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Retina  |   February 2015
Activation of Liver X Receptor Protects Inner Retinal Damage Induced by N-methyl-D-aspartate
Author Notes
  • Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China 
  • Correspondence: Bo Lei, Department of Ophthalmology, 1 You Yi Road, Yu Zhong District, Chongqing 400016, China; bolei99@126.com 
Investigative Ophthalmology & Visual Science February 2015, Vol.56, 1168-1180. doi:10.1167/iovs.14-15612
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      Shijie Zheng, Hongxia Yang, Zihe Chen, Changwei Zheng, Chunyan Lei, Bo Lei; Activation of Liver X Receptor Protects Inner Retinal Damage Induced by N-methyl-D-aspartate. Invest. Ophthalmol. Vis. Sci. 2015;56(2):1168-1180. doi: 10.1167/iovs.14-15612.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate whether activation of liver X receptors (LXRs) protects N-methyl-D-aspartic (NMDA)-induced retinal neurotoxicity in mice and to explore the underlying mechanism.

Methods.: Inner retinal damage was induced by intravitreal injection of NMDA. A synthetic LXR ligand TO901317 (TO90, 50 mg/kg/d) or vehicle was intragastrically administrated from 3 days before to 1 day or 7 days after NMDA injection. The severity of retinal damage was evaluated with histological analysis and TUNEL staining, and retinal functions were evaluated by ERG. The expressions of caspase-3, bax, bcl-2, TNF-α, and BACE1, the rate-limiting enzyme in the formation of amyloid β (Aβ), in the retina were examined by real-time PCR and ELISA. The levels of LXRs, NF-κB subunit p65, p-p38 mitogen-activated protein kinase (MAPK), and an LXR target gene ABCA1 were detected with real-time PCR and Western blotting. The localization and protein expression of Aβ in the retina was assessed by immunohistochemistry and Western blotting.

Results.: The NMDA enhanced the expression of LXRβ but not LXRα and ABCA1 in mouse retina. Nevertheless, administration of TO90 after NMDA injection not only enhanced the expression of LXRβ but also upregulated the level of ABCA1, suggesting retinal LXRs were activated in a ligand-dependent manner. The LXRα expression was unchanged in the vehicle and the TO90-treated groups. Activation of LXRβ with TO90 inhibited cell death in the ganglion cell layer (GCL) and inner nuclear layer (INL), preserved ERG b- and a-wave amplitudes, and the b/a ratio in the NMDA-treated mice. Meanwhile, TO90 suppressed the elevation of apoptosis factors caspase-3 and bax induced by NMDA and upregulated the level of an antiapoptotic factor bcl-2. The TO90 also inhibited the increase of p-p38 MAPK and proinflammatory cytokine TNF-α after NMDA injection. Furthermore, activation of LXR attenuated the activation of NF-κB, and reduced gene expression of BACE1 and accumulation of Aβ induced by NMDA.

Conclusions.: Activation of LXRβ with a synthetic LXR ligand TO90 protects the inner retinal damage induced by NMDA in mice. We speculate the protective effect is associated with inhibition of the NF-κB signaling pathway and reduction of Aβ formation in retina. The LXR agonists may become a new class of neuroprotective agent for retinal diseases associated with glutamate-induced excitotoxicity.

