Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

a typically unilateral developmental abnormality; chronic vitreous hemorrhage; retinoblastoma, a childhood cancer; Coats disease, a retinovascular condition that can lead to exudative retinal detachment; and infectious endophthalmitis, that can occur from systemic infection or conjunctivitis, as in the case of Pseudomonas,49 or after surgery. It should be noted that endophthalmitis can occur in preterm infants, whereas other conditions usually occur in full-term infants or children.

Pathology

In ROP, the peripheral avascular retina lacks retinal vascularization. At the junction of vascular and avascular retina, aberrant angiogenesis (stage 3 ROP) grows into the vitreous. The avascular retina that forms after hyperoxia-induced vaso-obliteration in the cat or mouse or after fluctuations in oxygen in the rat (see below) is hypoxic.50,51 The hypoxia is believed to occur because of reduced oxygen supply from an incompletely developed retinal vasculature in the face of a regressing hyaloid vasculature,52 increased metabolic and oxygen demand of maturing photoreceptors,53,54 and the possible inability of the choroid to adjust its oxygen concentration in the young animal even with increases in inspired oxygen level.51,55 Although several growth factors, including

erythropoietin, IGF-1, and hypoxia-inducible factor-1α (HIF-1α), have been reported to be involved in the pathomechanisms of ROP models,56–58 VEGF has also been shown to be consistently important in other human diseases with intravitreous neovascularization.59–61 The avascular retina has been shown to express VEGF in animal models.60,62 Also VEGF was overexpressed in the avascular retina of a human infant with stage 3 ROP.63 The junction of vascular and avascular retina was described as a proliferation of primitive vascular mesenchyme (possibly describing angioblasts) in a vanguard of advancing vasculature with an intraretinal band of endothelial cells in the rearguard, and extraretinal neovascularization extending into vitreous.64,65 With the change from a vascularly active process (stage 3 ROP) to a fibrovascular one (stage 4 ROP), there is often mesenchymal tissue that will fold upon itself to form a retinal detachment64,65 (Figure 72.8).

Etiology

Environmental risk factors

Although genetics plays a role, outside stimuli, such as nutrition and oxygen, are important in the development of ROP. Arginyl-glutamine and omega-3 fatty acids have been shown

Figure 72.8  (A) Cross-section of the eye showing elevated intravitreous neovascularization at the junction of vascular and avascular retina (stage 3 ROP). (B) Interactions with the vitreous collagen lead to fibrovascular contraction and elevation of the retina (stage 4 ROP). (C) Continued contraction leads to total retinal detachment and a retrolental fibrovascular membrane (stage 5 ROP).

to reduce intravitreous neovascularization or avascular retina, respectively, in the mouse oxygen-induced retinopathy (OIR) model.66,67

Role of oxygen: animal models of oxygen-induced retinopathy

After the initial appearance of ROP/RLF in the 1940s, the importance of high oxygen at birth was appreciated and animal models of OIR were developed. Most of these models exposed animals to high constant oxygen, which has been found both to cause capillary obliteration68 and to inhibit the differentiation of precursor cells into endothelial cells.69 Now, high oxygen at birth is avoided in NICUs in the USA. However, these models continue to provide important data in understanding the role of oxygen exposure in retinal vascular development and in pathologic angiogenesis. The cat model was used in initial observations of hyperoxia-induced vaso-obliteration9,70 and in describing the role of astrocytes in retinal vascular development.71,72 The mouse OIR model permits the study of mechanisms of hyperoxia-induced vaso-obliteration and relative hypoxia by permitting the use of genetically manipulable animals.68 No model reproduces stage 4 or 5 ROP. However, the beagle OIR model develops retinal folds similar to those reported in human fibrovascular ROP64 at the junction of vascular and avascular retina.73

The rat 50/10 OIR model74 provides the most relevant model of acute ROP in the USA today. Inspired oxygen extremes in the rat 50/10 OIR model led to rat arterial oxygen levels75 similar to the transcutaneous oxygen levels

568

measured in a preterm infant who developed severe ROP.12 Also,ratherthanconstantoxygenusedinothermodels,50,68,76,77 the 50/10 OIR model exposes pups to repeated fluctuations in oxygen, which increase the risk of severe ROP.12,14,78 The 50/10 OIR model reproducibly and consistently develops first avascular retina (analogous to zone of human ROP),75,79 then vessel tortuosity80 (analogous to plus disease in ROP), and later intravitreous neovascularization (analogous to stage 3 ROP).79,81

Other retinopathy models mimic conditions that occur in the preterm infant, including hypercarbia82 and metabolic acidosis.83 Newborn pups exposed to minute-to-minute oxygen fluctuations similar in extremes to what human preterm infants experience develop vascular abnormalities in the peripheral retinas,84 particularly if the fluctuations are performed around a hyperoxic rather than hypoxic mean.11

Similar in all animal models is the creation of avascular retina associated with intravitreous neovascularization at junctions of vascular and avascular retina at a time during development when oxygen supply is limited because of insufficiently developed retinal vascularization and a regressing hyaloidal vasculature while oxygen demand is increased from maturing photoreceptors.

