The effect of fingomolid (FTY720) protecting the retina of rats from light-induced retinal degeneration (LIRD)

doi:10.21203/rs.3.rs-4617110/v1

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The effect of fingomolid (FTY720) protecting the retina of rats from light-induced retinal degeneration (LIRD)

https://doi.org/10.21203/rs.3.rs-4617110/v1

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Object: Fingomolid (FTY720), an immunosuppressive agent, was found to protect the retina against light stress in our previous study. In this study, we investigate whether FTY720 has a protective effect on retinal degeneration and whether immune response is involved in photoreceptor apoptosis in light-induced retinal degeneration (LIRD) in rats.

Methods: Sprague-Dawley rats raised in cyclic dim light were exposed to 2700 lux white light for 6 hours to build LIRD animal models. FTY720 (10.0 mg/kg) or vehicle was administered intraperitoneally to rats 0.5 h before light exposure. Histology and F-ERGs analysis were used to evaluate the structure and function of retina, respectively. The apoptosis of retinal cells was detected by TUNEL assay. The immune T cells on light-damaged retina were measured by immunofluorescence analysis, and the expression of immune proteins was examined by western blot.

Results: After light exposure, significant reductions in ERGs response were observed in vehicle-treated (VLD) group, whereas there was no significant difference between FTY720-treated (FTY-LD) group and normal group. A slight thinning was observed in FTY-LD group, which was not reflected in the full field ERG responses. Pretreatment with FTY720 inhibited light-induced photoreceptor apoptosis and protected retinal structure and function against light damage. CD3⁺ and CD8⁺ T molecules were increased in the VLD group, but did not occur on the whole retina in rats treated with FTY720. The expression of CD3⁺ and CD8⁺ proteins were up-regulated by light exposure and suppressed by FTY720 pretreatment. Light stress activated the microglial cells, and FTY720 could suppress the activation.

Conclusion: FTY720 could inhibit apoptosis and suppress CD3⁺ and CD8⁺ T cells and microglial activation in light-damaged retina in rats, showing an obvious protective effect on photoreceptors. These results help to better understand the pathogenesis of LIRD, and FTY720 may provide therapeutic benefit for retinal diseases.

fingomolid (FTY720)

light-induced retinal degeneration

retina

apoptosis

immune cells

Light-induced apoptosis is an important process of cell death in retinal degenerations, including age-related macular degeneration and retinitis pigmentosa.[1, 2] With the increasing application of artificial light source and ophthalmic optical instruments, light-induced retinal degeneration (LIRD) has become one of the major causes of blindness worldwide. Excessive light exposure has been shown to cause photoreceptor and retinal pigment epithelial cell damage through various mechanisms in rats and mice.[3–5] However, a comprehensive understanding for its mechanism remains elusive due to its complex pathophysiology.

FTY720, also known as fingomolid, is an immunomodulating drug that was derived from the fungal metabolite myriocin.[6] FTY720 could inhibit lymphocyte egress from secondary lymphoid tissue into inflammatory sites by mediating sphingosine 1-phosphate (S1P) receptors on lymphocytes.[6–9] and increase their anti-inflammatory potential through modulating immune cell function.[6, 10] Recent reports further revealed that FTY720 limited the inflammatory response in microglia against ischemic injury.[11] Based on its immunosuppressive properties, the effect of FTY720 has been investigated on various types of autoimmune and neurodegenerative diseases, such as relapsing multiple sclerosis,[12, 13] Alzheimer’s disease,[14] Parkinson's disease,[15] uveitis.[9, 16] and optic neuritis.[17] The safety of FTY720 in animal models and in humans and its ability to cross the blood-brain barrier have been well documented.[12]

Increasing evidence over the past decade highlighted the involvement of the ocular immune response in LIRD. T lymphocytes recruitment has been reported to participate in the retina of mice following light damaging.[18] Furthermore, microglia, active components of the retinal immune system, was also reported to trigger a chronic inflammatory response in light-induced retinopathy model.[11, 19] In our previous study, retinal ceramide levels increased by de novo biosynthesis in the LIRD model, and systemic delivery of FTY720 could effectively prevent the increase of ceramide in the retina and protect photoreceptors from apoptotic cell death.[20] These results supported a strong therapeutic potential of FTY720 in retinal degenerations. However, the underlying mechanism of FTY720 related to immune response in LIRD is unknown.

