Tetrahydrofolate Attenuates Cognitive Impairment after Hemorrhagic Stroke by Promoting Hippocampal Neurogenesis via PTEN Signaling

THF promotes the proliferation of hippocampal NSCs after ICH. A, Schematic diagram of the ICH model, as well as experimental timeline for THF, EdU administration, and cell proliferation analysis. B, Representative images of brain section stained with EdU (red, Alexa Fluor 555), Sox2 (green, Alexa Fluor 488), GFAP (white, Alexa Fluor 647), and DAPI in the DG of ICH mice. Scale bar, 20 μm. C, Quantification of numbers of Sox2⁺GFAP⁻EdU⁺ and Sox2⁺GFAP⁺EdU⁺ cells from B. N = 5 for each group. D, Representative images of brain section stained with EdU (red, Alexa Fluor 555), DCX (green, Alexa Fluor 488), and DAPI in the DG of ICH mice. Scale bar, 20 μm. E, Quantification of numbers of EdU⁺DCX⁺/EdU⁺ cells from D. N = 5 for each group.

THF can alleviate ICH-induced abnormal neurogenesis. A, Schematic diagram of the ICH model, as well as experimental timeline for THF, EdU administration, and neurogenesis analysis. B, Representative images of brain section stained with EdU (red, Alexa Fluor 555), GFAP (white, Alexa Fluor 647), and genetically labeled GFP (green, Alexa Fluor 488) in the DG in ICH mice with or without THF treatment. Scale bar, 10 μm. C, Quantification of numbers of proliferated rNSCs (GFP⁺GFAP⁺EdU⁺) cells from B. N = 6 for each group. D, Representative images of brain section stained with EdU (purple, Alexa Fluor 647), Ki67 (red, Alexa Fluor 555), and genetically labeled GFP (green, Alexa Fluor 488) in the DG in ICH mice with or without THF treatment. Scale bar, 10 μm. E, Quantification of ratio of Ki67⁺EdU⁺ rNSCs/EdU⁺ rNSCs from D. N = 6 for each group. F, Experiments timeline for cell differentiation analysis of Sham, ICH + Vehicle, and ICH + THF groups. G, Representative images of brain section stained with EdU (green, Alexa Fluor 488) and NeuN (red, Alexa Fluor 555) in the DG in different group. Scale bar, 100 μm. H, Quantification of numbers of NeuN⁺EdU⁺ cells from G. N = 6 for each group. *p < 0.05; **p < 0.01; ***p < 0.001.

THF promotes neurogenesis by downregulating PTEN after ICH. A, Immunoblot bands showing the expression level of PTEN in group Sham, ICH + Vehicle, and ICH + THF, respectively. β-Actin was served as an internal control. B, Semiquantitative analysis of PTEN expression from A. N = 3 for each group. C, Schematic diagram of the ICH model, as well as experimental timeline for afzelin, EdU administration, and neurogenesis analysis. D, Immunoblot bands showing the expression level of PTEN in group ICH + Vehicle and ICH + Afzelin, respectively. β-Actin was served as an internal control. E, Semiquantitative analysis of PTEN expression from D. N = 3 for each group. F, Representative images of brain section stained with EdU (purple, Alexa Fluor 647), GFAP (red, Alexa Fluor 555), and genetically labeled GFP (green, Alexa Fluor 488) in the DG in ICH mice with or without afzelin treatment. Scale bar, 10 μm. G, Quantification of numbers of and GFP⁺GFAP⁺EdU⁺ cells from F. N = 6 for each group. H, Representative images of brain section stained with EdU (green, Alexa Fluor 488) and NeuN (red, Alexa Fluor 555) in the DG in different group. Scale bar, 50 μm. I, Quantification of numbers of NeuN⁺EdU⁺ cells from H. N = 6 for each group. *p < 0.05; **p < 0.01; ***p < 0.001.

Conditional knock-out of PTEN rejuvenates neurogenesis after ICH. A, B, Schematic showing the stereotaxic injection of AAV into the DG of adult Nestin-Cre mice, as well as experimental timeline for analysis. C, Immunoblot bands showing the expression level of PTEN in group ICH + Scramble and ICH + shPTEN, respectively. GAPDH was served as an internal control. Semiquantitative analysis of WB results. GAPDH was served as the internal control (n= 3 for each group). D, Sample images of virus-labeled mCherry cells costained with GFAP (green, Alexa Fluor 488) in the adult DG. Scale bar, 10 μm. E, Quantification of numbers of GFAP⁺mCherry⁺ cells from E; N = 6 for each group. F, Sample images of virus-labeled mCherry cells costained with NeuN (green, Alexa Fluor 488) in the adult DG. Scale bar, 10 μm. G, Quantification of numbers of NeuN⁺mCherry⁺ cells from D; N = 6 for each group. *p < 0.05; **p < 0.01; ***p < 0.001.

Administration of THF after ICH activated the downstream signaling pathway of PTEN. A, Immunoblot bands showing the expression level of p-Akt, Akt, p-mTOR, mTOR in group Sham, ICH + Vehicle, and ICH + THF, respectively. β-Actin was served as an internal control. B, Semiquantitative analysis of p-Akt, Akt, p-mTOR, and mTOR expression from A. N = 3 for each group. C, Immunoblot bands showing the expression level of p-Akt, Akt, p-mTOR, and mTOR in group ICH + Vehicle and ICH + Afzelin, respectively. GAPDH was served as an internal control. D, Semiquantitative analysis of p-Akt, Akt, p-mTOR, and mTOR expression from C. N = 3 for each group. *p < 0.05; **p < 0.01; ***p < 0.001.