Fig. 8
P2X7R/Ca2+/NFAT/NF-kB pathway mediates microglial inflammatory response. a BzATP-induced changes in intracellular Ca2+ concentrations ([Ca2+]i), indicated by F340/380, in microglial cells acutely isolated from control (Ctr) and COH retinas of the MacGreen mice at different post-operational times. b–e Bar charts summarizing the changes of peak value of F340/380 (b), delay time (c), time to peak (d), and recovery (e) in microglial cells of Ctr and cells of COH retinas at different post-operational times. n = 10 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Ctr using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (b–e). f Immunofluorescent images showing the BzATP treatment-induced translocation of NFAT and NFkB from cytoplasm to nuclei. Scale bar: 20 μm for all images. g, h Quantitative analyses of the immunofluorescent densities of NFAT (g) and NF-kB (h) in microglial cells under different conditions. The lines show the average values of 10 cells in each group. Nucleus were stained with DAPI. i Representative immunoblots showing the expression of phosphorylated CaMKII (p-CaMKII) and total CaMKII in retinal primary cultured microglia with or without 100 μM BzATP treatment for different times. j, k A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different treatment times is shown in bar charts (j for p-CaMKII; k for total CaMKII). l Bar chart showing the changes in TNF-α mRNA levels induced by BzATP (100 μM) treatment for 30 min in primary cultured microglia with or without the presence of the CaMKII inhibitors KN62 and KN93. m Bar chart showing the changes in TNF-α mRNA levels induced by BzATP (100 μM) treatment for 30 min in primary cultured microglia with or without the presence of either the NFAT inhibitor VIVIT or the NF-kB inhibitor PDTC. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Ctr using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (l, m); #P < 0.05, and ##P < 0.01 vs. DMSO group using unpaired two-tailed Student’s t test (m). All in vitro experiments: n = 3 biological replicates × 3 technical replicates