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

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

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

Microglia markedly increased in number in the lumbar motor region following thoracic SCI.

<p>(A-C) Representative images of lumbar spinal cord sections stained with an antibody recognizing the microglial marker Iba1 (dark brown) antibody in sham operation (A), at 3 weeks (3W) after injury (B), and at 3W after injury with treadmill training (TMT) (C). Immunostained sections were counterstained with eriochrome cyanine to differentiate the gray matter and white matters. Insets are magnified images of the regions in the white boxes in the ventral gray matter. Scale bars represent 100 µm. (D-F) Representative images of lumbar spinal cord sections colabeled with Iba1 (red) and MAP2 (green) antibodies in sham operation (D), at 3W after injury (E), and at 3W after injury with TMT (F). Note that Iba1 positive microglial cells are frequently associated with MAP2 positive neurons and dendritic neuropil areas. Scale bars represent 20 µm. (G-H) Quantification of stereological counting of Iba1-positive microglial cells in the gray matter (G) and white matter (H) of the lumbar spinal cord. *** represent <i>p</i><0.001 by one-way ANOVA followed by Tukey's post-hoc analysis. N = 5 for each group. Error bars represent SEM.</p

Validation of microarray data for inflammation-related genes using real time RT-PCR.

<p>(A-F) Quantification graphs of real-time RT-PCR for Lgals (Galectin3) (A), C3 (Complement component 3) (B), C1qa (complement component 1, q subcomponent, A chain) (C), C1qb (complement component 1, q subcomponent, B chain) (D), Itgb2 (integrin, beta 2, complement receptor 3 subunit) (E), and Timp 1 genes (F) in the sham operated group (sham), animals 1 week (1W), 3 weeks (3W) after injury without treadmill training (TMT), and with TMT. 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

Primer sequences used for real time RT-PCR.

<p>Primer sequences used for real time RT-PCR.</p

Genes upregulated by treadmill training.

<p>(A) Color-coded heatmaps of gene expression levels for the genes whose expression was higher with treadmill training (TMT) than without TMT at 3 weeks 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 5 clusters using a k-means clustering algorithm. The numbers shown at the left side of heatmaps indicate the cluster indexes to which the genes belong. Genes for which validation data are presented 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-F) 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

The complement pathway protein-protein interaction network.

<p>Nodes represent proteins and lines represent physical interactions between them. The color of the node indicates the fold-change in gene expression at 3 weeks relative to that in the control sham group. The color of the node border indicates the fold-change in expression at 1 week relative to that in the control sham group. Color scales are shown in the upper right. The fold-changes were log₂-transformed and only genes that showed significantly different expressions (<i>p</i><0.05 by Student’s t-test and absolute fold-change > 1.5) were color-mapped.</p

Number of animals in each group and at different time points for various experiments.

<p>SCI: spinal cord injury, TMT: treadmill training.</p

A list of genes that were regulated by treadmill locomotor training.

<p>A list of genes that were regulated by treadmill locomotor training.</p