The phagocytic response of microglia is impaired in CX3CR1^−/− mice after cuprizone treatment. TREM2 and CD11c expression levels were used as indicators of microglial phagocytic activity. (A) In situ hybridization signal for trem2 mRNA in the mouse brain 4 wk after the cuprizone treatment or nomal chow. (B) Flow cytometry analysis of CD11c surface protein expression in CD45⁺ CD11b⁺ cells in the CNS. (C) CD45⁺ cells were selected among live cells, which were then gated against CD11b⁺ and CD11c⁺, cells positive for both markers are reported. (D and E) Iba1-immunoreactive signal in the brain of WT and CX3CR1^−/− mice 5 wk after normal show or cuprizone supplemented diet. Representative electron microscopy images of microglia are shown in WT (D) and CX3CR1^−/− (E) mice. Unstained endosomes (red arrow), partially digested myelin products localized within the endosomes (green arrow), and cholesterol crystals inside of their cytoplasm (blue arrow) were abundant in WT (D) but not in CX3CR1^−/− microglia (E). One representative experiment out of two is shown. n = 3–6 mice/group. ****, P < 0.0001. Bars, 2 µm.