For transmission experiments, homogenate from frozen brain was injected into tg66 mice, and mice were followed as described above. By IHC PrPSc was detected in brain of 11 of 11 mice tested between 531 and 784 dpi (Table 4). At earlier time-points (two mice at 531dpi) PrPSc deposits appeared as linear axonal staining in the cerebral cortex (Fig. 4a), and gray matter vacuoles and microglia were noted in the same areas (Fig. 4b and c). In these same mice, PrPSc deposits were detected as small round objects consistent with axonal cross-sections in the Oriens layer of the ventral HC (Fig. 4d). At later time-points, such as 731 dpi, the deposits in the cerebral cortex had the same fine linear axonal pattern as at 531 dpi (not shown). However, at 731 dpi, PrPSc deposits in the Oriens layer of the dorsal hippocampus had a bulky coarse pattern (Fig. 5a and b). In these PrPSc-positive areas, vacuolation of neuropil (Fig. 5c) and astro- and micro-gliosis were observed (Fig. 5d and e).Table 4Transmission study of human, genetic mutant G131V PrP to tg66 transgenic mice expressing human PrPMouse numberDPIPrPSc IHCPrPSc western blotClinical TSE suspectClinical notes (reason for euth) & relevant necropsy findingsB300–379a–ntNonormalB339–379a–ntNonormalB307–1241a–ntNonormalB307–2241a–ntNonormalB308–3531a+ntNonormalB328–1531a+ntNonormalB633–1596+–NonormalB297–1650+–Noweight loss, hind limb weaknessB297–2731+–Yeshead bob, tremors, thinB298–1731+–Yesweak hind legs, thin, kyphosis, weepy eye, ataxia for 1 monthB297–3735+–Yesthin, slight wobble, rough coat, squinty eyes, decent nestB297–4735+–Yesthin, slight wobble, rough coat, squinty eyes, decent nestB632–1758+–Yesthin, rough coat, mild ataxiaB298–2769+–Yesthin, ataxicB303–1784+–Yesurine scalding, weak hind legs, tremora- Indicates mice that were stereotactically microinjected into the striatum. Mice injected using this technique were euthanized electively and tissues were processed to directly screen the injection needle track and adjacent brain by IHC for any PrP replication. Mice without asterisks were intracerebrally inoculated with a 30ul volume of brain homogenate and euthanized when they developed neurologic signs consistent with prion infection or conditions requiring euthanasia for humane reasonsFig. 4Immunohistochemistry and neuropathology of G131V-injected tg66 mice at 531 days post injection. In cerebral cortex: Linear axonal staining of PrPSc (arrow) was detected by antibody 3F4 (a). Vacuoles (arrow) shown by H&E staining (b) and microglia seen with anti-Iba1 staining (c) were also detected. In the Oriens layer of hippocampus of the same mouse shown in panels a, b, c, punctate 3F4 staining of PrPSc was detected in a pattern possibly indicating association with axonal cross-sections in this region (arrow) (d). As a negative control, in the same region of a mouse injected with Q227X brain homogenate no PrPSc deposits were observed at 529 dpi (e). The scale bar shown in panel a is 50 μm and applies to panels a-eFig. 5Immunohistochemistry and neuropathology of a G131V-injected tg66 mouse at 731 days post-injection. a-e Oriens layer of dorsal hippocampus. Coarse plaque-like staining of PrPSc by antibody 3F4 is seen at low (a) and high (b) magnification. Rectangular box in a denotes area seen in b. In the same area, gray matter vacuoles (arrow) were noted by H&E staining (c), and astrogliosis (d) and microgliosis (e) were observed (arrows) by IHC using antibodies to GFAP and Iba1. Panel f: No PrPSc deposits were seen in a control age-matched uninfected mouse in the same hippocampal region shown in (a) The scale bar in panel a is 200 μm and applies to panel f. In b the bar is 50 μm and applies to panels b-e