Lycium ruthenicum water extract preserves retinal ganglion cells in chronic ocular hypertension mouse models

Lycium ruthenicum Murray (LR), known as “black goji berry” or “black wolfberry”, is widely utilized in chinese herbal medicine. LR fruit showed its antioxidant and/or anti-inflammation activity in treating cardiac injury, experimental colitis, nonalcoholic fatty liver disease, fatigue, and aging. Glaucoma is the leading cause of irreversible blindness. Besides elevated intraocular pressure (IOP), oxidative stress and neuroinflammation were recognized to contribute to the pathogenesis of glaucoma. This study investigated the treatment effects of LR water extract (LRE) on retinal ganglion cells (RGCs) threatened by sustained IOP elevation in a laser-induced chronic ocular hypertension (COH) mouse model and the DBA/2J mouse strain. The antioxidation and anti-inflammation effects of LRE were further tested in the H2O2-challenged immortalized microglial (IMG) cell line in vitro. LRE oral feeding (2 g/kg) preserved the function of RGCs and promoted their survival in both models mimicking glaucoma. LRE decreased 8-hydroxyguanosine (oxidative stress marker) expression in the retina. LRE reduced the number of Iba-1+ microglia in the retina of COH mice, but not in the DBA/2J mice. At the mRNA level, LRE reversed the COH induced HO-1 and SOD-2 overexpressions in the retina of COH mice. Further in vitro study demonstrated that LRE pretreatment to IMG cells could significantly reduce H2O2 induced oxidative stress through upregulation of GPX-4, Prdx-5, HO-1, and SOD-2. Our work demonstrated that daily oral intake of LRE can be used as a preventative/treatment agent to protect RGCs under high IOP stress probably through reducing oxidative stress and inhibiting microglial activation in the retina.


Introduction
Lycium ruthenicum Murray (LR), also called "black goji berry", or "black wolfberry", has traditionally been utilized in medical practices to address conditions such as abnormal menopause, menstruation, and hypertension (Liu et al., 2020).LR fruit contains a rich assortment of compounds, such as anthocyanins, phenolic acids, polysaccharides, carotenoids, alkaloids, essential oils, and fatty acids.LR fruit has multifaceted functions including antioxidant, anti-fatigue, immuneenhancement, and anti-aging properties (Wang et al., 2018).LR extract (LRE) decreased the contents of lipid peroxidation and malondialdehyde (MDA) in serum and brain, accompanied by increased activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX), in D-galactose induced aging mice (Cui et al., 2023).Polyphenols in LRE had neuroprotective effects against acrylamide-induced neurotoxicity (Pang et al., 2023).Polysaccharides in LRE protected cortical neurons against oxygenglucose deprivation/reperfusion in neonatal hypoxic-ischemic encephalopathy (Deng et al., 2020).
Glaucoma is a leading cause of irreversible blindness, affecting an estimated 111.8 million people worldwide by 2040 (Allison et al., 2020).Progressive degeneration of retinal ganglion cells (RGCs) and subsequent visual field loss is the main symptom of glaucoma (Jayaram et al., 2023).Besides elevated intraocular pressure (IOP), mounting evidence suggests that additional mechanisms, such as oxidative stress and neuroinflammation, contribute to the pathogenesis and progression of glaucoma (Baudouin et al., 2021).In glaucoma, compromised retinal blood flow could trigger the generation of reactive oxygen species (ROS) in the retina (McMonnies, 2018;Wang et al., 2023).Elevated IOP can compress the optic nerve fiber and then reduce retrograde neurotrophin support for RGC axons, further contributing to ROS production in RGCs (McMonnies, 2018).Excessive ROS in RGCs directly induces their degeneration in glaucoma (Fernández-Albarral et al., 2024).ROS can also mediate microglial activationrelated inflammation and neurotoxicity, which is a significant contributor to the pathogenesis of glaucoma (Baudouin et al., 2021).The interaction between oxidative stress and inflammatory response in microglia is known to contribute to its activation, playing a role in neurodegenerative diseases including glaucoma (Simpson and Oliver, 2020).
In this study, we investigated the effects of LRE on the retina in 2 mouse models mimicking glaucoma: a laser-induced chronic ocular hypertension (COH) mouse model and the DBA/2J mouse strain.Our primary objectives were to evaluate the impact of LRE on the function and survival of RGCs and to assess its ability to modulate oxidative stress and microglial activation under high IOP mimicking glaucoma.