The role of supplemental oxygen

As discussed above, high constant oxygen caused vasoobliteration through apoptosis, resulting in hypoxic avascular areas of retina,50 and subsequent intravitreous neovascularization (see above). Likewise, fluctuations in oxygen caused apoptosis of endothelial cells, in part causing larger avascular retinal areas in the 50/10 OIR model.85

Some studies showed that supplemental oxygen reduced the severity of OIR.86 Mice raised in sustained hyperoxia beyond postnatal day (p)12 had less vaso-obliteration and neovascularization compared to mice exposed to the standard OIR model.87 Rats exposed to oxygen fluctuations and recovered in supplemental oxygen (28%) rather than room air had reduced intravitreous neovascularization at some time points.88 Despite these data, the Supplemental Therapeutic Oxygen for Prethreshold ROP (STOP-ROP) multicenter clinical trial did not find an overall significant benefit from supplemental oxygen given to infants with prethreshold ROP.89

In the 50/10 OIR model, supplemental oxygen increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, accounting partly for pathologic intra­ vitreous neovascularization, even though it also reduced neurosensory retinal VEGF.51 Furthermore, hypoxic retina, quantified by insoluble retinal pimonidazole (Hypoxyprobe), was not reduced with supplemental oxygen. These results show that increased oxygen in the retinal vasculature reduces the stimulus for VEGF production but does not overcome overall retinal hypoxia that may occur with increased metabolic demand of developing photoreceptors. Furthermore, the retinal hypoxia appears unmet by the choroid. It was previously found that, unlike in the adult rat, the choroid in the p15 rat was unable to support increased oxygen tension with supplemental oxygen.55 Therefore, supplemental oxygen may not have as beneficial an effect as hoped in the retina and it can also lead to greater complications of pulmonary disease.89

Pathophysiology

ROP is complex. Many changes occur from the in utero environment to that after birth, including oxygen concentration, growth factor relationships, and nutrition. The effects of prematurity and environmental factors necessary to keep premature infants alive on the pathophysiology of ROP are incompletely understood. Below is evidence of growth factor signaling in retinal vascular development and in the pathogenesis of ROP.

Signaling through VEGFA in development, as a survival factor, and in disease

VEGFA has emerged as one of the most important angiogenic factors in the development of human intravitreous neovascularization.59,61 (VEGFA is the most widely studied ligand of the VEGF family and will be referred to as VEGF in this chapter). Besides its role in pathology, however, it is important in retinal vascular development,71,72 and is also an endothelial and neuronal survival factor.90–92

To understand ROP, it is important first to understand the known processes in human retinal vascular development. It is believed that vasculogenesis or de novo development of central vasculature around the optic nerve occurs from angioblasts, endothelial cell precursors.93 Angioblasts do not have markers commonly thought to be present on endothelial cells like CD31, CD34, and von Willebrand’s factor but do express CD39 and CXCR4.94,95 Retinal vascular development is believed to be completed mainly through a process of angiogenesis,96 but the role of circulating endothelial precursors is being appreciated more.94 During angiogenesis, a front of migrating cells, e.g., astrocytes in cat72 or angioblasts in dog,97 sense physiologic hypoxia and express VEGF. The ensuing endothelial cells are attracted to VEGF and migrate to create blood vessels.72 The VEGF signaling pathway has been found to regulate and integrate several cell processes that are important during sprouting angiogenesis. Whereas VEGF concentration is thought to regulate endothelial cell division rate,98,99 the presentation of VEGF, as in a gradient, may regulate filopodia formation of endothelial tip cells at the migrating front and direct the growth of endothelial cells.100 The delta-like ligand 4/Notch1 (Dll4/Notch1) signaling pathway regulates VEGF-induced endothelial tip/stalk cells at the junction of vascular and avascular retina and permits ordered angiogenesis.101

VEGF signaling is also important in preventing hyperoxiainduced vaso-obliteration. Exogenous VEGF injected into the vitreous prior to hyperoxia reduced vaso-obliteration.102 Stimulation of VEGF receptor 1 (VEGFR1) with the specific ligand placental growth factor (PlGF-1), prior to hyperoxia, reduced vaso-obliteration,103 whereas stimulation of VEGFR2 with the specific ligand VEGFE did not. VEGFR1 has a higher affinity for VEGF than does VEGFR2, but its receptor tyrosine kinase is less active.104 In mouse development, expression of VEGFR1 RNA increased significantly more than did the expression of VEGFR2 RNA. In addition, VEGFR1 RNA was localized to the developing vasculature, thus supporting its role in vascular development, whereas VEGFR2 RNA was localized to the neuronal retina.105–107 VEGFR1 is believed to

Pathophysiology

limit excessive VEGF signaling through VEGFR2 during development98 and permit ordered angiogenesis.108

The hypoxic, avascular retina that occurs from hyperoxiainduced vaso-obliteration in the mouse OIR or from repeated oxygen fluctuations in the rat 50/10 OIR expresses excess VEGF62,109 and is causally related to pathologic intravitreous neovascularization,110,111 in part through the src kinase pathway.112 Compared to hypoxia alone, repeated fluctuations in oxygen, a risk factor for ROP,12–15 were shown to increase RNA expression of VEGF164, an isoform created through alternative splicing of the parent VEGF mRNA.109

VEGF164/165 was also reported to increase leukostasis, endothelial apoptosis, and avascular retina in the mouse OIR

model.113 An intravitreous neutralizing antibody made against VEGF164 at a dose that inhibited signaling through VEGFR2, but not VEGFR1, significantly reduced intravitreous neovascularization in the retina in the 50/10 OIR model.111 In addition, numerous studies have shown that inhibiting VEGF through other mechanisms reduced intra­ vitreous neovascularization.114

Although inhibition of VEGF alone does not inhibit intravitreous neovascularization completely, combinations of inhibitors that affect multiple receptors do.115,116 However, in the case of ROP in which development of the vasculature and retina is ongoing, complete inhibition of angiogenesis or removal of survival aspects of growth factors is not desirable. VEGF is important in development, survival, and also in the pathogenesis of ROP. Thus, data suggest that the amount, presentation, isoform expression, and timing of VEGF are important. Below are studies providing evidence of the role of other growth factors (Box 72.5).

