Typically, the hyaloid vasculature has totally regressed by the ninth month of gestation.

Formation of the Human Retinal Vasculature

Takes Place Through Vasculogenesis and

Angiogenesis

Blood vessels can be formed by one of two distinct mechanisms. Vasculogenesis is the de novo formation of vessels by the aggregation of endothelial precursor cells. Vessels develop from vascular precursor cells (VPCs) that aggregate into solid vascular cords, which then become patent and differentiate to form primitive endothelial tubes. Formation of vessels by angiogenesis occurs through budding from existing vessels; this process takes place through proliferation of vascular endothelial cells and serves to vascularize neighboring tissues. The process of angiogenesis is driven by signals that are produced in response to hypoxia, including vascular endothelial growth factor165 (VEGF165), whereas the process of vasculogenesis is independent of hypoxia.

Vasculogenesis: Vascular formation through transformation from VPCs

The first event in the development of the retinal vasculature is the de novo formation of vessels by vasculogenesis. This stage, detectable in the human retina before 12 WG, initiates with the migration of spindle-shaped cells of mesenchymal origin from the ONH. The individual cells can be Nissl stained and express CD39, vascular endothelial growth factor receptor 2 (VEGFR2), and ADPase, an ecto-enzyme found on the luminal surface of endothelial cells in the adult retinal vasculature. The VPCs migrate outward from the optic disk, as shown in Figure 2(a).

Patent vessels form through the transformation of solid vascular chords. The VPCs localize to the inner surface of the retina, between nerve fiber bundles, and orient their longitudinal axes along the direction of migration. The population of VPCs proliferates and differentiates to form a primordial vascular bed centered on the optic disk (Figure 2(b)). As early as 14–15 WG, vascular chords begin to coalesce on the surface of the retina behind the wave of spindle cells, beginning in the region proximal to the ONH and

progressing outward (Figure 2(c) and 2(e)). Figure 2(d) shows the transition from vascular chords and the discrete spindle cells located more peripherally.

Vascular chords begin to establish themselves as vessels that express CD34 and support blood flow as early as 18 WG (Figure 2(f )). These first primordial vessels formed through vasculogenesis are typically radial, have uniform diameter and have low capillary density (Figure 3(a)). The formation of primitive vessels lags behind the leading edge of VPCs by a distance of at least 1 mm. Vasculogenesis and the formation of the primordial vessel architecture is complete by 21 WG, at which point spindle cells are no longer detectable in the retina (Figure 3(b)).

Spindle cells (and the formation of cords and vessels behind the leading edge of spindle cells) migrate outward from the optic disk in a four-lobed pattern, as seen in Figure 3(b). The lobes extend the farthest in the temporal and superior directions, and they correspond to the future location of the four major artery–vein pairs in the adult retina (Figure 1(b)). The lobes curve around the region of the incipient fovea, leaving this area free of spindle cells and vascular cords. The fovea and perifoveal region remains avascular through 25 WG.

Although vasculogenesis is responsible for the primordial vessels that form in the inner two-thirds of the developing retina, this primitive network with its low capillary density is very inefficient in meeting the metabolic demands of the underlying retinal tissue. As a result, the remaining vessels in the retina form through the process of angiogenesis, which is driven by physiological levels of hypoxia. Angiogenesis is responsible for the increasing vascular density in the central retina (Figure 3(c)), vessel formation on the inner surface (the inner plexus) of the peripheral retina, and the formation of the outer, deep plexus and the radial RPCs near the ONH.

Angiogenesis. Angiogenesis in the retina is driven by physiological hypoxia, which occurs as a result of the increasing synaptic activity in the retinal tissues. As the retina thickens and the cell layers start to differentiate and become active, the tissue becomes hypoxic. In response to hypoxia, the astrocytes in the nerve fiber and ganglion cell layers (GCLs) express VEGF165, which in turn stimulates endothelial cell growth. Once these vessels become patent and can direct blood flow, the demand for oxygen is met and local VEGF expression decreases. It is important to note that the physiologic levels of hypoxia are too low to damage the surrounding tissue, but are sufficient to signal the need for increased oxygen supply to the tissue.

Inner plexus: Angiogenic filopodial extension is mediated by an astrocytic template and basal lamina components. As with vasculogenesis of the retina, formation of the superficial vascular plexus through angiogenesis begins in the region around the optic disk and spreads outward toward the periphery. As early as 17–18 WG, an exuberant network of broad capillaries starts to grow out from the primary

vasculature, filling the area that lies between the primordial vessels (Figure 3(d)). Between 18 and 30 WG, the sprouting of new vessels is led by the filopodia of endothelial tip cells (Figures 3(e) and 4(b)). These filopodia orient along the net-like framework that has been set up by astrocytes in the wake of the leading edge of APCs,

initially producing a capillary bed that closely follows the structure and organization of the astrocyte scaffold (Figure 4(a) and 4(c)). Although endothelial tip cells serve to orient the growth of the capillary network, the tip cells themselves do not divide. Instead, rapid cell growth occurs at the level of the trailing stalk cell (Figure 4(b)).

