Figure 1.
Number of proliferating cells and early lineage commitment in the SGZ.
A, experimental timeline. Cells were labeled with BrdU at 4 weeks after irradiation (i.e. at 3 months of age) with two injections (8 hours apart) and sacrificed 24 hours after the first injection. The expected cell identities (BrdU+ → BrdU+; Dcx+ → NeuN+), if were allowed to continue the maturation process for another month, were also depicted. B, the number of proliferating cells (BrdU+) in the SGZ. C, the number of immature neurons (Dcx+) in the SGZ. Two-way ANOVA with Bonferroni post test was carried out. Radiation was the major source of variation in BrdU (F(1,21) = 20.13, p = 0.0002) and Dcx (F(1,16) = 9.30, p = 0.0077) analyses, and there was no genotype×treatment (radiation) interaction. P values indicate results from Bonferroni post tests. Data are presented as mean ± SEM. Sample size (in the order of Sod2+/+/0 Gy, Sod2+/+/5 Gy, Sod2−/+/0 Gy, and Sod2−/+/5 Gy): BrdU, n = 6, 5, 7, and 7; Dcx, n = 6, 5, 4, and 5.
Figure 2.
Hippocampal-dependent learning and memory.
A, experimental timeline. Cells were labeled with BrdU at 4 weeks after irradiation (1 injection per day×5 days), evaluated for neurocognitive functions, and sacrificed after the completion of behavioral studies. B, novel location recognition task; comparison of time spent investigating an object in a familiar vs. a novel location. C, novel object recognition task; comparison of time spent investigating familiar objects vs. a novel object. D, radial-arm water maze; comparison of errors in arm entry in the beginning (block 1) vs. the end (block 10) of the test. E, radial-arm water maze; comparison of time spent finding the platform in the beginning (block 1) vs. the end (block 10) of the test. F, contextual fear condition; relative freezing to the baseline on day 1. G, cued conditioning; comparison of % time freezing in a novel environment during the pre-cue vs. the post-cue phase. P values from the Bonferroni post test are shown. ***, p<0.001. Data are presented as mean ± SEM. Sample size (in the order of Sod2+/+/0 Gy, Sod2+/+/5 Gy, Sod2−/+/0 Gy, and Sod2−/+/5 Gy): n = 5, 6, 5, and 4 for novel location recognition; n = 13, 12, 8, and 9 for novel object recognition; n = 18, 18, 13, and 14 for radial-arm water maze and contextual fear tests.
Figure 3.
Long-term survival of newly generated cells in the SGZ following behavioral studies.
A, total number of BrdU+ cells in the SGZ. Sod2 genotype (F(1,23) = 4.71, p = 0.041) and radiation treatment (F(1,23) = 14.28, p = 0.001) both contributed significantly to variations in BrdU numbers, but there was no interaction between genotype and treatment (F(1,23) = 0.44, p = 0.52). B, a representative image showing BrdU+/NeuN+ and BrdU+/GFAP+ cells in the SGZ of the dentate gyrus. C, the total number of newborn neurons. Genotype (F(1,16) = 4.66, p = 0.046) and treatment (F(1,16) = 13.76, p = 0.0019) both contributed significantly to variations in BrdU+/NeuN+ numbers, but there was no interaction between these two factors. D, the total number of newborn glial cells. Only genotype (F(1,16) = 6.97, p = 0.018) contributed significantly in the data variation, and there was an interaction between genotype and treatment (F(1,16) = 19.05, p = 0.0005). E, the percentage of newly born neurons. Only treatment (F(1,16) = 19.80, p = 0.0004) contributed significantly to the data variation, and there was an interaction between genotype and treatment (F(1,16) = 7.30, p = 0.0157). F, the percentage of newly born glial cells. Only treatment (F(1,16) = 11.79, p = 0.0034) contributed significantly to the data variations, and there was an interaction between genotype and treatment (F(1,16) = 10.27, p = 0.0055). Data are presented as mean ± SEM. Sample size (in the order of Sod2+/+/0 Gy, Sod2+/+/5 Gy, Sod2−/+/0 Gy, and Sod2−/+/5 Gy): BrdU, n = 8, 6, 7, and 6; BrdU+/NeuN+ and BrdU+/GFAP+, n = 5 each.
Figure 4.
Dendritic spine density following behavioral studies.
The number of dendritic spines was analyzed in secondary and tertiary dendrites of granule cells in the dentate gyrus after behavioral studies. Upper panel, representative photos from (left to right) Sod2+/+/0 Gy, Sod2+/+/5 Gy, Sod2−/+/0 Gy, and Sod2−/+/5 Gy mice. Lower panel, average spine densities. Sample size: n = 4, 6, 5, and 4 for Sod2+/+/0 Gy, Sod2+/+/5 Gy, Sod2−/+/0 Gy, and Sod2−/+/5 Gy, respectively. Only genotype (F(1,15) = 15.59, p = 0.0013) contributed significantly to the data variations, and there was a significant interaction between genotype and treatment (F(1,15) = 5.46, p = 0.0337). P values indicate Bonferroni post test results. Data are presented as mean ± SEM. Scale bar = 10 µm.
Figure 5.
Redox state and mitochondrial mass.
Levels of the advanced lipid peroxidation end product, 4-hydroxynonenal (4-HNE) adducts, (A) and protein nitration product 3-nitrotyrosine (3-NT) (B) were determined by ELISA to monitor the extent of oxidative stress in the hippocampus at 3 months and 5 months of age after a single dose of cranial irradiation (see timeline in Figure 3A). C, a representative western blot image from samples collected following behavioral studies showing APEX, VDAC, and MnSOD. D, relative protein levels of VDAC in the Sod2−/+ cohorts. In order to compare changes in 4-HNE, 3-NT, and VDAC levels in sham and irradiated Sod2−/+ from two different experimental stages – at 3 months of age before behavioral studies and at 5 months of age after behavioral studies – levels in Sod2−/+ mice were calculated as a percentage of their corresponding Sod2+/+ controls. Two-way ANOVA with Bonferroni post test was carried out. P values indicate Bonferroni post test results. There was no treatment×time interaction. Data are presented as mean ± SEM. Sample size: n = 5 each at 3 months of age, and 4 each at 5 months of age for sham and irradiated Sod2−/+.