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. 2023 Apr 21;146(10):4247–4261. doi: 10.1093/brain/awad132

Figure 4.

Figure 4

Effect of bumetanide on microglia phenotypes and morphological changes after CCI in the DG 3 days post-CCI. (A) Total area of Iba1+ cells soma, quantifications of Iba1+ process end points, attachment points and length in the ipsilesional DG. n = 5 animals, two slices per animal. (B) Iba1 immunostaining from sham, CCI-vehicle and CCI-bumetanide treated animals. (C) Quantification of the number of contacts between microglia (Iba1 staining) and PV interneurons at 3 days post-CCI. n = 5 animals per condition, three slices per animal. (D) Examples of PV and Iba1 immunostaining from sham-, CCI vehicle- and CCI bumetanide-treated animals in the ipsilesional DG, 3D representations (top) and stack (bottom). (E) Flow cytometry gating plots at 3 days post-CCI on the ipsilesional side. (F) Analysis of microglia phenotypes by detection of MHCII+ (pro-inflammatory) and CD206+ (pro-phagocytosis) markers by flow cytometry on the ipsilesional side at 3 days post-CCI. n = 5–6 animals per condition. (G) Quantity of IL-4, IL-10 and IL-6 produced in the ipsilesional hippocampus. Phenotype changes were analysed by t-test. Number of contacts and morphological analysis (quantified using the ImageJ plugins Neurphology and SynapCountJ) were analysed using one-way ANOVA with Dunnett's post hoc test. Interleukin production was analysed using a Brown–Forsythe ANOVA with Dunnett's post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ns = not significant.