Treatment with 11-cis-6mr-retinal protects photoreceptor cells from light-induced degeneration. Abca4^−/−Rdh8^−/− albino mice on a C57B6/J and 129SV background were treated with 11-cis-6mr-retinal (20 mg/kg b.wt.) or DMSO vehicle 30 minutes before bright-light exposure for 60 minutes and then kept in the dark. On day 3 after light exposure, TPM imaging was performed in intact mouse eyes immediately after enucleation to quantify enlarged photoreceptors and assess the effect of 11-cis-6mr-retinal. (A) Three-dimensional views of the photoreceptor-RPE interface. Enlarged photoreceptors are visible as fluorescent pillars with a diameter >2 µm (the diameter of unaffected photoreceptors was ∼1.2 µm). The center of the RPE layer was set at z = 0 µm, and the section through the photoreceptor cell layer is shown 10 µm below. (B) Representative, large-field images of the retina and RPE are shown. The RPE is visible at the edges of the images as the curvature of the intact eyeball brings it in focus. (C) Quantification of the enlarged photoreceptors. The number of enlarged photoreceptors caused by illumination with bright light was significantly decreased (P = 0.02) in mice treated with 11-cis-6mr-retinal before light exposure. Error bars represent S.D. Statistical significance was calculated with the Student’s t test, unpaired two-tailed.

Effect of 11-cis-6mr-retinal pretreatment on visual function in Abca4^−/−Rdh8^−/− mice exposed to bright light. (A) Single-flash ERG responses to increasing light intensity obtained under dark conditions in mice either unexposed to light or exposed to bright light after treatment with either DMSO or 11-cis-6mr-retinal at a dose of 20 mg/kg b.wt. (B) ERG responses compared between treatment groups revealed significant protective effects in 11-cis-6mr-retinal-treated mice before acute light illumination as compared with DMSO-treated mice in both scotopic a- and b-waves and in photopic b-waves (C). ERG measurements were carried out in 10 mice per group.

11-cis-6mr-retinal prevents bright light-induced retinal degeneration in WT mice. BALB/c mice were injected intraperitoneally with 11-cis-6mr-retinal (20 mg/kg b.wt.) 30 minutes before exposure to white light at 12,000 lux for 1 hour and then kept in the dark for 7 days before examination of retinal morphology and function. (A) Representative OCT images show substantial protection of the ONL in mice pretreated with 11-cis-6mr-retinal compared with DMSO-treated control mice. Asterisk indicates severely disrupted photoreceptor structures in DMSO-treated control mice. Scale bar, 50 µm. (B) Quantification of the ONL thickness in mice unexposed to light or in mice treated with either DMSO vehicle or 11-cis-6mr-retinal before exposure to bright light in five mice per each treatment group. Error bars indicate standard deviation. Statistical significance was calculated with one-way ANOVA and post hoc Student’s t test, unpaired two-tailed. (C) Representative SLO images show AF spots in the retina of mice unexposed to light or exposed to bright light after pretreatment either with DMSO vehicle or 11-cis-6mr-retinal. Scale bar, 50 µm. (D) Quantification of AF spots in five mice per treatment group. Error bars indicate standard deviations. Changes in the number of AF spots after treatment with 11-cis-6mr-retinal compared with DMSO-treated group were statistically significant (P < 0.001). No significant difference was observed between mice unexposed to light and those treated with 11-cis-6mr-retinal (P = NS). Statistical significance was calculated with one-way ANOVA and post hoc Student’s t test, unpaired two-tailed. (E) Examination of gross retinal morphology after H&E staining of paraffin sections from eyes collected from WT mice either unexposed to light or exposed to bright light after indicated treatments. Asterisk indicates severely disrupted photoreceptor structures in DMSO-treated control mice. Scale bar, 50 µm. (F) Examination of retinal morphology by IHC in cryosections prepared from eyes of BALB/c mice either unexposed to light or exposed to bright light after indicated treatments. Sections were stained with an anti-rhodopsin C terminus specific antibody (red) that indicates the structural organization of rod photoreceptors. PNA staining (green) denotes the health of cone photoreceptors, and DAPI staining (blue) reveals the nuclei. Asterisk indicates severely disrupted photoreceptor structures in DMSO-treated control mice. Scale bar, 50 µm. (G) Visual function determined by ERG responses. ERG recordings revealed significant protective effects in 11-cis-6mr-retinal-treated mice before acute-light illumination as compared with DMSO-treated mice in both scotopic a- and b-waves and in photopic b-waves. ERG measurements were carried out in five mice per group.

