Cell culture

NSCs were cultured from fetal rat cortex at embryonic day 14.5, as described previously [18]. Cells were expanded as monolayer cultures in serum-free DMEM/F12 medium (Life Technologies, Darmstadt, Germany) with N2 supplement (Gibco, Karlsruhe, Germany) and fibroblast growth factor (FGF2; 10 ng/ml; Invitrogen, Karlsruhe, Germany) for 5 days and were replated in a 24-well plate at 10,000 cells per cm2. FGF2 was included throughout the experiments.

Ar-turmerone (Fluka, Munich, Germany) was added to cultures at replating at concentrations of 0, 1.56, 3.125, 6.25, 12.5, and 25 μg/ml. All experiments were performed in triplicate. After 72 hours, representative pictures were taken by using an inverted fluorescence phase-contrast microscope (Keyence BZ-9000E). Three images were taken per well, and cells were counted by using the software ImageJ with a threshold of 20 px (National Institutes of Health, Bethesda, MD, USA, Version 1.47 k).

To determine the ratio of proliferating cells, 10 μM bromodeoxyuridine (BrdU; Fluka, Munich, Germany) was added to cultures for 6 hours, before cells were fixed with 4% PFA. Again, all experiments were performed in triplicate. Cells were stained with mAb against BrdU to identify proliferating cells (clone BU-33, dilution 1:100; Sigma-Aldrich, Munich, Germany). For antigen-retrieval before staining, sections were incubated in 2 N HCl for 30 minutes. For visualization, FITC-labeled anti-mouse IgG was used (Invitrogen); all cells were additionally counterstained with Hoechst 33342 (Life Technologies). To calculate the ratio of proliferating cells, BrdU-positive cells were divided by the total cell number in each sample, and mean values were established among equally treated cells.

To establish its effect on cell survival, ar-turmerone was added to NSC cultures for 24 hours. To discriminate between live and dead cells, the live/dead cell-mediated cytotoxicity kit (Life Technologies, cat. no. L7010) was used according to the manufacturer’s instructions. Both viable and dead NSCs were counted in n = 6 samples per condition, and a ratio of surviving cells was calculated for each field of view; mean values were calculated for each concentration tested.

To assess the differentiation potential of NSCs treated with ar-turmerone, mitogen was withdrawn during the expansion phase, followed by a differentiation phase of 10 days, in the absence (control) or presence of 6.25 μg/ml ar-turmerone. Immunocytochemistry with markers for young neurons (TuJ1), astrocytes (GFAP), and oligodendrocytes (CNPase) was used to verify all three differentiated fates of NSCs, whereas SOX2 marked undifferentiated NSCs.

Real-time quantitative PCR (RT-qPCR)

RNA from cells was isolated by using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA concentration and purity were evaluated photometrically. Total RNA was converted to c-DNA by reverse transcription with the Quantitect reverse transcription kit (Qiagen). The primer used for Ki67 was obtained from Biolegio (Nijmegen, The Netherlands). The sequences of the primers were as follows: (a) forward: TCTTGGCACTCACAGTCCAG, and (b) reverse: GCTGGAAGCAAGTGAAGTCC. The q-PCR reaction was carried out by using 10 ng total RNA in a 20-μl reaction (Quantitect Reagents, Qiagen) according to the manufacturer’s instructions. The samples were amplified and quantified on a Rotorgene 2000 (Corbett, Sydney, Australia) by using the following thermal cycler conditions: activation: 95°C 10 minutes; cycling: 50 cycles, step 1: 92°C, 15 seconds, step 2: 52°C, 15 seconds, and step 3: 72°C, 40 seconds. PCR product integrity was evaluated by melting-point analysis and agarose gel electrophoresis. Each sample and gene was normalized to RPL13a as reference gene [19]. Ki67 mRNA levels were normalized to endogenous RPL13a expression (ΔCT); normalized values were then expressed as 2-ΔCt. Mean values were calculated for treated and untreated cells.

Animals and surgery

All animal procedures were in accordance with the German Laws for Animal Protection and were approved by the local animal care committee (Buero der Tierschutzbeauftragten, MPIfNF, Cologne, Germany), as well as local governmental authorities (LANUV NRW 84–02.04.2012.A116). Spontaneously breathing male Wistar rats weighing 290 to 330 g were anesthetized with 5% isoflurane and maintained with 2.5% isoflurane in 65%:35% nitrous oxide/oxygen. Throughout surgical procedures, the body temperature was maintained at 37.0°C with a thermostatically controlled heating pad.

