Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Photoreceptors are the most fragile of retinal neurons in the face of genetic and environmental stress, and oxygen and oxidative stress are emerging as major environmental factors affecting their stability and contributing to retinal degeneration. This chapter addresses the question of why photoreceptors are vulnerable to a wider range of mutations than any other neuron class of the retina or brain. Increasing evidence suggests that this fragility results in great part from environmental factors such as tissue oxygen levels. Although studies of the mouse retina have been prominent in the growing literature on the subject, this chapter also considers relevant findings from other species, including humans.

Photoreceptors are selectively vulnerable not only to genetic mutations but also to a range of environmental stresses, metabolic toxins (Eells et al., 2003; Graymore and Tansley, 1959), hypoxia and hyperoxia (Wellard et al., 2005), trauma such as retinal detachment (Fisher et al., 2001; Mervin et al., 1999), and the cumulative stresses of aging (Gao and Hollyfield, 1992; Jackson et al., 2002). Genetic mutations act on a background of stress to and degeneration of photoreceptors. The phenotypes that emerge—the many forms of retinitis pigmentosa, the normal aging of the retina, the edge-specific degeneration that begins in childhood, the collapse of the macula with age— occur on a normal background. Appropriate therapy requires knowledge of both the normal and mutation-driven degeneration of photoreceptors and of the interaction between environmental and genetically induced stress.

In this chapter, we focus on the delivery of oxygen to the retina, the regulation and dysregulation of that delivery, and the consumption of oxygen by photoreceptors. Clinically, there are two reasons for this focus. First, tissue oxygen levels affect the stability of the normal retina, from neonatal life to advanced age. Second, the role of environmental stress in genetically driven degeneration has long gone unrecognized and provides an unexplored avenue for therapy of the degenerating retina.

The delivery of oxygen to the retina

The mouse retina has a retinal vasculature, like the human and many other mammalian retinas. Oxygen is delivered from two sources, the retinal and choroidal circulations. These circulations have distinct properties, and the interaction between them is critical in determining oxygen levels across the retina, and therefore the stability of photoreceptors.

The Retinal Circulation The retinal circulation is a conventional system of arteries, capillaries, and veins. It forms developmentally from the hyaloid artery, which forms to supply the lens vesicle well before the retina requires its own circulation. At birth in the rat (Cairns, 1959), the retina is almost avascular, but the hyaloid artery passes from the optic disc to the developing lens. Over the first 10 days of postnatal life, the hyaloid circulation regresses and the retinal circulation forms by growth from the hyaloid vessels, following a template of astrocytes that spread from the optic disc across the retinal surface (Chan-Ling et al., 1990; Stone et al., 1995), expressing potent angiogenic factors (reviewed in Stone and Maslim, 1997). In the mouse, the retinal circulation begins to form before birth (Connolly et al., 1988) and also forms along a preexisting astrocyte template (Dorrell et al., 2002). Once the branches of the hyaloid vessels that supply the lens have regressed, the stem vessels are termed retinal vessels, and supply the inner layers of the retina.

In the adult retina, arteries radiate from the optic disc across the inner surface of the retina and branch to form to arterioles and capillaries, which spread through the inner half of the retina. The capillaries space themselves through the inner layers of the retina and are described as concentrating in three layers, in the ganglion cell layer (GCL) and at the inner and outer surfaces of the inner nuclear layer (INL). Capillaries drain to venules, and these in turn drain to veins, which converge on the optic disc, to leave the eye.

Physiologically, arterial and venous oxygen levels have been measured in the human (Alm, 1992) and cat (Alm and Bill, 1970, 1972a, 1972b). The consistent outcome is that oxygen extraction from the retinal circulation is 30%–40%, a level of oxygen extraction similar to that in the cerebral circulation.

Rates of blood flow through retinal tissue have been estimated for a range of animals (reviewed in Paques et al., 2003), including rodents and primates. The rate of capillary flow in the mouse retinal circulation was estimated by Paques and colleagues at 1.26 mm/s, within the range reported for cat, rat, and monkey but higher than in other rodent tissues, including the rat brain (0.7 mm/s). Paques et al. note that the relatively high flow rate would be advantageous for oxygen delivery. Retinal vessels show blood-brain barrier properties, induced by their contact with neuroglial cells (Chan-Ling and Stone, 1992; Janzer and Raff, 1987; Tout et al., 1993; reviewed in Stone and Maslim, 1997).

