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. 2026 Aug 13;15(8):9. doi: 10.1167/tvst.15.8.9

Morphological and Functional Alterations in the Mouse Retina After Sodium Fluorescein Administration

Tianchang Tao 1, Jie Zheng 1, Xian Wang 2, Lun Liu 1,✉, Lixia Feng 1,✉
PMCID: PMC13489213  PMID: 42593203

Abstract

Purpose

Sodium fluorescein (SF) is commonly used to observe retinal and choroidal vasculature for clinical or scientific purposes, but few studies focused on concentration of fluorescein, volume injected, and rate of injection in animal experiments. The aim of this study was to investigate the effect of SF on structure and function of the retina in mice.

Methods

C57BL/6J mice were injected intraperitoneally with 0.7% saline solution, 5.0% SF, and 10.0% SF, morphological, functional, and molecular changes of the retina were evaluated by fluorescein angiography, electroretinography (ERG), histology, TUNEL assay, immunohistochemistry (IHC), and Western blot.

Results

Fluorescein intensity of 10.0% SF-treated mice was higher than 5.0% SF-treated mice. ERG showed early reduction of retinal function, followed progressive restoration of ERG responses. Histology revealed irregular or disorganized features of outer retina after SF administration. TUNEL assay revealed apoptosis in the outer nuclear layer. IHC demonstrated comparable expressions of Rhodopsin, M-opsin, and S-opsin in all groups, but the 10.0% SF-treated mice exhibited the higher immunoreactivity of glial fibrillary acidic protein (GFAP). Molecular analysis showed increased expressions of BAX, NRF2, and HO1, and decreased expression of BCL2 in the retina and retinal pigment epithelial (RPE).

Conclusions

Administration of a higher dose of SF could induce some abnormal alterations in the retina, which were not easily distinguishable from pathological changes in the development of ocular diseases in animal models.

Translational Relevance

The SF-induced retinal changes that are similar with some histopathological features could affect the authenticity of experimental data, which optimizing the dose of SF should not be ignored in experimental research on mice.

Keywords: sodium fluorescein (SF), adverse reactions (ARs), retinal alterations, mouse model

Introduction

Fundus fluorescein angiography (FFA) was first reported by Novotny and Alvis in 1961,1 and has become one of the common ophthalmic examinations to investigate the vascular changes in the retina and choroid, thus contributing to diagnostic procedure and clinical management of various ocular diseases.2–5 Besides, FFA is also useful as a method to learn the anatomic, physiological, and pathological features of retinal and choroidal circulation of animal models in experimental research.6–9

Currently, sodium fluorescein (SF) is utilized to perform FFA, this agent is an orange water-soluble dye with a molecular weight of 376.3 Da, and could emit light in the wavelength of 520 to 530 nm when it absorbs light in the wavelength of 465 to 490 nm. The tight junctions between retinal vascular endothelial cells (inner blood-retinal barrier) and the tight junctions between retinal pigment epithelial (RPE) cells (outer blood-retinal barrier) prevent SF from passing, and the choriocapillaris allows the diffusion of SF. After entering the circulation, approximately 80.0% of SF binds to proteins, the remaining SF is free in the blood,10 and produces most of the fluorescence.

Previous studies proposed the relationship between the dose of SF administration and the frequency of fluorescein angiography (FA)-associated adverse reactions (ARs).11–13 Moosbrugger et al. tried to compare FA-associated ARs and image quality using 2 mL and 5 mL SF in the context of high-resolution digital angiography, and found patients who received 2 mL of 10.0% SF produced inferior quality digital images compared with those produced by patients who received 5 mL of 10.0% SF, but all ARs-related events were reported in patients with 5 mL of 10.0% SF administration,14 indicating a controversial issue between higher quality digital images of FFA and increased frequency of FA-associated ARs.

On the basis of previous findings,11–14 we were inspired to explore the potential dose-related toxic effects of SF administration on the retina of animal models. This study used two different doses of SF to perform FFA on C57BL/6J mice to compare the quality of digital angiography, and tried to learn whether the mouse retina would differentially respond to different doses of SF.

