Cell Culture and Electrical Stimulation
DRG neurons were dissected from the spinal cords of embryonic day (E) 13.5 mice as described (Stevens et al., 1998
). Neurons were grown for w4 weeks in MEM medium supplemented with N3 containing 100 ng/ml of nerve growth factor and 5% heat-inactivated horse serum in the side compartment of three-compartment chambers equipped with stimulating electrodes (Fields et al., 1992
). Mitosis of nonneuronal cells was inhibited by a 4 day treatment with 13 μg/ml fluoro-2’-deoxyuridine (FUDR) beginning 1 day after plating. These cultures can be maintained indefinitely with half-volume changes of medium every 3 days.
Primary cultures of OPCs were obtained from cerebral cortices of E19-E20 rats or mice and plated into 75 cm2
tissue culture flasks. The resulting cultures were maintained at 5% CO2
at 37°C in medium containing 10% fetal bovine serum (Hyclone). After 11 days in culture, the flasks were shaken at 37°C for 3 hr to kill nonglial cells, and then the medium was changed, and the flasks shaken overnight to lift OPCs from the flask. The cell suspension was pelleted, resus-pended, and incubated in an uncoated culture dish for 30 min to enrich the population for OPCs. Contaminating cells, primarily endothelial cells, astrocytes, macrophages, and microglia adhere strongly to the plastic and can be separated out by this panning method. For some experiments, O1-positive oligodendrocytes were obtained by immunopanning (Gard and Pfeiffer, 1989
). Immunocytochemistry confirmed that these were mature oligodendrocytes without contaminating astrocytes or NG2-positive cells.
Astrocytes were obtained from cerebral cortices of E19-E20 rats or mice. After oligodendrocytes were isolated, the flasks (above) were shaken for 2 days to remove floating cells and cell debris. Adherent astrocytes were removed by 0.25% trypsin (5 min at 37°C) and plated in 35 mm dishes or multicompartment cultures as required.
For myelinated cocultures, purified OPCs (>90% OPCs) were counted and plated on 3 to 4 week old DRG cultures at a density of 40,000 cells per side compartment. After 3-4 hr, the media was removed and replaced with differentiating media (DMEM + N1 + 0.5% FBS [Stevens et al., 2002
]). 7 days later, cultures were treated with purinergic agonists and antagonists.
Action potentials were induced in DRG axons by 200 ms 5 V biphasic pulse through platinum electrodes in three-compartment chambers (Fields et al., 1992
). Only those axons growing into the central compartment beneath the high-resistant barriers are stimulated by this method. 10 Hz phasic (0.5 s at 10 Hz every 2 s) stimulus patterns were used.
ATP, adenosine, 2MeSATP, α-β methyl ATP, and UTP were obtained from Tocris. Adenosine, N'-Benzyl-5’-N-ethylcarboxamidoadenosine (NECA), apyrase, and TTX were obtained from Sigma. Antibody against mouse LIF was from R&D Systems, and human LIF was from Alomone. Cultures were treated with fresh agonist or antagonist solutions every 2-3 days for 2 weeks.
Sudan Black Stain for Compact Myelin
After 4% paraformaldehyde fixation, cultures were washed with PBS and postfixed for 1 hr with 0.1% osmium tetroxide. After two washes with PBS, the cultures were dehydrated with an ethanol series (25%, 50%, 70%) for 10 min each and incubated for 2 hr in a 0.5% Sudan black solution in 70% ethanol and washed three times in 3% ethanol and rehydrated in PBS. Nuclei were stained with Hoechst stain (1:2000) in PBS for 10 min.
