Figure 7
Activation of specific TLRs causes neurodegeneration and inflammation in the brain in vivo. (A, B) C57BL/6J mice were injected intrathecally with water (control, n = 7) or one of the four TLR ligands (10 μg LPS, n = 10; 40 μg Pam3CysSK4, Pam, n = 11; 136 μg loxoribine, Lox, n = 9; 10 μg CpG ODN, CpG, n = 10), as indicated. After 3 days, brains were analyzed by immunohistochemistry. (A) Representative micrographs of the cerebral cortex immunostained with NeuN Ab, neurofilament Ab, and Iba1 Ab to detect neurons, axons, and microglia, respectively. Scale bar, 100 μm, inset 50 μm. (B) NeuN+ cells of the cerebral cortex were quantified. The mean of six high power fields per cerebral cortex is expressed as NeuN+ cells per mm2; median with Kruskal-Wallis test followed by Bonferroni post-hoc test of control vs. treatment. P* <0.05; P** <0.005. (C) Representative micrographs of the cerebral cortex immunostained with an Ab against activated caspase-3. Cortical cells positive for activated caspase-3, as indicated by arrows, were quantified. The mean of six high power fields per cerebral cortex is expressed as caspase-3+ cells per mm2; median with Kruskal-Wallis test followed by Bonferroni post-hoc test of control vs. treatment. P*** <0.001. (D) C57BL/6J mice were injected intrathecally with 0.9% NaCl (control, n = 9) or one of the four TLR ligands (10 μg LPS, n = 9; 10 μg Pam3CysSK4, Pam, n = 9; 10 μg imiquimod, Imi, n = 8; 10 μg CpG ODN, CpG, n = 9), as indicated. After 12 h, cerebrospinal fluid (CSF) was obtained from the cistern magna. Leukocytes in the CSF were counted and are expressed as leukocytes per μL CSF. Median with Kruskal-Wallis test followed by Dunn’s multiple comparison test of control vs. treatment. P* <0.05; P** <0.005. (E) C57BL/6J mice were injected intrathecally with 0.9% NaCl (control, n = 4) or one of the four TLR ligands (10 μg LPS, n = 4; 10 μg Pam3CysSK4, Pam, n = 4; 10 μg loxoribine, Lox, n = 4; 10 μg CpG ODN, CpG, n = 4), as indicated. After 24 h, CSF was analyzed by flow cytometry-based multiple analyte detection for amounts of various cytokines/chemokines, as indicated. Median with Kruskal-Wallis test followed by Dunn’s multiple comparison test of control vs. treatment. P* <0.05.