Demyelination/remyelination and decreased P0 expression in dKO mice.

<p>(A) Electron micrographs of ultrathin cross sections of control, dKO, H1HTZ, H2HTZ, H1KO, and H2KO sciatic nerves, at 8 wk post-tamoxifen, and percentage of demyelinated/remyelinated axons (3 animals per group, at least 700 axons counted per mouse), identifying a demyelination/remyelination phenotype in dKO sciatic nerves. Asterisks indicate demyelinated axons and “M” macrophages. (B) CD68 (green) immunofluorescence in longitudinal cryosections of control and dKO sciatic nerves labeled with DAPI (blue = nuclei) showing increased presence of macrophages in dKO sciatic nerves, consistent with the demyelination phenotype. Arrows indicate macrophages. (C) In situ hybridization (ISH) of P0 on longitudinal cryosections of control and dKO sciatic nerves identifying a reduction of P0 at the transcript level at 5 wk post-tamoxifen, before the onset of demyelination and when macrophages are not present in the nerve. Pictures on the right are magnifications of black boxes depicted on the left. Black arrows show SC nuclei. (D) Western blot of P0 and quantification normalized to GAPDH (loading control) in sciatic nerve lysates of control and dKO mice at 8 wk post-tamoxifen (3 mice per group), showing reduced P0 protein levels in dKO sciatic nerves. (E) Confocal images of MBP (magenta) and P0 (red) coimmunofluorescence in paraffin cross sections of dKO mice at 8 wk post-tamoxifen, showing reduced P0 levels in most myelin rings, while MBP levels remain high. Nuclei are labeled in blue by DAPI. A single optical section is shown. White arrows indicate MBP positive/P0 negative myelin rings, the yellow arrowhead shows an MBP/P0 double negative SC, and blue arrowheads MBP/P0 double positive myelin rings. Three animals per group were used for each experiment. <i>P</i>-values (two-tailed unpaired (A) or paired (D) Student's <i>t</i> test): * = <i>p</i> < 0.05, ** = <i>p</i> < 0.01, *** = <i>p</i> < 0.001, error bars = SEM.