What Is the Role of Ischemia in AMD?

Early detection of ischemic damage may allow for the diagnosis of progression before changes manifest clinically.

Shani Golan, MD • Dov Weinberger, MD • Tami Livnat, PhD • Anat Loewenstein, MD

Age-related macular degeneration is the leading cause of legal blindness among older individuals in the developed world, occurring primarily in people aged 50 and older.^1-3 The Blue Mountain study group identified the 10-year incidences of early AMD and late AMD to be 10.8% and 2.8%, respectively.⁴ The Cardiovascular Health and Age-related Maculopathy study demonstrated that the risk of early AMD progression was between 3.4% and 4.7% per year and the risk of late AMD progression was 0.9% per year.⁵

It has been predicted that as the population ages, there will be a 50% increase in the incidence of AMD before 2020.⁶ In another study, AMD was reported to account for 54% of the cases of blindness among the Caucasian population in the United States, and the number of blind people could increase by as much as 70% by 2020.⁷

This review provides a brief summary of AMD's proposed pathogenesis, with an emphasis on ischemia. Supporting evidence for this theory is provided by imaging studies using the Retinal Function Imager (RFI).

REVIEW OF DEFINITIONS

As retinal specialists are well aware, AMD affects the retinal pigment epithelium and leads to a progressive photoreceptor degeneration in the central retina, with associated loss of central vision.^8-9 AMD can be divided into early, intermediate and late forms.^10-11

Figure 1. Quantification of retinal blood-flow velocity in the human retina. A. RFI image showing calculated velocity in mm/sec, obtained automatically. The image shows several arteries (reddish) and veins (pink) that were manually selected for quantification. A negative value indicates blood flow away from the heart, whereas a positive value indicates blood flow toward the heart. B. Single frame of a flow movie from the same human retina. Erythrocyte clusters (black) and gaps (white) move through the small vessels shown here at an average velocity rate of 2 to 3 mm/sec.

Drusen, the hallmark of early and intermediate AMD, can be found in about 85% of patients.¹⁰ These amorphous deposits accumulate between the RPE and the inner collagenous zone of Bruch's membrane. Ophthalmoscopically, drusen can be divided into hard (less than 63 µm) and soft (greater than 125 µm) drusen. Soft drusen are a major risk factor for developing more advanced stages of AMD, particularly when they are accompanied by pigment irregularities, such as hyper- and hypopigmentation.¹¹

Analysis has revealed many protein remnants and a number of inflammatory and immune-associated elements.^12-13

Intermediate AMD is characterized by the accumulation of more than six focal or diffuse drusen measuring greater than 125 µm and hyper- or hypopigmentation of the RPE.

Advanced AMD can be classified into either of two categories. The first of these comprises the geographic atrophy typical of nonexudative or “dry” AMD, which is characterized by a sharply delineated area of RPE atrophy measuring at least 175 µm along one dimension and including visible choroidal vessels.¹⁰ Alternatively, there is the neovascular or “wet” form of AMD, in which there is new vessel growth through Bruch's membrane from the underlying choroid. Wet AMD may involve some or all of the following: subretinal neovascular membranes, subretinal fluid, exudates, and hemorrhages. It causes acute central vision loss in about 15% of AMD patients.

RISK FACTORS

Although advancing age is the greatest risk factor associated with the development of AMD, environmental and lifestyle factors may significantly affect individual risk. Smoking is an important, modifiable factor. The risk of macular degeneration is two- to threefold higher in current smokers compared with individuals who have never smoked.¹⁴ Oxidative stress and antioxidant depletion have been implicated in retinal damage from smoking, although the precise mechanism remains unclear.^15,16 Other factors that have been reported to influence risk for AMD include sunlight exposure, alcohol consumption, cardiovascular disease, history of diabetes, increased plasma fibrinogen levels, diet, hypertension, body mass index and iris color.^11,17-19 Regarding diet, antioxidants, such as carotenoids, zinc, and vitamins A and E, may provide a protective benefit against AMD.

The condition is also thought to be more prevalent among Caucasians, compared with either African Americans or people of Hispanic origin.²⁰ Such ethnic differences may reflect genetic, as well as environmental, risk factors.

