Animals
All procedures involving, and care of, animals adhered to the guidelines of Animal Ethics Committee of Eye and ENT Hospital of Fudan University (Shanghai, China), and conformed to the standards of the National Institute of Health and Association for Research in Vision and Ophthalmology. Adult (6-8 weeks old), postnatal (PN) 3-day-old C57BL/6J mice were purchased from the Chinese Academy of Sciences (Shanghai, China).
Induction of retinal light damage and hematoxylin-eosin (HE) staining
Following a 1-day dark adaptation, C57BL/6J mice over the age of 6-8 weeks were subjected to 5,000 lux of cool white LED light, positioned around and on top of the light box, for 14 consecutive days. Atropine eye drop solution (1%; prepared at the EENT Hospital of Fudan University) was used daily for pupil dilation. The mice were dark-adapted again for 1 day before being returned to a normal environment. Mice in the control group were not exposed to light, and were maintained in a normal environment.
Retinal histology was evaluated using hematoxylin and eosin (HE). Briefly, tissues embedded in paraffin were sectioned at 5 μm, placed on glass slides, deparaffinized, and rehydrated, after which the retinas were stained with HE performed in accordance with a standard protocol. The thickness of outer nuclear layer (ONL) was quantified under a light microscope (Leica DM 4000B, German).
Müller cell culture
Müller cell cultures were prepared as described previously [26,27]. Briefly, mouse retina was dissected out and digested using a papain-based dissociation system (Worthington Biochemical; Lakewood, NJ) in accordance with the manufacturer's instructions. Dissociated cells were incubated at 37°C in Dulbecco's Modified Eagle's Medium/Ham's F-12 Medium (DMEM/F12) (Invitrogen; Carlsbad, CA) containing 1% penicillin-streptomycin (Invitrogen) and 10% fetal bovine serum (FBS, Invitrogen). Primary Müller cells were cultured, and adherent cells were passaged twice before being used in further experiments.The purity of Müller cells was evaluated by immunocytochemistry using immunolabeling with primary antibodies specific for glutamine synthetase (GS) [28] and vimentin [29], which are biomarkers of Müller cells.
Enzyme-linked immunosorbent assay (ELISA)
The levels of TNFα in mouse Müller-cell cultures were quantified using a TNFα mouse ELISA kit (Invitrogen) in accordance with manufacturer’s instructions. Absorbance was read at 450 nm using a microplate reader (please provide manufacturer’s information for this instrument.
Cellular proliferation assay
To assess cellular proliferation, Müller cells were dissociated into single-cell suspension using 0.25% EDTA-trypsin (Invitrogen), seeded at a density of 4,000 cells per well in 96-well culture plates containing DMEM/F12 medium supplemented with 10% FBS. TNFα (50 ng/ml; R&D systems, Minneapolis, MN) was added into the culture media of the treated groups of Müller cells, and the cells were allowed to incubate for 24 hours at 37℃.Cell proliferation was evaluated via Click-iT EdU Alexa Fluor 555 Imaging Kit (Invitrogen) [30,31] in accordance with manufacturer’s instructions. Briefly, EdU (10μΜ) was added to cell-culture media, and cells were incubated for 4 hours at 37℃; cells were then fixed and incubated with Hoechst 33342 (1:1000, Invitrogen) used to stain cell nuclei. Rabbit anti-TNFα monoclonal antibody (diluted 1:100; Abcam, Boston, MA) was added to the media used to culture Müller cell obtained from adult light-injured mice to specifically block the effect of TNFα. Five images were obtained per well, and the numbers of EdU+ cells and Hoechst + cells were counted using Image J 2.1.4.7 (NIH, Bethesda, USA). The ratio of EdU+ cells to Hoechst+ cells indicated the rate of Müller-cell proliferation.
RNA isolation
Müller cells were cultured at density of 106 cells/25 cm2 culture flask. Cells were then treated with 50 ng/ml TNFα for 24 hours, while control cells were treated with PBS. Cells were lysed using Buffer RLT (Qiagen, Valencia, CA) containing 1% β-mercaptoethanol (β-ME, Sigma, USA); RNA was then extracted in accordance with manufacturer’s instructions (Qiagen). NanoDrop 2000 (Thermo Scientific, Wilmington, DE) was used to measure RNA concentration.
