Profound Effect of Light on Cysts in X-Linked Retinoschisis

Abstract

Purpose

X-linked retinoschisis (XLRS), caused by RS1 pathogenic variants, leads to macular dystrophy. Patients with XLRS show diurnal changes in optical coherence tomography (OCT), with more schisis in the morning. We studied diurnal variation in Rs1-knockout (KO) mice retinal structure and electrical function.

Methods

Rs1-KO mice 2.5 to 4 months old (MO) had electroretinogram (ERG), OCT, and intraocular pressure (IOP) measurements collected at 5 AM and 5 PM on different days and under different experimental conditions. Mice were maintained under standard 12-hour light/dark cycle, reversed 12-hour light/dark cycle, continuous light, or continuous darkness. At study endpoint, eyes were collected and fixed for immunohistochemistry or harvested for Western blot analysis.

Results

Extended light exposure resolved cysts completely and improved ERG b-wave amplitudes, whereas darkness worsened schisis and ERG function. Synaptic staining confirmed disrupted photoreceptor–bipolar connections in dark-exposed retinas and reorganization after light exposure, without changes in synaptic protein expression or rhodopsin localization. IOP still followed a diurnal pattern under constant light or dark, whereas cyst fluctuation correlated with lighting rather than time of day.

Conclusions

Initial findings suggested a diurnal rhythm in cyst size but reversed light cycle experiments showed that light exposure—not time of day—drives retinal changes in Rs1-KO mice. RS1-deficient retinas are vulnerable to darkness, whereas light exposure preserves retinal structure and function. To ensure valid OCT and ERG comparisons in XLRS, measurements should be time- and lighting-stamped. Dark-adapted conditions may best reveal treatment effects. Controlled light exposure may be a therapeutic option for patients with XLRS.

X-linked retinoschisis (XLRS) is a hereditary retinal disorder caused by mutations in the RS1 gene which encodes retinoschisin-1 (RS1), a protein essential for maintaining retinal synaptic integrity and structural cohesion.^1–5 In the absence of RS1, retinal architecture is compromised, leading to schisis, or cystic separation of retinal layers. In humans, XLRS primarily affects the macula and results in progressive vision loss.^6,7 Retinoschisin knockout (Rs1-KO) mice recapitulate the human disease, making them a valuable model for studying disease mechanisms and potential interventions.^8–10

Currently, there is no approved treatment for XLRS. However, various approaches have been explored to mitigate macular schisis, including the use of topical and oral carbonic anhydrase inhibitors (CAIs).¹¹ Reports have shown inconsistent responses, and spontaneous resolution of cysts has also been documented in untreated eyes.¹²

A few reports in the literature suggest that diurnal variations play a role in cyst fluctuations, necessitating a deeper understanding of the underlying mechanisms.¹³ Studies have documented greater schisis severity in the morning with progressive reduction throughout the day.^13–15 These studies have primarily focused on retinal structure, using optical coherence tomography (OCT) and did not report corresponding electroretinogram (ERG) findings. Interestingly, a small case series of patients with XLRS found that sleeping in an illuminated room can lead to reduced macular thickness compared to sleeping in darkness.¹⁵ A study in Rs1-KO mice demonstrated that rearing light conditions influenced the development of retinal cavities and post-photoreceptor function.¹⁶ Given that the light/dark cycle plays a significant role in retinal homeostasis,¹⁷ investigating its impact on XLRS pathology could offer new therapeutic insights. To date, no study has systematically examined the effects of diurnal variation and light exposure on retinal structure and function in an XLRS mouse model.

This study investigates underlying diurnal and environmental effects on retinal structure, function, and synaptic integrity in Rs1-KO mice. Using a combination of OCT imaging, ERG recordings, intraocular pressure (IOP) measurement and histological analysis, we aim to elucidate the relationship among light exposure, retinal cyst dynamics, and functional outcomes. By integrating structural and functional assessments, this study seeks to address the knowledge gap regarding how diurnal regulation and light exposure affect the retina in XLRS, providing a foundation for potential therapeutic strategies targeting light-mediated retinal homeostasis.

Materials and Methods

Study Design

Rs1-KO Mice

Rs1-KO mice aged 2.5 to 4 months old (MO), the period when cyst severity and schisis are most advanced,¹⁰ were evaluated with ERG, OCT, and IOP measurements collected at 5 AM and/or 5 PM under different experimental light and timing conditions to assess structural and functional changes in the retina. Mice were evaluated under various conditions, including standard 12-hour light/dark cycles, reversed 12-hour light/dark cycles, prolonged light or dark exposure, and varying durations of dark adaptation. OCT images were analyzed to measure the cross-sectional cyst area and retinal thickness. Additionally, mouse eyes were collected and fixed for immunohistochemistry, whereas retinal tissue was harvested for Western blot analysis.

To investigate the potential impact of anesthesia, a group of 5 Rs1-KO mice were anesthetized twice daily with ketamine/xylazine (intraperitoneal 87.5 mg/kg ketamine and 12.5 mg/kg xylazine) for ERG and OCT at 5 AM and 5 PM on the same day, and again a week later with testing in the opposite order, starting at 5 PM and then at 5 AM. After an initial round of testing, the same mice were again tested at either 5 PM or 5 AM, then given a 3 to 5-day washout period to allow the ketamine/xylazine effect to wear off before the corresponding 5 AM or 5 PM repeat testing. This experiment aimed to determine whether the observed changes in retinal structure and function were influenced by ketamine/xylazine anesthesia.

A separate group of 5 mice was placed in a reversed 12-hour light/dark cycle for 14 days, that is, they were exposed to 12 hours of light overnight and 12 hours of darkness during the day. ERG and OCT testing were conducted on day 6 and day 14 to assess whether structural and functional retinal changes were driven by time of day or by a different factor.

The role of prolonged dark adaptation was examined by placing a group of 10 mice in total darkness for 24 hours. ERG and OCT testing were performed at 5 AM. After 3 more days in continuous darkness ERG and OCT were performed at 5 PM. This allowed the same mice to have morning and evening testing without having two doses of ketamine in the same day. To further explore a dose-dependent effect of light adaptation, another group of 3 mice was light-adapted for decreasing durations with 3 to 5-day washout periods between each test. The light adaptation durations tested were 11 hours and 35 minutes, 10 hours, 8 hours, 6 hours, 3 hours, and 0 hours of light. OCT and ERG measurements were taken at each time point to assess the progression of structural and functional changes with increasing darkness duration.

