Fig 1: Microglia restricted ischemia-induced astrocyte response and neural injury. Primary cortical neurons were subjected to 5 min OGD. Immediately after OGD, culture media was replaced with regular neurobasal medium mixed with equal volume of glia-conditioned medium for 24 h. Glia-conditioned medium was obtained from cultured microglia, astrocytes or mixed microglia and astrocytes (microglia/astrocyte) at 24 h after 30 min OGD. (a) Representative images show staining of beta III Tubulin (green), DAPI (blue) and TUNEL (red) in primary cortical neurons exposed to OGD and glia-conditioned medium as indicated. Primary cortical neurons without exposure to OGD or glia-conditioned medium were used as controls. Scale bars: 20 µm. (b) Quantification of TUNEL+ cells after exposure to indicated treatment. CM: conditional medium. (c) Bar graphs show the level of released LDH from primary cortical neurons after exposure to indicated treatment. LDH release was expressed by normalizing the released LDH in the medium to the total LDH (released + intracellular). CM: conditional medium. (d) Primary astrocytes were cultured separately or together with microglia (microglia/astrocyte). After 30 min OGD and subsequent 24 h recovery, intracellular staining of GFAP and indicated cytokines was performed and analyzed by flow cytometry. Bar graphs show the percentage of GFAP+ cells expressing IL-1a, IL-1ß, iNOS, CCL2, TNF-a and IL-6. Error bars represent SD.; *P < 0.05; **P < 0.01.