Preparation of Lycium ruthenicum water extract
Lycium ruthenicum water extract (LRE) was provided by Eu Yan Sang (HK) Ltd.LR from Qinghai, the People's Republic of China, was used for this study.To prepare the LRE, 500 g dried LR was separated into 10 equal portions.Then, one portion of LR was put into 250 mL de-ionized water at 50 °C-60 °C for 15 min.Subsequently, LR was removed, and the extract was filtered.The filtrate was added with de-ionized water to make up to a final volume of 250 mL.This 250 mL filtrate was used to extract the next portion of LR as above procedures until all 10 portions were processed.Each milliliter of the final LRE contained 2 g of the crude drug.The LRE was stored in a refrigerator at 4 °C.

Detection of anthocyanins and anthocyanidins in LRE
To test the anthocyanins and anthocyanidins contents, 39 g LRE was subjected to high-performance liquid chromatography (HPLC) (conducted by Eurofins Food Testing HK Ltd).The contents of anthocyanins and anthocyanidins are listed in Table 1.There were 0.0313% (w/w) anthocyanins and 0.0103% (w/w) anthocyanidins in the LRE.Delphinidine 3 glucoside was the major ingredient which was 0.027% (w/w) in the LRE and used as the standard for quality control.

Animals
CX3CR-1 GFP knock-in/knock-out mice (Jackson Laboratory, stock No. 005582), DBA/2J mice (Jackson Laboratory, stock No. 000671), and C57BL/6J mice were obtained from the Laboratory Animal Unit of the University of Hong Kong.The mice were housed in a controlled environment with a 12-h light/dark cycle, maintaining a pathogen-free setting.All animal procedures were conducted in accordance with the ARRIVE guidelines and approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong.

Laser photocoagulation induced COH mouse model
COH mouse model was constructed by laser photocoagulation on the corneal limbus according to an optimized protocol (Feng et al., 2013).Briefly, female CX3CR1 +/GFP mice at the age of 6 months were anesthetized by intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg).The right eyes were applied with 1% cyclopentolate hydrochloride (Mydriacyl, Alcon Labs, Inc., Fort Worth, TX, USA) for pupil dilation followed by proparacaine hydrochloride (0.5% alcaine, Alcon) for topical anesthesia.The anterior chamber was punctured with a 30 G syringe needle to drain the aqueous humor.Subsequently, 60-80 consecutive laser spots (Size, 500 μm; power, 800 mW; pulse duration, 50 msec) were delivered perpendicularly to the limbus surface, while sparing the nasal area, using a 532 nm laser (Lumenis Novus Spectra, Yokneam, Israel).About 10% of mice eyes exhibiting anterior chamber hemorrhage, cataract, or corneal ulcer following the induction of IOP elevation were excluded from the study.

Measurement of IOP
The IOP of mouse eyes was measured using a rebound tonometer (Icare ® TonoLab, Colonial Medical Supply, Franconia, NH).In the laser-induced COH model, IOP measurements were performed on awake mice without any eye drops, whereas measurements were conducted on general anesthetized DBA/2J mice under local corneal anesthesia.Each IOP value was determined by averaging six consecutive measurements and the IOP level of the mouse eye was represented by the average of three such values.

Animal feeding and grouping
The dose of LRE oral feeding at 2 g/kg body weight was chosen according to the previous LRE dose-response study in mice suffered radiation injury (Duan et al., 2015).For the laser-induced COH mice, daily LRE oral feeding started at 7 days before laser photocoagulation till 30 days after the induction of COH.Distilled water was fed as a placebo control.At the end of the experiment, there were 22 normal control eyes, 20 eyes with COH fed with water, and 22 eyes with COH fed with LRE for data analysis.For DBA/2J mice, daily LRE feeding started at 6 months of age for 4 months and ended at 10 months of age.C57BL/6J mice at 10 months of age were used as wild-type controls.There were 10 mice in each group, both eyes of the DBA/2J and C57BL/6J mice were used for analysis.