AMD has a significant genetic component,^21-22 as several twin studies have shown significantly higher concordance rates between monozygotic twins as compared to concordance between dizygotic twins.²³ Family members of individuals with AMD are at increased risk (2.4- to 19.8- fold) for developing the disease relative to individuals with no family history.²⁴

PATHOGENESIS

The etiology and pathophysiology of AMD are poorly understood.

Drusen are the histological markers of AMD. The cause of drusen may be linked to one or more of the following key processes that are implicated in AMD.

(1) Increased outer segment turnover.
(2) Free radical/oxidative damage.
(3) Aging and degeneration of elements of Bruch's membrane (eg, collagen and elastin).
(4) Reduced clearance of material from Bruch's membrane into the choriocapillaries. Specific alterations in Bruch's membrane composition that have been suggested to cause drusen include excessive lipid deposition and protein cross-linking as well as impaired permeability to nutrients.
(5) Deleterious immune system activation.²⁵
(6) Impaired activity/function of the RPE. Several changes in the RPE structure contribute to the development of AMD; among them are structural changes that occur in the tight-belted monolayer of the RPE and disrupt the outer blood retina barrier (oBRB). These changes contribute to the cascade of AMD development, especially the wet form of the disease.

These processes are triggered by several mechanisms; inflammation and ischemia are among the major pathologic mechanisms.

AMD AND THE DISRUPTION OF THE oBRB

The RPE is the major component of the oBRB, separating the neural retina from a network of fenestrated choriocapillaris at the innermost vascular layer of the choroid. This layer lies adjacent to Bruch's membrane and is composed of small but richly anastomotic capillaries. The choroidal vasculature layer is the main supporter of the metabolic and oxygen requirements of the RPE cells and photoreceptors.²⁶

The oBRB regulates the composition of the subretinal space, enabling the photoreceptors to carry out their functions. The RPE plays many essential roles including transport of nutrients, water and ions and the removal of metabolic waste. It is responsible for daily phagocytosis and renewal of the outer segment discs of the photoreceptors and plays a crucial role in the transport and recycle of fatty acids. It is also responsible for the removal of excessive water from the subretinal space toward the choriocapillaris.²⁷ AMD is often diagnosed by the appearance of subretinal fluid. This fluid creates a local detachment of the retina in the macular area resulting in decreased visual acuity.²⁸

The oBRB integrity is maintained by a series of tight junction (TJ) proteins, responsible for regulating RPE cell-to-cell contact and limiting the intercellular space, thus controlling the passive paracellular transport and enabling the active transport of needed materials. The TJ family includes more than 40 proteins, among them trans-membrane proteins, folding proteins and signal transduction proteins.²⁹ The TJ system is a dynamic system capable of regulating the intercellular space and controlling the passage of substances of varying size. The TJ belt divides the RPE membrane into its basal and epical sides, creating polarity in the RPE monolayer and moving its different functional needs in the direction of either the photoreceptors or the choroid.²

Various studies have connected the functional changes in TJ proteins of the RPE to the pathophysiology of AMD.^30,31 In addition, vascular endothelial growth factor can cause rapid post-translational modifications of TJ proteins by stimulating occludin phosphorylation and by inducing tyrosine phosphorylation of ZO-1.³¹ Increased intraocular VEGF production occurs in ischemic retinal diseases as well as choroidal vascular diseases, and it has a direct influence on the oBRB structure.

Disruption of the integrity of the RPE barrier also occurs when a neovascular membrane penetrates the RPE monolayer to invade the subretinal space, as found in the neovascular form of AMD. A recent work³² demonstrated that direct pathological contact between RPE and endothelial cells (which are normally separated) enhances the proangiogenic potential of the endothelial cells to proliferate and migrate, similar to the process induced by hypoxia. These findings support the linkage between pathological changes in the RPE and the development of angiogenesis involved in CNV formation.

ISCHEMIA AND HYPOXIA IN AMD DEVELOPMENT

Considering ischemia and hypoxia as key factors in the pathophysiology of AMD, these terms should be clarified. Choroidal ischemia is determined as insufficient blood supply to the retina. In ischemia, the partial pressure of oxygen within the tissue is reduced due to inadequate oxygen supply to the retina. Furthermore, hypoxia means reduced oxygen levels within the cells, leading to reductions in mitochondrial respiration and oxidative metabolism. A prolonged hypoxic state may lead to cell death. Ischemia always results in hypoxia; however, cellular hypoxia can occur without ischemia even in an environment rich in oxygen.