Introduction
Retinal neurodegeneration, especially the impairment of retinal ganglion cells (RGCs), is an early and critical event in several blinding diseases, including diabetic retinopathy and glaucoma.1,2 Although the underlying mechanism is not fully elucidated, it is known that excessive glutamate receptor activation plays an important role in the pathological process.3,4 In many instances, the neurotoxic effect of glutamate has been predominantly attributed to excessive stimulation of N-methyl-D-aspartic (NMDA) receptors.5,6 In NMDA receptor-mediated cell death, increase of intracellular Ca2+ concentration is thought to be the key event.7 In addition, upregulation of proinflammatory cytokines8,9 and accumulation of amyloid β (Aβ) in the retina are involved in this process.10 Thus, excessive activation of NMDA receptors appears to affect retinal neuronal cell survival by direct as well as indirect mechanisms. A single intravitreal injection of NMDA is commonly used in vivo to induce experimental RGC degeneration.11,12 By using the NMDA-induced neurotoxic model, we attempted to investigate whether activation of liver X receptors (LXRs) protects the neurotoxicity and to elucidate the possible molecular mechanisms. 
A hallmark of neurodegenerative diseases, such as Alzheimer's disease (AD), is the deposition of Aβ,13 and Aβ is also the major constituent of drusen from eyes of patients with AMD.14 The formation of Aβ is from a two-step proteolytic processing of the amyloid precursor protein (APP) by β- then γ-secretase. The APP is widely expressed in the central nervous system, including RGCs.15 Studies have shown that patients with AD present RGC loss similar to typical glaucomatous changes.16 In addition, Aβ has recently been reported to be implicated in the development of RGC apoptosis in glaucoma, with evidence of caspase-3–mediated abnormal APP processing and increased expression of Aβ in RGCs in experimental glaucoma.17 Furthermore, Aβ colocalizes with apoptotic RGCs in experimental glaucoma and induces significant RGC apoptosis in vivo in a dose- and time-dependent manner, and neutralizing Aβ with antibodies significantly attenuates RGC apoptosis.18 It also has been reported that sublethal NMDA receptor activation increased the production and secretion of Aβ.10 Thus, Aβ could be involved in the pathological process of NMDA-induced retinal neurotoxicity. 
Liver X receptors are known for their important roles in modulating cholesterol homeostasis.19 There are two isoforms (LXRα, LXRβ) of LXRs, which belong to the nuclear receptor superfamily of ligand-activated transcription factors. The LXRα is expressed predominantly in liver, kidney, intestine, and adrenal gland, whereas LXRβ is expressed ubiquitously. Our previous research has shown that both LXRα and LXRβ are expressed in mouse retina.20 The LXR target genes, including the ATP-binding cassette (ABC) transporters, ABCA1, ABCG1 and apolipoprotein E (ApoE), are associated with lipid metabolism and reverse cholesterol transport.19 In recent years, studies have described new functions of LXRs and their ligands, including regulation of inflammation and different aspects of the acquired immune response.20,21 The anti-inflammatory effect of LXRs has been attributed to inhibition of the transcription factor nuclear factor–κB (NF-κB) signaling.20,22 Previous study also demonstrated that NF-κB participates in NMDA-induced retinal neuronal cell death.23 Notably, LXRs and their ligands also have been proposed to prevent neurodegeneration in the adult nervous system,24 and LXR agonists reduce Aβ formation in neurons.25,26 Thus, we hypothesize that an LXR agonist may be a novel class of drug for retinal neurodegenerative diseases as it not only inhibits Aβ formation but also suppresses neuroinflammation and alleviates the pathology changes. 
In this study, we investigated the protective effect of systemic administration of an LXR agonist, TO901317 (TO90), on NMDA-induced retinal neurotoxicity and its potential mechanism. 
Materials and Methods
Animals
Six- to 8-week-old C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed in the Laboratory Animal Center of Chongqing Medical University (Chongqing, China). All experimental procedures conformed to the ARVO statement for the use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. 
NMDA-Induced Retinal Neurotoxicity
The C57BL/6J mice were randomly divided into three groups: a control group, an NMDA plus TO90–treated group, and an NMDA plus vehicle–treated group (30 mice were used in each group). Retinal neurotoxicity was induced by a single intravitreal injection of NMDA (Sigma-Aldrich Corp., St. Louis, MO, USA), as previously described.27 Briefly, mice were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg/kg; Fujian Gutian Pharmaceutical Co., Ltd., Ningde, Fujian, China) and xylazine (10 mg/kg; VEDCO, Inc., St. Joseph, MO, USA); pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine HCl solution. A single 2-μL amount of 10 mM NMDA in 0.01M PBS (pH7.40) was injected into the vitreous body under a microscope to avoid lens injury. The same volume of PBS was administrated as control. One drop of 0.01% levofloxacin ophthalmic solution was applied topically to the injected eyes after intravitreal injection. 
Treatment With TO90
The TO90 (Cayman, Ann Arbor, MI, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and then diluted with PBS to a final DMSO concentration of 2%, as described previously.20 The TO90 (50 mg/kg/d) or vehicle was intragastrically administrated from 3 days before to 1 or 7 days after NMDA injection. 
Histological Evaluation
Eyeballs were enucleated at day 1 and day 7 after intravitreal injection of NMDA and fixed with a solution containing 4% paraformaldehyde for at least 24 hours at room temperature. Fixed and dehydrated retinal tissues were embedded in paraffin and 5-μm sections were cut through the optic disc and then stained with hematoxylin and eosin. The images of each section were acquired with a light microscope. The number of cells of the ganglion cell layer (GCL) was manually counted in a region of 500 to 1000 μm from the center of the optic nerve head on both sides, and the thickness of inner nuclear layer (INL) was measured in three areas at a distance of 500 to 1000 μm from the edge of optic disc. At least four sections of each eye were measured for morphometric analysis, and data from four sections were averaged for each eye. All measurements and analysis were performed in a masked manner. 
Staining by TUNEL
Mice were euthanized with an overdose of sodium pentobarbital 24 hours after intravitreal injection of NMDA. The TUNEL staining was performed according to the manufacturer's instructions (In Situ Cell Death Detection Kit; Roche Diagnostics, Mannheim, Germany) to detect the apoptotic cells. Sections were reacted with 20 μg/mL proteinase K for 20 to 30 minutes at room temperature. After adding 50 μL TUNEL reaction mixture, sections were incubated in a moist chamber for 60 minutes at 37°C. Slides were then rinsed three times in PBS for 5 minutes, subsequently incubated with converter-POD at 37°C for 30 minutes. Then, 100 μL diaminobenzidine (DAB) was added and sections were kept at room temperature for an additional 3 to 10 minutes, and finally counterstained with hematoxylin. Light microscope images were photographed and the TUNEL-positive cells were counted between 500 to 1000 μm from the center of the optic disc on both sides in the GCL and INL. 
Immunofluorescence
Immunofluorescence was performed using previously described protocols.28 Briefly, eyes were collected 24 hours after NMDA injection and immersed in 4% paraformaldehyde for 4 hours, then embedded in optimum cutting temperature compound (Tissue-Tek OCT; Sakura Finetechnical Co., Ltd., Tokyo, Japan) in liquid nitrogen. Frozen sections (10-μm thick) were cut through the cornea-optic nerve axis, then immersed with 10% normal goat serum in PBS for 10 minutes and incubated overnight at 4°C with rabbit anti-NeuN (neuronal nuclei, NeuN) antibody (1:100; Abcam, Cambridge, MA, USA). The sections were washed and then incubated with secondary antibody for 1 hour at 37°C. The sections were counterstained with 4′,6′-diamino-2-phenylindole (DAPI). Images were captured with a fluorescence microscope (Leica, Bannockburn, IL, USA). Fluorescently labeled ganglion cells were counted between 500 to 1000 μm from the center of the optic disc on both sides in the GCL. 
Electroretinogram Analysis
Electroretinogram (RetiMINER System; AiErXi Medical Equipment Co., Ltd., Chongqing, China) was recorded at 7 days after NMDA injection. Mice were dark-adapted overnight and anesthetized with the mixture of ketamine and xylazine, and pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine HCl. Full-field ERG was recorded after inserting a ground electrode near the tail and a reference electrode on the back subcutaneously. A golden-ring electrode was gently positioned on the cornea.29 All procedures were performed under dim red light. Responses to brief flashes were analyzed primarily by measuring the amplitudes of the a- and b-waves. The amplitude of the a-wave was measured from the baseline to the maximum a-wave peak, and the b-wave was measured from the nadir of the a-wave to the apex of the b-wave peak. We also analyzed the ratio of b-wave amplitude to a-wave amplitude. 
Real-Time PCR Analysis
The mRNA expressions of LXRα, LXRβ, ABCA1, Caspase-3, Bax, Bcl-2, TNF-α, p65, and BACE1 at 24 hours after NMDA injection were detected by real-time PCR as previously described.20 Briefly, total RNA was extracted with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) from mouse retinas. After quantifying by a Nanodrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), cDNA was synthesized from 1 μg total RNA with PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China). To quantify the cDNA, real-time PCR (SYBR Green) was performed on an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). The PCR amplification was conducted in a volume of 20 μL using all-in-one quantitative PCR mix (Applied Biosystems, Carlsbad, CA, USA). The cycling protocol consisted of one cycle of 10 minutes at 95°C followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. To determine the mRNA expression, all samples were tested in duplicate and the average Ct values were used for quantification. The mRNA expression was normalized to the endogenous reference gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative quantification was achieved by using the 2−ΔΔCt method, where Δ Ct = (Ct target gene − Ct GAPDH), and ΔΔ Ct = (Δ Ct [experimental group] − Δ Ct [the mean of control group]). The relative fold changes of mRNA of target gene X in the experimental group compared with the control group was calculated as 2−ΔΔCt. The primers sequences are listed in the Table
Table
 