Materials and Methods

Animals

This study used C57BL/6J female mice aged 8 weeks, the mice were divided into 3 groups randomly, and were injected intraperitoneally with 0.7% saline solution, 10.0% SF, and 5.0% SF at the volume of 50 µL/20g. All mice were housed and maintained in room temperature, 55% ± 5% humidity, and a constant 12-hour light-dark cycle, with ad libitum access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Anhui Medical University (LLSC20230420) and were performed in compliance with the statement of Association for Research in Vision and Ophthalmology for the use of animals in ophthalmic and vision research.

Fundus Fluorescein Angiography

For FFA, the 10.0% SF-treated and 5.0% SF-treated mice were anesthetized by intraperitoneal phenobarbital (50 mg/kg) and xylazine (5 mg/kg), and pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine, then their eyes were adjusted to suitable position, and the FFA images of the right eyes were obtained at 1 minute after SF administration using MICRON IV retinal imaging system (Phoenix Research Labs, USA) as previously described.9,15 In addition, the fluorescein intensity was analyzed using ImageJ software to compare the color measurement for fluorescein from FFA images in two groups, as previously described.16 The FFA images were imported into ImageJ software and underwent individual color channels separation, in which red and blue channels were removed, and all areas or intensity values were measured from the green channel, then the fluorescein intensity values of the middle or lateral FFA images were measured.

The retinal vasculature in FFA images was evaluated using Angio Tool software (National Institutes of Health National Cancer Institute, Gaithersburg, MD), as previously described,8,15,17 including the percentage of the retinal vessel area, the total number of retinal vascular junctions, and the average retinal area without detectable vasculature (Mean E Lacunarity), to learn the differences of qualities in FFA images between 10.0% SF-treated and 5.0% SF-treated groups.

Electroretinography

The full-field flash electroretinography (ERG) was performed on mice before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection using an ERG recording system (RETIport, Roland Consult, Germany), as described previously.15,18 Following 4 hours of dark adaptation, mice were anesthetized, and their pupils were dilated, and 0.1% sodium hyaluronate eye drops were topically administered to keep the ocular surface moist. The reference electrodes were placed in the forehead, a needle ground electrode was placed at the tail, and the recording gold electrodes were placed directly onto the cornea. The ERG responses were recorded in a dark room under dim red lighting. In the scotopic ERG, flash intensities included 0.001, 0.003, 0.3, and 3.0 cd*s/m2; in the photopic ERG, flash intensities included 1.0, 3.0, and 10.0 cd*s/m2. Each recording was averaged three times at different luminance levels.

Histological Analysis and TUNEL Assay

Mice were euthanized at 3 days postinjection, and the eyeballs were harvested and fixed in FAS eyeball fixative solution (Servicebio, Wuhan, China) over 24 hours. Paraffin-embedded eyeballs were cut into 5-µm thick sections and stained with hematoxylin and eosin (H&E), as previously described,15,19 then, the microscopic examination was used to observe the morphological features of the mouse retina.

In addition, the thickness of the inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor layer (PRL), and RPE layer were measured at the middle of a posterior quadrant or at the middle of an anterior quadrant of the retina to represent the central or peripheral regions of the retina of 0.7% saline solution-treated, 5.0% SF-treated, and 10.0% SF-treated mice, and the number of nuclei rows in central or peripheral ONL was also measured. TUNEL assay was performed to observe the apoptotic cells in the retina, as previously described,20 nuclei were labeled with 4, 6-diamidino-2-phenylindole (DAPI) staining solution (Beyotime, Shanghai, People's Republic of China), the number of TUNEL-positive cells in the retina through the optic nerve were counted and averaged from six sections per group.

Immunohistochemistry

The eyeballs were fixed in 4% paraformaldehyde for 2 hours, and were dehydrated in 30% sucrose overnight. Optimal cutting temperature compound-embedded eyeballs were cut into 10-µm thick sections, and were blocked with 3% bovine serum albumin for 30 minutes, then, the sections were incubated with the following primary antibodies: anti-glial fibrillary acidic protein (GFAP; 1:500; Cell Signaling Technology), anti-Rhodopsin (1:500; Santa Cruz Biotechnology), anti-S-opsin (1:200; Sigma-Aldrich), anti-M-opsin (1:200; Sigma-Aldrich). After washing in PBS, sections were stained with goat anti-rabbit IgG (1:500; Abcam) or goat anti-mouse IgG (1:500; Thermo Fisher Scientific). Nuclei were labeled with DAPI staining solution (Beyotime, Shanghai, People's Republic of China). Antibodies for immunohistochemistry (IHC) are listed in Supplementary Table S1. The number of M-opsin- or S-opsin-positive cells, and the level of fluorescence density of GFAP or Rhodopsin staining in the retina through the optic nerve were measured and averaged from six sections per group.

