Supplementary Figure 4: Microgliosis following transgene suppression occurs in cortex and hippocampus in addition to SC, though without a concurrent change in astrocytosis.
(a-c) Representative images of cortex, hippocampus, and lumbar SC of control rNLS8 mice (a), disease rNLS8 mice at 6 weeks off DOX (b), and early recovery rNLS8 mice (c) show a slight increase in the number of activated microglia (IBA-1, red) during recovery relative to controls in all regions, with changes in the SC being most pronounced. Similar results were observed in n = 3 animals per treatment group. (d) GFAP staining (green) on SC cryosections at multiple time-points during disease and recovery shows no obvious increase in astrocytosis at any time-point, relative to control rNLS8 mice maintained on DOX. Tissue was processed from n = 4 animals for disease time-points, and n = 5 animals for recovery time-points. All scale bars = 100 μm. (e-g) Consistent with this, we did not find significant changes in expression of genes associated with the A1 (Serping1 and Fkbp5) or A2 (Emp1) astrocyte phenotype by qPCR in RNA isolated from whole lysate of rNLS8 SC before (0 weeks off DOX, n = 4), during (4 weeks off DOX, n = 5), or after disease (6 weeks off DOX + 1 week on DOX, n = 6). Whiskers in box plots show minima and maxima, and box shows Q1-Q3 with median indicated, with individual points represented by black circles overlaid. Analysis by one way ANOVA, F2,14 = 0.63, p = 0.55 (e), F2,14 = 1.73, p = 0.22 (f), and F2,14 = 0.95, p = 0.42 (g).