In this study, we used a rat LIRD model to measure the protective efficacy of intraperitoneal injection of FTY720 on retina. Our results revealed that pretreatment with FTY720 could effectively protect retina against light damage by preventing photoreceptor apoptosis and suppressing the immune T cells in retina.

Animal

All procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals Ophthalmic and Vision Research. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committees of Sichuan Provincial People’s Hospital. Sprague-Dawley (SD) albino rats (male and female) were born and raised under a dim cyclic light environment (5–10 lux, 12 h on/off) at 22 to 24°C before the experiments.

FTY720 treatment and light exposure model

FTY720, purchased from Selleck Chemicals (Houston, TX, USA), was dissolved in a vehicle containing sterile 100% DMSO and 0.9% saline 1:1(v/v) and administered at a dose of 10 mg/kg. The dosage was determined based on our previous report.[20] Eight to ten week-old SD rats were randomly divided into three groups: FTY720-treated light-damaged (FTY-LD), vehicle-treated light-damaged (VLD) and control. The FTY-LD group was intraperitoneally injected with FTY720 (10 mg/kg) 0.5 h before light exposure, and VLD group was injected with an equal volume of vehicle 0.5 h before light exposure. Then rats in both groups were exposed to damaging light for 6 hours (9 am to 3 pm), with an average light intensity of 2700 lux. After light exposure, the rats were returned to their dim cyclic room (5–10 lux) until subsequent analysis. Cyclic light reared rats without light exposure were served as normal controls. Retinal samples were collected 1, 3, 5 and 7 days after light stimulation.

Flash- electroretinograms (F-ERGs)

Seven days after light exposure, flash ERGs were recorded using the Espion Visual Electrophysiology System (Diagnosys LLC, Lowell, MA) to evaluate the retinal function. Rats were dark-adapted overnight, and subsequent procedures were carried out under dim red light. Then they (10 animals per group) were anesthetized by intramuscular injection of Ketamine (120 mg/kg) combined with Xylazine hydrochloride (6 mg/kg). After the pupils were completely dilated with 0.5% tropicamide and 5% Phenylephrine hydrochloride, a reference electrode was inserted subcutaneously into the forehead between two ears and a ground electrode into the tail, and two gold electrodes were lightly contacted to the central cornea. To assess the rod photoreceptor function, scotopic ERGs were measured in a Ganzfeld with flash intensities ranging from 0.003 to 20.0 cd•s/m². The latency and amplitude of a- and b- were measured, and the average values were applied to statistical analysis. Hyaluronic acid sodium eye drops were used to protect the cornea.

Histology

After F-ERGs recordings, rats were euthanized by carbon dioxide asphyxiation; eyes were harvested and marked with a slit tattoo dye at the 12 o’clock position on the limbus, fixed overnight with freshly-prepared fixative (4% (w/v) paraformaldehyde, 20% (v/v) isopropyl, 2% (v/v) zinc chloride, 2% (v/v) trichloroacetic acid and 72% (v/v) distilled water), and embedded in paraffin, as previously reported[20, 21]. All tissues were cut into 5-µm sections parallel with the vertical meridian through the optic nerve, and stained with hematoxylin and eosin (H&E). The thickness of the outer nuclear layer (ONL) and the numbers of rows of photoreceptors in the ONL were measured at 500-µm distances from the optic nerve to the superior and inferior ora serrata.

Immunohistochemistry

For immunohistochemistry and TUNEL analyses, eyes were enucleated at 1, 3, 5 and 7 days after light exposure, fixed with 4% paraformaldehyde in PBS (PH 7.4) for 2 h at 4°C, washed with PBS, and then soaked in 30% sucrose for 2 h for dehydration. After removal of the cornea, tissues were embedded in optimal cutting temperature (OCT), and cut into 10-µm cryosections for analysis. Immunolabeling of frozen sections was carried out as described by our previous report[20]. The following primary antibodies were used: anti-CD3⁺ (Abcam, England, 1:500 dilution), anti-CD8⁺ (Abcam, England, 1:1000 dilution). Alexa Fluor 488 and Alexa Fluor 594 (Sigma-Aldrich, St. Louis, MO, USA, 1:200 dilution) secondary antibodies and DAPI (Invitrogen, Carlsbad, CA, USA, 1:2000 dilution) were also used. Images were captured under the same light condition by LSM 800 confocal microscope (Carl Zesis, Jena, Germany).