Flash electroretinography (ERG)
The function of RGCs was evaluated using flash ERG, following the standard protocol of the International Society for Clinical Electrophysiology of Vision.Following general anesthesia, 1% cyclopentolate hydrochloride (Mydriacyl, Alcon) was applied to the eyes to dilate the pupil and then proparacaine hydrochloride (0.5% alcaine, Alcon) was applied as topical anesthesia for 5 min.Afterward, ERG signals were recorded using an ERG system (Espion E2 Electrophysiology System, Diagnosys LLC, USA).Full-field flash ERG test was conducted at a photopic intensity of 3.0 and 10.0 cd s.m -2 to detect the photopic negative response (PhNR) which represents the RGCs activity.The acquired data were

Retinal ganglion cell counting on flatmounted retina
In the COH mice study, the RGC survival was evaluated by counting the number of Brn-3a+ cells on the flat-mounted retina.Mice were euthanized, and the eyeballs were enucleated and fixed in 4% paraformaldehyde (PFA) for 1 h.Subsequently, the retina was dissected from the sclera and flat mounted with the RGC layer faceup under a stereo microscope.To stain the RGCs, the retinas were rinsed with PBS, followed by blocking with a solution containing 10% normal donkey serum and 0.1% Triton X-100 in PBS.The retinas were then incubated overnight at 4 °C with goat anti-Brn-3a (1:500, Santa Cruz, Dallas, USA).After thorough washing, the retinas were incubated with a secondary antibody, Alexa-568 fluorescent-conjugated donkey anti goat IgG secondary antibody (1:500; Thermo Fisher Scientific, Waltham, MA, USA), for 2 h at room temperature.To visualize cell nuclei, the retinas were counterstained with 4',6-Diamidino-2-phenylindole (DAPI) (1: 1000).Confocal images were acquired using a ZEISS LSM 800 confocal microscope (Carl Zeiss Microscopy GmbH, Germany), and the number of Brn-3a+ cells was quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Retinal section histological analysis
In the DBA/2J mice study, the RGC survival was evaluated by counting the nuclei in the RGC layer.After standard eyeball fixation, dehydration, and paraffin embedding, retinal cross sections containing optic nerve were collected for further analysis.These sections were stained with hematoxylin and eosin (H&E) and mounted using DPX mounting medium.Images were captured using an optical microscope (Nikon Eclipse 80i, Tokyo, Japan).The number of nuclei in the RGC layer was quantified using ImageJ software.
For the experiments, IMG cells were seeded in 96-well plates (1 × 10 4 cells/well) or 12-well plates (1.5 × 10 5 cells/well) in DMEM with high glucose supplemented with 1% FBS and allowed to incubate overnight before the treatments.To test the dose-response cytotoxicity (LDH test) induced by H2O2 or LRE, cells were treated with 50, 100, 200, 300, 400, and 800 µM of H2O2 (Merck Millipore, Burlington, MA, USA) or 100, 200, 400, and 800 μg/mL of LRE for 24 h followed by LDH test.The antioxidant effect of LRE was first evaluated in the pretreatment test.The IMG cells were incubated with LRE for 2 h at 10, 50, 100, and 200 μg/mL before 300 µM H2O2 stress.In the further experiments, the timing for 200 μg/mL LRE to be applied were evaluated 2 h before (pretreatment), at the same time (simultaneous), or 2 h after (post-treatment) the H2O2 stimulation in the IMG cells.To explore the mechanism of LRE antioxidative effect, the expressions of H2O2 decomposition related enzymes including catalase, GPX-1, GPX-4, peroxiredoxin (Prdx)-1, Prdx-2, Prdx-3, Prdx-4, Prdx-5, and Prdx-6 were detected at 2 h after 200 μg/mL LRE treatment in IMG cells.Furthermore, LRE pretreatment effect on the antioxidant genes such as heme oxygenase (HO)-1 and SOD-2 were evaluated.Untreated cells were applied as controls.Each experiment was repeated three times.

Cytotoxicity assay
The cytotoxicity of IMG cells was assessed using a Pierce LDH Cytotoxicity Assay Kit (Cat.88953, Thermo Fisher Scientific).Cells were seeded in a 96-well plate.After 24 h of treatment with H2O2 and/ or LRE, the cell culture supernatant was collected and mixed with LDH assay buffer in a new 96-well plate.After incubation for 30 min, the reaction was stopped by adding the stop solution to the sample wells.The absorbance at 490 nm and 680 nm was measured using a spectrometry (EnSpire Multimode Plate Reader, PerkinElmer, Waltham, MA, USA).The LDH activity was determined by subtracting the absorbance at 680 nm from the absorbance at 490 nm.

