The electromagnetic spectrum covers a continuum of electromagnetic waves from radio waves, microwaves, infrared, visible and ultraviolet (UV) radiations, through to X-rays and gamma-rays, the photon energy increasing with decreasing wavelength [figure 1]. Sunlight comprises 5-10% UV (100-380nm), ~40% visible radiation (380-780nm), and 50-55% infrared radiation. These are either absorbed or transmitted by the successive layers of the eye, modulating the light reaching the retina.1

Figure 1: Visible light (380-780nm) in the electromagnetic specturm. HEV – high energy visible; LEV – low energy visible

UV waves are harmful to the anterior part of the human eye. In a healthy adult’s eye no UV radiation actually reaches the retina. UVC (100-280nm) from sunlight are filtered by the atmosphere, while most UVB (280-315nm) are absorbed by the cornea. Residual UVB and most UVA (315-380nm) are then absorbed by the crystalline lens. In contrast, visible light reaches the retina in high proportions.2

In addition to allowing us to perceive the world around us in terms of shape, contrast and colour, visible light also plays an important role in various non-visual functions of the body, controlling many rhythmic biological functions. High energy visible light (380-500nm), commonly known as blue light, accounts for ~25 to 30% of the sunlight within the visible range. It includes both harmful blue-violet radiations (415-455nm) which can be damaging to the retina, but also beneficial blue-turquoise radiations (465-495nm), essential for normal physiological functioning during the day. Although transmission of blue light to the retina decreases with age, as a protection, it nonetheless remains present at significant levels.

Fundamentals of the retinal visual cycle

To reach the retina, light passes first through the cornea, the aqueous humour, the crystalline lens and then the vitreous humour. From here, it crosses the retinal ganglion cells and then several cell layers before reaching the outer retina. The outer retina is composed of retinal pigment epithelium (RPE) cells plus the outer segments of the visual photoreceptors (rods and cones) [figure 2].

Figure 2: Visual pigments in photoreceptors outer segments

The discs of the photoreceptor outer segments (POS) contain visual pigments formed by covalent binding between 11-cis-retinal (a photosensitive derivative of vitamin A) and a transmembrane opsin signalling protein.

Absorbed photons transmit energy to the photoreceptors via the opsin, triggering isomerisation of the 11-cis-retinal which causes a conformational change to all-trans-retinal [figure 3]. The all-trans-retinal is released from the activated opsin into the cytoplasm and is then rapidly reduced to its non-oxidised form all-trans-retinol,3-5 in a healthy retina. This crosses the sub-retinal space and enters the RPE where it is converted back to 11-cis-retinal which returns back to the photoreceptors, binding with opsin, and completing the visual cycle [figure 3]. The RPE plays a critical role in vision; in addition to the constant renewal of 11-cis-retinal, it is also responsible for the phagocytosis of the POS discs and providing nutriments and oxygen to the photoreceptors. The visual cycle is the fundamental basis of our vision, and its dysfunction triggers irreversible retinal damage.

Figure 3: The visual cycle in rods

Eye damage and retinal pathologies

Chronic eye exposure to solar UV waves is associated with the pathogenesis of numerous diseases of the anterior part of the eye, such as pterygium and pingueculum. It is also associated with crystalline lens pathologies, in particular the development of cataract. While the visual cycle can be progressively disrupted with ageing, this process is known to be accelerated by light. Retinal damage can originate from photomechanical, photothermal or photochemical reactions.

Optical radiation (UV, visible and infrared) has the potential to cause photomechanical and photothermal damage from brief and extreme exposure, while photochemical damage is more commonly due to cumulative and prolonged exposure and is also wavelength-dependent being blue-violet light specific for the outer retina. The cumulative harmful effect of light on the retina depends on the irradiance (ie the power received on a given surface per unit area). Retinal irradiance is in turn dependent not only on the light source radiance (ie the power of the light source per unit area per unit angle), but also on pupil size (decreases with age and brighter light) and anterior ocular media transmittance.

Among known retinal pathologies, the most preoccupying is age-related macular degeneration (AMD). Along with age, genetics, smoking and diet, blue-violet light is known to contribute to accelerated ageing of the outer retina and is thus a risk factor for AMD.6-13 AMD involves the degeneration of RPE cells and then the photoreceptors, and is associated with chronic inflammation and oxidative stress. In developed countries, it is the leading cause of irreversible visual impairment, with 17.8 million cases in the US14 and estimated as 265 million worldwide over the next 30 years. Prevention of retinal damage caused by blue-violet light via photoprotection is an important aspect of retinal health management.

The changing profile of light exposure

Light exposure profiles vary considerably among individuals, influenced by a multitude of factors; the nature and number of light sources, their localisation, spatial distribution, as well as radiance, spectral exposure duration and frequency.

