Figure 3

CX3CR1 deficiency attenuates infiltration and leads to a different M1/M2 polarization pattern on microglia /macrophages in ipsilateral hemisphere of middle cerebral artery occlusion mice. (A) Staining with anti-Iba-1 antibody in the ipsilateral hippocampus, striatum, cortex and peri-infarct zone of CX3CR1^-/- and wild-type (WT) mice 72 hours after middle cerebral artery occlusion (MCAO). (B) Quantification of Iba-1 positive cells. CX3CR1^-/- mice have fewer Iba-1 positive cells (macrophages/microglia) in the ipsilateral hippocampus, striatum, cortex and peri-infarct zone, while no differences are observed in these sites of the contralateral hemisphere. P < 0.0001 by two-way analysis of variance for genotype, localization and interaction. *P < 0.05, **P < 0.01 by Bonferroni post-hoc tests. n = 4 per group. Scale bars = 50 μm. (C) Flow cytometry analysis of CD45^hi/CD11b⁺ and CD45^low/CD11b⁺ cells isolated from the ipsilateral and contralateral hemispheres of CX3CR1^-/- and WT mice 72 hours after MCAO by gating on Ly6G^- events (Ly6G vs FSC). (D) Quantification of the number of events in the CD45^hi/CD11b⁺/Ly6G^- (left) and CD45^low/CD11b⁺ /Ly6G^– (right) gate. Statistical analysis shows CX3CR1^-/- mice have fewer CD45^hi/CD11b⁺/Ly6G^- as well as CD45^low/CD11b⁺ /Ly6G^– cells in the ipsilateral hemisphere. CD45^hi/CD11b⁺/Ly6G^-: P = 0.0001 for genotype, P < 0.0001 for localization and interaction by two-way analysis of variance. CD45^low/CD11b⁺/Ly6G^-: P = 0.0112 for genotype, P < 0.0001 for localization, and P = 0.0152 for interaction by two-way analysis of variance. **P < 0.01 by Bonferroni post-hoc tests. n = 4 per group. (E) Microglia/macrophage in ischemic hemisphere of CX3CR1^-/- brain polarize toward the M2 phenotype. Real-time reverse-transcription polymerase chain reaction was performed using total RNA extracted from sorted CD45⁺/CD11b⁺/Ly6G^- microglia/macrophage at 72 hours after MCAO. Data are expressed as fold change vs sham-operated controls. **P < 0.01 with Student’s t-test for Ym1, Mcr1, and iNOS, respectively. n = 4 per group.