NMDA-induced neuronal injury caused microglial accumulation in the injured areas.

<p><b>A, B:</b> Representative images of EGFP (green)-expressing microglia before (pre) or 1–7 days after treatment with NMDA (<b>A</b>) or vehicle (<b>B</b>). Injured cells at one day after the treatment are visualized by PI fluorescence (red). Scale bar = 500 µm. <b>C:</b> Accumulation of microglia in the injured areas was assessed by comparing the fluorescent intensity between the pyramidal cell layer and stratum radiatum. Fluorescent intensity in an area of 100 µm×200 µm (shown by white squares in the inset) was quantified by analyzing the captured fluorescent images using ImageJ software. The ratio of fluorescence intensity of the pyramidal cell layer (P) to that of the stratum radiatum (R) (designated as the P/R ratio) was calculated and used as an index of microglial accumulation in the injured areas. Microglial accumulation in the injured areas after treatment with NMDA (closed circle) or vehicle (open circle) was shown by the P/R ratio. Data are expressed as the mean ± SEM from seven slices. **<i>P</i><0.01, ***<i>P</i><0.001 versus pre (0 day) (one-way ANOVA, followed by Bonferroni <i>post hoc</i> test). <b>D:</b> The number of Iba1-immunopositive cells in the pyramidal cell layer (P) and stratum radiatum (R) at day 4 after NMDA treatment. Accumulation of microglia in the injured areas was assessed by counting the number of Iba1-immunopositive cells. Slice cultures at day 4 after treatment with vehicle (C) or NMDA (N) were subjected to immunostaining with anti-Iba1 antibody and then the number of Iba1-immunopositive cells in the areas of 200 µm×200 µm at three different z-positions (5-µm intervals) was counted. Data are expressed as the mean ± SEM from twelve areas of four slices. **<i>P</i><0.01 versus pyramidal cell layers of vehicle-treated control slices (Welch’s <i>t</i>-test).