Exposure to UV and blue light from the sunlight varies depending on the time of day, geographic location, season, etc, but is also affected by social influences (skin cancer awareness, sunglasses’ quality, and social norms relating to skin tanning).

Artificial light sources also contribute to retinal light exposure, altering the light exposure profile with more light sources, longer and repetitive exposure, higher radiance and energy, and at shorter distances. Exposure occurs in people of all ages, and at increasingly younger ages. Solid-state lighting (SSL) now dominates domestic lighting, with incandescent bulbs being phased out this year, and the European Lighting Industry estimating that over 70% of light sources will be based on SSL by 2020. Current ‘cold-white’ light emitting diodes (LEDs) include up to 35% of blue light within the visible range, compared to incandescent lamps which have less than 5%.15 ‘Warm-white’ light has less than 10% of blue light but also has lower luminous efficacy. Thanks to their compact form and wide spectral range, LEDs are now extensively used in everyday self-illuminating applications including mobile phones, tablets, computers, TVs and even in toys and clothes. Radiance from LEDs can be up to 1,000 times higher than that of traditional incandescent lamps. Combined with the fact that the chronic toxic effect of a light source depends strongly on exposure duration and repetition, this could make LEDs a potential contributor to long-term retinal damage.16-18

Background research on retinal photodamage

Impact of UV on the anterior part of the eye

In the healthy adult’s eye, UV radiations are almost totally filtered out by the cornea and the crystalline lens and do not reach the retina. In vitro, in vivo, and epidemiological data demonstrate that chronic eye exposure to UV radiation is associated with the pathogenesis of numerous corneal and crystalline lens pathologies. The role of UV in corneal damage was shown as early as the mid-1950s when Kerkenezov reported its involvement in the development of pterygium.19 Since then, numerous in vivo and in vitro studies using corneas and crystalline lenses from several species (including humans) have demonstrated the higher the wavelength, the higher the UV light damage threshold and thus the lower the toxic effect.20-24 Weighting the UV hazard spectrum by the sunlight spectral distribution, the greatest danger of UV is in between UVA and UVB with a maximum at around 315nm.

The mechanisms behind blue-violet light retinal damage

Photochemical damage is mainly associated with long-term and repetitive exposure to moderate irradiances, arising when a photosensitive molecule or chromophore undergoes physico-chemical changes after photon absorption. Damage is dependent on the balance between light exposure and the body’s retinal repair systems which manage oxidative stress. These systems are affected by age, genetic and/or environmental factors that can decrease their efficiency. In the presence of oxygen, high-energy photons can react with photosensitive compounds to produce photochemical reactions and then reactive oxygen species (ROS) including singlet oxygen (O2), superoxide anion (O2–.), hydrogen peroxide (H2O2) and hydroxyl radicals (HO–). These ROS are highly toxic and can cause protein oxidation, lipid peroxidation, mutagenesis, etc.25 They are naturally derived from numerous intracellular sources including the mitochondria, enzymatic systems or photosensitisers and can occur as a result of exogenous influences such as light, smoking or diet poor in antioxidants.

As one of the highest oxygen-consuming structures in the body,26 the retina is extremely susceptible to oxidative stress. Combined with an abundance of photosensitizers in the outer retina, prolonged visible light exposure and a high energy demand, this gives fertile ground for oxidative stress. The two major photosensitizers in the retina are 11-cis-retinal in the outer segments of the photoreceptors and lipofuscin, a ‘wear and tear’ pigment which accumulates with age in RPE cells.25 Other photosensitive molecules which may also play a role include cytochrome C, flavins and flavoproteins. Three major natural antioxidant systems supporting retinal health are superoxide dismutase (SOD), catalase and glutathione [figure 4]. SOD alternately catalyses the partitioning of the (O2-) radical into ‘safe’ (O2) or (H2O2). (H2O2), which is also dangerous, is in turn converted into water (H2O) and (O2) by the catalase enzyme or by the glutathione peroxydase enzyme which also converts reduced glutathione (GSH) into oxidized glutathione (GSSG).

Figure 4: Simplified normal processing of ROS. ROS in red. Antioxidant defences in green. (Adapted from Jarrett et al 2012)

When exposed to blue-violet light, all-trans-retinal (which accumulates in the POS), is highly photoreactive and induces oxidative stress, with decreasing sensitivity between 400 and 450nm. In the absence of sufficient antioxidant activity, the POS progressively oxidises and their renewal within RPE becomes more challenging, generating an accumulation of residual lipofuscin in the RPE10. Lipofuscin contains a photosensitizer with a maximum absorption in the blue-violet spectral range at 440 nm. Accumulation of lipofuscin in the RPE is a key feature of ageing and AMD.28,31 The RPE cells become progressively clogged with age-related waste products, ultimately resulting in apoptosis. Deprived of their support cells, the photoreceptors deteriorate, leading to permanent retinal damage.

