Proliferation of NG2+ glial progenitor cells.

<p>(A–C) Representative images of spinal cord sections doubly stained with BrdU (dark brown) and NG2 (dark blue) cells from PBS (A), F3 (B), and F3.VEGF (C) groups. Arrows indicate NG2+/BrdU+ cells and arrow heads NG2 single positive cells. Scale bars; 50 um. (D) The number of NG2+/BrdU+ cells per 1 mm³ of spinal cord tissue was stereologically counted and compared between the three groups at 2 weeks after injury. (E) The number of NG2+/BrdU+ cells was separately counted at the epicenter (designated as E) and 1.8 mm rostral and caudal (designated as R and C, respectively) to the epicenter. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 by one-way ANOVA followed by Tukey's <i>posthoc</i> analysis. White, hatched, and black bars represent PBS (N = 5), F3 (N = 5), and F3.VEGF (N = 5) groups, respectively.</p

Promotion of angiogenesis by transplantation of F3.VEGF.

<p>(A–F) Representative images of spinal cord sections (6 weeks after injury) stained with blood vessel marker vWF from PBS (A, D), F3 (B, E), and F3.VEGF (C, F) groups. Figures in the lower panel (D, E, F) are high power images of the boxed regions in (A, B, C), respectively. Scale bars in the upper panel; 200 um. Scale bars in the lower panel; 50 um. (G) The fraction of areas occupied by vWF immunoreactive vessels was compared between the three groups at the epicenter (designated as E) and 1.8 mm rostral and caudal (designated as R and C, respectively). ** <i>p</i><0.01, *** <i>p</i><0.001 by one-way ANOVA followed by Tukey's <i>posthoc</i> analysis. White, hatched, and black bars represent PBS (N = 5), F3 (N = 5), and F3.VEGF (N = 5) groups, respectively.</p

Locomotor recovery assessed by Basso, Beattie, and Bresnahan (BBB) test.

<p>Hind limbs locomotor function was scored as from 0 to 21 (flaccid paralysis to normal gait). Rats with transplantation of F3.VEGF showed significantly improved locomotor behavior up to 6 weeks after SCI. *** <i>p</i><0.001 compared to PBS group, ^#<i>p</i><0.05, ^###<i>p</i><0.001 compared to F3 group, by one-way ANOVA followed by Tukey's <i>posthoc</i> analysis. N = 8 for each group.</p

<i>Ex vivo</i> delivery of VEGF carried by human neural stem cells (NSCs) to the injured spinal cord.

<p>(A–C) Detection of transplanted NSCs by human specific mitochondria (hMito) staining at 1 week after transplantation (2 weeks after injury). Surviving F3 (B) and F3.VEGF (C) cells were observed around the lesion cavities. Control PBS group did not show any immunoreactivity to hMito (A). (D) Most of the transplanted NSCs did not incorporate BrdU. An arrow indicates a transplanted NSC colocalized with BrdU. Asterisks indicate lesion cavities at the epicenter. Scale bars; 100 um. (E) Levels of VEGF production in spinal cord tissue were measured by ELISA at 2 and 6 weeks after injury (1 and 5 weeks after transplantation). White, hatched, and black bars represent PBS (N = 5), F3 (N = 5), and F3.VEGF (N = 5) groups, respectively. * <i>p</i><0.05, *** <i>p</i><0.001 by one-way ANOVA followed by Tukey's <i>posthoc</i> analysis. (F) Western blot analysis of VEGF receptor flk-1 and phosphorylated flk-1 (p-flk-1).</p

Long term fate of early proliferating glial progenitor cells.

