P-Glycoprotein Exacerbates Brain Injury Following Experimental Cerebral Ischemia by Promoting Proinflammatory Microglia Activation

Figure 4

P-glycoprotein regulates GR nuclear translocation and further CCL2 degradation in experimental ischemic stroke. Mice were intracerebroventricularly injected with P-glycoprotein (P-gp) siRNA or negative control (NC) siRNA (1.5 μL/10 g body weight), P-gp p-AAV or NC p-AAV (2.5 μL/10 g body weight), 48 hr or 7 days prior to MCAO/R surgery. Twenty-four hours after the surgery, brains were harvested for RT-PCR, ELISA, western blotting, and immunofluorescence assays. (a) mRNA expression levels of CCL2 and CCR2 measured via RT-PCR assay as fold changes relative to sham treatment (n = 4). (b) Contents of CCL2 and CCR2 determined by ELISA assay (n = 6). (c, d) Immunostaining and western-blotting quantification of cytoplasm GR and nucleus GR expressions (n = 4). (e) Representative immunofluorescence images (left) and quantification (right) of nuclear and cytoplasmic localization (ratio of nuclear to cytoplasmic location) of GR (n = 4). Scale bars, 100 μm. (f) Coronal brain diagrams showing locations of regions for immunofluorescence staining analysis in infarct cortex. Mann–Whitney test for (a). One-way ANOVA followed by the post hoc least significant difference test or Games Howell test for (b), (d), and (e). All data are mean ± SD; , between two groups.

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