Animals Used in the Study
Mice: two groups of p75+/+
mice were used for the study. For the injury study, a congenic C57/BL6 line that carries a mutation in exon 3 of the p75 gene (Lee et al., 1992
) was purchased from the Jackson Laboratory (Bar Harbor, ME). For the culture study, the p75-/-
mice were obtained from heterozygote mating as littermates. Their genotype was determined by PCR analyses of tail DNA according to Bentley and Lee (Bentley and Lee, 2000
Rats: adult female Long-Evans hooded rats were obtained from Simonsen Labs (Los Angeles, CA).
Spinal Cord Injuries
Mice were anesthetized with isoflurane and the spinal cord was exposed at T10. A Beaver blade was used to produce a dorsal hemisection of the cord, including the dorsal columns and the dorsal part of the lateral funiculus. Contusion injuries in rats were made using the NYU device (Gruner, 1992
). Under pentobarbital anesthesia, the spinal cord was exposed at T10 and a 10g weight was dropped from 25 mm onto the dural surface as previously described (Basso et al., 1996
). All procedures were approved by the Institutional Laboratory Animal Care and Use Committee and followed the NIH Guidelines for the proper use and care of laboratory animals.
Under deep anesthesia (80 mg/kg ketamine, Fort Dodge Animal Health, Fort Dodge, IA; 10 mg/kg xylazine, Vedco, Inc., St. Joseph, MO), rats and mice were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde fixative. The spinal cords were removed, and three adjacent blocks were cut from the cords, each being either 3 mm long for the mouse cords or 5 mm long for the rat cords. In each case, one block was centered on the lesion, and the others were rostral and caudal to that block. The blocks were sectioned at 20 μm thickness on a cryostat.
Sections were incubated in blocking solution containing 10% goat serum, 10% horse serum, 1% BSA, and 0.3% Triton X-100 in 0.1M PB for 2 hr at room temperature. For double-staining for p75 and oligodendrocyte cell bodies, the sections were incubated simultaneously with an anti-p75 antibody, 9651 (Huber and Chao, 1995
), and CC1 antibody (Bhat et al., 1996
; Crowe et al., 1997
) in 5% goat serum, 5% horse serum, and 0.1% BSA in 0.1M PB at room temperature overnight. 9651 recognizes the extracellular domain of p75. CC1 antibody recognizes the APC gene product, which is expressed in rat oligodendrocyte somata and proximal processes (Bhat et al., 1996
). Although CC1 antibody can detect GFAP+
astrocytes, less than 0.5% of cells were positive for both CC1 and GFAP in our rat and mouse spinal cord tissue. Sections were then incubated with biotinylated anti-rabbit antibody (Vectorlabs) for p75 stain and an anti-mouse antibody conjugated to Alexa 488 (Molecular Probes) for CC1. P75 staining was visualized using Extravidin Cy3 (Sigma). For double-staining for p75 and active caspase 3, 192 anti-mouse anti-p75 antibody was used simultaneously with active caspase 3 anti-rabbit antibody (Cell Signaling, Beverly, MA). For 192 immuno-staining, a biotinylated anti-mouse secondary and an anti-mouse antibody conjugated to Alexa 488 (Molecular Probes) was used, and for active caspase 3, an anti-rabbit secondary conjugated to Cy3 (Jackson ImmunoResearch, West Grove, PA) was used. For double-staining for CC1 and active caspase 3, a biotinylated anti-mouse secondary and an anti-mouse antibody conjugated to Alexa 488 (Molecular Probes) was used for CC1. Active caspase 3 was detected with anti-rabbit secondary conjugated to Cy3 (Jackson ImmunoResearch, West Grove, PA). The sections were mounted with Vectashield containing DAPI to label the nuclei (Vector Labs). For confocal microscopy, BioRad MRC 1024 attached to a Nikon Optiphot-2 was used.
For cell counts shown in and 5A, CC1 staining of rat and mouse tissues were done in the same way as the fluorescence staining, except that the positive staining was visualized using DAB and the Vectastain ABC kit (Vector Labs).
All the counts were done blind to mouse genotype or lesion condition. Cell counts were made on rat and mouse coronal sections at a series of rostral and caudal locations relative to the contusion or hemisection lesions. Counts and reference volumes were estimated using procedures specified in the Stereologer ™ program (Systems Planning and Associates, Inc., Alexandria, VA). For rat contusion injuries, three sections were randomly sampled from 1 mm blocks taken from 13 and 7 mm rostral and 7 and 13 mm caudal to the lesion epicenter. A pilot study was run to determine the optimal disector size and spacing to allow for counts of at least 100 cells per block. CC1 positive cells were only counted when the cell body and proximal processes were darkly labeled and were within the inclusive zone of each disector frame. Results are reported as total number of oligodendrocytes and as density (number per mm3). Data were gathered from rats with 25 mm spinal cord injury surviving for 5 or 8 days (n = 4/time point) or 3 or 6 weeks (n = 3/time point), and control uninjured rats (n = 3). Mouse CC1+ cells were counted in a similar fashion at 8 days postinjury, except that the distance from the lesion center sampled was 1.2 mm and 1.8 mm rostral (R1 and R2) and 1.2 mm and 1.8 mm caudal (C1 and C2). The number of mice analyzed was n = 6 for p75+/+ and n = 5 for p75-/-. For quantification of CC1+/active caspase 3+ cells in , p75+/+ (n = 5) and p75-/- (n = 5) mice were analyzed at 5 days postinjury, using rostral 4 mm blocks.