Western Blot

Retinal tissues and RPE/choroid complexes were harvested, and RPE cells were isolated when RPE/choroid complexes were incubated in PBS without Ca2+ and Mg2+ containing 1% Dispase Ⅱ (Wako Pure Chemical, Osaka, Japan) for 30 minutes at room temperature. The retinas and RPE cells were fully lysed using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail, then the protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime, Shanghai, People's Republic of China). Samples were heated for 10 minutes at 100°C, and were subjected to gel electrophoresis (SDS-PAGE), then they were transferred onto nitrocellulose membranes (Millipore, Burlington, MA). Membranes were blocked for 1 hour at room temperature in Western Blocking Solution (Beyotime, Shanghai, People's Republic of China) and incubated overnight at 4°C with the following primary antibodies: anti-NRF2 (1:6,000; Proteintech); anti-HO1 (1:3,000; Proteintech); anti-BCL2 (1:3,000; Proteintech); anti-BAX (1:3,000; Proteintech); and anti-Beta Actin (1:20,000; Proteintech). The membranes were washed with Tris-buffered saline containing 0.1% Tween-20 and then incubated with anti-rabbit secondary antibody (1:5,000; LI-COR Biosciences) or anti-mouse secondary antibody (1:1,000; Santa Cruz Biotechnology) for 1 hour at room temperature. The protein bands were visualized using the LI-COR Odyssey CLx imaging system (LI-COR Biosciences), and were quantified using ImageJ software. Antibodies for Western blot are listed in Supplementary Table S1.

Statistical Analysis

Data are presented as mean ± SD and statistical analyses were performed using SPSS software, 1-way ANOVA with Bonferroni post hoc test was used for comparing more than two groups, and Student's t-test was used for differences between two groups of data. A P value < 0.05 was considered statistically significant.

Results

Quantitative Analysis and Comparison of In Vivo Retinal Imaging

Compared with the 5.0% SF-treated group, the middle or lateral FFA image of the right eyes from the 10.0% SF-treated group exhibited the higher green fluorescein intensity (Figs. 1A, 1B). Angio Tool software also demonstrated that there was significant difference in the quantification of the percentage of vessel area, the total number of vascular junctions, or the non-perfused area of FFA images of the right eyes from the two groups. The “vessels percentage area” and “total number of junctions” in the middle or lateral image of 10.0% SF-treated mice was statistically higher than that of 5.0% SF-treated mice (Figs. 1C, 1D), and the “Mean E Lacunarity” in the middle or lateral image of 10.0% SF-treated mice was lower than that of 5.0% SF-treated mice significantly (Fig. 1E). The data of these FFA-associated indexes from two groups are listed in Supplementary Table S2.

Figure 1.

Figure 1.

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Comparison of the quality of digital angiography of right eyes from mice (n = 3 eyeballs of 3 mice per group) receiving 5.0% and 10.0% sodium fluorescein (SF). Representative images of fundus fluorescein angiography (FFA) photographed at 1 minute after SF administration of right eyes from 5.0% SF-treated and 10.0% SF-treated groups (A). Quantification of the fluorescein intensity of FFA images from 5.0% SF-treated and 10.0% SF-treated groups (B). Quantification of the “vessels percentage area” of FFA images from 5.0% SF-treated and 10.0% SF-treated groups (C). Quantification of the “total number of junctions” of FFA images from the 5.0% SF-treated and 10.0% SF-treated groups (D). Quantification of the “Mean E Lacunarity” of FFA images from 5.0% SF-treated and 10.0% SF-treated groups (E). Data are presented as mean ± SD were compared using Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Electrophysiological Changes of Retinal Function

To investigate the retinal functional alterations after 0.7% saline solution or SF administration, we performed full-field flash ERG under dark-adapted and light-adapted conditions, and the results of scotopic 0.001 ERG, scotopic 3.0 ERG, and photopic 3.0 ERG were obtained to evaluate the rod, mixed rod-cone, and cone function in the retina of mice (Figs. 2A–C).

