Fig. 4

Isolated retinal microglia respond to P2X7 receptor stimulation with rapid retraction, gene activation but not migration. a Images of isolated retinal microglial cells taken before (left) and ~ 7min after (right) application of isotonic solution (Control), 250 μM BzATP (BzATP), or 250 μM BzATP (BzATP) ± 10 μM A839977 (A83) suggests P2X7 receptor leads to process retraction in vitro. Similar responses were found in > 7 experiments. Bar = 10 μm, real time in min on image; solution added at minute 3:00; see Fig. S4 for video. Elevated expression of Nos2 and Arg1 was detected in cultured retinal microglial cells following 4 h exposure to 1 mM ATP (b, n = 9 wells from 3 culture preparations) or 200 μM BzATP (c, n = 3 wells from 1 culture preparation; unpaired Student’s t test). d Representative images of isolated retinal microglial cells with Hoechst-stained nuclei after passing through a Boyden chamber, indicate that microglia migrate towards a 1 mM ATP gradient. Bar = 50 μm. e Correlation between number of Hoechst-stained nuclei in brain microglia per well and fluorescence at 340ex/527em (Pearson’s correlation r = 0.9396 with p = 0.0001; 1= 17 wells from 1 culture preparation). f Migration of retinal microglia towards 1 mM ATP was inhibited by exposure to 10 μM P2Y12 inhibitor AR-C 69931 (ARC) in the presence of ATP but not 1 μM A839977 (A83) in the presence of ATP. (1-way ANOVA with Sidak’s MC test; n = 17 Ctrl, 20 ATP, 20 ARC, 19 A83 wells from 4 culture preparations). Statistical significance shown as **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant