All test substances were diluted in Dulbecco’s modified Eagle medium (canine glia, DMEM Gibco-Invitrogen, Karlsruhe, Germany) or DMEM/F12 (NT2 cells, Gibco-Invitrogen) containing 10% fetal bovine serum (Invitrogen), 1% penicillin and streptomycin. The cAMP analogue 8-Br-cAMP (8-Bromoadenosine 3′,5′-cyclic monophosphate), the cGMP analogue 8-Br-cGMP (8-Bromoguanosine 3′,5′-cyclic monophosphate) were purchased from Sigma (Taufkirchen, Germany). The protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate purchased from Alomone Labs (Jerusalem, Israel). Unless stated otherwise, all chemicals were obtained from Sigma.
All animals used for cell isolation were treated according to the legal and ethical requirements of the University of Veterinary Medicine Hannover (Germany). The procedures complied with the guidelines of animal welfare as laid down by the German Research Council (DFG). Purified primary OEC and Schwann cell cultures were generated as described previously .
OM-OEC cultures were isolated from the caudal regions of the nasal septum and conchae. All Schwann cells were removed immediately from primary cell suspensions by using anti-HNK-1 (OM-OECs) and anti-p75NTR antibodies (OM-OECs, OB.OECs) and magnet-activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Schwann cells were obtained from the fibular nerve after removal of the epineural sheath . Trypsin, hyaluronidase type IV, and collagenase type XI (0.5% each, Sigma Aldrich, Taufkirchen, Germany) were used to digest nerve preparations after 5–7 days of pre-degeneration in vitro by incubating nerve fibers . Canine glia were cultured in T75 culture flasks with DMEM containing additionally 20 ng/ml recombinant human FGF-basic (fibroblast growth factor, Peprotech). Cells were trypsinized (Trypsin-EDTA, PAA, Marburg, Germany) twice a week and transferred to new T75 flasks and cultured under standard conditions (37°C, 5% CO2). Purity of glia cell cultures was about 95% as earlier reported in Ziege et al. . NT2/D1 precursor cells (NT2) were purchased from the American Type Culture Collection (ATTC, Manassas, VA20108, USA). Neuronal differentiation was done as previously described [75, 76]. After grown to confluence NT2 precursor cells were trypsinized (Trypsin-EDTA, Gibco-Invitrogen) and cultured in 95 mm, bacteriological grade Petri dishes (Greiner, Hamburg, FRG) at a density of 5 × 106 cells/dish in 10 ml Dulbecco’s modified Eagle medium (DMEM/F12,) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin and 10 μM retinoic acid to start neuronal differentiation. After 7–10 days, cells were trypsinized again and transferred to T75 culture flasks with retinoic acid at a density of 4 × 107 cells / flask. After additional 7–10 days, cells were trypsinized and cultured 2 days with DMEM/F12 until they were transferred to T175 flasks with DMEM/F12 containing mitotic inhibitors (1 μM 1-6-D-arabinofuranosylcytosine, 10 μM 2′-deoxy-5-fluorouridine and 10 μM 1-β-D-ribofuranosyluracil). During culturing of cells with medium containing mitotic inhibitors, neurons became visible after 7–10 days. Neurons were selectively trypsinized, counted and used in up-coming experiments. Neurons termed as 2wk RA representing a model for developing human neurons were cultured for additional 7 days with 10 μM retinoic acid in petri dishes instead of being transferred to T175 flasks with inhibitor medium . After two weeks of retinoic acid treatment in petri dishes cells were dispersed, counted and used for neurite outgrowth experiments.For all cell culture experiments, canine glia of passage 7 (OB-OECs, OM-OECs and SCs), 2wkRA neurons of passage 20–30 and hNT2 passage 22–31 were used.
Scratch wound assay
Migration of glial cells (passage 7) and human NT2 precursor (passage 22–30) cells was measured by the scratch wound assay as described by Liang et al. . Olfactory ensheathing cells, Schwann cells and NT2 precursor were plated at a density of 150.000 -200.000 cells to poly-L-lysine coated (glia) and uncoated (NT2 precursor) 24-well-plates (Corning Costar, Kaiserslautern, Germany) 24 hours prior to the scratch. The next day a cell free area spanning approximately 600 μm in diameter was scratched using a crystal 10 μl pipette tip (Figure 1A). The wells were washed with medium to remove cellular debris. Cells were incubated with DMEM (canine glia) or DMEM/F12 (NT2) containing the cGMP analogue 8-Br-cGMP (10, 100, 500 μM), the cAMP analogue 8-Br-cAMP (10, 100, 500 μM) and PKC activator phorbol-12-myristate 13-acetate (TPA, 1, 10, 100 nM). The scratch was photographed 0 h and 8 h after its generation using a Zeiss Axiovert 200 microscope equipped with a Cool Snap camera (Photometrics, Tucson, Ariz., USA) and Meta Morph software (Molecular Devices, Sunnyvale, Calif., USA). Gap width was measured using ImageJ 1.46d (NIH, http://rsbweb.nih.gov/ij/, USA).
