Antioxidants reduce cone cell death in a model of retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is a label for a group of diseases caused by a large number of mutations that result in rod photoreceptor cell death followed by gradual death of cones. The mechanism of cone cell death is uncertain. Rods are a major source of oxygen utilization in the retina and, after rods die, the level of oxygen in the outer retina is increased. In this study, we used the rd1 mouse model of RP to test the hypothesis that cones die from oxidative damage. A mixture of antioxidants was selected to try to maximize protection against oxidative damage achievable by exogenous supplements; alpha-tocopherol (200 mg/kg), ascorbic acid (250 mg/kg), Mn(III)tetrakis (4-benzoic acid) porphyrin (10 mg/kg), and alpha-lipoic acid (100 mg/kg). Mice were treated with daily injections of the mixture or each component alone between postnatal day (P)18 and P35. Between P18 and P35, there was an increase in two biomarkers of oxidative damage, carbonyl adducts measured by ELISA and immunohistochemical staining for acrolein, in the retinas of rd1 mice. The staining for acrolein in remaining cones at P35 was eliminated in antioxidant-treated rd1 mice, confirming that the treatment markedly reduced oxidative damage in cones; this was accompanied by a 2-fold increase in cone cell density and a 50% increase in medium-wavelength cone opsin mRNA. Antioxidants also caused some preservation of cone function based upon photopic electroretinograms. These data support the hypothesis that gradual cone cell death after rod cell death in RP is due to oxidative damage, and that antioxidant therapy may provide benefit.

Fig. 1.

The change in cone cell density over time in different regions of the retina in rd1 mice in a C57BL/6 genetic background. At several time points between P21 and P180, C57BL/6 mice, either wild-type or homozygous for the rd1 mutation (n = 6 for each at each time point), had cone cell density quantified in 0.0529-mm² bins 1 mm superior, inferior, temporal, or nasal to the center of the optic nerve, as illustrated. Each point shows the mean (±SEM) calculated from measurements in six mice. In wild-type mice, there was no difference over time in cone cell density and no difference in different regions of the retina. In rd1 mice, there was a rapid decline in cone cell density between P21 and P35 and then a more gradual decline over the next 2 months. Cones were still detectable at P91 but not at P180. The rate of decline was greatest in the inferior and nasal parts of the retina and least in the superior part of the retina.

Fig. 2.

A mixture of antioxidants prevents progressive lipid peroxidation in the retinas of rd1 mice between P18 and P35. rd1 mice were killed at P18 or divided into two groups and given daily injections between P18 and P35 of vehicle or vehicle containing a mixture of four antioxidants, α-tocopherol, MnTBAP, ascorbic acid, and α-lipoic acid. Ocular sections were stained with an antibody that specifically recognizes acrolein–protein adducts (a biomarker for lipid peroxidation) and a secondary FITC-labeled antibody (column 2), rhodamine-conjugated PNA that stains cones (column 1), and Hoechst, which stains all cell nuclei (column 4; to conserve space, the outer nuclear layer is labeled cones, but there are still some rods remaining at P18). All of the sections shown in column 2 were stained at the same time, and the results were identical in two mice from each group. There was some mild staining for acrolein in P18 rd1 mouse retina (column 2, row 2), and it was increased in P35 rd1 mouse retina (column 2, row 3). There was no detectable acrolein staining in cones at P18, but there was strong staining in remaining cones at P35 (columns 2 and 3, row 3) and increased staining in the inner nuclear layer (INL). Treatment with antioxidants between P18 and P35 resulted in a marked decrease in acrolein staining in rd1 mouse retina at P35 (column 2, row 4). The cones in vehicle-treated retinas from P35 rd1 mice are yellow in merged images (column 3, row 3), indicating oxidative damage in cones, and the lack of yellow staining in antioxidant-treated P35 rd1 mice (column 3, row 4) indicates that the oxidative damage in cones was substantially reduced by the antioxidant treatment.

Fig. 3.

A mixture of antioxidants significantly reduces carbonyl adducts on proteins in the retinas of rd1 mice. rd1 mice were killed at P18 or divided into two groups and given daily injections between P18 and P30 of vehicle or vehicle containing a mixture of four antioxidants, α-tocopherol, MnTBAP, ascorbic acid, and α-lipoic acid. The retina was dissected from one eye of each mouse, and retinal lysates were assayed for carbonyl adducts by ELISA (a quantitative measure of protein oxidation), as described in Materials and Methods. The bars show the mean (±SEM) carbonyl content (nmol per mg protein of retina) calculated from five mice. There was no significant difference in carbonyl content in the retinas of P18 rd1 mice and the retinas of rd1 mice treated with vehicle between P18 and P21 or between P18 and P30. The amount of carbonyl adducts on proteins in retinas of rd1 mice treated with antioxidants was significantly less than their corresponding vehicle control at P21 and P30 (∗, P < 0.02; ∗∗, P < 0.0005 by unpaired Student’s t test).

Fig. 4.