To confirm the effect of light exposure on the retinal phenotype, a group of 5 Rs1-KO mice was placed in a constant light environment in a room illuminated by a fluorescent lamp (Sylvania OCTRON XP 32W 4100K USA F032/841/XP/5003, 45–4660 lux; Supplementary Fig. S1), the lamp exhibits a non-continuous spectral power distribution with different peaks at approximately 435 nm (blue region), 545 nm (green region), and 610 nm (orange-red region). The strongest emission occurs around the green wavelength. This pattern is consistent with the typical emission spectra of fluorescent light sources, which are designed to optimize energy efficiency while covering key regions of the visible spectrum. Two cohorts of mice were placed in the illuminated room for 14 days with no dark period. ERG and OCT tests were performed on day 6 and day 14 with one cohort tested at 5 AM and a littermate cohort tested at 5 PM of the same day to determine whether prolonged light exposure could prevent or reverse retinal cyst formation and improve retinal function.

This experimental design allowed for a comprehensive assessment of the impact of ketamine/xylazine, diurnal variation, and light or dark exposure on Rs1-KO mice, helping to elucidate the underlying mechanisms driving retinal structural and functional changes.

Animal Ethics Statement

This study adhered to the guidelines outlined in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. All animal procedures were conducted in full compliance with the approved Institutional Animal Care and Use Committee (IACUC) protocol #4031421 at the University of Iowa. The Rs1-KO mouse model was generously supplied by Paul Sieving, MD, PhD, at the National Eye Institute. Detailed characterization of this model has been described elsewhere.⁸ Animals were housed in accordance with IACUC guidelines. The mouse colonies were generated by mating Rs1-KO male mice with either Rs1-KO or heterozygous female mice. Both hemizygous male and homozygous Rs1-KO female mice were utilized as affected mice and wildtype (WT) male mice, or heterozygous (WT/HET) female mice were used as controls. Euthanasia was performed through carbon dioxide inhalation, followed by cervical dislocation. All measures were taken to minimize animal suffering.

Genotyping

Genotyping was conducted using Taq Polymerase (New England Biolabs, Ipswich, MA, USA) with the primers as described previously.^9,10

Statistical Analysis

Statistical analysis was conducted using GraphPad PRISM version 10.2.3 (GraphPad Software, Inc., San Diego, CA, USA). Paired t-tests were used to compare data from two time points within one cohort of mice. One-way ANOVA was used to compare more than two time points or variation between cohorts and testing conditions followed by multiple comparisons (Tukey's test). Simple linear regression was used for correlation of cyst area and ERG amplitudes. A mixed effects model was used to identify the relationships among light exposure, ketamine exposure, and cyst area within a cohort of mice. This was performed with the lme4 package on RStudio software.¹⁸

Electroretinography

The ERG was obtained using the Celeris system from Diagnosys (Diagnosys LLC, Lowell, MA, USA). Prior to testing the mice received a dark adaptation period ranging from 25 minutes to 24 hours depending on the experiment. A dim red light was used during testing to maintain dark adaptation. Mice were anesthetized via an intraperitoneal (IP) injection of a ketamine/xylazine mixture (87.5 mg/kg ketamine and 12.5 mg/kg xylazine.) A 1% tropicamide solution was applied to each eye for dilation prior to testing, followed by applying a (2.5%) hypromellose solution as an eye lubricant to maintain corneal moisture. An impedance below 10 kΩ was maintained for each test. A modified International Society for Clinical Electrophysiology of Vision (ISCEV) protocol was utilized,¹⁹ as described in our previous work.¹⁰

Optical Coherence Tomography

The OCT was obtained and quantified using Bioptigen InVivoVue software (Leica Biosystems Inc., Buffalo Grove, IL, USA), as previously described.¹⁰

Cystic regions within the retina were quantified using manual segmentation in Adobe Photoshop. For each OCT image, retinal cysts were identified visually based on hypo-reflective (dark) spaces within the retinal layers. The borders of each cyst were then carefully outlined using the magnetic lasso tool, and the enclosed area was measured in pixels using the measurement log feature. These pixel values were subsequently converted to square millimeters (mm²) using the scale bar provided in each OCT image.

Total retinal thickness was also measured manually using Photoshop. OCT images were captured from multiple regions of the retina—including central, superior, inferior, and peripheral (side) scans—to ensure comprehensive coverage. This approach allowed us to assess the entire retina rather than focusing on a single region, reducing the risk of overlooking cyst formation or resolution in any specific area. Representative images were obtained under 2 light exposure conditions: 24 hours of light (Supplementary Fig. S3a) and 24 hours of darkness (Supplementary Fig. S3b)

Intraocular Pressure

The IOP was measured with the Tonolab iCare Tonometer (Colonial Medical Supply Company, Inc., Windham, NH, USA). IOP was measured immediately following anesthesia via IP injection of a ketamine/xylazine mixture (87.5 mg/kg ketamine and 12.5 mg/kg xylazine). The average of six readings was recorded for each eye.

Western Blotting

Mice were euthanized by CO₂ asphyxiation and confirmed with cervical dislocation. Mouse eyes were collected via enucleation, and micro-dissecting scissors were used to remove the anterior chamber and lens. Retinas were separated and isolated, snap-frozen in liquid nitrogen, and stored at −80°C until Western blotting, as described previously.⁹ Proteins were detected by primary antibodies: PSD-95 recombinant rabbit monoclonal antibody (sr38-09; Thermo Fisher Scientific, dilution 1:1000). Ribeye antibody A-domain-192 103 (SYSY antibodies, dilution 1:1000). β-Actin antibody monoclonal IgG sc-47778 (Santa Cruz Biotechnology, dilution 1:1000), mGluR6 Rabbit IgG RA13105 (Neuromics, dilution 1:1000), and Rhodopsin polyclonal antibody PA1-729 (Thermo Fisher Scientific, dilution 1:1000). Secondary antibodies were added following the primaries including: IRDye 800CW goat anti-rabbit IgG and 680RD goat anti-mouse IgG, dilutions 1:10000 (LI-COR). Images were taken with LI-COR Licor Odyssey CLx 9140 Imaging System Unit2 Pred DLx—AV.

Immunohistochemistry

Immunostaining and imaging techniques have been previously described¹⁰ and images were captured using a THUNDER Imager Leica DM6B microscope equipped with a Leica DFC9000 GT camera.

