Age-progressive Aβ pathology in 5xFAD mice. 6E10 immunostaining and quantification in age-matched WT and 5xFAD for both ages. A, Representative images of 6E10 amyloid antibody-immunostained plaques and neurons in young and middle age-matched WT and 5xFAD mice. The four panels show coronal brain sections. The large white squares show an enlarged view of the SUB outlined by the smaller white squares. DAPI staining is in blue, and the 6E10 + amyloid signal is in red. B–E, Quantification of extracellular and intraneuronal 6E10 signals in the SUB. More amyloid plaques were detected in 5xFAD middle-age mice relative to 5xFAD young, while none were detected in both age groups of WT mice. 5xFAD middle-age mice show slightly higher plaque density and significantly greater plaque size and intensity relative to 5xFAD young mice (B, density, 5xFAD young, 2,151 ± 214.3; 5xFAD middle-age, 2,698 ± 141.8; Wilcoxon rank-sum test, p = 0.0676; C, size, 5xFAD young, 73.59 ± 1.645; 5xFAD middle-age, 139.9 ± 2.247; LME model, p = 1.86 × 10−12; D, intensity, 5xFAD young, 3,018 ± 33.42; 5xFAD middle-age, 3,761 ± 30.54; LME model, p = 9.13 × 10−7). E, 5xFAD young mice have significantly greater intraneuronal 6E10 density relative to 5xFAD middle-age (density, 5xFAD young, 7,188 ± 548.1; 5xFAD middle-age, 3,554 ± 396.7; Wilcoxon rank-sum test, p = 7.58 × 10−5). The mouse numbers for each of the four groups of WT and 5xFAD mice of both ages are n = 10. F–J, Sex-specific comparison of 6E10 immunostaining. F, Representative images of DAPI and 6E10 antibody immunostaining in 5xFAD mice of both age groups and genders. The first row of panels shows coronal brain sections with DAPI and 6E10 amyloid antibody immunostaining. The second row of panels shows an enlarged view of the SUB outlined by the smaller white squares in the first row. The third row of panels shows an even larger view of the white squares in the second row. G–J, Quantification of sex differences in SUB 6E10-stained amyloid plaques and neurons. G, There is a significant increase in extracellular plaque density in young female 5xFAD and middle-age male 5xFAD compared with male 5xFAD young mice. There is a significant age-dependent increase in extracellular plaque density in male mice, but not in female mice (density, male 5xFAD young, 1,601 ± 94.84; female 5xFAD young, 2,702 ± 214.6; male 5xFAD middle-age, 2,432 ± 157.5; female 5xFAD middle-age, 2,963 ± 148.6; Wilcoxon rank-sum test, male 5xFAD young vs female 5xFAD young, p = 0.0079; male 5xFAD young vs male 5xFAD middle-age, p = 0.0159; male 5xFAD middle-age vs female 5xFAD middle-age, p = 0.0571). H, For both genders of 5xFAD mice, the middle-age mice have significantly greater extracellular plaque size relative to the young mice, revealing that plaque size has an age-dependent increase within the same sex (size, male 5xFAD young, 68.69 ± 2.734; female 5xFAD young, 74.3 ± 2.022; male 5xFAD middle-age, 130.4 ± 2.917; female 5xFAD middle-age, 149.8 ± 3.421; LME model, male 5xFAD young vs male 5xFAD middle-age, p = 2.63 × 10−11; female 5xFAD young vs female 5xFAD middle-age, p = 5.81 × 10−6). I, There is a significant age-dependent increase in extracellular plaque intensity within the same sex and a significant sex-dependent increase only in 5xFAD young mice (male 5xFAD young, 1,985 ± 35.98; female 5xFAD young, 3,473 ± 37.11; male 5xFAD middle-age, 3,718 ± 37.67; female 5xFAD middle-age, 3,805 ± 48.52; LME model, male 5xFAD young vs male 5xFAD middle-age, p = 1.39 × 10−23; female 5xFAD young vs female 5xFAD middle-age, p = 0.01184; male 5xFAD young vs female 5xFAD young, p = 6.76 × 10−16). J, There is a significant sex- and age-dependent decrease in intraneuronal 6E10-stained neuron density in 5xFAD mice. In both male and female 5xFAD mice, middle-age mice have significantly less intraneuronal neuron density than young mice. In 5xFAD mice of the same age group (young or middle), the female mice have significantly less intraneuronal neuron density (density, male 5xFAD young, 8,479 ± 293.9; female 5xFAD young, 5,896 ± 656.4; male 5xFAD middle-age, 4,603 ± 323.5; female 5xFAD middle-age, 2,506 ± 232.0; Wilcoxon rank-sum test, male 5xFAD young vs female 5xFAD young, p = 0.0317; male 5xFAD middle-age vs female 5xFAD middle-age, p = 0.0079; male 5xFAD young vs male 5xFAD middle-age, p = 0.0079; female 5xFAD young vs female 5xFAD middle-age, p = 0.0079). The mouse numbers for each of the eight groups (2 age groups, male and female, WT and 5xFAD) are 4–5. One representative section was used for quantification for each mouse brain (Fig. 1B–E,G–J). Sample sizes for the LME model are based on both the mouse numbers and the number of individual plaques measured from each mouse brain (Fig. 1C,D,H,I; 5xFAD young male, n = 432; 5xFAD young female, n = 980; 5xFAD middle male, n = 1,321; 5xFAD middle female, n = 1,255). Scale bars are included in the figure panels. The units for plaque size and plaque density are μm2 and per mm2, respectively, and the intensity is AU of optical density. Statistical significances are denoted by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.
