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

Mean values and standard deviations of data shown in Figure 1b.

<p>* = p<0.05 between Naïve mice and respective time point after FTY720 (n = 5 per group).</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 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

FTY720 does not alter infarct volume in alternative experimental protocols.

<p>(<b>A</b>) Representative images of brain sections stained by the silver-staining technique from coagulation-MCAO and transient 60 min filament-MCAO as used for determination of infarct volumes. (<b>B</b>) Brain ischemia was induced by 60 min reversible MCAO and infarct volumes were determined at 24 h after ischemia in animals receiving daily oral administration of FTY720 or PBS starting 48 h before stroke induction (n = 12, p = 0.42, 3 individual experiments). (<b>C, D</b>) FTY720 or PBS were administered by daily i.p. injection starting 48 h berfore MCAO. (<b>C</b>) MCAO was induced by transcranial permanent coagulation and infarct columes determined at 7d after MCAO (n = 10, p = 0.36, 2 individual experiments). (<b>D</b>) MCAO was induced by transient 60 min filament-occlusion of the MCA and infarct volume measured at 24 h after MCAO (n = 9, p = 0.86, 2 individual experiments).</p

FTY720 does not affect basal cardiovascular parameters.

<p>Mean arterial pressure (<b>A</b>) and cerebral blood flow (<b>B</b>) were recorded for 10 min before and 180 min after oral administration of 1 mg/kg FTY720 in 100 µl PBS or 100 µl PBS alone. Values are expressed as relative units in relation to baseline values before the respective treatment (n = 3 per group, each mouse was an individual 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