HIF-1alpha

Stabilization of HIF-1α occurs under hypoxic conditions or secondary to reactive oxygen species (ROS) generated from NADPH oxidase, nitric oxide, mitochondria, and other enzymes.117 HIF-1α binds to the hypoxia response elements to cause transcription of several genes, including angiogenic factors, VEGF, and erythropoietin.117 A knockout to HIF-1α is lethal but a knockout to the HIF-1α-like factor (HLF)/HIF-2α provided evidence that erythropoietin was a major gene involved in intravitreous neovasculari­ zation after relative hypoxia from hyperoxia-induced vaso-obliteration.56

Box 72.5  Vascular endothelial growth factor (VEGF)

Erythropoietin is angiogenic, erythropoietic, and neuroprotective. It is upregulated after stabilization of HIF-1α in response to hypoxia.117 Besides being a target for oxygeninduced retinopathy in the mouse model,56 clinical studies have shown an association between the number of administrations of erythropoietin for anemia of prematurity and the prevalence of severe ROP.118,119 These studies link erythropoietin use in preterm infants and the experimental findings from HLF/HIF-2α knockout mice.56

IGF-1 and IGF-BP3

IGF-1 is important in physical growth and influences birth weight. IGF-1 levels that occur in utero are not maintained upon birth in preterm infants.120 Low serum IGF-1 correlated with increased avascular retina in human ROP.36 Furthermore, transgenic mice expressing a growth hormone antagonist gene, or wild-type mice treated with an inhibitor to growth hormone, had reduced retinal neovascularization in the mouse OIR model of hyperoxia induced vaso-oblitera- tion and angiogenesis.57,58 IGF-1 was also found to be important for maximal signaling through the mitogen-activated protein (MAP) kinase pathway, which is important in cell proliferation. In addition, VEGF and IGF-1 synergistically triggered Akt, which is important in cell survival.36 Based on these findings, it is theorized that IGF-1, which is low in the preterm infant, is necessary for early retinal vascular survival and growth, but can result in later intravitreous neovascularization in ROP. However, timing and dose appear critical.

A hypoxia-regulated binding protein of IGF-1, IGF-1BP3, was shown to be important in reducing hyperoxia-induced vaso-obliteration and in promoting vascular regrowth into the retina.121,122 IGF-BP3 was shown to promote differentiation of endothelial precursor cells into endothelial cells and in promoting angiogenic processes, such as cell migration and tube formation.122

Mechanisms of avascular retina

The size of the avascular zone of retina is directly associated with poor outcomes from severe ROP.18,123 As discussed earlier, both hyperoxia-induced vaso-obliteration and fluctuating oxygen can lead to increased avascular retina in OIR models.81 The ischemic microenvironment created by avascular regions50,51 produces angiogenic factors, like VEGF,7,124 that cause intravitreous neovascularization.110,111 Studies support the concept that increased apoptosis leads to increased avascular areas; for example, through inflammatory leukostasis125,126 or, in bcl-2 knockout mice, through a defect in protection against apoptosis.127 Furthermore, growth factors, including VEGF, IGF-1, IGF-1BP3,102,103,121,122 and nutritional supplements, including omega 3 fatty acids,66,67 can reduce the apoptosis of endothelial cells from newly formed capillaries if given prior to the hyperoxic insult in the mouse OIR model. Finally, the use of antioxidants has shown benefit in reducing the size of the avascular areas of retina in animal models.85,128–130

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Oxidative stress has been proposed to be important in the development of ROP,131,132 because the retina is susceptible to oxidative damage given its high metabolic rate and rapid rate of oxygen consumption.133 In addition, the premature infant has a reduced ability to scavenge ROS,134 increasing its vulnerability to oxidative stress. End-products of ROS, lipid hydroperoxides (LHP), were significantly increased in the 50/10 OIR model at time points corresponding to intravitreous neovascularization.85 When injected into the vitreous, LHP caused intravitreous neovascularization in the rabbit.135 Also, ROS can trigger signaling pathways relevant to apoptosis136 or angiogenesis,137 both important in the pathogenesis of ROP.

Treatment of pups in the 50/10 OIR model or humans with ROP using a broad antioxidant, n-acetylcysteine,138,139 failed to show reduction in clock hours of intravitreous neovascularization, or avascular retinal area.85,140 In a clinical trial in preterm infants, there was no difference in the incidence in ROP between those receiving n-acetylcysteine or control.140 However, reduction in ROS with preparations of vitamin E128,129 or liposomes containing the antioxidant enzyme, manganese superoxide dismutase (MnSOD), reduced OIR severity.130 Also, a meta-analysis of infants treated with vitamin E showed a significant reduction in severity of ROP. Reducing the activation of NADPH oxidase, an enzyme that produces ROS, can also reduce the size of the avascular areas85 and subsequent intravitreous neovascularization in certain OIR models.51