Results

Diurnal Variations in Cyst Size and Dark-Adapted ERG Amplitudes in Rs1-KO Mice (Standard 12-Hour Light/Dark Cycle)

WT/HET controls, and male and female Rs1-KO mice aged 2.5 to 4 MO were dark-adapted (DA) overnight under a standard 12-hour light/dark cycle, and OCT and ERG were performed at 2 time points: early morning (5 AM) following 12 hours of DA, and late afternoon (5 PM) following 12 hours of light exposure Mice were housed in a standard mouse facility overnight where the lights were turned off and the room was completely dark starting at 5 PM except for a shaded window on the door that is exposed to a hallway light. At 5 AM, the mice were in standard laboratory lighting emitted by OCTRON 841XP bulbs. Initially, we performed the 5 AM OCT in darkness except for a dim red bulb to preserve the 12 hour overnight dark adaptation. We then performed an experiment in which mice had OCT imaging in darkness with a dim red bulb, then again after 10 minutes in ambient light. There was no difference in cyst size or central macular thickness (CMT) after this brief light exposure, therefore all subsequent OCTs after dark exposure were done following 10 minutes of room light.

WT/HET controls displayed normal retinal architecture with well-defined retinal layers at both time points, with no observable structural changes (Fig. 1A). In contrast, Rs1-KO mice exhibited significant retinal schisis, with large cystic cavities and disruptions between the outer nuclear layer (ONL) and inner nuclear layer (INL) at the 5 AM time point (Fig. 1B). By 5 PM, repeat testing of the same mice showed almost complete resolution of the cystic cavities seen earlier in the day, indicating a diurnal variation in retinal structure (see Fig. 1B). Statistical analysis confirmed a significant difference in cyst size between Rs1-KO mice examined at 5 AM (following overnight DA) and those examined at 5 PM (following daytime light), with P < 0.0001 (Fig. 1C). There was no statistical change in the retinal thickness of the WT/HET retinas at the two different time points (P = 0.99; Fig. 1D). However, Rs1-KO mice tested at 5 AM had statistically significant retinal thickness compared to the Rs1-KO group tested at the 5 PM time point (P = 0.007; see Fig. 1D).

Figure 1.

Diurnal variations in cyst sizes and dark-adapted ERG amplitudes in Rs1-KO mice (standard 12-hour light/dark cycle) . (A) Representative OCT images of HET female mice at 5 AM and 5 PM showing normal retinal architecture with no structural differences between timepoints. (B) Representative OCT images of Rs1-KO mice showed prominent retinal schisis with large cystic cavities at 5 AM (after 12 hours overnight dark adaptation), which largely resolved by 5 PM (after 12 hours of light adaptation). (C) Quantification of cyst area showing a significant reduction in Rs1-KO mice cysts between 5 AM and 5 PM. (D) Retinal thickness measurements showing no significant difference between WT/HET mice at different time points, but Rs1-KO mice exhibited significantly reduced retinal thickness at 5 PM compared to 5 AM. Central macular thickness (CMT) was significantly thicker in Rs1-KO retinas than WT/HET at 5 AM after 12 hour DA, however, Rs1-KO CMT was not thicker than WT/HET at 5 PM after 12 hours of light exposure. (E) Representative 0.01 DA ERG waveforms from WT mice recorded at 5 AM and 5 PM showed no functional differences. (F) The 0.01 b-wave ERG amplitudes between WT/HET mice at 5 AM and 5 PM showing no statistical difference. (G) Representative waveforms of 0.01 DA ERG at 5 AM and 5 PM timepoints in Rs1-KO mice. (H) Rs1-KO mice displayed significantly lower 0.01 DA ERG b-wave amplitudes at 5 AM, which improved at 5 PM. (I) Cyst area was assessed in the same three Rs1-KO mice across 2 consecutive 12-hour light/dark cycles on August 11, 2024, and again on August 20, 2024. OCT imaging was performed at 5 AM following 12 hours of dark adaptation (DA) and at 5 PM following 12 hours of light exposure on each day. Each color represents a different mouse. Rs1-KO, retinoschisin-1 knockout; WT, wild-type; HET, heterozygous mice; µV, Microvolt, *P < 0.05, **P < 0.01, and ****P < 0.0001, ns, not significant.

ERGs were recorded at 5 AM (after 12 hours of DA) and 5 PM (after 12 hours of light exposure) in the same mice on the same day to assess rod photoreceptor function. Representative 0.01 DA ERG waveforms from a WT mouse recorded at 5 AM and 5 PM are shown in Figure 1E. WT/HET mice tested at 5 AM and 5 PM displayed no significant changes in retinal function (Fig. 1F). Rs1-KO mice tested at 5 AM displayed significantly lower b-wave amplitudes in the 0.01 DA ERG test (63.72 ± 6.2 µV; Figs. 1G, 1H). However, mice tested at 5 PM on the same day exhibited a marked increase in b-wave amplitudes (131.7 ± 10.5 µV, P = 0.012; see Figs. 1G, 1H), suggesting that functional improvements coincide with structural cyst resolution. We followed the same animals for multiple 12-hour light/dark cycles to determine if this pattern persisted. As shown in Figure 1I, 3 different mice were followed through 4 consecutive 12-hour light/dark cycles. OCT imaging was performed starting at 5 AM after 12 hours of DA overnight and then 5 PM after 12 hours in light during the day. We repeated the same imaging schedule a week later at the same time points. We consistently observed the same pattern: large cysts were present in the morning, whereas minimal or no cysts were detected in the evening.

Figure 3.

Structural and functional retinal changes in Rs1-KO mice are not dependent on time of day (reversed light cycle) . (A) Representative OCT images of Rs1-KO mice after 6 days on a reversed light/dark cycle (12 hours of light overnight and 12 hours of dark during the day). (B) Quantification of cyst area in Rs1-KO mice after 6 days in the reversed light cycle showing significantly fewer cysts at 5 AM after light exposure compared to 5 PM after DA. (C, D) Corresponding 0.01 DA ERG recordings revealed significantly higher b-wave amplitudes in mice tested at 5 AM after light exposure compared to 5 PM after DA. (E, F) After 14 days on the reversed cycle, representative OCT images E again showed significantly fewer cysts at 5 AM after light exposure compared to 5 PM after DA F. (G, H) ERG recordings at 14 days confirmed higher b-wave amplitudes at 5 AM after light exposure versus 5 PM after DA. *P < 0.05, **P < 0.01, and ***P < 0.001.

Together, these findings indicate that Rs1-KO retinas undergo significant diurnal fluctuations in both structure and function, with cyst resolution and improved rod bipolar cell response occurring after 12 hours in the light (at the 5 PM time point). However, additional contributing factors may also play a role in this effect and will be explored in the following experiments.

