Percent light-saturated ERG response.

<p>The mean (±S.E.M) of the maximal photopic b-wave amplitude for non light-damaged and light-damaged eyes are shown as a percent of the maximal amplitude from untreated, non light-damaged rats (100% response). n = 6 rats. An asterisk (*) denotes a significant difference (p<0.05) when compared to the untreated, non light-damaged group. A pound sign (#) denotes a significant difference (p<0.05) when compared to the light-damaged, <b>4</b>-treated group.</p

Histological sections of the ONL.

<p>Representative microscope images taken at 1 mm inferior and superior to the ONH of H&E stained sections through the optic nerve of retinas treated with/without compounds 4 and 8. Left: Non light-damaged (−) retinas treated with/without compounds 4 and 8. Right: ONL thickness of light-damaged (+) retinas treated with/without compounds 4 and 8. The white point (background) in each image was set using Adobe Photoshop prior to analysis of ONL thickness using Image-Pro Plus software.</p

Dark-adapted (scotopic) ERG responses.

<p>Representative average maximal ERG scotopic response from untreated, control rats (A,B), 4-treated rats (C,D), and 8-treated rats (E,F). The a-wave was measured from baseline to trough, while the b-wave was measured from the trough of the a-wave to the peak of the wave.</p

Expression of thioredoxin (TRx) in the neural retina.

<p>Top: Comparison of expression of TRx normalized to levels of GAPDH in rats treated with/without compounds <b>4</b> and <b>8</b> suggest that treatment with multi-functional compounds reduce induction of TRx. Retinal protein homogenate was subjected to Western blotting with anti-TRx and anti-GAPDH antibodies. Bottom: Lanes 1, control diet, non light-damaged; Lane 2, control diet, light-damaged; Lane 3, <b>4</b>-treated, non light-damaged; Lane 4, <b>4</b>-treated, light-damaged; Lane 5, <b>8</b>-treated, non light-damaged; Lane 6, <b>8</b>-treated, light-damaged. Lanes of interest were cropped from entire western blot scan prior to be auto leveled using Adobe Photoshop and analyzed by ImageJ software. Mean ± S.D.; n = 6. An asterisk (*) denotes a significant difference (p<0.05) when compared to the untreated, non light-damaged group. A double dagger (‡) denotes a significant difference (p<0.05) when compared to the untreated, light-damaged group.</p

HPLC-MS analysis of neural retinal levels of 4 and 8.

<p>Analysis confirms accumulation of investigational compounds <b>4</b> and <b>8</b> in the neural retina following two week feeding of diet supplemented with 0.05% compounds <b>4</b> and <b>8</b>. Mean ± S.E.M.; n = 6. The pound (#) sign denotes a significant difference (p<0.05) when compared to the <b>4</b>-treated group.</p

Expression of thioredoxin reductase (TRxR) in the neural retina.

<p>Top: Comparison of expression of TRxR normalized to levels of GAPDH in rats treated with/without compounds <b>4</b> and <b>8</b> suggest that treatment with multi-functional compounds reduce induction of TRxR. Retinal protein homogenate was subjected to Western blotting with anti-TRxR and anti-GAPDH antibodies. Bottom: Lanes 1, control diet, non light-damaged; Lane 2, control diet, light-damaged; Lane 3, <b>4</b>-treated, non light-damaged; Lane 4, <b>4</b>-treated, light-damaged; Lane 5, <b>8</b>-treated, non light-damaged; Lane 6, <b>8</b>-treated, light-damaged. Lanes of interest were cropped from entire western blot scan prior to be auto leveled using Adobe Photoshop and analyzed by ImageJ software. Mean ± S.D.; n = 6. An asterisk (*) denotes a significant difference (p<0.05) when compared to the untreated, non light-damaged group. A double dagger (‡) denotes a significant difference (p<0.05) when compared to the untreated, light-damaged group.</p

Protective effects of compounds 4, 8 and Trolox against Fenton-generated ROS.

<p><b>A</b>. Protection against apoptosis detected by TUNEL staining of retinal ganglion RGC-5 cells cultured for 4 h in oxidative conditions generated by 1 mM H₂O₂ and 1 mM Fe(II). Mean ± SD, n = 4. <b>B</b>. Cell viability detected by the spectrophotometric MTS assay of 661w photoreceptor cells after 5 hr exposure to ROS generated by 1 mM H₂0₂ and 1 mM Fe(II). Mean ± SD, n = 6.</p

Properties of compounds <b>4</b> and <b>8</b><i>in vitro</i>.

<p>*Stoichiometry (Compound ∶ Metal Ion) of complexes formed in solution as determined by Job Plots <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021926#pone.0021926-Jin1" target="_blank">[23]</a>.</p>#<p>Percent viable human lens epithelial cells (HLECs) and retinal pigmented epithelium (RPE) cells after 2 hour exposure to 1 mM Fenton reagents (1 mM H₂O₂ and Fe²⁺) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021926#pone.0021926-Jin1" target="_blank">[23]</a>.</p

Percent dark-adapted ERG response.

<p>The mean (±S.E.M) of the maximal scotopic a-wave and b-wave amplitudes for non light-damaged and light-damaged eyes are shown as a percent of the maximal amplitude from untreated, non light-damaged rats (100% response). n = 6 rats. An asterisk (*) denotes a significant difference (p<0.05) when compared to the untreated, non light-damaged group.</p

Comparison of 4-hydroxynonenal (HNE)-histidine adduct levels and nitrotyrosine adduct levels in the neural retina.

<p><b>A</b>. HNE-histidine adduct levels, as determined by ELISA, in rats treated with/without compounds <b>4</b> and <b>8</b> suggests that HNE levels were reduced by treatment with multi-functional compounds. <b>B</b>. Similar conclusions were obtained with ELISA measurements of nitrotyrosine-modified proteins in rats treated with/without compounds <b>4</b> and <b>8</b>. Mean ± S.D.; n = 6. An asterisk (*) denotes a significant difference (p<0.05) when compared to the untreated, non light-damaged (NLD) group.</p