Light was proposed to be important in ROP development through photo-oxidation of polyunsaturated fatty acids within photoreceptor outer segments. On the other hand, during the dark, photoreceptors are more metabolically active. A clinical trial testing the effect of light or shade on the development of ROP showed no significant difference.141

Inflammatory aspects

Resident macrophages and microglia are necessary for retinal vascular development142 and in remodeling of the retinal vasculature,126 but can also be involved in pathologic angiogenesis and photoreceptor apoptosis.126,143,144 Macrophages produce ROS from activated NADPH oxidase, oxidoreductases, or nonenzymatically as side products of reactions utilizing electron transfer.136 Macrophages can produce nitric oxide, which is involved in angiogenic signaling,136,145 and can express inflammatory cytokines in response to hypoxia146 and release VEGF.147 Furthermore, VEGF164 is upregulated by fluctuations in oxygen in the 50/10 OIR model109 and was found to be proinflammatory by increasing adhesion molecules in vessels and by attracting monocytes in vivo.113,148

Summary

The role of VEGFA and its receptors, as well as the effects and interactions with other factors, is complex when considering the pathogenesis of ROP. The concentration, presentation, isoform expression, and coordination in development

are important. Also the effect and type of oxygen stress appear important. Since fluctuations in oxygen as well as absolute oxygen concentration are important, growth factors and signaling pathways that are involved in the relative hypoxia-induced angiogenesis following hyperoxia require further study. Broad inhibition of VEGF, use of antioxidants, or reduction in inspired oxygen concentration may be detri-

Key references

mental to the developing preterm infant. Also the systemic concentration from intravitreous drugs is greater in infants than adults because of an approximate 10-fold difference in the vitreous/blood volumes. New treatments for ROP are on the horizon but these issues must be considered in the developing preterm infant.

1.International Committee. An international classification of retinopathy of prematurity. Br J Ophthalmol 1984;68:690–697.

2.Early Treatment for Retinopathy of Prematurity Cooperative Group. Revised indications for the treatment of retinopathy of prematurity: results of the early treatment for retinopathy of prematurity randomized trial. Arch Ophthalmol 2003;121:1684–1694.

3.Cryotherapy for Retinopathy of Prematurity Cooperative Group. Multicenter trial of cryotherapy for retinopathy of prematurity: Snellen visual acuity and structural outcome at

512 years after randomization. Arch Ophthalmol 1996;114:417–424.

12.Cunningham S, Fleck BW, Elton RA, et al. Transcutaneous oxygen levels in retinopathy of prematurity. Lancet 1995;346:1464–1465.

19.McColm JR, Hartnett ME. Retinopathy of prematurity: current understanding based on clinical trials and animal models. In: Hartnett ME (ed.) Pediatric Retina. Philadelphia, PA: Lippincott Williams & Wilkins, 2005:387–409.

36.Hellstrom A, Peruzzu C, Ju M, et al. Low IGF-1 suppresses VEGF-survival signaling in retinal endothelial cells: direct correlation with clinical

retinopathy of prematurity. Proc Natl Acad Sci USA 2001;98:5804–5808.

43.Coats DK. Retinopathy of prematurity: involution, factors predisposing to retinal detachment, and expected utility of preemptive surgical reintervention. Trans Am Ophthalmol Soc 2005;103:281–312.

44.Hartnett ME, McColm JR. Retinal features predictive of progression to stage 4 ROP. Retina 2004;24:237–241.

47.Trese MT, Capone A. Retinopathy of prematurity: evolution of stages 4 and 5 ROP and management. A. Evolution to retinal detachment and physiologically based management. In: Hartnett ME (ed.) Pediatric Retina. Philadelphia, PA: Lippincott Williams & Wilkins, 2005:387–409.

48.Capone A Jr, Hartnett ME, Trese MT. Treatment of retinopathy of prematurity: peripheral retinal ablation and vitreoretinal surgery. In: Hartnett ME (ed.) Pediatric Retina. Philadelphia, PA: Lippincott Williams & Wilkins, 2005:387–409.

54.Berkowitz BA, Roberts R, Penn JS, et al. High-resolution manganese-enhanced MRI of experimental retinopathy of prematurity. Invest Ophthalmol Vis Sci 2007;48:4733–4740.

55.Cringle SJ, Yu PK, Su EN, et al. Oxygen distribution and consumption in the developing rat retina. Invest Ophthalmol Vis Sci 2006;47:4072– 4076.

68.Smith LEH, Wesolowski E, McLellan A, et al. Oxygen induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 1994;35:101–111.

75.Penn JS, Henry MM, Tolman BL. Exposure to alternating hypoxia and hyperoxia causes severe proliferative retinopathy in the newborn rat. Pediatr Res 1994;36:724–731.

82.Holmes JM, Zhang S, Leske DA, et al. Carbon-dioxide induced retinopathy in the neonatal rat. Curr Eye Res 1998;17:608–616.

88.Berkowitz BA, Zhang W. Significant reduction of the panretinal oxygenation response after 28% supplemental oxygen recovery in experimental ROP. Invest Ophthalmol Vis Sci 2000;41:1925–1931.

111.Geisen P, Peterson L, Martiniuk D, et al. Neutralizing antibody to VEGF reduces intravitreous neovascularization and does not interfere with vascularization of avascular retina in an ROP model. Mol Vis 2008;14:345–357.

Clinical background (Box 73.1)

Altered retinal oxygenation plays a central role in the pathogenesis of many retinal diseases and a hypothesized role in others.1 Unfortunately, despite decades of work in some cases, the precise role of oxygen in certain complex diseases remains elusive.

