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Published in final edited form as: Exp Eye Res. 2024 Dec 20;251:110221. doi: 10.1016/j.exer.2024.110221

Testosterone promotes photoreceptor degeneration in the sodium iodate model

Timothy T Lee a, Brent A Bell a, Ying Song a, Joshua L Dunaief a,b
PMCID: PMC11798707  NIHMSID: NIHMS2044427  PMID: 39710099

Abstract

Previously, we found that retinas of young male mice were more damaged than those of young female mice in the sodium iodate (NaIO3) model. The purpose of this study was to test whether reducing testosterone levels would be retina-protective. Male C57Bl/6J mice underwent surgical castration or sham surgery, then were given an intraperitoneal injection of NaIO3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Surgical castration partially protected photoreceptors, which was indicated by less photoreceptor layer thinning exhibited in OCT images compared to the sham surgery group. Consistent with this, qRT-PCR of castration group neural retinas revealed less reduction of rhodopsin mRNAs, and less upregulation of antioxidant as well as glucose transporter 1 mRNAs. ERG results also demonstrated partial preservation of both cone and rod function. These results indicate that surgical castration provided structural and functional protection to photoreceptors against NaIO3. These neuroprotective effects suggest that testosterone may be harmful to the stressed retina. Further investigation of this pathway could lead to a better understanding of the mechanisms involved in retinal degeneration.

1. Introduction

Sodium iodate (NaIO3) is an oxidizing agent that selectively damages the retina and predominantly affects the retinal pigment epithelium (RPE), followed by photoreceptor damage (Berkowitz et al., 2017; Chowers et al., 2017). Due to NaIO3’s ability to quickly induce oxidative stress to the retina and cause death of both RPE and photoreceptors within a week of injection, it has been used commonly to model features of age-related macular degeneration (AMD).

Our recent study demonstrated sex differences in the NaIO3 model, showing that male mice had more photoreceptor loss than female mice (Anderson et al., 2024). These results prompted the current investigation to determine whether endogenous testosterone potentiates photoreceptor loss or endogenous estrogen provides protection in this model.

Testosterone has been suggested to promote neurodegeneration in other models, promoting neuronal apoptosis and other neurotoxic effects (Cunningham et al., 2009; Estrada et al., 2006; Willson, 2023). Furthermore, men experience twice the risk of Parkinson’s disease development compared to women (Baldereschi et al., 2000; Cerri et al., 2019; Solla et al., 2012).1

Sex effects have also been demonstrated in retinal degeneration models, with males being more impacted than females. In a light-induced retinal degeneration mouse model, activating toll-like receptor 2 (TLR2) provided protection for both male and female mice against light damage; however, male mice were still more severely affected by the light damage (Hooper et al., 2018). Additionally, sex differences have been witnessed in the rate of RPE degeneration within a liver-specific hepcidin knockout mouse model, where more severe retinal degeneration was experienced by male mice (Baumann et al., 2019). The mechanisms of these sex differences are undetermined, but sex hormones could be a potential explanation.

The removal of testes through surgical castration leads to a significant decrease in testosterone production (Nishiyama, 2014; Valkenburg et al., 2016). Therefore, the primary purpose of this study is to test whether lowering the levels of endogenous testosterone by surgical castration of male mice can minimize the damage, both structurally and functionally, induced by NaIO3.

2. Materials and Methods

2.1. Animals

Male C57Bl/6J mice (2 months old) were acquired from Jackson Laboratory (strain number 000664; Bar Harbor, ME) and fed a standard laboratory diet. These mice were maintained on a 12:12 hours light/dark cycle. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania (Philadelphia, PA) and were performed in accordance with the ARRIVE guidelines and the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Surgical castration and sham surgeries were performed by the surgical services team at Jackson Laboratory as described: An incision was made, the testicles were visualized (and either removed or not), and the incision was closed. All other aspects of the procedure were identical between the sham and castration procedure. The surgeries were performed at 6 weeks of age. The mice were shipped at 8 weeks of age.

Surgical ovariectomy were also performed on female C57Bl/6J mice by the surgical services team at Jackson Laboratory as described: An incision was made, the ovaries were visualized (and either removed or not), and the incision was closed. All other aspects of the procedure are identical between the sham and ovariectomy procedure. The surgeries are performed at 6 weeks of age. The mice were shipped at 8 weeks of age.

