Histopathology of retina from control and NaIO₃-treated αB crystallin knockout (αB-/-) mice.

<p>Three weeks after tail vein injection of 20/kg NaIO₃, eyes were enucleated and frozen sections were stained with H&E. The four experimental groups of mice were PBS-treated WT (A), NaIO₃-treated WT (B), PBS-treated αB-/- (C), and NaIO₃-treated αB-/- (D). The RPE layer in αB-/- mice with NaIO₃ injection were discontiguous and damaged. Only two out of seven WT mice showed discontiguous and damaged RPE (E). Bar graph showing retinal thickness (µm) with and without NaIO₃ in WT and αB-/- mice. (F). Retinal thickness was significantly lower (P<0.01) in NaIO₃ treated αB-/- mice when compared to the corresponding WT group. No significant differences in the number of nuclei in the ganglion cell layer, outer nuclear layer or inner nuclear layer were found between the NaIO₃-injected and PBS-injected WT mice (G–H). The number of nuclei per unit area in the outer nuclear layer of NaIO₃ injected αB-/- mice showed a significant decrease (P<0.01) as compared to control without NaIO₃ injection (I). RPE: RPE cell layer; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Data are mean ± SEM, n = 7/group, **P<0.01. Bar equals 75 µm.</p

Caspase 3 activation with NaIO₃ treatment and increased activation with knockdown of αB crystallin siRNA in human RPE cells.

<p>Forty-eight hours after scrambled siRNA or αB crystallin siRNA transfection, human RPE cells were treated with 200 µg/ml NaIO₃ for 24 hours. Cleaved caspase 3 staining was performed in scrambled siRNA-transfected human RPE cells (A, B) and αB crystallin siRNA-transfected human RPE cells (C, D). Treatment with 200 µg/ml of NaIO₃ resulted in increased amount of cleaved caspase 3-positive cells with αB crystallin siRNA-transfection vs. scrambled siRNA controls (E). Data are mean ± SEM from three individual experiments, αB siRNA refers to αB crystallin siRNA in panels C and D. **P<0.01. Scale bar = 40 µm.</p

Increased production of reactive oxygen species (ROS) in mouse primary RPE cells and human RPE cells transfected with αB crystallin (αB-/-) siRNA after NaIO₃ treatment.

<p>Confluent mouse RPE cells from αB-/- and WT mice were treated with 200 µg/ml NaIO₃ for 24 h. In panels A–D, DAPI is shown in blue, ROS staining in green and mitotracker in red. ROS staining was observed in WT RPE cells treated with NaIO₃ (A–B). RPE cells from αB-/- mice (C–D, see white arrows) showed increased accumulation of ROS that partially colocalized with mitochondria. For studies with human RPE cells, αB crystallin was knocked down (∼80%) by siRNA transfection (Fig. 5E). Increased ROS production was observed in αB crystallin si-RNA transfected human RPE cells with NaIO₃ as compared to scrambled siRNA transfected cells which showed negligible ROS staining (I–K). (F–H). Scale bars represent 20 µm for A–D and 10 µm for F–K, respectively.</p

Occurrence of necrosis with high doses of NaIO₃ in human RPE cells.

<p>Forty-eight hours after scrambled siRNA or αB crystallin siRNA transfection, RPE cells were treated with 200, 500, 1000 µg/ml NaIO₃ for 24hours. Propidium iodide (PI) staining positive cells were determined in scrambled siRNA-transfected RPE cells (A–D) and αB crystallin siRNA-transfected human RPE cells (E–H) treated with NaIO₃. Treatment with 500 and 1000 µg/ml of NaIO₃ resulted in increased PI positive cells both in control RPE cells and αB crystallin siRNA pretreated human RPE cells (I). However, no significant differences were found between the number of PI positive cells in control RPE groups and αB crystallin siRNA-transfected group (I). Data are mean ± SEM from three individual experiments, αB siRNA refers to αB crystallin siRNA. *P<0.05. Scale bar = 40 µm.</p

Fundus photograph of control and NaIO₃-treated αB crystallin knockout mice (αB-/-).

<p>Three weeks after tail vein injection of 20/kg NaIO₃, fundus photograph was taken in PBS-treated wild type (WT) (A), NaIO₃-treated WT (B), PBS-treated αB-/- (C), and NaIO₃-treated αB-/- (D) mice. Arrows indicate sites of retinal degeneration. Thirteen NaIO₃-treated αB-/- mice showed patchy retinal degeneration three weeks after NaIO₃ injection (E). Only three out of fourteen eyes of NaIO₃-treated WT mice showed retinal degeneration (E). A statistically significant difference was found between NaIO₃-treated αB-/- and NaIO₃-treated WT mice (P<0.001).</p

H & E staining of retina showing dose dependent effect of NaIO₃ in 129S6/SvEvTac wild type (WT) mice.

