Glutamate

glutamate clearance capacity was measured using Glutamine and glutamate Determination Kit (Sigma) following manufacturer’s protocol[28 ]. In brief, the culture medium was replaced with 1.5 mL of serum-free HBSS containing 100 mM glutamate (Abcam). Cells were returned to the incubator for two hours at 37 °C, 12.5 μL of supernatant was transferred to 96-well plate, and remaining glutamate in the medium was determined. The absorbance at 340 nM was obtained using BioTek Synergy HT Microplate Reader (BioTek, Winooski, VT). The concentration in the supernatant was calculated using the standard curve, and change in glutamate concentration was calculated. As a control, 293FT cells were treated the same way as the astrocytes. The concentration of total cell protein was determined using BCA Protein Assay Kit and used as a reference (Pierce, Rockland, IL).

For the in vitro analysis, R28 cells were seeded in 6-well plates at a density of 3 × 10⁵ cells/well and incubated overnight before treatment with different concentrations of glutamate (Abcam, Cambridge, UK) and for different durations. GSK872 (Selleck, Houston, TX, USA) and Nec-1 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in DMSO and diluted in culture medium. The cells were divided into five groups according to different treatments: Control group (without treatment), glutamate-treated, glutamate + DMSO treated, glutamate + GSK872 treated, and glutamate + Nec-1 treated groups. Then the cells were incubated for 24 h before being analyzed.
For the in vivo analysis, mice received an intravitreal injection of 1 μL solution containing NMDA and GSK872 (80 μM in saline) or NMDA and Nec-1 (40 μM in saline). The group treated with the same volume of solution containing NMDA and DMSO was used as control. All treatments were given intravitreally in both eyes. Five days after injection, eyes were enucleated and processed for further analysis.

Standard immunohistochemical procedures were used. Transverse sections of mouse brains sliced at 20 µm thickness were incubated with the following antibodies: GABA (rabbit anti-GABA, 1:500, Sigma), GABA_A (rabbit anti-GABA_A, 1:100, Millipore), GABA_B, (guinea pig anti- GABA_BR2, 1:500, Chemicon) and glutamate (rabbit anti-glutamate, 1:500; Abcam). Following 2 hour incubation with the primary antibodies at room temperature, the incubation with the appropriate secondary antibodies were performed for 1 hour. Then the sections were mounted with ProLong antifade reagent (Invitrogen). Each set of immunohistochemistry was performed on all tissue sections concurrently to control for variations in processing and allow quantitative analysis of staining intensity. ROIs were manually selected around individual cell bodies and/or processes. Optical density was calculated as a difference in ROI intensity and average intensity of the entire image divided by average intensity of the image, and represented as a percentage.