RMECs culture
Rat primary RMECs were purchased from the Cell Biologics Company (catalogue no. RA-6065; Chicago, IL, USA). The cell line was recovered and cultured in accordance with the supplier’s instructions. Cells from passages 4–10 were used in this study.
Primary retinal microglia culture
Microglia were isolated from the retinas of newborn Sprague–Dawley rats. Newborn Sprague–Dawley rats (1–3 days old) were obtained from the Shanghai SLAC Laboratory Animal Company, and were sacrificed by decapitation. Treatment of animals was complied with the rules of the “Instruction and Administration of Experimental Animals”, and was approved by the Eye and ENT Hospital of Fudan University. The dissected retinas were collected and digested with 0.25 mg/mL trypsin at 37 °C. After 5 min, the trypsin was deactivated with DMEM/F12 (Gibco, Carlsbad, CA, USA) containing 20% FBS (Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich, Billerica, MA, USA). The digested tissues were mechanically dissociated into a single cell suspension and collected by centrifugation. The cells were then resuspended in a DMEM: F12 (1:1) solution containing 20% FBS and plated in T75 cell culture dishes at 106 cells/dish. After 12 days, the supernatant containing microglia was collected and centrifuged, and the cells were resuspended at the appropriate density, depending on the experiment. The microglia were identified by immunocytochemical staining using microglia-specific antibodies, OX42 (targeting CD11b/c) and ED1 (targeting CD68). The morphology of the isolated microglia was examined by phase-contrast microscopy (Nikon) and fluorescence microscopy (Leica Microsystems).
Flow cytometry
Isolated microglia were collected in DMEM/F12 containing 20% FBS and centrifuged for 10 min. After washing in PBS, the cell precipitate was resuspended in blocking solution (PBS containing 5% FBS and 1% BSA) and labelled with Alexa-Fluor-647-conjugated mouse anti-OX42 (1:100, Abcam, Cambridge, MA) for 15 min at 4 °C. The OX42-positive cells were counted by flow cytometry (Coulter Epics XL, Beckman-Coulter, Fullerton, CA).
Immunofluorescence assay
For immunofluorescence assays, cell cultures were fixed with 4% paraformaldehyde in 0.01 M PBS for 10 min and then washed with PBS. After washing, the cultures were incubated in blocking buffer (5% normal goat serum) for 30 min at 37 °C. The cells were then incubated overnight at 4 °C with primary antibodies specific for markers of microglia (mouse OX42, 1:100, and mouse ED1, 1:100; Abcam, Cambridge, MA, USA) or RMECs (mouse anti-von Willebrand factor (vWF, 1:100; Abcam). After washing with PBS, the cells were incubated with the appropriate secondary antibodies for 30 min at 37 °C and counterstained with DAPI (1:1000; Molecular Probes/Thermo Fisher Scientific, Waltham, MA, USA). The labelled cells were examined by fluorescence microscopy (Leica Microsystems).
Activation of microglia by LPS
The harvested microglia were seeded at 106 cells/well in a six-well culture plate pre-coated with 20 μg/mL poly-d-lysine (Sigma-Aldrich). Twenty-four hours after seeding, each well was washed three times with 0.1 M PBS and incubated with culture medium containing 0, 0.1, 1, 10, 100, or 1000 ng/mL lipopolysaccharide (LPS) (Escherichia coli OB4:1111; Sigma-Aldrich) for 24 h.
Assessment of microglial viability
The effects of LPS on the viability of microglia were measured using the cell proliferation reagent water soluble tetrazolium-1 (WST-1; Roche, Basel, Switzerland). The WST-1 assay is based on the cellular reduction of WST-1 by viable cells. Microglia were seeded in a 96-well microplate at 4 × 103 cells/well in 100 μL culture medium containing 0, 0.1, 1, 10, 100, or 1000 ng/mL LPS. The cells were incubated for 48 h at 37 °C in 5% CO2, and 10 μL WST-1 reagent was added to each well and incubated for 4 h at 37 °C in 5% CO2. The plate was thoroughly shaken for 1 min on a shaker. To detect the production of formazan, the absorbance of each well at 420–480 nm was measured relative to the blank wells on a microplate reader.