Further, the capillaries of the retinal circulation show the conventional property of autoregulation (Alm and Bill, 1970, 1972a, 1972b; Eperon et al., 1975); that is, they constrict, reducing flow, when oxygen levels in the tissue they supply rise, and dilate when oxygen levels fall. The mechanism of constriction is believed to lie in muscular control of arteriolar diameter, or in the control of capillary flow by pericytes (Peppiatt et al., 2006). Paques and colleagues (2003) note that the architecture of the mouse retinal circulation (which may be representative of mammalian retinal circulations) has features that favor control of capillary flow by local tissue conditions. Some superficial capillaries, for example, connect directly to large veins, and could provide a shunt past the deeper capillary beds.

The one distinctive feature of the retinal circulation is that its capillaries reach only halfway across the thickness of the retina. From arteries at the inner surface of the retina, capillaries extend radially as far as the outer plexiform layer (OPL), and never (normally) into the outer nuclear layer (ONL). Their growth into that layer is robustly inhibited by a mechanism that has been explored in the mouse. The development of the retinal circulation is severely disturbed by antibodies to R-cadherin (Dorrell et al., 2002; reviewed in Stone et al., 2006), which presumably prevent the normal adherence of vessels to cells that express cadherin. Cadherin is strongly expressed in the OPL of the mouse, and Dorrell and colleagues suggest that vessels growing radially outward in the retina are diverted to follow this line of expression, along the OPL. Vessels grow past the OPL into the ONL only if this barrier is disturbed (by masking cadherin with antibodies) or overcome by an abnormal expression of angiogenic factors by cells of the ONL (Tobe et al., 1998). As a consequence, the ONL and the layers of inner and outer segments—effectively, the photoreceptors—are normally avascular, the only avascular parts of the CNS. Oxygen

diffuses to the photoreceptors from the deepest capillaries of the retinal circulation, but the major oxygen supply to photoreceptors reaches them from the external surface of the retina, by diffusion (Yu and Cringle, 2001). This dual supply of oxygen to photoreceptors is distinctive and an important factor in their relative fragility.

The Choroidal Circulation In all vertebrates, a rich vascular bed forms just external to the neural retina. Known as the choroidal circulation, this vascular bed is highly distinctive structurally and functionally. Anatomically, the choroidal circulation is a rich vascular bed that forms the tunica vasculosa of the eyeball, between the retina (tunica nervosa) and sclera (tunica fibrosa). The capillaries of the choroidal circulation (the choriocapillaris) are coarse and profuse and form a lake of blood at the outer surface of the retina. The choriocapillaris abuts the outer surface of the basement membrane of the retinal pigmented epithelium (RPE) (Bruch’s membrane), and oxygen diffuses across Bruch’s membrane, across the RPE and subretinal space to reach the mitochondria of the inner segments.

The capillaries of the choriocapillaris do not have barrier properties but are highly fenestrated and permeable (Bill et al., 1980). Further, the rates of blood flow through the choroidal circulation are very high. When expressed in milliliters per minute per gram of tissue supplied, the choroidal blood flow is several times higher than in other high-rate tissues, such as the cortex of the kidney, cardiac muscle, the inner retina, and cerebral cortex (see Alm, 1992, fig. 6.15 therein). Veins converge into distinctively convoluted vortex veins, which emerge from the eyeball near its equator and join orbital veins.

As a result of the very high rates of blood flow through the choroidal circulation, the partial pressure of oxygen in vortex vein blood is only 3%–4% below arterial levels (Alm, 1992; Bill et al., 1983); that is, the level of oxygen extraction is much lower than in retinal and cerebral circulations. As a consequence, the capillary bed of the choroidal circulation (the choriocapillaris) forms a lake of near-arterial blood around the outer surface of the retina, which is the main source of oxygen to photoreceptors.

An important feature of the anatomy of the choroidal circulation is that its capillaries do not lie in the tissue they supply and do not respond to conditions in that tissue. Choroidal blood flow shows some evidence of regulation, in response, for example, to experimental increases in arterial pressure (Riva et al., 1997), but is not responsive to oxygen or metabolite levels in the outer retina, the tissue it supplies. As a consequence, oxygen levels in the outer retina can fluctuate more widely than in any other retinal layer, and this fluctuation underlies the pathogenesis of a range of retinal diseases, among them retinopathy of prematurity, retinopathy of detachment, and retinitis pigmentosa.

