Wild type and GFAP:GFP transgenic mice (FVB/N-Tg(GFAPGFP)14Mes/J) (Zhuo et al., 1997
) were purchased from the Jackson Laboratory (Bar Harbor, ME). PEDF null mice (Doll et al., 2003
) were obtained from Northwestern University. Two to four month old male and female adult mice were used in this study. They were housed in a pathogen-free facility. All animal procedures were performed according to the Institutional Animal Care and Use Committee of the University of California Davis, and National Institutes of Health guidelines.
SVZ dissociation and neurosphere culture
SVZ tissue was microdissected from coronal slices of wild type and GFAP:GFP transgenic mouse brains, minced with a razor blade, and incubated for 30min at 37°C in HBSS medium (Invitrogen) containing 0.05% trypsin (Invitrogen) and 60U/ml Dnase I (Sigma). Cell suspensions were generated by gentle trituration using a fire-polished Pasteur pipette and then centrifuged for 5min at 400g. Single cells were resuspended and plated in non-coated culture flasks (BD) containing neurosphere medium [DMEM/F12 (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), 20ng/ml EGF (Millipore) and 20ng/ml bFGF (R&D)]. The cultures were maintained at 37°C, 5% CO2 and supplemented with growth factors every two days for 6 days to generate primary neurospheres. The spheres were then spun and enzymatically dissociated into single cells. The dissociated cells were plated at a density of 5 cells/μl in neurosphere medium in the absence or presence of 50ng/ml PEDF (Bioproducts MD) to generate control or PEDF-treated secondary neurospheres.
Flow cytometry and fluorescence activated cell sorting (FACS)
Control and PEDF-treated secondary GFAP:GFP neurospheres were dissociated into single cells and positive GFP expression was determined using wild-type SVZ neurosphere cells as a negative control. For flow cytometric analysis of percentages of NG2+ cells within the GFAP:GFP+ cell population, single cells dissociated from control and PEDF-treated secondary GFAP:GFP neurospheres were incubated with anti-NG2 antibody (1:500) for 20min followed by incubation with goat anti-rabbit secondary antibody conjugated with allophycocyanin (1:1000, R&D) for 15min at RT, and then analyzed using a Cyan flow cytometer (Dako). For FACS isolation of GFP+NG2- and GFP+NG2+ single cells, primary GFAP:GFP neurospheres were dissociated into single cells, immunolabeled for NG2 expression as described above, and isolated using a FACS Aria II (BD Biosciences). The FACS-isolated cells were plated at a density of 5 cells/μl in neurosphere medium in the absence or presence of PEDF to generate secondary neurospheres.
Immunocytochemistry on neurospheres and FACS isolated cells
For immunocytochemical characterization of wild type control and PEDF-treated secondary neurospheres, the spheres were directly plated or mechanically dissociated into single cells before plating onto poly-D-lysine (PDL) (20μg/ml, Sigma)-coated chamber slides (Nunc). After 1hr, cells were fixed with 4% paraformaldehyde (PFA) for 15min, washed three times with PBS, and blocked in 8% normal goat serum in PBS for 30min at RT. Cells were then incubated for 3hr at RT or overnight at 4°C with primary antibodies as follows: anti-nestin (1:250, Millipore), anti-NG2 (1:400, Millipore), anti-O4 (1:200, Millipore), or anti-Tuj1 (1:400, Covance). Cells were washed three times with PBS, and then incubated with Alexa Fluor-conjugated secondary antibodies (1:800, Invitrogen) for 1hr at RT. For nestin and Tuj1 immunostaining, permeabilization was done with 0.1% Triton in PBS (v/v) for 10min before blocking and primary antibody incubation. Stained cells were mounted with Prolong Gold containing DAPI (Invitrogen) and images were acquired using a Zeiss fluorescence microscope.
For differentiation assays of control and PEDF-treated secondary neurospheres derived from FACS purified GFAP:GFP+NG2- or GFAP:GFP+NG2+ cells, the spheres were plated on PDL (20μg/ml, Sigma)- and laminin (5μg/ml, Sigma)-coated chamber slides in differentiation medium (DMEM/F12 supplemented with N2 and B27 but without EGF, bFGF or PEDF) for 3 days. Cells were then fixed with 4% PFA and immunostained for NG2, O4, or Tuj1 as described above.
For validation of GFAP expression in GFAP:GFP+ or GFAP:GFP− cell fractions, GFP− and GFP+ cells that had been FACS-isolated from GFAP:GFP neurospheres were plated on PDL-coated chamber slides for 1hr before fixation and GFAP immunostaining using anti-GFAP (1:1000, Dako). The staining procedure was done in the same way as for the Tuj1 staining described above.
Proliferation assay in neurosphere cultures
To assess proliferation of GFAP:GFP+NG2+ cells, BrdU (10μM) was added to control and PEDF-treated GFAP:GFP secondary neurospheres for 12hr prior to cell dissociation and NG2 labeling. FACS-purified GFAP+NG2+ cells were plated on PDL coated chamber slides for 1hr before fixation with 4% PFA. Cells were then permeabilized with 0.1% Triton in PBS (v/v) for 10min and blocked with 8% normal goat serum in PBS (v/v) for 30min. DNA denaturation was done with 0.07N NaOH for 10min and cells were washed three times with PBS. Cells were then incubated with anti-BrdU (1:100, Dako) overnight at 4°C. After primary antibody incubation, cells were washed three times with PBS and incubated with Alexa Fluor-conjugated secondary antibodies for 1hr at RT.
