Effects of aluminum on the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in the dentate gyrus of D-galactose-treated mice via increasing oxidative stress

Abstract

Aluminum (Al) accumulation increases with aging, and long-term exposure to Al is regarded as a risk factor for Alzheimer's disease. In this study, we investigated the effects of Al and/or D-galactose on neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons in the hippocampal dentate gyrus. AlCl3 (40 mg/kg/day) was intraperitoneally administered to C57BL/6J mice for 4 weeks. In addition, vehicle (physiological saline) or D-galactose (100 mg/kg) was subcutaneously injected to these mice immediately after AlCl3 treatment. Neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons were detected using the relevant marker for each cell type, including nestin, Ki67, doublecortin, and NeuN, respectively, via immunohistochemistry. Subchronic (4 weeks) exposure to Al in mice reduced neural stem cells, proliferating cells, and differentiating neuroblasts without causing any changes to mature neurons. This Al-induced reduction effect was exacerbated in D-galactose-treated mice compared to vehicle-treated adult mice. Moreover, exposure to Al enhanced lipid peroxidation in the hippocampus and expression of antioxidants such as Cu, Zn- and Mn-superoxide dismutase in D-galactose-treated mice. These results suggest that Al accelerates the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in D-galactose-treated mice via oxidative stress, without inducing loss in mature neurons.

Keywords: D-galactose; adult neurogenesis; aluminum; hippocampus; oxidative stress.

Fig. 1. Changes in body weight (A), food intake (B), and blood glucose levels (C) in the vehicle, aluminum (Al), D-galactose (D-gal), and aluminum-D-galactose (Al-D-gal) groups (n = 8 per group; ^*p < 0.05 indicates a significant difference between vehicle/vehicle-D-gal and Al/Al-D-gal, ^†p < 0.05 indicates a significant difference between vehicle/vehicle-Al and D-gal/Al-D-gal). Data are presented as the mean ± standard error of the mean (SEM).

Fig. 2. Immunohistochemistry of nestin in the dentate gyrus of the vehicle (A), Al (B), D-gal (C), and Al-D-gal (D) groups. Nestin-immunoreactive cells and fibers decreased in the Al-D-gal and D-gal groups compared to the vehicle group. Nestin-immunoreactive cells and fibers decreased significantly in the Al-D-gal group compared to the vehicle group. (E) Relative optical density (ROD) demonstrated as percentages of vehicle group in nestin immunoreactivity per section in all groups (n = 8 per group). Data are presented as the mean ± SEM. GCL, granule cell layer; PL, polymorphic layer; ML, molecular layer. Scale bar = 50 µm.

Fig. 3. Immunohistochemistry for Ki67 in the dentate gyrus of the vehicle (A), Al (B), D-gal (C), and Al-D-gal (D) groups. Ki67-immunoreactive nuclei are observed in the subgranular zone (arrows) of the dentate gyrus. Note that Ki67-immunoreactive nuclei decrease slightly in the Al and D-gal groups compared to the vehicle group. Ki67-immunoreactive nuclei are significantly reduced in the Al-D-gal group compared to the vehicle group. (E) Mean number of Ki67-positive nuclei per section in all groups (n = 8 per group; ^*p < 0.05 indicates a significant difference between vehicle/vehicle-D-gal and Al/Al-D-gal, ^†p < 0.05 indicates a significant difference between vehicle/vehicle-Al and D-gal/Al-D-gal). Data are presented as the mean ± SEM. Scale bar = 50 µm.

Fig. 4. Immunohistochemistry for doublecortin (DCX) in the dentate gyrus of the vehicle (A and B), Al (C and D), D-gal (E and F), and Al-D-gal (G and H) groups. DCX-immunoreactive neuroblasts are detected in the subgranular zone (arrows) of the dentate gyrus. Note the decreased DCX-immunoreactive neuroblasts in both the Al and D-gal groups compared to the vehicle group. DCX-immunoreactive neuroblasts are also significantly decreased in the Al-D-gal group compared to the vehicle group. (I) ROD demonstrated as a percentage of the vehicle group in DCX immunoreactivity per section in all groups. (J) Relative mean number of DCX-immunoreactive neuroblasts per section demonstrated as percentage of vehicle group (n = 8 per group; ^*p < 0.05 indicates a significant difference between vehicle/vehicle-D-gal and Al/Al-D-gal, ^†p < 0.05 indicates a significant difference between vehicle/vehicle-Al and D-gal/Al-D-gal). Data are presented as the mean ± SEM. Scale bars = 50 µm (A, C, E and G), 25 µm (B, D, F and H).

Fig. 5. Immunohistochemistry for NeuN in the dentate gyrus of the vehicle (A), Al (B), D-gal (C), and Al-D-gal (D) groups. NeuN-immunoreactive cells are mainly located in GCL of the dentate gyrus. NeuN-immunoreactive cell numbers are similar in all groups. (E) Relative number of NeuN-immunoreactive mature neurons demonstrated as percentage of vehicle group per section in all groups (n = 8 per group). Data are presented as the mean ± SEM. Scale bar = 50 µm.

Fig. 6. Immunohistochemistry for 4-hydroxynonenal (4-HNE) in the dentate gyrus of the vehicle (A), Al (B), D-gal (C), and Al-D-gal (D) groups. 4-HNE immunoreactivity is mainly observed in GCL of the dentate gyrus. 4-HNE immunoreactivity is abundantly detected in the Al-D-gal group. (E) ROD demonstrated as percentage of vehicle group in 4-HNE immunoreactivity per section in all groups (n = 8 per group; ^*p < 0.05 indicates a significant difference between vehicle/vehicle-D-gal and Al/Al-D-gal, ^†p < 0.05 indicates a significant difference between vehicle/vehicle-Al and D-gal/Al-D-gal). Data are presented as the mean ± SEM. Scale bar = 50 µm.

Fig. 7. Immunohistochemistry for superoxide dismutases (SOD)1 in the dentate gyrus of the vehicle (A), Al (B), D-gal (C), and Al-D-gal (D) groups. SOD1 immunoreactivity is mainly observed in GCL of the dentate gyrus. SOD1 immunoreactivity is increased in the GCL of the Al-D-gal group. (E) ROD demonstrated as a percentage of vehicle group in SOD1 immunoreactivity per section in all groups (n = 8 per group; ^*p < 0.05 indicates a significant difference between vehicle/vehicle-D-gal and Al/Al-D-gal, ^†p < 0.05 indicates a significant difference between vehicle/vehicle-Al and D-gal/Al-D-gal). Data are presented as the mean ± SEM. Scale bar = 50 µm.

Fig. 8. Immunohistochemistry for SOD2 in the dentate gyrus of the vehicle (A), Al (B), D-gal (C), and Al-D-gal (D) groups. SOD2 immunoreactivity is mainly observed in GCL of the dentate gyrus. SOD2 immunoreactivity is increased in the GCL of the Al-D-gal group. (E) ROD demonstrated as a percentage of vehicle group in SOD2 immunoreactivity per section in all groups (n = 8 per group; ^*p < 0.05 indicates a significant difference between vehicle/vehicle-D-gal and Al/Al-D-gal, ^†p < 0.05 indicates a significant difference between vehicle/vehicle-Al and D-gal/Al-D-gal). Data are presented as the mean ± SEM. Scale bar = 50 µm.