Measurement of microglial cytokine concentrations
After exposure to LPS, the culture media was collected and centrifuged. Aliquots of the supernatant (50 μL) were collected to measure the concentrations of TNFα and IL-1β using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA).
Transwell co-culture of microglia and RMECs
Freshly collected microglia were seeded onto 12-well Transwell collagen-coated membrane inserts (Corning Co., Corning, NY, USA). Separately, RMECs were grown to confluence in a collagen-coated 12-well plate. The microglia and RMECs were incubated in basal media for 24 h before co-culturing, and the Transwell inserts containing the retinal microglia (treated with or without LPS for 24 h) were placed into the wells containing RMECs. The 0.4 μm pore size of the Transwell prevents direct cell–cell interactions but allows the diffusion of soluble factors across the membrane (Fig. 1). After 24 h, the co-cultured cells were separated and cultured in fresh culture mediums for another 24 h, and then, the supernatant and cells were collected for further research. The experiment was divided into three groups: Con (RMECs without microglia), MG: (RMECs with static microglia), LPS-MG (RMECs with activated microglia).
Levels of angiogenesis-related growth factors in RMECs measured by ELISA
The supernatant of RMECs was collected and subjected to ELISA. The levels of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) were evaluated by sandwich ELISA (human VEGF-A and PDGF-BB ELISA kits, Abcam) according to the manufacturer’s instructions. Colorimetric analysis was performed and the absorbance was measured using an ELISA plate reader.
Tube formation assays
Tube formation assays were conducted on Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). A 96-well plate was coated with 50 μL/well Matrigel at 37 °C for 30 min. After co-culturing with microglia for 24 h, RMECs were seeded on the Matrigel at 1.5 × 104 cells/well in 100 μL medium. After 4 h, tube formation was observed and photographed with a microscope (Leica Microsystems). Images were analyzed using ImageJ (NIH public domain), and four parameters were measured for quantification of tube formation: total tube length, number of nodes, number of branches, and total branch length.
Wound healing assays
RMECs were plated at a density of 104 in 24-well plates and grown to 100% confluence. To investigate the migration capability of RMECs, a wound was created by scratching the confluent monolayer down the middle of each well using a 10 μL pipette tip. Scratched RMECs were washed with PBS, and then 500 μL of fresh DMEM without serum was added to each well. Cells were imaged immediately after stimulation (0 h) and then at 6, 12, and 18 h after wounding. ImageJ software was utilized to determine the percentage of wound closure.
Assessment of RMEC proliferation
RMECs were collected and seeded in 96-well plates at 1 × 104 cells/well and treated with WST-1 for 4 h. The absorbance of the wells was read at 450 nm using the Benchmark Plus microplate reader to evaluate RMEC proliferation.
Phenotype and tight junction of RMECs
ZO-1 and occludin are markers of tight junctions in RMECs.CD31 and CD34 are markers of endothelial cells. RMECs were harvested, and the protein expression levels of zonula occludens-1(ZO-1), occludin, CD31 and CD34, were determined by Western blotting. After the proteins were transferred onto a PVDF membrane, the blots were incubated with primary antibodies (mouse anti-ZO-1, 1:100; mouse anti-occludin, 1:100; mouse anti-CD31, 1:100; mouse anti-CD34, 1:100; all from Abcam) and probed with a horseradish-peroxidase-conjugated secondary antibody. Protein expression was detected using an enhanced chemiluminescence kit. The density of each band was quantified using ImageQuant software (ImageQuant TL v. 7.0, GE Healthcare, Piscataway, NJ, USA). All samples were assayed in triplicate.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 5.0. Measurement data were presented as‾x ± s. Differences between groups were evaluated using unpaired one-way ANOVA and LSD post-hoc tests. Each experiment was repeated three times. P < 0.05 was considered statistically significant.