Effect of Ketamine/Xylazine Double Dosing on Cyst Size in Rs1-KO Mice

After observing structural and functional differences in Rs1-KO mice across different time points, we sought to determine whether these changes were driven by time-of-day effects (diurnal rhythm), anesthesia exposure, light exposure, or a combination of factors. Because the same mice were anesthetized twice daily for ERG and OCT testing in the initial experiments above—once in the morning at 5 AM and once in the evening at 5 PM—we investigated whether ketamine/xylazine, our anesthesia agents, contributed to the observed retinal differences (Fig. 2).

Figure 2.

Effect of ketamine double dosing on cyst size in Rs1-KO mice. This figure shows data for all mice tested in this experiment. (A1-2) Representative OCT images of Rs1-KO mice and comparisons after a standard 5 AM (after 12 hours of dark exposure) and 5 PM (after 12 hours of light exposure), with ketamine/xylazine administered prior to each time point 3 and 4. A week later, the anesthesia order was reversed: mice were anesthetized first at 5 PM (after light exposure) and then at 5 AM (after overnight dark exposure). Time point 5 and 6, after a 3 to 5-day washout period to eliminate cumulative ketamine/xylazine effects. DA, dark exposure. (B-D) Statistical analysis of effects and interaction of exposure to ketamine/xylazine 12 hours prior to OCT testing versus no ketamine exposure 12 hours prior to OCT. B Scatter plot shows the effect of prior ketamine/xylazine exposure, with cyst area significantly reduced in mice that received ketamine/xylazine 12 hours prior to the experiment. C Scatter plot shows the effect of light exposure, with cyst area significantly smaller after 12 hours of light compared with darkness. D Scatter plot shows the interaction between ketamine/xylazine and light exposure; both factors reduce cyst area independently, and no significant interaction is observed.

As expected, large cysts were observed in the morning after receiving the first dose of ketamine/xylazine (without ketamine/xylazine anesthesia in the 12 hours prior; see Fig. 2A.1), whereas cysts were substantially reduced by the evening after receiving a second dose of ketamine/xylazine (ketamine/xylazine anesthesia within 12 hours prior) within the same day (see Fig. 2A.2).

One week later, we reversed the order of testing. In Figure 2A.3, the mice received the first dose of ketamine/xylazine at 5 PM, after spending the day in light, and underwent OCT. They were then placed in the dark for 12 hours overnight and received a second dose of ketamine/xylazine at 5 AM the following morning for imaging (see Fig. 2A.4). In this case, cysts found in the evening at time point 3 were larger than the cysts found in the evening at time point 2, when mice were imaged at the same time of day but had received previous ketamine/xylazine anesthesia within the prior 12 hours. Similarly, cysts found at the 5 AM time point 4 were smaller than those found at time point 1, which is without ketamine/xylazine anesthesia in the 12 hours prior. These results suggest that receiving previous ketamine/xylazine anesthesia within a 12-hour window has the effect of reducing cyst size in the retina. Remarkably, cysts were reduced in the morning despite being in the dark after prior ketamine/xylazine anesthesia at time point 4.

Importantly, the evening light-exposed cyst size without ketamine/xylazine anesthesia in the 12 hours prior at time point 3 was larger than the evening light-exposed cyst size with previous ketamine/xylazine exposure at time point 2, and the morning dark-exposed cyst size after previous ketamine/xylazine exposure at time point 4 was smaller than the morning cyst size after a single dose at time point 1, suggesting that ketamine/xylazine exposure contributes to cyst resolution.

To remove the confounding effect of ketamine/xylazine anesthesia, we performed OCT imaging on the same mice at 5 AM the following week (after overnight dark adaptation) and then included a washout period of 3 to 5 days between ketamine/xylazine exposures. After this washout, mice underwent OCT imaging at 5 PM (after light exposure during the day) without receiving ketamine/xylazine twice in the same day (time points 5 and 6). Cyst areas at time point 5 were roughly equivalent to those at time point 1. Cysts were larger in the morning after dark exposure at time point 5 and smaller in the evening after light exposure at time point 6. Notably, the evening cyst size after a washout period without ketamine/xylazine at time point 6 was larger than the evening cyst size after previous ketamine/xylazine treatment at time point 2 but smaller than the evening cyst size after a single dose at time point 3.

In a linear mixed-effects model, both previous ketamine/xylazine administration and 12-hour light exposure were associated with substantially smaller cysts (see Figs. 2B, 2C). Relative to mice with no prior ketamine/xylazine exposure, animals that received ketamine/xylazine 12 hours prior showed a 0.025 ± 0.006 mm² decrease in the cyst area (P = 4.83 × 10⁻⁵; see Fig. 2B). Similarly, exposure to 12 hours of light led to a 0.016 ± 0.004 mm² reduction in the cyst area compared with darkness (P = 3.46 × 10⁻⁵; see Fig. 2C).

These findings suggest that, when the confounding effect of prior ketamine administration is mitigated, cysts are consistently larger in the morning (after dark exposure) compared to in the evening (after light exposure). Although ketamine/xylazine contributes to cyst resolution in Rs1-KO mice—a finding that warrants further study—it is not the sole driver of this effect. Both light exposure and prior ketamine/xylazine treatment independently produced significant reductions in the cyst area.

The interaction between ketamine/xylazine treatment and light exposure was not significant (P = 0.216; see Fig. 2D), indicating that the magnitude of the effect of light on the cyst area did not differ depending on whether ketamine was administered, and vice versa. These statistical patterns were reflected in the data visualizations: both prior ketamine/xylazine and light exposure independently shifted the distributions toward smaller cyst areas, whereas the interaction plot showed almost parallel slopes across treatment groups, consistent with additive rather than synergistic effects.

Structural and Functional Retinal Changes in Rs1-KO Mice are not Dependent on Time of Day (Reversed Light Cycle)

To explore whether diurnal effects were circadian, we conducted a reversed light cycle experiment. This allows us to isolate time of day from different illumination conditions. Mice were exposed to 12 hours of light overnight and 12 hours of DA during the day. Mice were acclimated to this reversed light cycle for 2 weeks, with OCT and ERG testing conducted after 6 days and 14 days.

After 6 days on the reversed cycle, OCT imaging revealed that Rs1-KO mice tested at 5 AM—following 12 hours of light—had significantly fewer cystic cavities compared with those tested at 5 PM—following 12 hours of DA (and after the washout period, P < 0.0001; Figs. 3A, 3B). Similarly, ERG recordings showed that mice tested at 5 AM after light exposure had significantly higher b-wave amplitudes in the 0.01 DA ERG test compared with those tested at 5 PM after DA (P = 0.0105; Figs. 3C, 3D). We also investigated the 3.0 Standard Combined Response (SCR), which is another scotopic test to see if it shows a similar pattern. SCR b-wave amplitudes in Rs1-KO mice were also significantly higher after 12 hours of light, P = 0.0012. These results indicate that the structural and functional changes observed in Rs1-KO mice are not dependent on the time of day (morning versus evening) but rather are linked to lighting conditions.

