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In Vivo Expression of Reprogramming Factors Increases Hippocampal Neurogenesis and Synaptic Plasticity in Chronic Hypoxic-Ischemic Brain Injury

View Article: PubMed Central - PubMed

ABSTRACT

Neurogenesis and synaptic plasticity can be stimulated in vivo in the brain. In this study, we hypothesized that in vivo expression of reprogramming factors such as Klf4, Sox2, Oct4, and c-Myc would facilitate endogenous neurogenesis and functional recovery. CD-1® mice were induced at 1 week of age by unilaterally carotid artery ligation and exposure to hypoxia. At 6 weeks of age, mice were injected GFP only or both four reprogramming factors and GFP into lateral ventricle. Passive avoidance task and open field test were performed to evaluate neurobehavioral function. Neurogenesis and synaptic activity in the hippocampus were evaluated using immunohistochemistry, qRT-PCR, and/or western blot analyses. Whereas BrdU+GFAP+ cells in the subgranular zone of the hippocampus were not significantly different, the numbers of BrdU+βIII-tubulin+ and BrdU+NeuN+ cells were significantly higher in treatment group than control group. Expressions of synaptophysin and PSD-95 were also higher in treatment group than control group. Importantly, passive avoidance task and open field test showed improvement in long-term memory and decreased anxiety in treatment group. In conclusion, in vivo expression of reprogramming factors improved behavioral functions in chronic hypoxic-ischemic brain injury. The mechanisms underlying these repair processes included endogenous neurogenesis and synaptic plasticity in the hippocampus.

No MeSH data available.


The number of new neurons in the hippocampus increased by in vivo expression of reprogramming factors. At postnatal 6 weeks, mice were injected with viral vector expressed GFP only (control group) or the four reprogramming factors and GFP (treatment group). To identify newly generated cells, mice were injected daily with 5-bromo-2-deoxyuridine (BrdU) up to 12 days. Eight weeks after injection, histological evaluations were performed. (a) The density of BrdU+ cells in the hippocampus was significantly higher in the treatment group than in the control group (t = 3.528, p < 0.05). (b-c) The density of newly generated neurons was determined through confocal microscopy by calculating the density of cells triple positive for DAPI (blue, nuclei), BrdU (green), and cell type-specific markers such as βIII-tubulin and NeuN. The densities of BrdU+βIII-tubulin+ (b) and BrdU+NeuN+ (c) cells were significantly higher in the treatment group than the control group (t = 2.450, p < 0.05 and t = 2.297, p < 0.05, resp.). (d–g) Confocal microscope images are immunohistochemistry results. (e, g) Cells with triple positive for DAPI, BrdU, and cell type-specific markers are indicated in the yellow box at the right panel. Scale bars = 50 μm. ∗p < 0.05.
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fig3: The number of new neurons in the hippocampus increased by in vivo expression of reprogramming factors. At postnatal 6 weeks, mice were injected with viral vector expressed GFP only (control group) or the four reprogramming factors and GFP (treatment group). To identify newly generated cells, mice were injected daily with 5-bromo-2-deoxyuridine (BrdU) up to 12 days. Eight weeks after injection, histological evaluations were performed. (a) The density of BrdU+ cells in the hippocampus was significantly higher in the treatment group than in the control group (t = 3.528, p < 0.05). (b-c) The density of newly generated neurons was determined through confocal microscopy by calculating the density of cells triple positive for DAPI (blue, nuclei), BrdU (green), and cell type-specific markers such as βIII-tubulin and NeuN. The densities of BrdU+βIII-tubulin+ (b) and BrdU+NeuN+ (c) cells were significantly higher in the treatment group than the control group (t = 2.450, p < 0.05 and t = 2.297, p < 0.05, resp.). (d–g) Confocal microscope images are immunohistochemistry results. (e, g) Cells with triple positive for DAPI, BrdU, and cell type-specific markers are indicated in the yellow box at the right panel. Scale bars = 50 μm. ∗p < 0.05.

Mentions: To determine the density of proliferating cells in the subgranular zone of the hippocampus, BrdU+ cells were counted. The number of BrdU+ cells in the treatment group (17.6 ± 1.9 × 103 cells) was significantly 2.2 times higher than in the control group (8.1 ± 2.0 × 103 cells) (t = 3.528, p < 0.05; Figure 3(a)). Meanwhile, to evaluate the cell fate of proliferating cells in the hippocampus, double-staining of cells with BrdU and cell type-specific markers such as βIII-tubulin (Tuj1, early neuronal marker), NeuN (mature neuronal maker), or GFAP (astrocyte marker) was performed. The numbers of BrdU+βIII-tubulin+ cells in the treatment group (9.2 ± 2.3 × 103 cells) were significantly 3.1 times higher than in the control group (3.0 ± 1.6 × 103 cells) (t = 2.450, p < 0.05; Figures 3(b), 3(d), and 3(e)), indicating that the in vivo expression of reprogramming factors enhanced neurogenesis. The number of BrdU+NeuN+ cells in the treatment group (13.2 ± 5.0 × 103 cells) was also significantly 6.2 times higher than in the control group (2.1 ± 1.5 × 103 cells) (t = 2.297, p < 0.05; Figures 3(c), 3(f), and 3(g)), suggesting that the newly generated neurons differentiate into mature neurons. However, there were no BrdU+GFAP+ cells in the hippocampus in either group suggesting that neurogenesis in the hippocampus is towards neurons, not astrocytes. The number of BrdU+ cells of two weeks after the surgical treatment groups is shown in Figure S1. The above values are described in Table 1.


