P90-110 mice were deeply anesthetized with fentanyl (0.05 mg/kg) and mounted into a stereotaxic frame, then were subjected to either single saline or LPS (lipopolysaccharide from Escherichia coli O26:B6; 200 nl of 5 mg/ml, rate = 200 nl/10 minutes; #L8274, Sigma-Aldrich) injections using a glass micropipette [86 (link)]. The coordinates for the injection were anterior–posterior −2.5 mm, lateral +1.5 mm, and ventral −0.25 mm from the bregma. Bumetanide, a specific inhibitor of NKCC1 in the brain, was coinjected with LPS (50 μM; #3108, Tocris). At 24 hours, mice were transcardially perfused with saline, and approximately 0.5 × 0.5 × 0.5 cm sized tissue pieces from the center of each injected cortical region were cut off and collected for cytokine array and flow cytometric analysis. For tissue sectioning, mice were perfused with saline followed by 4% paraformaldehyde in PBS. For real-time quantitative PCR (qRT-PCR) experiments to assess the effect of NKCC1 deficiency on microglial expression of genes, which contribute to ion homeostasis, membrane potential, cell volume regulation, or inflammation, LPS (5 μg dissolved in ACSF) was administered into the cisterna magna in 5 μl final volume, using a glass capillary. At 24 hours, mice were transcardially perfused with saline followed by CD11b+ magnetic cell sorting.
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