Control and PEDF-treated wild type neurospheres were pulsed with BrdU for 12hr before plating on PDL-coated chamber slide and fixation with 4% PFA. Staining was performed as described above. For Ki67 staining, control and PEDF-treated wild type neurospheres were directly plated on PDL-coated chamber slides for 2–3hr and fixed with 4% PFA. Cells were permeabilized with 0.1% Triton in PBS (v/v) for 10min before incubation with anti-ki67 (1:400, vector lab) overnight at 4°C and Alexa Fluor conjugated secondary antibody for 1hr at RT.
RNA isolation and RT-PCR
For RNA isolation, neurospheres were spun and lyzed with RNA lysis buffer, and total RNA was extracted using absolute microRNA prep kit (Stratagene). During RNA extraction, DNase I was included to prevent genomic DNA contamination. cDNA was synthesized from 0.5μg to 1μg total RNA using oligo-dT primer and superscript III First-Strand cDNA synthesis kit (Invitrogen) in 20μl reactions. For detection of PEDF receptor, also known as patatin-like phospholipase domain-containing protein 2 (PNPLA2; also referred to as desnutrin) (Notari et al., 2006
), 2μl of cDNA was used as a template, and PCR amplification was conducted using AmpliTaq Gold PCR master mix (AB Applied Biosystems). The oligonucleotide primers for the PEDF receptor were: forward 5' ACAGTGTCCCCATTCTCAGG 3'; and reverse 5' TGGTGAAGGACACTGCACTC 3'. PCR conditions were as follows: 95°C for 5min; 30 or 36 cycles of [95°C for 15s, 55°C for 15s, 72°C for 1min]; 72°C for 10min. The PCR products were resolved on a 2% agarose gel and photographed using a Kodak Gel Logic Digital Imaging System. For quantitative analysis of oligodendroglial lineage-associated mRNAs, real-time PCR was performed on a LightCycler 480 (Roche) using SYBR green qPCR kit (Roche) with the following parameters: 95°C for 5min, 40 cycles of [95°C for 10s, 55°C for 20s, 72°C for 30s]. Each primer set showed single peak in the melting curve analysis ensuring the absence of false signaling. Glyceraldehyde-3-phosphatase-dehydrogenase (GAPDH) was used as an internal control for normalization of mRNA levels. The oligonucleotide primer sets used in this assay are listed as follows.
Olig1 forward 5' CTTGCTCTCTCCAGCCAAAC 3'; reverse 5' CAGAACTGGGAGTGGAGAGG 3'.
Olig2 forward 5' AGCAATGGGAGCATTTGAAG 3'; reverse 5' TTCCATATCGGGACTTTTGG 3'.
Sox 10 forward 5' AGGCAGGAAGGGTTAGGGTA 3'; reverse 5' GCGGAGAAAGGATCAGAGTG 3'.
PDGFrα forward 5' TGGCATGATGGTCGATTCTA 3'; reverse 5' CGCTGAGGTGGTAGAAGGAG 3'.
GAPDH forward 5'ATTCAACGGCACAGTCAAGG 3'; reverse 5' TGGATGCAGGGATGATGTTC 3'.
PEDF in vivo infusion and lysolecithin lesioning
PEDF (Bioproducts MD) was dissolved in 0.9% saline and continuously administered (300ng per day) into the lateral ventricle of adult GFAP:GFP (Figures 3,6) or wild type mice (Figure 5) for 7 days via mini-osmotic pumps (Alzet 1007D and brain infusion kit #3; Durect). Control animals received 0.9% saline alone. Stereotaxic coordinates for cannula implantation of the pumps into the lateral ventricle were 0.2 mm posterior and 1.1 mm lateral to bregma, and 2.5 mm deep from the skull surface. For BrdU labeling of the SVZ (Figure 6), BrdU (100mg/kg body weight ip) was daily injected into adult GFAP:GFP transgenic mice during day 5 to 7 of intraventricular saline or PEDF infusion. For the BrdU labeling followed by PEDF infusion described in Figures 7 and 8, adult wild type mice first received BrdU (100mg/kg body weight ip) three times at 6hr intervals to label cycling OPCs in the corpus callosum. Twenty-four hours after the first injection, animals were either sacrificed (Figure 7, day 0), or subjected to saline or PEDF infusion (300ng per day) using mini-osmotic pumps into the corpus callosum for 9 days (Figure 7), or 2 or 5 days (Figure 8). The cannula was placed at the following coordinates for corpus callosum infusion: 0.2 mm posterior and 1.1 mm lateral to bregma, and 1.5 mm deep from the skull surface. For the experiment described in Figure 8, EdU (100mg/kg body weight ip) was injected into the animals 5hr prior to sacrifice to label proliferating cells.
