Animals and reagents
Wildtype B6.129SF2/J and C57BL/6J mice, transgenic eGFP mice (003291, background C57BL/6J), and TNF (background B6.129SF2/J) and TNFR1 (background C57BL/6J) knockout mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Recombinant TNF was obtained from R&D Systems (Minneapolis, MN). PDGF and basic FGF were from PeproTech (Rocky Hill, NJ). Recombinant GFP adenovirus was from Gene Transfer Vector Core, University of Iowa. Rabbit polyclonal antibodies against Iba1 were purchased from Wako Chemicals (Richmond, VA). Rat antimouse TNF (clone MP6-XT22) was obtained from eBioscience (San Diego, CA). Olig2 antibody was a generous gift from Dr. Richard Lu (University of Texas Southwestern Medical Center). Lactate dehydrogenase (LDH) cytotoxicity kit was from Roche Applied Science (Indianapolis, IN). Unless specified otherwise, all other reagents were from Sigma (St. Louis, MO).
Primary cell cultures
Primary preOLs, microglia, astrocytes and mixed glial cultures were prepared from the forebrains of 1 to 2-d-old Sprage-Dowley rats or mice using a differential detachment method as detailed previously (Li et al. 2008
). Briefly, mixed glial cultures were grown in poly-D-lysine coated culture plates or in flasks for individual cell type isolation. Microglia were isolated by shaking mixed glia-containing flasks for 1 h at 200 rpm. The purity of microglia at this stage was consistently >95%. To obtain highly purified microglial monocultures (>99%), cells were further plated into regular petri dishes for 1 h and then detached from the plates with ice cold EBSS. PreOLs isolated from mixed glial cultures were maintained in growth medium, i.e., the serum-free Basal Defined Medium (BDM) (DMEM, 0.1% bovine serum albumin, 50 µg/ml human apo-transferrin, 50 µg/ml insulin, 30 nM sodium selenite, 10 nM D-biotin and 10 nM hydrocortisone) supplemented with PDGF (10 ng/ml) and bFGF (10 ng/ml) for 5–9 days. The OL mono-cultures consisted of OL precursors [O4+
] and were of at least 95% pure. Mature OLs were obtained by culturing preOLs in differentiation medium (BDM supplemented with 10 ng/ml CNTF and 15 nM T3) for 7–10 days.
Astrocytes were purified from the astrocyte layer in the flask that has been exposed to the specific microglia toxin L-leucine methyl ester (1 mM) for 1 h, followed by 1~2 cycles of subculture and repeated exposure to L-leucine methyl ester. The enriched astrocytes were consistently more than 95% pure with preOLs being the major contaminating cells. For astrocytes and preOLs co-cultures, astrocytes (2.4 × 105 cells per well) were plated into 24-well culture plates 1~2 days before seeding preOLs (6 × 104 cells per well), and the co-cultures were maintained in growth medium for 1–2 days before use. For bridged no cell contact co-cultures, preOLs grown on PO-coated coverslips with mini PDMS columns attached at four corners were transferred to 24-wells containing astrocytes. The distance between the astrocyte layer and the preOL layer was 1mm. Unless stated otherwise, all cell treatments were carried out in the growth medium. Mouse mixed glial cultures and various glia monocultures were prepared with the same methods as described above from wildtype (WT) and TNF and TNFR1 knockout (KO) mice. Mosaic mouse mixed cultures using cells from WT and KO mice were prepared by seeding isolated astrocytes (4 × 104/well) in poly-D-lysine coated 8 well chamber slide (Nunc, Rochester, NY), followed by microglia seeding (2 × 104/well) on the next day, and preOLs plating (0.8 × 104/well) plating the following day. Five to seven days after preOLs plating, these mixed glial cultures were subjected to vehicle or LPS treatment for 48 h and preOL survival was determined by counting the number of O4+ cells.
Cell viability determination
Cell death was induced by exposure to LPS (Escherichia coli
O111:B4, Sigma) or TNF as specified in the figure legend. Survival of preOLs was determined by counting O4 positive cells with normal nuclei. Briefly, cells were treated in triplicate as specified for 24–48 h. After washing with PBS and fixation with 4% paraformaldehyde, cells were immunostained with O4 antibody (1:500). Total number of cells was revealed by staining all nuclei with Hoechst 33258. Five random, consecutive fields were counted in each coverslip under 200× magnification with a total of >1000 cells counted in the control conditions. Cell survival is expressed as mean ± SD. When monocultures of preOLs were used, cell viability was evaluated by Alamar blue, a tetrazolium dye that is reduced by living cells to a colored product, as described previously (Li et al. 2003
). Cell toxicity was also assessed by lactate dehydrogenase (LDH) release assay according to the manufacturer’s protocol (Roche Diagnostics, Indianapolis, IN).
Infection of preOLs with recombinant adenovirus
Purified rat preOLs were infected with adenovirus containing green fluorescent protein (GFP) cDNA (AdGFP) as described previously (Baud et al. 2004
). Briefly, cells were exposed to 1 × 108
pfu/ml of AdGFP overnight in BDM followed by a complete medium change the next day. Cells were allowed to recover for 48 h. The infection rate was consistently more than 90% with minimum toxicity. Cells were then trypsinized off the culture dish and seeded at density of 1–2 × 104
per well into established mixed glial cultures. The next day, the cultures were treated with either vehicle or LPS for 24 h, and immunostained for Iba1 and GFP.
Analysis of TNF production
The levels of TNF in the culture media of cells treated as specified were measured using a commercially available ELISA kit according to the manufacturer’s instruction (eBioScience, San Diego, CA). Absorption at 450 nm was determined in a microplate reader (Fluostar Optima, BMG Labtech). The detection limit of the ELISA was 8 pg/ml.