Finally, the choroid develops early, well before the neural differentiation of the retina. Choroidal development has been described in most detail for the human (Allende et al., 2006). It shows a centroperipheral pattern of formation and forms well before the photoreceptors (which in the normal adult retina consume all the oxygen delivered by the choroid) have begun to function. The development of the retinal circulation occurs much later, in the mouse in the first 2 weeks of postnatal life (Connolly et al., 1988), well after vessels invade the cerebral cortex (in midgestation; Stone and Maslim, 1997), and is strongly influenced by oxygen reaching the retina from the choroid.

Oxygen consumption in the retina

Oxygen Consumption in the Adult The consumption of oxygen by the retina has been scrutinized with a range of technologies. For the mouse, the most direct and recent data are oxygen tension measurements obtained in vivo with oxygen-sensitive electrodes. These data (figure 46.1A) demonstrate that in the mouse (Yu and Cringle, 2006), as in the rat, cat, and monkey (reviewed by Yu and Cringle, 2001), oxygen flows into the retina from two sources, one in inner retina and corresponding to the retinal circulation and the other at the outer surface of the retina and corresponding to the choriocapillaris.

Oxygen is consumed in the retina at locations corresponding to the sites of mitochondria. Most of the oxygen flowing from the choriocapillaris is consumed by the dense concentration of mitochondria in the inner segment. In the darkadapted retina, the oxygen tension in the ONL is near zero;

Figure 46.1 Oxygen levels within the retina, measured with oxygen-sensitive electrodes stepped through the retina, and the effect of depletion. In all three graphs the inner surface of the retina is at left, and the outer (choroidal) surface is at right. A, In the mouse, oxygen levels are maximal at the outer surface, close to the choriocapillaris. A second area of increased oxygen level is apparent at the inner surface, representing oxygen delivered by the retinal circulation. Asterisk marks a minimum in the region of the ONL; the sharp fall in tissue oxygen tension between the choroidal maximum and this minimum results from intense oxygen consumption by photoreceptor inner segments. B, Corresponding measurements in the naturally degenerative RCS rat. This is a developmental series. The minimum produced by photoreceptor oxygen consumption is evident in the youngest age studied (P20) but disappears as the animals age and the photoreceptor population is exhausted. C, Comparison of oxygen tension profiles obtained from a nondegenerative Sprague-Dawley (SD) rat and the degenerative P23H-3 transgenic strain. The minimum produced by photoreceptor consumption of oxygen is absent from the P23H-3 strain. (A, From Yu and Cringle, 2006. B, From Yu et al., 2000. C, From Yu et al., 2004.)

oxygen from the choriocapillaris does not reach beyond the inner segments. The oxygen sinks of the inner retina are more distributed, in ganglion cells, in the IPL and OPL.

As already noted, the flow of blood through the choriocapillaris and the level of oxygen in choroidal blood do not vary with the level of oxygen in the tissue being supplied. As a consequence, oxygen flows from the choroid to the outer

Track distance through retina (m m)

Track distance from inner retinal minimum (mm)

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C: P23H vs SD rat

40 P23H-3

Track distance through retina (mm)

retina (i.e., to photoreceptors) following physical gradients; there is no mechanism to regulate that flow in response to the needs of photoreceptors.

The Developmental Onset of Oxygen Consumption

The onset of oxygen consumption by photoreceptors has been well documented in rodents. Graymore (1959, 1960, 1963) used as a measure of metabolism the retina’s production of lactic acid, an end product of glycolysis. He showed that the retina’s production of lactate is low at birth and increases dramatically, in the rat between P15 and P20. This period corresponds to the onset of several measures of photoreceptor function, including the development of the electroretinogram (Fulton et al., 1995), the growth of the inner and outer segments of photoreceptors (Weidman and Kuwabara, 1968), the onset of oxygen consumption by the photoreceptors (Cringle et al., 2006), and the onset of a period of physiological hypoxia (Chan-Ling et al., 1995a), which induces the expression of angiogenic factors in glial templates on which the retinal blood vessels form (Dorrell et al., 2002; Stone et al., 1995).

