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Search for a Medical Therapy: Focus on Oxidative Stress
Digital Journal of Ophthalmology 1999
Volume 5, Number 5
August 1, 1999
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Alan M. Laties, M.D. | University of Pennsylvania, Scheie Eye Institute

The search for an effective treatment for retinal degenerations is not new. The recommendation "remedies of night blindness in the eyes; liver of ox, roasted and crushed, is given against it. Really excellent." was recorded in the Ebers Papyrus over 3500 years ago [1]. This recommendation was then incorporated INTO Greek medicine and lasted for millennia. Only in this century did the pace quicken as numerous new therapies have been proposed. Whether medical or surgical, most proposed treatments were based on a glimmer of scientific rationale; uniformly they lacked a sound laboratory or clinical demonstration of efficacy. And sadly, none lived up to the claims made for them or to the expectations they aroused.

Just in the last few years the situation has changed dramatically for the better. First, experiments conducted in the laboratory with growth factors and neurotrophins [2] and second, a report of a clinical trial of vitamin A supplementation [3] have each put forth convincing proofs that the course of a retinal degeneration can be moderated. Moreover, advances in cell biology and molecular genetics have opened up the prospects wider still for other promising medical therapeutic initiatives. These have a wide range, extending FROM gene therapy and antisense modulation of gene function at one extreme to helpful dietary advice at the other. In addition, there are early indications that cellular transplants as a therapeutic modality might well be feasible in the future.

Clearly no one can predict which approach to therapy will enjoy greatest success; hence it is important to pursue several DISTINCT therapeutic avenues simultaneously. Not only does a broad-based strategy promise the greatest chance of success but there are reasons to believe that efforts in DISTINCT disciplines might well be mutually supportive: an understanding achieved in one effort can enable a second.

Several initiatives towards a Medical Therapy for retinal degeneration are now underway, one at the University of Pennsylvania. Today I will present to you as part of that initiative, a survey of oxidative stress undertaken for laboratory planning purposes. The survey emphasizes the importance of oxidative stresses in general and the appropriateness of attention to susceptibility to oxidative damage in retinal degenerations in particular.
Oxidative Stress and Photoreceptors
Oxidative damage defines the consequences of a mismatch between the production of the reactive oxygen species and the ability to defend against them. For the most part reactive oxygen species originate in the body. They are many and include free radicals such as superoxide, nitric oxide and hydroxyl ion as well as the oxygen derived species such as singlet oxygen, hydrogen peroxide and hypochlorous acid. Major sources include mitochondrial oxidative metabolism, phospholipid metabolism and proteolysis. As noted below, the photodynamic effect of light absorbed by photosensitizing chromophores in the retina can add to the burden by generating singlet oxygen. Environmental exposure, especially inhalation of oxidizing air pollutants such as tobacco smoke, oxides of nitrogen FROM motor vehicle exhaust and ozone add to the total. Lastly and usually ignored, pro-oxidant effects of certain vitamin supplements can at times be a cause for concern.

Raised against the challenges are an extensive and highly effective array of protective agents and defense mechanisms. These comprise numerous small molecular weight antioxidants to forestall initiation of oxidative damage and/or LIMIT its propagation; enzymes that convert reactive oxygen species; enzymes to REPAIR oxidative damage when it occurs, plus mechanisms to route damaged molecules for destruction and replacement. For mitochondria where reactive oxygen intermediates inevitably leak FROM the electron transport chain to cause local damage, rapid turnover and replacement of the organelle is recognized. In fact, the need for rapid regeneration of mitochondria is often used to EXPLAIN its independent store of DNA. Lastly, outer segments have a turnover mechanism based on disc renewal and shedding to supplement local repair.

A series of special conditions imposed upon photoreceptors puts them in what can only be termed a high risk, proxidant environment. Chief among these risks is a partial pressure of oxygen several fold higher than that usually found elsewhere in the body [4]. The photoreceptor outer segment harbors a high proportion of polyunsaturated fatty acids that are readily oxidized. Coincident is the need for a large supply of ATP to power the active ion pumps and substantial metabolic demands of an active photoreceptor. As a result the inner segment contain a considerable number of mitochondria. In turn, they utilize a great amount of glucose and oxygen as substrate, inevitably leaking a small but significant fraction of the newly formed superoxide produced during the 1-electron stepwise reduction of oxygen. Under normal conditions the production of superoxide is met by an ample supply of the detoxifying enzyme, superoxide dismutase. Catalase and glutathione reductase are then readily available to handle the hydrogen peroxide so produced. These enzymes in concert with antioxidant proteins and small molecular reductants such as a -tocopherol, ascorbic acid, reduced glutathione and urea form an effective antioxidant defense within the the photoreceptor in normal circumstances. When overwhelmed, lipid peroxidation within the outer segment results.

The need for photoreceptors to function under illumination imposes a second special condition. The antioxidant defense system needs to quench reactive oxidative species such as singlet oxygen generated as energetic photons enter the eye. In this regard, as Dillon et al. [5] have pointed out, chromophores essential to vision such as the Schiff base of retinal can act as photosensitizers. The extra burden imposed by light tests the antioxidant resources of the retina, a concept stemming largely FROM the work of Noell and others on light damage. Of special note in this regard of the twin observations by Remé and colleagues [6,7]. Light damage at threshold occurs first at the tips of the photoreceptor outer segment. If it is correct that the most distal (and aged) outer-segment discs are at greatest hazard, it is also true that these are the ones most easily dispensed. Also light damage is accompanied by the local release of arachidonic acid. Arachidonic acid is subject to rapid conversion to several biologically active and pro-inflammatory mediators by cycloxygenases. It can also induce lipid peroxidative reactions leading to the formation of superoxide. Since release of arachidonic acid follows the activation of phospholipase A2 [8], excess activity of this enzyme might well represent an indicator for cell damage. In this respect assessment of its activity in tissues subject to oxidative stress could well prove useful. Similarly since it is now known that stable isoprostanes form by the non-enzymatic, free radical catalyzed oxidation of arachidonic acid, these again can serve as a marker for oxidative stress [9].

