Representative pictures of Golgi Cox-impregnated layer V pyramidal neurons of the control, CIMT and G-CSF-treated group in the peri-infarct cortex (A) and the corresponding contralateral cortex (B), respectively.

<p>Note the increase of the basilar dendritic arbor in the peri-infarct cortex of G-CSF treated rats. Scale bar: 50 μm.</p

Sholl analysis of Golgi Cox-impregnated layer V pyramidal neurons in the peri-infarct cortex and in the corresponding contralateral cortex.

<p>Note that corresponding to the increased length of basilar dendrites (c.f. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146679#pone.0146679.g003" target="_blank">Fig 3</a>) a significantly increased number of intersections of higher order in the G-CSF group of the peri-infarct cortex were detectable (B) which paralleled the functional outcome as recently published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146679#pone.0146679.ref006" target="_blank">6</a>] (A, B, D, E significant difference compared with control, CIMT, CIMT+G-CSF, CIMT/G-CSF, respectively. Values are expressed as mean number of intersections / μm ± SEM; p<0.05).</p

Topographic localization of the photothrombotic infarct.

<p>Note that infarct was mirrored on the contralateral side to analyze Golgi Cox-impregnated cortical layer V pyramidal neurons in the ipsilateral peri-infarct cortex and in the corresponding contralateral cortex (Insets: representative Golgi Cox-impregnated cells).</p

Quantitative analysis of Golgi Cox-impregnated layer V pyramidal neurons in the peri-infarct cortex and in the corresponding contralateral cortex.

<p>There were no differences in the number of basilar dendrites between the various experimental groups neither in the peri-infarct cortex nor in the corresponding contralateral cortex (Values are expressed as mean number of basilar dendrites ± SEM).</p

Flow cytometric analysis of brain immune cells at 5d after permanent MCAO.

<p>Cells per one hemisphere (Mean±SD). N = 6 mice per group. Data of 2 individual experiments.</p

FTY720 changes serum cytokine levels.

<p>Serum cytokine concentrations of the pro-inflammatory cytokines IL-6, IFN-γ and TNF-α (<b>A–C</b>) and anti-inflammatory cytokines TGF-β and IL-10 (<b>D,E</b>) were measured in naïve mice and at 24 h and 5d after FTY720 or control treatment. Each assay was performed in duplicate (n = 5, serum sampling as 2–3 individual experiments, assays as one experiment). * P<0.05 between treatment groups at the respective time point.</p

FTY720 does not influence brain edema and blood-brain-barrier permeability.

<p>(<b>A</b>) Brain water content was analyzed as the percentage water content per hemisphere by the “wet/dry weight” method and is shown as the water content ratio of ischemic/non-ischemic hemispheres. Open bars represent water content ratios in animals at 3d after Sham operation (“Sham”) and black bars in mice at 3d after permanent coagulation MCAO (“MCAO”) receiving FTY720 or PBS treatment by oral gavage (n = 5 per group, 2 individual experiments). (<b>B</b>) Blood-brain-barrier permeability was tested using the Evans blue assay for dye extravasation at 3d after permanent coagulation MCAO in mice receiving FTY720 or PBS treatment by oral gavage. Data is shown as the ratio for ischemic/non-ischemic hemisphere fluorescence intensity at 680 nm (n = 5, one experiment).</p

FTY720 decreases cerebral lymphocyte invasion.

<p>(<b>A</b>) Brain sections were stained 5d after MCAO for T cells (CD3), B cells (B220), granulocytes (MPO) and activated microglia/macrophages (IBA1). (<b>B</b>) Analysis of absolute cell counts of T cells, B cells, granulocytes and microglia/macrophages per total hemisphere in PBS and FTY720 treated animals at 5d after MCAO (n = 6–10 per group, 2–3 individual experiments).</p

FTY720 does not reduce infarct volume after cortical permanent ischemia.

<p>Animals in the treatment groups (FTY720) received daily administrations of 1 mg/kg FTY720 by oral gavage starting at 48 h before infarct induction.I Infarct volumes were determined (<b>A</b>) at 3d (n = 8 per group, p = 0.73, 2 individual experiments) and (<b>B</b>) at 7d (n = 17, 0.54, 4 individual experiments) after infarct induction. Control animals received daily PBS injections. (<b>C</b>) Animals were treated daily with either FTY720 or PBS, starting from 3 h after MCAO and infarct volumes were determined at 7d after brain ischemia (n = 10, p = 0.43, 2 individual experiments). (<b>D</b>) Mice received a single dose of FTY720 or PBS at 48 h before brain ischemia and infarct volumetry was performed at day 7 (n = 10, p = 0.27). Behavioural dysfunction and recovery after experimental stroke was assessed in FTY720 pretreated animals (daily treatment starting 48 h before MCAO) or in control animals by the (<b>E</b>) “cylinder test” (n = 12, 3 individual experiments) and the (<b>F</b>) “corner test” (n = 12, 3 individual experiments).</p

FTY720 treatment induces leukopenia.

<p>(<b>A</b>) Differential blood cell counting was performed in normal mice (Naive) and 6 h, 24 h, 3d and 7d after daily administration of FTY720. Mean values (n = 5 per time point) are depicted for total leukocytes, granulocytes, lymphocytes and monocytes as cells per µl whole blood. (<b>B</b>) Leukocyte subpopulations were further characterized by specific epitope markers for T cells (CD3, CD4, CD8), B cells (B220), regulatory T cells (Foxp3) and monocytes (CD11b) at the indicated time points in blood, spleen and mesenteric lymph nodes (n = 5 per group). Each experiment was performed 2–3 times.</p