Representative high-power photomicrographs from the hippocampus of a 17-month-old APP/PS-1 double-transgenic mouse (A) and a 17-month-old control mouse (B), stained with tomato lectin histochemistry for chronically activated microglia. Note the strong activation of microglia in the hippocampus of the APP/PS-1 double-transgenic mouse related to plaques (white arrows) but also within the pyramidal cell layer (CA3, black arrows) and within the molecular layer (arrowheads). Microglial activation was not observed in the hippocampus of 4.5-month-old APP/PS-1 double-transgenic mice or in the hippocampus of the PS-1 single-transgenic mice (data not shown). Bar, 50 μm.

Results of stereologic examinations. Control mice (C), transgenic mice expressing human mutant presenilin-1 (PS-1), and transgenic mice expressing human mutant amyloid precursor protein and human mutant PS-1 (APP/PS-1) were compared. A, C, E, G, and I: Results obtained for the left hippocampal granule cell layer (dentate gyrus). B, D, F, H, and K: Results obtained for the left hippocampal pyramidal cell layer (CA1–3). A and B: Volumes of the cell layers. C and D: Numbers of neurons. E and F: Correlation between numbers of neurons (y-axis) and the percentage of the volume of the corresponding cell layer occupied by aggregated Aβ and surrounding astrocytes in the APP/PS-1 double-transgenic mice (x-axis). G and H: Correlation between numbers of neurons (y-axis) and the percentage of the volume of the entire hippocampus occupied by aggregated Aβ and surrounding astrocytes in the APP/PS-1 double-transgenic mice (x-axis). I and K: “Reconstructed” numbers of neurons, calculated assuming that in the hippocampus of the APP/PS-1 double-transgenic mice, the space within the corresponding cell layer occupied by aggregated Aβ and surrounding astrocytes would have contained neurons at the same mean neuronal density as the other parts of the corresponding cell layer. Dots represent individual data; lines represent mean data. M4.5, 4.5-month-old animals; M17, 17-month-old animals. *, P < 0.05; **, P < 0.01; ***, P < 0.001. In E to H, the curves indicate the upper and lower boundaries of the 95% confidence intervals of the corresponding regression lines. In all cases, the slope of the regression line was not statistically significantly different from zero (P > 0.05). Differences between groups were tested with analysis of variance (analysis of variance), followed by post-hoc Bonferroni’s multiple comparison tests for pair-wise comparisons. Statistical significance was established at P < 0.05.

Age-related astrocytosis, aggregation of Aβ aggregates, and loss of neurons in APP/PS-1 double-transgenic mice. Immunohistochemical analysis of the hippocampus of control mice (A and B), transgenic mice expressing human mutant presenilin-1 (PS-1 M146L, HMG promoter; C and D), and transgenic mice expressing human mutant amyloid precursor protein (APP) 751 (KM670/671NL and V717I, Thy1 promoter) and human mutant PS-1 (E to L). A, C, and E: Representative low-power photomicrographs from the hippocampus of 4.5-month-old mice, showing immunohistochemical staining for GFAP (red) and NeuN (green). B, D, and F: Representative low-power photomicrographs from the hippocampus of 17-month-old mice, showing immunohistochemical staining for GFAP (red) and NeuN (green). In all pictures, the dentate gyrus is seen on the left, and the CA3 and hilar region on the right. Note the strong increase in GFAP immunoreactivity in the APP/PS-1 double-transgenic mice. Furthermore, neuron loss was seen in the pyramidal cell layer of the 17-month-old APP/PS-1 double-transgenic mice (arrows in F). G and H: Representative low-power photomicrographs from the hippocampus of a 4.5-month-old (G) and a 17-month-old (H) APP/PS-1 double-transgenic mouse showing immunohistochemical staining for NeuN (red) and β-amyloid 40–42 (Aβ, green). Note the age-related extracellular Aβ aggregation and neuron loss in the APP/PS-1 double-transgenic mice, particularly in the CA3 region. I, K, and L: Representative high-power photomicrographs from the CA2 region of 17-month-old APP/PS-1 double-transgenic mice, showing immunohistochemical staining for NeuN (red) and Aβ 40–42 (green) (I), GFAP (red) and Aβ 40–42 (green) (K), or GFAP (red) and NeuN (green) (L). There were regions free of neurons in the vicinity of Aβ aggregations, but neuron loss was also found at distance from Aβ aggregations (arrows in I). The regions surrounding the Aβ aggregations were partly occupied by astrocytes (shown in K). However, the amount of neuron loss exceeded the accumulation of aggregated Aβ and surrounding astrocytes (arrows in L). Bar, 250 μm in A to F, 150 μm in G and H, 20 μm in I to L.

Representative low-power photomicrographs of 30-μm thick sagittal sections of the hippocampus of 17-month-old control mice (A), mice transgenic for human mutant presenilin-1 (B), and mice double-transgenic for human mutant APP751 and human mutant presenilin-1 (C), respectively. Sections were stained with cresyl violet and represent the same parasagittal level in the mouse brain (bar, 200 μm). Insets, high-power photomicrographs of the granule cell layers (dentate gyrus, DG) and the pyramidal cell layers (area CA3) representative of the magnification at which the stereological estimates were made (bar, 15 μm). Small rectangles depict the positions at which the inset photomicrographs were taken. Sections like these were used for quantification of hippocampal volumes and the total number of hippocampal pyramidal cells and granule cells.

A cell (arrow) immunopositive for both NeuN (green; A) and TUNEL (red; B; merged in C) in the immediate vicinity of an Aβ aggregate (asterisk) in a 17-month-old APP/PS-1 double-transgenic mouse, suggesting that neuronal death could be directly associated with accumulation of Aβ. Both pictures were taken at identical focal depth. Bar, 25 mm.