Additional file 1: Figure S1. of MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response

(A) Normalized melt curves in duplicates showing the melt behavior of methylation standards (red=0 %, pink=25 %, blue=50 %, green=75 %, orange=100 %) and an unknown sample (black). (B) Regression model used for MGMT promoter methylation quantification. Area under the curve (AUC) from the normalized melt curves are used and regressed to the known methylation level of the standards. The linear regression model was chosen for quantification (R² > 0.98). (C) Pyrogram of the MGMT promoter of a patient tumor sample with a mean methylation of 31.4 %. (D) Typical pyrogram obtained from a grade III patient tumor sample indicating a heterozygous G-to-A point mutation of the IDH1 gene resulting in a mutation at codon 132 (R132H). (E) Relationship between MGMT protein activity and promoter methylation (r3) in 14 GBM cell lines. Figure S2. (A) Pyrogram of the whole HRM (R4) assay region from a patient sample. (B) Dot-plot of the methylation values obtained in a subsample by HRM and pyrosequencing showing a high correlation of this two methods. The dotted line indicates the 95 % CI. Figure S3. Overlay of Kaplan-Meier estimates of PFS and OS according to MGMT promoter methylation status. Kaplan-Meier estimates for (A) PFS and (B) OS of 65 high-grade glioma patients determined by HRM (red lines), MSP (blue) and PSQ (green). The solid and the dashed lines indicate the group as being categorized unmethylated (UM) or methylated (ME), respectively. Table S1. Primers used for HRM and pyrosequencing. Table S2. MGMT promoter methylation status determined by HRM, MSP, and PSQ in dependence of the IDH1 status. Table S3. ROC curves were plotted for 15 methylation cut-off scores (1-15 %) for predicting PFS ≥ 12 months and OS ≥ 18 months. (PPTX 312 kb

Acute effects on perfusion, cortical signaling and ICP.

<p>A: sample traces of rCBF of the three experimental groups immediately after injection. B: sample ECoG traces of the three experimental groups immediately after injection. C: ICP in SAH and saline groups before, during and until 1 minute after injection. Curves of the two groups do not significantly differ from each other. Both groups display significant elevation from baseline in the first 20 seconds after injection in paired samples t-test. Interestingly, although no longer statistically significant, in both groups, ICP settles at a new plateau that is higher than baseline for the rest of the recording period.</p

Cerebral and cerebellar rCBF impairment during DCI.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114946#pone-0114946-g003" target="_blank">Fig. 3</a> summarizes impairment of regional cerebral blood flow and the concentration of moving blood cells in the S1 and cerebellar cortex related to pre-injection values at time points 6, 12, 24 and 72 hours after injection. A: relative rCBF in the S1 cortex. B. concentration of moving blood cells in the S1. C. relative rCBF in the cerebellar cortex. D. concentration of moving blood cells in the cerebellar cortex. Note the delayed perfusion impairment in the SAH group 6 and 12 hours after injection and interestingly, the significant and long-lasting hyperperfusion that is caused by saline injection.</p

ECoG spectrum following SAH, saline and sham injection.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114946#pone-0114946-g004" target="_blank">Fig. 4</a> depicts the effect of the three experimental conditions on the ECoG spectrum. A: power spectrum of the SAH group. Injection of blood causes a drastic reduction of absolute power of almost all frequency bands lasting several minutes. Fast ripples were not detectable before SAH, but emerge instantly thereafter. B: power spectrum of the saline-injected group. C: power spectrum of the sham-injection group. Fast ripples were not detectable in saline or sham injected groups. D: Total ECoG power was significantly reduced in the SAH group by an average of 65%.</p

Exemplary ECoGs during acute SAH.

<p>A: Induction of SAH causing immediate severe perfusion impairment corresponds to complete cessation of ECoG signal in some animals lasting several minutes with activity returning in burst suppression patterns. Interburst intervals became continuously shorter until pre-injection levels of total power were reached. B: Bandpass (0.5–2 Hz) filtered ECoG. Recurrent short episodes of depression, potentially corresponding to cortical spreading depression were recorded in the low-frequency range in SAH (n = 3) but not in either control group. C: Bandpass (100–500 Hz) filtered ECoG. In SAH animals, short bursts in the ultra-high frequency spectrum occurred about 24 hours after SAH.</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

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 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