Figure 2.

Induction of DTA expression in CX3CR1-expressing cells in TG mice results in complete and sustained depletion retinal microglia. A, TAM was administered orally to TG mice to induce CreER-mediated expression of DTA in microglia per the following schedule: two initial doses of TAM 1 d apart (500 mg/kg per oral dose) at Day −2 and Day 0, followed by an equal repeated dose every 5 d (Day 5, Day 10, etc) up to Day 35. B, Flat-mounted retinas from TAM-administered TG mice were immunostained for Iba1 to determine the presence and density of retinal microglia; insets show expanded views in the boxed areas. In control TG mice, which were administered corn oil without TAM (TG Control), retinal microglia were present throughout the retina in the normal distribution. In TAM-administered TG mice, Iba1+ microglia at Day 10 were absent in most retinal areas, with only isolated rare Iba1+ cells detected (inset). Near-complete depletion of retinal microglia was sustained by repeated TAM administration up to Day 35. C, Quantification of total number of retinal microglia indicated that although microglial numbers were similar between WT and TG Control animals, TAM-administered TG animals resulted in sustained and near-complete depletion (>95%) for up to 30 d (data are represented as mean ± SEM; n = 3 female 8-week-old animals at each group; *p < 0.05 on one-way ANOVA with Dunnett's multiple-comparisons test). D, Immunohistochemical localization of microglia performed with antibodies to CD11b (red),EYFP (green), and Iba1 (blue) confirmed microglial depletion across all retinal areas; the panels show a rare residual microglial cell that was typically immunopositive for all three markers. Scale bar, 40 μm. E, mRNA expression levels were assessed using NGS and separately verified by RT-PCR in independent replicates. Comparisons were made between TG mice gavaged with corn oil without TAM (TG Control), and animals depleted of microglia with continuous administration of TAM in corn oil for 15 and 30 d. Levels of microglia-expressed genes were significantly reduced in general, indicating microglial depletion. (Graphical data are represented as mean ± SEM; *indicates comparisons with control for which p < 0.05, one-way ANOVA, n = 3 male, 8-week-old animals per group).