Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease

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Abstract

We review the cellular and physiological mechanisms responsible for the regulation of blood flow in the retina and choroid in health and disease. Due to the intrinsic light sensitivity of the retina and the direct visual accessibility of fundus blood vessels, the eye offers unique opportunities for the non-invasive investigation of mechanisms of blood flow regulation. The ability of the retinal vasculature to regulate its blood flow is contrasted with the far more restricted ability of the choroidal circulation to regulate its blood flow by virtue of the absence of glial cells, the markedly reduced pericyte ensheathment of the choroidal vasculature, and the lack of intermediate filaments in choroidal pericytes. We review the cellular and molecular components of the neurovascular unit in the retina and choroid, techniques for monitoring retinal and choroidal blood flow, responses of the retinal and choroidal circulation to light stimulation, the role of capillaries, astrocytes and pericytes in regulating blood flow, putative signaling mechanisms mediating neurovascular coupling in the retina, and changes that occur in the retinal and choroidal circulation during diabetic retinopathy, age-related macular degeneration, glaucoma, and Alzheimer's disease. We close by discussing issues that remain to be explored.

Introduction

The retina has the highest metabolic demands of any tissue in the body (Saari, 1987; Buttery et al., 1991). Studies utilizing oxygen microelectrodes (Alder et al., 1990) and immunohistochemical visualization of the activity of the enzyme cytochrome oxidase (Buttery et al., 1991) have shown that the outer segments of the photoreceptors are the most metabolically active layer of the retina. Because of the high metabolic activity of the retina, the ability to regulate blood flow is an essential feature of the mammalian retina. The conflicting requirements of sufficient blood supply and minimal interference with the light path to the photoreceptors have been met by the evolution of two vascular supplies – inherent intra-retinal vessels supply the inner two-thirds of the retina, while the choroidal circulation supplies the photoreceptors in the outer one-third of the retina. Further, in the retinas of primates, an avascular region at the fovea further facilitates high acuity vision.

The human retinal vasculature is comprised of the central retinal artery, which enters the optic disc through the lamina cribrosa, where it branches into four principal intra-retinal arteries (Fig. 1A). Whilst termed retinal arteries, even the central retinal artery is only of a caliber of an arteriole and if accurate terminology is used, only retinal arterioles exist, not arteries. The arterioles bifurcate to form smaller arteriole branches and terminal arterioles, which feed into a capillary bed as they extend toward the peripheral retina. Retinal arterioles, due to the higher oxygen content of the blood they carry, are typically surrounded by a capillary-free zone, approximately 30–50 μm in diameter in monkeys (Okada and Ohta, 1994). The venous system of the retina has a similar arrangement with the central retinal venule leaving the eye through the optic disc to drain venous blood into the cavernous sinus. The terminal branches of the vessels, pre-capillary arterioles and post-capillary venules, are linked through anastomotic capillaries. Retinal capillaries are organized in an interconnecting two-layer network. A superficial layer is located in the nerve fiber and ganglion cell layers and a second lies deeper, in the inner nuclear and outer plexiform layers.

In the mammalian retina, the vasculature in the nerve fiber and ganglion cell layers is known as the inner (or superficial) plexus, while the inner nuclear and outer plexiform layers receive blood from the deeper plexus located at the junction between them (Figs. 1B and 2A). The superficial plexus, contains arterioles, venules, and capillaries, while the deep vascular bed consists predominantly of capillary-sized vessels. Both the superficial and deep retinal plexus reach almost to the edge of the human (Fig. 2C) (Hughes et al., 2000; Chan-Ling et al., 2004a), cat (Chan-Ling et al., 1990), rat (Fig. 2D) (Stone et al., 1995) and mouse retina (Dorrell et al., 2002), except for a small avascular rim. The fovea, found only in primates, is also avascular; the thinness of the retina in this region permits adequate retinal oxygenation via the choroidal circulation (Engerman, 1976). The superior and inferior temporal vessels deviate in their paths to bypass the fovea and minimize their density in the temporal raphe region (Fig. 1A).