In the normal eye, oxygen diffuses from the choriocapillaris through Bruch's membrane and RPE towards the avascular outer retina and is consumed mainly by the photoreceptors. The level of oxygen in the RPE is usually very high because of the close proximity between the RPE and the choriocapillaris. The level of oxygen decreases almost linearly as the distance from the choriocapillaris to the inner portion of the photoreceptors increases.

Pathological changes, such as drusen accumulation, thickening of Bruch's membrane, water accumulation, pigment epithelial detachment and retinal detachment, increase the distance between the choriocapillaris and the retina,^33-35 reduce the oxygen flux to the photoreceptors and induce hypoxia in the retinal cells. Low oxygen levels in the cellular environment induce elevation in hypoxia-inducible factor, a transcription factor that further activates VEGF gene expression, thus leading to increases in permeability and angiogenesis.

The theory that choroidal and retinal blood flow abnormalities play a role in AMD is also well established. Vascular deficits due to reduced choroidal and retinal blood flow have been identified in early and late AMD using fluorescein angiography, Doppler flowmetry and Doppler imaging.^36-40 Reduced blood flow in the choroid and retina causes chronic ischemia in Bruch's membrane, the RPE and the neuroretina.^36,41 Both drusen and RPE abnormalities are associated with decreased choroidal blood flow.³⁶ Impaired perfusion might cause impaired RPE function that then results in decreased degradation of photoreceptor disc membranes, reduced antioxidant capacity and an increased number of drusen in Bruch's membrane.

Figure 2. Invisible vasculature (in red-free images) revealed noninvasively by the RFI. A. Red-free image of a healthy subject. B. The RFI's capillary perfusion map, showing fine microvasculature detail (scale bar = 1 mm).

Compromised RPE cell function causes a diminution of vascular growth factors such as vascular endothelial growth factor from the choroid. VEGF depletion induces choroidal atrophy⁴² and further delays perfusion. The thickening of Bruch's membrane due to drusen not only leads to reduced diffusion of oxygen and thus pronounced neuroretinal ischemia but also to impaired transport capacity of important metabolites of the retinoid cycle (such as vitamin A).⁴³

Feigl et al. proposed the ischemia postreceptoral hypothesis.⁴¹ They suggested that photoreceptors are more resistant to ischemic insult than postreceptoral neurons, owing to their proximity to metabolic reserves available in the RPE and the choriocapillaris. The postreceptoral neurons are particularly vulnerable, as they are located in the watershed zone between two blood supplies,⁴¹ the choroidal and retinal vascular systems. This hypothesis is supported by histopathogical findings. When the RPE degenerates, the postreceptoral outer plexiform and inner nuclear layers are first affected.⁴⁴ It is still not clear which retinal layer is first targeted by ischemia in humans; whether it is the outer⁴⁵ or the inner retina^46,47 needs to be confirmed.

RETINAL FUNCTION IMAGER

In recent years, several attempts have been made to develop new retinal imaging technologies.^48-51 However, most systems available for clinical use monitor structural changes in the retina and do not provide direct information about the retina's functioning. The Retinal Function Imager (RFI) is a digital imaging system designed to capture accurate retinal images via a standard fundus camera. It offers a noninvasive diagnostic approach to retinal function assessment and provides easy, direct, qualitative and quantitative imaging of four parameters: (1) blood-flow velocity,^52-54 (2) capillary perfusion maps,^52-54 (3) blood oximetry ^52-54 and (4) metabolic intrinsic state.^52-54 All of these parameters directly assess retinal function and are thought to be modified in the course of retinal diseases. Most importantly, as retinal functional changes precede structural changes, quantitative imaging of these four parameters provides a useful tool for early detection of retinal diseases in general and AMD in particular.^53,54

The four key parameters offered by the new imager are:

1. Blood flow. Under green illumination, hemoglobin within the erythrocytes in the bloodstream provides a natural, high-contrast chromophore for tracking blood flow. Cross-correlation matching of moving erythrocyte patterns within an image series gives a direct measure of velocity. Reregistration and differential processing of a series of images taken at 55 Hz produces a “flow movie,” in which it is possible to follow the motion of individual clusters of red blood cells — or even single red blood cells — in vessels as small as capillaries. Flow movies provide a tool for qualitative assessment of retinal blood-flow rates.