The distance was calculated by subtracting the average gap width after 8 h from the average gap width at 0 h divided by 2 (Figures 1E and F). BrdU incubation caused the labelling of only a few cells in the scratch wound (data not shown). Due to the short time interval of 8 h, we can rule out a significant contribution of cell proliferation to the closure of the gap. Contrast enhancement and image overlay was done with ImageJ.
Phagocytic activity was determined for olfactory ensheathing cells and Schwann cells in vitro using latex microsphere beads. Glial cells were seeded to poly-L-lysine (100 μg/ml) coated Nunc® Lab-Tek® 8 Chamber Slides™ (Sigma-Aldrich, Taufkirchen, Germany) at a density of 30,000 cells per well. Cells were incubated at 37°C and 5% CO2 in DMEM medium containing 5% FBS. After 12 hours yellowgreen fluorescent fluoresbrite carboxylated microspheres (1 μm diameter, Polysciences Europe GmbH, Eppelheim, Germany) were coated with FBS for 60 minutes at room temperature before they were diluted with DMEM medium to a final concentration of 2.8×107 microspheres/ well. After 60 to 120 minutes at 37°C in the incubator, cells were fixed with 4% PFA (paraformaldehyde) for 15 minutes at room temperature. Remaining microspheres were rinsed off with PBS and 0.1% Triton X-100. As a negative control glial cells were incubated at 4°C. Afterwards Chamber Slides™ were stained for p75NTR and 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Karlsruhe, Germany) to determine co-localization of YG microspheres and glial cells. Phagocytized microspheres were localized taking z-axis series of single cells on the Chamber Slides™, using a Leica TCS SP5 AOBS confocal microscope. Using the confocal series we created XY, XZ and YZ images to check whether the microspheres are located on the inside of the cell or on the exterior (Figure 5).
Neurite outgrowth assay
To monitor effects of glial cells on neurite outgrowth of human neurons we seeded hNT2 (passage 22–31) neurons and 2wk RA (passage 23–30) neurons on different substrates. As control coatings we used poly-D-lysine (10 μg/ml) and poly-D-lysine/laminin (100 μg/ml). Glial cells of passage 7 were seeded 1 hour prior to neuron seeding at a density of 25,000 cells/well to poly-L-lysine coated 96-well-plates (Corning Costar, Kaiserslautern, Germany). After attachment of glial cells to the substrate, neurons were seeded at a density of 10,000 cell/well to the OB-OECs, OM-OECs and the Schwann cells with a 1:1 ratio of DMEM and DMEM/F12 medium. Neurons, cultured for 24 h under 37°C and 5% CO2, were fixed and stained for β-III-Tubulin to evaluate the neurite outgrowth. For each neuron all neurites were measured using ImageJ. Furthermore the longest neurite and number of extending processes for each neuron were determined. For evaluation of the effect on neurite outgrowth only neurons with an apparent neurite were taken into account. Each experiment was performed at least in six wells and repeated three to five times with at least 74 neurons (2wkRA neurons) and 50 neurons (hNT2 neurons), per experiment, respectively.
Immunocytochemistry was performed on mature hNT2 neurons and dispersed 2wk RA neurons as previously described . Human neurons were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. The monoclonal antibody β-III-Tubulin (1:10000, Sigma, Taufkirchen, Germany) was applied overnight at 4°C. The biotinylated secondary antibody (Vector, Burlingame, Mass., USA) was applied for at least 1 h at room temperature before Streptavidin coupled Cy3 was added for 1 h at room temperature to detect immunofluorescence. Nuclei were visualized using DAPI (4′6-diamidino-2′henylindoldihydrochloride, 0.1 μg/ml, Sigma, Taufkirchen, Germany) as counterstain.
Graph Pad Prism was used for statistical evaluation. Data are expressed as the mean ± SEM. Significant differences were determined by one-way-ANOVA and the Newman-Keuls Multiple Comparison Test (scratch wound assay) or the Kruskal-Wallis Test and the Dunns Test (neurite outgrowth assay). Quantitative results are usually based on 3–5 independent experiments performed at least in triplicate. Significant levels are: * < 0.05, ** < 0.01, *** < 0.001.