Figure 2.

Figure 2.

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The waveforms of scotopic and photopic electroretinography (ERG) responses of control, 5.0% sodium fluorescein (SF)-treated, and 10.0% SF-treated mice. Representative scotopic ERG recordings evoked by the flash intensity of 0.001 cd*s/m2 before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection (A). Representative scotopic ERG recordings evoked by the flash intensity of 3.0 cd*s/m2 before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection (B). Representative photopic ERG recordings evoked by the flash intensity of 3.0 cd*s/m2 before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection (C).

The scotopic and photopic ERG responses showed an obvious decline at 3 days after intraperitoneal injection of 10.0% SF (Figs. 3A–C), and a gradual restoration was observed at 6 and 9 days postinjection. Although there was a trend toward decreased amplitudes of scotopic b-wave at 0.001 cd*s/m2 through 9 days (see Fig. 3B), the amplitudes of scotopic a-wave and b-wave at 3.0 cd*s/m2 both decreased at 3 days after injection but increased at 6 and 9 days after injection of 10.0% SF (see Figs. 3A, 3B). The amplitudes of photopic b-wave at 3.0 cd*s/m2 at 3 days postinjection were reduced by approximately 50% as compared to baseline, but showed noticeable increases at 6 and 9 days after 10.0% SF administration, even the amplitudes of b-wave at 9 days postinjection were comparable with those of baseline (see Fig. 3C).

Figure 3.

Figure 3.

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Alterations in the amplitudes of scotopic and photopic electroretinography (ERG) responses of mice (n = 12 eyeballs of 6 mice per group) after 0.7% saline solution or sodium fluorescein (SF) administration. The amplitudes of scotopic a-waves, scotopic b-waves, and photopic b-waves at 0.001, 0.003, 0.3, and 3.0 cd*s/m2 before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection (A-C). The amplitudes of scotopic b-waves at 0.001 cd*s/m2 of control, 5.0% SF-treated, and 10.0% SF-treated mice before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection (D). The amplitudes of scotopic a-waves at 3.0 cd*s/m2 of control, 5.0% SF-treated, and 10.0% SF-treated mice before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection (E). The amplitudes of scotopic b-waves at 3.0 cd*s/m2 of control, 5.0% SF-treated, and 10.0% SF-treated mice before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection (F). The amplitudes of photopic b-waves at 3.0 cd*s/m2 of control, 5.0% SF-treated, and 10.0% SF-treated mice before 0.7% saline solution or SF administration (baseline), and 3 days, 6 days, and 9 days postinjection (G). Data are presented as mean ± SD were compared using 1-way ANOVA with Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Compared with the 10.0% SF-treated group, the scotopic and photopic ERG responses showed a slight reduction at 3 days after 5.0% SF administration (see Figs. 3A–C), and such electrophysiological functions restored to original levels of baseline 3 days later (6 days postinjection). The amplitudes of scotopic b-wave at 0.001 cd*s/m2 significantly decreased at 3 days postinjection, and the amplitudes at 6 and 9 days postinjection were not statistically different from the baseline level (see Fig. 3B), which is inconsistent with the attenuation of scotopic 0.001 ERG responses after 10.0% SF administration. Moreover, the alterations of mixed rod-cone or cone-dominated responses also shared similarities with those of rod-dominated responses in the 5.0% SF-treated group (see Figs. 3B, 3C). The data of ERG responses from all groups are listed in Supplementary Tables S3 to S5, and comparative analysis of scotopic 0.001 ERG, scotopic 3.0 ERG, and photopic 3.0 ERG responses between all groups are provided in Figures 3D to 3G and Supplementary Tables S6 to S9.