For evaluating the retinal inflammatory reaction after light exposure, a commonly used macrophage/microglial marker (ionized calcium binding adaptor molecule 1 [Iba1]) was applied to deparaffinized sections as described in previous study.[22] Briefly, sections were immersed in the blocking buffer (10% normal goat serum) for 30 min at room temperature and incubated with anti-Iba1 Ab (Abcam, England, 1:800 dilution) overnight at 4°C. Then sections were washed with PBS and incubated with biotinylated secondary antibody for 30 min at 37°C. Labeling was visualized with 3,3′-diaminobenzidine for 5 min. Cell nuclei were counterstained with Gill’s hematoxylin.

Apoptosis detection by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

The TUNEL staining was performed on retinal cryosections using a commercial kit (Alexa Fluor 640, Yeasen Biotech, Shanghai, China) according to manufacturer’s protocol. Frozen sections of rat retina collected at different time points after light-damage were used for the assay along with non-light-damaged controls. The Fluorescence images were captured by LSM 800 confocal microscope (Carl Zesis, Jena, Germany).

Western Blot

Retinas were separated under microscopic view, lysed and homogenized in RIPA lysis buffer containing phosphatase and protease inhibitor cocktails (Roche, IN, USA), and centrifuged at 12,000g for 10 min at 4°C to obtain the supernatants. Equal amounts of protein (20 µg) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then electrophoretically transferred to nitrocellulose (NC) membranes. After the membrane was blocked for 1 h at room temperature (8% non-fat milk), it was incubated with monoclonal anti-CD3⁺ (1:500), anti-CD8⁺ (1:1000) and anti-β-actin (1:5000) antibodies overnight at 4°C. The membrane was rinsed three times with 1x TBST buffer and incubated with the appropriate peroxidase-linked secondary antibody for 1 h at room temperature. The blots were exposed with the ECL enhanced chemifluorescent reagent (Thermo Scientific, Rockford, IL, USA). The intensities of bands were quantified by image analysis software.

Statistical Analysis

Statistical analysis was conducted by one-way ANOVA and Student’s and paired t-tests using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA). Quantitative data are expressed as mean ± standard deviation (SD) for each group. A value of P < 0.05 was considered significant.

FTY720 effectively protects retina from light-induced retinal degeneration

In previous study, we found that FTY720 administered at a dose of 10 mg/kg at 0.5 h before light exposure (2,700 lux for 6 h) provided maximal protection of retinal structure and function.[20] Similar experimental conditions have been reproduced here. Retinal function was examined by F-ERGs at 7 days after light exposure. In vehicle-treated light-damaged (VLD) group, we observed significant reductions in scotopic a- and b- amplitudes compared to normal group (*, P < 0.01) (Fig. 1). For the rats treated with 10mg/kg FTY720 at 0.5 h before light exposure (FTY-LD), the a- and b- responses were preserved significantly compared to the vehicle group (**, P < 0.001), and there was no significant difference between the FTY-LD group and normal group (Fig. 1).

H&E-stained sections of retina were prepared after F-ERGs recordings to analyze retinal structure and effect of light-damage. After light exposure, the dead retinal cells were cleared by macrophages, and the remaining nuclei in the outer nuclear layer (ONL) represent the surviving photoreceptors. As shown in the representative images, in the VLD group, the number of photoreceptor cell nuclei after light damage was significantly lower than that in control at central retina (close to optic nerve head. ONH). And FTY720 treatment at 0.5 h before light damage effectively protected the photoreceptor cells (Fig. 2A). By quantitative morphometry of the ONL thickness, we found that light damage caused a significant decrease in the inferior and superior photoreceptor cells from rats pre-treated with vehicle, with the superior hemisphere showing more sensitivity towards acute light-induced damage (Fig. 2B).These results indicated that FTY720 significant protection (almost completely) against light-induced loss of retinal function and structure and support our previous results with remarkable reproducibility.

FTY720 prevented retinal photoreceptor cells apoptosis

In previous study, we did not measure cell death at various time points following light damage. Photoreceptor cells are known to undergo apoptosis following light damage and that results in light-induced retinal degeneration, which could be studies by TUNLE assay. As expected, the number of TUNEL-positive cells in the VLD group was markedly higher than that in the normal group after light exposure, and TUNEL-labeled nuclei were mainly located in the ONL, indicating the effect is restricted to the photoreceptor cells. At the 3th, 5th, 7th days after light exposure, the apoptotic nuclei in the ONL were decreased gradually. However, rats pre-treated with FTY720 (10 mg/kg) presented few to none TUNEL positive nuclei at any stage (Fig. 3). These results suggested that FTY720-mediated protection of retina works through inhibition of light-induced photoreceptor apoptosis.