Sequences of Primers for Real-Time PCR
Table
 
Sequences of Primers for Real-Time PCR
Gene Forward Primer Reverse Primer
Caspase-3 5′-AACCAGATCACAAAATTCTGCAAA-3′ 5′-TGGAGTCCAGTGAACTTTCTTCAG-3′
Bax 5′-CCAGGATGCGTCCACCAAG-3′ 5′-AAGTAGAAGAGGGCAACCAC-3′
Bcl-2 5′-TGGGATGCCTTTGTGGAACTAT-3′ 5′-AGAGACAGCCAGGAGAAATCAAAC-3′
BACE1 5′-TCCGGCGGGAGTGGTATTATGAA-3′ 5′-ATCCGGGAACTTCTCCGTCGA-3′
TNA-α 5′-GCCTCTTCTCATTCCTGCTT-3′ 5′-CTCCTCCACTTGGTGGTTTG-3′
NF-κB p65 5′-AGGCTTCTGGGCCTTATGTG-3′ 5′-TGCTTCTCTCGCCAGGAATAC-3′
LXRα 5′-AGGAGTGTCGACTTCGCAAA-3′ 5′-CTCTTCTTGCCGCTTCAGTTT-3′
LXRβ 5′-AAGCAGGTGCCAGGGTTCT-3′ 5′-TGCATTCTGTCTCGTGGTTGT-3′
ABCA1 5′-TCCTCATCCTCGTCATTCAAA-3′ 5′-GGACTTGGTAGGACGGAACCT-3′
GAPDH 5′-GTATGACTCCACTCACGGCAAA-3′ 5′-GGTCTCGCTCCTGGAAGATG-3′
Western Blotting Analysis
Western blotting analysis was performed as previously described.30 Briefly, retinas were dissected from enucleated eyeballs at 24 hours after intravitreal injection, then homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1% proteases inhibitor (Beyotime), and the protein concentration was determined with a bicinchoninic acid (BCA) assay kit (Beyotime). Then, 60 μg protein was separated by 6% to 10% SDS-polyacrylamide gel and the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies against LXRα, LXRβ, ABCA1 (1:500; Abcam), NF-κB p65 (1:1200, Abcam), Aβ (1:1000, Abcam), p-p38 or p38 (1:500; Cell Signaling Technology, Beverly, MA, USA), and β-actin (1:500; Abcam) overnight at 4°C, and then washed and incubated with horseradish peroxidase conjugated secondary antibody (1:3000; Abcam) for 1 hour at room temperature. Signals were visualized by ECL kit (Advansta, Menlo Park, CA, USA), and band densitometry was performed using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). The measurements were repeated three times in each experiment, and the β-actin and p-38 were used as loading controls. 
Enzyme-Linked Immunosorbent Assay
The concentration of the TNF-α protein was determined by ELISA kits for mice (Invitrogen), according to the manufacturer's protocols. Retinas were collected from mice at 24 hours after NMDA injection and homogenized and solubilized in RIPA lysis buffer containing 1% protease inhibitor. The absorbance at 450 nm wavelength was measured using a multifunction microplate reader (Molecular Devices, Sunnyvale, CA, USA). 
Immunohistochemistry
Eyes were collected at 24 hours after NMDA injection and fixed with 4% paraformaldehyde for 24 hours. After deparaffinization and rehydration, sections were permeated with 0.3% H2O2 methanol solution for blocking the endogenous peroxidase activity. Tissue sections were subsequently treated for antigen retrieval by boiling samples for 15 minutes, and blocked with 5% BSA for 30 minutes. Then sections were incubated with anti-Aβ antibody (1:100; Abcam) overnight at 4°C. After triple washing with PBS for 5 minutes, the slides were incubated with horseradish peroxidase– and secondary antibody–conjugated polymer, EnVision+ (Dako, Glostrup, Denmark). The color reaction was developed in DAB solution and counterstained with hematoxylin. Negative controls were obtained by replacing the primary antibody with serum or PBS. 
Statistical Analysis
All data were presented as mean ± SEM. Statistical analysis was undertaken using the GraphPad Prism software (GraphPad Prism Software, Inc., San Diego, CA, USA). The results were analyzed by one-way ANOVA followed by Bonferroni correction for multiple comparisons; P less than 0.05 was considered statistically significant. 
Results
The TO90 Activated an LXR Target Gene ABCA1
Consistent with our previous report,20 both LXRα and LXRβ were expressed in the mouse retina. Intravitreal injection of NMDA resulted in a significant increase in LXRβ expression at both mRNA (P = 0.04) and protein (P < 0.001) levels, whereas LXRα expression appeared unaffected. After TO90 treatment, the level of LXRβ was increased (P < 0.01) but the level of LXRα remained unchanged compared with the control group (Figs. 1A, 1B). The expression of LXRβ was not significant between the NMDA plus vehicle group and the NMDA plus TO90 group. We also detected the expression of an LXR target gene, the transporter ABCA1 in mouse retina to determine whether LXR was activated and functional after TO90 treatment. Real-time PCR and Western blotting analysis showed that the expression of ABCA1 was not changed in NMDA-injected mice but was potently induced after administration of TO90 (P < 0.01) (Fig. 1C), suggesting that retinal LXRs were activated in a ligand-dependent manner. 
Figure 1
 
Expression of LXRα and LXRβ receptor isoforms and an LXR target gene, ABCA1 in mouse retina. Real-time PCR and Western blotting were detected at 24 hours after NMDA injection. Liver X receptor α and LXRβ were expressed in the retina of mice (A, B), and the expression of LXRβ was significantly increased after NMDA injection with or without TO90 treatment at mRNA and protein levels (B). There was no significant difference of LXRβ levels between the NMDA plus vehicle group and NMDA plus TO90 group, but the expression of LXRα was unchanged (A). The level of ABCA1 was robustly increased after TO90 treatment, whereas it was unaffected in the vehicle-treated mice after intravitreal injection of NMDA, and demonstrated that LXR was activated by intragastric administration of TO90. The results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Seven mice were used in each group.
Figure 1
 
Expression of LXRα and LXRβ receptor isoforms and an LXR target gene, ABCA1 in mouse retina. Real-time PCR and Western blotting were detected at 24 hours after NMDA injection. Liver X receptor α and LXRβ were expressed in the retina of mice (A, B), and the expression of LXRβ was significantly increased after NMDA injection with or without TO90 treatment at mRNA and protein levels (B). There was no significant difference of LXRβ levels between the NMDA plus vehicle group and NMDA plus TO90 group, but the expression of LXRα was unchanged (A). The level of ABCA1 was robustly increased after TO90 treatment, whereas it was unaffected in the vehicle-treated mice after intravitreal injection of NMDA, and demonstrated that LXR was activated by intragastric administration of TO90. The results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Seven mice were used in each group.
The TO90 Protected NMDA-Induced Inner Retinal Damage
Excitotoxic damage induced by NMDA is an acute model of RGC death. The onset of inner retinal damage begins soon after NMDA injection and reaches its peak at the 24th hour. The number of lost cells becomes relatively stable at 7 days post injection. Therefore, we investigated the effects of TO90 on NMDA-induced retinal toxicity in mice at 1 day and 7 days after NMDA injection. In the control mice, the cell number in the GCL was 121.7 ± 7.5 cells/mm and the thickness of the INL was 45.71 ± 5.5 μm (n = 6). Intravitreal injection of NMDA significantly decreased cell number in the GCL at day 1 (81.94 ± 6.2 cells/mm, P < 0.001) and day 7 (55.8 ± 6.6 cells/mm, P < 0.001), and the thickness of the INL at day 7 (29.8 ± 2.7 μm, P < 0.001) (n = 6). Hematoxylin and eosin staining revealed that intragastric administration of TO90 not only significantly rescued cell loss in GCL at day 1 (104.7 ± 4.4 cells/mm) and day 7 (80.8 ± 7.4 cells/mm, P < 0.001) (n = 6) after NMDA injection, but also prevented the reduction of INL thickness at day 7 (36.6 ± 3.2 μm, P = 0.029) (n = 6) after intravitreal injection (Fig. 2). 
Figure 2
 
