Effects of Fluoxetine on Hippocampal Neurogenesis and Neuroprotection in the Model of Global Cerebral Ischemia in Rats

Abstract

A selective serotonin reuptake inhibitor, fluoxetine, has recently attracted a significant interest as a neuroprotective therapeutic agent. There is substantial evidence of improved neurogenesis under fluoxetine treatment of brain ischemia in animal stroke models. We studied long-term effects of fluoxetine treatment on hippocampal neurogenesis, neuronal loss, inflammation, and functional recovery in a new model of global cerebral ischemia (GCI). Brain ischemia was induced in adult Wistar male rats by transient occlusion of three main vessels originating from the aortic arch and providing brain blood supply. Fluoxetine was injected intraperitoneally in a dose of 20 mg/kg for 10 days after surgery. To evaluate hippocampal neurogenesis at time points 10 and 30 days, 5-Bromo-2'-deoxyuridine was injected at days 8-10 after GCI. According to our results, 10-day fluoxetine injections decreased neuronal loss and inflammation, improved survival and functional recovery of animals, enhanced neurogenesis, and prevented an early pathological increase in neural stem cell recruitment in the subgranular zone (SGZ) of the hippocampus without reducing the number of mature neurons at day 30 after GCI. In summary, this study suggests that fluoxetine may provide a promising therapy in cerebral ischemia due to its neuroprotective, anti-inflammatory, and neurorestorative effect.

Keywords: CA1; antidepressants; dentate gyrus; fluoxetine; global brain ischemia; hippocampus; inflammation; neural stem cells; neurogenesis; rats.

Figure 1

Fluoxetine effect on neuronal loss in the hippocampus after GCI. (a) Micrographs of the whole hippocampus of the sham-operated animals, positive controls, and fluoxetine-treated animals after GCI at days 11 and 31 after surgery, 10× magnification. Brain sections were stained with NeuN. (b) Comparison of total number of neurons in hippocampal regions per 100 × 100 µm² between the groups. Significant differences between the groups, according to ANOVA after Bonferroni’s correction for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 1

Fluoxetine effect on neuronal loss in the hippocampus after GCI. (a) Micrographs of the whole hippocampus of the sham-operated animals, positive controls, and fluoxetine-treated animals after GCI at days 11 and 31 after surgery, 10× magnification. Brain sections were stained with NeuN. (b) Comparison of total number of neurons in hippocampal regions per 100 × 100 µm² between the groups. Significant differences between the groups, according to ANOVA after Bonferroni’s correction for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Fluoxetine effect on cell proliferation and inflammation. (a) Micrographs of the CA1 field of the hippocampus of the sham-operated animals, positive controls, and fluoxetine-treated animals at day 11 after GCI. Brain sections were stained with BrdU, NeuN and DAPI (top row) and BrdU, Iba1 and DAPI, 63× magnification; (b) Comparison of BrdU+ cells in hippocampal regions between the groups at days 11 (b, left) and 31 (b, right) after surgery; (c) Comparison of Iba1+ cells in the CA1 field between the groups at days 11 and 31 after surgery; (d) The number of Iba1\BrdU double-positive cells in the CA1 field at days 11 and 31 after surgery compared to the total number of BrdU+ cells. Significant differences between the groups, according to ANOVA after Bonferroni’s correction for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Fluoxetine effect on cell proliferation and inflammation. (a) Micrographs of the CA1 field of the hippocampus of the sham-operated animals, positive controls, and fluoxetine-treated animals at day 11 after GCI. Brain sections were stained with BrdU, NeuN and DAPI (top row) and BrdU, Iba1 and DAPI, 63× magnification; (b) Comparison of BrdU+ cells in hippocampal regions between the groups at days 11 (b, left) and 31 (b, right) after surgery; (c) Comparison of Iba1+ cells in the CA1 field between the groups at days 11 and 31 after surgery; (d) The number of Iba1\BrdU double-positive cells in the CA1 field at days 11 and 31 after surgery compared to the total number of BrdU+ cells. Significant differences between the groups, according to ANOVA after Bonferroni’s correction for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Fluoxetine effect on neural stem cell recruitment in the DG. (a) Micrographs of the DG of the hippocampus in the sham-operated animals, positive controls, and fluoxetine-treated animals at day 11 after GCI. Brain sections were stained with BrdU, GFAP and DAPI, 63× magnification. NSCs are indicated by arrows; (b) Comparison of GFAP+\BrdU+ cells in hippocampal regions between the groups at day 11 (b) after surgery; (c) Comparison of GFAP+\BrdU− (quiescent NSC) cells and GFAP+\BrdU+ (recruited NSC) cells in the SGZ between the groups at days 11 and 31 after surgery. Significant differences between the groups, according to ANOVA after Bonferroni’s correction for multiple comparisons: ** p < 0.01, * p < 0.05.

Figure 3

Fluoxetine effect on neural stem cell recruitment in the DG. (a) Micrographs of the DG of the hippocampus in the sham-operated animals, positive controls, and fluoxetine-treated animals at day 11 after GCI. Brain sections were stained with BrdU, GFAP and DAPI, 63× magnification. NSCs are indicated by arrows; (b) Comparison of GFAP+\BrdU+ cells in hippocampal regions between the groups at day 11 (b) after surgery; (c) Comparison of GFAP+\BrdU− (quiescent NSC) cells and GFAP+\BrdU+ (recruited NSC) cells in the SGZ between the groups at days 11 and 31 after surgery. Significant differences between the groups, according to ANOVA after Bonferroni’s correction for multiple comparisons: ** p < 0.01, * p < 0.05.

Figure 4

Fluoxetine effect on neurogenesis in the DG. (a) Micrographs of the DG of the hippocampus in the sham-operated animals, positive controls, and fluoxetine-treated animals at day 31 after GCI. Brain sections were stained with DCX and DAPI, 20× magnification; (b) Comparison of the number of DCX+ cells in the SGZ between the groups and time points. Significant differences between the groups according to ANOVA after Bonferroni’s correction for multiple comparisons: *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 4

Fluoxetine effect on neurogenesis in the DG. (a) Micrographs of the DG of the hippocampus in the sham-operated animals, positive controls, and fluoxetine-treated animals at day 31 after GCI. Brain sections were stained with DCX and DAPI, 20× magnification; (b) Comparison of the number of DCX+ cells in the SGZ between the groups and time points. Significant differences between the groups according to ANOVA after Bonferroni’s correction for multiple comparisons: *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 5

Fluoxetine effect on maturation of newborn neurons. (a) Micrographs of the DG of the hippocampus in the sham-operated animals, positive controls, and fluoxetine-treated animals at day 31 after GCI. Brain sections were stained with BrdU, NeuN and DAPI, 63× magnification; (b) Comparison of the number of NeuN+\BrdU+ cells in the regions of the hippocampus between the groups. No significant differences between the groups.

Figure 6

Summary of fluoxetine effects on neuronal loss, inflammation, and neurogenesis in the DG after GCI. (a) Mean percentage changes in the number of NeuN+ and Iba1+ cells in the CA1 field at days 11 and 31 after GCI; (b) Mean percentage changes in the number of recruited NSCs, and proliferation at day 11 after surgery, and in total number of NSCs, neurogenesis, and mature neurons at day 31 after surgery.

Figure 7

(a) Experimental design of the study; (b) Scheme of NeuN+ cells counting to quantify severity of the ischemic lesion in experimental animals.