Isolation of GRPs and generation of GDAs
GRPs were isolated by fluorescence activated cell sorting (FACS) of dissociated cell suspensions from spinal cords of embryonic day (E)13.5 transgenic Fischer 344 rat embryos expressing the gene for human placental alkaline phosphatase (hPAP) under the control of the ROSA26 promoter (TgN(R26ALPP)14EPS) [72
]. GRPs were maintained on a fibronectin/laminin substrate at 4 × 103
to 2 × 104
in Dulbecco's modified eagle medium (DMEM)/F12 Sato-medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF). Passage number, days in vitro
, cell density and media conditions were tightly controlled for experimental replicates. To differentiate GRPs before transplantation, 10 ng/ml of human recombinant BMP-4 (R&D Systems) or 10 ng/ml human recombinant CNTF (Peprotech) were added to the culture media for 7 days to differentiate them into astrocytes – GDAsBMP
) and GDAsCNTF
), respectively. For in vitro
induction experiments, GRPs were seeded at 5,000 cells/cm2
on a fibronectin/laminin substrate in DMEM/F12 Sato-medium with 10 ng/ml bFGF. After 18 h, cell culture conditions were switched as indicated and cells were allowed to differentiate into astrocytes for up to 7 days. Medium was changed every 2 days. Parallel cultures were used for Western blot and immunofluorescent analysis.
In vitro immunofluorescence
Cells grown on fibronectin/laminin-coated glass coverslips were fixed for 5 minutes in 2% formaldehyde, rinsed and blocked using 5% normal goat serum in Hanks balanced salt solution (HBSS) with Hepes pH 6.8. For Olig2 and GFAP labeling, cells were permeabilized using 0.1% Triton-X100 in phosphate buffered saline (PBS) for 15 minutes. Anti-Olig2 (1:4000, Chemicon) and anti-GFAP (1:400, Cell Signaling) were incubated at 4°C for 18 h. Anti-NG2 (Chemicon, 1:2000) staining was performed on live cells in growth medium for 30 minutes prior to fixation with formaldehyde. Fluorescently labeled, secondary anti-Ig antibodies (Alexa 488 and 568 conjugates, Invitrogen) were used at a 1:2000 dilution for 1 h at room temperature. Coverslips were mounted on glass slides with ProLong Gold and viewed using a Nikon 80i microscope equipped with a Spot RT camera. Monochrome images of parallel samples were captured using identical exposure times and gain settings, and merged as pseudo-colored images. Both BMP- and CNTF-induced GDAs were uniformly immunoreactive for human alkaline phosphatase in vitro.
Western blot analysis
After treatment of cultures for 5 days with conditions as indicated, PBS-washed cells were harvested in XDP buffer (1% Triton X100, 0.5% sodium deoxycholate in PBS pH 7.2) supplemented with Complete Mini Protease Inhibitor Cocktail (Roche). The protein concentration of cleared lysates was determined using the Biorad DC protein assay. Samples (25 μg of protein per sample) were fractionated using NuPage 4–12% gradient gels (Invitrogen) and then transferred to polyvinylidene difluoride (PVDF) membranes (Perkin Elmer). Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (Sigma) and then incubated with primary antibodies at 4°C for 18 h. Antibodies and dilutions used: NG2 (Chemicon, 1:1000), anti-phosphacan (Developmental Studies Hybridoma Bank, 1:1000), β-tubulin (Santa Cruz Biotechnology, 1:1000). Horseradish-peroxidase-conjugated anti-mouse (PerkinElmer) or anti-rabbit (Invitrogen) antibodies were applied to washed blots and visualized using Luminol reagent (Santa Cruz Biotechnology) and Kodak X-OMAT LS X-ray film. Film was developed using a Kodak X-OMAT 3000RA processor. Densitometric analysis of scanned film images was performed using NIH Image-J software. Expression levels of phosphacan (320–340 kDa band) and NG2 (270–300 kDa band), respectively, were normalized for each sample to β-tubulin (52 kDa) expression. All Western blot experiments were conducted in triplicate and results were compared using the Student's t-test, p < 0.05.
Homogeneity of cell populations for transplantation
To confirm cell phenotype and homogeneity before transplantation, small volumes of cell suspensions were plated onto glass coverslips and labeled with A2B5 and anti-GFAP antibodies. GRP cell suspensions occasionally contained a small number (average of 2.1%) of A2B5+/GFAP+ cells, and GDACNTF cell suspensions included a small number (average 1.3%) of A2B5+/GFAP- cells. To ensure that GDABMP suspensions for transplantation did not contain undifferentiated GRPs or cells with the phenotype of CNTF-induced astrocytes (A2B5+/GFAP+), potential contaminating cell types were removed from the suspension by immunopanning with the A2B5 antibody. For transplantation, GRPs or GDAs were suspended in HBSS at a density of 30,000 cells/μl.