Intracerebroventricular injections

One group of animals (n = 3) underwent a single intracerebroventricular (i.c.v.) injection of 3 mg ar-turmerone at a concentration of 1 mg/μl. For control, n = 6 rats were vehicle-injected with the identical volume of normal saline. Under anesthesia with 1.5% isoflurane, each rat’s skull was fixated in a stereotaxic frame in plane orientation. After incision of the skin, the bregma was exposed, and a burr hole was drilled over the right lateral ventricle by using the following stereotaxic coordinates: bregma, AP −0.9 mm; ML, −1.4 mm; and VD, +3.8 mm. Ar-turmerone dissolved in normal saline, or respectively, pure saline as control, was injected at 1 μl/min. After injection, the needle was left in place for another 5 minutes to allow a distribution of the solution within the ventricles. The needle was thereafter withdrawn slowly, and the skin sutured with nonabsorbing silk.

After each procedure, all animals were allowed to recover from anesthesia and were put back into their home cages, where they were given access to food and water ad libitum.

BrdU injections

In all animals, the tracer bromodeoxyuridine (BrdU) was injected intraperitoneally for 5 days, starting on the day of i.c.v. injection, at a concentration of 50 mg/kg per injection, as described previously [18]. This regimen resulted in a cumulative dose of 250 mg/kg BrdU per animal.

Positron emission tomography (PET)

[18F]-fluoro-L-thymidine ([18F]FLT) was synthesized as described previously [20]. Seven days after i.c.v. injection of ar-turmerone or placebo, respectively, PET imaging was performed on a microPET Focus 220 scanner (Concorde Microsystems, Inc., Knoxville, TN, USA; 63 image planes; 1.5-mm full width at the half maximum). Animals were anesthetized with 5% isoflurane, maintained with 2% isoflurane in a 65%:35% nitrous oxide/oxygen atmosphere, and placed in the scanner. Temperature was monitored by using a rectal probe and maintained at 37°C ± 0.5°C by a thermostatically controlled water-flow system (Medres, Cologne, Germany). After a 10-minute transmission scan for attenuation correction, rats received an intravenous bolus injection of [18F]FLT (1.0 to 2.2 mCi/rat), and emission data were acquired for 60 minutes. PET data were reconstructed in two time frames of 1,800 seconds. The last frame (that is, minutes 31 to 60 after tracer injection) was used for image analysis.

Image analysis

PET images were co-registered to anatomic data of a 3D rat-brain atlas constructed from the brain slices presented by Swanson [21]. Based on the 3D anatomic data, ellipsoid volumes of interest (VOIs) measuring 4 mm3 were placed to cover the subventricular zone (SVZ) as well as the dentate gyrus region of the hippocampus. A standard uptake value (SUV) was calculated for each VOI, dividing maximal VOI activity by the decay-corrected injected radioactive dose per body weight. SUVs were individually determined and then averaged between animals within each group.

Immunohistochemistry

After PET imaging, or 7 days after ar-turmerone treatment, rats were deeply anesthetized and decapitated. The brains were rapidly removed, frozen in isopentane, and stored at −80°C before further histologic and immunohistochemical processing. Ten-μm-thick adjacent serial coronal brain sections were cut at 500-μm intervals and stained with anti-BrdU to identify proliferating cells (mAb clone BU-33, dilution 1:200; Sigma-Aldrich), or with anti-doublecortin (DCX) to identify neuroblasts (rabbit polyclonal, dilution 1:1,000, Sigma-Aldrich). For antigen-retrieval before BrdU staining, sections were microwave-heated in 0.01 M citrate buffer, pH 6.0, for 5 minutes, followed by 2 N HCl at 37°C for 30 minutes. For visualization, the ABC Elite kit (Vector Laboratories with diaminobenzidine (Sigma-Aldrich) as the final reaction product was used.

To quantify the width of the SVZ and of the dentate gyrus of the hippocampus, it was measured on three consecutive BrdU-stained slices per animal, and an average was calculated per animal. To quantify the number of neuroblasts in the SVZ, their number was counted on three consecutive DCX-stained slices within a standardized field-of-view for each animal. For both schemes of quantification, mean values were calculated for each group of animals.

Statistical analysis

Descriptive statistics were performed with Microsoft Excel 2003 (Microsoft Corp., Redmond, WA, USA). One-way ANOVA tests (followed by Holm-Sidak post hoc test) were performed with SigmaPlot 11.0 for Windows (Systat Software Inc., San Jose, CA, USA). Statistical significance was set at P < 0.05.