Figure 1: Prion-infected organotypic slice cultures exhibit distinct biochemical and neurodegenerative changes.

(A) Lysates from organotypic cerebellar slices exposed to NBH or RML scrapie and cultured for 35 days were subjected to PK treatment and then Western blotted (undigested samples were loaded after diluting 10-fold to prevent over-exposure on blots). RML-treated slices accumulate the PK-resistant PrP signal, but NBH-treated slices did not. (B) An equal amount of brain homogenates from WT mice was infected with RML scrapie either undigested or digested with proteinase K (PK) to detect PK resistance (designated as PrP^Sc) on immunoblot. Membranes were probed with anti-PrP (α-helix 1) mouse monoclonal antibody, POM1. The banding profile showed differential migration with banding profiles consistent with PrP^C for undigested and PrP^Sc for digested samples. (C,D) Propidium Iodide (PI) incorporation to visualize dead cells in 49-dpi slice cultures. Quantification of PI⁺ cells from NBH or RML scrapie-infected slices using ImageJ. RML scrapie-infected cerebellar slices showed significantly higher cell death when compared to NBH-treated brain slice cultures. Data were analyzed using a two-tailed t-test; n = 6 biological replicates. Data are mean ± SEM (*p < 0.05, **p < 0.01, ***P < 0.001). (E,F) Slices were stained with NeuN to determine any neuronal loss at 42 dpi. RML scrapie-infected slice cultures suffered significant neuronal loss as evidenced by NeuN⁺ area of percent NBH-treated slices. (G) RML scrapie-infected slice cultures show pro-apoptotic signaling as evidenced by both upregulation and proteolytic activation of PKCδ and augmented cleaved caspase 3 when compared to controls. All the samples were normalized to the loading control β-actin.