Fig. 1. A mixture of nitric oxide synthase (NOS) inhibitors prevents S-nitrosylation of cysteine thiols in proteins in the retinas of rd1 mice. Between postnatal day (P) 18 and P30, rd1 mice were given twice daily intraperitoneal injections of vehicle or vehicle containing a mixture of four NOS inhibitors, L-NNA, L-NAME, L-NMMA, and aminoguanidine. Ocular sections were stained for S-nitrosocysteine (SNO-Cys, column 1) and Hoechst, which stains all cell nuclei (column 2). In rd1 mouse retina, there was minimal staining for SNO-Cys at P18 (row 1) that was substantially increased in P30 vehicle-treated mice throughout the entire retina including the area of remaining photoreceptors (row 2). This increase was blunted by treatment with NOS inhibitors (row 3). In the absence of primary antibody, there was essentially no staining in the retinas of P30 vehicle-treated mice. Results were identical in 2 mice for each time point/condition. Scale bar = 50 μm.

Fig. 2. A mixture of NOS inhibitors reduces staining for S-nitrosocysteine (SNO-Cys) and nitrotyrosine in the retinas of rd1 mice in the presence of high levels of superoxide radicals. Ocular sections were stained for SNO-Cys or nitrotyrosine in P30 wild-type C57BL/6 mice or P30 rd1 mice in a C57BL/6 background that had been given twice daily intraperitoneal injections of PBS or PBS containing a mixture of four NOS inhibitors, L-NNA, L-NAME, L-NMMA, and aminoguanidine between P18 and P30 (n = 4 for each group) and fluorescence intensity was measured as described under Materials and methods. There was very little staining for nitrotyrosine (A) or SNO-Cys (B) in the retinas of P30 wild-type C57BL/6 mice. There was increased staining for nitrotyrosine (C, arrows) and SNO-Cys (D, arrows) in cells of the inner nuclear layer of the retinas of P30 rd1 mice that had received PBS injections. This staining was markedly reduced in P30 rd1 mice that had received injections of NOS inhibitors between P18 and P30 (E and F). Staining of sections from the same mouse shown in (C) with anti-nitrotyrosine antibody that had been preincubated with nitrotyrosine-BSA eliminated the staining (G). Staining of sections from the same mouse shown in (D) with anti-SNO-Cys antibody that had been preincubated with NO-cysteine-G-BSA eliminated the staining (H). Staining was quantified by measurement of fluorescence intensity with image analysis (n = 4 for each group) in the area of interest and normalized to background staining on the same slide. Each bar shows the mean (± SD) normalized intensity (I and J). The staining intensity of outer retina for sections from mice treated with NOS inhibitors was significantly less than that for sections from mice treated with PBS (P < 0.05;P < 0.001 by unpaired Student t test) confirming that the mixture of NOS inhibitors significantly reduced staining for nitrotyrosine and SNO-Cys in rd1 mouse retinas. Scale bar = 50 μm.

Fig. 3. An inhibitor of NADP(H) oxidase reduces superoxide radicals and protein nitrosylation in the retinas of rd1 mice. There was minimal fluorescence in the retinas of P30 wild-type mice after systemic injection of hydroethidine (A–C), but the retinas of P30 rd1 mice showed prominent fluorescence in the remaining outer retina (D–F). Rd1 mice treated with intraperitoneal injections of PBS between P15 and P30 also showed strong fluorescence in the retina (G–I), but rd1 mice treated with intraperitoneal injections of apocynin between P15 and P30 showed only mild fluorescence (J–L). Rd1 mice treated with intraperitoneal injections of PBS between P15 and P30 showed strong staining for nitrotyrosine in cells of the inner and remaining outer nuclear layer (M–O, arrows), but rd1 mice treated with apocynin showed mild staining (P–R). Scale bars = 50 μm.

Fig. 4. Nitric oxide synthase (NOS) inhibitors promote cone survival in rd1 mice. Starting at P18, rd1 mice received twice daily intraperitoneal injections of vehicle or vehicle containing a mixture of four NOS inhibitors, L-NNA, L-NAME, L-NMMA, and aminoguanidine. At P35, cone density was measured in 0.0529 mm² bins 1 mm superior, inferior, temporal, and nasal to the optic nerve, or the retina was used for real-time RT-PCR. Confocal images appeared to show a higher density of cone inner segments, particularly superior and temporal to the optic nerve (A). Three-dimensional reconstruction of confocal images showed preservation of cone outer segments (OS) in some NOS inhibitor-treated mice (B, bottom row), whereas all vehicle-treated mice showed small flattened inner segments and no OS (B, top row). Cone density was significantly higher in the NOS inhibitor group (n = 9) in all 4 regions of the retina (C, P < 5.0 × 10^− 6; P < 5.0 × 10^− 5; P < 5.0 × 10^− 8; P < 0.01 by unpaired Student t test for difference from corresponding vehicle control (n = 17). The mean (± SE) amount of m-cone or s-cone opsin mRNA per retina was normalized to the P35 vehicle-treated group, which was set to 1.00. The amount of m-cone opsin mRNA, but not s-cone opsin mRNA, was significantly greater in NOS inhibitor-treated mice compared to vehicle-treated mice (D, P < 0.02 by unpaired Student t test). Photopic electroretinograms done on rd1 mice treated with NOS inhibitors at P25 showed greater b-wave amplitudes than those seen in vehicle-treated mice (E). Scale bars = 50 μm for (A) and 20 μm for (B).

Fig. 5. 7-Nitroindazole, but not aminoguanidine, reduces cone cell death in rd1 mice. At P18, rd1 mice were given intraperitoneal injections twice a day of high-dose aminoguanidine (1250 mg/kg/injection in PBS, n = 10) or PBS (n = 10). Another group of P18 rd1 mice was given intraperitoneal injections twice a day of 7-nitroindazole (30 mg/kg/injection in DMSO, n = 10) or DMSO (n = 10). At P35, cone density was measured in 0.0529 mm² bins 1 mm superior, inferior, temporal, or nasal to the center of the optic nerve. Compared to PBS-treated mice, there was no significant difference in cone density in any region of the retina in mice treated with aminoguanidine (A), but cone density was significantly higher in 2 of 4 regions of the retina (P < 0.02, P < 0.05 by unpaired Student t test) in mice treated with 7-nitroindazole compared to those treated with DMSO.