Effects of TO90 on retinal damage induced by intravitreal injection of NMDA. Representative graphs showing histology of the control (A, D), NMDA plus vehicle–treated (B, E), and NMDA plus TO90–treated (C, F) groups at day 1 (AC) and day 7 (DF) after intravitreal injection of NMDA. Retinal damage was evaluated by counting the cell number in the GCL (G, I) and measuring the thickness of the INL (H, J) at day 1 and day 7 after intravitreal injection. Data are shown as mean ± SEM (n = 6). *P < 0.05, ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 2
 
Effects of TO90 on retinal damage induced by intravitreal injection of NMDA. Representative graphs showing histology of the control (A, D), NMDA plus vehicle–treated (B, E), and NMDA plus TO90–treated (C, F) groups at day 1 (AC) and day 7 (DF) after intravitreal injection of NMDA. Retinal damage was evaluated by counting the cell number in the GCL (G, I) and measuring the thickness of the INL (H, J) at day 1 and day 7 after intravitreal injection. Data are shown as mean ± SEM (n = 6). *P < 0.05, ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
The TO90 Prevented NMDA-Induced Apoptosis in Mouse Retina
The apoptosis of RGCs is thought to play an important role in inner retinal degeneration.12 To further elucidate whether TO90 protected retinal cell apoptosis mediated by NMDA excitotoxicity, we performed TUNEL staining on retina sections at 24 hours after intravitreal injection of NMDA. The TUNEL-positive cells in the retina were observed and counted in the GCL and INL. No TUNEL-positive cells were observed in the control group, whereas numerous TUNEL-positive cells were found in the GCL (36.1 ± 6.8 cells/mm) and INL (55.5 ± 8.7 cells/mm) after NMDA injection (P < 0.001) (Figs. 3A–C). Compared with the vehicle-treated animals, mice treated with TO90 had a significantly lower number of TUNEL-positive cells in both GCL (24.7 ± 2.5 cells/mm) and INL (33.8 ± 5.1 cells/mm) (P < 0.001) (n = 6) (Figs. 3D, 3E), suggesting that TO90 presented an antiapoptotic effect in NMDA-induced neuronal death. 
Figure 3
 
Effects of TO90 on NMDA-induced apoptosis at 24 hours after intravitreal injection of NMDA. Representative graphs of retinal cross sections of the control (A), NMDA plus vehicle–treated (B), and NMDA plus TO90–treated (C) groups. The TUNEL-positive cells with hyperchromatic nuclei are indicated by arrows. The TO90 significantly decreased the number of TUNEL-positive cells in GCL and INL (D, E). The results are mean ± SEM (n = 6). ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 3
 
Effects of TO90 on NMDA-induced apoptosis at 24 hours after intravitreal injection of NMDA. Representative graphs of retinal cross sections of the control (A), NMDA plus vehicle–treated (B), and NMDA plus TO90–treated (C) groups. The TUNEL-positive cells with hyperchromatic nuclei are indicated by arrows. The TO90 significantly decreased the number of TUNEL-positive cells in GCL and INL (D, E). The results are mean ± SEM (n = 6). ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
The TO90 Prevented RGC Loss Induced by NMDA
Retinal neurodegeneration, especially loss of RGCs, is a critical event in several blinding diseases, including diabetic retinopathy and glaucoma.1,2 To further investigate whether TO90 prevented RGC loss induced by NMDA, we quantitatively analyzed RGCs in cross sections of the retina with immunofluorescence using a NeuN marker (Fig. 4). Compared with the control mice (96 ± 15.6 cells/mm), the numbers of NeuN-positive RGCs (49 ± 7.5 cells/mm, P < 0.001) was significantly decreased in NMDA-injected mice without TO90 treatment. However, TO90 treatment significantly rescued RGC loss (80.3 ± 10.3 cells/mm, P < 0 .001) after NMDA injection (Fig. 4J). Thus, the number of NeuN-labeled RGCs suggested that TO90 presented a potent neuroprotective effect in NMDA-induced RGC loss. 
Figure 4
 
Effects of TO90 on NMDA-induced RGC loss at 24 hours after intravitreal injection of NMDA. The RGCs were labeled with NeuN. Representative graphs of the control (AC), NMDA plus vehicle–treated (DF), and NMDA plus TO90–treated (GI) groups. Staining by NeuN (green; [A, D, G]), DAPI nuclear staining (blue; [B, E, H]), and merge images (C, F, I). Arrows indicate NeuN-labeled RGCs. The TO90 significantly inhibited RGC loss induced by NMDA (J). The results are mean ± SEM (n = 6). ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 4
 
Effects of TO90 on NMDA-induced RGC loss at 24 hours after intravitreal injection of NMDA. The RGCs were labeled with NeuN. Representative graphs of the control (AC), NMDA plus vehicle–treated (DF), and NMDA plus TO90–treated (GI) groups. Staining by NeuN (green; [A, D, G]), DAPI nuclear staining (blue; [B, E, H]), and merge images (C, F, I). Arrows indicate NeuN-labeled RGCs. The TO90 significantly inhibited RGC loss induced by NMDA (J). The results are mean ± SEM (n = 6). ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
The TO90 Modulated Cell Death/Survival Signals After NMDA Injection
We further investigated the mechanism of cell death and the protective effects of TO90 in retina by measuring the mRNA levels of caspase-3, bax, and bcl-2 at 24 hours after intravitreal injection of NMDA. Bax and bcl-2 are members of the bcl-2 family and are found to be proapoptotic or antiapoptotic proteins that interact with caspase signals. As shown in Figure 5, the levels of caspase-3 (P = 0.002) and bax (P < 0.001) significantly increased after NMDA injection but decreased after TO90 treatment (P = 0.006, P = 0.003, respectively) (Figs. 5A, 5B). However, the bcl-2 mRNA level decreased markedly after NMDA injection (P = 0.016) and increased in the TO90-treated group (P = 0.003) (Fig. 5C). These data suggest that TO90 downregulated the expression of proapoptotic factors and upregulated the antiapoptotic factor. 
Figure 5
 
Effects of TO90 on the expression of apoptosis factors at 24 hours after intravitreal injection of NMDA. The TO90 downregulated the mRNA expression levels of proapoptotic factors caspase-3 (A) and bax (B), and upregulated the level of antiapoptotic factor bcl-2 (C) in NMDA-induced inner retinal damage mouse retina. Data are shown as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Four mice were used in each group.
Figure 5
 