(a) (c)

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In the human fetal retina, Pax-2 expression is limited to cells of the astrocyte lineage. Glial fibrillary acidic protein (GFAP) is expressed in mature astrocytes. Pax-2+/GFAP- astrocyte precursor cells (APCs) can be detected at the ONH around 12 WG. This population of cells appears at a slightly later time than the VPCs, and can readily be distinguished from VPCs by their rounded shape and by the expression of Pax-2. Like the VPCs, the APCs migrate outward from the optic disk. The leading edge of APCs is immediately peripheral to the leading edge of vessel formation, preceding the region of vessel formation by no more than 120 mm (Figure 4(c)). Beginning at 18 WG, astrocytes loosely ensheath the newly formed vessels, where they play a role in the induction of the BRB. APCs migrate outward from the optic disk behind the spindle cells. Similar to the spindle cells, they also remain

excluded from the incipient fovea as they migrate outward. VPCs (and the primordial vasculature formed from these cells) are limited to the inner two-thirds of the retina (Figures 2(b) and 3(b)) but APCs continue to migrate toward the peripheral retina. As they mature, APCs begin to express GFAP. The Pax-2+/GFAP+ astrocyte cells reach the edge of the retina around 26 WG.

In addition to driving the formation of capillaries in the space between the primitive vascular network, angiogenesis also controls the peripheral spread of vessels beyond the inner two-thirds of the retina. The growth of vessels in this region closely follows the central-to-peripheral migration of astrocytes. Figure 4(c) and 4(d) show the relationship between patent vessel formation, APCs, and differentiated astrocytes. Again, the increasing metabolic demands of the maturing tissues lead to hypoxia and the

production of VEGF by the network of astrocytes that has formed on the surface of the retina; the dense capillary mesh forms along the astrocyte scaffold. Conversely, the endothelium may also influence astrocytic differentiation, as vascular endothelial cells have been shown to induce the expression of GFAP in APCs.

Outer plexus: Angiogenic growth is driven by neuronal maturation. The outer or deep layer of vessels also forms by the process of angiogenesis. Beginning around 25–26 WG, the superficial vessels start to bud and grow radially

down from the inner plexus of the retina (Figure 5). At the junction of the inner nuclear layer and the outer plexiform layer, the vessels start to ramify among the two layers. This stage in fetal development coincides with the peak period of eye opening, when the visually evoked potential, indicative of a functional visual pathway and photoreceptor activity, is first detectable in the human infant. The radial growth of these descenders is not preceded by either VPCs or APCs. VEGF expression in this region correlates with the soma of Mu¨ller cells, suggesting

that these cells produce the VEGF that stimulates and guides endothelial cell growth in the outer vascular plexus.

Unlike the inner plexus, the formation of the outer plexus is centered around the fovea. Formation of the outer plexus exactly matches the pattern of maturation of the neuronal retina, suggesting that increased metabolic demands from active neurons produce local, physiological hypoxia and drive the growth of vessels to these tissues (Figure 5). In this region, the vasculature is limited to capillary-sized vessels; larger vessels are not found in the outer plexus.

Foveal and perifoveal region. The vasculature of the inner plexus surrounds, but does not enter, the fovea; the fovea is supported by the outer plexus alone. The area of greatest cone density in the retina lies in the center of the avascular area, in a region called the fovea centralis (Figure 6(a)–6(c)). The absence of surface vessels in this region allows for maximal light penetration with minimal shadowing of the densely packed cones. The fovea itself, an oval of 500 – 600 mm in diameter, remains completely avascular through 25 WG (Figure 6(d)). The developmental cues that direct this aspect of retinal vascularization are not well understood, but recent evidence suggests that the developing fovea expresses antiangiogenic factors

including pigment epithelium derived factor (PEDF), natriuretic peptide precursor B, and collagen type IVa2.

More recently, it has been shown that expression of Eph-A6 directs vascularization in the fovea and perifoveal region. Immunohistochemistry from fetal primate retinas suggests that a gradient of Eph-A6 is centered at the fovea. This gradient, which is highest at the inner GCL and lowest near the inner plexiform layer, serves to repel astrocytes and angiogenesis from the inner layer of the retina toward the inner plexiform layer, where the vessels are found in the primate fovea. Because the incipient fovea lacks an astrocytic scaffold to set up the framework for vascularization, the pattern of formation of vessels in the outer plexus of the fovea and perifoveal region is different from that of the capillary networks in the inner plexus. As shown in Figure 6(e), from the time they are formed, the vessels are much more regular in their size and spacing than are newly formed capillary networks elsewhere in the retina (Figure 6(f )).

Radial peri-papillary capillaries. The third vascular plexus of the retina, the RPCs, begins to form around 21 WG (Figure 5). These capillary-sized vessels typically extend radially from the ONH (Figures 5 and 7(a)) and cover a small rim surrounding the optic papilla (ONH); for this

reason, they have been given the name RPCs. These vessels extend from the inner vasculature to supply the nerve fiber layer near the ONH (Figures 5 and 7(b)). The RPCs form a very limited plexus, extending no further than 1 mm from the ONH throughout human fetal development (Figure 5). As with other retinal vessels that are formed through angiogenesis, the timing and extent of RPC formation suggests that it is driven by hypoxia-mediated VEGF resulting from increased metabolic demands of the underlying tissues. Their superficial location and lack of mural cell ensheathment, leading to a thin vascular wall, makes RPCs particularly prone to hypoxia resulting from raised

intraocular pressure in glaucoma (the ischemic model of glaucoma susceptibility).

Lack of Involvement of VEGF in Early Stages

of Vascularization

Although retinal angiogenesis is driven by hypoxiainduced VEGF expression, vasculogenesis in the human retina is independent of metabolic demand and hypoxiainduced VEGF expression. Most of the vessels formed by vascularization develop prior to the expression of VEGF. By 18 WG, the inner plexus covers more than half of the