Article Figures & Data
Figures
- Figure 1.
In vivo mouse model of acute light exposure-induced photoreceptor injury involves photoreceptor apoptosis, retinal atrophy, and loss of photoreceptor cell function. Young adult (2–3 months old) C57BL6J mice were dark reared for 1 week before being subjected to pupillary dilation and exposure to ambient white light at 20 × 103 lux for 2 h. The effects on retinal structure and function were evaluated at time points from 2 h to 14 d after LI. A, B, Retinal thickness and lamination was evaluated in vivo using OCT; 1.4 mm wide scan fields centered on the optic nerve were obtained. Heat maps (A) representing total retinal thickness of OCT images taken at baseline (before LI) and 7 d after LI demonstrated retinal thinning that was most marked in the superior temporal retina. Individual OCT B-scans from the superior temporal retina (B) show progressive thinning of the ONL from 3 d after LI. C–E, Histological analysis of retina in the superotemporal quadrant (1.25 mm from the optic nerve) revealed prominent emergence of apoptotic photoreceptors (as marked by TUNEL, red) in the ONL starting at 1 d after LI. Significant thinning of the ONL was observed starting at 3 d after LI. Plots in D and E show the time course of changes in the density of TUNEL-positive photoreceptors and ONL thickness after LI (column heights and error bars represent mean and SEM; n = 3–4 animals per time point). F, G, Representative ERG recordings demonstrating functional decreases at 7 d after LI relative to uninjured control mice for a- and b-wave amplitudes in dark- and light-adapted responses.
- Figure 2.
Tamoxifen administration before acute LI results in near complete rescue of structural and functional retinal damage. Adult mice were administered daily oral tamoxifen (added to standard animal chow, 500 mg/kg chow, estimated intake of 80 mg/kg body weight/d) 1 week before LI and thereafter; control animals were fed standard chow without tamoxifen at all time points. A, Total retinal thickness in the central retina (radius 600 μm around optic nerve) as measured by OCT are depicted in heat maps; control animals demonstrate extensive retinal thinning (blue areas) at 7 d after LI that was absent in tamoxifen-treated animals. B, Individual OCT B-scans in the superior retina demonstrated the following: (1) loss of outer segments, (2) thinning of the ONL layer (white box), and (3) emergence of areas of retinal detachment (white arrow); these features were absent in tamoxifen-treated animals (red box). C, Quantification of mean OCT-derived total (top row) and outer retina thickness (bottom row) in inner (pink quadrants) and outer (yellow quadrants) in the horizontal (left) and vertical (right) axes demonstrate marked thinning 7 d after LI in control animals (black lines) but no significant changes from baseline in tamoxifen-treated animals (red lines) (n = 27 eyes from 14 animals of mixed gender for each group, 2-way ANOVA). D, Prevalence of localized retinal detachments, scored at 7 d after LI, was significantly lower in tamoxifen-treated versus control groups (n = 27 eyes in each group in 3 independent experiments, χ2 statistic). E, ERG measurements of uninjured animals administered standard chow (control diet) versus oral tamoxifen for 14 d demonstrated that tamoxifen did not exert a large change on the magnitude of response amplitudes (2-way ANOVA, n = 6 animals in each group, 3 male and 3 female, 2.5–3 months old). F, ERG measurements in the LI model demonstrated that a- and b-wave amplitudes for dark- and light-adapted functional responses obtained 7 d after LI in tamoxifen-treated animals (red lines) were significantly greater than those in untreated controls (black dashed lines). a-wave amplitudes for both light- and dark-adapted responses in tamoxifen-treated animals were statistically similar to uninjured controls (p > 0.99 for both comparisons), indicating full protection. b-wave amplitudes approached those in uninjured controls, but did not reach full protection (p < 0.05 for both comparisons). Top, Data points and error bars represent mean and SEM; n = 12, 7, and 7 animals for uninjured control, untreated control 7 d after LI, and tamoxifen-treated animals 7 d after LI, respectively. Bottom, Difference in column means between the three groups, all comparisons made with two-way ANOVA, error bars indicate 95% confidence intervals.
