All experiments were carried out in accordance with the guidelines established by the National Council on Animal Care and were approved by the local Animal Care Committee of the Universidad Autónoma of Madrid (UAM, Madrid, Spain) or the University of Basque Country (Spain) Animal Ethics committee, as relevant, following European Communities Council Directive of 22 September 2010 (2010/63/EU). Every possible effort was made to minimize animal suffering and the number of animals used.
Isolation and culture of rat hippocampal cells
Pregnant Sprague–Dawley rats (Charles River Laboratories International, Inc., Wilmington, USA) were sacrificed by CO2 inhalation and 18-day embryos (E18) were immediately removed by caesarean section. Hippocampi were dissected rapidly by using a Leica CLX 150× stereomicroscope (Leica Microsystems, Barcelona, Spain) under sterile conditions in cold (4°C) PBS. The tissue was digested with 0.5 mg/ml papain (Sigma-Aldrich, Madrid, Spain) and 0.25 mg/ml DNAase (Sigma-Aldrich, Madrid, Spain). The enzymes were dissolved in a Ca2+- and Mg2+-free PBS solution containing 1 mg/ml BSA (Sigma-Aldrich, Madrid, Spain) and 10 mM glucose (Sigma-Aldrich, Madrid, Spain) at 37°C for 20 min. The papain solution was replaced with 5 ml Neurobasal™ A Medium (Life Technologies Corporation, Paisley, UK) supplemented with 2% B27 Supplement (Life Technologies Corporation, Paisley, UK), 1% L-glutamine solution 200 mM (Sigma-Aldrich, Madrid, Spain) and 1% penicillin/streptomycin solution (Sigma-Aldrich, Madrid, Spain). The digested tissue was gently triturated by suction using a glass pipette flamed on the tip to avoid cellular damage. The cell suspension was centrifuged for 4 min at 1,200 rpm. The supernatant was removed and the cells resuspended in 5 ml of medium and seeded on both PLL- and nECM-coated multiwell plates in the same maintenance medium at a density of 120,000 cells/ml. Cells were maintained in these conditions in a 5% CO2 incubator at 37°C.
Isolation and differentiation of rat SVZ progenitor cells
Neurosphere cultures were prepared from 4 to 7-day-old Sprague–Dawley rat pups (Charles River Laboratories International, Inc., Wilmington, USA). Subventricular zone (SVZ) tissue from 2–3 rat brains were isolated and minced with a McIllwain tissue chopper (H. Saur Laborbedarf, Reutingen, Germany) and digested for 10 min at 37°C in 5 ml of 0.25% trypsin/EDTA solution (Sigma-Aldrich, Madrid, Spain). Digestion was stopped by adding an equal volume of trypsin inhibitor (Sigma-Aldrich, Madrid, Spain) and 0.01% DNAse I for 5 min at room temperature. Cell suspension was centrifuged for 10 min at 600 g and the pellet was mechanically dissociated 25 times in NeuroCult medium (Stem Cell Inc., Grenoble, France) using a glass Pasteur pipette and 20 times using 1 ml pipette tips. Non-dissociated cells were decanted and the single cell suspension was counted using the Neubauer method. Cells were seeded in proliferation medium: NeuroCult with 10% neural stem cell factors (both from Stem Cell Inc., Grenoble, France), 1% L-glutamine solution 200 mM (Sigma-Aldrich, Madrid, Spain), 1% penicillin-streptomycin solution (Sigma-Aldrich, Madrid, Spain), 20 ng/ml EGF and 10 ng/ml FGF2 (both from Promega, Madrid, Spain), at a density of 10,000 cells/cm2 and cultivated in suspension for 7 days at 37°C with 5% CO2. EGF and FGF2 were added fresh every 2–3 days.
The differentiation of neurospheres on 12 mm-diameter PLL- and nECM-coated coverslips (10 neurospheres/coverslip) was carried out in Neurobasal™ A Medium supplemented with 2% B27 Supplement and 1% N2 Supplement (all from Life Technologies Corporation, Paisley, UK), 10% FBS (LGC Standards S.L.U., Barcelona, Spain), 1% L-glutamine solution 200 mM and 1% penicillin-streptomycin solution (both from Sigma-Aldrich, Madrid, Spain). Every two days, half of the medium was replaced and supplemented gradually and at different time periods up to 35 days with 50 ng/ml rat recombinant nerve growth factor β (NGFβ) (Sigma-Aldrich, Madrid, Spain), 50 ng/ml human recombinant brain derived neurotrophic factor (BDNF) (Sigma-Aldrich, Madrid, Spain), 200 ng/ml human recombinant Sonic hedgehog (Shh) (R&D, Minneapolis, USA) and 200 ng/ml mouse recombinant fibroblastic growth factor 8b (FGF8b) (R&D, Minneapolis, USA).