Literature review on retinal blue-violet light damage

Retinal damage by blue light has been studied for a half century, starting with the landmark paper published by Noell et al, describing blue retinal phototoxicity in rodents exposed to white fluorescent lamps.30 In vitro studies on immortalised RPE cells loaded with purified lipofuscin showed lower toxicity thresholds with violet/blue-green light (390-550nm) versus yellow-red light (550-800nm).31 Similarly, human RPE cells loaded with A2E (a well-characterised chromophore in lipofuscin) were approximately seven-fold more sensitive to blue light than to green light.32 Phototoxicity was not observed without any photosensitizer, and increased with increasing photosensitizer concentrations. This was confirmed in several animal models.33-36 The role of broadband blue light in oxidative stress was shown in cultured human RPE cells causing lipofuscin-dependent protein oxidation, lipid peroxidation, mitochondrial DNA damage, lysosomal changes and cell death.9,31,37

Research has been taken a step further with an increasing body of literature studying the impact of LED lighting on the outer retina. A recent in vitro study on human RPE cells reported decreased cell viability by up to 99%, increased apoptosis up to 89%, and increased ROS production and DNA damage, after bright exposure to white or blue LED lighting.12 A similar study on primary human RPE cells reported that cold-white LEDs disrupted the expression of inflammatory markers (VEGF-A, IL-6, IL-8 and MCP-1) and pathological cytokines, and activated relevant signal pathways.38 A recent in vivo study in rats confirmed blue-light dependent damage with a range of coloured LEDs with loss of photoreceptors and activation of apoptosis.39 Supportive data are found in numerous epidemiological studies suggesting a correlation between blue light exposure from the sun and AMD40; in a recent meta-analysis of 14 epidemiology studies, 12 reported an increased risk of AMD with greater sunlight exposure, six of which were significant.13 Studies of human macular pigment density and the risk of AMD progression following cataract surgery lend further weight to the hypothesis that blue light exposure has a role in AMD pathogenesis, with a three-fold increased risk of AMD progression directly attributed to a dramatic increase in blue light exposure41-44 after surgery.

In vitro modelling of blue light toxicity on the outer retina (cell death)

While these studies leave little doubt that the outer retina sustains photochemical injury from blue light mediated by the visual pigment for the photoreceptor outer segments and by lipofuscin in the RPE cells, many of the published in vitro studies in this field suffer limitations. These include a lack of precision in terms of the light dose and/or use of very high irradiances that can trigger acute light-toxicity mechanisms rather than reflecting lifelong cumulative exposure damage which is more accurately represented by moderate irradiances and longer exposure, particularly in the context of AMD.

In 2011, a fruitful collaboration was developed between researchers at the Paris Vision Institute and at Essilor to address these issues. A well-established in vitro AMD model and innovative cell illumination protocol and device were used to evaluate the precise phototoxicity action spectrum (cell apoptosis) occurring under conditions mimicking physiological retinal exposure to sunlight.

Primary swine RPE cells were cultured in the absence of any photosensitizer, then photosensitised with A2E and finally exposed to 10nm-wide illumination bands across the blue-green range (from 390 to 520nm in 10nm increments) plus an additional band centred at 630nm for 18 hours, using an innovative LED-based fibred device. After light exposure, cells were maintained in darkness for 6 hours then analysed. Moderate irradiances (<1.6mW/cm² for 630nm and <1.3mW/cm² below 460nm) normalised to the daylight spectrum reaching the retina after being filtered by the ocular media were used. Cell necrosis (reflecting acute light toxicity) and apoptosis (reflecting long-term cumulative light toxicity) were measured.44

Figure 5: Light toxicity spectrum (apoptosis) in 20μM A2E-loaded RPE cells after 18 hours light exposure.

What they found was that firstly, none of the light exposures evaluated altered the necrosis rate compared to cells maintained in darkness, supporting that moderate light irradiance is not associated with acute toxicity. Secondly, decreased cell viability was detected with very low A2E concentrations at 420, 430 and 440nm, corresponding to blue-violet light. Finally, apoptosis was significantly induced between 415-455nm [figure 5], and increased with increasing A2E concentrations. These findings delivered a very precise definition of action spectrum.45

Coralie Barrau is a research engineer in optics and photonics, Amélie Kudla is R&D innovations programs co-ordinator and Mélanie Tessieres is an R&D optical engineer for Essilor.

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