Fig. 6.

Metformin enhances neurogenesis and proliferation in the unirradiated brain. Representative confocal images (A–F) of BrdU, NeuN, and DAPI immunostaining in the dentate gyrus of the hippocampus for unirradiated (VEH-0 Gy, n = 9; MET16-0 Gy, n = 3, MET19-0 Gy, n = 4) or irradiated (VEH-7 Gy, n = 8; MET16-7 Gy, n = 4, MET19-7 Gy, n = 4) mice at P42 (after BrdU labeling from P28-P35). Confocal images show cells with immunoreactivity for BrdU (first column), NeuN (second column), DAPI (third column), and merged (fourth column). Arrows indicate BrdU-positive, NeuN-positive cells and arrowheads indicate BrdU-positive and NeuN-negative cells (G). Scale bars represent 100 and 10 µm (A–F and G, respectively). BrdU- and BrdU/NeuN-positive cell counts within the dentate gyrus (H, I) demonstrate a significant radiation-induced loss in proliferative cells and new neurons at 42 days of age (P < .05, 2-way ANOVA; H, I). In unirradiated mice, an increase in the number of BrdU- and BrdU/NeuN-positive cells is observed with MET16 treatment compared with VEH controls. In irradiated mice, a significant decrease in double BrdU/NeuN-positive cells is observed with MET16 treatment compared to VEH controls (I). MET19 treatment, on the other hand, was not different from VEH in either irradiated or unirradiated mice for any cell counts. Colored bars represent means, whereas small black circles and triangles represent individual male or female mice, respectively. Error bars are SEM. Abbreviations: 0 Gy, unirradiated; 7 Gy, irradiated with a 7-Gy dose; MET16, treated with metformin starting at P16; MET19, treated with metformin starting at P19; VEH, vehicle-treated mice. *P < .05, **P < .01, Student t test.