Figure 5.

Intraperitoneally injected Aβ seeds are detectable shortly after the injection in macrophages in the peritoneal cavity, blood, liver, and spleen. A, Electrochemiluminescence-conjugated immunoassay for human Aβ (Aβ₄₀ and Aβ₄₂ combined) in the cellular fraction of the peritoneal fluid 3 h, 24 h, 1 week, or 1 month after intraperitoneal inoculation with Tg or WT extract. Male or female 4- to 8-month-old APP23 mice, n = 5 per group and time point except n = 4 for the 1 month time point. Separate statistical analysis did not indicate a gender or age difference. ANOVA revealed significant main effects and a significant extract × post-inoculation time interaction (F_(3,30) = 14.02; p < 0.001). Post hoc Tukey's tests showed significantly more Aβ in the Tg extract-treated mice at 24 h compared with 3 h (p < 0.05) and 1 week (p < 0.001). Wilcoxon's comparisons revealed similar significances. Indicated is the mean ± SEM. B, Pappenheim's stain (purple) combined with Aβ immunostaining (dark blue) of lavaged peritoneal cells from a Tg extract-inoculated mouse disclosed Aβ-positive monocytes (arrowheads). No Aβ immunoreactivity was found in WT extract-inoculated mice. C, Electrochemiluminescence-conjugated immunoassay for human Aβ in the blood cellular fraction 1–24 h, 1 week, or 1 month after intraperitoneal inoculation. Male or female 4- to 8-month-old APP23 mice, n = 16 (Tg extract) and n = 11 (WT extract) for the 1–24 h time point and n = 5 per group for other time points. (Animals were added to the 1–24 h time period in an unsuccessful effort to reduce the high variability of Aβ measurements.) Separate statistical analysis did not indicate a gender or age difference. ANOVA indicated no significant differences, but Wilcoxon's test revealed a significant difference between Tg extract-treated and WT extract-treated mice in the 1–24 h post-inoculation group (median, 0.495 vs 0; p < 0.05). D, Aβ-positive monocytes (arrowheads) in a blood film from a Tg extract-inoculated mouse (Pappenheim's stain, purple; Aβ immunostain, dark blue). E, Double-immunofluorescence staining revealed that Aβ-positive (red) cells are positive for CD11b and CD45 (green; maximum projection of 10 μm z-stack). Scale bars, 10 μm. The same cells were also positive for CD68 (data not shown). F, Immunoprecipitation of the blood cellular fraction with an antibody against human Aβ followed by immunoblot revealed a robust 4 kDa Aβ band in Tg extract-inoculated mice, as well as weaker oligomeric Aβ bands (indicated by asterisks) and APP, very similar as seen in the Tg extract (Langer et al., 2011). Only a weak signal corresponding to monomeric Aβ was seen in WT extract-inoculated mice, possibly because of residual human endogenous Aβ from the plasma fraction. IG heavy chain (HC) and light chain (LC) signals are attributable to coelution of the antibodies used for immunoprecipitation. G, The amyloid-specific dye pFTAA stains Aβ in peritoneal monocytes isolated from Tg-extract inoculated mice. DAPI is used as a nuclear counterstain (scale bar, 5 μm). H, I, Peripheral organs of representative mice measured and stained in (A–D) were immunostained for Aβ and counterstained with nuclear fast red. Aβ-positive cells with the morphological characteristics of macrophages were found in the liver of Tg extract-inoculated mice (n = 3 analyzed mice) but not WT inoculated mice (n = 2 analyzed mice) 1 d after inoculation. Scale bar: 50 and 10 μm. In a separate study, non-Tg B6 mice were injected with Tg and WT extract (n = 2 for Tg and WT each) and analyzed 1 h, 1 d, 1 week, and 1 month later. Again, Aβ-positive cells with a macrophagic appearance were found after 1 and 7 d but not 1 month after inoculation (results not shown).