Antioxidants promote cone survival in rd1 mice. (A) Retinal sections or flat mounts stained with PNA show cone inner segments (IS) in P21 wild-type and rd1 mice. Retinal sections are counterstained with Hoechst to show the cell layers including the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), and the outer nuclear layer (ONL). The cone IS are markedly flattened in the retinas of rd1 mice compared to wild-type mice, and the ONL is reduced to a single layer of predominantly cone cell bodies. 3D reconstruction of confocal images of a PNA-stained retina from a wild-type mouse (column 1, row 3) shows normal cone IS. In rd1 mice, the cone IS are flattened, and their arrangement is somewhat disorganized (column 2, row 3). OS, outer segment. (B) At P18, rd1 mice started receiving daily injections of vehicle or vehicle containing a mixture of four antioxidants, α-tocopherol, MnTBAP, ascorbic acid, and α-lipoic acid. Mice were killed at P21 or P35, and one eye was used for retinal flat mounts, which were stained with PNA; the other retina was dissected and used for real-time RT-PCR (Fig. 5). Confocal images of cone inner segments in 0.0529-mm² bins 1 mm superior, inferior, temporal, or nasal to the center of the optic nerve are shown. At P21, all of the images looked very similar, and so only those from the superior region of the retina of vehicle- and antioxidant-treated mice are shown. At P35, compared to vehicle-treated mice, antioxidant-treated rd1 mice appeared to have greater cone density in all four regions of the retina. (C) Quantification of cone density in each of the four 0.0529-mm² bins for each retina was done as described in Materials and Methods. Each bar represents the mean (±SEM) calculated from measurements in 10 mice. There were no significant differences between vehicle- and antioxidant-treated mice at P21, but at P35, cone density was significantly higher in antioxidant-treated mice in all four regions of the retina. ∗, P < 1.0 × 10⁻⁶; ∗∗, P < 1.0 × 10⁻¹¹; ∗∗∗, P < 1.0 × 10⁻¹² by unpaired Student’s t test for difference from corresponding vehicle control.

Fig. 5.

Antioxidants reduce loss of cone-specific mRNAs in retinas of rd1 mice. At P18, rd1 mice started receiving daily injections of vehicle or vehicle containing a mixture of four antioxidants, α-tocopherol, MnTBAP, ascorbic acid, and α-lipoic acid. Mice were killed at P21 (n = 10 for each group) or P35 (n = 10 for each group), and one eye was used to isolate total retinal RNA. Real-time RT-PCR was done using primers specific for m- or s-cone opsin mRNA. Each bar represents the mean value (±SEM) calculated from 10 mice and normalized to the value for the P21 rd1 vehicle group, which was set to 1.00. Values for P21 rd1 antioxidant-treated mice were nearly identical to the corresponding P21 vehicle group for both mRNAs. At P35, the level of m-cone opsin mRNA was significantly higher in antioxidant- compared to vehicle-treated mice (∗, P < 0.01 by unpaired Student’s t test), but there was not a statistically significant difference in s-cone opsin mRNA levels between the two groups.

Fig. 6.

Daily injections of α-tocopherol or α-lipoic acid between P18 and P35 promote cone survival in rd1 mice. Four independent experiments were done in which, starting at P18, rd1 mice were given daily injections of one of four antioxidants (n = 10 for each), and each of the four groups was compared to its own vehicle-injection group (n = 10 for each). The four antioxidants were α-tocopherol (200 mg/kg in olive oil; A), MnTBAP (10 mg/kg in PBS; B), ascorbic acid (250 mg/kg in PBS; C), and α-lipoic acid (100 mg/kg in PBS containing 30% ethanol; D). At P35, mice were killed, and the retina from one eye was flat-mounted and stained with PNA; cone density was quantified in 0.0529-mm² bins 1 mm superior, inferior, temporal, or nasal to the center of the optic nerve using confocal microscopy as described in Materials and Methods. Each bar represents the mean (±SEM) calculated from values obtained from 10 mice. Mice treated with α-tocopherol showed significantly higher cone density in three of four regions of the retina (∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 5.0 × 10⁻⁵ by unpaired Student’s t test). Compared to their respective vehicle controls run in parallel, there was no significant difference in cone density in any region of the retina in mice treated with MnTBAP or ascorbic acid. Mice treated with α-lipoic acid had significantly higher cone densities in three of four regions of the retina compared to corresponding vehicle control mice (†, P < 0.05; ††, P < 0.01, by unpaired Student’s t test). The values for the α-lipoic acid vehicle control group were lower than those for the other three vehicle control groups, suggesting there may have been a deleterious effect for the PBS containing 30% ethanol vehicle.

Fig. 7.

Antioxidants provide partial preservation of cone function in rd1 mice. rd1 mice were given daily injections between P18 and P27 of vehicle or vehicle containing a mixture of four antioxidants, α-tocopherol, MnTBAP, ascorbic acid, and α-lipoic acid. Photopic ERGs were done at P27, as described in Materials and Methods, with investigator masked with respect to treatment group. Representative wave forms are shown for the antioxidant (A) and vehicle groups (B), and measurements showed that mean b-wave amplitudes were significantly higher in the antioxidant group by unpaired Student’s t test.