Effect of 11-cis-6mr-retinal on the retinoid cycle. WT C57BL/6J mice were injected intraperitoneally with DMSO vehicle, 11-cis-6mr-retinal at a dose of 20 mg/kg b.wt., or retinylamine [a potent inhibitor of the visual cycle (Golczak et al., 2005)] at a dose of 4 mg/kg b.wt., 30 minutes before illumination with white light at 2000 lux for 5 minutes. Scotopic ERG responses were recorded immediately after exposure to light and every 5 minutes thereafter for a total of 60 minutes. Increasing amplitudes of both a-waves (A) and b-waves (B) after light illumination in mice treated either with DMSO or 11-cis-6mr-retinal were noted but were much slower in mice treated with retinylamine. Five mice per group were used for these ERG measurements. Error bars represent standard deviation.

Effect of 11-cis-6mr-retinal on photoreceptor generation of ROS in Abca4^−/−Rdh8^−/− mice exposed to bright light. (A) Dark-adapted Abca4^−/−Rdh8^−/− mice were injected intraperitoneally with the ROS probe DHE 1 day after illumination with 10,000 lux white light for 30 minutes. These mice were treated with either DMSO or 11-cis-6mr-retinal (20 mg/kg b.wt.) 30 minutes before light exposure. Mice unexposed to bright light also were treated with the DHE probe. Eyes were harvested 1 hour later, fixed, and processed for cryosectioning. The ROS signal detected on sections from different experimental groups, was obtained with a fluorescence microscope. DAPI staining was used to visualize nuclei and the retinal layers. Scale bar, 50 µm. (B) Quantification of fluorescence intensity in arbitrary units obtained from various regions of the photoreceptor cell layers (mean ± standard deviation). Changes in fluorescence intensity observed in the photoreceptor layers after treatment with 11-cis-6mr-retinal compared with the DMSO-treated group were statistically significant (P < 0.001). No significant difference was observed between mice unexposed to light and those treated with 11-cis-6mr-retinal (P = NS). Statistical significance was calculated with the one-way ANOVA and post hoc Student’s t test, unpaired two-tailed.

Detection of 11-cis-6mr-retinal-bound pigment in mouse eyes. 11-cis-6mr-retinal (20 mg/kg b.wt.) was administered to Abca4^−/−Rdh8^−/− or WT mice 30 minute before their exposure to bright light for 30 minutes. After illumination, mice were kept in the dark. This procedure was repeated three times. On the 4th day (24 hours after the third treatment), eyes were harvested, and pigment was purified by 1D4 anti-rhodopsin immunoaffinity chromatography. Additionally, Lrat^−/− mice were injected intraperitoneally with 11-cis-6mr-retinal for three consecutive days, and 24 hours after the last injection, eyes were collected and used for pigment purification. UV-visible spectra of the rhodopsin or rhodopsin/11-cis-6mr-retinal-bound opsin mixture purified from Abca4^−/−Rdh8^−/− mice (A), WT mice (B), or Lrat^−/− mice (C) that were either untreated (black spectrum) or treated with 11-cis-6mr-retinal (green spectrum), left panel. Difference spectra were obtained by subtracting the UV-visible spectrum of the light-illuminated sample from the spectrum recorded in the dark; right panel.