Fig 1.
Cell proliferation at the SVZ in a rat model of SAH.
Coronal sections of forebrain were immunostained with anti-Ki67 antibody (red, proliferating cell), anti-nestin antibody (green, NSC) and Hoechst 33342 (blue, nucleus).The level of cell proliferation in the SVZ was determined by Ki67/ Hoechst 33342 co-staining. (A)-(T): photomicrograph showing the distribution of Ki67+ (A, E, I, M, and Q) and nestin+ (B, F, J, N, and R) signals and as merged images (D, H, L, P, and T) in the SVZ of animals at different SAH time course (1, 3, 5, and 7 days after SAH) and sham control. Scale bar = 50 μm. (U)-(X): Images of Ki67+ /nestin+ NSCs in the SVZ on day 7 post SAH showing Ki67 (U) and nestin (V) immunoreactivity separately or as merged image (X). Scale bar = 7.5 μm. (Y): Percentage of NSCs (Nestin+ of Hoechst+ cells) in the SVZ. (Z): Percentage of proliferating cells (Ki67+ of Hoechst+ cells) in the SVZ. Means ± SD, n = 6 for each time-point. *, P < 0.05; ***, P < 0.001, one way ANOVA with Dunnett's multiple comparisons test.
Fig 2.
NSC differentiation and neuroblast migration around the SVZ after SAH.
Coronal sections of forebrain were immunostained with anti-DCX (green: neuroblast), anti-GFAP (red: astrocyte) and Hoechst 33342 (blue: nucleus) to analyze the effect of SAH on NSC differentiation at the SVZ and neuroblast migration toward the striatum. (A)-(T): photomicrograph showing the distribution of DCX+ (A, E, I, M, and Q) and GFAP+ (B, F, J, N, and R) signals and as merged images (D, H, L, P, and T) at the SVZ of animals with different SAH time course (1, 3, 5, and 7 days after SAH) and sham control. Scale bar = 50 μm. (U)-(X): Images showing DCX+ (U) and GFAP+ (V) cells or as merged image (X) in the striatum on day 7 post SAH. Scale bar = 10 μm. (Y): Percentage of DCX+ area (DCX+ area/ total area) at the SVZ; (Z): Percentage of DCX+ area (DCX+ area/ total area) in the striatum 50 μm away from the SVZ; (Aa) Percentage of GFAP+ area (GFAP+ area/ total area) in the striatum. Means ± SD, n = 6 for each time-point. **, P < 0.01; ***, P < 0.001, one way ANOVA with Dunnett's multiple comparisons test.
Fig 3.
Treatment with post-SAH CSF promotes cell proliferation of cultured neurospheres.
Cells were immunostained with anti-nestin antibody (green: NSC), Ki67 antibody (red: proliferating cell) and Hoechst 33342 (blue: nucleus) to determine the level of cell proliferation of cultured neurospheres. (A)-(T): photomicrograph showing the distribution of Ki67+ (A, E, I, M, and Q) and nestin+ (B, F, J, N, and R) signals and as merged images (D, H, L, P, and T) in the neurospheres treated with and without CSF. Scale bar = 50 μm. (U): Percentage of proliferating cells (Ki67+ of Hoechst+ cells) in the neurospheres. Means ± SD, 12 neurospheres were counted for each condition. ns: non-significant; *, P < 0.05; ***, P < 0.001, one way ANOVA with Tukey's multiple comparisons test.
Fig 4.
Treatment with post-SAH CSF enhances cell differentiation of cultured neurospheres.
Cell differentiation were determined by immunocytostaining of cultured neurospheres with anti-DCX antibody (green: neuroblast), GFAP antibody (red: astrocyte) and Hoechst 33342 (blue: nucleus). (A)-(T): photomicrograph showing the distribution of DCX+ (A, E, I, M, and Q) and GFAP+ (B, F, J, N, and R) signals and as merged images (D, H, L, P, and T) in the neurospheres treated with and without CSF. Scale bar = 50 μm. (U)-(X): Images showing DCX+ (U) and GFAP+ (V) cells or as merged image (X) in the neurospheres treated with CSF collected from rats 7 days post SAH. Scale bar = 16 μm (Y): Percentage of DCX+ area (DCX+ area/ total area); (Z): Percentage of GFAP+ area (GFAP+ area/ total area). Means ± SD, 12 neurospheres were counted for each condition. ns: non-significant; *, P < 0.05; ***, P < 0.001, one way ANOVA with Tukey's multiple comparisons test.
Fig 5.
Concentration of trophic factors in CSF from SAH rats.
ELISA analysis was applied to measure the concentration of trophic factors in the CSF. The concentration of BDNF was significantly increased in the CSF collected from rats on days 5 and 7 post SAH as compared to that in the CSF from rats without SAH. The concentration of bNGF, EPO and EGF was increased in the CSF from rats on day 3 or 5 post SAH. Results of ELISA were analyzed by one way ANOVA with Tukey's multiple comparisons test. *, P < 0.05; **, P < 0.01, ***, P < 0.001 compared with CSF without SAH. ND, non-detectable.
Fig 6.
BDNF promotes cell proliferation, differentiation, and migration of cultured neurospheres.
Neurospheres were immunostained with Ki67, nestin, DCX and GFAP antibodies to determine the effects of recombinant BDNF (5 pg/ml) on neurogenesis of cultured neurospheres. The groups of neurospheres treated with 0.5% CSF (collected from rats on day 7 post SAH) and without treatment (control) were used for comparison. (A)-(I): photomicrograph showing the distribution of Ki67+ (A, D, and G) and nestin+ (B, E, and H) signals and as merged images (C, F, and I); (J)-(R): photomicrograph showing the distribution of DCX+ (J, M, and P) and GFAP+ (K, N, and Q) signals and as merged images (L, O and R). Scale bar = 50 μm. (S): Percentage of proliferating cells (Ki67+ of Hoechst+ cells) in the neurospheres. (T): Percentage of DCX+ area (DCX+ area/ total area). (U): Percentage of GFAP+ area (GFAP+ area/ total area). Means ± SD, 12 neurospheres were counted for each condition. ns: non-significant; ***, P < 0.001, one way ANOVA with Tukey's multiple comparisons test.
Fig 7.
BDNF expression and its cellular localization in the SVZ of SAH rats.
Coronal forebrain sections were immunostained with anti-BDNF, nestin, GFAP, Iba-1 (microglia) antibodies to determine the expression of BDNF after SAH. (A)-(J): photomicrograph showing the distribution of BDNF+ (A, C, E, G, and I) signal and as merged images (B, D, F, H, J) in the SVZ of animals with different SAH time course (1, 3, 5, and 7 days after SAH) and sham control. Scale bar = 50 μm. (K)-(V): Images showing BDNF+ (K, L and M), nestin+ (N), GFAP+ (O) and Iba-1+ (P) immunoreactivity and as merged images (T, U and V) on day 7 post SAH in the SVZ. Scale bar = 16 μm. (W): Percentage of BDNF+ area (BDNF+ area/ total area) in the SVZ. Means ± SD, n = 6 for each time-point. *, P < 0.05; one way ANOVA with Tukey's multiple comparisons test.