Histological Characterization of Retinal Morphology

H&E staining was performed in the control, 10.0% SF-treated, and 5.0% SF-treated mice to observe the histological changes of the retina. In the central region of the retina, the thickness of IPL, INL, OPL, and PRL in control mice was comparable with that of 10.0% or 5.0% SF-treated mice, but the thickness of central ONL in 10.0% SF-treated mice was significantly higher than that of control mice (Supplementary Fig. S1A). Meanwhile, the peripheral retina in three groups also showed similar thickness of IPL, INL, OPL, and PRL, and control or 5.0% SF-treated mice showed the lower thickness of peripheral ONL compared with 10.0% SF-treated mice (Supplementary Fig. S1B). The thickness of the RPE layer in the central retina of 10.0% or 5.0% SF-treated mice were significantly lower than that of control mice, the thickness of the RPE layer in the peripheral retina of 5.0% SF-treated group were higher than that of the other two groups statistically, and 10.0% SF-treated mice showed lower thickness of RPE layer in the peripheral retina compared with control or 5.0% SF-treated mice (Supplementary Fig. S1C). The nuclei rows in the central or peripheral ONL of the 10.0% or 5.0% SF-treated mice were significantly lower than that of control mice (Supplementary Fig. S1D). The ONL in the central or peripheral retina of control mice showed dense and regular distribution of photoreceptor nuclei, no signs of abnormal structure or organization were observed (Fig. 4), and the ONL of 5.0% SF-treated mice demonstrated slight disorganization compared with the control group, such as the sparse distribution of photoreceptor nuclei (see Fig. 4). After administration of 10.0% SF, remarkable disorganization of photoreceptor nuclei and vacuole-like changes were observed in the ONL of the central or peripheral retina (see Fig. 4). It is noteworthy that the RPE layer in the central or peripheral region of both 5.0% and 10% SF-treated mice became relatively hypopigmented (see Fig. 4).

Figure 4.

Figure 4.

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Representative images of total retina, ONL, and RPE in the central and peripheral regions of control, 5.0% sodium fluorescein (SF)-, and 10.0% SF-treated mice stained with hematoxylin and eosin. Scale bar = 20 µm.

H&E staining also revealed abnormal findings in the partial region of the outer retina in 10.0% SF-treated mice (Supplementary Figs. S1E–G), including the accumulation of pigment granules or the formation of deposit-like lesion in RPE, the discontinuity or absence of the RPE layer, and the subtle disorganization of photoreceptor outer segment (POS).

Evaluation on Apoptosis in the Retina

We performed the TUNEL assay to investigate the apoptotic cells in the retina of mice after SF administration, and found the apoptosis occurred in the ONL, INL, and the retinal ganglion cell (RGC) layer of 10.0% or 5.0% SF-treated group (Fig. 5A), quantitative analysis showed that 10.0% SF administration significantly increased the number of TUNEL-positive cells in the retina, compared with the 5.0% SF-treated group and the control group (Fig. 5B).

Figure 5.

Figure 5.

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Representative images of TUNEL staining (A), and quantification of the number of TUNEL-positive cells in the retina of control, 5.0% sodium fluorescein (SF)-, and 10.0% SF-treated mice (n = 6 sections from 6 eyeballs of three mice per group) (B). Representative images of immunohistochemical expression of GFAP (C), and quantification of the fluorescence density level of GFAP staining in retina of control, 5.0% SF-, and 10.0% SF-treated mice (n = 6 sections from 6 eyeballs of 3 mice per group) (D). Scale bar = 50 µm. Data are presented as mean ± SD were compared using 1-way ANOVA with Bonferroni post hoc test. ***P < 0.001, ns, no significance.

Immunohistochemical Characterization of Retinal Morphology

To further characterize the morphology of the retina after SF administration, we performed IHC to investigate the expression of GFAP, M-opsin, S-opsin, and Rhodopsin. The immunoreactivity of GFAP was exhibited in the RGC layer in 5.0% SF-treated group or control group, and increased GFAP staining in the inner retina was observed in 10.0% SF-treated group compared with other groups (Fig. 5C), quantitative analysis showed that the GFAP fluorescence density of 10.0% SF-treated group was significantly higher than that of 5.0% SF-treated group and the control group (Fig. 5D). SF administration did not significantly affect the immunostaining of rod cells (Rhodopsin) or cone cells (M-opsin and S-opsin) in the retina of mice, compared with the control group, quantitative analysis showed that the Rhodopsin fluorescence density and the number of M-opsin or S-opsin–positive cells did not differ among all groups (Figs. 6A–F).

Figure 6.

Figure 6.