The development of the retinal vasculature is thus driven by the development of photoreceptor function (reviewed in Stone and Maslim, 1997). The mechanism that links photoreceptor function to vascular development is hypoxia; the onset of photoreceptor function and oxygen consumption reduces the flow of choroidal oxygen to the inner retina, and the resulting hypoxia serves as a signal for vasogenesis.

Neuroglobin: An Oxygen-Binding Globin Prominent in the Retina Neuroglobin is a member of the globins, heme proteins found in plants, bacteria, fungi, and animals, with the property of reversibly binding oxygen. Neuroglobin was first described in the mouse and human (Burmester and Hankeln, 2004) and was named for its specific location in brain tissue (Burmester and Hankeln, 2004; Sun et al., 2001); it is a candidate protein for providing an oxygen store in brain tissue, as hemoglobin does in blood. Neuroglobin expression is upregulated in hypoxia (Sun et al., 2001) and is expressed roughly 100 times more strongly in the retina than in brain tissue (Schmidt et al., 2003), arguably reflecting the high oxygen utilization of the retina. The distribution of neuroglobin in the retina, in the IPL and OPL, and in the inner segments of photoreceptors correlates with the distribution of mitochondria, the organelles in which oxygen is recruited in the energy-producing mechanisms of oxidative phosphorylation (Schmidt et al., 2003). Correspondingly, neuroglobin levels are low in the inner retina of the guinea pig (Bentmann et al., 2005), in which species a retinal vasculature is lacking and oxygen metabolism cannot be detected (Yu and Cringle, 2001). The cellular and laminar distribution of neuroglobin in humans (Rajendram and Rao, 2007) and dogs (Ostojic et al., 2006) is similar to

that observed in mice, and its amino acid sequence is highly conserved within mammals (Zhang et al., 2002).

Consequences of lack of regulation of oxygen supply to the outer retina

As we have argued previously (Chan-Ling and Stone, 1993; Lewis et al., 1999; Stone and Maslim, 1997; Stone et al., 1999), the inability of the choroidal circulation to regulate its delivery of oxygen to the retina underlies the pathogenesis of several retinal diseases, including retinopathy of prematurity, retinopathy of detachment, and the photoreceptor degenerations. The following sections trace the underlying mechanisms.

Inhaled Oxygen Reaches the Retina Selectively The lack of regulation of the choroidal circulation is evident in the effect that raising oxygen levels in inhaled air has on body tissues. The two body tissues most directly affected by hyperoxia are the epithelium of the lung (where hyperoxia induces a dysplasia; Barazzone et al., 1998; O’Brodovich and Mellins, 1985) and the retina, where effects include a narrowing of retinal vessels and the death of photoreceptors. Other tissues appear to be protected from hyperoxia by autoregulatory mechanisms induced by the hyperoxia. The contrast between autoregulation and the lack of it is evident in studies of the effect of hyperoxia on tissue oxygen levels across the retina, where there is a retinal circulation (Yu and Cringle, 2001; figure 46.2). As the level of oxygen in inhaled air is increased, oxygen levels rise throughout the retina, but much more so in the outer retina.

The flow of higher than normal levels of oxygen from the choroid to the retina has two spectacular effects on the structure of the retina. It causes the death of photoreceptors, and it causes the constriction and in some circumstances the closure of vessels of the retinal circulation.

Inhaled oxygen kills photoreceptors. Because inhaled oxygen reaches the outer retina from the choroid, simply breathing oxygen-enriched air can kill photoreceptors. The first evidence of this toxicity came from Noell’s brief report in 1955 that inhalation of 100% oxygen causes the total degeneration of photoreceptors in the rabbit retina within 3 days. The inner retinal layers appeared unaffected. The selective vulnerability of photoreceptors was subsequently confirmed in the mouse (Walsh, Bravo-Nuevo, et al., 2004; Yamada et al., 2001) and rat (Geller et al., 2006; Wellard et al., 2005) (figure 46.3). The effect takes longer (2 weeks) in rodents than in the rabbit, but it is also highly specific to photoreceptors and is accompanied by an upregulation of stress-inducible proteins (fibroblast growth factor-2 [FGF-2], glial fibrillary acidic protein [GFAP]). Wellard et al. (2005) reported that the vulnerability of photoreceptors to hyperoxia is part

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Figure 46.2 Effects of inhaled oxygen and light on intraretinal oxygen, measured in the eye of the rat (left) and cat (right). Left, The lower-most curve is equivalent to the SD data in figure 46.1C, the oxygen tension profile across the retina, in normoxia. As the proportion of oxygen in the air inhaled increases, oxygen levels rise all across the retina. The rise is much greater near the choriocapillaris (at right) than in the inner retina; this greater rise results from the lack of regulation of the choroidal circulation (From Cringle et al., 2002). Right, The lower pair of curves shows the oxygen tension profiles across the cat retina, in normoxic conditions, lightand dark-adapted. The dark-adapted curve shows the same general

of a wider pattern in which photoreceptors are maximally stable at 21% oxygen (room air) and are destabilized (i.e., the rate of their death is increased) by both hypoxia and hyperoxia.

