Antibodies
Monoclonal antibodies included mouse anti-human-p75NTR antibodies (clone HB8737; American Tissue Culture Collection; Krudewig et al., 2006) used as hybridoma supernatants (1:5), mouse anti-CD57/HNK-1 antibodies (1:500; C6680;
Sigma Aldrich, Saint Louis, Missouri, USA; Bock et al., 2007) and rabbit anti-βIII-tubulin antibodies (1:1,000; Covance, Emeryville, California, USA; Ziege et al., 2012).
Experimental animals
Tissue was obtained from six adult dogs (Beagle n=5, and Labrador retriever n=1) with a mean age of 15 month that did not suffer from diseases affecting the nervous and respiratory system, as determined by clinical and histopathological examination.
Dorsal root ganglia (DRG) were obtained from neonatal rats (n=40; Sprague Dawley, Hannover Medical School). All animals were treated according to the legal and ethical requirements of the University of Veterinary Medicine Hannover.
Tissue isolation and single cell dissociation
Olfactory mucosa (OM), olfactory bulb (OB), and fibular nerve was collected as described previously (Bock et al., 2007; 2009; Krudewig et al., 2006; Ziege et al., 2012). Tissue was sequentially rinsed in ProntoVet® disinfectant (B. Braun Melsungen AG, Melsungen, Germany) and phosphate-buffered saline without calcium and magnesium (PBS−) containing penicillin/streptomycin (1%; PAA, Marburg, Germany). After removal of meninges and white matter under binocular control, OB and OM were cut into small pieces (Bock et al., 2007; Krudewig et al., 2006). Tissue was treated with hyaluronidase type IV (0.5%; OM, OB; H-3884), collagenase type XI (0.5%; OM, OB; C-9407), and collagenase type IV (0.5%; OM;
C-5138; all from Sigma Aldrich, Taufkirchen, Germany; Ziege et al., 2012). Epineural sheath was removed and teased nerve preparations were prepared using fine
forceps (Wewetzer et al., 1997). Tissue was incubated with trypsin (T-8003), hyaluronidase type IV (H-3884) and collagenase type XI (0.5%, each; C-9407; all from Sigma Aldrich, Taufkirchen, Germany). Incubation was done for all preparations at 37°C for 45min. Single cell dissociation was car ried out in the presence of DNase I (0.05%; Roche Diagnostics, Mannheim, Germany) using a fire-polished Pasteur pipette (Omar et al., 2011).
Purification of adult canine glia
Highly purified and Schwann cell (SC)-free adult canine OM- and OB-olfactory ensheathing cell (OEC) preparations were generated using a recently introduced novel two-step procedure (Fig.5-1; Ziege et al., 2012). Magnet-activated cell sorting (MACS) of the freshly-dissociated tissue (Fig.5-1A) using antibodies against HNK-1 and p75NTR was carried out to collect myelinating and non-myelinating SCs, respectively, in the bound fraction (Fig.5-1B). After up-regulation of p75NTR expression in OECs of the non-bound fraction (Fig.5-1C) during five to seven days in vitro (Fig.5-1D), anti-p75NTR antibodies and MACS were used to separate OECs (Fig.5-1F) from fibroblast-like cells (Fig.5-1E). For details of the MACS procedure, see Ziege et al. (2012). Purification of SCs from fibular nerve was essentially done as previously described (Ziege et al., 2012).
Figure 5-1:
Establishment of Schwann cell (SC)-free cultures of olfactory mucosa-derived olfactory ensheathing cells (OM-OECs). Immediately after tissue dissociation, myelinating (green) and non-myelinating (red) SCs of the primary cell suspension (A) were targeted by anti-HNK-1 and anti-p75NTR antibodies, respectively, and magnet-activated cell separation (MACS) was used to collect both cell types in the bound fraction (B), while OECs (dark grey) negative for both markers remained in the non-bound fraction (C). During culturing of the non-bound fraction (D), OECs up-regulated p75NTR-expression (red) and were separated from fibroblasts (light grey) using a second MACS step (E,F). Purification of olfactory bulb-derived OECs (OB-OECs) followed the same protocol except that HNK-1 was not used for depletion. (A-C): HNK-1+ myelinating SCs (green), p75NTR+ non-myelinating SCs (red), OECs (dark grey), fibroblasts (light grey); (D-E): p75NTR+ OECs (red), fibroblasts (light grey). Modified according to Ziege et al. (2012).
OM-OECs, OB-OECs, and SCs were seeded on poly-L-lysine-coated (PLL, 100µg/ml; P1274; Sigma Aldrich, Taufkirchen, Germany) flasks (Nunc, Roskilde, Denmark) in complete medium consisting of Dulbecco´s modified Eagle (DME) medium (Life Technologies GmbH, Darmstadt, Germany) supplemented with fetal calf serum (FCS, 10%; PAA Marburg, Germany), penicillin/streptomycin (1%; PAA) and sodium pyruvate (1%; PAA). The three cell types were expanded under identical conditions using human basic fibroblast growth factor-2 (FGF-2, 20ng/ml; Peprotec, Tebu, Frankfurt, Germany) as a mitogen (Ziege et al., 2012). For all of the experiments, cells from six independent preparations were pooled to minimize inter-individual differences (Ziege et al., 2012).
