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. 2023 Aug 10;10:1184029. doi: 10.3389/fmolb.2023.1184029

FIGURE 2.

FIGURE 2

Bioassay of LS-PMCA generated prions. (A) C57BL/6J mice were intracerebrally injected with brain-derived RML and in vitro generated RML by either LS-PMCA or standard PMCA. As controls, RML BH at the dilution of 10–19, the unseeded PMCA product (with normal mouse BH as substrate), and the seeded PMCA product (with PrP knockout mouse BH as substrate) were injected as well. The graph shows the survival time, measured as the number of days it took for animals to reach the terminal stage of the disease and to be humanely euthanized. Animals which did not show any clinical symptoms, were kept for 500 days. (B). Analyses of PrPres from injected animals. Symptomatic animals were sacrificed at terminal stage (RML, RML (LS-PMCA), and RML-PMCA). Of the control groups, one animal was sacrificed at 300 dpi (RML 10–19, No Seed, and PrP0/0 substrate). The brains were collected, homogenized, and digested with 50 μg/mL PK. N is 5 µL of mouse BH used as migration control. (C) Vacuolation analyses after H&E staining. Representative images from different brain regions from control, RML, and LS-PMCA RML animals. Tha: Thalamus, Hip: Hippocampus, CWM: Cerebellar White Matter. Scale bar: 100 µm. (D) Immunohistochemical analyses. Representative images of PrPSc (detected by 6H4 antibody) deposition in the cerebellum from control, RML, and LS-PMCA RML animals. Representative images of reactive astrocytes (detected by anti-GFAP antibody) in the Thalamus from control, RML, and LS-PMCA RML animals. Left panels: control; middle panels: RML; right panels: LS-PMCA RML. Scale bar: 100 µm.