Finally on this point, it has been reported that in mice, the vulnerability of photoreceptors to oxygen is strain dependent (Walsh et al., 2004a), being less in the Balb/c strain than in the C57BL/6 strain. These strain differences are currently being used in our laboratory to identify genes that regulate the oxygen vulnerability of photoreceptors.

Inhaled oxygen blocks vasogenesis and thins or obliterates formed vessels. The impact of choroidal oxygen on the retinal vasculature was first detected in the analysis of the causes of retinopathy of prematurity, a neovascularizing disease of the infant retina associated with the use of oxygen to relieve respiratory difficulties in prematurely born infants (reviewed in Chan-Ling and Stone, 1993). Work in mouse (Browning et al., 1997; Smith et al., 1994), rat (Penn et al., 1994), and cat (Chan-Ling and Stone, 1992, 1993; Chan-Ling et al., 1992; Stone et al., 1996) models established that oxygen flowing from the choroid in the neonatal retina inhibits the

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features as reported for other mammals (see figure 46.1); in this graph the choroidal side of the retina is to the right. Light adaptation causes a rise (shown by the bracket) in tissue oxygen tension in the outer retina, the result of a light-induced reduction in the consumption of oxygen by photoreceptors. The upper pair of curves shows the oxygen tension profiles obtained when the animal breathed oxygen-enriched air. Oxygen tension is raised all across the retina, but again more markedly so at the choroidal side, and light adaptation increases tissue oxygen tension further (bracketed area). That is, the effects of hyperoxia and light on oxygen tension are additive. (From Linsenmeier and Yancey, 1989.)

normal development of retinal vessels and causes the thinning or obliteration of vessels already formed. When the baby or pup or kitten is returned to room air, the inner retina, lacking its normal complement of vessels, becomes abnormally hypoxic, triggering the death of astrocytes (the cells that normally express angiogenic factors such as vascular endothelial growth factor, VEGF) and upregulation of the same factors by neurons (Stone et al., 1996). The resulting rapid growth of vessels in the absence of astrocytes, which both induce barrier properties in new vessels and form part of the inner limiting membrane, results in the formation of leaky vessels, which grow into the vitreous humor. The use of supplemental oxygen to allow the late, nearly normal growth of vasculature has been explored (Chan-Ling et al., 1995b). In recent decades, the incidence of retinopathy of prematurity has been greatly reduced with the careful monitoring of arterial oxygen levels.

Vessels in the developing retina are particularly vulnerable to oxygen. In the rat, molecular events associated with the spread of pericytes at the end of the first postnatal month make adult vessels resistant to obliteration (Benjamin et al., 1998).

Figure 46.3 Oxygen is selectively toxic to photoreceptors. A–D, Sections across the retina of C57BL/6 mice exposed to high oxygen levels in inhaled air. Before exposure, dying (red, TUNEL-labeled) cells were rare. Exposure to hyperoxia for 14–35 days (B–D) increased the frequency of dying cells, selectively in the ONL (o). By 35 days, the ONL had thinned and TUNEL+ debris appeared

Retinal Detachment Causes Intense Hypoxia of the Outer Retina: The Use of Oxygen Relieves the

Retinopathy of Detachment The viability of the retina is threatened when it becomes detached from the outer walls of the eyeball. This detachment can be spontaneous or trauma induced; typically the neural retina detaches from the RPE, separating photoreceptors from their major source of oxygen. The results of detachment have been well studied (Fisher et al., 2001) and include photoreceptor death and proliferation and hypertrophy of neuroglia, which in humans can result in the total loss of retinal function.