Oxidative Stress and Retinal Degenerations
Unfortunately a direct and reliable, non-invasive measure of the oxidative balance of the retina is lacking. At present there is no accepted technique to signal when oxidative stress overmatches antioxidant capacity. Several methods, some quite ingenious, have been proposed to measure oxidant-antioxidant imbalance but none have found their way INTO standard clinical usage. In the absence of direct measurement of oxidative stress in the retina, reliance must be placed on indirect measurements and circumstantial evidence. For hereditary retinal degeneration an enhanced susceptibility to retinal photodynamic injury in animal models has been convincingly established [10]. In fact, there are indications that in some mouse models of hereditary retinal degeneration even moderate ambient light levels can be deleterious to photoreceptor survival [11]. In the clinic, it is common for patients with retinitis pigmentosa to refuse ever to undergo a second fluorescein angiogram, claiming severe and relatively long lasting visual discomfort and disability FROM successive flashes of bright light incurred FROM the first angiogram.

For macular degeneration several correlations have been established to oxidative stress. In one report there was a greater susceptibility to iron-induced lipid peroxidation at the macula than at the retinal periphery [12]. Of equal pertinence, the older the patient the greater was the susceptibility to lipid peroxidation. Reports also exist that retinal levels of antioxidant enzymes are lower in patients with age-related macular degeneration when compared to age-matched controls [13]. This has been paralleled in direct assay of enzyme levels in the retinal pigmented epithelium [14]. Unfortunately, study of the relationships between light exposure and macular degeneration have not been helpful to date. They are both complex and problematic. Short-term exposure to a bright light in early macular degeneration does cause discomfort, and is accompanied by a delayed recovery of normal vision. But serial attempts to find a correlation between life-long light exposure and retinal disability has yielded mixed results: in fact the case has been hard enough to prove as to undermine the hypothesis [15-17]. Were such studies to encompass a reliable measure of the antioxidant capacity of the retina for the individuals involved, case stratification would make evaluation far more sensitive and perhaps yield a more definitive result. Tobacco smoking is an exception to this generalization. It is an acknowledged oxidant stress, one that is reflected in lowered plasma levels of antioxidants: smokers do experience a substantially greater risk of macular degeneration than do non-smokers [18]. One further set of observation appear credible. A two-fold variation in risk of age-related macular degeneration was found in a large and authoritative nutritional study. In the multi-center eye disease case-control study reported by Seddon et al, [18] the quintile ingesting the greatest amount of carotenoids in the diet were half as likely to experience macular degeneration as those in the lowest quintile. A less clear-cut result issued FROM a smaller case-control study reported by Mares-Perlman [19]. However, even in this study for one carotenoid, lycopene, the level in plasma had a clear and favorable correlation with a lower prevalence of macular degeneration.

Carotenoids and Retinal Degeneration
The findings of protective effects of ingested carotenoids against macular degeneration merit consideration in relation to hereditary retinal degenerations in general. An improved definition of the role(s) played by carotenoids in the retina follows FROM several recent research reports and authoritative reviews [20-22]. In summary, the case can be made that they likely act as multifaceted retinoprotectives. Although a central retinal yellow spot was recognized and named macula lutea over 200 years ago, it is just in the last 15 years that intensive biochemical attention has defined its biochemical characteristics. To the greatest surprise it has been found that beta carotene, the best known of the carotenoids, and one present in high concentration in plasma, does not find its way to the retina [23]. Further beta carotene is also absent FROM the retinal pigmented epithelium [24]. Instead the oxygenated xanthophyll carotenoids, lutein and zeaxanthin have been consistently recognized in the retina by sensitive assays. Since retinal concentrations for numerous xanthophyll pigments differ sizably FROM their plasma concentrations, a specific uptake mechanism for lutein and zeaxanthin and an exclusion mechanism for beta-carotene to the retina is implied. Although most easily recognized by their high concentration in the central retina, both lutein and zeaxanthin are present at lower concentrations in peripheral retina as well. In the macular area, these pigments are found in Henle’s fiber layer, apparently bound to tubulin [25]. In addition, van Kuijk has recently asserted that a substantial proportion of retinal xanthophyll pigment is present in the rod outer segment [26].

The presence of xanthophyll pigments at the macula is susceptible of several interpretations. Exclusion of the most highly refracted blue rays based on their shorter wave length clearly enhances the clarity of the image. Moreover, immediately the concept of light-damage emerged, it was realized that selective absorbtion of the most energetic, shorter wavelength photons represented a retinoprotective function [27]. This idea is reinforced by the fact that carotenoids radiate energy away as heat rather than by forming dangerous oxyradicals. More recently, evidence has been developed that lutein and zeaxanthin can even function as antioxidants independent of light stimulation [28]. Specifically, not only are they able to quench photodynamically originated singlet oxygen but they also can react with peroxyl radicals, thus limiting free radical propagation. In confirmation the work of Dorey et al in the quail model of light-damage demonstrated that the addition of lutein to the diet leads both to raised retinal levels and photoprotection [29]. Based on these observations, studies are now underway at our institution to see whether addition of carotenoids such as lutein to the diet will moderate the progress of hereditary retinal degenerations in mice.
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