Effects of TO90 on the expression of apoptosis factors at 24 hours after intravitreal injection of NMDA. The TO90 downregulated the mRNA expression levels of proapoptotic factors caspase-3 (A) and bax (B), and upregulated the level of antiapoptotic factor bcl-2 (C) in NMDA-induced inner retinal damage mouse retina. Data are shown as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Four mice were used in each group.
The TO90 Inhibited Retinal Function Loss Induced by NMDA
By 7 days after injection, there was a remarkable cell loss in the GCL and a significant reduction of INL thickness. Thus, to determine the effect of TO90 on retinal dysfunction induced by NMDA, dark- and light-adapted ERG was performed at day 7 after intravitreal injection of NMDA. The amplitudes of a-wave, which represents the function of the photoreceptors, and the b-wave, which represents the function of bipolar cells, were significantly decreased after NMDA injection. Intragastric administration of TO90 significantly rescued the amplitudes of the a- and b-wave (P < 0.05) (Fig. 6). To distinguish whether the reduction in b-wave amplitude mainly originated from impairment of photoreceptor activity or through impairment of bipolar cell function, we compared the dark-adapted b- and a-wave amplitude ratio at 1.0 log cd-m/s2 flash intensity. Figure 6D showed a significant decrease in the b/a-wave ratio after NMDA injection, suggesting NMDA mainly caused inner retinal damage rather than photoreceptor dysfunction. This finding indicated that TO90 treatment preserved inner retinal function after NMDA insult. 
Figure 6
 
Effects of TO90 on retinal function insult induced by intravitreal injection of NMDA. Retinal functions were evaluated by ERG at 7 days after NMDA injection. Representative ERG responses in the control, NMDA injection plus vehicle–treated, and NMDA injection plus TO90-treated (50 mg/kg/d, oral) groups (A). The ERG amplitude versus flash intensity for dark-adapted a-wave (B), dark-adapted b-wave (C), light-adapted a-wave (E), and light-adapted b-wave (F). The dark-adapted b/a-wave amplitude ratio at 1.0 log cd-m/s2 flash intensity (D). Intragastric administration of TO90 significantly prevented the reductions when compared with the vehicle-treated group. Data are shown as mean ± SEM (n = 10–14). #P < 0.05, ##P < 0.01, ###P < 0.001 versus control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle group.
Figure 6
 
Effects of TO90 on retinal function insult induced by intravitreal injection of NMDA. Retinal functions were evaluated by ERG at 7 days after NMDA injection. Representative ERG responses in the control, NMDA injection plus vehicle–treated, and NMDA injection plus TO90-treated (50 mg/kg/d, oral) groups (A). The ERG amplitude versus flash intensity for dark-adapted a-wave (B), dark-adapted b-wave (C), light-adapted a-wave (E), and light-adapted b-wave (F). The dark-adapted b/a-wave amplitude ratio at 1.0 log cd-m/s2 flash intensity (D). Intragastric administration of TO90 significantly prevented the reductions when compared with the vehicle-treated group. Data are shown as mean ± SEM (n = 10–14). #P < 0.05, ##P < 0.01, ###P < 0.001 versus control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle group.
The TO90 Reduced the Expression of TNF-α Induced by NMDA
As a potent inflammatory mediator, abundant increase of TNF-α was observed in the retina after NMDA injection.31,32 We investigated the effect of TO90 on TNF-α at gene expression and protein levels by real-time PCR and ELISA at 24 hours after intravitreal injection of NMDA. Compared with the control group, NMDA injection resulted in an increase of TNF-α in retina (P < 0.01) but the release of TNF-α was attenuated significantly by TO90 treatment (P = 0.046) (Fig. 7). 
Figure 7
 
Effects of TO90 on the expression of TNF-α in the mouse retina injected with NMDA. Real-time PCR ([A]; n = 4) and ELISA ([B]; n = 5) were performed at 24 hours after NMDA injection and the results showed that both the mRNA and protein levels of TNF-α significantly increased after NMDA injection and were suppressed by TO90. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7
 
Effects of TO90 on the expression of TNF-α in the mouse retina injected with NMDA. Real-time PCR ([A]; n = 4) and ELISA ([B]; n = 5) were performed at 24 hours after NMDA injection and the results showed that both the mRNA and protein levels of TNF-α significantly increased after NMDA injection and were suppressed by TO90. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
TO90 Downregulated NF-κB p65 and Phosphorylation of p38 Mitogen-Activated Protein Kinase (MAPK) After NMDA Injection
Previous studies have shown that activation of NF-κB and phosphorylation of p38 MAPK were involved in NMDA-induced neurotoxicity and inhibition of NF-κB activation and MAPK phosphorylation protected neurons in the GCL and INL.23,33 We investigated the effect of TO90 on NF-κB and p38 MAPK signal pathways with real-time PCR and Western blotting at 24 hours after NMDA injection. A significant increase of phosphorylation of p38 (p-p38) (P = 0.002) (Figs. 8A, 8B), as well as NF-κB p65 (P < 0.001) (Figs. 8C–E), was observed in the vehicle-treated retina after NMDA injection. The TO90 significantly suppressed the elevation of p65 and p-p38. 
Figure 8
 
Effects of TO90 on the expression of NF-κB p65 and p-p38 MAPK at 24 hours after intravitreal injection of NMDA. Western blotting showed that NMDA induced an increase in p-p38 protein level, and TO90 significantly suppressed the elevation of p-p38 expression ([A, B]; n = 4). Western blotting and real-time PCR showed both protein ([C, D]; n = 3) and mRNA ([E]; n = 4) levels of NF-κB subunit p65were significantly increased after NMDA injection and suppressed by TO90. Error bars are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Six mice were used in each group.
Figure 8
 
Effects of TO90 on the expression of NF-κB p65 and p-p38 MAPK at 24 hours after intravitreal injection of NMDA. Western blotting showed that NMDA induced an increase in p-p38 protein level, and TO90 significantly suppressed the elevation of p-p38 expression ([A, B]; n = 4). Western blotting and real-time PCR showed both protein ([C, D]; n = 3) and mRNA ([E]; n = 4) levels of NF-κB subunit p65were significantly increased after NMDA injection and suppressed by TO90. Error bars are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Six mice were used in each group.
The TO90 Inhibited the Formation of Amyloid β in the Mouse Retinas After NMDA Injection
Recent evidence indicates that activation of NMDA receptor increases the production and secretion of Aβ, and the deposition of Aβ participates in the development of RGC apoptosis.10,18 We examined whether TO90-mediated upregulation of ABCA1 had an impact on the level of Aβ in the retina with real-time PCR, Western blotting, and immunohistochemistry. Samples were collected at 24 hours after intravitreal injection of NMDA. Compared with the control group, the NMDA-injected group showed a high gene expression of β-secretase BACE1 (β-site amyloid precursor protein cleaving enzyme) (P = 0.033), which catalyzes the rate-limiting step in the production of the Aβ by cleaving the APP, whereas systemic administration of TO90 significantly suppressed this elevation (P < 0.001). Immunohistochemical analysis showed that Aβ was located in the GCL and INL of the retina. Meanwhile, quantitative analysis with Western blotting showed a significant increase of Aβ deposition in the NMDA-injected group when compared with the control (P = 0.013). Activation of LXR with TO90 significantly reduced the formation of Aβ (P = 0.009) (Fig. 9). 
Figure 9
 