Dorsal root ganglion neuron-glia cocultures
Neonatal rat DRG neurons were isolated as described previously (Wewetzer et al., 1997). Neurons were seeded in serum-free DME/F12 medium (Life Technologies GmbH, Darmstadt, Germany) containing additives (Giulian and Baker, 1986) and bovine serum albumin (BSA, 0.25%; Sigma Aldrich, Munich, Germany) at a density of 100 neurons/well on PLL-coated (100µg/ml; P1274; Sigma Aldrich, Taufkirchen, Germany) 96well microtiter plates (Nunc, Wiesbaden, Germany). Canine OM-OECs, OB-OECs and SCs were immediately added (20,000 cells/well) in serum-free DME/F12 medium containing additives and BSA (see above). Cocultures were maintained under standard conditions (5% CO2, 37°C) for 18h.
For morphometric analysis of neurite growth, total area of neurites, length of the longest neurites from 200 neurons (n=200) and the percentage of neurite distance in association with either glial cells or PLL substrate (n=50) were measured using analySIS® Imaging Software (3.1; Olympus Soft Imaging Solutions GmbH, Münster, Germany). The number of primary neurites and branching points (n=200) were counted and the percentage of branching points either on canine glial cells or on the PLL-coated plastic dishes (n=50) were calculated. Data were collected from four independent experiments.
Immunofluorescence
Cell surface staining of viable OM-OECs in vitro with antibodies to p75NTR was essentially performed as described previously (Wewetzer et al., 2005). Cells were incubated with hybridoma supernatant (1:5) diluted either in serum-free DME/F12 medium containing 0.25% BSA (Sigma Aldrich) or in complete medium containing 10% FCS for 20min (37°C, 5% CO2). Secondary antibodies (Cy3-coupled goat anti-mouse; Jackson Immuno-Research Laboratories, Dianova, Hamburg, Germany) were applied for 20min at (1:200) under the same conditions (Ziege et al., 2012).
Cultures were fixed with paraformaldehyde (PFA, 4%) at room temperature (RT).
Canine glia and DRG neurons in coculture were visualized by sequential immunostaining of viable cells for p75NTR and fixed cells for βIII-tubulin, respectively (Ziege et al., 2012). After fixation, cell cultures were permeabilized and treated with normal goat serum (5%) in PBS-Triton X-100 containing 3% BSA (Sigma Aldrich) to reduce non-specific antibody binding, nuclei were stained using bisbenzimide H33258 (Wewetzer et al., 2005).
Cell proliferation assay
5´-bromo-2′-deoxy-uridine (BrdU; FLUOS BrdU Proliferation Kit; Roche Diagnostics, Mannheim, Germany) was used to assay proliferation of OM-OECs, OB-OECs, and SCs (all of 5th passage). Cells were seeded in quadruplicate (4,000 cells/well) on PLL-coated (100µg/ml) 96well microtiter plates (Nunc) and maintained in the absence of growth factors for 48h in serum-free DME/F12 medium (see above).
Recombinant human FGF-2, epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), human β-nerve growth factor (NGF; all from Peprotec, Tebu, Frankfurt, Germany), and heregulin-1β (HRG-1β, EGF domain; R&D Systems, Wiesbaden, Germany) were applied at 40ng/ml for three days. Proliferation of OM-OECs was additionally assayed in serum-containing complete medium using the same protocol. BrdU (10µM) was applied for 12h and cell cultures were fixed and immunostained according to the manufacturer´s instructions (Krudewig et al., 2006;
Techangamsuwan et al., 2008). Nuclei were counterstained using bisbenzimide
H33258 (Wewetzer et al., 2005). The percentage of BrdU-positive cells was calculated by counting a strip across the 96well. Immunopositive cells were then related to the total number of bisbenzimide-stained cells. Each experiment was repeated at least twice. Data represent the median, minimum and maximum (Fig.5-6, Fig.5-8). To compare growth factor effects between the different cell types, the ratio of BrdU-positive cells in treated to the BrdU-positive cells in controls were calculated (fold-change; Fig.5-7).
Illustrations
Cell culture images were acquired with an inverted fluorescence microscope (Olympus IX-70; Olympus Life Science Europe GmbH, Hamburg, Germany) and photographs were taken using a digital camera (Olympus DP72; Olympus Life Science Europe GmbH, Hamburg, Germany) and a Cell^F Imaging Software (Olympus Soft Imaging Solutions GmbH, Münster, Germany) at 100x magnification.
Slides were stored as tiff-files and Adobe® Photoshop® (version 7.0; Adobe Systems, Inc., San Jose, CA, USA) was used to prepare the figures, with uniform adjustment of contrast, brightness and sharpness, if necessary.
Statistical analysis
Statistical analysis was done using SPSS® for windows (version 16 and 20; SPSS Inc., Chicago, IL, USA) employing the Mann-Whitney U-test. Statistical significance was designated as P<0.05.