M1-like pro-inflammatory microglia are transiently identified in the NMDA-damaged acute injury model. Double-transgenic fish Tg(mfap4:tdTomato;tnfα:GFP) were used to monitor M1-like pro-inflammatory microglia/macrophages. Confocal images show 3 wpf control retinas possess tdTomato-positive ramified microglia (arrows), while amacrine cells express GFP (A,B–B”). NMDA-injured retinas were evaluated at 12, 24, 48, and 96 h post-injection (hpi, C–J). At 12 h following NMDA injection, mfap4:tdTomato-positive microglia/macrophages start to co-express GFP (arrowheads). GFP-positive microglia/macrophages are detectable up to 72 hpi, but they are no longer detectable by 96 hpi. White boxes in Panels A, C, E, G, I show the area magnified in Panels B, D, F, H, J. White arrows: tdTomato-positive, GFP-negative microglia/macrophages; yellow arrows: tdTomato-negative, GFP-positive microglia/macrophages; arrowheads: tdTomato and GFP double-positive microglia/macrophages. Histogram displays the number of microglia/macrophages in 300 μm of the central region of the NMDA-damaged retinas (K). Bars and lines indicate mean ± SEM, n = 8–12 per group. Black bars: total number of tdTomato-positive cells; purple bars: tdTomato and GFP double-positive microglia/macrophages. Statistical analysis was performed with a One-way ANOVA with Tukey’s multiple comparisons test was applied (*p < 0.05; **p < 0.001; ***p < 0.001).
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