Effects of TO90 on the formation of Aβ at 24 hours after NMDA injection. Expression of Aβ in the mouse retina of the control (A), NMDA plus vehicle–treated (B), and NMDA plus TO90–treated (C) groups. Negative controls were obtained by replacing the primary antibody with PBS (D). There was a considerable increase in Aβ immunoreactivity in the GCL and INL in the NMDA–treated retinas (arrows), and TO90 significantly reduced the protein expression of Aβ (E, F). The mRNA level of BACE1 was analyzed by real-time PCR, the expression of BACE1 was significantly upregulated by NMDA but was downregulated in the retina of NMDA-injected mice treated with TO90 (G). Data are shown as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 9
 
Effects of TO90 on the formation of Aβ at 24 hours after NMDA injection. Expression of Aβ in the mouse retina of the control (A), NMDA plus vehicle–treated (B), and NMDA plus TO90–treated (C) groups. Negative controls were obtained by replacing the primary antibody with PBS (D). There was a considerable increase in Aβ immunoreactivity in the GCL and INL in the NMDA–treated retinas (arrows), and TO90 significantly reduced the protein expression of Aβ (E, F). The mRNA level of BACE1 was analyzed by real-time PCR, the expression of BACE1 was significantly upregulated by NMDA but was downregulated in the retina of NMDA-injected mice treated with TO90 (G). Data are shown as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Discussion
Excessive activation of NMDA receptor, a subtype of glutamate receptor, is strongly implicated in the progress of RGC death.3,4,34 In the present study, we demonstrated that an LXR agonist, TO90, significantly prevented NMDA-induced inner retinal degeneration, including morphological and functional damage, upregulated inflammatory cytokine and apoptosis factors, and increased apoptotic cells and RGC loss. The protective effect of TO90 was partially related to suppressing the expression of NF-κB p65 and p38 MAPK, and inhibiting the formation of Aβ. 
Liver X receptors are nuclear receptors involved in lipid and cholesterol metabolism, as well as exert anti-inflammatory effects.1921 It is well known that lipid metabolism disorder and inflammation are involved in the pathogenesis of neurodegenerative diseases. Recently, the potent neuroprotective effect of LXR agonist has been confirmed in many disease models, such as AD, stroke, splanchnic ischemia, and reperfusion injury.3537 More recently, it was reported that regulated LXR and the target gene ABCA1 could modulate the outcome of diabetic retinopathy, experimental autoimmune uveitis (EAU) and AMD.20,38,39 However, whether LXRs play a role in inner retinal neurodegeneration is still unclear. Here, we demonstrated that not only was LXR associated with the pathogenesis of retinal damage induced by NMDA, but also activation of LXRβ could prevent the damage. 
Our previous research has shown that LXRα and LXRβ were expressed in mouse retina,20 and in EAU mice, LXRα may play a major role in intraocular inflammation. In this study, we found that mRNA and protein expression of LXRβ were significantly elevated after intravitreal injection of NMDA, but the expression of LXRα was unchanged. It suggested that LXRβ could be associated with response to neurodegeneration induced by the excessive activation of glutamate receptor. These findings are consistent with previous studies that LXRβ is more associated with central nervous system pathologies.40,41 
The TO90 is an exogenous LXR ligand. Compared with the NMDA plus vehicle–treated mice, we found the mRNA and protein levels of LXRα and LXRβ in the NMDA plus TO90–treated mice were unchanged. For this reason, we further examined the expression of an LXR target gene ABCA1 in the retina to identify whether LXR was functional. The results of real-time PCR and Western blotting showed the expression of ABCA1 was significantly increased in the TO90-treated mice but not in the vehicle-treated mice, demonstrating that TO90 activated the LXRβ-ABCA1 axis and the LXRβ was operational. Although the level of LXRβ was increased in the vehicle-treated group, the ABCA1 was inactive and the retinal damage was not alleviated. Thus, we presumed that the increase of LXRβ in the vehicle-treated group was not functional and might be a compensatory response to the retinal damage induced by NMDA. It was interesting that after TO90 treatment, the change of LXRα was inconsistent with LXRβ; this might be interpreted with the different distribution and function of the two isoforms. Because the LXRα remained unaffected by TO90, we proposed that activation of LXRβ played a major role of neuroprotection in retinal damage induced by NMDA. However, we could not completely exclude the possibility that LXRα also played a role in this neuroprotection, the activation of LXRα might be mediated by nuclear translocation and not necessarily by the elevation of total protein level in the retina. Thus, further in-depth research is needed to establish the role of LXRα in NMDA-induced retinal damage. 
As one of the most ubiquitous transcription factors and a key component in the inflammatory response,23,42 NF-κB is ubiquitously expressed in neurons and glia,43,44 and it can be activated by various stimuli, such as oxidative45 and glutamate.46 Recent reports show that NF-κB participates in glutamate-induced neurotoxicity, and it could act as a regulator in neurodegeneration.23 It was shown that retinal inflammation contributed to retinal neurotoxicity, and NMDA-induced retinal inflammation was evident by marked upregulation of proinflammatory gene TNF-α expression. To investigate the underlying mechanisms by which TO90 attenuates the development of NMDA-induced neurotoxicity, we evaluated the expression of NF-κB and a proinflammatory gene TNF-α, which was induced by activation of NF-κB and could facilitate the excitotoxic damage.31,47 Our data showed that TO90 treatment blocked the enhanced expression of NF-κB and the release of TNF-α. Moreover, the results provided further support to our previous study showing that TO90 also inhibited NF-κB signaling in EAU mice.20 To the best of our knowledge, the present study is the first to demonstrate the neuroprotective effects of LXR agonist by modulating NF-κB, TNF-α in a retinal neurotoxicity model. 
The deposition of Aβ has been shown to play a crucial role in the pathogenesis of progressive neurodegenerative disorders such as AD.13 A growing body of evidence has indicated that the accumulation of Aβ was implicated in the pathogenesis of glaucoma and AMD, and neutralizing antibodies to Aβ can significantly attenuate RGC apoptosis in experimental glaucoma.18,48 Because excessive activation of glutamate receptors is strongly implicated in the development of RGC loss and apoptosis,3,4,34 we examined the level of Aβ in the retina after intravitreal injection of NMDA. We found that the level of Aβ in the retina was significantly increased in the vehicle-treated mice but was suppressed in the TO90-treated mice after NMDA injection. Several studies have reported that ABCA1 was involved with increasing cholesterol levels and deposition of amyloid plaques and ABCA1-deficient mice exhibited increased amyloid load and Aβ levels in the brain.49 In contrast, overexpression of ABCA1 reduced amyloid deposition.50 In accordance with these findings, our study suggested that the suppression of Aβ could be attributed to upregulation of ABCA1 with TO90 and subsequently the downregulation of BACE1, which catalyzes the rate-limiting step in the production of the Aβ by cleaving the APP. Moreover, a recent report has shown that the accumulation of Aβ induced NF-κB p65 nuclear translocation in microglia cells, and this process also could be blocked by TO90.35 Thus, activation of LXR receptors appears to inhibit the expression of NF-κB by direct as well as indirect mechanisms. However, further studies are necessary to elucidate the detailed mechanism for activation of LXR in anti-inflammation and cholesterol metabolism. 
To investigate the molecular mechanisms with which TO90 inhibited the apoptosis induced by NMDA in the retina, we evaluated the phosphorylation of p38 MAPK, which can be induced by oxidative stress and inflammatory signals.51,52 It has recently been reported that NMDA-induced apoptosis was partially dependent on the activation of p38 MAPK in rat cortical cells53 and NMDA also activated p38 MAPK in RGCs, and inhibition of this process protected against NMDA-induced cell death.12 Our results showed a significant increase in phosphorylation of p38 MAPK, and TO90 treatment decreased the phosphorylation level significantly. Moreover, we have demonstrated that TO90 treatment attenuated the apoptosis by TUNEL staining. After treatment with TO90, apoptotic transcriptional changes, including downregulation of proapoptotic factors caspase-3 and bax, upregulation of antiapoptotic factor bcl-2 was observed. The neuroprotective effects of TO90 were evident by preventing activation of the apoptotic pathway, significant reduction of TUNEL-positive cells in GCL and INL, and restoration of inner retinal functions. 
In conclusion, the present study demonstrated that activation of LXRβ with a synthetic LXR agonist TO90 exerted a potent neuroprotective effect against NMDA-induced retinal neurotoxicity in mice. The protective effect may be associated with the inhibition of the NF-κB signaling pathway and reduction of Aβ formation in retina. Our results suggest LXR agonists may become a new class of candidate pharmacological interventions for preventing the development of retinal neurodegenerative diseases. 
Acknowledgments
Supported partially by National Natural Science Foundation of China Grants 81271033 and 81470621, Chongqing Science and Technology Commission (2014pt-sy10002), and National Key Clinical Specialties Construction Program of China. 
Disclosure: S. Zheng, None; H. Yang, None; Z. Chen, None; C. Zheng, None; C. Lei, None; B. Lei, None 
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Figure 1
 