Spinal cord injury models and cell transplantation
Adult female Sprague Dawley rats (3 months old, Harlan) were used in all in vivo
spinal cord injury experiments (see Table for numbers of rats used per experiment) and were anesthetized by injection of a cocktail containing ketamine (42.8 mg/ml), xylazine (8.2 mg/ml), and acepromazine (0.7 mg/ml). For dorsal column injuries (Figure ), the right-side dorsal column was unilaterally transected between cervical vertebrae 1 and 2 using a 30-gauge needle as a blade (see also [18
]). Injuries extended to a depth of 1 mm and extended laterally 1 mm from the midline. For rubrospinal tract injuries, unilateral transections of the right-side dorsolateral funiculus including the rubrospinal pathway were conducted at the C3/C4 spinal cord level with Fine Science Tools micro-scissors. Injuries extended to a depth of 1 mm and extended medially 1 mm from the lateral pial surface of the spinal cord (Figure ). Transection spinal cord injuries were used instead of contusion injuries in order to minimize axon sparing and permit more accurate quantification of axon growth across injury sites bridged with GDACNTF
or GRPs. The use of an intervertebral surgery approach in combination with discrete transection injuries of the dorsolateral funiculus also results in highly consistent deficits in grid-walk locomotor performance and atrophy of red nucleus neurons [14
Numbers of animals per experimental group in vivo
A total of 6 μl of GDACNTF, GDABMP or GRP suspensions (30,000 cells/μl; 180,000 cells total) per animal were acutely transplanted into six different sites in dorsal column injuries; that is, two injections each into medial and lateral regions of the rostral and caudal injury margins, and two injections into medial and lateral regions of the injury center (Figure ). All dorsal column injury experiments were conducted in the absence of immunosuppressants. Transplants of either GDAsBMP, GDAsCNTF or undifferentiated GRPs were injected in an identical pattern into injuries of the dorsolateral funiculus and a total of 6 μl of GDA or GRP cell suspension (30,000 cells/μl; 180,000 cells) injected per injury site. Control injured rats were injected with 6 μl HBSS. All control or cell transplanted rats in the dorsolateral funiculus injury groups were given daily injections of cyclosporine (1 mg/100 g body weight) beginning the day before injury/transplantation through to experimental endpoints.
Adult DRG neuron transplantation
Single-cell suspensions of adult mouse sensory neurons were prepared from 10–12-week-old transgenic mice expressing the gene for EGFP [73
] as previously described [25
]. No growth factors were added to the neuron suspension. Five hundred nanoliters of the neuron suspension (approximately 1,500 neurons/μl) were acutely microtransplanted into dorsal column white matter approximately 500 μm caudal to the injury site (Figure ).
At 4 days, 8 days and 5 weeks post-surgery animals were deeply anesthetized and transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde in 0.1 M PBS. Dissected spinal cords were cryoprotected in a 30% sucrose/PBS solution at 4°C overnight. Tissue was embedded in optimal cutting temperature (OCT) medium (Sakura Finetek) and quickly frozen. Serial 25-μm-thick frozen sections were cut in the sagittal plane and air dried onto gelatin-coated glass slides. All tissue sections were washed in PBS, blocked with 4% normal goat serum in solution with 0.1% Triton/PBS for 30 minutes, then incubated with appropriate primary antibodies in the blocking solution overnight at 4°C. Secondary antibody incubations were for 45 minutes at room temperature.
The following primary antibodies were used: monoclonal anti-GFAP (Sigma) and polyclonal anti-GFAP (Sigma); polyclonal anti-NG2 (Chemicon); monoclonal anti-neurocan (clone 1F6, Developmental Studies Hybridoma Bank); polyclonal anti-GFP (Molecular Probes); monoclonal anti-hPAP (Sigma); polyclonal anti-hPAP (Fitzgerald); polyclonal anti-Olig2 (Chemicon); polyclonal anti-CGRP (Chemicon). Cy5, Cy2 (Jackson), Alexa-488 and Alexa-594 (Molecular Probes) conjugated secondary antibodies were used to visualize primary antibody binding. All secondary antibodies were pre-absorbed against rat serum. To control for nonspecific secondary antibody binding, adjacent sections were also processed as described above without primary antibodies. Some sections were counterstained with DAPI to show nuclei. Labeled sections were examined and imaged using a Zeiss Observer Z1 fluorescence light microscope or a Zeiss 510 Meta confocal microscope. Antigen co-localization and cellular associations were determined with Zeiss Confocal image analysis software. Spinal cord white matter rostral to the injury site is shown to the left in all figures with images of sections cut in the sagittal plane.
Tracing and quantification of endogenous ascending dorsal column axons
In the dorsal column injury model, ascending endogenous axons were traced by injection of 10% biotinylated dextran amine in sterile PBS (BDA, Molecular Probes) at 8 days prior to an experimental endpoint. BDA tracer was injected to a depth of 0.5 mm into the right-side, cuneate and gracile white matter at the C4/C5 spinal level (Figure ). For histological analysis of BDA-labeled axons, 25-μm serial sagittal sections were collected and processed for immunohistochemistry as described above. BDA was visualized by incubating tissue sections with the Vectastain ABC solution (Vector Labs), and further intensified with the Tyramide-Alexa 488 reagent (Molecular Probes).