- Figure 3.
Tamoxifen-mediated rescue of photoreceptor degeneration in LI model is not dependent on the sex of experimental animals. Young adult (2–3 months old) female and male mice were subjected to LI with and without pretreatment with tamoxifen. OCT evaluation of total retinal thickness (top) and outer retinal thickness (bottom) demonstrated that significant protection was conferred in both male and female animals treated with tamoxifen relative to sex-matched untreated controls (p-values from 2-way ANOVA, n = 17 treated and 17 untreated control female animals, 10 treated and untreated 10 control male animals).
- Figure 4.
Tamoxifen administration reduced microglial activation and infiltration induced by acute LI. A, In vivo fundus autofluorescence (FAF) imaging after LI revealed the emergence of a punctate hyperautofluorescent pattern centered in the superotemporal quandrant at the level of the subretinal space that peaked in intensity at 7 d after LI; this pattern of fundus autofluorescence was not observed in tamoxifen-treated animals across the same time points after LI. B, Immunohistochemical analysis of RPE flat mounts in control animals 14 d after LI demonstrated that the subretinal hyperautofluorescent spots (green) observed in FAF imaging in vivo and on histological analysis corresponded to Iba1-immunopositive microglia (red), indicating that microglial infiltration into the outer retina was induced by LI. C, Histological analysis of retinal sections from experimental animals after LI showed early (1 d after LI) infiltration of the ONL by Iba1-positive microglia (green) by microglia from the inner retina; this occurred concurrently with the emergence of TUNEL staining (red) in the ONL. Microglia infiltration increased at subsequent time points with additional accumulation of Iba1+ cells in the subretinal space (arrow). In comparison, microglia infiltration into the outer retina was markedly decreased by tamoxifen treatment. D, Infiltrating microglia in the outer retina showed expression of the activation marker, CD68 (red) in untreated control animals, which were largely absent in tamoxifen-treated animals. E, F, Quantitative histological analyses showing the time course of photoreceptor atrophy (as revealed by ONL thinning) (E) and photoreceptor apoptosis (as revealed by TUNEL staining) (F) in control animals, which were both markedly reduced in tamoxifen-treated animals. Morphological rescue of photoreceptors in tamoxifen-treated animals was correlated with decreases in the numbers of infiltrating microglia in the ONL (G) and subretinal space (H) and activated CD68-immunpositive microglia (I). n = 3–5 animals per time point in control and treated groups, p-values correspond to comparisons between control and treated groups, 2-way ANOVA. J, Histological analysis of retinal sections from animals 3 and 7 d after LI in control versus tamoxifen-treated groups revealed a low-level induction of GFAP immunopositivity in Müller cell processes in control animals that was absent in tamoxifen-treated animals.
- Figure 5.