Expression of LXRα and LXRβ receptor isoforms and an LXR target gene, ABCA1 in mouse retina. Real-time PCR and Western blotting were detected at 24 hours after NMDA injection. Liver X receptor α and LXRβ were expressed in the retina of mice (A, B), and the expression of LXRβ was significantly increased after NMDA injection with or without TO90 treatment at mRNA and protein levels (B). There was no significant difference of LXRβ levels between the NMDA plus vehicle group and NMDA plus TO90 group, but the expression of LXRα was unchanged (A). The level of ABCA1 was robustly increased after TO90 treatment, whereas it was unaffected in the vehicle-treated mice after intravitreal injection of NMDA, and demonstrated that LXR was activated by intragastric administration of TO90. The results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Seven mice were used in each group.
Figure 1
 
Expression of LXRα and LXRβ receptor isoforms and an LXR target gene, ABCA1 in mouse retina. Real-time PCR and Western blotting were detected at 24 hours after NMDA injection. Liver X receptor α and LXRβ were expressed in the retina of mice (A, B), and the expression of LXRβ was significantly increased after NMDA injection with or without TO90 treatment at mRNA and protein levels (B). There was no significant difference of LXRβ levels between the NMDA plus vehicle group and NMDA plus TO90 group, but the expression of LXRα was unchanged (A). The level of ABCA1 was robustly increased after TO90 treatment, whereas it was unaffected in the vehicle-treated mice after intravitreal injection of NMDA, and demonstrated that LXR was activated by intragastric administration of TO90. The results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Seven mice were used in each group.
Figure 2
 
Effects of TO90 on retinal damage induced by intravitreal injection of NMDA. Representative graphs showing histology of the control (A, D), NMDA plus vehicle–treated (B, E), and NMDA plus TO90–treated (C, F) groups at day 1 (AC) and day 7 (DF) after intravitreal injection of NMDA. Retinal damage was evaluated by counting the cell number in the GCL (G, I) and measuring the thickness of the INL (H, J) at day 1 and day 7 after intravitreal injection. Data are shown as mean ± SEM (n = 6). *P < 0.05, ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 2
 
Effects of TO90 on retinal damage induced by intravitreal injection of NMDA. Representative graphs showing histology of the control (A, D), NMDA plus vehicle–treated (B, E), and NMDA plus TO90–treated (C, F) groups at day 1 (AC) and day 7 (DF) after intravitreal injection of NMDA. Retinal damage was evaluated by counting the cell number in the GCL (G, I) and measuring the thickness of the INL (H, J) at day 1 and day 7 after intravitreal injection. Data are shown as mean ± SEM (n = 6). *P < 0.05, ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 3
 
Effects of TO90 on NMDA-induced apoptosis at 24 hours after intravitreal injection of NMDA. Representative graphs of retinal cross sections of the control (A), NMDA plus vehicle–treated (B), and NMDA plus TO90–treated (C) groups. The TUNEL-positive cells with hyperchromatic nuclei are indicated by arrows. The TO90 significantly decreased the number of TUNEL-positive cells in GCL and INL (D, E). The results are mean ± SEM (n = 6). ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 3
 
Effects of TO90 on NMDA-induced apoptosis at 24 hours after intravitreal injection of NMDA. Representative graphs of retinal cross sections of the control (A), NMDA plus vehicle–treated (B), and NMDA plus TO90–treated (C) groups. The TUNEL-positive cells with hyperchromatic nuclei are indicated by arrows. The TO90 significantly decreased the number of TUNEL-positive cells in GCL and INL (D, E). The results are mean ± SEM (n = 6). ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 4
 
Effects of TO90 on NMDA-induced RGC loss at 24 hours after intravitreal injection of NMDA. The RGCs were labeled with NeuN. Representative graphs of the control (AC), NMDA plus vehicle–treated (DF), and NMDA plus TO90–treated (GI) groups. Staining by NeuN (green; [A, D, G]), DAPI nuclear staining (blue; [B, E, H]), and merge images (C, F, I). Arrows indicate NeuN-labeled RGCs. The TO90 significantly inhibited RGC loss induced by NMDA (J). The results are mean ± SEM (n = 6). ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 4
 
Effects of TO90 on NMDA-induced RGC loss at 24 hours after intravitreal injection of NMDA. The RGCs were labeled with NeuN. Representative graphs of the control (AC), NMDA plus vehicle–treated (DF), and NMDA plus TO90–treated (GI) groups. Staining by NeuN (green; [A, D, G]), DAPI nuclear staining (blue; [B, E, H]), and merge images (C, F, I). Arrows indicate NeuN-labeled RGCs. The TO90 significantly inhibited RGC loss induced by NMDA (J). The results are mean ± SEM (n = 6). ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 5
 
Effects of TO90 on the expression of apoptosis factors at 24 hours after intravitreal injection of NMDA. The TO90 downregulated the mRNA expression levels of proapoptotic factors caspase-3 (A) and bax (B), and upregulated the level of antiapoptotic factor bcl-2 (C) in NMDA-induced inner retinal damage mouse retina. Data are shown as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Four mice were used in each group.
Figure 5
 