2.2. NaIO3 administration

NaIO3 (Sigma-Aldrich, St. Louis, MO) was administered at a 25 mg/kg dose and injected intraperitoneally (IP) 4 weeks post-surgery. This timepoint of 4 weeks after surgical castration was used because serum testosterone was significantly reduced (to approximately 10% of normal levels) relative to intact adult C57Bl/6J mice one month following surgical castration (Davidyan et al., 2021). Administering NaIO3 4 weeks post-surgery ensured the reduction of systemic testosterone levels and provided mice ample time to recover from the stress of the surgery. Additionally, a similar timepoint was used for ovariectomized mice because the concentration of estradiol is expected to significantly decrease to around 20–30% of baseline levels two weeks following surgical ovariectomy (Luengo-Mateos et al., 2024). The dose optimization, preparation, and administration of NaIO3 were performed as described in (Anderson et al., 2024). The NaIO3 dose was adjusted based on the weight of the mice so that they were all given the same relative dose of 25 mg/kg.

2.3. In vivo Electroretinography

One week post-administration of NaIO3, electroretinography (ERG) testing of a-, b-, and c-waves was performed on a range of white light (6500k) flash stimuli. The ERG testing was conducted as described in (Lee et al., 2024).

2.4. In vivo imaging and analysis

In vivo retinal imaging was performed on mice immediately after ERG testing as described (Lee et al., 2024). Color heat maps and standard Early Treatment Diabetic Retinopathy Study (ETDRS) grids of whole retina were extracted from 1.4 × 1.4 mm Volume Scans (1000 A-scans/B-scan × 100 B-scans × 1 Frame) using Bioptigen InVivoVue Diver 2.1 auto-segmentation software (Dysli et al., 2015; Sergeys et al., 2019). Briefly, volume scans were loaded into Diver and locked within the image analysis window with the optic nerve centrally positioned. A fixed 3 × 3 grid was overlaid on the en face view highlighting 9 cardinal locations (super, super-nasal, nasal, nasal-inferior, etc.) used to manually identify various retinal lamina in preparation for automated segmentation. Once lamina training was completed at each cardinal location, the segmentation algorithm automatically provided color thickness maps showing the overall retinal thickness from the Inner Limiting Membrane to the Retinal Pigment Epithelium (ILM-RPE) across the 1.4mm2 fundus area acquired by the volume scan. Additionally, ETDRS grids were also generated showing the mean retinal thicknesses for superior, nasal, inferior and temporal quadrants with radii of 300 and 600 micrometers from the center of the en face view.

2.5. qRT-PCR

Following in vivo imaging, mice were euthanized with CO2 asphyxiation followed by cervical dislocation while they were still under general anesthesia. Neural retina tissues were isolated as described in (Anderson et al., 2024).

Gene expression changes in the neural retina were evaluated using the following TaqMan probes (Applied Biosystems, Foster City, CA): Gapdh (Mm99999915_g1), Rho (Mm00520345_m1), Opn1sw (Mm00432058_m1), Opn1mw (Mm00433560_m1), Hmox1 (Mm00516005_m1), Sod1 (Mm01700393_g1), Sod2 (Mm01313000_m1), Cat (Mm00437992_m1), Gpx4 (Mm00515041_m1), Glut1 (Mm00600697_m1), C3 (Mm01232779_m1), Iba1 (Mm00479862_g1), and Tfrc (Mm00441941_m1).

All reactions were performed and data was analyzed as described in (Anderson et al., 2024).

2.6. Statistical analysis

All statistical analyses were performed as described in (Lee et al., 2024), with the exception that Mean ± standard error of the mean (SEM) was used for all graphs rather than standard deviation. However, Mean ± standard deviation (SD) was used for the colored heat maps.

A one-way ANOVA analysis was run followed by Tukey’s multiple comparisons test for graphs describing analyses of OCT thickness and qRT-PCR data. For the ERG analysis, generalized linear models were used to compare each measurement under every light intensity among 3 groups. Both eyes were analyzed from each mouse, so a generalized estimation equation (GEE) as described in (Liang and Zeger, 1986) was used to account for the inter-eye correlation. The statistical analyses were then performed in SAS v9.4 (SAS Institute Inc., Cary, NC). Two-sided P < 0.05 was defined as statistically significant.