In Vivo Expression of Reprogramming Factors Increases Hippocampal Neurogenesis and Synaptic Plasticity in Chronic Hypoxic-Ischemic Brain Injury
The number of new neurons in the hippocampus increased by in vivo expression of reprogramming factors. At postnatal 6 weeks, mice were injected with viral vector expressed GFP only (control group) or the four reprogramming factors and GFP (treatment group). To identify newly generated cells, mice were injected daily with 5-bromo-2-deoxyuridine (BrdU) up to 12 days. Eight weeks after injection, histological evaluations were performed. (a) The density of BrdU+ cells in the hippocampus was significantly higher in the treatment group than in the control group (t = 3.528, p < 0.05). (b-c) The density of newly generated neurons was determined through confocal microscopy by calculating the density of cells triple positive for DAPI (blue, nuclei), BrdU (green), and cell type-specific markers such as βIII-tubulin and NeuN. The densities of BrdU+βIII-tubulin+ (b) and BrdU+NeuN+ (c) cells were significantly higher in the treatment group than the control group (t = 2.450, p < 0.05 and t = 2.297, p < 0.05, resp.). (d–g) Confocal microscope images are immunohistochemistry results. (e, g) Cells with triple positive for DAPI, BrdU, and cell type-specific markers are indicated in the yellow box at the right panel. Scale bars = 50 μm. ∗p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: The number of new neurons in the hippocampus increased by in vivo expression of reprogramming factors. At postnatal 6 weeks, mice were injected with viral vector expressed GFP only (control group) or the four reprogramming factors and GFP (treatment group). To identify newly generated cells, mice were injected daily with 5-bromo-2-deoxyuridine (BrdU) up to 12 days. Eight weeks after injection, histological evaluations were performed. (a) The density of BrdU+ cells in the hippocampus was significantly higher in the treatment group than in the control group (t = 3.528, p < 0.05). (b-c) The density of newly generated neurons was determined through confocal microscopy by calculating the density of cells triple positive for DAPI (blue, nuclei), BrdU (green), and cell type-specific markers such as βIII-tubulin and NeuN. The densities of BrdU+βIII-tubulin+ (b) and BrdU+NeuN+ (c) cells were significantly higher in the treatment group than the control group (t = 2.450, p < 0.05 and t = 2.297, p < 0.05, resp.). (d–g) Confocal microscope images are immunohistochemistry results. (e, g) Cells with triple positive for DAPI, BrdU, and cell type-specific markers are indicated in the yellow box at the right panel. Scale bars = 50 μm. ∗p < 0.05.
Mentions: To determine the density of proliferating cells in the subgranular zone of the hippocampus, BrdU+ cells were counted. The number of BrdU+ cells in the treatment group (17.6 ± 1.9 × 103 cells) was significantly 2.2 times higher than in the control group (8.1 ± 2.0 × 103 cells) (t = 3.528, p < 0.05; Figure 3(a)). Meanwhile, to evaluate the cell fate of proliferating cells in the hippocampus, double-staining of cells with BrdU and cell type-specific markers such as βIII-tubulin (Tuj1, early neuronal marker), NeuN (mature neuronal maker), or GFAP (astrocyte marker) was performed. The numbers of BrdU+βIII-tubulin+ cells in the treatment group (9.2 ± 2.3 × 103 cells) were significantly 3.1 times higher than in the control group (3.0 ± 1.6 × 103 cells) (t = 2.450, p < 0.05; Figures 3(b), 3(d), and 3(e)), indicating that the in vivo expression of reprogramming factors enhanced neurogenesis. The number of BrdU+NeuN+ cells in the treatment group (13.2 ± 5.0 × 103 cells) was also significantly 6.2 times higher than in the control group (2.1 ± 1.5 × 103 cells) (t = 2.297, p < 0.05; Figures 3(c), 3(f), and 3(g)), suggesting that the newly generated neurons differentiate into mature neurons. However, there were no BrdU+GFAP+ cells in the hippocampus in either group suggesting that neurogenesis in the hippocampus is towards neurons, not astrocytes. The number of BrdU+ cells of two weeks after the surgical treatment groups is shown in Figure S1. The above values are described in Table 1.

View Article: PubMed Central - PubMed

ABSTRACT

Neurogenesis and synaptic plasticity can be stimulated in vivo in the brain. In this study, we hypothesized that in vivo expression of reprogramming factors such as Klf4, Sox2, Oct4, and c-Myc would facilitate endogenous neurogenesis and functional recovery. CD-1&reg; mice were induced at 1 week of age by unilaterally carotid artery ligation and exposure to hypoxia. At 6 weeks of age, mice were injected GFP only or both four reprogramming factors and GFP into lateral ventricle. Passive avoidance task and open field test were performed to evaluate neurobehavioral function. Neurogenesis and synaptic activity in the hippocampus were evaluated using immunohistochemistry, qRT-PCR, and/or western blot analyses. Whereas BrdU+GFAP+ cells in the subgranular zone of the hippocampus were not significantly different, the numbers of BrdU+&beta;III-tubulin+ and BrdU+NeuN+ cells were significantly higher in treatment group than control group. Expressions of synaptophysin and PSD-95 were also higher in treatment group than control group. Importantly, passive avoidance task and open field test showed improvement in long-term memory and decreased anxiety in treatment group. In conclusion, in vivo expression of reprogramming factors improved behavioral functions in chronic hypoxic-ischemic brain injury. The mechanisms underlying these repair processes included endogenous neurogenesis and synaptic plasticity in the hippocampus.

No MeSH data available.