A third intra-retinal plexus, reported in the cat and human retina and known as the radial peripapillary capillaries (RPCs), is located in the nerve fiber layer in a small rim surrounding the optic nerve head (Chan-Ling et al., 1990; Hughes et al., 2000). These RPCs are located superficially around the optic nerve head, where the nerve fiber bundles are thickest, prior to exiting the retina (Henkind, 1967). Fig. 2C shows the extent of the RPC's surrounding the optic nerve head in the human. For detailed reviews of the retinal vasculature, see Chan-Ling (2008), Pournaras et al. (2008), Riva et al. (2011).

In the human, the walls of the largest arterioles, near the optic disc, are comprised of five to seven layers of smooth muscle cells (tunica media). Smooth muscle actin filaments extend circumferentially around the retinal arterioles (Fig. 3A). After several branchings of the vascular network, the number of layers diminishes to just one or two in the retinal periphery. In retinal arterioles, the smooth muscle cells are orientated both circularly and longitudinally, each being surrounded by a basal lamina that contains an increasing amount of collagen toward the acellular adventitia (the tunica externa); reviewed in Pournaras et al. (2008). Endothelial cells (part of the tunica interna) are orientated longitudinally along the axis of the vessel and share their basement membrane with adjacent smooth muscle cells and pericytes. This basement membrane is composed of collagen IV, fibronectin, laminin, matrix metalloproteinases (MMPs-2, MMPs-9) and serine proteinase urokinase (UPA) and acts as an important regulatory matrix for the passage and sequestration of vasoactive agents and pro-survival growth factors; reviewed in Archer et al. (2007). In the smallest pre-capillary arterioles, the distribution of smooth muscle cells is frequently sporadic. Contrary to other vascular networks, the human retina lacks pre-capillary sphincters (Henkind and De Oliveira, 1968) and therefore the retinal capillaries are continuously perfused.

The capillary unit consists of a continuous endothelium and intramural pericytes, which extend longitudinally along the capillary (Hughes and Chan-Ling, 2004) (Fig. 3B). Both cell types are in direct communication via gap junctional complexes (Oku et al., 2001) and share a common basement membrane. Regarding pericyte-to-endothelial cell ratios, a recent study of the human retina, utilizing ultrastructural criteria, showed a 94.5% frequency of pericyte coverage on human retinal capillaries (Chan-Ling et al., 2011b). Therefore, the retinal microvasculature is characterized by a uniquely high density of pericytes, substantially greater than that of human choriocapillaris, with an 11% relative frequency of pericyte coverage (Tilton et al., 1985; Chan-Ling et al., 2011b) or cerebral capillaries (Frank et al., 1987). In addition to numerous morphological characteristics, venules can be distinguished from arterioles by the size of the capillary-free zone around them; the zone is narrower around venules (Hogan and Feeney, 1963). The fine structure of venular muscle cells is similar to that of pericytes. Collagen fibrils are also seen in the outer layers of the basement membrane of these venules, and tend to increase in amount in the larger vessels (Ishikawa, 1963). Retinal microvessels are not-fenestrated and possess tight junctional complexes between the endothelial cells on their luminal aspect. The tight junctions represent the structural component of the inner blood-retinal barrier; for review see Chan-Ling (2006). Retinal arterioles, venules, and capillaries are closely ensheathed by macroglia. The superficial retinal vasculature is ensheathed by both astrocytes (Fig. 3C) and Müller cells, whilst the deep vascular plexus is ensheathed solely by Müller glia (Fig. 3D) (Holländer et al., 1991).

The choroidal circulation is derived primarily from the long and short ciliary arteries with some contribution from the anterior ciliary arteries. Histologically, the choroid is divided into five layers. Starting from the retinal side, these include Bruch's membrane, three vascular layers (the choroicapillaries, Sattler's layer and Haller's layer) and the suprachoroidea (Figs. 1B and 2B). Haller's layer includes large arteries and veins, while Sattler's layer is composed of medium and small arterioles that feed the capillary network of the choriocapillaris and venules. The choroidal arteries arise from the long and short posterior ciliary arteries and branches of Circle of Zinn (around the optic disc). The choriocapillaris is a highly anastomosed network of capillaries (with little or no basement membrane material), forming a dense capillary network opposed to Bruch's membrane. Drainage of blood from the choroid is thought to occur exclusively through the vortex veins that ultimately merge with the ophthalmic vein (Ruskell, 1997). In contrast to the retina (Fig. 4G), choroidal microvessels are fenestrated (Bill et al., 1980), although the fenestrae are not as frequent in choroidal capillaries as in capillaries of other tissues (Chan-Ling et al., 2011a) (see Fig. 4H inset b). Unlike retinal vessels, the choroidal circulation is under neurogenic control. Sympathetic innervation includes noradrenergic and neuropeptide fibers (Bruun et al., 1984), whereas the parasympathetic nerves are primarily cholinergic (Bill and Sperber, 1990). For a comprehensive review on structure and function of the choroid see Nickla and Wallman (2010).

