*2*.*6*. *Immunohistochemistry*

Immunohistochemical staining was performed as described previously [20,30,31]. The brain was fixed with 4% phosphate-buffered paraformaldehyde. Coronal brain sections (8 μm thick) were incubated with 3% hydrogen peroxide for 40 min at room temperature to inhibit endogenous peroxidase and then incubated with blocking buffer (4% Block Ace, Dainippon Sumitomo Pharma, Osaka, Japan) for 2 h. Then, the slices were incubated with polyclonal rabbit anti-IL-1β antibody (1:300, Santa Cruz Biotechnology, Dallas, TX, USA), polyclonal rabbit anti-TNF-α antibody (1:200, Rabbit mAb, Hycult Biotech, Uden, The Netherlands), polyclonal rabbit anti-cleaved caspase-3 antibody (1:100, Cell Signaling Technology, Danvers, MA, USA), or monoclonal mouse anti-MPO (1:100, Hycult Biotech, Uden, The Netherlands), polyclonal rabbit anti-SVCT2 antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, USA), polyclonal rabbit anti-GLUT1 antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) in 0.01 mol/L phosphate-buffered saline overnight at 4 °C. In a double-immunohistchemical study for determination of the cell types of expressing SVCT2 or GLUT1, monoclonal mouse anti-neuronal nuclei (NeuN) antibody (1:300, Millipore, Billerica, MA, USA) and monoclonal mouse anti-rat endothelial cell antigen (RECA1) antibody (1:200, Abcom, Cambridge, UK) were used. After washing with phosphate-buffered saline, the slices were incubated with either Cy3- or FITC-conjugated secondary antibody (1:200, Millipore, Billerica, MA, USA) for 2 h at room temperature. Finally, the sections were incubated with the nuclear stain TO-PRO-3 (1:10,000, Invitrogen, Carlsbad, CA, USA) in phosphate-buffered saline for 10 min at room temperature with gentle agitation. Immunofluorescence was visualized using the laser scanning confocal microscope and the intensity was measured using the imaging software. Three sections per rat and 3–4 rats per group were used for the analyses.