There are three different categories of situations in which oxygen can play a critical role in disease. First, and most obvious, are situations involving ischemia. Because oxygen is usually the limiting substrate for metabolism, and cannot be stored, even a short-term loss of oxygen leads to devastating consequences in conditions such as central or branch retinal artery occlusion (Chapter 63). Ischemia is certainly involved in other cases, such as after capillary dropout in diabetes (Chapters 65 and 66). These conditions involve anoxia, because there is essentially no redundancy or safety factor in the retinal circulation where one arteriole or capillary can take over oxygenation of a region ordinarily supplied by another vessel. The choroidal circulation is usually not affected in the same diseases, but cannot compensate for the loss of retinal circulation when an individual is breathing air.2–5

The extent to which substrates other than oxygen are involved in ischemic diseases is not generally clear. During retinal artery or capillary occlusion, one might expect that glucose from the choroid could diffuse into the inner retina to allow glycolytic metabolism, but this is uncertain. Purely glycolytic metabolism also leads to acidosis, which is observed in animal models of retinal artery occlusion.6 Interestingly, in primates and cats, retinal function does return to a large extent if circulation is restored within about 1.5 hours,7,8 which is far longer than the brain could survive. The long survival time probably does not reflect an inherent difference in retinal and brain neurons, but rather the small reservoir of glucose in the vitreous and the remaining supply from the choroid, neither of which has an analog in the brain. Elevating glucose prior to ischemia in rats prolongs the survival of the electroretinogram during ischemia, providing some support for this idea.9

Some investigators believe that less severe reductions in blood flow through the retinal or choroidal circulation, which produce milder hypoxia, play a role in diabetes,10,11 glaucoma,12,13 and age-related macular degeneration.14,15 In diabetes, for example, hypoxia could be caused by the upreg-

ulation of adhesion molecules and leukostasis that is known to occur.10,16 In many tissues, hypoxia leads to a dramatic elevation in the level of the transcription factor hypoxiainducible factor 1α (HIF-1α), not because synthesis is increased, but because degradation is prevented during hypoxia. HIF-1α increases in the retina in many situations.17 HIF-1α has multiple downstream effects, including the upregulation of vascular endothelial growth factor (VEGF), which is a hallmark of diabetic retinopathy and wet macular degeneration. Complicating the picture, however, are findings that VEGF and HIF-1α can increase as a result of factors other than hypoxia.18,19

The second role for oxygen in disease is the situation where there is nothing wrong with the circulation per se, but there is a mismatch between oxygen supply and metabolic demand. An important and underappreciated example of this is in retinal detachment (Chapter 71), where the choroid is functional, but is too far from the photoreceptors to supply their needs fully.20,21 Theoretically, metabolic starvation of the inner-segment mitochondria would occur with even very small separations between the choroid and photoreceptors,20 as would occur in the presence of large drusen, so it is possible that a supply–demand mismatch contributes to the loss of photoreceptors in dry age-related macular degeneration, which occurs directly over drusen.22 In these cases the mismatch is caused by insufficient supply. Only one case is known at present where the mismatch is caused by increased metabolic rate. This is the situation that leads to the loss of photoreceptors in some hereditary degenerations (Chapter 76). In rd mice23 and in some cases of human retinitis pigmentosa,24 phosphodiesterase (PDE) activity is reduced. This is expected to lead to increased cyclic guanosine monophosphate (cGMP) levels, which in turn would lead to increased numbers of open channels in the photoreceptors, increased influx of Na+ and Ca2+, and a greater demand for adenosine triphosphate (ATP) to operate Na+ and Ca2+ pumps than the cell can meet. Whether metabolic overload causes loss of photoreceptors or other retinal neurons in other situations is not known.

The third situation is one in which there is too much oxygen. This has long been recognized as a fundamental problem in retinopathy of prematurity25 (Chapter 72), preventing the growth of retinal blood vessels in the neonate, and more recently it has been recognized to be the reason why retinal blood vessels ultimately disappear or are seri-

Box 73.1  Selected roles of hypoxia in retinal disease

Retinal diseases are complex, but hypoxia contributes to many:

•Diabetic retinopathy: hypoxia due to capillary loss in preproliferative retinopathy contributes to neovascularization. Possible hypoxia in background retinopathy

•Retinopathy of prematurity: hypoxia following end of oxygen supplementation contributes to neovascularization

•Central or branch retinal artery occlusion: choroidal supply cannot compensate for the lack of retinal circulation, leading to cell death in the inner retina

•Retinal detachment: separation of retina from choroidal circulation reduces flux of oxygen to photoreceptors

•Age-related macular degeneration: reduced choroidal blood flow and impaired transport may contribute to upregulation of vascular endothelial growth factor

•Glaucoma: reduced blood flow to the optic nerve head may contribute to hypoxia and the loss of retinal ganglion cells

ously attenuated in all forms of retinitis pigmentosa.26,27 Oxidative damage is also thought to play a role in reperfusion injury in the retina.28

The presence of the choroidal circulation offers possibilties for therapy, because it is rarely compromised at the same time as the retinal circulation. The leading hypothesis for the success of panretinal photocoagulation in proliferative diabetic retinopathy is that laser destruction of the photoreceptors removes their oxygen demand, allowing choroidal oxygen to reach the inner retina. This relieves hypoxia and reduces angiogenesis.11 Intraretinal Po2 measurements show that O2 levels do increase after photocoagulation.29 Choroidal oxygen is probably also responsible for the benefit of hyperbaric oxygen in cystoid macular edema.30 We and others have also argued that treatment with hyperoxia would be beneficial in arterial occlusion7 and retinal detachment,21,31 and that attempts at using hyperoxia previously have misunderstood some of the key features of retinal oxygenation outlined below.