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Representative images of immunohistochemical expression of S-opsin (A), and quantification of the number of S-opsin-positive cells in the retina of control, 5.0% sodium fluorescein (SF)-, and 10.0% SF-treated mice (n = 6 sections from 6 eyeballs of 3 mice per group) (B). Representative images of immunohistochemical expression of M-opsin (C), and quantification of the number of M-opsin-positive cells in retina of control, 5.0% SF-, and 10.0% SF-treated mice (n = 6 sections from 6 eyeballs of 3 mice per group) (D). Representative images of immunohistochemical expression of Rhodopsin (E), and quantification of the fluorescence density level of Rhodopsin staining in retina of control, 5.0% SF-, and 10.0% SF-treated mice, the fluorescence density level of Rhodopsin staining in retina were measured and averaged from 6 sections (n = 6 sections from 6 eyeballs of 3 mice per group) (F). Scale bar = 50 µm. Data are presented as mean ± SD were compared using 1-way ANOVA with Bonferroni post hoc test. NS, no significance.

Alterations of Expressions of Factors Associated With Apoptosis and Oxidative Stress

To learn the molecular mechanism through which SF induces retinal alterations of mice, we initially performed Western blot to analyze the protein expression of apoptosis- and oxidative stress-associated factors in the retina and the RPE. Our study showed the protein level of BCL2 was statistically lower in the retina of 10.0% SF-treated mice compared with the control group (Figs. 7A, 7B), and was also decreased significantly in the RPE of 5% or 10.0% SF-treated mice (Figs. 7C, 7D). Then, we found a significant increase in the protein level of BAX in the retina or the RPE after 10.0% SF administration (see Figs. 7A–D). In addition, the protein levels of NRF2 and HO1 were increased statistically in the retina or the RPE of 10.0% SF-treated mice compared with the control group (see Figs. 7A–D).

Figure 7.

Figure 7.

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Protein levels of apoptosis- and oxidative stress-related genes in the retina/retinal pigment epithelium (RPE) of control, 5.0% sodium fluorescein (SF)-, and 10.0% SF-treated mice. Representative Western blots and density plots for BCL2, BAX, NRF2, and HO1 in the retina (A, B). Representative western blots and density plots for BCL2, BAX, NRF2, and HO1 in RPE (C, D). Data are presented as mean ± SD were compared using 1-way ANOVA with Bonferroni post hoc test. Three biological replicates for Western blot were performed per group. In each experiment per group, three protein samples of retina/RPE were extracted from three mice (2 eyeballs per mouse) respectively, and were used to analyze the protein relative expression of BCL2 (26 kDa), BAX (21 kDa), and (NRF2 [110 kDa] + HO1 [33 kDa]), respectively. Thus, three mice per group were used to analyze the relative expression of each protein across three biological replicates for Western blot (n = 3 mice for each protein per group), and a total of 18 eyeballs of nine mice per group were used across three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001. NS, no significance.

Discussion

SF was commonly used to visualize the retinal and choroidal vasculature for both diagnostic and scientific purposes, several studies have examined concentration of fluorescein, volume injected, and rate of injection to evaluate the FA-associated ARs of patients in clinical practice,12,14 but few studies focused on such details of SF administration in experimental procedure for animal models. Here, we investigated the functional, histological, and molecular characterizations in the retina of SF-treated mice, which exhibited some aberrant alterations compared with the control group, and provided one insight into the effect of SF on the retina of mouse models.

In this study, an electrophysiological test showed the gradual decrease in scotopic ERG responses at 0.001 cd*s/m2 in 10.0% SF-treated group, but the general ERG responses recovered with the increased intensities at 9 days postinjection. We also found the affected ERG responses in 5.0% SF-treated group presented earlier restoration than 10.0% SF-treated group, suggesting a dose-dependent functional effect for SF, and this finding is similar to the effect of sodium iodate (SI) on retinal function, which the ERG responses in 20 mg/kg SI-injected group decreased temporarily and recovered by 14 days, and the higher doses of SI resulted in the irreversible and immediate reduction in the response magnitude.21,22 It is noteworthy that scotopic ERG responses revealed slower recovery compared with photopic ERG responses, which might be associated with the differences of susceptibility of rod function and cone function to SF administration. In addition, most of SF would be eliminated 24 hours after administration, it might be traced for approximately a week in the urine,23 some studies also found SF could stain the retina or vitreous body,24–26 DiLoreto et al. used SF as a marker of human fetal neural retina for subretinal transplantation,24 suggesting the possibility of residual SF in retinal tissue, which could explain the early reduction and gradual restoration of retinal functions over time with metabolism or excretion of SF.