FTY720 inhibited the lymphocyte on light-damaged retina

FTY720 is approved as a therapeutic drug for multiple sclerosis because of its immunosuppressive properties. Treatment with FTY720 led to lymphopenia by significantly reducing peripheral blood lymphocyte counts, which is correlated with the lymphocyte homing and trafficking[23, 24]. Therefore, we determined whether FTY720 suppresses the immune response in LIRD by examining the immune T cells in the retinas. In contrast with the results observed in normal rats, the processing of CD3⁺ T molecules across the whole retina was found to increase from the first day after light stimulation, and showed a trend with decrease gradually in the VLD group. The CD8⁺ T molecules started to increase at the 3th day after light damage, and reached the peak at 5th day (n = 5) (Fig. 4). Besides, it showed that the retinal photoreceptor cells decreased gradually with the progressing of time. For rats pre-injected with 10mg/kg FTY720, CD3⁺ and CD8⁺ T cells were not detected on the whole retina during the 7 days after light stimulation. And loss of retinal photoreceptor cells did not occur (n = 5).

Effect of FTY720 on the expression of immune proteins on light-damaged retina

To further investigate the immune mechanism of LIRD and the protective mechanism of FTY720, Western blot was used to test the expression of CD3⁺ and CD8⁺ protein of retina obtaining from 1, 3, 5 and 7 days after light exposure (Fig. 5). In vehicle pre-treated group, higher levels of activated CD3⁺ and CD8⁺ proteins were detected after light stress compared to normal group (n = 5). Immune proteins of CD3⁺ and CD8⁺ in FTY720 pre-treated group were significantly lower than that in vehicle pre-treated group (n = 5). The results of Western Blot assays were consistent with those observed in immunohistochemistry analysis of CD3⁺ and CD8⁺ T cells.

FTY720 suppresses microglial activation on light-damaged retina

Activated microglia were reported to play a major role in retinal degeneration, thus, we measured the activation of microglia in rats retina. Three days after light exposure, the number of microglia increased, and these cells were activated, became amoeboid and migrated to the outer retina in the vehicle-treated group (Fig. 6B). For rats pre-injected with FTY720, activated microglial cells were not detected in the retina, which was similar to those in normal group (Fig. 6A, Fig. 6C). These data suggested that FTY720 could suppress the activation of microglia after light stress.

Retinal degeneration is a heterogeneous group of diseases that generally characterized by apoptosis of photoreceptor cells.[5] Intense visible light exposure can lead to retinal photoreceptor cells damage, and light-induced apoptosis is the main pathway of cell death in these diseases.[25] However, owing to its complex pathophysiology associated with genetic and environmental factors, very few effective therapies exist for these diseases. Here, in order to investigate the effect of FTY720 on retinal degeneration, we used the LIRD model and established that pretreatment with FTY720 at a dose of 10 mg/kg effectively protected the retina by inhibiting apoptosis and suppressing CD3⁺ and CD8⁺ T cells in light-damaged retina.

LIRD is an ideal model for retinal degeneration research, such as retinitis pigmentosa and age-related macular degeneration.[26] Since its discovery in 1966 by Noell et al.,[26] this animal model has been widely used to discover the underlying mechanisms of photoreceptor function and to screen various putative neuroprotective compounds.[3] However, the precise molecular mechanisms of LIRD are poorly understood.

There is now strong evidence that microglial reactivity is an early and common hallmark of retinal degeneration.[27] Zeng et al. pointed out that activated microglia and their signaling molecules (TNF-α and IL-1β) were involved in the photoreceptor cell death.[28] In the normal retina, microglial cells were exclusively localized to the ganglion cell layer (GCL), inner plexiform layer (IPL) and inner retina, but not observed in the outer retina and the subretinal space. These cells were quiescent and typically ramified with small cell bodies and slender, varicose processes.[29] After light exposure, microglial cells were activated and migrated to the outer retina, and locally produced complement deposition, that triggered a chronic inflammatory process.[22] Light stimulation could affect the differentiation and survival of postnatal Müller cells, which could induce the immune response of the retina.[30] Moreover, the numbers of CD3⁺ and CD8⁺ T lymphocytes were reported to increase in light-damaged retina of old mice.[18] These findings supported a role for local immune response in the pathogenesis of retinal degeneration.