Quantitative reverse transcription polymerase chain reaction
Total RNA from mouse retinas or IMG cell samples was extracted using an RNA extraction kit (Cat.74106, QIAGEN, Hilden, Germany).Subsequently, the RNA was reverse transcribed into cDNA using a reverse transcription kit (Cat. 205413, QIAGEN).The resulting cDNA samples were then subjected to real-time PCR analysis using an SYBR Green PCR Kit (Cat. 208056, QIAGEN).The amplification process consisted of an initial incubation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 15 s.Melting curve analysis was performed to ensure amplification specificity.The primer sequences of the tested genes are listed in Table 2.The gene expression levels of target genes were normalized to housekeeping gene β-actin.The 2 −ΔΔCT formula was applied for calculation purposes.

Statistical analysis
GraphPad Prism 8.0 software (GraphPad software, San Diego, California, USA) was utilized to create statistical graphs.Data analysis was performed using SPSS software for Windows (version 20.0; SPSS, Inc., IL, USA).To compare multiple groups, a one-way ANOVA was conducted, followed by Fisher's Least Significant Difference (LSD) test for multiple comparisons or Dunnett's test when the variances of all groups were not equal.When comparing two groups, two-tailed Student's t-tests were employed.The data was presented as mean ± SD, and a significance level of *p < 0.05 was considered statistically significant.
The antioxidant properties of LRE treatment were further investigated in DBA/2J mice.Similar to the changes in COH eyes, there was increased 8-OHdG expression in the inner retina of water-fed DBA/2J eyes (Figure 4D).Semi-quantification revealed a significant increase in 8-OHdG expression in the retina of waterfed DBA/2J mice compared to the C57BL/6J mice (***p < 0.001,

LRE upregulated antioxidant enzymes in microglial cells under H2O2 stress
The antioxidation mechanisms of LRE were investigated by adding LRE to H2O2-treated IMG cells in vitro.H2O2 induced significant cytotoxicity in IMG cells in a concentration-dependent manner, starting from 200 μM.H2O2 at 300 μM increased cytotoxicity to IMG by 2.12 ± 0.28 folds than control (***p < 0.001, Figure 6A).Therefore, 300 μM was chosen to be the oxidative stress stimulator in the following experiments.LRE can also be a stressor to IMG cells.Below 200 μg/mL, there was no significant difference between LRE and no treatment control.However, when the LRE concentration increased to 400 and 800 μg/mL, there was significant cytotoxicity in IMG cells (Figure 6B).The protective effect of LRE was first evaluated by pretreating IMG cells with LRE from 10 to 200 μg/mL for 2 h and then challenging the cells with 300 μM H2O2.LRE significantly prevented H2O2 induced cytotoxicity in IMG cells in a concentration-dependent manner.200 μg/mL LRE maintained IMG cell's reaction at a level similar to no treatment control (Figure 6C).Following this, the best time for LRE application was further evaluated.200 μg/mL LRE treatment at 2 h before H2O2 stimulation (pretreatment) significantly reduced the cytotoxicity to 0.85 ± 0.09 folds (***p < 0.001 vs. H2O2) and simultaneous administration of LRE with H2O2 also had a protective effect (**p = 0.003 vs. H2O2, Figure 6D).But when LRE was applied at 2 h after H2O2 stimulation (post-treatment), there was no protective effect.