To determine whether this effect persisted over time, we extended the reversed light cycle for a full 14 days and repeated the testing. Once again, we observed statistically significant differences between the 5 AM and 5 PM groups. Mice tested at 5 AM after light exposure had significantly fewer cyst cavities (P = 0.0043; Figs. 3E, 3F) and exhibited higher b-wave amplitudes in the 0.01 DA ERG test (P = 0.0325; Figs. 3G, 3H) compared with those tested at 5 PM after DA. Thus, the effect is not transient but remains stable with prolonged exposure to the altered cycle.

Impact of Prolonged Dark Adaptation on Cyst Size and Dark-Adapted ERG Amplitudes

To determine the effect of prolonged darkness on the retinal phenotype of Rs1-KO mice, we conducted an experiment in which mice were dark-adapted overnight and then also throughout the entire following day. Mice underwent ERG and OCT recordings in the morning after 12 hours of overnight DA, then OCT and ERG testing were performed. After 3 to 5 days’ washout, mice were DA again overnight and for an additional 12 hours during the day (total of 24 hours of darkness) and were then tested in the evening.

The results demonstrated a striking effect of extended darkness: at the 5 PM time point, OCT revealed significantly larger cyst cavities compared to the morning (Figs. 4A, 4B, P = 0.012). ERG recordings also showed a trend toward reduced b-wave amplitudes in the 0.01 DA test, although this difference did not reach statistical significance (Figs. 4C, 4D, P = 0.61). These findings confirm that light exposure—rather than just time of day or ketamine/xylazine anesthesia—plays a critical role.

Figure 4.

Impact of prolonged dark adaptation on cyst size and dark-adapted ERG amplitudes . Dark adaptation during the day, deviating from the normal light cycle. (A, B) Representative OCT images of Rs1-KO mice dark-adapted for 12 hours (5 AM) and 24 hours (5 PM) A revealed significantly larger cyst cavities at 5 PM compared to 5 AM after 24 hours of darkness (B). (C, D) ERG recordings showed reduced b-wave amplitudes after prolonged dark adaptation C, although the difference was not statistically significant D. (E) Quantification of cyst size after varying light adaptation durations (11 hours and 35 minutes, 10, 8, 6, and 0 hours) showed a light dose-dependent decrease in cyst cavity size, with significant enlargement by 0 hours LA (F). Corresponding 0.01 DA ERG recordings revealed progressively reduced b-wave amplitudes with shorter periods of light exposure. ns, not significant.

We next investigated whether the duration of light exposure had a dose-dependent effect on retinal changes. Rs1-KO mice were dark-adapted overnight in the standard 12-hour light/dark cycle and then were exposed to light for varying lengths of time—11 hours and 35 minutes, 10 hours, 8 hours, 6 hours, 3 hours, and 0 hours—with anesthesia washout periods between each testing session, before undergoing OCT and ERG.

Cyst cavities progressively increased in size with shorter light exposure periods, starting from minimal cyst presence at 11 hours and 35 minutes of light exposure (0.0005 ± 0.0004 mm²) and reaching significantly larger cavities at 0 hours of light (0.04 ± 0.001 mm²; Fig. 4E). Similarly, functional deficits in b-wave amplitudes became more pronounced with shorter light exposure durations (Fig. 4F), reinforcing the link between amount of light exposure and worsening retinal pathology in Rs1-KO mice.

Taken together, these findings confirm that prolonged darkness exacerbates both structural and functional impairments in Rs1-KO mice. These results provide strong evidence that cyst sizes vary according to light exposure, rather than time of day.

Extended Light Exposure Confirms Light's Protective Role in Rs1-KO Mice

Having established that prolonged darkness exacerbates retinal abnormalities in Rs1-KO mice, we next sought to determine whether extended light exposure could have the opposite effect. To test this hypothesis, we placed 2.5 to 4 MO Rs1-KO mice in continuous light exposure with standard fluorescent overhead lighting (see Supplementary Fig. S1). ERG and OCT were assessed at 2 time points: after 6 days and after 14 days of constant light.

After 6 days of continuous light, the effects were striking. OCT imaging revealed that the cysts had completely disappeared at both the 5 AM and 5 PM time points (Fig. 5A). The b-wave amplitudes in the 0.01 DA ERG test were markedly elevated compared to previous experiments, with amplitudes reaching 210.4 ± 13.98 µV at 5 AM and 169.8 ± 11.03 µV at 5 PM (Figs. 5B, 5C), which is significantly different than 0.01 DA ERG amplitudes of Rs1-KO mice that are tested after 12 hours of darkness (P < 0.0001). SCR b-wave amplitudes showed the same pattern as the 0.01 DA ERG b-waves — an increase after light exposure.

Figure 5.

Extended light exposure confirms light's protective role in Rs1-KO mice. (A) Representative OCT imaging after 6 days of constant light (45–4000 lux) showed complete resolution of cyst cavities in Rs1-KO mice at both 5 AM and 5 PM time points. (B, C) ERG recordings revealed significantly elevated 0.01 DA b-wave amplitudes after 6 days of continuous light exposure at both 5 AM and 5 PM time points. (D) Rs1-KO retinas after 14 days of continuous light, OCT confirmed sustained absence of cyst cavities in the OCT images. (E, F) ERG responses remained high. (G) Exposure to dim red light (670 nm, 0–9.2 lux) for 12 hours did not reduce cyst size, as OCT imaging showed no significant difference compared to dark-adapted mice. ns, not significant.

When we compared 24 hours of light exposure to 24 hours of darkness, the SCR b-wave amplitudes were significantly higher in the light-exposed group than in the dark-exposed group (P = 0.002; Supplementary Fig. S4a), and the a-wave amplitudes were also significantly higher (P = 0.03; Supplementary Fig. S4b). The b/a-wave ratio is particularly relevant in XLRS, as a healthy retina exhibits a b-wave larger than the a-wave (ratio >1), whereas an “electronegative” waveform—where the b-wave is reduced relative to the a-wave (ratio <1)—is characteristic of disease. In our study, the b/a ratio was significantly higher in the light-exposed group compared with the dark-exposed group (P = 0.02; Supplementary Fig. S4c), suggesting that light exposure enhances overall retinal function and may partially normalize the waveform pattern in Rs1-KO mice. To assess whether this effect was sustained over time, we tested the mice again after 14 days of constant light exposure. The protective effect persisted. OCT confirmed the continued absence of cysts (Fig. 5D), and ERG recordings showed that b-wave amplitudes remained high (5 AM = 206.5 ± 12.97 µV, 5 PM = 210.7 ± 11.88 µV; Figs. 5E, 5F).

