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. 2014 Dec 11;10(12):e1004531. doi: 10.1371/journal.ppat.1004531

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© 2014 Sorce et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Microglial proliferation was analyzed by immunohistochemistry with the IBA1 antibody on brains of Nox2^+/Y (A I-VI) and Nox2^-/Y (A VII-XII) mice injected i.c. with NBH or prions and culled at 4, 8, 12, and 16 weeks post inoculation (wpi) or at the terminal stage of disease. Cerebellar areas are displayed in the pictures. Scale bar for A I-XII: 250 µm (displayed in panel XII). Dashed boxes in images VI and XII are shown at higher magnification in panels VI′-VI″ and XII′-XII″; scale bar: 100 µm (displayed in panel XII″). (B) Microglia cell number quantification in cerebellar cortex of Nox2^+/Y and Nox2^-/Y mice injected i.c. with NBH or 22 L prions and culled at different time points during disease incubation or at terminal stage. Each dot corresponds to one mouse (average of 3–5 sections per mouse). Nox2^+/Y, n = 4–7; Nox2^-/Y, n = 3–6; no significant difference between Nox2^+/Y and Nox2^-/Y mice; two-way ANOVA followed by Bonferroni's post-hoc test. (C) Detection of ROS production in vivo was performed by injecting i.p. the DHE probe and analyzing the fluorescence of its oxidation products in cerebellar homogenates. Dot plots show relative fluorescent units (RFU) per mg of proteins; each dot corresponds to one mouse (Nox2^+/Y, n = 5–8; Nox2^-/Y, n = 4–8): ***, P<0.001; two-way ANOVA followed by Bonferroni's post-hoc test. (D) Representative images of hematoxylin and eosin-stained sections from Nox2^+/Y and Nox2^-/Y mice injected i.c. with NBH or 22 L prions. Scale bar for D I-VI: 250 µm (displayed in panel VI). Dashed boxes in images I to VI are shown at higher magnification in panels I′-VI′; scale bar: 100 µm (displayed in panel VI′). (E) Number of spongiform vacuoles was quantified in cerebellar cortex of Nox2^+/Y and Nox2^-/Y mice injected i.c. with NBH or 22 L prions and culled at different time points during disease incubation or at terminal stage. Each dot corresponds to one mouse (average of 7–12 sections per mouse). Nox2^+/Y, n = 4–7; Nox2^-/Y, n = 3–6; ***P<0.0001; two-way ANOVA followed by Bonferroni's post-hoc test.