Discussion
Neuroprotection, antioxidation, and anti-inflammation effects of LR were summarized by Lee and Choi (2023).Our study investigated the effects of LRE in alleviating RGC degeneration in mouse models mimicking glaucoma.Daily LRE oral feeding significantly preserved RGC function, reduced apoptosis, and promoted RGC survival in the laser-induced COH mouse model Glaucoma is characterized by progressive RGC loss which leads to irreversible blindness.Current glaucoma treatment relies primarily on IOP lowering surgery/medication, however, was not enough to halt the disease progression (Saifi et al., 2023).Previous studies investigating the pathogenesis of glaucoma highlighted the critical contributions of oxidative stress and microglial activation (Wei et al., 2019;Fan Gaskin et al., 2021).IOP-independent neuroprotective treatments thus are warranted for the future development of glaucoma therapies (Jayaram et al., 2023).Neuroprotective treatments in conjunction with IOP lowering methods might slow down the disease progression especially if the diagnosis is confirmed at early stage.Two mouse models mimicking glaucoma were used in this study with different LRE treatment starting time.In the laser-induced COH model, the LRE oral feeding started at 7 days before the IOP increase.LRE was used as a preventative supplement aiming to potentiate the retinal resilience against high IOP.In the DBA/2J mice, the pigment dispersion in the anterior chamber was detectable from 5-6 months of age and became prominent at 9 months of age (Libby et al., 2005).The dispersed iris pigment obstructs the trabecular meshwork, resulting in secondary IOP elevation in DBA/2J eyes.Libby et al. (2005) found that IOP in DBA/2J eyes started to increase from 6 months of age, reached the highest level at 10 months of age, and then declined at 12 months of age.In this DBA/2J mice, oral feeding of the LRE started at 6 months of age indicating early interference in glaucoma.Oral taking of LRE at a dose of 2 g/kg improved the RGC function and survival without affecting the IOP elevation in both the COH and the DBA/2J mouse models.
LRE showed its antioxidant and anti-inflammatory effects in dextran sulfate sodium induced murine experimental colitis, exhaustive exercise-induced cardiac injury, high-fat diet-induced nonalcoholic fatty liver disease, and radiation injury (Duan et al., 2015;Lin et al., 2015;Hou et al., 2019;Lu et al., 2020;Zong et al., 2020).LRE administration was indicated to enhance the expressions of antioxidant enzymes such as SOD, GPX, and catalase in affected tissues, countering oxidative stress.In fact, anthocyanins, polyphenols, and polysaccharide from LR could activate the Nrf2/ HO-1 signaling pathway which regulates a host of antioxidant enzymes (Deng et al., 2020;Tian et al., 2021;Gao et al., 2022).The anthocyanins and anthocyanidins in the LRE might be the key bioactive agents for the protective effect on RGCs under high IOP, they occupied 0.0416% (w/w).LRE oral feeding ameliorated the oxidative stress marked by 8-OHdG increase dominantly in the inner retina of the COH and the DBA/2J eyes.LRE pretreatment of IMG for 2 h successfully upregulated the H2O2 decomposition related enzyme GPX-4 and Prdx-1 gene expression.When H2O2 was added to the LRE primed IMG cells, antioxidant genes (HO-1 and SOD-2) expression increased even higher than the LRE treatment control group.Upregulated GPX-4, Prdx-1, HO-1, and SOD-2 in the LRE pretreatment group successfully prevented the H2O2 induced LDH increase to a level similar to the control group.While this in vitro finding is consistent with the reports in other systems, the finding in the COH model that HO-1 and SOD-2 returned to normal levels with LRE oral feeding was unexpected.We postulated that retinal tissue upregulated the antioxidant genes to combat oxidative stress induced by IOP elevation.Daily LRE intake, on the other hand, potentiated the antioxidant ability of retinal tissue, reducing ROS levels and restoring the retinal microenvironment to normalcy, obviating the need for further antioxidant gene increase.The inconsistency between our in vivo findings and others may stem from variations in pathological conditions and their temporal dynamics, which necessitates validation by further investigations.
The anti-inflammatory effect of LRE in the glaucoma models was evaluated by counting the Iba-1 positive cells in the retina.Elevated IOP caused significantly increased microglial activation.LRE as a pretreatment agent decreased Iba-1 positive cells in the COH eyes.In the IMG cell culture, the cytotoxicity of H2O2 can be prevented when LRE was applied as pre-or simultaneous but not post-treatment.In the DBA/2J mice, the RGC function started to be impaired from 3 months of age when the IOP remained at a normal level (Saleh et al., 2007;Harazny et al., 2009).Activation of retinal microglia in the DBA/2J mice is much earlier than 6 months when the LRE oral feeding started.LRE as a post-treatment did not reduce Iba-1 positive cells in the DBA/2J eyes.This differential effect of LRE on microglial activation under high IOP in COH and DBA/2J mice could be induced by the intricate genetic background and the considerable variation in disease progression (Turner et al., 2017).We demonstrated that daily LRE feeding preserved the function of RGCs and enhanced their survival under the threat of sustained IOP elevation using two chronic glaucoma mouse models.This protective effect was likely attributed to reduced oxidative stress in the retinal neurons by LRE treatment, while inhibition of microglial activation could also contribute.In vitro study found that LRE pretreatment protected IMG cells from H2O2 induced damage by priming these microglial cells into an antioxidative status with upregulated GPX-4, Prdx-5, HO-1, and SOD-2.LRE may also confer neuroprotection to other retinal diseases such as retinitis pigmentosa and age-related macular degeneration in which oxidative stress in the ONL was featured (Murakami et al., 2020;Jabbehdari and Handa, 2021).LRE contains various bioactive components, such as anthocyanins, polyphenols, and polysaccharides.Future studies investigating the roles of specific LRE components in treating glaucoma would enhance the translation of LRE treatment to patients.