Histological examination showed 10.0% SF-treated mice revealed similar but milder destruction of retinal morphology compared with that of SI-treated mice,21,27–29 and SI-induced retinal alterations mainly included pigmentary disruption or atrophy of RPE, disorganization or loss of photoreceptors, and irregular rows of nuclei in the ONL, which SI is thought to target primarily the RPE cells and result in subsequent injury of photoreceptors and additional retinal impairments,21,27–29 it is unclear in regard to whether SF contributing to RPE abnormalities precedes disorganizations of the ONL or POS and increased apoptosis of the ONL. Given the restriction of the inner or outer blood-retinal barrier (BRB) and the permeability of choriocapillaris for SF, the SF could enter into the intercapillary space of the choroid, and might affect the Bruch's membrane and induce influences of oxidative stress on RPE cells via photosensitization,30–33 which could elucidate SF affecting the RPE layer with secondary effects on photoreceptors.

Immunostaining did not reveal significant difference among any of all groups in markers of photoreceptors, although SF could indirectly affect the RPE and photoreceptors and photosensitized oxidation, these findings seemed to be inconsistent with the alterations in scotopic and photopic ERG responses, suggesting that SF did not induce highly cytotoxic effect on photoreceptors. It has been reported that integrity of junction between photoreceptor inner segment (PIS) and POS is essential for visual improvement in retinal pathophysiology.34–36 Based on anatomy of photoreceptors, the maintenance of the visual cycle was also associated with the integrity of PIS/POS junction,37,38 which was preserved in the retina after SF administration in our study, this morphological characterization may help to explain the involvement of Rhodopsin and cone opsins in the biochemical process was not severely affected by SF. The early decrease of scotopic and photopic ERG responses followed by gradual restoration of electrophysiology, along with the stable expressions of Rhodopsin and cone opsins, our findings may indicate the flexibility of structure and function in rod or cone cells in terms of resistance to SF.

Immunostaining also observed the GFAP signal in the retina of the 5.0% SF-treated group was comparable with that of the control group, and the GFAP signal in the retina of the 10.0% SF-treated group was relatively higher than other groups, in which the signal was primarily restricted to the RGC layer, indicating the gliosis of macroglial cells in the retina after SF administration. One dynamic regulation, called neuroglio-vascular cross-talk, gets involved in integrity of retinal microvessels, and pericytes could communicate with adjacent endothelial cells, microglia, astrocytes, and neurons in the neurovascular unit.39,40 In our study, the overdose of SF could flow into the superficial capillary plexus in inner retina, and generation of superoxide anions by SF photosensitization might contribute to increased production of peroxynitrite, stimulating pericytes to attract and activate astrocyte or Müller cells,41 which might be associated with the relatively higher expression of GFAP in the inner retina of the 10.0% SF-treated group.

BCL2 and BAX belong to the BCL2 family, BCL2 acts as an antiapoptotic factor, and BAX can form a polymer with BCL2 to antagonize the antiapoptotic effect of BCL2, which this process is involved in regulating mitochondrial permeability to promote apoptosis. Besides, NRF2 is one of the important regulators in the protective response to oxidative stress, as the generation of ROS induces the release of NRF2 from KEAP1-mediated proteasomal degradation,42 then, NRF2 enters into the nucleus and accumulates to promote the expression of HO1,43,44 which is reactive to external insults, such as oxidative damage, inflammation, heat shock, and ischemia-reperfusion injury. HO1 could decompose heme to biliverdin with concurrent release of free iron or carbon oxide, and these end products exert potent inhibitory effect on apoptosis or oxidation,45,46 indicating the significant role of HO1 in endogenous compensatory response. Moreover, it appears to be controversial whether abundance of HO1 confers beneficial effects in retinal pathophysiology, as some research showed that overexpression of HO1 improved the activity of antioxidant enzyme and inhibited the production of ROS,45,47 but some research found upregulation of HO1 elicited the accumulation of ferrous ion and stimulated ferroptosis in photoreceptors and RPE,43,44 that further studies are required to validate the possibility of regulating HO1-associated signaling pathways to alleviate oxidative impairment in retinal diseases. On the one hand, the increase in BAX and the decrease in BCL2 levels reveal that the apoptosis was enhanced in the retina and RPE of the 10.0% SF-treated group. On the other hand, the NRF2 and HO1 levels in the retina and RPE increased statistically in the 10.0% SF-treated group, but the alterations in expressions of NRF2 and HO1 in RPE were more prominent than the retina, this finding is paralleled by the morphological abnormalities of the RPE layer, and is also similar to that of Sachdeva et al., who reported the increased transcript levels of NRF2 and HO1 in RPE cells of 2-month old mice after SI administration,48 our results suggest the activation of NRF2/HO1 signaling pathway in response to excessive SF, and the higher sensitivity of RPE to SF-induced oxidative stress compared with the retina.

