Analysis of preclinical prion-inoculated EAM / control mice with either Prnp0/0
or wt LRS. a) NaPTA blots of muscle and Western blots of spleen for PrPSc over time in EAM versus control mice. As positive controls for NaPTA blots, RML is used as a spike (S) in untreated tissue homogenate and is either PK-digested or undigested. As positive controls for spleen, brain homogenate from a terminally diseased wild type mouse is loaded with and without PK-digestion. As a negative control, PK-digested normal tissue homogenate is used. Muscular PrPSc can only be found in EAM mice with PrPC expressing LRS at day 35. At day 90, no PrPSc could be detected in both EAM and control mice. In spleen PrPSc is detectable in all analyzed samples. b) Histological staining with H&E evidences signs of myositis in the muscle of both chimeric mice with EAM but not controls. The staining for marker CD3 shows that most of the infiltrating cells are T-cells. Anti-PrPC staining could not be detected most probably because its expression is under the detection limit of the method. In brain tissue in both cohorts no gross pathological hallmarks of prion disease like spongiosis or gliosis could be detected by immunohistochemical staining for astrocyte marker GFAP. c) Bioassay to determine prion infectivity titers reveal high infectivity titers for all analyzed spleen samples. In muscle, prion titers were under the detection limit of the assay at day 35 irrespective of PrPC expression in the LRS. At day 90 borderline infectivity (attack rate of 25%) could be detected in mice with PrPC-deficient LRS irrespective of myositis.