Primary Oligodendrocyte Cultures
The p75 knockout and the wild-type mice were obtained from heterozygote mating as littermates. For spinal cord oligodendrocytes (), mouse pups at postnatal days 12-14 and for cortical oligodendrocytes (), mouse pups at postnatal days 15-16 were used. Cell suspension obtained from the triturated tissues was loaded onto a 36% Percoll gradient, and oligodendrocytes were isolated following centrifugation at 10,000 g (Fuss et al., 2000
; Lubetzki et al., 1991
). Isolated oligodendrocytes were resuspended in 10% FBS in DMEM and plated onto poly-D-Lysine coated 4-well slide dishes at 0.1 × 106
per well. The following day, the medium was changed to a differentiation medium with no serum, as previously described (Yoon et al., 1998
). The culture was kept for 4 days before NGF was added at 100 ng/ml for the indicated amount of time. Rat oligodendrocytes were cultured as described (Harrington et al., 2002
Quantification of Apoptotic Oligodendrocytes in Culture
For quantification of apoptotic mouse oligodendrocytes, cells were fixed at indicated times after NGF treatment and incubated with anti-myelin basic protein (MBP) antibody (Boehringer-Mannheim). Cells were then stained for TUNEL and processed for visualization of MBP stain using an anti-mouse secondary antibody conjugated to Alexa 488 (Molecular Probes, Eugene, OR).
The spinal cords were homogenized in a lysis buffer containing 1% Nonidet P-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 10 mM Na2P2O7, 10 mM NaF, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM vanadate, and 1 mM phenylmethylfulfonyl fluoride. Induction of p75 by spinal cord injury was detected on Western analyses using an anti-rabbit, anti-p75 antibody from Co-vance (Berkeley, CA). For detection of proNGF and mature NGF, anti-mouse anti-NGF from Chemicon International (Temecula, CA) was used, but the same data were obtained with anti-rabbit anti-NGF from Cedarlane (Hornby, Ontario). The samples for neurotrophin Western analyses were prepared in Laemli buffer that was supplemented with 20 mM DTT and 100 mM iodoacetamide to prevent any potential dimeric interaction between mature NGFs. BDNF and NT3 antibodies were from Promega (Madison, WI).
The lysates were subjected to two rounds of immunoprecipitation using proNGF antiserum. The supernatants resulting from immuno-precipitation were analyzed in Western analyses with NGF antibody (Chemicon International, Temecular, CA) to assess the extent of depletion. The lysates taken before immunodepletion was used as undepleted controls in .
Generation of Recombinant ProNGF and Mature NGF
The cDNA of murine NGF was amplified by RT-PCR and sequenced in both directions for any errors. To improve translation initiation, 11 bases from the mouse untranslated region of murine NT-3, including the Kozak consensus site, was exchanged for the murine NGF sequence. PCR-mutagenesis was performed to add six histidine (His) residues at the C terminus, and residues RR (bp 1008-1013) near the C terminus were mutated to AA to impair cleavage of the His tag. To generate proNGF with impaired furin cleavage (proNGF), the KR (bp 651-657) was mutated AA. After bidirectional sequencing, the constructs were cloned into pcDNA, and stable 293 transfectants expressing pcDNA, pcDNA-proNGF, and pcDNA-mature-NGF were isolated following G418 treatment. For purification, cells were cultured for 18 hr in serum-free media, and the resulting media were collected after removing cells by centrifugation. His-tagged mature or cleavage resistant proNGF was purified using Ni-bead chromatography (Xpress System Protein purification, Invitrogen) as per the manufacturer's instructions using imidazole (350 mM) for elution. Medium from cells stably transfected with pcDNA vector alone was harvested and purified in parallel. The concentration of proNGF or mature NGF was estimated by silver stain, using known concentrations of mature NGF (Harlan Bioproducts for science) in parallel.
Generation of ProNGF- and ProBDNF-Specific Antibodies
GST fusion proteins encoding amino acids 23-81 (asp23 to arg81) of human proNGF or amino acids 25-90 (asn25 to asp90) of human proBDNF were generated in bacteria and purified by chromatography with glutathione-sepharose. Rabbits (using GST-proNGF) or chickens (using GST-proBDNF) were immunized to generate antisera. Specific antisera were purified by first incubating whole serum with GST to adsorb GST-specific immunoreactivity and then by adsorption to and elution from a glutathione column to which GST-proBDNF or GST-proNGF had been irreversibly coupled.
Antibody Blocking Experiments Using Injured Spinal Cord Extracts
The extracts from injured spinal cord were added at 0.14 μl in volume, which was estimated to give a final proNGF concentration of 14 ng/ml based on Western analyses. Extracts from sham-operated spinal cord were used at the same volume. For the dose curves in , rat oligodendrocytes were treated with column-purified recombinant proNGF, column-purified recombinant mature NGF, injured spinal cord extracts from mice, sham extracts, or vehicle at the indicated concentrations. For the vehicle control for the recombinant NGFs, the elution buffer containing 350 mM imidazole was used. The final concentration of imidazole therefore ranged from 250 μM to 5 mM. Following a 24 hr incubation period, samples were processed for TUNEL and MBP staining as described. For antibody blocking experiments, either the injured or sham extracts (0.14 μl) were preincubated with mature NGF (5 μl; Chemicon), proNGF (5 μl), proBDNF (10 μl) antibodies, or pre-immune serum (10 μl) for 2 hrat 4°C. The extract and antibody mix was then added to oligodendrocytes for 24 hr before they were processed for TUNEL and MBP stain.
A two-way ANOVA (site × time) was used for the cell counts in the rat and a Student's t test in the mice to evaluate the number of surviving and apoptotic oligodendrocytes.