Suppression of Light-Induced Oxidative Stress in the Retina by Mitochondria-Targeted Antioxidant

Abstract

Light-induced oxidation of lipids and proteins provokes retinal injuries and results in progression of degenerative retinal diseases, such as, for instance, iatrogenic photic maculopathies. Having accumulated over years retinal injuries contribute to development of age-related macular degeneration (AMD). Antioxidant treatment is regarded as a promising approach to protecting the retina from light damage and AMD. Here, we examine oxidative processes induced in rabbit retina by excessive light illumination with or without premedication using mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl)decyltriphenyl-phosphonium). The retinal extracts obtained from animals euthanized within 1⁻7 days post exposure were analyzed for H₂O₂, malondialdehyde (MDA), total antioxidant activity (AOA), and activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) using colorimetric and luminescence assays. Oxidation of visual arrestin was monitored by immunoblotting. The light exposure induced lipid peroxidation and H₂O₂ accumulation in the retinal cells. Unexpectedly, it prominently upregulated AOA in retinal extracts although SOD and GPx activities were compromised. These alterations were accompanied by accumulation of disulfide dimers of arrestin revealing oxidative stress in the photoreceptors. Premedication of the eyes with SkQ1 accelerated normalization of H₂O₂ levels and redox-status of lipids and proteins, contemporarily enhancing AOA and, likely, sustaining normal activity of GPx. Thus, SkQ1 protects the retina from light-induced oxidative stress and could be employed to suppress oxidative damage of proteins and lipids contributing to AMD.

Keywords: SkQ1; age-related macular degeneration; antioxidant activity; disulfide dimerization of proteins; glutathione peroxidase; light-induced retinal damage; mitochondria-targeted antioxidant; oxidative stress; superoxide dismutase; visual arrestin.

Figure 1

Normalized outer nuclear layer (ONL) thickness in the illuminated retinas of rabbits with or without premedication with 7.5 μM SkQ1. ONL thickness in the intact retinas is taken as 100%. Each bar represents the data obtained from six animals (12 eyes). * p ≤ 0.05 compared with the values measured in the intact animals; ** p ≤ 0.05 compared with the values measured in the control animals.

Figure 2

Malondialdehyde (MDA) concentration in homogenates of intact and illuminated rabbit retinas with or without premedication with 7.5 μM SkQ1. MDA concentration was determined by thiobarbituric acid assay. Each bar represents the data obtained from six animals (12 eyes). * p ≤ 0.05 compared with the values measured in the control animals.

Figure 3

Hydrogen peroxide content in extracts of intact and illuminated rabbit retinas with or without premedication with 7.5 μM SkQ1. Hydrogen peroxide concentration was measured by Fe²⁺/o-dianisidine/xylenol orange colorimetric assay. Each bar represents the data obtained from six animals (12 eyes). * p ≤ 0.05 compared with the values measured in the control animals.

Figure 4

Total antioxidant activity (AOA) in extracts of intact and illuminated rabbit retinas with or without premedication with 7.5 μM SkQ1. AOA was determined by hemoglobin/H₂O₂/luminol assay. Each bar represents the data obtained from six animals (12 eyes). * p ≤ 0.05 compared with the values measured in the control animals.

Figure 5

Antioxidant enzyme activity in extracts of intact and illuminated rabbit retinas with or without premedication with 7.5 μM SkQ1. (a) Superoxide oxidase (SOD) activity; (b) glutathione peroxidase (GPx) activity. Enzyme activities were determined using standard colorimetric assays as described in the Materials and Methods section. Each bar represents the data obtained from six animals (12 eyes). p ≤ 0.05 compared with the values measured in the control animals.

Figure 6

Monitoring of visual arrestin forms in extracts of intact and illuminated rabbit retinas with or without premedication with 7.5 μM SkQ1. (a) Western blotting of retinal extracts (normalized by total protein content) under non-reducing conditions using anti-visual arrestin antibodies; (b) weight fractions of disulfide dimers of arrestin determined from the Western blotting data. Each bar represents the data obtained from three animals (6 eyes). * p ≤ 0.05 compared with the values measured in the control animals.