M1-like microglia are modulated by dexamethasone and LPS treatment in acute and chronic damaged retinas. At 3 wpf, Tg(mfap4:tdTomato;tnfα:GFP) fish, which allow visualization of M1-like pro-inflammatory retinal microglia/macrophages, were intravitreally injected with buffer (A,C,E) or NMDA (B,D,F). A group of fish were placed in Dexamethasone, intravitreally injected with either buffer or NMDA 24 h later, and collected 72 h following the injections for immunostaining (C,D). Alternatively, the fish were intravitreally injected with LPS, intravitreally injected 3 h later with either buffer or NMDA and collected 69 h later for immunostaining (E,F). Control retinas possess some ramified tdTomato-positive microglia/macrophages (A, arrow). NMDA-injured retinas show significantly greater number of tdTomato-positive microglia/macrophages and tdTomato-positive cells that co-express GFP from the tnfα:GFP transgene (B, arrowhead; M). Dexamethasone treatment significantly reduces the number of tdTomato-positive microglia/macrophages and tdTomato-positive cells that co-express GFP (D, arrowhead; M). LPS treatment significantly increases the number of tdTomato-positive microglia/macrophages and tdTomato cells that colabel with GFP in control and NMDA-damaged retinas (E,F, arrowheads; M). gosh mutants display ameboid tdTomato-positive microglia/macrophages, and some also co-express GFP (H, arrowheads; N). Dexamethasone treatment significantly reduces the number of tdTomato-positive microglia/macrophages and tdTomato-positive cells that co-express GFP in control siblings (I,N) and gosh mutants (J,N). LPS injection significantly increases the number of tdTomato-positive microglia/macrophages and GFP/tdTomato-double-positive cells in controls (K, arrows; N) and gosh mutants (L, arrowheads; N). White arrows: tdTomato-positive, GFP-negative microglia/macrophages; yellow arrows: tdTomato-negative, GFP-positive microglia/macrophages; arrowhead: tdTomato, GFP double-positive microglia/macrophages. Histograms represent the number of tdTomato-positive microglia/macrophages in 300 μm of the central region of the NMDA-damaged retinas (M) or the entire section of the gosh retinas (N). Bars and lines indicate mean ± SEM, n = 10–13 per group. Black bars: total number of tdTomato-positive cells; purple bars: tdTomato and GFP double-positive microglia/macrophages. Statistical analysis was performed with One-way ANOVA with Tukey’s multiple comparisons test on both graphs (*p < 0.05; **p < 0.01, ***p < 0.001).
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