Microglia ablation on CD11b-HSVTK COCS enhanced prion-induced neurotoxicity. (A and B) Tga20^TK+ or tga20^TK− slices were treated with NBH or RML6 and cultured in the optional presence of GCV for 49 dpi. Representative COCS stained for NeuN (green) and DAPI (blue; A) and analyzed by NeuN morphometry (B). Bars, 200 µm. n = 10–11. ***, P < 0.001; n.s, P > 0.05. Bars labeled Co denote co-culture of slices divided by a grease barrier. Red bars indicate microglia depletion. (C) NeuN morphometry of RML6 infected tga20^TK+ COCS optionally treated with GCV and reconstituted with PLCs prepared from tga20 mice (∼70,000 cells per COCS). Red: microglia-depleted, Blue: microglia-depleted and PLC reconstituted. n = 10; ***, P < 0.001; n.s, P > 0.05. (D) NeuN morphometry of RML6 infected tga20^TK+ COCS optionally treated with GCV and reconstituted with 70 × 10³ CD19⁻CD11b⁺ cells per slice. Red: microglia-depleted, Blue: microglia-depleted and CD19⁻CD11b⁺ cells reconstituted. n = 10. ***, P < 0.001. (E) PrP^Sc Western blot of cultures prepared from Prnp^−/−, tga20^TK+, or tga20^TK− mice exposed to NBH, RML6, or 22L and cultured until 35 dpi in the optional presence of GCV. Samples were digested with PK as indicated and detected with POM1. (F–I) qRT-PCR of cytokines and chemokines in tga20^TK+ slices treated with NBH or RML6 at 42 dpi ± GCV. (J–M) qRT-PCR of cytokines and chemokines in tga20^TK− slices treated with NBH or RML6 at 42 dpi ± GCV. (F and J) TNF mRNA, (G and K) IL-1β mRNA, (H and L) RANTES mRNA, and (I and M) MCP-1 mRNA. n = 3–5. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, P > 0.05. Relative expression normalized to GAPDH expression and represented as fold change compared with noninfected, non-GCV samples ± SEM. Statistical significance in B–D and F–M was determined using one-way ANOVA with Tukey’s post-test for multicolumn comparison. Histology, Western blot, and qRT-PCR results represent at least three independent experiments.