Microglia and astrocyte monocultures were plated onto poly-D-lysine coated 6-well plates at a density of 1.0 × 106 cells per well in DMEM containing 10% fetal bovine serum. After 24 h, cells were rinsed twice with BDM media, and stimulated with LPS (0–1.0 µg/ml) or vehicle for 0–24 h. RNA was extracted using an RNeasy kit according to the manufacturers’ instructions (Qiagen, Valencia, CA). Residual DNA was removed by incubating RNA samples with DNase I for 15 min at room temperature followed by DNase inactivation at 65°C for 10 min according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The samples were reverse transcribed to cDNA using the reverse transcription system kit (Promega, Madison, WI) and random primers in the presence or absence of reverse transcriptase for 10 min at 25°C followed by 15 min at 45°C, 5 min at 95°C, and 5 min at 4°C. RT-PCR was used to test for the presence of TNF, IL-1β, IL-10, TLR2, TLR3, TLR4, GFAP, Iba1 and β-actin cDNA using the following specific primers: TNF, forward-GCCCACGTCGTAGCAAAC, reverse-GCAGCCTTGTCCCTTGAA; IL-1β, forward-TGACCCATGTGAGCTGAAAG, reverse-AGGGATTTTGTCGTTGCTTG; IL-10, forward-GAGTGAAGACCAGCAAAGGC, reverse-TTGTCCAGCTGGTCCTTCTT; TLR2, forward-AGCTGGAGAACTCTGACCCA, reverse-CAAAGAGCCTGAAGTGGGAG; TLR3, forward-TGCGATTGGCAAGTTATTCG, reverse-GCGCAGGCTGTTGTAGGAAA; TLR4, forward-TGCTCAGACATGGCAGTTTC, reverse-TCAAGGCTTTTCCATCCAAC; GFAP, forward-CAGCTT CGAGCCAAGGAG, reverse-TGTCCCTCTCCACCTCCA; Iba1, forward-CTTTTGGACTGCTGAAAGCC, reverse-GTT TCTCCAGCATTCGCTTC; and β-actin, forward- AGACTTCGAGCAGGAGATGG, reverse- CCATCATGAAGTGTGACGTTG. Briefly, after an initial denature step at 95°C for 10 min, 100 ng cDNA per reaction was subjected to 30 cycles of PCR (95°C 15 sec, 56°C 15 sec, 72°C 30 sec) followed by a final elongation step at 72°C for 5 min. Products were electrophorezed on 2% agarose gels and visualized under UV light using a Bio-Rad Chemidoc XRS gel documentation system and Quantity-one software.
Immunocytochemistry and fluorescence microscopy
Cells were fixed with 4% paraformaldehyde in PBS for 10 min and blocked with TBST (50 mM Tris·HCl, pH 7.4/150 mM NaCl/0.1% Triton X-100) or TBS (for O4 immunostaining) containing 5% goat serum. The coverslips were then incubated with antibody O4 (1:500) or antibodies against Iba1 (1:1000) or GFAP (1:1000) overnight at 4°C. After washes, secondary antibody conjugated with either Alexa Fluor 488 or Alexa Fluor 594 (1:1000 dilution, Molecular Probes, Eugene, OR) was incubated with the coverslips for 1 h at room temperature. Following more washes, nuclei were stained with Hoechst 33258 at a final concentration of 2 µg/ml for 1 min. The coverslips were then washed 2–3 times and mounted onto glass slides with FluoroMount and kept in the dark at 4°C. Cell images were captured with a fluorescence microscope equipped with an Olympus DP70 digital camera (Olympus IX71).
For intracellular TNF staining, mixed glial cultures from C57BL/6 mice were washed twice with media and then treated with LPS at 0, 0.1 or 1.0 µg/ml for 8 or 24 h. During the last 5 h of stimulation, protein secretion was blocked using the protein transport inhibitor Brefeldin A (eBioscience, San Diego, CA) diluted 1:1000 in BDM alone or in BDM containing LPS. Cells were then fixed, permeabilized, blocked, and immunostained as described above.
Cell surface phosphatidylserine (PS) was detected with an annexin-V-FITC apoptosis detection kit (Sigma) according to the manufacturer’s instructions with slight modification. Cells were treated with LPS (1.0µg/ml) or media for 2, 6, 8, 12, 24 and 48h, at which time an aliquot of the supernatants was removed and stored at −80°C for subsequent TNF ELISA and LDH analyses. The cells were washed twice with sterile filtered TBS, and 500µl of binding buffer containing 5µl annexin-V-FITC was added to each well for exactly 10 min. The cells were then fixed with 2% PFA and stained for O4.
Western blotting analysis
Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis followed by electrotransferring of separated proteins to PVDF membrane. Nonspecific binding was blocked with TBS-T (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat milk for 1 h at room temperature. Anti TNFR1 (1: 500 dilution, Rabbit IgG, Santa Cruz Biotech, Santa Cruz, CA.), and β-actin (1:10,000 dilution) antibodies were diluted in TBS-T containing 5% non-fat milk and incubated overnight with the membrane at 4°C. After washing 3–5 times with TBS-T, the membrane was incubated with horseradish peroxidase conjugated secondary antibody (1:2000) for 1 h and was visualized by chemiluminescence using the SuperSignal detection kit (Thermo Scientific, Rockford, IL).
All cell culture treatments were performed in triplicate. Unless specified otherwise, data were presented as mean ± SD. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test to determine statistical significance. Comparison between two experimental groups was based on two-tailed t test. P<0.05 was considered statistically significant.