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. 2017 Nov 28;7:16528. doi: 10.1038/s41598-017-16466-3

Figure 1.

Figure 1

Age-related decline in hippocampal neurogenesis. (A) Experimental design used to analyze the neurogenic cascade in young (1 and 2 m) and mature (6 and 12 m) mice. Mice received an injection of BrdU every 2 h for 6 h (4x total) and were sacrificed (SAC) at different time points after the last injection. (B) Representative confocal z-stacks of the dentate gyrus (DG) of 1, 2, 6 and 12 m mice at 2 h, 2d, 4d, 10d, and 30d after the BrdU injections (magenta). DAPI staining indicates cell nuclei and the outline of the DG. Scale bar = 100 µm; z = 16.5 µm. (C) Absolute number of BrdU+ cells per hippocampus along the BrdU time course. N = 5 for 1mo and 2mo mice at 2 h, 2d, 4d, 10d, 30d; N = 4 for 6mo mice at 2 h, 2d, 4d, 10d, 30d; N = 7 for 12mo mice at 2 h, 2d, 4d and 10d, and N = 10 for 12mo mice at 30d. 2-way ANOVA (time after injection × age) showed a significant interaction between the two variables F(12, 88) = 122.982, p < 0.001. Thus 1-way ANOVA was used to analyze statistical differences due to the time after BrdU injection at each age: F(4, 20) = 199.587, p < 0.001 for 1 m; F(4,20) = 145.638, p < 0.001 for 2 m; F(4,15) = 97.491, p < 0.001 for 6 m; and F(4,33) = 43.665, p < 0.001 for 12 m. Holm-Sidak was used as a posthoc test. a, b, c and d represent significance compared to the prior time point for 1 m, 2 m, 6 m and 12 m mice, respectively. One symbol represents p < 0.05, two: p < 0.01, and three: p < 0.001. The number of BrdU+ cells in the septal hippocampus is shown in Fig. S2A. Raw data is shown in Table S1. (D) Percentage of BrdU+ cells per hippocampus. Absolute cell count was normalized to the number of BrdU+ cells found at 2 h. N as in Fig. 1C. 2-way ANOVA (time after injection × age) showed no significant interaction between the two variables: F(12, 88) = 1.463, p = 0.154; no significant effect of age: F(3,88) = 0.553, p = 0.647; and a significant effect of the time after injection: F(4,88) = 237.431, p < 0.001. While no overall effect of age was found, we analyzed statistical differences due to the age at each time point after the injection using 1-way ANOVA: F(3,17) = 0.33, p = 0.814 at 2d; F(3,17) = 3.329, P = 0.045 at 4d (although posthoc analysis by Holm-Sidak did not reveal any significant differences); F(3,17) = 1.364, p = 0.287 at 10d; F(3,20) = 91.088, p < 0.001 at 30d. Holm-Sidak was used as a posthoc test. The symbols indicate p < 0.01 between 2 m vs. 12 m; and *, #, $ represent p < 0.001 between 1 and 2 m vs. 6 m, 1 and 2 m vs. 12 m, and 6 m vs. 12 m, respectively. The percentage of BrdU+ cells in the septal hippocampus is shown in Fig. S2B. The percentage of BrdU+ cells expressing Ki67 at 2d is shown in Fig. S2C,D. N = 10 at 1 m, 2 m; N = 8 at 6 m; N = 14 at 12 m. Raw data is shown in Table S4. Points represent mean ± SEM.