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Fig. 5
Immunomodulatory control of rod cell regeneration kinetics. (A) Schematic of assay timelines and Dex treatment paradigms for data shown in B–F. (B–B′′) Whole-retina intravital time-lapse sequences from Mtz-treated 6-dpf rho:YFP-NTR;mpeg1:Tomato larvae and microglia migration patterns quantified ± preablation Dex exposure. (B) Exposing larvae to Dex before Mtz-induced rod cell death suppressed microglia responsiveness. (B′) Representative dragon-tail microglia migration data from the preablation Dex-treated retina group. (B′′) False-colored image showing microglia (yellow) and macrophages (red) at t = 0. (C and D) Retinal sections were processed for PCNA immunoreactivity and imaged at the indicated time points (dots in the Histo line in A). (C and D) Quantification of proliferative MG/progenitor cells in the INL (C) and ONL (D) from uninjured control retinas (dark green boxes), Mtz-treated retinas (light green boxes), and Mtz+Dex (preablation)-treated retinas (red boxes) normalized to the number of cells per 1 mm of retina (measured at the OPL). Dex pretreatment causes a decrease in proliferation in the INL and ONL after 24 h Mtz and at +1 d recovery. (E) Plate reader-based (i.e., ARQiv) quantification of rod cell regeneration kinetics (i.e., YFP intensity) in individual rho:YFP-NTR larvae at +4 d of recovery following Mtz-induced rod cell death and ± Dex exposure (pre- or postablation). Comparisons between larvae treated with Mtz alone (green boxes) or with Mtz +Dex (red boxes) preablation (Left) or postablation (Center), or with Mtz + Dex-P postablation (Right) showed that pretreatment with Dex inhibited rod cell replacement, whereas posttreatment with Dex (or Dex-P) accelerated rod cell regeneration kinetics. (Note that for logistical reasons associated with ARQiv assays, Mtz was applied at 5 dpf for this experiment.) Statistics (effect sizes, 95% confidence intervals, and P values for paired comparisons) are provided in Table S4; bolded letters denote pairs showing reproducible differences; italicized letters denote pairs with no discernible differences.