In a study of AMD patients, the RFI detected reduced blood-flow velocity in exudative AMD eyes compared with fellow dry AMD eyes (in all sizes of veins and in small arteries; mean age, 78.5 ± 4.5 years).⁵⁵ Drug effects on the retina have also been studied with the RFI. Following intravitreal bevacizumab injections, a distinctive pattern of change in patient retinal blood-flow velocity was noted in responders vs nonresponders.⁵⁵

A recent study assessed the short-term effects of intravitreal bevacizumab on retinal blood-flow velocity compared to clinical outcomes assessed by optical coherence tomography and tests of visual acuity.⁵⁶ Eight patients receiving intravitreal injection of bevacizumab for CNV were included. All were imaged by the RFI preinjection and at one and seven days postinjection. VA and OCT were recorded preinjection and at one month postinjection. The researchers found a good correlation between the one-month change in VA and OCT measurements and the short-term change induced in bloodflow velocity. Arterial and venous velocity changes one day after the injection correlated with the VA change (P<.05). The one-day arterial velocity changes correlated with total macular volume (P=.02) and venous velocity changes correlated with central macular thickness (P=.04).

In a different study, Landa et al.⁵⁷ compared venous blood-flow velocity with central retinal volume in patients with various ocular diseases and observed a strong correlation between venous retinal blood-flow velocity and central retinal volume. The RFI may thus be a useful tool for evaluating changes in retinal thickening.

2. Capillary perfusion maps. The ability to directly witness the motion of red blood cell aggregates provides a tool to map the surface retinal vasculature in detail. Following image registration, pixel value distribution parameters are analyzed to locate blood motion, tracing microvasculature based on motion contrast rather than on total reflectance contrast. The RFI's noninvasive capillary perfusion map (nCPM) shows vessels that are indistinct or invisible on even sharp red-free images. Indeed, small vessels often appear as clearly as larger ones on these maps because the red cell clusters moving in these vessels are more distinct and less motion blurring occurs.⁵² Recently, in studies with an improved algorithm,⁵⁵ as much and often more microvascular detail could be seen on the nCPM than on corresponding fluorescein angiography images. The RFI can assist in cases where adverse reactions to fluorescein injection have been observed.⁵⁸

3. Oximetry. The balance of oxygen supply and demand in the retina is closely regulated to maintain the processes of visual perception. Alterations in either oxygen supply or consumption might directly indicate the early onset of retinal abnormalities.^59-60 Thus, it is important to have a tool for qualitative and quantitative assessment of oxygen utilization in the tissue. The difference between the absorption spectra of oxyhemoglobin and deoxyhemoglobin can be used to determine the oxygenation of blood with multispectral imaging methods. In multispectral imaging mode, the RFI can perform spectroscopic decomposition to qualitatively assess the oximetric state of the retina.

4. Metabolic function. The RFI is capable of imaging outside the absorption range of photoreceptors under near-infrared light (750 – 840 nm), and can be used to optically monitor retinal activity in response to a well-defined visual stimulus (562 ± 20 nm). The difference between the post-stimulated and prestimulated images is used to determine the metabolic state of the retinal compartments.

CONCLUSION

AMD is a multifactorial disease, and ischemic damage plays a major role in its development and progression.

Detection of functional parameter abnormalities may permit diagnosis of disease progression before anatomic abnormalities become evident, allowing treatment intervention before irreversible retinal damage occurs. The ongoing advances in the treatment of AMD, in conjunction with better diagnostic tools, will improve outcomes for patients suffering from this devastating disease. Furthermore, treatment can be improved by noninvasive follow-up.

The RFI's multimodal approach to functional imaging also opens research and drug development opportunities with regard to a wide range of retinal diseases, beyond the capabilities of structural imaging. RP

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