The effects of detachment are greatly reduced by giving supplemental oxygen (i.e., enriching the inhaled air with

in the inner nuclear layer (i), probably ingested by Müller cells. The blue dye is a DNA label (bisbenzamide). E and F, When the sheaths of cones were labeled with PNA lectin, the nuclei of some were TUNEL labeled (red), indicating that cones as well as rods were oxygen sensitive. See color plate 48. (From Geller et al., 2006.)

oxygen). Photoreceptor death is reduced (Mervin et al., 1999), glial proliferation and hypertrophy are limited (Lewis et al., 1999), and the neurochemistry of the retina is less disturbed. This indicates that hypoxia plays a major role in inducing the pathological changes, and that hypoxic damage occurs because the choroid has no mechanism to detect or respond to abnormal hypoxia in the outer layers of the detached (or attached) retina.

A Reduction in Photoreceptor Metabolism Raises Oxygen Levels in the Outer Retina Once it is accepted that the flow of oxygen from the choroid to the outer retina is unregulated, it follows that any reduction in the oxidative

metabolism of photoreceptors will, because the choroidal circulation cannot autoregulate, lead to an increase in the oxygen reaching the retina (Stone et al., 1999). This prediction was formalized in models of oxygen supply and consumption in the retina developed from the laminar analysis of oxygen distribution in the retina (Haugh et al., 1990; Yu and Cringle, 2001). These models include a major oxygen sink at the inner segments of photoreceptors. If that sink weakens in intensity, then, the models predict, oxygen from the choroid will flow into the cellular layers of the retina. Further, the thinning of the outer retina caused when photoreceptors degenerate will reduce the required diffusion path and add to this effect.

Experimentally, light raises oxygen levels in the outer retina. Photoreceptors generate an electrical response to light (beginning the coding of vision) by reducing their dark current. Molecules generated when rhodopsin absorbs light sequester cyclic guanosine monophosphate (cGMP) from the cytoplasm, closing cGMP-gated Na+ channels, and reducing the need for Na+ pumps in the inner segment to expel Na+ from the cell. These pumps utilize adenosine triphosphate (ATP), so that light reduces the photoreceptors’ requirement of ATP, and therefore for oxygen, which is used in oxidative phosphorylation to produce ATP.

As a consequence, a maintained increase in light absorbed by photoreceptors causes a maintained increase in oxygen tension in the outer retina (reviewed in Wangsa-Wirawan and Linsenmeier, 2003; see figure 46.2). The rise is reversible; decreasing light falling on the retina decreases retinal oxygen tension. The dynamics of the oxygen change are relatively rapid; oxygen levels rise and fall with increases and decreases in light, with a time constant of seconds with dim light, and of minutes with bright (photopic) levels of light (see Linsenmeier, 1986, fig. 46.4 therein).

Experimentally, photoreceptor depletion raises oxygen levels in the outer retina. A sustained rise in oxygen tension in the outer (photoreceptor) layers of retina has now been demonstrated in three animal models of photoreceptor degeneration, the RCS rat (Yu et al., 2000) (see figure 46.1B), P23H-3 rat (Yu et al., 2004) (see figure 46.1C ), and Abyssinian cat (Padnick-Silver et al., 2006). These degenerations are all genetic in origin, but the mutations are diverse. In the RCS rat, a mutation in the receptor tyrosine kinase Mertk is associated with failure of the RPE to phagocytose the discarded membrane of photoreceptors (D’Cruz et al., 2000); in the P23H-3 rat, a transgene generates rhodopsin with a single amino acid substitution (Machida et al., 2000). The mutation in the Abyssinian cat (Ehinger et al., 1991) remains unidentified but appears not to involve rhodopsin (Gould and Sargan, 2002). All these studies relate the rise in oxygen levels in the outer retina to a decrease in oxygen consumption by photoreceptors.

Clinically, photoreceptor degeneration protects against retinal neovascularization. Confirmation of the idea that, when photoreceptors degenerate, oxygen from the choroidal circulation reaches the inner layers of retina comes from reports that retinal degeneration is protective against hypoxic neovascularizing diseases of the retina, in particular diabetic retinopathy (Arden, 2001; Sternberg et al., 1984). The protective effect has been confirmed (Lahdenranta et al., 2001) in a mouse model of photoreceptor degeneration; these authors noted that the normal hypoxia-induced expression of the angiogenic factor VEGF during development, which is critical for vessel formation, does not occur in the degenerative mouse retina.

