Fig. 1.

Hippocampal cell loss and microgliosis precede infiltration of CCR2⁺ monocytes. Two-month-old CCR2-RFP male mice received intraperitoneal KA (30 mg/kg) or saline solution control injection and were killed 1, 3, or 14 d later. (A) The mice subject to KA showed neuronal cell loss in the CA3 hippocampal region at all time points examined. This was accompanied by microgliosis, which was robust at the 3-d time point. Anti-RFP immunohistochemistry revealed isolated CCR2-RFP⁺ cells at the 1- and 14-d time points. The 3-d time point showed massive infiltration of CCR2-RFP⁺ monocytes. Few CCR2-RFP⁺ cells were encountered in the saline solution-treated animals. (Scale bar: 100 μm.) (B) Stereological analysis of total hippocampal Iba1⁺ cells showed more than threefold increase at the 3- and 14-d time points. There was an approximately twofold increase 1 d after KA injection (n = 5 for each condition; one-way ANOVA followed by Dunnett’s post hoc tests). (C) Stereological analysis revealed ∼80,000 monocytes at the 3-d time point in the hippocampus. ANOVA followed by Tukey's post hoc tests revealed a significant difference of total hippocampal CCR2-RFP⁺ monocytes between 1 d and 3 d as well as 3 d and 14 d (***P < 0.001; n = 5). (D) Most CCR2-RFP–expressing cells (red) were also CD11b⁺ (green; arrows). A CCR2-RFP⁺;CD11b⁻ cell was occasionally encountered (arrowhead). Ramified CCR2-RFP⁻ CD11b⁺ microglia were also observed (asterisk). (Scale bar: 50 μm.) (E) All mice treated with KA showed a seizure severity score of at least 5.