Approximately 10% of NaIO3 injections fail to result in any damage to RPE or photoreceptors and such outliers were identified based on the mouse’s RPE morphology in OCT images. These outliers were then removed from statistical analysis.

3. Results

To determine whether testosterone is harmful to the retina in the NaIO3 model, surgical castration was performed on mice followed by testing to determine whether a decrease in endogenous testosterone affected retinal damage.

3.1. Surgical castration leads to less photoreceptor layer thinning in the NaIO3 model.

To determine if surgical castration provides protection of photoreceptor structure, four weeks after castration or sham surgery, mice were injected with 25 mg/kg NaIO3 IP and OCT images were then taken one week post-injection. After centering OCT images on the optic nerve, retinal thickness measurements were taken. Significant retinal thinning was observed in mice injected with NaIO3 compared to the control saline injection group (Fig. 1A). Castrated mice had less thinning than those that received a sham surgery (Fig. 1B–L).

Fig. 1. SD-OCT imaging of mouse retinas.

Fig. 1.

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(A–C) Representative horizontal (OCT-H) and vertical (OCT-V) retinal OCT b-scans from mice that received (A) an intraperitoneal saline injection, (B) a sham surgery and intraperitoneal 25 mg/kg NaIO3 injection, and (C) a castration surgery and intraperitoneal 25 mg/kg NaIO3 injection. (A) The red brackets and arrowhead indicate reflective OCT lamina representing the inner nuclear layer (INL), outer nuclear layer (ONL), and Bruch’s membrane (BrM), respectively. (B–C) Red arrows indicate hypertrophic RPE cells in the superior region of OCT-V b-scans. OCT-H (yellow) and OCT-V (blue) b-scan locations are shown overlaid on Volume Intensity Projection (VIP) en face views obtained from high-density SD-OCT volume scans (D–F). Volume scans processed by InVivoVue Diver segmentation software auto-generated colored heat maps (G–I) and ETDRS grids (J–L) that represent the total retinal thickness spanning the Inner Limiting Membrane to the RPE (ILM-RPE). Colored heat maps show relative differences in the amount of retinal degeneration (Average ± SD) between “Sham + NaIO3” (H) and “Castrated + NaIO3” (I) retinas compared to the “No Surgery, No NaIO3” (G). ETDRS grids (J–L) centered on the optic nerve (ON, inner-ring) head and encircling diameters of 0.6 mm (mid-ring) and 1.2mm (outer-ring) show mean retinal thickness separated into various retinal locations including superior, nasal, temporal and inferior regions.

Since the ONL was difficult to define in NaIO3-injected mice, retinal thickness measurements were obtained from the Bruch’s membrane to the innermost inner nuclear layer (INL), adjacent to the inner plexiform layer (Fig. 1A, see top of bracket labeled “INL”). The NaIO3-injected, sham castration group had retinal thinning to 80.6 ± 1.9 μm compared to the saline injected, no surgery controls that had a retinal thickness of 141.7 ± 2.5 μm (Fig. 2A). The NaIO3-injected castration group also had some retinal thinning to 117.2 ± 2.0 μm, but not as much as the NaIO3-injected sham surgery group (Fig. 2A). Retinal thinning was reduced by 59.9% in the castrated mice compared to the sham surgery mice. Furthermore, these differences were seen throughout varying locations of the mouse retina, which is highlighted through “spider graphs” (Fig. 2B and C). For the horizontal OCT images spider graph, the retinal thickness at each specified distance from the optic nerve for each experimental group was statistically different from one another with a p-value of less than 0.001 (Supplementary Table 1). For the vertical OCT images spider graph, the retinal thickness at each specified distance from the optic nerve for each experimental group was statistically different from one another with a p-value of less than 0.001, with the exception that the no NaIO3, no surgery group was significantly different from the NaIO3-injected castration group with a p-value of 0.02 at one location (Supplementary Table 2).

Fig. 2. OCT analysis of mice protected against NaIO3 from surgical castration.

Fig. 2.

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(A–C) OCT image quantification through measurements taken from the INL/inner plexiform layer junction to Bruch’s membrane where measurements were taken every 50 pixels, which equates to 109 μm. Mean thickness ± SEM is reported with the distribution of “No Surgery, No NaIO3” (n = 5), “Sham + NaIO3” (n = 9), and “Castration + NaIO3” (n = 8). (A) A single data point per mouse was produced through the averaging of twelve measurements from each mouse’s four image quadrants (OD horizontal, OD vertical, OS horizontal, and OS vertical). (B–C) Each data point represents the mean thickness at the specified distance from the optic nerve among all mice within the designated treatment group ± SEM. (B) One data point per location per treatment group was produced by calculating the mean thickness at each specified location in the horizontal OCT images. (C) One data point per location per treatment group was produced by calculating the mean thickness at each specified location in the vertical OCT images. Error bars represent SEM. * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001).