In recent years, increasing attention has been focused on anti-inflammatory drugs in retinal degenerations, including a variety of antioxidants and immunosuppressants.[5, 8] FTY720, a structural analog of Sphingosin (Sph), can be phosphorylated to FTY720-P in vivo by sphingosine kinase 2 (SPHK2) to mimic the action of S1P, and then FTY720-P mediates S1P receptor signaling in lymphocyte homing and trafficking.[31] FTY720 served as an immunosuppressive agent and led to a significant reduction in blood lymphocyte counts by accelerating the homing of lymphocytes into lymph nodes.[32] FTY720 has been tested on various diseases, such as multiple sclerosis.[12, 13] However, the protective effect of FTY720 on retinal degenerations is not clearly known. Previously, our study found that systemic delivery of FTY720 effectively inhibited de novo ceramide biosynthesis and led to a 50% reduction of the number of circulating lymphocytes, providing a remarkable protection of retinal structure and function.[20]

In the current study, we found that 10 mg/kg FTY720 administered 0.5 h before light exposure had significant protection of retinal structure and function by histology analysis and F-ERGs test which is consistent with our previous results. Rats pre-treated with FTY720 have few TUNEL-positive nuclei in the ONL, indicating that FTY720 could effectively prevent retinal photoreceptor cells apoptosis following light damaging. Compared with the FTY720 pre-treated group, the processing of CD3⁺ and CD8⁺ T molecules were increased, and their relevant proteins were also highly expressed in vehicle-treated light-damage group, which was consistent with Camelo et al.[18]. Furthermore, three days after light exposure, microglial cells increased in number, and they were activated, became amoeboid and migrated toward the outer retina, whereas no activated microglial cells were observed in the FTY720-treated group. Thus, we speculated that our model of LIRD was significantly related to local immune response and microglial activation, and that FTY720 might inhibit this process by modulating the localization and expression of immune T cells and suppressing the activation of microglia after light stress.

In conclusion, light stress could induce photoreceptors apoptosis, and activate ocular immune response by increasing the processing of CD3⁺ and CD8⁺ T cells and the expression of CD3⁺ and CD8⁺ proteins in rats retina, and microglial activation. Systemic administration of FTY720 (10 mg/kg) 0.5 h before light exposure could effectively protect the retina by inhibiting the apoptosis and suppressing immune T cells and microglial activation in retina.

Funding Declaration

This study was supported by grants from the National Basic Research Program of China (973 Program, No. 2015CB554100). The authors thank all the participants in this study.

Disclosure of interest

The authors have reviewed this manuscript and declared no competing financial or non-financial interests of this paper.

Data Availability

All data generated or analyzed during this study are are available from the corresponding author upon request.

Beneficial effect of FTY treatment on retinal electrophysiology by ERG analysis.

The scotopic a- and b- amplitudes were reducted significantly in vehicle-treated light-damaged (VLD) group compared to normal group (*, P < 0.01). For the rats treated with FTY, the a- and b- responses were preserved significantly compared to the vehicle group (**, P < 0.001), and there was no significant difference between the FTY-LD group and normal group.

FTY720 could suppress the activation of microglia after light stress.Three days after light exposure, the number of microglia increased and activated, became amoeboid and migrated to the outer retina in the VLD group (Fig. 6B). For rats pre-rejected with FTY720, activated microglial cells were not detected in the retina, which was similar to those in normal group (Fig. 6A, Fig. 6C).

Author Contribution

Qian Zhang and Hui Chen performed the experiment and wrote the main manuscript text. Menghan Zhao and Xingshang Zhang analyzed the dataa. All authors reviewed the manuscript.

Acknowledgement

The authors have reviewed this manuscript and declared no competing financial or non-financial interests of this paper.

Data Availability

All data generated or analyzed during this study are are available from the corresponding author upon request.

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You are reading this latest preprint version

The effect of fingomolid (FTY720) protecting the retina of rats from light-induced retinal degeneration (LIRD)

Status:

Version 1

Abstract

Figures

Introduction

Methods

Animal

FTY720 treatment and light exposure model

Flash- electroretinograms (F-ERGs)

Histology

Immunohistochemistry

Apoptosis detection by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Western Blot

Statistical Analysis

Results

FTY720 effectively protects retina from light-induced retinal degeneration

FTY720 prevented retinal photoreceptor cells apoptosis

FTY720 inhibited the lymphocyte on light-damaged retina

Effect of FTY720 on the expression of immune proteins on light-damaged retina

FTY720 suppresses microglial activation on light-damaged retina

Discussion

Disclosure of interest

Data Availability

Declarations

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1