Clinically, laser burns limit retinal neovascularization. The still current use of laser burns to peripheral retina to protect central retina from neovascularization, particularly in diabetic retinopathy, provides further evidence that photoreceptor depletion results in increased oxygen levels in the retina. Laser treatment was developed initially without a theory of its mechanism (reviewed in Benson et al., 1988). Several authors (Stefansson et al., 1981, 1986; Stone and Maslim, 1997) have argued that the well-established protective effects of laser treatment arise from depletion of the photoreceptor layer at the site of the burn and a consequent flow of oxygen from the choroid to the inner retina. This oxygen counteracts the hypoxia caused in diabetic retinopathy by capillary closure, reducing or delaying the damaging neovascularization. The recent study of Yu and colleagues (2005) addressed this question, demonstrating that partial laser burns at the level of the RPE and outer segments in rabbit retina caused increased retinal oxygen levels internal to the lesions.

Depletion-Induced Hyperoxia Is Inhibitory to Vessels and May Be Toxic to Rods and Cones If the increases in retinal oxygen levels known to be induced by photoreceptor depletion are sufficient to influence the cell biology of the retina, then effects similar to those induced by inhaling oxygen (the death of photoreceptors and the thinning of vessels) should be apparent.

Retinal vessels are thinned. Evidence of depletion-induced thinning of retinal vessels is substantial. Clinicians had long noted that vessels in the degenerating human retina are abnormally thin (Heckenlively, 1988), and thinning of the retinal vessels has been documented in several animal models of photoreceptor degeneration, including the rd mouse (Blanks and Johnson, 1986), the P23H-3 mouse (Penn et al., 2000), and the Abyssinian cat (Nilsson et al., 2001). Penn and colleagues noted that the effect was reduced by hypoxia, confirming that the thinning is caused by hyperoxia. Nilsson and colleagues noted further that as the degeneration of

photoreceptors progresses in the Abyssinian cat, the volume of blood passing through the retinal circulation decreases markedly, while the choroidal circulation continues without reduction.

Cones are damaged by rod dysfunction. Evidence that the depletion of rods can damage surviving photoreceptors is clearest in the rod-cone dystrophies, in which photoreceptor death occurs first among rods, perhaps owing to mutations in a protein expressed only in rods (e.g., rhodopsin), and later involves cones. Arguably, the tendency of macular degeneration to follow relatively severe loss of rod function in normal human retina (Curcio et al., 2000; Jackson et al., 2002) represents a form of rod-cone dystrophy.

Two explanations, one specific and one general, have been developed to explain the impact of rod loss on cone viability. The specific explanation is that rods produce a cone survival factor (Hicks and Sahel, 1999; Mohand-Said et al., 1998), for want of which cones die when rods are depleted. The more general explanation is the oxygen toxicity hypothesis (Stone et al., 1999), namely, that the depletion-induced hyperoxia will damage all surviving photoreceptors, rods, and cones. These two mechanisms are not exclusive.

Shen and colleagues (2005) reported evidence, from a rod-degenerative pig model, that surviving cones are subject to oxidative stress, which they related to the depletion of rods, giving support to the idea that hyperoxia may be the factor linking rod death to cone damage. There is no reason, moreover, to expect surviving rods to be immune to the oxidative stress caused by rod depletion, and evidence is available to demonstrate that rods surviving in a rod-specific mutant degeneration, such as the P23H-3 rat, are damaged. Comparing nondegenerative (Sprague-Dawley) and P23H-3 retinas raised in identical conditions, Walsh and colleagues (2004b) showed that the P23H-3 photoreceptors have shorter, more disorganized outer segments, with a higher rate of cell death and a smaller a-wave. Evidence that this damage to P23H-3 photoreceptors is at least partly due to an environmental factor came from the demonstration that, when the ambient light to which the retina is exposed was raised, damage to surviving rods increased, and the damage could be reversed by reducing ambient illumination ( Jozwick et al., 2006).

Light Accelerates Many Forms of Photoreceptor

Degeneration The effect of light experience on the progress of retinal degenerations varies with the underlying mutations. Light exposure has been known for many years to accelerate retinal degenerations in the RCS rat (Dowling and Sidman, 1962; Kaitz and Auerbach, 1978, 1979), in the P23H-3 mouse (Naash et al., 1996), and in the P23H-3 rat (Walsh et al., 2004b). Evidence in the human is limited but suggests that light restriction may slow degenerations in

some cases (reviewed in Stone et al., 1999). The variability in the human response to light exposure has yet to be related to the underlying genetic defect. In animal rodent models as in humans, light either exacerbates the degeneration or has no effect (Paskowitz et al., 2006); there appear to be no reports of light being protective to photoreceptors.

