Figure 5. Microglial repopulation occurs between 48 and 72 hours after CSF1R inhibitor withdrawal.

A) To investigate early repopulation events, 2 month-old CX3CR1-GFP^+/− mice were treated with PLX3397 for 7 days. The inhibitor was then withdrawn and groups of mice sacrificed 1, 2, and 3 days later (n = 4 per group). B–F) Representative sections shown from the hippocampus of each of these groups, in which microglia express GFP. G) Flow cytometry of GFP+ cells from the liver and spleen of these animals. H) Quantification of whole brain sections for microglial numbers shows a reduction of ~70% with 7 days PLX3397 treatment. Microglial numbers continue to decline for 2 days after inhibitor withdrawal, but rapidly recover between days 2 and 3, highlighting a crucial time period in repopulation. I) Brain levels of PLX3397 show rapid clearance of the drug from the CNS. J) GFP⁺ cells strongly express nestin at the 2- and 3-day recovery timepoints. 63X Z-stacks obtained by confocal microscopy and maximal projections are shown. Scale bar represents 20 μM. Separate channels and merge are shown for each of the timepoints in the panels below. K) Western blots of whole brain homogenates show significant increases in nestin and CSF1R with repopulation, but no changes in CCR2. L) Quantification of K) normalized to GAPDH as a loading control. Same capital letters above conditions indicates no significance (p>0.05) via one-way ANOVA with post-hoc Newman-Keuls Multiple Comparison Test. Error bars indicate SEM.