Microarray hybridization and analysis
Müller cells, obtained from postnatal 3-day-old mice, were treated with TNFα (50 ng/ml) for 24 hours. To acquire biotin-labeled cRNA, two groups of RNA (with three samples per group) were amplified, labeled, and purified using GeneChip 3' IVT PLUS Reagent Kit (Affymetrix, Santa Clara, CA, US) in accordance with the manufacturer’s protocol. Array hybridization and washing procedures were performed using Affymetrix Gene Chip Mouse Genome 430 2.0 Hybridization, Wash and Stain Kit (Affymetrix) in Hybridization Oven 645 (Affymetrix) and Fluidics Station 450 (Affymetrix) according to the manufacturer’s instructions. Subsequently, GeneChip Scanner 3000 (Affymetrix) and Command Console Software 4.0 (Affymetrix) at default settings were used to scan the slides. After normalization, GeneSpring GX 11.5 Software was used for data analysis and hierarchical clustering. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for gene annotation and function/pathway analysis.
Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR
Selected gene-expression data, generated via microarray, were confirmed using real-time PCR (qPCR). For qPCR, RNA was reverse-transcribed into cDNA using SYBR Green real-time PCR kit (Takara, Osaka, Japan) according to the manufacturer’s instructions. Quantitative PCR was performed using a ViiA 7 Real-Time PCR System (Life Technologies, Pleasanton, CA). Primer sequences were designed and synthesized by Shenggong Company (Shanghai, China) and are shown in Table 1. Relative mRNA expression was normalized to that of β-actin used as endogenous control, and expressed as fold change calculated using the comparative CT method (2-ΔΔCT) with ViiA 7 Software (Life Technologies). The primers shown in Table 1 were also used for conventional RT-PCR, which was carried out at the annealing temperature of 60°C for 32 cycles.
Western blotting
Cells or tissues from 10 mice were pooled together and lysed in radioimmunoprecipitation (RIPA) buffer (Beyotime, Shanghai, China) on ice for 30 min. The lysates were centrifuged at 1000 rpm at 4°C for 10 min to obtain the supernatants. After boiling for 5 min, each 2 μg/μl sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (PVDF, 0.45 μm; Millipore, Bedford, MA). The membrane was blocked using 5% bovine serum albumin (BSA, Beyotime) for 1 hour at room temperature and incubated with the following monoclonal primary antibodies: rabbit anti-TNFα (Abcam, Boston, MA), rabbit anti-GAPDH (CST, Massachusetts State, USA), rabbit anti-Stat3 (CST), and mouse anti-phospho-Stat3 (CST) diluted 1:1000 in 2% BSA ) overnight at 4°C. After washing thrice with PBS containing 0.1% Tween-20 (Beyotime, Shanghai, China), the membranes were probed with secondary antibodies (diluted 1:5000 in 2% BSA). Jak inhibitors ruxolitinib or tofacitinib (at 10 μM; Selleck, Houston, USA) were added 2 hours, and exogenous TNFα (R&D systems) was added 30 min, before the cells were harvested. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control.
Simple western analysis
Simple western analysis was performed using 12-230 kDa Wes Separation Module and 8x25 capillary cartridges in accordance with the manufacturer’s protocol (ProteinSimple, California, USA). Briefly, after preparing standard pack reagents, samples, and primary antibodies (diluted 1:50) in accordance with manufacturer’s instructions, the samples and biotinylated ladder were denatured in a heating block for 5 min and stored on ice until use. Luminol-S and peroxide, supplied in the detection module, were combined in a microcentrifuge tube. The biotinylated ladder, samples, primary antibodies, secondary antibodies, and reagents were dispensed into the assay plate using volumes shown in the plate diagram, and the plate was centrifuged for 5 minutes at 2500 rpm and RT. After completion of the analysis, results were evaluated using Compass software v3.1The following monoclonal primary antibodies were used in this procedure: rabbit anti-GAPDH (CST), rabbit anti-p44/42 MAPK (Erk1/2, CST), and rabbit anti-Phospho-p44/42 MAPK (CST). ERK inhibitor FR180204 (10 μM, Selleck) was added 2 hours, and exogenous TNFα (R&D systems) was added 30 min, before the cells were harvested.
Statistical analysis
All data were analyzed using SPSS 19.0 (IBM Corporation, Armonk, NY), and Student's t-test was used to compare differences between two groups All results are shown as mean ±SD; p <0.05 was considered statistically significant.