Figure 2.

CCR2 deficiency in monocytes reduces monocyte infiltration to the ischemic tissue. We conditionally targeted Ccr2 gene expression by Cre-mediated recombination crossing LysMcre⁺ mice with floxed CCR2 mice (Ccr2^fl/fl) that express reporter eGFP.³¹ (a) The study was conducted in littermate Ccr2^fl/flLysMcre⁺ and Ccr2^fl/flLysMcre^– mice (control). (b) We verified that blood eGFP⁺ cells in the Ccr2^fl/flLysMCre⁺ mice were monocytes: CD11b⁺CD115⁺Ly6G^–. (c) Gating strategy to study monocytes and neutrophils in the contralateral and ipsilateral cortex. Images correspond to 1 day after ischemia. We separated monocytes with high Ly6C expression (Ly6C^hi) from monocytes with dim or negative Ly6C expression (collectively termed Ly6C^lo). (d) We studied monocyte subsets infiltrating the brain tissue at different time points after induction of ischemia, i.e. 1, 4 and 15 days, by flow cytometry in mice of both genotypes (n = 5–12 mice per time point and genotype). Ly6C^hi and Ly6C^lo monocytes increased after ischemia in the ipsilateral (ischemic) but not the contralateral cortex. The % of CD45^hiCD11b^hiLy6G^–Ly6C^hi cells is lower in Ccr2^fl/flLysMcre⁺ mice one day post-ischemia (Two-way ANOVA by genotype and time point followed by Sidak’s multiple comparisons test, ***p < 0.001). The number of Ly6C^hi monocytes decreases in the ipsilateral cortex in Ccr2^fl/flLysMCre⁺ mice at day 1 (two-way ANOVA by genotype and brain region followed by Sidak’s multiple comparisons test; ***p < 0.001). The % of CD45^hiCD11b^hiLy6G^–Ly6C^lo cells also tend to decrease in Cre⁺ mice (two-way ANOVA by genotype and time point, genotype effect p = 0.06), and the absolute cell number at day 1 is lower in Ccr2^fl/flLysMCre⁺ mice (two-way ANOVA by genotype and brain region followed by Sidak’s multiple comparisons test; ***p < 0.001). However, the numbers of infiltrating Ly6G⁺ neutrophils are no different between genotypes. (e) Ccl2 mRNA expression in the ischemic brain tissue at different time points after ischemia shows the highest increase at day 1 versus 4 or 15 days, but there are no differences between genotypes (n = 8–14 mice per time group and genotype). Values are expresses as fold versus non-ischemic contralateral hemisphere of the control genotype.