The susceptibility of the retina to photochemical damage from visible light

Abstract

The photoreceptor/RPE complex must maintain a delicate balance between maximizing the absorption of photons for vision and retinal image quality while simultaneously minimizing the risk of photodamage when exposed to bright light. We review the recent discovery of two new effects of light exposure on the photoreceptor/RPE complex in the context of current thinking about the causes of retinal phototoxicity. These effects are autofluorescence photobleaching in which exposure to bright light reduces lipofuscin autofluorescence and, at higher light levels, RPE disruption in which the pattern of autofluorescence is permanently altered following light exposure. Both effects occur following exposure to visible light at irradiances that were previously thought to be safe. Photopigment, retinoids involved in the visual cycle, and bisretinoids in lipofuscin have been implicated as possible photosensitizers for photochemical damage. The mechanism of RPE disruption may follow either of these paths. On the other hand, autofluorescence photobleaching is likely an indicator of photooxidation of lipofuscin. The permanent changes inherent in RPE disruption might require modification of the light safety standards. AF photobleaching recovers after several hours although the mechanisms by which this occurs are not yet clear. Understanding the mechanisms of phototoxicity is all the more important given the potential for increased susceptibility in the presence of ocular diseases that affect either the visual cycle and/or lipofuscin accumulation. In addition, knowledge of photochemical mechanisms can improve our understanding of some disease processes that may be influenced by light exposure, such as some forms of Leber's congenital amaurosis, and aid in the development of new therapies. Such treatment prior to intentional light exposures, as in ophthalmic examinations or surgeries, could provide an effective preventative strategy.

Figure 1

The visual cycle. There are many thorough reviews of the visual cycle (e.g. (Lamb and Pugh. 2004, Sparrow et al. 2010, Travis et al. 2007)). The visual cycle is initiated as 11-cis-retinal, from the chromophore within an opsin molecule, is photoisomerized to all-trans-retinal and the process of converting it back into 11-cis-retinal begins. The all-trans-retinal molecule is moved from the photoreceptor disc membrane to the cytoplasmic space partly by ATP-binding cassette retina (ABCA4) and is then reduced by all-trans-retinol dehydrogenase (RDH) into all-trans-retinol. With an inter-photoreceptor retinol binding protein (IRBP) as a chaperone, all-trans-retinol is transported to the cytoplasm of the RPE where it is then chaperoned by cellular retinol binding protein (CRBP). The alcohol is esterified by the enzyme lecithin retinol acyl transferase (LRAT) to form all-trans-retinyl ester. Multiple all-trans-retinyl ester molecules can group together to form lipid bodies in which they are stored until needed (Imanishi et al. 2004). As the regulation of the visual cycle occurs by means of changing the rate of production of 11-cis-retinal within the RPE, a retinyl ester storage particle (REST; also known as a retinosome) can change in concentration and hence volume depending on the state and requirement of the visual system (Imanishi et al. 2004). To continue the visual cycle, all-trans-retinyl ester is isomerised to 11-cis-retinol by the RPE65 protein. The 11-cis-retinol is then oxidised into 11-cis-retinal by 11-cis retinol dehydrogenase (11-cis RDH) and transported back into the photoreceptors (chaperoned by cellular retinaldehyde binding protein (CRALBP) and IRBP). The 11-cis-retinal binds to an opsin in the outer segment membrane, becoming ready to absorb a photon of light and restart the visual cycle.

Figure 2

In vivo images of RPE AF photobleaching and disruption. A series of repeated images in the living macaque eye obtained using a fluorescence-enabled adaptive optics scanning light ophthalmoscope (AOSLO) shows the sequence of light-induced changes in RPE AF and photoreceptor reflectance that was observed following 568 nm light exposure. The honeycomb mosaic of discrete RPE cells can be seen because the cell nucleus does not contain lipofuscin and appears dark, whereas the cytoplasm surrounding the nucleus appears bright due to lipofuscin AF. The white outline in the pre-exposure image indicates the region of the retina exposed to either 788 J/cm² or 210 J/cm². The abrupt reduction in RPE AF intensity is visible in the immediately post-exposure images. There are no immediate changes in the appearance of the photoreceptor mosaic. Six days post-exposure, a long-term disruption in the RPE mosaic and an alteration in the photoreceptor reflectance (origin unknown) is seen with 788 J/cm². Although less pronounced these changes persist over time, as seen 19 days post exposure. No long term changes were observed in the RPE or photoreceptors for 210 J/cm² exposures.

Figure 3

Models of AF photobleaching. Schematic representation depicting AF ratio outcomes if a single fluorophore (a) or if multiple fluorophores (b) are involved in AF photobleaching. The arrows indicate the colour of the light exposure and are matched to the corresponding colour in the AF excitation spectra and AF ratio plots.

Figure 4

AF photobleaching involves multiple molecules. Average RPE AF ratio immediately post exposure to 88 J/cm² of 488 nm (solid circles) or 130 J/cm² of 568 nm (open circles) light plotted versus the wavelength used for AF excitation in the pre and post exposure images. Error bars represent the standard error of the mean.

Figure 5

Ex vivo AF photobleaching. Pre (left) and immediately post 568 nm exposure (right) images of the ex vivo human RPE cells exposed to 30.6 J/cm² (top) and the A2E-laden ARPE19 cells exposed to 106.4 J/cm² (bottom). The exposure locations are outlined in white in the pre exposure images and are clearly visible in the post exposure images.

Figure 6

Quantifying ex vivo AF photobleaching and partial recovery. The reduction and partial recovery over 3 days of RPE AF is quantified by the AF ratio in A2E-laden ARPE-19 cells exposed to 14.2 J/cm² (solid circles) or 106.4 J/cm² (open circles) of 568 nm light. Pre and post exposure images were taken in a 2° field using 568 nm light. Each location received only one exposure and none of the regions overlapped. Error bars represent the standard error of the mean of 2 measurements.

Figure 7

Histology of RPE disruption. Fluorescence images of 3 light exposed locations in macaque retina seen in both wholemounted retina (a, c, e) and 6 µm paraffin sectioned (b, d, f) macaque retina, 12 days (a, b, e, f) and 6 months (c, d) post-exposure to 568 nm light with retinal radiant exposures of 788 J/cm² (a–d) and 247 J/cm² (e, f). In c, the central black region is the fovea and the bright region on the left (white arrow) is the location of the exposure showing RPE disruption. In the images of the paraffin sections, only the 788 J/cm² exposures (edges denoted by white arrows in b and d) produced detectable changes in the photoreceptors and RPE. Scale bar is 130 µm.