Although we designate vessels as arteries, arterioles, capillaries, venules and veins, the truth of the matter is that each segment of a vessel represents a continuum of vascular phenotype where the physiological characteristics as well as the proteins expressed and the cellular associations vary continuously along the vessel (Hughes and Chan-Ling, 2004). Thus, there are vessel segments that have characteristics of both arterioles and capillaries in certain parts of the retinal and choroidal vascular bed.

Section snippets

Development of retinal and choroidal circulation

Concomitant with the maturation of retinal neurons, the retina's vasculature develops to form an elaborate vascular tree that is well matched to the metabolic needs of the tissue (Chan-Ling et al., 1990). The formation of the intra-retinal vessels takes place via two distinct cellular processes under different molecular cues. Formation of the primordial superficial vessels of the central one-third of the human retina takes place via the process of vasculogenesis, the de novo formation of

Retinal blood flow

A variety of techniques have been developed to monitor retinal and choroidal blood flow. The Doppler effect has been employed in measuring the velocity, volume, and flux of blood through the capillaries and larger vessels of the optic nerve head and superficial retina. The reader is referred to reviews by Feke (2006), Riva and Falsini (2008). In the laser Doppler technique, the frequency of reflected laser light changes when scattered by red blood cells moving through vessels. The magnitude of

Regulation of retinal blood flow

The retina tends to maintain a constant blood flow in the face of variations in perfusion pressure, blood gasses and intraocular pressure. This is an intrinsic autoregulatory response since the potential influence of autonomic innervation can be excluded (Laties, 1967; Ye et al., 1990) and the contribution of circulating hormones and neurotransmitters on retinal vascular resistance is generally assumed to be negligible due to the blood-retinal barrier (Delaey and Van De Voorde, 2000b).

Mechanisms of functional hyperaemia

The mechanisms that mediate functional hyperemia remain controversial. For many years, metabolic feedback mechanisms were thought to mediate the regulation of blood flow in response to changes in neuronal activity. As originally proposed by Roy and Sherrington, metabolic feedback would work as follows: Increases in metabolism which accompany neuronal activity would lower O2 and glucose levels and produce vasoactive metabolites (Roy and Sherrington, 1890). These metabolites would elicit

Blood flow regulation in the choroid

In contrast to the retina, the choroidal circulation is controlled by extrinsic autonomic innervation. Decreases in choroidal blood flow are mediated by activation of sympathetic efferent nerves that release noradrenaline, activating alpha 1-adrenoceptors on vascular smooth muscle cells (Alm, 1977; Kawarai and Koss, 1998). In turn, increases in choroidal blood flow are mediated by parasympathetic efferent nerves which act via NO signaling (Nilsson, 1996). The choroid also receives rich

Regulation of ocular blood flow in disease

Normal retinal and choroidal blood flow is altered in a number of disorders that affect the eye, including diabetic retinopathy, glaucoma, age-related macular degeneration, and Alzheimer's disease. Changes in responses to flickering light, as well as altered rates of basal blood flow, are observed under pathological conditions. The following sections detail changes in ocular blood flow that are observed under a number of conditions.

Future directions

While much is known about the development of the retinal and choroidal vasculature, the structure of the vascular unit and the contractile machinery of vascular smooth muscle cells, mechanisms mediating neurovascular coupling, and the responses of retinal and choroidal vessels to visual and autoregulatory stimuli, many questions remain to be answered. Several key issues that remain to be addressed are outlined in the following paragraphs.

Although there is a detailed understanding of the growth

Acknowledgments

The authors thank Mr. Sam Adamson for assistance with digital imaging. Supported by Fondation Leducq of France, the National Institutes of Health of the United States (EY004077), the National Health and Medical Research Council of Australia (#571100, 1005730), the Rebecca L. Cooper Medical Research Foundation, the Brian M Kirby Foundation – Gift of Sight Initiative, and the NSW Optometrist Registration Board – Best Practice Grant.

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