This chapter gives an overview of retinal energy metabolism and substrate supply, providing a basis for understanding the potential roles of these topics in the etiology of the diseases covered in more detail elsewhere.

Pathology

There are many studies showing detrimental effects of reduced oxygen and substrates in acute experiments, but only a few studies investigating whether reduced supply of substrates, in the absence of any disease, can cause retinal pathology, because chronic, moderate changes that would mimic disease are more difficult in an experimental setting. However, the experiments that have been done reveal aspects of pathology similar to those that would be observed in disease. For example, in birds, it has been shown that reduced choroidal blood flow can cause photoreceptor loss and upregulation of GFAP,32 as well as decreased acuity.33 In rat retina, chronic systemic hypoxia (10% inspired oxygen) leads to apoptosis of photoreceptors.34 Hypoxia can also itself lead to neovascularization.35

Pathophysiology

Box 73.2  Differences between the retinal and

choroidal circulations

Retinal

•Modest flow rate (40–50 ml blood/100 g tissue/min)

•High oxygen extraction (venous saturation 8 vol% below arterial saturation)

•Metabolic local control by neural activity, tissue Po2, tissue CO2, pH, and lactate

•Good autoregulation in the face of changing perfusion pressure

•Microcirculation similar to that of brain, but more pericytes in capillary walls

Choroidal

•Unusually high flow rate (1200 ml blood/100 g tissue/min)

•Low oxygen extraction (venous saturation 1 vol% below arterial saturation)

•Control exclusively by autonomic system

•Modest ability to autoregulate in the face of pressure changes

•Microcirculation with large fenestrated capillaries

Etiology

Apart from the comments under clinical background, consideration of oxygen in the etiology of different diseases is left to the chapters on individual diseases.

Pathophysiology

A number of properties make the retina unique, in terms of:

(1) the supply of oxygen and glucose to the retina by its circulations; (2) the utilization of the substrates to provide ATP; and (3) the relative importance of the cellular processes that use the ATP. A great deal is known about the first two topics. The third has been more difficult to investigate and our knowledge is mainly about photoreceptors.

Supply of oxygen and glucose from the circulation (Box 73.2)

The retina of humans and many other mammals is supplied by two circulations, both of which are essential (Figure 73.1). The choroidal circulation lies behind the retinal pigment epithelium and the retinal circulation lies within the inner half of the retina, except in the central part of the fovea, where the retinal circulation is absent. Because the two circulations have such different physiological properties, they must be treated separately. The retinal circulation is like

the circulation of the brain in most respects. The flow rate is modest, on the order of 25 l/min, or 20–25 ml/(100 g-min)

of retina if normalized to the whole retinal weight,36–44 or about 40–50 ml/(100 g-min) if normalized by the inner retinal weight (half the total retinal weight), which is what the retinal circulation normally supplies. This is called the “nutrient flow rate” below. The retinal blood flow rate is controlled by tissue oxygen and metabolite levels, with hypoxia and hypercapnia increasing flow, and hyperoxia

Autoregulation Low flow rate

Low venous oxygen saturation

No autoregulation Very high flow rate Very high venous oxygen saturation

Capillary Arteriole

Venule

Inner limiting membrane

Optic nerve fibers

Ganglion cell layer

Inner plexiform layer

Inner nuclear layer

Outer plexiform layer

Outer nuclear layer

Rod and cone layer

Pigment epithelium

Choriocapillaris

Layer of large choroidal vessels

Suprachoroid

Sclera

Episcleral artery and vein Vortex vein

Figure 73.1  Schematic of the retinal and choroidal circulations. (Modified from Kaufman P, Alm A (eds) Alder’s Physiology of the Eye, 10th edn. St. Louis, MO: Mosby, 2003.)

decreasing flow.37,45–49 Increased activity of retinal neurons caused by visual stimuli can increase retinal blood flow,50 but the blood flow in steady illumination is only transiently increased relative to the flow in the dark.51 There is no autonomic control of the retinal vessels after the retinal artery enters the eye at the center of the optic nerve.52

In contrast, the choroidal circulation has a flow rate of 700–900 l/min,37,38,43,44 or 1200–1500 ml/(100 g-min) if normalized by choroidal weight,37,39,42,43 or still slightly higher if normalized by the outer retinal weight. The choroidal flow rate changes little in response to hypoxia or hyperoxia, although it does increase in hypercapnia.37,53–55 Instead, autonomic control is very strong,52,56,57 with sympathetic influences decreasing flow, and facial nerve stimulation increasing flow.58,59 Flow rate increases somewhat in response to illumination,60 but this is when the photoreceptors need less oxygen, so it cannot be a metabolically dependent effect.