We compared ERG waveforms from WT mice under standard conditions to those from Rs1-KO mice exposed to light for 14 days. Representative traces from both groups appeared similar (Supplementary Fig. S5a); however, quantitative analysis across multiple animals revealed that WT responses under standard conditions remained significantly higher than those of Rs1-KO mice following 14 days of light exposure (Supplementary Fig. S5b). To assess whether wavelength of light plays a role in cyst resolution, Rs1-KO mice were exposed to a red light from an ERG illuminator (670 nm, 9.2 lux measured just outside the wire cage) for 12 hours during the day following typical overnight dark-adaptation. OCT performed at 5 PM following red-light exposure showed no significant reduction in cyst size (0.029 ± 0.008 mm², P = 0.94), suggesting that 670 nm red light with an intensity of 9.2 lux does not contribute to cyst resolution in Rs1-KO retinas (Fig. 5G). The 670 nm dim red light is commonly used for visibility while performing DA ERG testing as it does not appreciably bleach the rods.

These findings provide compelling evidence that light exposure plays a critical protective role in Rs1-KO mice. Not only does light prevent the formation of cysts, but it also enhances retinal function in the rod-driven 0.01 DA ERG test. This suggests that the retinoschisis observed in Rs1-KO mice is not simply a result of their genetic mutation but are highly influenced by environmental lighting conditions. By removing darkness entirely, we were able to eliminate structural abnormalities and significantly improve retinal function in the dark.

Cone ERG Does Not Change Based on Light Exposure

During the 6-day reversed light cycle, the light-adapted (LA) tests — including the 3.0 flash and 5 hertz (Hz) flicker, which assess cone function — showed no significant difference in Rs1-KO mice between morning and evening measurements. After 12 hours of light exposure, at 5 AM, the 3.0 flash amplitude was 22.47 ± 4.54 µV and the 5 Hz flicker amplitude was 22.11 ± 3.39 µV, compared to at 5 PM after 12 hours of DA, the 3.0 flash amplitude was 14.90 ± 2.62 µV, P = 0.92 and the 5 Hz flicker was 14.59 ± 2.04 µV, P = 0.83; Supplementary Figs. S2a, S2b). Following 6 days of constant light exposure, the 3.0 flash and 5 Hz flicker tests showed no change in the amplitudes (Supplementary Figs. S2c, S2d).

LA ERG amplitudes measured after 24 hours of darkness or after 24 hours of constant light demonstrated no significant differences between these conditions for either the 3.0 flash (P = 0.09; Supplementary Fig. S2e) or the 5 Hz flicker (P = 0.1; Supplementary Fig. S2f). Together, these results reinforce that the functional changes observed under DA conditions are primarily rod mediated.

Functional and Structural Correlation in Rs1-KO Mice (ERG, OCT, and Synaptic Changes)

A crucial aspect of our study was establishing a novel correlation between retinal structure and function in Rs1-KO mice. Given the dynamic changes in cyst size and ERG amplitudes observed under different lighting conditions, we sought to determine whether a direct relationship exists between these structural abnormalities and retinal electrical function measured by ERG. To explore this, we performed a correlation analysis between cyst areas measured via OCT and the b-wave amplitudes recorded in the 0.01, 3.0 SCR DA and 3.0 flash and 5 HZ flicker LA ERG tests. The testing was done at 5 AM after 12 hours of dark adaptation.

A strong inverse correlation between cyst size and retinal function was present. OCT images showed a clear distinction between mice with large cyst cavities and those with minimal or no cysts (Fig. 6A). Corresponding ERG waveforms demonstrated that mice with larger cysts exhibited significantly reduced b-wave amplitudes specifically in the 0.01 DA ERG test (black waveform; Fig. 6B), whereas those with smaller cysts had higher amplitudes (pink waveform; see Fig. 6B). Our quantitative analysis confirmed this relationship for the 0.01 DA test with a R² value of 0.71 (P < 0.0001) and for the 3.0 SCR test with R²: 0.54, P = 0.001, indicating a strong correlation between cyst severity and functional impairment (Fig. 6C). We also found an inverse correlation between LA amplitudes and cyst severity (3.0 flash: R² = 0.48, P = 0.001, 5 Hz flicker: R² = 0.54, P = 0.001; Fig. 6D). These results suggest that cyst resolution is directly linked to improved retinal function, reinforcing the idea that structural integrity plays a vital role in maintaining normal visual signaling in Rs1-KO mice. When the eye is exposed to light, the photoreceptor hyperpolarize and generate the a-wave, then photoreceptors synapse with ON bipolar cells which in turn depolarize and generate the b-wave. Schisis cysts disrupt the connection between the photoreceptor and bipolar cells.

Figure 6.

Functional and structural correlation in Rs1-KO mice (ERG, OCT, and synaptic changes) . (A) Representative OCT images showing retinal structure in Rs1-KO mice with large cysts versus minimal or absent cysts. (B) Corresponding ERG waveforms demonstrating that eyes with large cyst cavities exhibit lower 0.01 DA b-wave amplitudes (black waveform) compared with eyes with minimal cysts that show higher amplitudes (pink waveform). (C) Quantitative analysis showing a strong inverse correlation between cyst area and 0.01 DA b-wave amplitude (R² = 0.71, P < 0.0001), and 3.0 SCR DA b-wave amplitude (R² = 0.54, P = 0.0016) indicating that larger cysts are associated with worse retinal function. (D) Quantitative analysis showing an inverse correlation between cyst area and 3.0 flash LA b-wave amplitude (R² = 0.48, P = 0.001), and 5 Hz flicker amplitudes (R² = 0.54, P = 0.0016) indicating that larger cysts are associated with worse retinal function. (E) Immunohistochemical staining of synaptic markers PSD95 (photoreceptor presynaptic terminals) and PKCα (bipolar cell postsynaptic markers) in WT retinal sections reveals normal, organized synaptic layering. (F) In Rs1-KO mice retinal sections with large cysts, significant disruption of synaptic architecture is evident, with bipolar cells stretched across cystic spaces. (G) In Rs1-KO mice with cyst resolution, retinal sections show signs of synaptic reorganization, with bipolar cells re-establishing connections to photoreceptor terminals. DA, dark-adaptation.

