The late stages of age-related macular degeneration (AMD), neovascularization and geographic atrophy, are the most common causes of severe visual loss and legal blindness in persons over 60 years of age in the United States. Clinical and epidemiological studies that report significant associations of AMD with cataracts, prior cataract surgery, cumulative exposure to sunlight and pigmentation support the hypothesis that chronic photochemical injury plays a critical role in the aging of the macula and in the pathogenesis of AMD. Histopathology and recent in vivo noninvasive measurements suggest that in normal populations there is an increasing accumulation of potentially damaging photoproducts with age in the retinal pigment epithelium (RPE)/Bruch?s Membrane (BM) complex. Preventive intervention of these light-driven processes could be accomplished by spectral filtering of light reaching the macula. Recent in vitro and animal experiments have shown that RPE lipofuscin photosensitizes singlet oxygen generation, with an action spectrum quite similar to that for acute photochemical RPE injury in primates. Additionally, lipofuscin granules contain at least 10 different fluorescent species that originate largely from photochemical reactions involving A2E (N-retinylidene-N-retinylethanolamine), its epoxides and other as yet chemically unidentified A2E-related fluorophores which can be both cytotoxic and phototoxic. The precursors of the fluorophores found in RPE lipofuscin granules originate from reactions of all-trans-retinal within the rod outer segment (ROS) discs during periods associated with significant rhodopsin bleaching (i.e., normal daylight). Although RPE lysosomal processing enzymatically digests >99% of the shed ROS discs contents by mass, A2E and related fluorophores are not digested, but are concentrated into ~1 micron lipofuscin granules. By age 60 years, the average concentration of A2E within RPE cells reaches 400uM in normal eyes. However, A2E is toxic to cellular membranes at much lower concentrations. We hypothesize that segregation of A2E into lipofuscin granules and prevention of its redistribution into critical membranes is required for RPE health. Specific mechanisms for photochemical injury related to AMD progression have been proposed: 1) accumulation of toxic photochemicals such as A2E or A2E epoxides in critical membranes of the RPE/BM complex, 2) chronic photo-oxidative stress via singlet oxygen generation by short-wavelength activation of accumulated A2E-related photosensitizers either within lipofuscin granules or in more sensitive targets, and 3) all-trans-retinal photosensitized ROI generation leading to rod disc rim proteins damage. Wehave developed a biophysical model that uses a variety of published average normal values of pupil size(age,corneal irradiance), lens transmission (age,l), and rod dark adaptation time constant trh(age) to determine average retinal spectral irradiance and steady state rod bleaching as a function of age and ambient light intensity. Our model relates retinal spectral irradiance as a function of age and lens status to the relative rates of rod bleaching, steady-state concentration of all-trans-retinal, all-trans-retinal photosensitization of oxidative damage in the rod outer segments, all-trans-retinal reactions to form A2E-related species, and A2E photo-oxidation within lipofuscin granules. Given the predicted 30% decrease in the action spectra-weighted short wavelength macular irradiance with each decade, repair mechanisms would have to fall precipitously at advanced age for either all-trans-retinal or lipofuscin photo-sensitization of ROI to explain late onset macular degeneration in phakic individuals. Furthermore, if such photo-oxidative damage is driving any stage of AMD progression, then the dramatic increases in short wavelength macular irradiance following conventional cataract surgery shouuuld lead to dramatically increased risk. Similarly, the model of Sun and Nathans for all-trans-retinal photoaggregation of rim proteins in the rod ROS should be a much stronger effect in the first few years of life and then rapidly diminish with age. Our model predicts a nearly constant production rate of A2E-related fluorophores in the RPE during the first 60 years, but falling significantly thereafter. A similar age dependence of total lipofuscin granule volume and total fluorescence per RPE cell was reported recently by Okuba in histopathology of human cadaver eyes. However, the rates of lipofuscin increase with age are slower than the rate of decrease in short-wavelength macular irradiance in the phakic eye with age. Therefore, our model predicts 1O2 photosensitization in the RPE should also fall with increasing age. Spectral confocal imaging of human tissue sections exhibit intense characteristic lipofuscin fluorescence localized to the granules. Photo-oxidative stress in the outer retina might arise from the smaller amounts of A2E-related fluorophores observed in critical membranes of the RPE/BM complex. However if the RPE/BM complex were the site of photo-oxidative injury driving AMD progression, the magnitude and rate of this oxidative injury would be expected to increase dramatically following cataract removal and IOL implantation. We could not find a mechanism of photo-oxidative injury that would increase with advancing age in our general population model (as does AMD), but not be greatly enhanced by cataract surgery and IOL implantation. Consequently, we propose a contrarian view that singlet oxygen generation by RPE lipofuscin is useful in allowing the chemical alteration in A2E, thereby limiting the steady-state levels of A2E ([A2E]ss) in the RPE and preventing A2E chemical toxicity. Given the short diffusion distances (~10nm) of singlet oxygen (4msec lifetime), singlet oxygen photochemically generated in lipofuscin granules will react with A2E, which avidly quenches singlet oxygen, to form A2E epoxides which are themselves highly reactive. Thus A2E once localized to a lipofuscin granule would be photo-oxidized, and the resulting epoxides would then chemical react to form increasingly complex cross-linked material that is insoluble. Using spectral confocal microscopy, we determined that ~13J/cm2 @430nm is required to bleach or photo-oxidize A2E in such granules. Using a standardized ambient-light-exposure distribution, the macular RPE [A2E]ss in our model increases in the normal phakic eye with age, even after 60 years when rod bleaching and A2E production decreases significantly. This modeled [A2E]ss varies only slightly with as the mean irradiance was varied by more than 20-fold at any given age. Our theoretical model of macular aging reproduces the normal age dependence of lipofuscin (total A2E-related material) and of A2E determined by in vivo fluorescence techniques. It also provides for a primary cytotoxic mechanism in which, once [A2E] reaches a maximum level in the RPE cell, A2E concentration rises in critical membranes (i.e., lysosomal, mitochondrial, and other including the RPE basement membrane) and causes damage with or without additional photo-activation. The coupling of light to both the rate of production of A2E and its photo-degradation (i.e., chemical alteration) allows the balancing the A2E level for a wide range of cumulative light exposures. In the model, it is primarily the yellowing of the lens with age that distorts the original spectral balance between rate of production and rate of photo-oxidation found in youth, and allows the [A2E]ss to rise with age. We are evaluating noninvasive retinal imaging methods that might permit clinical validation of our predictions of photochemical changes following cataract surgery and our predictions of the benefits of specific spectral photo-protective filters.