3.2. ERG analysis shows mild protection of photoreceptor function by castration

To determine if surgical castration provides functional protection in addition to structural protection, ERG analysis was performed. NaIO3 administration led to a significant decrease of rod a and b wave, cone b wave, and c-wave responses (Fig. 3A–D). Castration resulted in significant preservation of rod a-wave responses at higher light intensities (Fig. 3A). Castration also provided functional protection of cone b-waves (Fig. 3D). Castration also resulted in a slight increase in rod-b waves, but this difference in comparison to sham surgery mice was not significant (Fig. 3B). In contrast, castration provided no protection for the RPE c-waves (Fig. 3C).

Fig. 3. ERG analysis of mice protected against NaIO3 through surgical castration.

Fig. 3.

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(A–D) ERG analysis using various light stimuli for (A) rod a-wave, (B) rod b-wave, (C) C-wave, (D) cone b-wave function. A comparison was made for each measurement at every light intensity among the three study groups. Because the mean responses of both eyes from each mouse were measured, a generalized estimation equation (GEE) was utilized in order to take into account the inter-eye correlation. Error bars represent SEM. Significance only between groups “Sham + NaIO3” and “Castration + NaIO3” is indicated * (P < 0.05), ** (P < 0.01), and *** (P < 0.001).

3.3. mRNA quantification highlights protection against NaIO3 toxicity by surgical castration

qRT-PCR was conducted in order to understand the effects of surgical castration on mRNA levels. Because we observed the RPE to be focally hypertrophic in both the “Sham + NaIO3” and “Castration + NaIO3” groups as well as no functional protection of the RPE c-waves through castration, only the neural retina RNA was tested.

There was a decline in rhodopsin (Rho) mRNA levels for both NaIO3 groups (Fig. 4A). However, the castrated mice experienced a smaller decrease in Rho, which aligns with the protection of rod photoreceptors. NaIO3 administration also led to a downregulation of short-wave cone opsin (Opn1sw) and medium-wave cone opsin (Opn1mw) mRNA levels, but castration failed to provide protection to these cone-specific genes (Fig. 4B and C).

Fig. 4. qRT-PCR analysis of neural retina samples.

Fig. 4.

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(A–L) Fold change analysis of mRNA levels in the neural retina of (A) rhodopsin [Rho], (B) opsin 1 short-wave-sensitive [Opn1sw], (C) opsin 1 medium-wave-sensitive [Opn1mw], (D) heme oxygenase 1 [Hmox1], (E) superoxide dismutase 1 [Sod1], (F) superoxide dismutase 2 [Sod2], (G) catalase [Cat], and (H) glutathione peroxidase 4 [Gpx4], (I) Glucose transporter type 1 [Glut1] (J) complement component 3 [C3], (K) ionized calcium binding adaptor molecule 1 [Iba1], and (L) transferrin receptor [Tfrc]. Mean ± SEM is reported with the distribution of “No Surgery, No NaIO3” (n = 3), “Sham + NaIO3” (n = 9), and “Castration + NaIO3” (n = 8). Error bars represent SEM. Only one eye from each mouse was used for qRT-PCR and each data point represents an individual mouse analyzed in triplicate. All statistical analyses were completed using one-way ANOVA with correction for multiple comparisons. * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001).

Both NaIO3 groups had increased mRNA levels of the antioxidant gene heme oxygenase 1 (Hmox1), but the castrated group experienced reduced upregulation (Fig. 4D). This decreased upregulation suggests that the neural retina of castrated mice experienced less oxidative stress. Administration of NaIO3 led to no significant change in mRNA levels of superoxide dismutase 1 (Sod1) but surgical castration resulted in a significant decrease relative to the sham surgery group (Fig. 4E). Downregulation of mRNA levels of this antioxidant gene that provides protection against superoxide radicals also suggests that castrated mice experienced less oxidative stress. Additionally, castration and NaIO3 administration led to a downregulation of superoxide dismutase 2 (Sod2) mRNA levels relative to the saline-injected controls, but no significant difference was seen between the sham surgery and castrated mice (Fig. 4F). There was upregulation of catalase mRNA levels (Cat) from NaIO3, but surgical castration had no influence on the upregulation of this antioxidant gene (Fig. 4G). Glutathione peroxidase 4 (Gpx4) mRNA levels were also examined and both NaIO3 administration as well as surgical castration had no impact on expression levels of this antioxidant gene (Fig. 4H). Although some of the markers of oxidative stress such as Hmox1 and Sod1 demonstrated modulation from castration, this was not seen in all of the markers.