To investigate the impact of cysts at the synaptic level, we performed immunohistochemical staining of key synaptic proteins. We used PSD95, a presynaptic marker that labels the synaptic terminals of photoreceptors, and PKCα, a postsynaptic marker that labels bipolar cells. In WT retinal sections, the synaptic architecture appeared well-organized, with well-defined retinal laminations (Fig. 6E). However, in Rs1-KO retinal sections with large cysts, there was evident disruption of the synaptic connections between photoreceptors and bipolar cells, with bipolar cells stretched across the cystic spaces (Fig. 6F). This suggests that cyst formation physically distorts and misaligns the normal synaptic organization, leading to impaired signal transmission and lower ERG amplitudes.

Interestingly, in Rs1-KO mice that initially had large cysts but later exhibited cyst resolution, we observed a closer approximation of bipolar cell and photoreceptor markers (Fig. 6G), suggesting partial structural reorganization within the outer plexiform layer. This structural change correlated with functional improvement, as ERG recordings from these same eyes showed higher b-wave amplitudes following cyst resolution. Thus, retinal function in Rs1-KO mice appears closely tied to structural integrity, although the underlying mechanisms may include changes in extracellular signaling or ionic concentrations rather than synaptic plasticity.

Together, these results establish a strong link among cyst formation, synaptic organization, and retinal function in Rs1-KO mice.

Light Does Not Change Synaptic Protein Expression or Rhodopsin Localization in Rs1-KO Retina

Previous studies on Rs1-KO retinas have shown that synaptic organization is initially normal but by P21, synaptic proteins, such as PSD95 and mGluR6, become mislocalized, and by 4 months, their expression levels decline significantly.²⁰ This decline in synaptic protein levels parallels reductions in b-wave amplitudes.

We investigated whether the structural and functional improvements observed in Rs1-KO mice under specific conditions were associated with changes in synaptic protein expression. We performed Western blot analysis to assess the levels of key synaptic proteins, including PSD95, Ribeye, and mGluR6, under different experimental conditions (Figs. 7A, 7B). Our results showed that although PSD95 expression was reduced in Rs1-KO mice compared with WT/HET controls, levels did not differ between conditions in which cysts were present (DA) versus in those in which cysts had resolved (LA; see Fig. 7A). Similarly, Ribeye and mGluR6 expression remained unchanged between Rs1-KO and WT/HET controls and were not influenced by light exposure or cyst resolution (see Figs. 7A, 7B). These findings suggest that whereas synaptic protein expression is altered in Rs1-KO mice, the structural improvements induced by light do not restore synaptic protein levels.

Figure 7.

Light does not change synaptic protein expression or rhodopsin localization in Rs1-KO retina . (A) Western blot analysis showing reduced PSD95 expression in Rs1-KO retinas compared with WT controls, with no significant differences between dark-adapted (cystic) and light-adapted (cyst-resolved) conditions. Ribeye expression levels are comparable between Rs1-KO and WT retinas and are not influenced by lighting conditions. (B) The mGluR6 expression levels did not change compared with WT levels. Rhodopsin (RHO) levels were decreased compared to WT levels but not between DA and LA Rs1-KO mice, despite cyst resolution. (C) Immunohistochemical labeling of rhodopsin in WT retinas showing normal outer segment localization and organization. (D) Rs1-KO retinal section in dark conditions, rhodopsin is correctly localized to the outer segments but exhibits structural disorganization compared to WT retinas. (E) Light exposure does not correct the rhodopsin disorganization in Rs1-KO retinas, indicating that the functional recovery observed under light-adapted conditions is not due to changes in rhodopsin protein localization or expression.

Western blot analysis revealed that rhodopsin expression was lower in Rs1-KO mice compared with WT/HET controls, consistent with previous reports. However, its expression remained unchanged between conditions where cysts were present versus resolved (see Fig. 7B), indicating that light exposure did not alter rhodopsin protein levels. Immunolabeling further confirmed previous findings that, in Rs1-KO retinas, rhodopsin is translocated to the outer segments, albeit with a disorganized structure compared to WT retinas (Figs. 7C, 7D). Importantly, our results indicate that light exposure does not correct this disorganization (Fig. 7E).

Together, these findings suggest that although light exposure significantly improves retinal structure and function in Rs1-KO mice, these effects are not mediated by changes in synaptic protein expression or rhodopsin localization. Instead, the retinal dark cycle must require RS1 protein and, when lacking, retinoschisis results in the dark. During the light cycle, RS1 protein is not necessary for maintaining retinal structure.

Intraocular Pressure Under Different Lighting Conditions in Rs1-KO and WT/HET Mice

IOP is fluid pressure in the eye, and it exhibits a diurnal fluctuation which is influenced by circadian rhythms.²¹ It is reported that IOP measures higher in the morning and lower in the afternoon in humans.^22–27 In mice on a 12-hour light/dark cycle, IOP is higher during the dark phase (peaking at approximately 18.2 millimeters of mercury [mm Hg] at 9 PM) and lower during the light phase (trough at approximately 13.3 mm Hg at noon).²⁸ We measured IOP in 2 different lighting conditions, after 6 days of constant dark and after 6 days of constant light. As shown in Table 1, after 6 days of constant dark, the IOP measurements of the average of both eyes were lower in WT/HET during the day (light phase, 5 PM) compared to during the dark phase (5 AM) with a decrease of 6.08 mm Hg (30.6%, P = 0.2). The Rs1-KO mice showed a similar pattern of lower IOP in the light phase compared to the dark phase with a decrease of 5.67 mm Hg (28.11%, P = 0.14).

Table 1.

IOP Measurement of Rs1-KO and WT/HET Mice After 6 Days of Constant Dark

	IOP Morning at 5 AM	IOP Evening at 5 PM
WT	19.83 ± 2.57 mm Hg	13.75 ± 0.75 mm Hg
Rs1-KO	20.17 ± 4.65 mm Hg	14.50 ± 2.27 mm Hg

Table 2 shows the IOP measurements of the average of both eyes of WT/HET and Rs1-KO mice under the constant light condition. The IOP in WT/HET mice continued to be lower in the light phase (5 PM) compared to the dark phase (5 AM) with a decrease of 3.5 mm Hg (16.27%, P = 0.21). Rs1-KO mice had also lower IOP measurements in the light phase compared to dark phase with a reduction of 3.95 mm Hg (18.96%, P = 0.07). These findings indicate a consistent pattern of IOP reduction in the light phase (5 PM) in both WT/HET controls and Rs1-KO mice, which is also consistent with previous reports of humans and mice. Importantly, these results suggest that the lighting conditions do not alter the natural diurnal pattern of IOP.