Conclusion
LRE oral feeding provided antioxidative effect, preserving the RGCs function and survival as a neuroprotective measure for glaucoma.The 4 months continuous oral feeding in DBA/2J mice that ended at 10 months of age is a precious indication for clinical application of LRE as a supplement to the current glaucoma treatment strategies which focus on IOP control.

FIGURE 1
FIGURE 1 LRE preserved RGC function in the eyes of the laser-induced COH and DBA/2J mice without affecting the elevated IOP.(A) A schematic representation of the experimental flows in the laser-induced COH mouse model (upper lane) and the DBA/2J mouse strain (lower lane) (Diagram is created in BioRender.com).(B) IOP measurement on the awake mice in the laser-induced COH mice after 30 days of laser photocoagulation.(C) IOP measurement on the anesthetized C57BL/6J and DBA/2J mice at the age of 10 months.(D-G) Representative ERG waves were shown on the assessment of RGC function using a photopic ERG test with flash strengths of 3.0 and 10.0 cd s.m -2 in the laser-induced COH, DBA/2J, and control eyes after LRE treatment.(H-K) Bar chart figures demonstrated that LRE oral feeding significantly increased the PhNR amplitudes in the COH eyes at photopic 10.0 cd s.m -2 and in the DBA/2J mice eyes at photopic 3.0 cd s.m -2 .*, p < 0.05; **, p < 0.01; ***, p < 0.001; n. s., not significant.

FIGURE 2
FIGURE 2 LRE promoted RGC survival in the eyes of the laser-induced COH and DBA/2J mice.(A) Representative central retina (upper lane) and peripheral retina (lower lane) images of Brn-3a stained RGCs (red) in the retinal flat-mounts from the normal, water-fed, and LRE-fed COH mice.The density of RGCs markedly reduced in the water-fed COH eyes (middle column).Scale bar, 25 µm.(B) 30 days of LRE oral feeding significantly increased the RGC number in the central and peripheral retina of the COH eyes.(C) Representative images of H&E-stained retinal sections from the C57BL/6J control, water-fed, and LRE-fed DBA/2J mice.There were fewer cells in the RGC layer in the water-fed DBA/2J mice both at the central and peripheral retina.Scale bar, 25 µm.(D) 4 months of LRE oral feeding significantly increased nucleus density in the RGC layer of both the central and peripheral retina of the DBA/2J mice.*, p < 0.05; **, p < 0.01.

FIGURE 3 LRE
FIGURE 3 LRE inhibited apoptosis in the RGC layer of the laser-induced COH and DBA/2J mice.(A) Representative images of caspase-3 positive cells (red, arrows) in the retinal sections from the normal, water-fed, and LRE-fed COH mice.Scale bar, 25 µm.(B) LRE significantly decreased the number of caspase-3 positive cells in the RGC layer of the COH eyes.(C) Representative images of caspase-3 positive cells (red, arrows) in the retinal sections from the C57BL/6J control, water-fed, and LRE-fed DBA/2J mice.Scale bar, 25 µm.(D) LRE significantly decreased the number of caspase-3 positive cells in the RGC layer of the DBA/2J mice.*, p < 0.05; ***, p < 0.001.

FIGURE 5 LRE
FIGURE 5 LRE affected microglial activation in the retina of the laser-induced COH eyes.(A) Representative images of Iba-1 positive cells (red, arrows) in the retinal sections from the normal, water-fed, and LRE-fed COH mice.Scale bar, 25 µm.(B) LRE significantly decreased the number of Iba-1 positive microglia in the retina of COH eyes.(C) The gene expressions of IL-1β, IL-6, IL-10, and CX3CR1 in the retina of normal, water-fed, and LRE-fed COH eyes.(D) Representative images of Iba-1 positive cells (red, arrows) in the retinal sections from the C57BL/6J, water-fed, and LRE-fed DBA/2J mice.Scale bar, 25 µm.(E) LRE did not change the Iba-1 positive microglial cell number in the retina of DBA/2J mice.*, p < 0.05; **, p < 0.01; ***, p < 0.001.

TABLE 1
Identification of anthocyanins and anthocyanidins in LRE by HPLC.

TABLE 2
Primer sequences used for real-time PCR.