Figure 2

HSP60 triggers neurodegeneration in the cerebral cortex dependent on TLR4 and MyD88 in vivo . (A) Forty micrograms of HSP60 or 40 μg of SA were injected intrathecally into 8–12 week-old C57BL/6 J (WT, HSP60 n = 11, SA n = 8), TLR4^−/− (HSP60 n = 11, SA n = 8), and MyD88^−/− (HSP60 n = 11, SA n = 8) mice. Naïve mice received no injection (WT n = 6, TLR4^−/− n = 4, MyD88^−/− n = 4). Injection of LPS into WT mice served as a positive control for TLR4 activation (n = 10). Injection of water into WT mice served as a carrier control for LPS, HSP60, and SA injection (n = 7). After 3 d, the cerebral cortex was analyzed by immunostaining with NeuN antibody. Scale bar, 100 μm. (B) Quantification of cortical NeuN+ cells of WT, TLR4^−/−, and MyD88^−/− mice injected intrathecally, as described above. Median, Mann–Whitney U test for indicated groups. (C) Brain sections containing the corpus callosum of WT, TLR4^−/−, and MyD88^−/− mice injected as described above were immunostained with a neurofilament antibody. Scale bar, 50 μm. Quantification of TUNEL+ cells (D) and DAPI-stained nuclei displaying apoptotic hallmarks including irregular shape, shrinkage, and fragmentation (E) in representative sections of the cerebral cortex of WT, TLR4^−/−, and MyD88^−/− mice injected intrathecally with HSP60 or SA, as indicated. (D, E) Median, Mann–Whitney U test for indicated groups.