Table 2.

IOP Measurement of Rs1-KO and WT/HET Mice After 6 Days of Constant Light

	IOP Morning at 5 AM	IOP Evening at 5 PM
WT	21.50 ± 1.32 mm Hg	18.00 ± 0.71 mm Hg
Rs1-KO	20.83 ± 2.75 mm Hg	16.88 ± 1.44 mm Hg

Discussion

XLRS is the most common cause of genetic macular dystrophy in male patients, so finding an effective treatment could impact many lives. In this study of the Rs1-KO mouse, we found that the diurnal variations in schisis noted in humans between morning and afternoon are recapitulated in the mouse and are amenable to controlled manipulation. We have reported previously the unexpected finding that subretinal injection of hypertonic buffer ameliorates these cysts,¹⁰ and, in this paper, we report another surprising potential treatment: light exposure.

Through carefully planned consecutive experiments we were able to demonstrate that prolonged darkness (12 hours or more) induces huge cystic cavities and reduced dark adapted ERG b-wave amplitudes in the Rs1-KO mouse, whereas exposure to at least 11 hours and 35 minutes of light from a common fluorescent bulb resolves the cysts completely and increases ERG b-waves. Our results indicate that the effect of light exposure on cyst resolution in Rs1-KO mice follows a time-dependent pattern. The decrease in cyst area becomes evident within the first few hours of light exposure and approaches a half maximal effect at approximately 6 hours. This pattern suggests that light exposure influences retinal structure in a graded manner, possibly reflecting progressive physiological adjustments in retinal cells or the extracellular environment that stabilize the tissue architecture. Thus, while practically speaking, light exposure may be considered as a treatment, the most fascinating finding is that lack of the Rs1 protein alone does not lead to schisis; darkness appears to be required.

Mice were anesthetized prior to ERG and OCT testing using ketamine/xylazine at doses appropriate for their weight, with careful monitoring throughout. We applied a second dose of ketamine/xylazine to perform the second ERG/OCT, and because we found that ketamine/xylazine itself can reduce retinal cyst size, we included a 3 to 5-day washout period between experiments to eliminate confounding effects. Studies have shown that repeated ketamine/xylazine can cause mild short-term effects on well-being, particularly in female mice, but corticosterone metabolites suggest habituation over time, with no cumulative negative impact. These precautions ensured that subsequent measurements reflected true retinal function rather than anesthetic effects.²⁹

Mammalian vision requires adaptation to a wide range of light intensity occurring over the 24-hour day.³⁰ This adaptation relies on rhythms in cellular and molecular processes orchestrated by a network of circadian clocks synchronized to the day/night cycle. These clocks regulate sleep/wake cycle, gene expression, metabolic activity, and neuronal signaling. Recent studies implicate circadian mechanisms in both retinal development and aging, further emphasizing the importance of diurnal regulation in maintaining retinal integrity.³⁰ By rearing mice in a reversed light cycle we showed that both retinal morphology and ERG responses reflected the previous 12-hour light environment rather than the time of testing. On the other hand, IOP measurements in Rs1-KO mice followed the expected circadian rhythm, with higher values at 5 AM compared to 5 PM indicating that circadian rhythm remained intact. Importantly, these fluctuations in IOP were not correlated with changes in retinal cyst formation or resolution. This supports the notion that cyst dynamics in XLRS are not driven by variations in IOP or circadian rhythm but are instead influenced by light-dependent physiological mechanisms intrinsic to the retina.

Exposure to bright light, especially short wavelength, can damage the retina through oxidative stress, photoreceptor apoptosis, and altered metabolic activity in the retinal pigment epithelium (RPE).³¹ Although these effects raise concerns about potential exacerbation of retinal pathologies with light exposure, controlled lighting, especially long wavelength red light, has been explored as a therapeutic strategy for retinal disorders such as diabetic retinopathy and retinal degenerations.^32–37 In our mice, 650 nm dim red light did not ameliorate cysts, whereas mixed wavelength white light did. We are further exploring which wavelengths of light have the most effect.

Light exposure had a protective effect on the scotopic (DA) ERG function in Rs1-KO mice; however, the LA ERG tests, including the 3.0 flash and 5 Hz flicker responses, showed no significant differences across lighting conditions. Thus cone-mediated responses are more resilient to darkness compared with rod-mediated responses in Rs1-KO mice. In the dark, the photoreceptors are under high demand due to the retinoid cycle, regenerating visual pigment (rhodopsin).^38,39 Darkness is also when transducin is concentrated in the rod outer segment (ROS), ready to trigger the phototransduction cascade as soon as light hits.⁴⁰ In Rs1-KO mice, the ROS are structurally immature and short at P21, unable to properly support transducin localization or phototransduction; it has been reported that in the Rs1-KO mouse, transducin needs higher intensity light to be translocated compared to WT.⁴⁰

Based on our findings that light reduces the cyst area and increases the 0.01 DA ERG b-wave amplitude—and given that transducin is displaced from the ROS under light exposure—we propose that photoreceptors become less dependent on an intact outer segment. As a result, the structural abnormalities in Rs1-KO rods are less detrimental under light conditions.

Because the stress on photoreceptor-RPE interaction is reduced, the absence of RS1 is less detrimental in light. Our data suggest that the functional improvements observed in light-exposed Rs1-KO mice are mediated not by molecular-level changes in protein abundance, but rather by structural realignment of the retinal architecture. Together, these results underscore a model in which light promotes functional recovery through cellular organization rather than direct biochemical modulation of synaptic components.

Our findings have important implications for the development and interpretation of therapeutic strategies in XLRS. The discovery that environmental factors such as light exposure and ketamine/xylazine anesthesia can significantly influence retinal structure and function highlights the need to carefully control and report these variables in experimental reports in both humans and murine models. For example, if the pretreatment OCT were performed at 8 AM after the patient had been in the dark and the post-treatment OCT were performed at 8 PM after spending the day in the light, the cysts may be resolved at the post-treatment time point due to light rather than the intervention, leading to a false positive result. The opposite case could obscure a treatment effect.

Further studies are needed in human patients to determine whether visual acuity follows these same patterns. Prior studies in our laboratory demonstrated that when cysts were reduced early in life in Rs1-KO mice through subretinal buffer or gene therapy, the retinas retained more cones long term.¹⁰ Thus, keeping cysts small during childhood may prolong central macular cone function if the human eye responds as the mouse eye does. Leveraging both molecular and environmental strategies may improve outcomes for patients with XLRS.