Protective role of carotenoids in the visual cycle

Abstract

Exposure to light and accumulation of aberrant visual cycle by-products causes stress in the retina. The physical and chemical properties of carotenoids may provide protection against such scenario. These pigments exist in retinas of many vertebrates, including humans. However, the absence of carotenoids in mice, the preferred ophthalmologic animal model, hindered molecular and biochemical examination of the pigments' role in vision. We established a mouse model that accumulates significant amounts of carotenoids in the retina due to inactivating mutations in the Isx and Bco2 genes. We introduced a robust light damage protocol for the mouse retina using green (532 nm) and blue (405 nm) low-energy lasers. We observed that blue but not green laser light treatment triggered the formation of aberrant retinaldehyde isomers in the retina. The production of these visual cycle by-products was accompanied by morphologic damage in inferior parts of the mouse retina. Zeaxanthin supplementation of mice shielded retinoids from these photochemical modifications. These pigments also reduced the extent of the damage to the retina after the blue laser light insult. Thus, our study discovered a novel role of carotenoids in the visual cycle and indicated that vertebrates accumulate carotenoids to shield photoreceptors from short-wavelength light-induced damage.-Widjaja-Adhi, M. A. K., Ramkumar, S., von Lintig, J. Protective role of carotenoids in the visual cycle.

Keywords: light damage; retinoids; zeaxanthin.

Figure 1

Blue laser light treatment induces formation of aberrant visual cycle intermediates. Representative HPLC traces at 360 nm of lipid extracts from mouse retina bleached with green (532 nm) or blue (405 nm) laser light are shown. Geometric isomers of RAL were converted to corresponding RAL oximes (ROXs, syn, and anti) during extraction. Chemical identity of different peaks is indicated in diagram; spectral characteristics of different geometric ROX (syn) isomers are shown below the diagram.

Figure 2

Retinoid composition of mouse retina after green and blue laser light irradiation. Mice were subjected to green laser light irradiation for 3 s. After 60 s in dark, mice were irradiated with green or blue laser light for 10 s. Ocular retinoid composition was determined immediately after second irradiation or after indicted time points. Graphs display amounts of all-trans (A), 11-cis (B), 9-cis (C), and 13-cis (D) geometric RAL isomers after green/green (green squares) and green/blue (violet circles) bleaching.

Figure 3

Blue but not green laser light irradiation damages mouse retina. Mice were subjected to green laser light irradiation for 3 s. After 60 s in dark, mice were irradiated with green (A–C) or blue (D–F) laser light for 10 s. SD-OCT images of 2 representative retinas (A, D) after green/green (A) and green/blue (B) laser irradiation. Histologic sections through retinas after green/green (B) and green/blue (E) laser irradiation. Imaging of green/green (C) and green/blue (F) laser light–irradiated retinas underwent SLO imaging with 55-degree lens. Near-IR reflectance image (IR mode, 820 nm laser) was used to align fundus camera relative to pupil and thus acquire evenly illuminated fundus images. Images are displayed with superior on top, inferior on bottom, temporal on left, and nasal on right. Damaged areas in retinas are indicated by yellow dashed circles. INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments; RPE, retinal pigmented epithelium. Scale bars, 25 μm (left); 100 μm (right) (B, E).

Figure 4

DKO mice accumulate carotenoids in retina and serum. A) HPLC traces (HPLC system 2) at 460 nm (top) and 360 nm (bottom). Peak 1 displays spectral characteristics of 3,3′-di-oxo-ε,ε-carotene; peak 2 displays spectral characteristics of zeaxanthin. In addition to zeaxanthin metabolites, retina contains 11-cis- and all-trans-RAL which were converted to corresponding syn and anti ROX during extraction. B) HPLC traces (HPLC protocol 1) at 460 nm (orange trace) and 325 nm (blue trace) of DKO mouse serum detects 3,3′-di-oxo-ε,ε-carotene (peak 1), zeaxanthin (peak 2), and all-trans-retinol (peak 3). C) qPCR for SOD2 mRNA expression in retina of control and zeaxanthin-supplemented DKO mice.

Figure 5

Carotenoids shield RAL from blue laser light–induced geometric isomerization. A) Spectral characteristics of 11-cis-RAL (green), all-trans-RAL (red), and zeaxanthin (blue). Arrow indicates wavelength of blue laser light. B) All-trans-RAL solution (10 μM) was exposed to blue laser light for 2 s in presence and absence of increasing amounts of zeaxanthin as described in Materials and Methods. Blue trace, no laser light; red trace, laser light in absence of zeaxanthin; green trace, laser light in presence of 5 μM zeaxanthin; purple trace, laser light in presence of 100 μM zeaxanthin. C) Quantification of amount of all-trans-RAL (blue circles) and cis-RAL isomers (orange squares) upon blue laser light irradiation in presence of increasing amounts of zeaxanthin. Solid black circles and squares represent nonirradiated control sample.

Figure 6

Carotenoids reduce formation of aberrant visual cycle intermediates. A) HPLC traces at 360 nm of ocular retinoids of nonsupplemented (gray) and zeaxanthin-supplemented (orange) DKO mice. Geometric composition of RAL isomers is indicated. RAL was converted during extraction to corresponding oximes (syn and anti). B) Quantification of percentage composition of different RAL stereoisomers of blue laser light–irradiated eyes of nonsupplemented (control) and zeaxanthin-supplemented DKO mice after bleaching. Graph represents mean ± sd of 6 mouse eyes. Significance values (2-tailed Student’s t test) are indicated in graph.

Figure 7

Expression levels of mRNA of marker genes for light damage. All mouse eyes were irradiated with green laser light for 3 s. After 60 s in dark, nonsupplemented control mice were irradiated with green laser light (green bars). Nonsupplemented (blue bars) and zeaxanthin-supplemented (orange bars) DKO mice were irradiated with blue laser light to induce damage. Sixteen hours after light insult, RNA was isolated from retinas, and qPCR was performed. Data represent mean ± sd from 3 replicates from pooled retinas (n = 5/condition). Data are normalized to mRNA expression levels in green laser light–only treated mice. Statistical significance compared to control group was assessed by 2-tailed Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001 (threshold of significance).

Figure 8

Carotenoids ameliorate retinal damage caused by blue laser light irradiation. DKO mice were raised with and without zeaxanthin supplementation. They were then subjected to green laser light irradiation for 3 s. After 60 s in dark, mice were irradiated with blue laser light for 6 s. A, D) SD-OCT images of 2 representative retinas of nonsupplemented (A) and supplemented (D) mice. B, E) Histologic sections through retinas of nonsupplemented (B) and supplemented mice (E). C, F) Imaging of nonsupplemented (C) and supplemented (F) mice was performed with confocal SLO and 55-degree lens. Near-IR reflectance image (IR mode, 820 nm laser) was used to align fundus camera relative to pupil and thus acquire evenly illuminated fundus images. Images are displayed with superior on top, inferior on bottom, temporal on left, and nasal on right. Damaged areas in retinas are indicated by yellow dashed circles. INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments; RPE, retinal pigmented epithelium. Scale bars, 100 μm (B, E). G) Quantification of damaged area (square inch) of SD-OCT scans. H) Representative picture of 3-dimensional SD-OCT scan. Arrow indicates damaged area of retina. I) Quantification of volume of damaged area in OCT scans. Values (G, I) represent means ± sd of analysis of 5 mouse eyes per supplementation group. Statistical significance compared to group was assessed by Scheffé tests. **P < 0.01, ***P < 0.001 (threshold of significance).