Effect of neural precursor proliferation level on neurogenesis in rat brain during aging and after focal ischemia

Abstract

The observed age-related decline in neurogenesis may result from reduced proliferation or increased death rate of neuronal precursor cells (NPCs). We found that caspase-3, but not caspase-6, -7, or -9, was activated in NPCs in neurogenic regions of young, young-adult, middle-aged and aged rat brains. The number of capase-3-immunoreactive cells was highest in young and lowest in aged rats. Surprisingly, intraventricular administration of a caspase-3 inhibitor failed to restore the number of BrdU-positive cells in the aged dentate gyrus, suggesting that the age-related decline in neurogenesis may be attributable primarily to reduced proliferation. Additionally, we also found that NPCs in the subventricular zone of young-adult and aged rat brain were increased after focal cerebral ischemia, suggesting that the increase in neurogenesis induced by ischemia may result from an increase in the rate of NPC proliferation, but not from a decrease in NPC death. Thus, our results suggest that age-related and injury-induced changes in the rate of neurogenesis are controlled at the level of NPC proliferation. Furthermore, our results may imply that the mechanisms that maintain a stable population of NPCs in the normal adult and in the ischemic brain, which account for the observed age-dependent reduction or injury-induced increases in neurogenesis, impinge on the regulation of cell division at the NPC level.

Fig. 1

Caspase activation in neuroproliferative regions of young-adult rat brain. (A) Cleaved caspase-3-positive cells (brown) were seen in the SVZ, RMS, DG SGZ, and OB of rat brain. (B) Cleavage products of capase-6, caspase-7, and caspase-9 were not detected in SVZ (or DG, not shown). Data shown are representative of at least four experiments per panel.

Fig. 2

Phenotypic features of cleaved caspase-3-positive cells in young-adult rat brain. (A) Co-localization of nuclear BrdU (red) and cytoplasmic DCX (green) in SVZ. (B) Co-localization of nuclear cleaved caspase-3 (green) and cytoplasmic DCX (red) in hippocampal SGZ. (C) Co-localization of nuclear cleaved caspase-3 (green) and cytoplasmic DCX (red) in some (green arrow) but not other (red and yellow arrows) cells in SVZ; DAPI (blue) was used to stain nuclei. High magnification view of a merged image (insert, top right) shows co-localization of caspase-3 and DCX. (D) Lack of nuclear co-localziation of BrdU (red) and cleaved caspase-3 (green) in SVZ of normal brain, indicating that most BrdU labeling is not a manifestation of cell death. Data shown are representative of at least three experiments per panel.

Fig. 3

Age-dependent decline in NPC death in neuroproliferative zones of rat brain. Compared to 5-day-old (5D) rats (A), 24-month-old rats (B) show reduced numbers of cells with capase-3 cleavage product (brown) in DG, SVZ, RMS and OB. The number of cleaved caspase-3-immunopositive cells in DG SGZ (C) and SVZ (D) were also quantified for rats aged 5 days (5D), 3 months (3M), 11 months (11M) and 24 months (24M), which also showed an age-dependent reduction in this index of NPC death. Data shown are representative fields (A and B) or mean values ± SEM (C and D) from at least three experiments per panel.

Fig. 4

The numbers of TUNEL-positive cells in DG of young-adult and aged rat brain. Few TUNEL-positive cells (green) were found in the DG of young-adult (A) and aged rat brain (B). DAPI (blue) was used for counterstaining of nuclei. Paraffin sections from focal cerebral ischemic brain (3-month-old) were used as positive controls (C). A number of TUNEL-positive cells were observed in the infarction region (CPu), but only few TUNEL-positive cells were found in the SVZ. Left penal: low-magnification. Right penal: high-magnification. LV: lateral ventricle; CPu: caudate putamen. Images are representative of results from three experiments.

Fig. 5

Effect of caspase inhibition on numbers of BrdU-positive cells in DG of young-adult and aged rat brain. (A) The caspase inhibitor DEVD (20 mg/ml) was infused at 1 μl/hr for 3 days into the left lateral ventricle of young-adult (3M) and aged (24M) rats, and BrdU (50 mg/kg) was given twice i.p. on the last day. Immunocytochemistry showed that DEVD reduced the number of cells showing caspase-3 cleavage, without affecting the number of cells that incorporated BrdU (P>0.05). (B) The number of BrdU-positive cells in the hippocampal SGZ after infusion of DEVD into the left lateral ventricle of young-adult (3M) and aged (24M) rat brain for 3 days and BrdU was given twice on the last day. Animals were killed one day later. Images (A) are representative of results from three experiments.

Fig. 6

NPC proliferation and death in the SVZ after focal ischemia. The percentage of BrdU-immunoreactive cell (A) & caspase-3-positive cells (B) in the SVZ of sham-operated and ischemic brains in young-adult and aged rats is shown. The number of cleaved caspase 3-positive cells was significantly higher both in young (3M) and aged (24M) ischemic rat SVZ (P < 0.001 and P < 0.001 respectively). The ischemia-induced increase in caspase-3-positive cells in young-adult and aged brains, however, was not significantly different (P > 0.05). Pearson’s correlation analyses showed a positive correlation between numbers of BrdU-positive cells and numbers of cleaved caspase-3-positive cells in the SGZ in all experimental conditions examined (P < 0.0001, r2 = 0.7225) (C). Data shown are mean values ± SEM from at least three experiments per panel.