For the lysolecithin-induced corpus callosum demyelination studies (Figure 9), 1ul of 1% lysolecithin (Sigma) was sterotaxically injected into the corpus callosum of adult mice at 0.2 mm posterior, 1.1 mm lateral to bregma, and 1.7 mm deep from the skull surface. Saline or PEDF (300ng per day) was then continuously infused via mini-osmotic pumps into the corpus callosum for 3 or 7 days. The cannula was placed at the following coordinates for saline or PEDF intracerebral infusion: 0.2 mm posterior and 1.1 mm lateral to bregma, and 1.5 mm deep from the skull surface.
For Western blot analysis (Figure 10), 1ul of saline or 1% lysolecithin was sterotaxically injected into the corpus callosum of adult mice at 0.2 mm posterior, 1.1 mm lateral to bregma, and 1.7 mm deep from the skull surface. Corpus callosum tissues were harvested at 2, 5, or 7 day post-injection. PEDF (300ng per day) was infused into the corpus callosum of intact, non-lesioned adult mice for 2 days at the following coordinates: 0.2 mm posterior and 1.1 mm lateral to bregma, and 1.5 mm deep from the skull surface.
Tissue preparation, immunohistochemistry and quantification
After the saline or PEDF infusion, mice were anesthetized with ketamine (150mg/kg body weight ip) and xylazine (16mg/kg body weight ip) injection, and transcardially perfused with PBS, followed by 4% PFA in PBS. Brain tissues were isolated and post-fixed with 4% PFA in PBS overnight at 4°C, and washed with PBS. Tissues were then cryoprotected by sequential immersions in 15% and 30% sucrose solutions (v/v) before embedding in cryostat mounting media (Tissue-Tek OCT, Sakura Finetek).
For immunostaining, cryostat tissue sections were air-dried at RT before blocking with 8% normal serum (v/v) and 0.1% Triton (v/v) in PBS for 1hr at RT. Sections were then incubated with primary antibodies overnight at 4°C in PBS containing 5% normal serum (v/v) and 0.1% Triton (v/v), followed by incubation with Alexa Fluor-conjugated secondary antibodies (1:800, Invitrogen) for 1hr at RT. Primary antibodies were used as follows: anti-GFP (1:500, Rockland), anti-PDGFrα (1:200, BD), anti-NG2 (1:300, Millipore), anti-Olig1 (1:500, Millipore), anti-Olig2 (1:100, R&D), anti-Sox10 (1:100, Santa cruz), anti-BrdU (1:100, Santa Cruz), anti-CC1 (1:200, Calbiochem), anti-CNP (1:500, Millipore), anti-MBP (1:200, Novus Biologicals), or anti-PNPLA2/PEDF receptor (1:20, R&D). Sections were mounted in Prolong Gold containing Dapi (Invitrogen). For BrdU immunostaining, tissue sections were treated with 2N HCL for 30min at 37°C to denature DNA, followed by 10min incubation with 0.1M sodium borate pH 8.5 for neutralization. Subsequent immunostaining steps were performed as described above. EdU staining was performed by using Click-iT EdU assay kit (Invitrogen) following the manufacturer's instructions. Tunel staining was performed by using DeadEnd TUNEL assay kits (Promega) following the manufacturer's instructions. For BrdU/Tunel double labeling, Tunel staining was first performed, and tissues were then fixed for 8min with 4% PFA followed by BrdU staining as described above. For Tunel/Olig2 and Tunel/NG2+ staining, Olig2 or NG2 immunolabeling was first performed, followed by Tunel staining.
All fluorescent images were captured by laser scanning confocal microscopy (Nikon C1) and projected with 5–6μm thick Z-series stacks (Nikon EZ-C1 software). For quantifications, at least 10 coronal sections were analyzed for each marker, and cells were identified by their DAPI-labeled nuclei. Cell counts were performed in the dorsal corner of the SVZ (Figures 3,6), with the total number of cells analyzed ranging between 998 and 1484, in the ipsilateral corpus callosum (Figures 7,8), and in the lesion core determined by MBP immunostaining and cortical needle tract (Figure 9).
Western blot analysis
Control and PEDF-treated secondary neurospheres derived from the SVZ of adult wild type mice (Figure 2) and corpus callosum tissues (Figure 10) were collected and lysed in a RIPA buffer containing protease inhibitors (Thermo Scientific). Protein concentration was determined by BCA assay (Thermo Scientific). Protein separation was done on SDS polyacrylamide gels (Invitrogen) and transferred to PDVF membranes (Millipore). Membranes were blocked and then incubated with primary antibodies: Olig1 (1:3000, Millipore), Olig2 (1:3000, R&D), Sox10 (1:1000, Millipore), PDGFrα (1:500, Santa Cruz), and PEDF (1:100, R&D). Protein bands were detected using ECL (Thermo Scientific) with horseradish peroxidase-conjugated secondary antibodies (1:5000, GE Healthcare). Protein loading was determined by using antibody against GAPDH (1:5000, Millipore).
Paired comparisons were analyzed by two-tailed Student's t tests, with p<0.05 required for statistical significance