Tamoxifen administration suppresses microglial activation and inflammatory cytokine production and reduces microglial toxicity to photoreceptors. A, Effect of tamoxifen (TMX) on retinal microglia activation and inflammatory cytokine expression was assessed in vitro. Microglia cultured from the retinas of young adult mice (1–2 months of age, 2.5 × 105 cells/well in a 6-well plate) were exposed to tamoxifen (0, 1, 5, and 10 μg/ml) for 2 h, followed by 1 μg/ml LPS for 16 h, and then assessed for protein expression of inflammatory cytokines. In the absence of LPS, protein levels of inflammatory cytokines in microglia cell lysates were at low basal levels and were unchanged with exposure to tamoxifen (5 μg) alone. LPS-induced expression of cytokines in microglia was significantly reduced by tamoxifen pretreatment in a dose-dependent manner (n = 3 repeats per condition, *p < 0.05 for comparisons relative to the LPS only group, 1-way ANOVA). B, Ability of tamoxifen treatment to influence inflammatory cytokine production in the retina in vivo was evaluated in the acute LI model. Inflammatory cytokines levels were assessed in retinal tissue from 2- to 3-month-old control animals (not subject to LI), light-injured controls not treated with tamoxifen (3 d after LI), and tamoxifen-treated animals (3 d after LI). Although comparisons did not reach statistical significance, multiple cytokines demonstrated increases after LI in the absence of tamoxifen that were reduced to close to baseline levels with tamoxifen treatment (n = 3–6 animals in 2 separate trials). C, Ability of tamoxifen to decrease microglia-mediated neurotoxicity was evaluated in a microglia-photoreceptor interaction model. 661W photoreceptors cultured in 96-well plate (4 × 104 cells/well) were exposed to conditioned medium from LPS-stimulated (50 ng/ml) BV2 microglia (cultured in 6-well plate with a cell density of 4 × 105 cells/well) for 48 h. Before LPS stimulation, BV2 microglia were pretreated with varying doses of tamoxifen (1, 5, or 10 μg/ml). Cell viability of 661W photoreceptors were assessed using a MTT assay. Pretreatment with tamoxifen (5 and 10 μg/ml) significantly reduced neurotoxicity of microglia-conditioned medium. *p < 0.0001, 1-way ANOVA, comparison with LPS-only control, n = 16 independent replicates. Exposure of 661W photoreceptors to vehicle (0.5% ethanol) or tamoxifen (5 μg/ml) alone had no effect on viability.
- Figure 6.
Tamoxifen administration provides structural and functional rescue of photoreceptor degeneration in rd10 mouse model for RP. rd10 mice were provided either standard chow (control group) or tamoxifen-supplemented chow from P21 and evaluated at different time points during photoreceptor degeneration. A, In vivo OCT imaging at P42 and P49 demonstrated in control rd10 animals a marked thinning of the ONL and the emergence of local retinal detachments (white arrow). Comparison of equivalent retinal areas in tamoxifen-treated rd10 animals revealed a greater preservation of ONL thickness (insets show magnified views of the ONL and retinal detachment). B, Quantification of OCT-derived outer retinal thicknesses showed significantly greater preservation of the photoreceptor layer in tamoxifen-treated animals (red lines) versus control animals (black lines) (p-values from 2-way ANOVA). C, ERG evaluation demonstrated significantly greater b-wave amplitudes in light- and dark-adapted responses at both P29 and P50 in tamoxifen-treated animals (red lines) versus control animals (black lines). p-values from 2-way ANOVA. D, Visual acuity capabilities of P49 rd10 animals were evaluated under photopic conditions by automated assessment of optomotor responses. Sinusoidal gratings, rotating in a virtual cylinder at 12°/s, were presented at different spatial frequencies to each awake and unrestrained animal tested and resulting optomotor responses were quantitated from the tracking of head movements as the ratio of the time during which head movement occurred in the same direction with stimulus movement to the time during which it occurred in the opposite direction (Tcorrect/Tincorrect). Data points (top) indicate median ratios at each grating spatial frequency, with the colored areas indicating the upper and lower quartiles of the dataset. Comparison of responses indicated that tamoxifen-treated animals had greater optomotor responses to moving stimuli relative to control animals over a range of spatial frequencies presented. Estimations of visual threshold (bottom), defined as the spatial frequency corresponding to 25% of the maximum optomotor response, significantly higher for tamoxifen-treated animals (p-value from unpaired t test with Welch correction), indicating that tamoxifen treatment resulted in a greater preservation of visual acuity function relative to controls.
- Figure 7.