<p>(A–B) Confocal images of BrdU incorporated cells colocalized with mature oligodendrocyte marker CC1 (A) and astrocyte marker GFAP (B). Scale bars; 10 um. (C–D) The number of CC1+/BrdU+ (C) and GFAP+/BrdU+ (D) cells per 1 mm³ of spinal cord tissue was stereologically counted and compared between the three groups at 6 weeks after injury. * <i>p</i><0.05 by one-way ANOVA followed by Tukey's <i>posthoc</i> analysis. N = 5 for each group.</p

Sparing of spinal cord tissue at 6 weeks after injury.

<p>(A–I) Representative images of erichrome stained sections at the 1.8 mm rostral to the epicenter (A, D, G), epicenter (B, E, H), and 1.8 mm caudal to the epicenter (C, F, I). (A–C) PBS, (D–F) F3, and (G–I) F3.VEGF groups. Scale bar; 500 um. (J–L) The volumes of spared spinal cord tissue (J), white matter (K), and lesion cavities (L) were calculated by Cavelieri's method and compared between the three groups. ** <i>p</i><0.01, followed by Tukey's <i>posthoc</i> analysis. N = 8 for each group.</p

Transplantation of F3.VEGF enhanced cellular proliferation.

<p>(A–C) Representative images of BrdU stained sections from PBS (A), F3 (B), and F3.VEGF (C) groups. Scale bars; 50 um. (D) The number of BrdU+ cells per 1 mm³ of spinal cord tissue was stereologically counted and compared between the three groups at 2 weeks after injury. (E) The number of BrdU+ cells was separately counted at the epicenter (designated as E) and 1.8 mm rostral and caudal (designated as R and C, respectively) to the epicenter. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 by one-way ANOVA followed by Tukey's <i>posthoc</i> analysis. White, hatched, and black bars represent PBS (N = 8), F3 (N = 8), and F3.VEGF (N = 8) groups, respectively.</p

Validation of changes in protein expression from the Arc and Adam8 genes.

<p>(A, C) Representative western blots of Arc (A) and Adam8 (C). β-actin was used to normalize differences in loading amounts. (B, D) Quantification graphs of Arc (B) and Adam8 (D) western blots. Data are presented as fold-changes relative to the normal protein expression level after normalization with β-actin. N = 3 to 4 animals for each group. * and ** represent <i>p</i><0.05 and <i>p</i><0.01, respectively, by one-way ANOVA followed by Tukey's post-hoc analysis.</p

Treadmill training promotes locomotor recovery following contusive injury.

<p>Locomotor recovery was assessed by Basso, Beattie, and Bresnahan (BBB) locomotor scoring over the 8-week period after thoracic contusive injury. Animals subjected to treadmill training (TMT) showed enhanced locomotor recovery compared to those without TMT (control group). Arrows indicate the two time points chosen for microarray gene analysis. *, **, and *** indicate <i>p</i><0.05, <i>p</i><0.01, and <i>p</i><0.001, respectively, by Bonferroni post-hoc tests following repeated measures two-way ANOVA. N = 8 animals per group.</p

Genes downregulated by treadmill training.

<p>(A) Color-coded heatmaps of gene expression levels for genes whose expression was lower with treadmill training (TMT) than without TMT at 3 week after injury. Fold changes in gene expression level relative to the sham group expression level were log₂-transformed and color-coded based on the color scale shown at the bottom. Genes were grouped into 4 clusters using a k-means clustering algorithm. The numbers shown at the left side of the heatmaps indicate the cluster indexes to which the genes belong. Genes for which validation data are represented are shown in blue. (B) Expression patterns of genes in each cluster. Gray lines indicate expression levels of individual genes and black lines indicate the average expression level of all genes in the cluster. Numbers on the Y-axis indicate log₂-transformed fold changes relative to the expression level in the sham group. (C-E) Graphs of real-time RT-PCR results for genes selected from the list in (A) (shown in blue). N = 4 animals for each group. *, **, and *** represent <i>p</i><0.05, <i>p</i><0.01, and <i>p</i><0.001, respectively, by one-way ANOVA followed by Tukey's post-hoc analysis. Error bars represent SEM.</p