Figure 1.
Protective effect of idebenone against complex I inhibition in RGC-5 cells in vitro.
(A) Scheme of the experimental design. RGC-5 cells were pre-incubated with idebenone or vehicle (DMSO) for 1 or 2 days before the complex I inhibitor rotenone was applied to the cells for 6 hours. Cell viability was then measured after 1 day post-incubation with idebenone or vehicle. (B) Treatment with rotenone caused a 59% reduction in cell viability in vehicle-treated RGC-5 cells. (C) Idebenone pre-treatment for 1 (white bars) and 2 days (black bars) significantly rescued cell viability (expressed as % rescue). Data are expressed as mean ± SD (n = 6 wells per group). Statistical significance relative to vehicle group: p≤0.05 (*), p≤0.001 (***).
Figure 2.
NQO1 expression in RGCs and the inner segment of photoreceptors.
Distribution of NQO1 in the mouse retina co-stained with rabbit anti-NQO1 and mouse monoclonal anti-Brn3a primary antibodies. Anti-rabbit Alexa 488-conjugated and anti-mouse Alexa 495-conjugated secondary antibodies were used for fluorescent visualization of NQO1 (green) and RGCs (red) in the retina. The white rectangle in (A) depicts the region of interest shown at high magnification in (B). The different retinal cell layers are shown by nuclear staining using DAPI (blue). Scale bars represent 30 µm. (OS: outer segment, IS: inner segment, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer (incl. nerve fiber layer).
Figure 3.
Pharmacokinetic analysis of idebenone in eye fluids.
Concentrations of idebenone in aqueous humor (open circle) and vitreous humor (filled triangle) following once daily administration of idebenone in the diet for 21 days (A) and following single oral administration of idebenone at 60 mg/kg (B) were determined. Concentrations of idebenone in the eye fluids are expressed as ng/ml (left y axis) and nM (right y axis). For (A), sampling time was more than 8h after last dose of idebenone, the values therefore represent trough levels.
Figure 4.
Experimental approach to study the protective effect of idebenone in a LHON mouse model.
(A) Image of a normal retinal section showing RGCs (red) and Müller glial cell projections (green) using anti-Brn3a and anti-GFAP primary antibodies respectively. The different retinal cell layers are shown by nuclear staining using DAPI (blue). (Scale bar = 25 µm). (B) To investigate the efficacy of idebenone in protecting against complex I deficiency in the retina in vivo, we performed intravitreal injection of rotenone (15 mM) in mice pre-treated for 21 days with idebenone or vehicle in diet. Retinal ganglion cell number, retinal thickness and reactive gliosis were evaluated based on histological analysis of retinal sections 7 days after rotenone challenge.
Figure 5.
Idebenone prevents rotenone-induced RGC death. Analysis of RGCs in the mouse retina 7 days after intravitreal injection of rotenone or DMSO (Sham).
Mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-Brn3a primary antibody to visualize RGCs. Scale bar = 25 µm. (B) Quantification of RGCs (RGC number/mm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data expressed as mean ± SEM.
Figure 6.
Idebenone protects against rotenone-induced decrease in retinal thickness.
For analysis of retinal thickness 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-beta 3 tubulin primary antibodies to visualize inner and outer retinal layers. The arrows indicate retinal thickness measured from the pigment epithelium to the nerve fiber layer. Scale bar = 30 µm. (B) Quantification of total retinal thickness (µm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data are expressed as mean ± SEM.
Table 1.
Dose-effect of idebenone treatment on multiple endpoints in an in vivo mouse model for LHON.
Figure 7.
Idebenone protects against rotenone-induced gliosis.
For analysis of gliosis 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-GFAP primary antibody to visualize Müller glial cell projections (white arrows). White arrowheads indicate outer plexiform layer and also illustrate rotenone induced reduction in retinal thickness (i.e. distance between white arrow heads and ganglion cell layer at the top of the images). Scale bar = 15 µm. (B) Quantification of gliosis (GFAP signal based on relative fluorescence units, RFU) following idebenone treatment and rotenone injection (n = 3 to 7 per group). Data are expressed as mean ± SEM.
Figure 8.
Idebenone time-dependently restores rotenone-induced loss of vision.
Visual acuity was evaluated by counting the number of head movements at a velocity of 3 rpm 7 days prior injection (day -7) and 1 to 70 days after injection (day 1, 7, 21, 35, 70) of rotenone (5 mM). Mice were treated with dietary idebenone 2000 mg/kg or vehicle for the time of the experiment. (A) Schematic representation of the experimental setup. After vehicle (DMSO) injection, head movements in both directions of drum rotation (clockwise: CW; counterclockwise CCW) remain intact. Injection of rotenone into the left eye however, affects only the CW responses (dashed arrow) whereas CCW responses remain unaffected. (B) Quantification of clockwise (CW) head movement (number of head movements/2 min) following vehicle treatment and rotenone injection (vehicle + rotenone, n = 10 animals), and idebenone 2000 mg/kg treatment and rotenone injection (IDE 2000+ rotenone, n = 11 animals). Data are expressed as mean ± SEM. Statistical significance relative to vehicle + rotenone group: p≤0.05 (*); (C) Percentage of mice showing clockwise (CW) head movements for each treatment group 70 days after injection. Responder: group of mice showing head movements; Non-responder: group of mice without head movements.