Effects of TO90 on the expression of apoptosis factors at 24 hours after intravitreal injection of NMDA. The TO90 downregulated the mRNA expression levels of proapoptotic factors caspase-3 (A) and bax (B), and upregulated the level of antiapoptotic factor bcl-2 (C) in NMDA-induced inner retinal damage mouse retina. Data are shown as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Four mice were used in each group.
Figure 6
 
Effects of TO90 on retinal function insult induced by intravitreal injection of NMDA. Retinal functions were evaluated by ERG at 7 days after NMDA injection. Representative ERG responses in the control, NMDA injection plus vehicle–treated, and NMDA injection plus TO90-treated (50 mg/kg/d, oral) groups (A). The ERG amplitude versus flash intensity for dark-adapted a-wave (B), dark-adapted b-wave (C), light-adapted a-wave (E), and light-adapted b-wave (F). The dark-adapted b/a-wave amplitude ratio at 1.0 log cd-m/s2 flash intensity (D). Intragastric administration of TO90 significantly prevented the reductions when compared with the vehicle-treated group. Data are shown as mean ± SEM (n = 10–14). #P < 0.05, ##P < 0.01, ###P < 0.001 versus control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle group.
Figure 6
 
Effects of TO90 on retinal function insult induced by intravitreal injection of NMDA. Retinal functions were evaluated by ERG at 7 days after NMDA injection. Representative ERG responses in the control, NMDA injection plus vehicle–treated, and NMDA injection plus TO90-treated (50 mg/kg/d, oral) groups (A). The ERG amplitude versus flash intensity for dark-adapted a-wave (B), dark-adapted b-wave (C), light-adapted a-wave (E), and light-adapted b-wave (F). The dark-adapted b/a-wave amplitude ratio at 1.0 log cd-m/s2 flash intensity (D). Intragastric administration of TO90 significantly prevented the reductions when compared with the vehicle-treated group. Data are shown as mean ± SEM (n = 10–14). #P < 0.05, ##P < 0.01, ###P < 0.001 versus control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle group.
Figure 7
 
Effects of TO90 on the expression of TNF-α in the mouse retina injected with NMDA. Real-time PCR ([A]; n = 4) and ELISA ([B]; n = 5) were performed at 24 hours after NMDA injection and the results showed that both the mRNA and protein levels of TNF-α significantly increased after NMDA injection and were suppressed by TO90. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7
 
Effects of TO90 on the expression of TNF-α in the mouse retina injected with NMDA. Real-time PCR ([A]; n = 4) and ELISA ([B]; n = 5) were performed at 24 hours after NMDA injection and the results showed that both the mRNA and protein levels of TNF-α significantly increased after NMDA injection and were suppressed by TO90. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 8
 
Effects of TO90 on the expression of NF-κB p65 and p-p38 MAPK at 24 hours after intravitreal injection of NMDA. Western blotting showed that NMDA induced an increase in p-p38 protein level, and TO90 significantly suppressed the elevation of p-p38 expression ([A, B]; n = 4). Western blotting and real-time PCR showed both protein ([C, D]; n = 3) and mRNA ([E]; n = 4) levels of NF-κB subunit p65were significantly increased after NMDA injection and suppressed by TO90. Error bars are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Six mice were used in each group.
Figure 8
 
Effects of TO90 on the expression of NF-κB p65 and p-p38 MAPK at 24 hours after intravitreal injection of NMDA. Western blotting showed that NMDA induced an increase in p-p38 protein level, and TO90 significantly suppressed the elevation of p-p38 expression ([A, B]; n = 4). Western blotting and real-time PCR showed both protein ([C, D]; n = 3) and mRNA ([E]; n = 4) levels of NF-κB subunit p65were significantly increased after NMDA injection and suppressed by TO90. Error bars are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Six mice were used in each group.
Figure 9
 
Effects of TO90 on the formation of Aβ at 24 hours after NMDA injection. Expression of Aβ in the mouse retina of the control (A), NMDA plus vehicle–treated (B), and NMDA plus TO90–treated (C) groups. Negative controls were obtained by replacing the primary antibody with PBS (D). There was a considerable increase in Aβ immunoreactivity in the GCL and INL in the NMDA–treated retinas (arrows), and TO90 significantly reduced the protein expression of Aβ (E, F). The mRNA level of BACE1 was analyzed by real-time PCR, the expression of BACE1 was significantly upregulated by NMDA but was downregulated in the retina of NMDA-injected mice treated with TO90 (G). Data are shown as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Figure 9
 
Effects of TO90 on the formation of Aβ at 24 hours after NMDA injection. Expression of Aβ in the mouse retina of the control (A), NMDA plus vehicle–treated (B), and NMDA plus TO90–treated (C) groups. Negative controls were obtained by replacing the primary antibody with PBS (D). There was a considerable increase in Aβ immunoreactivity in the GCL and INL in the NMDA–treated retinas (arrows), and TO90 significantly reduced the protein expression of Aβ (E, F). The mRNA level of BACE1 was analyzed by real-time PCR, the expression of BACE1 was significantly upregulated by NMDA but was downregulated in the retina of NMDA-injected mice treated with TO90 (G). Data are shown as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 50 μm. Six mice were used in each group.
Table
 
Sequences of Primers for Real-Time PCR
Table
 
Sequences of Primers for Real-Time PCR
Gene Forward Primer Reverse Primer
Caspase-3 5′-AACCAGATCACAAAATTCTGCAAA-3′ 5′-TGGAGTCCAGTGAACTTTCTTCAG-3′
Bax 5′-CCAGGATGCGTCCACCAAG-3′ 5′-AAGTAGAAGAGGGCAACCAC-3′
Bcl-2 5′-TGGGATGCCTTTGTGGAACTAT-3′ 5′-AGAGACAGCCAGGAGAAATCAAAC-3′
BACE1 5′-TCCGGCGGGAGTGGTATTATGAA-3′ 5′-ATCCGGGAACTTCTCCGTCGA-3′
TNA-α 5′-GCCTCTTCTCATTCCTGCTT-3′ 5′-CTCCTCCACTTGGTGGTTTG-3′
NF-κB p65 5′-AGGCTTCTGGGCCTTATGTG-3′ 5′-TGCTTCTCTCGCCAGGAATAC-3′
LXRα 5′-AGGAGTGTCGACTTCGCAAA-3′ 5′-CTCTTCTTGCCGCTTCAGTTT-3′
LXRβ 5′-AAGCAGGTGCCAGGGTTCT-3′ 5′-TGCATTCTGTCTCGTGGTTGT-3′
ABCA1 5′-TCCTCATCCTCGTCATTCAAA-3′ 5′-GGACTTGGTAGGACGGAACCT-3′
GAPDH 5′-GTATGACTCCACTCACGGCAAA-3′ 5′-GGTCTCGCTCCTGGAAGATG-3′
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