Effects of agonism and antagonism of ERs and ERR on BV2 microglia-mediated neurotoxicity to 661W photoreceptors. 661W photoreceptors cultured in 96-well plate (4 × 104 cells/well) were exposed to conditioned medium from LPS-stimulated (50 ng/ml) BV2 microglia (cultured in 6-well plate with a cell density of 4 × 105 cells/well) for 24 h. Before LPS stimulation, BV2 microglia were pretreated with tamoxifen (TMX, 5 μg/ml). Cell viability of 661W photoreceptors was assessed using an MTT assay. Pretreatment with tamoxifen significantly reduced neurotoxicity of microglia-conditioned medium. Similar pretreatment with the ER agonist estradiol (E2, 100 nm and 10 μm), the ERα agonist PPT (1 μm), and the ERβ agonist DPN (10 nm) did not significant alter the neurotoxicity of microglia-conditioned media. Addition of ER agonist (E2, 10 μm) or antagonist (ERα/β antagonist, ICI, 1 μm; ERα antagonist, MPP, 1 μm; ERβ antagonist PHTPP, 1 μm), together with TMX (5 μg/ml), did not result in 661W survival levels that were significantly different from when TMX was applied alone. Pretreatment of diethylstilbestrol (DES, 1 μm), an inhibitor of ERRs, did not alter the neurotoxicity of activated microglia, nor did it significantly change the effect of TMX when applied in combination. n.s. indicates p > 0.05, 1-way ANOVA, Sidak's multiple-comparison test, n = 6 independent replicates.
- Figure 8.
Evaluation of the effects of tamoxifen on photoreceptor survival and function. A, Measurement of ERG recovery after a moderate bleaching flash in animals pretreated for oral tamoxifen for 7 d compared with age-matched control fed a standard control diet. The amplitudes of the a-wave of ERGs after bleach were recorded every 2 min at a flash intensity of 10 cd/s/m2 for the two groups of animals and mean a-wave recovery with time was plotted. Mean a-wave amplitudes recovered to baseline conditions at similar times and the dynamics of recovery were not significantly different between the 2 groups (2-way ANOVA, p = 0.493), indicating that tamoxifen pretreatment did not affect photoreceptor dark adaptation function. B, Direct effect of tamoxifen on photoreceptor survival was evaluated in vitro using a light-induced injury model. 661W photoreceptors cultured in a 96-well plate (4 × 104 cells/well) were subjected to two regimens of photic injury; the conditions were as follows: (1) control conditions (no LI), (2) under 35 × 103 lux for 90 min (severe injury), and (3) under 60 × 103 lux for 30 min (moderate injury). Cultured photoreceptors were either cultured under standard conditions or pretreated with tamoxifen (5 μg/ml for 24 h) before LI. Cell survival was measured 12 h after injury using an MTT assay. Tamoxifen pretreatment did not result in any significant improvements in photoreceptor survival relative to controls under any condition. n = indicates independent replicates, n.s. indicates p > 0.05, 1-way ANOVA, Sidak's multiple-comparisons test. C, Effect of tamoxifen pretreatment on the ability of 661W photoreceptors to resist the deleterious effects of BV2 microglia-conditioned medium was evaluated. 661W photoreceptors were cultured in the absence or presence of tamoxifen (1 and 5 μg/ml) for 24 h and then transferred into conditioned medium from LPS-treated BV2 microglia for another 48 h. Cell viability was assessed using an MTT assay and is expressed as a percentage of the control. Tamoxifen pretreatment of 661W photoreceptors did not confer protection from BV2 conditioned medium at 1 μg/ml and conversely decreased survival at 5 μg/ml. *p < 0.05, Kruskal–Wallis 1-way ANOVA with Dunn's multiple-comparisons test, n = 8 replicates in each condition. D, Direct effect of tamoxifen on the viability of 661W photoreceptors in vitro. 661W photoreceptors were cultured in the absence and presence of tamoxifen (1–15 μg/ml) for 24 h and viability of 661W photoreceptors assessed using an MTT assay. The experiment was repeated in an independent replicate and the results were pooled. n = 20 wells in 2 independent replicates, *p < 0.05, Kruskal–Wallis test with Dunn's multiple-comparisons test. Tamoxifen decreased the survival of 661W photoreceptors at a concentration of 15 μg/ml.