Cells were fixed with 4% paraformaldehyde for 5 min and washed three times in PBS. Nonspecific protein binding was blocked with 3% NGS, 0.1% Triton X-100 for 1 hr at RT. The following primary antibodies were used in overnight incubations at 4°C: anti-NG2 (Chem-icon, 1:1000), anti-O4 (Chemicon, 1:1000), anti-O1 (Chemicon, 1:500), anti-GFAP (Dako, Carpinteria, CA, 1:2000), anti-MBP (Sternberger Monoclonals, 1:1000), anti-OX-42 (Serotec, Oxford, UK), and anti-mouse LIF (R&D Systems, 1:200). Anti-MOG antibody was kindly provided by Dr. Barres (Stanford University). FITC- and Rhodamine-conjugated secondary antibodies against the appropriate species were applied for 30 min at RT and viewed by epifluorescence or confocal microscopy. Similar procedures were used for immunocytochemical staining of mouse optic nerve after 4% paraformaldehyde fixation and cryosectioning. Cultures were rinsed in PBS, treated with 0.02% sodium azide in PBS to prevent internalization of lectin, and incubated in 0.02 mg/ml FITC labeled tomato lectin (Sigma) for 30 min at RT and fixed with 4% paraformaldehyde to visualize microglia.
Quantification of Myelin and Cell Numbers
The number of Sudan-black-positive or MOG-positive myelin profiles was counted by observers without knowledge of the treatment condition. Ten or more random fields were selected from each culture dish or side compartment of the Campenot chamber, and the total number of myelin profiles and Hoechst-positive nuclei in each field was recorded. Statistical analysis was performed by using Mini-tab software with the significance of differences tested by ANOVA and errors reported as SEM.
Conditioned media was collected from cultures of DRG neurons, oligodendrocytes, astrocytes, and cocultures of DRG neurons and oligodendrocytes. The conditioned media from approximately eight cultures was collected, flash frozen, and subsequently concentrated with glycerine-coated Microsep tubes (Pall Life Sciences) to a volume of approximately 100 ml. At least two replicate 50 ml aliquots from each sample were assayed for LIF content on ELISA plates according to the manufacturer’s instructions (R&D Systems). LIF concentration values were obtained by linear regression of absorbance readings made with a microtiter plate reader, against known concentrations of LIF measured in serial dilution on each microtiter plate. The ELISA does not cross react with other cytokines, and our tests confirmed no cross reactivity with CNTF, a closely related cytokine to LIF.
Western Blot Analysis
Western blot was performed for the analysis LIF in DRG neurons, astrocytes, and oligodendrocytes. The cells were washed with PBS and lysed by adding M-PER mammalian protein extraction reagent (Pierce, Rockford, IL). Protein concentration was determined by Coomassie protein assay kit (Pierce, Rockford, IL). Electrophoresis was performed on 14% or 10%-20% gradient polyacrylamide gels (Invitrogen, Carlsbad, CA). As a positive control, recombinant mouse LIF (Chemicon) was used. Proteins were transferred to Immobilon transfer membranes (Millipore, Bedford, MA), and blots were blocked with 5% skim milk in 0.1% tween-20 tris buffer (TBST) overnight at 4°C. The blots were probed with anti-mouse LIF antibody (R&D Systems, Inc., Minneapolis, MN, 1:5,000) and anti-AnkyrinG antibody (Zymed Lab, Inc., 1:1,000) as a loading control overnight at 4°C, followed by horseradish peroxidase (HRP)-coupled donkey anti-goat secondary antibody (Chemicon) and sheep anti-mouse (Amersham Biosciences Buckinghamshire, UK). Blots were analyzed by ECL plus Western blotting detection system (Amersham Biosciences).
RNA Preparation and RT-PCR
Rat astrocytes were prepared as described above and plated onto axons of 3 to 4 week old cultures of DRG neurons 3 days prior to electrical stimulation in Campenot chambers. Medium was replaced with serum and NGF-free media 12 hr prior to electrical stimulation; 30 U/ml of apyrase was added if appropriate. DRG axons were electrically stimulated with a phasic pattern at 10 Hz for 24 hr. Total RNA was prepared by the TRIzol (Invitrogen) extraction method with modifications (Lee et al., 2004
). cDNA was prepared from 2 mg total RNA with Superscript II (Invitrogen) according to manufacturer’s instructions. PCR reactions were carried out by Platinum PCR super-mix high fidelity (Invitrogen) for 35 cycles, and reaction products analyzed on a 2% TAE agarose gel, visualized with SYBR I green staining (Invitrogen).