<p>The intravenous NaIO₃ doses used were 20, 35, 50 and 70 mg/kg body weight. Mice were euthanized after 3 weeks of treatment, eyes were enucleated, frozen sections of retinal were obtained, and H & E staining was performed. Most mice treated with 20 mg/kg showed an intact layer of contiguous RPE although occasional mice showed focal RPE degeneration and loss at the end of 3 weeks (as shown in this figure). The higher doses showed moderate and severe damage to the RPE cell layer, and outer nuclear layer. GCL: ganglion cell layer; ONL: outer nuclear layer; INL: inner nuclear layer; IS: inner segment; OS: outer segment. Bar equals 75 µm.</p

Detection of reactive oxygen species (ROS) in the presence and absence of PPAR_γ ligand/antagonist.

<p>Increased production of ROS was observed in RPE cells transfected with αB crystallin siRNA upon NaIO₃ treatment as compared to control group. A-PAF, a PPAR_γ ligand, suppressed ROS production significantly (P<0.01) in αB crystallin siRNA transfected cells treated with NaIO₃ (A). On the other hand, GW9662, a PPAR_γ antagonist significantly increased ROS positive cells in αB crystallin siRNA transfected cells treated with NaIO₃. ROS positive cells were quantified from four random images derived from 4 individual experiments. αB siRNA refers to αB Crystallin siRNA. (B). Scale bar = 40 µm. **P<0.01.</p

Western blot analysis showing changes in expression of signaling molecules with NaIO₃.

<p>Forty-eight hours after scrambled siRNA or αB crystallin siRNA transfection, human RPE cells were treated with 200 µg/ml NaIO₃ for 24 hours. Cells were lysed and western blot was performed. (A) Phosphorylated level of AKT at serine 473 increased after the treatment of low dose (200 µg/ml) NaIO₃ (B). The increase of phospho-AKT was lower in αB crystallin siRNA-transfected RPE cells treated with NaIO₃. Similar trends were found for phospho-GSK 3β and phospho-c-Raf, and PPAR_γ and quantification is shown for PPAR_γ in (C). However, no significant changes were found in phospho-PDK1, a kinase upstream of AKT. All experiments were performed three times and representative gels are presented. Protein expression levels were measured by Bandscan software, normalized to GAPDH, and labeled under each corresponding band. The bars for group B and C for pAKT and PPAR_γ are derived from 3 individual experiments. αB siRNA refers to αB crystallin siRNA. pαB in panel A refers to phosphorylated αB crystallin. **P<0.01.</p

Reduced ERG amplitudes in NaIO₃-treated αB crystallin knockout (αB-/-) mice.

<p>Three weeks after tail vein injection of 20/kg body weight NaIO₃, mesopic ERG responses were recorded in PBS-treated WT, NaIO₃-treated WT, PBS-treated αB-/-, and NaIO₃-treated αB-/- mice (representative ERGs shown in A). The amplitudes of <i>a</i> wave of the ERG of NaIO₃-treated αB-/- mice decreased by 68.3% compared with that of PBS-treated αB-/- mice (B). The amplitudes of <i>b</i> wave of the ERG of NaIO₃-treated αB-/- mice decreased by 55.3%, compared with that of PBS-treated αB-/- mice (C). Data are mean ± SEM, n = 5/group, *P<0.05, **P<0.01.</p

Inhibition of MRP1 significantly decreases basal and apoptotic GSH efflux.

<p>GSH efflux in ARPE-19 cells incubated with two MRP1 inhibitors (75 µM MK571 and 5 mM sulfinpyrazone [SP]) for 5 h (A). ARPE-19 cells were transfected with MRP1 siRNA at the indicated concentrations. RNA and protein were extracted 48 h post transfection. MRP1 mRNA and protein expression was significantly (P<0.01 vs scrambled control) decreased when compared to control or scrambled siRNA alone (B,C). GSH release from MRP1-silenced ARPE-19 cells incubated with 150 µM H₂O₂ for 5 h in serum free medium. A significant decrease (P<0.001 vs scrambled control) in GSH release was observed in 5 h. A decrease in GSH release only in control cells with no additional change in MRP1 inhibited cells was found and H₂O₂ treatment did not cause any further change (D). LDH release measured in MRP1 inhibited cells was not affected by any treatment (E). Values are mean± SE (n = 3–4). LDH- Lactate dehydrogenase. ** Significantly different from control cells, P<0.01.</p