Compared to optical coherence tomography (OCT) or OCT angiography (OCTA), FFA has the advantage of detecting and quantifying localized vascular leakage in retina.49 Although the differences in fluorescein intensity would not disturb observing some apparent morphological alterations in vessels, including retinal vascular deformity, choroidal neovascularization, and diffuse vascular leakage,6,50,51 as the FFA images in our study revealed the higher Mean E Lacunarity in the retina of the 5.0% SF-treated group compared with the 10.0% SF-treated group, it might be hard to differentiate between low fluorescence visibility and vascular filling defect, especially the lack of capillary perfusion. Currently, the dose of fluorescein injected has been evaluated in clinical investigations on humans, but is still unclear in experimental research on animals, so it is better to optimize the dose of SF, which depends on types of ocular diseases, severities of vascular impairments, or regulations of blood flow in vessels, to minimize the effect of SF on retina as much as possible.

The limitations of this study should be mentioned. First, the FFA on the mouse model was performed with intraperitoneal injection of SF, and was not undertaken using intravenous injection, different delivery routes influence not only the appearance time of retinal vasculature but also the intensity of the hyperfluorescent. Wigg et al. reported intraperitoneal administration of SF postponed arrival into the retinal vasculature and prolonged the peak florescence duration, at the expense of choroidal neovascularization definition and increased background fluorescence of choroid, thus precluding the intensity quantification due to the altered perfusion rates, but the better definition of hyperfluorescent areas could be observed using intravenous administration of SF via the lateral tail vein,16 so it would be interesting to compare the effect of SF on the mouse retina between intraperitoneal injection and intravenous injection. Second, the number of study groups was relatively small, because the doses of SF for FFA were variable in different experimental research,6,16,52–55 establishing several groups receiving a wide range of SF concentrations is required to further explore whether administration of different doses of SF induce functional or morphological alterations in the retina of a mouse model. Third, this procedure of investigating the effect of SF on mouse retina terminated at 9 days postinjection, although SF-treated mice exhibited early reduction and subsequent restoration of ERG responses during the experimental procedure, but histological and molecular characterizations indicated the impairment of higher doses of SF on the retina, so it is necessary to establish more follow-up times for long-term observation on functional, morphological, or molecular alterations of the mouse retina after SF administration.

Conclusions

We used the C57BL/6J mice to characterize the retinal morphology and function after intraperitoneal injection of SF, these changes might be indistinguishable from some histopathological features associated with the development or progression of disorders, and even disturb the authenticity of experimental data, so the dose of SF should not be ignored in experimental procedures for mouse models of ocular diseases in future studies.

Supplementary Material

Supplement 1
tvst-15-8-9_s001.docx (1.2MB, docx)
Supplement 2
tvst-15-8-9_s002.docx (18.3KB, docx)
Supplement 3
tvst-15-8-9_s003.docx (18.1KB, docx)
Supplement 4
tvst-15-8-9_s004.docx (27KB, docx)
Supplement 5
tvst-15-8-9_s005.docx (27.2KB, docx)

Acknowledgments

Supported by the National Natural Science Foundation of China Grant (82301229 and 82371094).

Disclosure: T. Tao, None; J. Zheng, None; X. Wang, None; L. Liu, None; L. Feng, None

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1
tvst-15-8-9_s001.docx (1.2MB, docx)
Supplement 2
tvst-15-8-9_s002.docx (18.3KB, docx)
Supplement 3
tvst-15-8-9_s003.docx (18.1KB, docx)
Supplement 4
tvst-15-8-9_s004.docx (27KB, docx)
Supplement 5
tvst-15-8-9_s005.docx (27.2KB, docx)

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