As Paskowitz and colleagues note, the effect of light is now well understood in a number of animal models, down to the molecular level. This evidence (which is beyond our present scope) makes clear how photoreceptor signaling is disturbed by mutations in proteins related to phototransduction, but the link between the disturbed signaling and cell death remains elusive.

Recent analyses of the P23H-3 rat model, which is light sensitive (Jozwick et al., 2006; Walsh et al., 2004b), showed that the damaging effect of light can occur quickly (within a week) and in response to very modest increases in just the daylight part of the day-night cycle. Further, the effects were reversible; within 2–5 weeks of a reduction in ambient light, the P23H-3 photoreceptors rebuilt their outer segments and partially (30%–40%) regained their sensitivity to light. We have recently demonstrated (Chrysostomou et al., 2008) that the same modest rise in ambient illumination that caused severe rod damage in the P23H-3 retina also damaged cones, and the damage to cones was also reversible. Since the transgene is in rhodopsin and cones do not express rhodopsin, the toxic factors that damaged cones must all have been environmental. One possibility, as we argued when proposing the idea of oxygen toxicity in 1999 (Stone et al., 1999), is that the toxic effect of increased ambient light on both rods and cones is mediated by a rise in oxygen tension in the outer retina induced by light.

The Edge of the Normal Retina Degenerates The normal (wild-type) retina degenerates at its edge. The degeneration has been shown in rodent models to begin as soon as photoreceptors begin to function (Mervin and Stone, 2002b; reviewed in Stone et al., 2005) (figure 46.4). In humans, a cystoid degeneration of the edge of the retina becomes evident in childhood and progresses with age until it involves the peripheral several millimeters of retina (Bell and Stenstrom, 1983).

There is considerable evidence that this “normal” degeneration of the edge of the retina is driven by hyperoxia. The structural factors that control retinal oxygen levels in the retina are perturbed at the edge of the retina by the sharp discontinuity of the retina itself. Specifically, the choroidal circulation extends past the edge, and as a result, oxygen can be predicted to reach the edge of the retina by diffusing around the edge. Working with the C57BL/6 mouse, Mervin (Mervin and Stone, 2002a, 2002b) demonstrated that the adult mouse retina shows an edge-specific degeneration. Retinal layers are thinned at the edge; photoreceptor mor-

Figure 46.4 Photoreceptors at the most peripheral margin of the retina are subject to stress from early postnatal life. A–C, The edge of the retina of C57BL/6 mouse retina, with dying cells (bright red, TUNEL labeled) clustering at the edge of P14, P16, and P18. All are in the ONL. D and E, This cluster of dying cells colocalizes with the upregulation of two stress-induced proteins. GFAP is normally expressed (red) only at the inner surface of the retina, by astrocytes (E and G). At the edge, GFAP is upregulated also in Müller cells. FGF-2 is strongly upregulated (green) in photorecep-

phology is disturbed, and stress-sensitive proteins (FGF-2, GFAP) are sharply upregulated (figures 46.4D–G). Second, a focus of accelerated photoreceptor death was detected at the edge, occurring in early postnatal life, between P12 and P20, as photoreceptors start to function. Third, the acceleration was slowed by hypoxia, indicating that it is caused by

tors, in a gradient that is maximal at the end. H, Quantification of the frequency of dying cells in the peripheral-most 100 μm of retina. I, Oxygen influences this distribution of dying cells at the edge of the early postnatal retina. Hypoxia increases photoreceptor death throughout most of the retina; at the edge, however, hyperoxia reduces death, suggesting that the edge-related death is driven by oxygen stress (see the text). Hyperoxia causes a small increase in the frequency of dying cells at the edge of the retina. See color plate 49. (From Mervin and Stone, 2002a, 2002b.)

hyperoxia. Throughout the rest of the retina, by contrast, hypoxia increased the rate of photoreceptor death. The oxygen status of the most peripheral 100 μm of the retina is distinctive, however; the region is degenerative and the degeneration is progressive, and related to high levels of oxygen.