Development of neural extracellular matrix (nECM)-coated subtrates
Neural extracellular matrix (nECM)-coated substrates were developed as described . Briefly, 12 mm-diameter coverslips were incubated with a solution of Cultrex® basement membrane extract (BME) without phenol red (8.32 mg/ml; Trevigen, Gaithersburg, USA; it is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm tumor; the major components of this BME include laminin, collagen IV, entactin, and heparin sulfate proteoglycan), hyaluronan of low molecular weight (31 kDa, HLMW) (7.5 mg/ml; R&D, Minneapolis, USA), Netrin-G1a and Netrin-4 (25 μg/ml; R&D, Minneapolis, USA) in phosphate-buffered saline (PBS) (Life Technologies Corporation, Paisley, UK). For some experiments, chondroitin 4-sulfate glycosaminoglycan (Sigma-Aldrich, Madrid, Spain) was added to the mix together with hyaluronic acid. In order to polymerize and adsorb the matrix on the exposed glass surface they were kept for 24 h at 37°C in sterile conditions. Finally, excess PBS was eliminated and coverslips left to dry up for 1 h under laminar flow and UV light. As a control, PLL-coated coverslips were prepared by incubation of 12 mm-diameter coverslips with a 0.01% solution of poly-L-lysine (Sigma-Aldrich, Madrid, Spain) in distilled water, followed by PBS washing and air drying under laminar flow for 1 h.
Immunofluorescence and confocal microscopy analyses
Differentiating cells were washed with PBS and fixed in 4% paraformaldehyde solution (Electron Microscopy Sciences, Hatfield, UK) for 20 min at room temperature. Cells were further washed with PBS and permeabilized for 1 hour in 0.3% Triton® X-100 (Sigma-Aldrich, Madrid, Spain) in PBS (PBS-T) and 5% normal donkey serum (Sigma-Aldrich, Madrid, Spain). After that, cells were incubated with the appropriate primary antibody diluted in PBS-T for 2 h at room temperature. Primary antibodies used were anti-Sox2 rabbit polyclonal (used at 1:1,000 dilution, Millipore #AB5603, Billerica, USA), anti-nestin mouse monoclonal IgG1 (1:200, Millipore #MAB353, Billerica, USA), anti-NeuN mouse monoclonal IgG1 (1:200, Millipore #MAB377, Billerica, USA), anti-GFAP mouse monoclonal IgG1 (1: 200; Dako, #M0761, Denmark), anti-MAP2 mouse monoclonal IgG1 (1:200; Millipore #MAB3418, Billerica, USA) and anti-tyrosine hydroxilase rabbit polyclonal (TH; 1:1,000; Millipore #AB152, Billerica, USA) antibodies. After 3 washes (5 min each) fixed cells were incubated with the appropriate secondary antibody diluted in PBS-T for 1 h at room temperature. Secondary antibodies used were donkey anti-mouse Alexa Fluor® 488 (1:1,000) and donkey anti-rabbit Alexa Fluor® 546 (1:1,000) (both from Life Technologies Corporation, Paisley, UK). Prior to embedding in Mowiol® mounting medium (Sigma-Aldrich, Madrid, Spain), cells were counterstained with 10 μg/ml Hoechst 33258 (Sigma-Aldrich, Madrid, Spain) for 5 min and washed with distilled water. Fluorescence images were obtained by using a Nikon Eclipse E600 FN microscope (objectives 10× and 20×) coupled to Nikon Digital Sight and analysed with Nikon NIS-Elements. Confocal images were generated with a Zeiss LSM 510 microscope coupled to Zeiss Axion camera and analyzed with Zeiss ZEN image analysis software (2008; SP1.1; Carl Zeiss MicroImaging, S.L., Barcelona, Spain). Z stack was analyzed with the plug in 3D viewer of Image J software. In both cases, at least ten different fields out of three independent experiments (n = 3) were quantified.
Quantification of cell viability with the MTT assay
Cell viability was assessed by quantitative colorimetric assay with (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as previously described . Briefly, a final concentration of 0.5 mg/ml MTT labelling reagent (Sigma-Aldrich, Madrid, Spain) in Neurobasal™ A Medium was added to each well containing embryonic hippocampal cells cultured for 8 days on control nECM- and chondroitin 4-sulfate containing nECM-coated coverslips, and incubated for 2 h. After that, colorimetric determination of MTT reduction was measured at 540 nm with a Kontron Instruments UVIKON 922 spectrophotometer. These experiments were repeated after treatment of embryonic hippocampal cells with several cytotoxic agents such as glutamate, oligomycin-rotenone (O/R) and amyloid beta 25:35 (Aβ) (all from Sigma-Aldrich, Madrid, Spain) for 24 h. These experiments were performed with cells from day 7 to day 8 in culture. For statistical analyses, cells in each nECM-coated well in absence of toxic were taken as 100% of viability.
Scanning electron microscopy analyses
For Scanning Electron Microscopy (SEM) analyses, substrates with cells were fixed with Vitrosec® 70 (Panreac Química S.A.U., Barcelona, Spain) for 20 min and stained with May-Grünwald’s eosin methylene blue solution (Merck, Madrid, Spain). Samples were dried and mounted onto aluminium disks and coated with gold-argon. Imaging was performed on a JEOL JSM-5910-LV scanning electron microscope using a 20 kV acceleration voltage. Samples were analyzed by an INCA-300 energy dispersed system (EDS) coupled to the microscope.
Data are expressed as the mean ± SD of the number of independent experiments (n). Statistical significance of the results was assessed by using GraphPad Prism software (version 5.01 for Windows). Two-way analysis of variance with subsequent pairwise multiple comparison procedures (Bonferroni’s posttests) was used to assess statistical significance between means. The statistical significance (*) was established at P-values <0.05 (** P < 0.01; *** P < 0.001).