Castrated mice also had less upregulation of the glucose importer, glucose transporter type 1 (Glut1), mRNA compared to sham surgery mice (Fig. 4I), potentially indicating less metabolic dysregulation. NaIO3 administration resulted in an increase of innate immunity gene complement factor 3 (C3) mRNA, but the castration group did not show any difference from sham (Fig. 4J). NaIO3 treatment increased the expression of ionized calcium binding adaptor molecule 1 (Iba1) mRNA, which increases with macrophage and microglial activation, but castration had no discernible effect (Fig. 4K). A decrease in mRNA levels of transferrin receptor (Tfrc) resulted from NaIO3, indicating increased iron levels, and surgical castration had no effect on altering this downregulation (Fig. 4L).

3.4. Surgical ovariectomy does not affect photoreceptor layer thinning

To determine whether estrogen may also have an effect on photoreceptor survival in the NaIO3 model, surgical ovariectomy was performed on female mice prior to NaIO3 injection. Based on OCT analysis, the ovariectomy group and the sham surgery group experienced thinning to a similar extent (Fig. 5A). No discernable difference between these two groups was seen throughout varying regions of the mouse retina, and this is indicated through “spider graphs” (Fig. 5B and C). These results demonstrate that ovary-generated estrogen had no significant effect on photoreceptor survival.

Fig. 5. OCT analysis of retina thickness in mice treated with surgical ovariectomy vs sham followed by NaIO3.

Fig. 5.

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(A–C) OCT image quantification through measurements taken from the INL/inner plexiform layer junction to Bruch’s membrane where measurements were taken every 50 pixels, which equates to 109 μm. Mean Thickness ± SEM is reported with the distribution of “Sham + NaIO3” (n = 5) and “Ovariectomy + NaIO3” (n = 5). (A) A single data point per mouse was produced through the averaging of twelve measurements from each mouse’s four image quadrants (OD horizontal, OD vertical, OS horizontal, and OS vertical). (B–C) Each data point represents the mean thickness at the specified distance from the optic nerve among all mice within the designated treatment group ± SEM. (B) One data point per location per treatment group was produced by taking the mean at each specified location in the horizontal OCT images. (C) One data point per location per treatment group was produced by taking the mean at each specified location in the vertical OCT images. For a no NaIO3 control reference, see (Fig. 2). Error bars represent SEM.

4. Discussion

In this study, surgical castration provided both structural and functional protection in the NaIO3 model, with castrated mice demonstrating partial rescue against photoreceptor thinning and experiencing neural retinal protection at the mRNA level. Furthermore, surgical castration resulted in slight preservation of both rod-a wave and cone-b wave function relative to mice that underwent sham surgery, especially at higher light intensities. This finding of rod-a wave responses being preserved by surgical castration but not rod-b wave is reminiscent of congenital stationary night blindness where there exists normal a-wave functionality and severely diminished b-wave (Kabanarou et al., 2004; Kim et al., 2022; van Genderen et al., 2009). This may result from impaired photoreceptor to bipolar cell signaling or bipolar cell defects.

Although castration provided both structural and functional protection, the structural protection seen in the neural retina was more pronounced than the functional protection. This difference may stem from the extensive damage to the RPE, an essential facilitator of the visual cycle, which is not protected by castration.

Selectivity in NaIO3’s initial site of damage may explain why castration provides some protection to photoreceptors but not to the RPE. The cells damaged first by NaIO3 are the RPE cells and this may be due to the drug having an affinity for these cells (Berkowitz et al., 2017; Chowers et al., 2017). Because NaIO3 is delivered systemically through an IP injection, the drug has easy access to the RPE through the fenestrated choriocapillaris (Strauss, 2005). The photoreceptors may then be damaged secondarily through inflammation or oxidative stress. Testosterone may increase the stress induced in photoreceptors; surgical castration helps mitigate this stress.