Oxygen supply

The retina is critically dependent upon a continuous supply of oxygen, with loss of vision occuring only 5–10 seconds after intraocular pressure is raised to a level that occludes all circulation to the retina.61,62 It is known that oxygen is the important factor, because vision can be slightly, but not usefully, prolonged if an individual breathes oxygen before the occlusion.61,62 Levels of oxygen in the retina have been measured by a variety of techniques,1,63 including optical and magnetic resonance measurements that are valuable because they are noninvasive,64–66 but here we focus on microelectrode measurements within the retina and measurements of oxygen in the two circulations, which yield information useful for determining metabolic rate.67

The choroidal circulation is normally responsible for most of the oxygen needed by the photoreceptors, while the retinal circulation provides most or all of the oxygen needed

574

Retinal depth (%)

Figure 73.2  Oxygen profiles from cat retina in dark adaptation and at a steady illumination sufficient to saturate the rod system, and a schematic diagram of retinal structure. Retinal depth is defined to be zero at the vitreal–retinal border, and 100% at the choriocapillaris. (Modified from Wangsa-Wirawan ND, Linsenmeier RA. Retinal oxygen: fundamental and clinical aspects. Arch Ophthalmol 2003;121:547–557.)

by the inner retina. Both circulations are required, and neither can compensate for the loss of the other under ordinary conditions. One might expect that there would be a minimum Po2 at the depth where the supply from the two circulations meets. There is such a location, but it varies with illumination, and may be different in different animals. In darkness in cat,67 monkey,68 and presumably human retina, the minimum Po2 is not at the middle of the retina, but is far distal, in the photoreceptor inner-segment layer, about 35 m from the choroid (Figure 73.2). Despite the distal location of this minimum, the retinal circulation only pro-

vides about 10% of the oxygen used by the photoreceptors.67,68 During strong illumination, in cat67 and rat,69 the oxygen gradient extends further toward the inner retina, and generally provides all of the oxygen for the photoreceptors, and possibly a little for the outer plexiform layer. However, in monkey, the change in the shape of the gradient is less dramatic,69 and the choroid does not provide all the oxygen for the photoreceptors during illumination.

One of the often confusing aspects of retinal oxygenation is why choroidal blood flow should be so high. By employing the Fick principle, a mass balance on oxygen, it is possible to calculate the total amount of oxygen extracted from each circulation:

QO2c = Fc (SaO2 − SvcO2 ) for the choroidal circulation, and

QO2r = Fr (SaO2 − SvrO2 ) for the retinal circulation.

where Qo2 is the oxygen utilization of the tissues supplied by the choroidal (c) and retinal (r) circulations, F is the nutrient flow rate discussed above, Sao2 is arterial oxygen saturation (about 20 vol% or 0.2 ml O2/ml blood), and SvO2 is venous saturation. Because of the technical difficulty of measuring flow rates and choroidal venous saturation, wide variations in Fc, Fr, and SvcO2 have been reported. Nevertheless, it is instructive to use reasonable in vivo values, which come from monkeys, cats, pigs, and humans: Fc = 1400 ml/ (100 g-min),37,39,42–44,58 Fr = 45 ml/(100 g-min),36 Svco2 = 0.19–0.195 ml O2/ml blood,44,70 and Svro2 = 0.13 ml O2/ml blood.71–73

Qo2c = 1400 × (0.2 − 0.195) = 7.00mlO2(100g - min)

Qo2r = 45 × (0.2 − 0.13) = 3.15ml O2(100g - min)

These averaged values do not account for regional variation across the retina, and would be slightly different if all the values were from a single animal (e.g., Figure 73.3).74 No matter how the calculation is done, however, both circulations contribute substantially to the oxygen supply of the retina. In addition, these equations reflect clearly that there can be a tradeoff between flow rate and arteriovenous difference to achieve a particular metabolic rate. Moreover they make clear why the high choroidal flow rate is required. The high flow rate allows a low oxygen extraction, and in turn, the high choriocapillaris and venous Po2, seen in oxygen profiles.20 A high Po2 at the choroid is essential in providing a large driving force for oxygen diffusion to the photoreceptor inner segments, which begin about 30 m from the choroid and have an unusually high oxygen demand. High choroidal blood flow may also assist in heat removal.

Glucose supply

Aside from oxygen, the most critical substrate for the retina is glucose, which can also be analyzed by the Fick principle. About one-sixth as much glucose as oxygen is needed for oxidative metabolism on a molar basis, but much larger quantities of glucose are used whenever glycolysis ends with lactate production. Using the Fick principle to investigate glucose utilization is difficult, because the arteriovenous differences of glucose and lactate are small, and because they require blood collection from a retinal vein or vortex vein,

Pathophysiology

Figure 73.3  Production of lactate and consumption of oxygen and glucose in pig inner and outer retina. Orange (lefthand) bars are for dark adaptation; lavender (righthand) bars are for light adaptation. (Modified from Wang L, Tornquist P, Bill A. Glucose metabolism of the inner retina in pigs in darkness and light. Acta Physiol Scand 1997;160:71–74.)

but this has been done for both circulations in pig44,73 and for the choroidal circulation in cat.43 Results for pig are shown in Figure 73.3. Both circulations deliver glucose. There is a separation of inner and outer retina in terms of glucose supply, as there is in oxygen supply, and it appears that the retinal circulation provides almost all the glucose needed in the inner half of the retina. A small amount of lactate appears in the retinal circulation, but it is not clear that any of it is produced in the inner retina. It may instead diffuse to the inner retina from the large amount of lactate that is normally produced in the outer retina. As Figure 73.3 illustrates, glucose consumption in the outer retina is greater than oxygen consumption, especially in darkness, which implies that a great deal is used for aerobic glycolysis, e.g., lactate production. Depending on how the computation is done, the values in Figure 73.3 for dark adaptation imply that 60–85% of the outer retinal glucose is used for lactate production. In vitro experiments on rat and rabbit support the conclusion that a large percentage of glucose is used for lactate production in the outer retina.75,76