Additionally, compared to the sham surgery group, the castration group experienced less variation for most of the genes investigated. Similarly, the no surgery, no NaIO3 group also had less variation. It has been previously established that the NaIO3 model has some inter-mouse variation (Anderson et al., 2024). There may be less variation when there is less damage induced, as seen with the castration surgery.

The mechanism of retinal protection from surgical castration could arise from the prevention of testosterone’s activation of the androgen receptor (AR). Single-cell RNA sequencing data from the Spectacle platform, using both the “IOWA integrated retina, RPE, and choroid” and “Single-Cell RNA Sequencing in Human Retinal Degeneration Reveals Distinct Glial Cell Populations” data sets, demonstrate that rods highly express AR, while cones display no expression (Voigt et al., 2020). Stimulation of AR in the NaIO3 model may promote rod death. Our observed protection of the cone b-wave may occur because rods can increase viability of cones (Aït-Ali et al., 2015).

While reducing endogenous estrogen through ovariectomy had no impact on the damage induced by NaIO3, tamoxifen has been shown to provide photoreceptor protection in the NaIO3 model (Lee et al., 2024). This suggests endogenous levels of estrogen may not be enough to provide protection of the neural retina, but supraphysiological estrogen receptor agonism through tamoxifen administration may explain the protection. Receptor selectivity of tamoxifen could also play an important role. Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) constitute the two classes of estrogen receptors (Eyster, 2016). While estrogen activates both ERα and ERβ, tamoxifen can specifically activate ERβ (Ivanova et al., 2011). The selective activation of ERβ might promote photoreceptor neuroprotection.

Collectively, these findings indicate that sex hormone receptor modulation influences the degree of photoreceptor degeneration in the NaIO3 model. This provides a foundation for further investigation of the influence of sex hormones on photoreceptor survival.

Supplementary Material

1

Highlights.

  • Testosterone promotes structural and functional photoreceptor degeneration in NaIO3.

  • Testosterone promotes NaIO3 oxidative stress in the neural retina.

  • Ovariectomy does not affect photoreceptor degeneration in NaIO3.

Acknowledgments

The authors thank the University of Pennsylvania Libraries’ Biotech Commons for 3D printing several pieces needed for the in vivo imaging process. We also thank Gui-Shuang Ying and Yinxi Yu for reviewing and making editorial comments on our statistical methods.

Funding sources

This research was funded by the Research to Prevent Blindness, the Paul MacKall and Evanina Bell MacKall Trust, F.M. Kirby Foundation, and National Institutes of Health grants (RO1EY015240, RO1EY028916, and RO1EY036292 to J.L.D., S10OD026860 Shared Instrumentation Grant, and P30EY001583 Core Grant for Vision Research).

Footnotes

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Declaration of competing interest

None.

CRediT authorship contribution statement

Timothy T. Lee: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing. Brent A. Bell: Investigation, Methodology, Writing – review & editing. Ying Song: Methodology, Writing – review & editing. Joshua L. Dunaief: Conceptualization, Funding acquisition, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

1

Abbreviations

AMD, age-related macular degeneration; AR, androgen receptor; BrM, Bruch’s membrane; C3, complement factor 3; ERα, estrogen receptor alpha; ERβ, estrogen receptor beta; ERG, electroretinography; ETDRS, Early Treatment Diabetic Retinopathy Study; GEE, generalized estimation equation; Glut1, glucose transporter type 1; Hmox1, heme oxygenase 1; Iba1, ionized calcium binding adaptor molecule 1; ILM-RPE, inner limiting membrane to the retinal pigment epithelium; INL, inner nuclear layer; IP, intraperitoneally; NaIO3, sodium iodate; Nrf2, nuclear factor erythroid-2-related factor 2; OCT, optical coherence tomography; OCT-H, optical coherence tomography horizontal scans; OCT-V, optical coherence tomography vertical scans; ONL, outer nuclear layer; Opn1mw, medium-wave cone opsin; Opn1sw, short-wave cone opsin; qRT-PCR, quantitative real-time PCR; Rho, rhodopsin; RPE, retinal pigment epithelium; SD, standard deviation; SEM, standard error of the mean; Tfrc, transferrin receptor; TLR2, toll-like receptor 2; VIP, volume intensity projection

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