Localization and mechanisms of ATP generation

Oxidative metabolism (Box 73.3)

Oxidative metabolism is not uniform throughout the retina, as one can appreciate from the distribution of mitochondria.77 The retinal pigment epithelial cells are located in a region with high Po2, and have numerous mitochondria, but exhibit lactate production rates similar to78 or higher than79 their oxygen consumption rates. The outer segments and outer nuclear layer have no mitochondria, but the inner seg-

Box 73.3  Retinal oxygenation

•Inner and outer retina both consume large amounts of O2 (3–5 ml O2/100g/min)

•Outer retinal Qo2 confined to the inner segments and synaptic regions of photoreceptors

•Inner retinal Qo2 probably highest in inner plexiform layer, but more uniform than in outer retina

•High local Qo2 of inner segments keeps their Po2 at just a few mmHg in darkness

•Light reduces photoreceptor oxygen utilization by 30–50%

•Inner retinal Po2 is slightly below 20 mmHg on average

•Steady light does not influence inner retinal metabolism, but flashing light probably increases metabolism

Table 73.1  Comparison of macaque and cat retinal oxygenation

Values are means ± sd. Where more than one value is given, they are from different original studies. Qo2, oxygen utilization; Qav is Qo2 averaged over outer retina; Pao2, arterial partial pressure of O2; Pc, average choroidal Po2.

*Values from regression of Qav on PC at PC = 50 mmHg.

Data from Birol G, Wang S, Budzynski E, et al. Oxygen distribution and consumption in the macaque retina. Am J Physiol 2007;293:H1696–H1704.

ments have many. Mitochondria occupy 54–66% of the inner-segment volume in primate rods and 74–85% of inner-segment volume in primate cones.80 The Qo2 of the inner segments of rods is extremely high, about 20 ml/ (100 g-min) in darkness, based on microelectrode measurements and a mathematical model of oxygen diffusion in cats and monkeys.67,68 Inner segments comprise only about 20– 25% of the outer half of the retina, so if the high value of Qo2 in the inner segments is averaged together with the lack of any oxygen consumption over the other 75–80% of the outer retina, the total photoreceptor Qo2 is 4–5 ml/ (100 g-min),68 similar to the value obtained from the Fick principle above. A summary of the parameters of cat and monkey retinal oxygenation, showing their fundamental similarity, is given in Table 73.1.

At a particular retinal eccentricity, the inner segments of cones have a total mitochondrial amount that can be 10 times greater than that in rods.80 The foveal cones, although

576

Box 73.4  Retinal lactate production

•Photoreceptors generate a substantial amount of lactate, even when the retina is well oxygenated

•As much as 90% of the glucose used by the photoreceptors goes to lactate production

•Lactate production by photoreceptors increases further during hypoxia or anoxia (Pasteur effect), compensating for the loss of oxidative metabolism

•Lactic acid production makes the outer retina acidic (pH about 7.2 at the lowest)

•Lactate production in the inner retina of humans and animals with a retinal circulation is very low

thin, are also richer in mitochondria than rods. While it has not been possible to measure individual cone oxygen consumption, it is highly unlikely that their metabolic rate is 200 ml/(100 g-min), as the relative amounts of mitochondria alone might suggest. Further, recent measurements show that foveal Qo2 is lower than Qo2 of parafoveal retina,68,81 even though the total mitochondrial density is higher in the fovea. Thus, it seems that mitochondrial amounts in the retina cannot be taken alone as an index of metabolic rate, and it has been argued that cone mitochondria may serve an optical function as well as a metabolic one.80

In the inner retina, the plexiform layers are richer in mitochondria than the nuclear layers.77 These layers also tend to be the location of the capillaries, and Po2 profiles (Figure 73.2) often have peaks in these regions that reflect those oxygen sources. Measurements in cat82 and rat83 by different techniques reinforce the conclusion that the inner and outer retina have similar overall rates of oxygen utilization. In most cases it has not been possible to tease apart Qo2 of different layers of the inner retina, but one group has attempted to analyze the Qo2 of just the oxygen-consuming layers in rat.84,85

For many years it has been clear that photoreceptor metabolism decreases during illumination (Figure 73.3), which is seen in oxygen profiles as an increase in the Po2 of the distal retina during illumination (Figure 73.2). The magnitude of the change is dependent on the level of illumination and on the species, with the maximum change being about a factor of two. This has been best studied in roddominated animals and rod-dominated regions of the retina, but clearly also occurs in cones.68 The decrease in metabolism with light is relatively rapid, occurring with a time constant of about 25 seconds in primates.86 The metabolism of the inner retina is independent of the level of steady illumination,73,74,82 but deoxyglucose measurements suggest that it probably increases in response to time-varying illumination.74

Aerobic and anaerobic glycolysis (Box 73.4)

Unfortunately, no technique is available to measure gradients of glucose within the retina, which would reveal local glucose utilization in the way that oxygen measurements reveal local oxidative metabolism. However, other lines of evidence point to the photoreceptors as the most active site of glycolysis. In addition to the measurements of Figure 73.3,73 and in vitro work to isolate the site or sites of glycolysis,75,76 two types of study have provided further localization.