For quantification of axon regeneration, the number of BDA-labeled axons was counted in every third tissue section spanning the medial-lateral extent of dorsal column injury sites at the following locations: 0.5 mm caudal to the injury; directly at the injury center; 0.5 mm, 1.5 mm and 5 mm rostral to the injury site; and within the dorsal column nuclei. To control for differences in axon tracing/labeling efficiency between animals, the numbers of BDA-labeled axons counted within the injury center and at all rostral sites were normalized to the number of BDA-labeled axons detected 0.5 mm caudal to the injury site for each tissue section examined. The normalized values from each tissue section for each separate animal (control, GRP-, GDABMP- and GDACNTF-transplanted rats) were averaged to generate values for each animal. The values for each animal (n = 5 per group) were then averaged and displayed graphically. ANOVA or t-tests were performed as appropriate, p < 0.01.
Quantification of CGRP c-fiber sprouting
For quantifying changes in the density of CGRP immunoreactivity in rats that had received right-side dorso-lateral funiculus transection injuries, 20-μm-thick serial cross-sections were labeled with anti-CGRP antibody. Images were captured of the right-side dorsal horn (ipsilateral to the injury/transplantation site) from five randomly chosen sections at the C6 spinal level from five animals in each experimental group. Analysis was conducted at the C6 spinal level because this is the level that maps to the dermatome as tested for forepaw mechanical sensitivity in rats [75
]. All images were captured at the same magnification, resolution and exposure time. Using ImagePro image analysis software, lamina III of the dorsal horn was selected as the region of interest and the number of pixels within lamina III that were CGRP-positive was recorded. The total number of pixels within each region of interest was also recorded and used to normalize CGRP pixel counts between sections and thus permit comparison of the density of CGRP immunoreactivity between experimental groups. Data are presented as the average percentage of CGRP-positive pixels per area sampled (number of CGRP-positive pixels divided by the total number of pixels per region of interest) and analyzed by ANOVA followed by Tukey's post test. An ANOVA analysis was also carried out to ensure that the total area sampled between groups was not significantly different (p
Grid-walk behavioral analysis
Two weeks before surgery, rats were trained to walk across a horizontal ladder (Foot Misplacement Apparatus, Columbus Instruments) and only rats that consistently crossed without stopping were selected for experiments. The grid-walk test is a sensitive measure of the ability of rats to step rhythmically and coordinate accurate placement of both fore and hind limbs [27
]. For analysis of recovery of locomotor function in GDABMP
- and GDACNTF
-transplanted versus untreated injured controls, trained rats were randomly assigned to one of three groups: RST injury + GDABMP
+ cyclosporine (n
= 9); RST injury + GDACNTF
+ cyclosporine (n
= 9); RST injury + suspension media + cyclosporine (n
= 9). One day before surgery (baseline) and at 3, 7, 10, 14, 17, 21, 24 and 28 days post-surgery, each rat was tested three times and the number of mis-steps from each trial was averaged to generate a daily score for each animal. Two-way repeated measures ANOVA and Tukey post test (p
< 0.05) were applied to analyze the data.
Mechanical and thermal sensitivity were measured the day before injury/transplantation (baseline) and then at 2, 3, 4 and 5 weeks post-injury. To test for changes in mechanical sensitivity, graded Von Frey filaments (Stoelting) were applied in ascending order to the plantar surface of the right forepaw. The lowest force that caused paw withdrawal accompanied by licking, paw-guarding behavior or vocalization at least three times per five trials was determined to be the mechanical threshold. Thermal sensitivity was tested with the hot-plate analgesia instrument (Stoelting). The temperature of the plate was held constant at 55°C, rats were placed on the plate, and the latency (in seconds) to licking of paws or vocalization was recorded. ANOVA followed by Tukey's post test analysis was applied to determine statistical significance of any change from baseline behavior (p < 0.05). Analysis was conducted on the same GDABMP-treated, GDACNTF-treated, and medium-injected control rats with spinal cord injury that were used for grid-walk analysis. An additional group of GRP-transplanted rats with dorsolateral funiculus injuries (n = 9) was also similarly tested for mechanical allodynia and thermal hyperalgesia at times ranging from 2 to 5 weeks after injury/transplantation.
Quantification of red nucleus neurons
At 5 weeks after injury/transplantation, animals were euthanized and 25-μm serial frozen sections were cut in the coronal plane from the brains of rats that had undergone behavioral analysis. Every third section through the rostro-caudal extent of the red nucleus was stained with 0.2% cresyl violet. Standard, design-based stereology methods (CAST software, Olympus) were used to quantify numbers of neurons in both red nuclei in six out of nine RST-injured rats per group that had received GDACNTF, GDABMP, or GRP transplants or control injections of culture medium. An optical fractionator was applied to left and right side red nuclei from every sixth section. Cell bodies greater than 20 μm in diameter and with characteristic neuronal morphology were counted. The numbers of neurons counted in the left-side (injured) red nucleus were normalized to counts obtained for the uninjured right-side nucleus for each animal. The values for each animal within a group were averaged and displayed graphically. A t-test was performed to determine the statistical significance of the difference between the groups (p < 0.01).
All procedures were performed under guidelines of the National Institutes of Health and approved by the Institutional Animal Care and Utilization Committee (IACUC) of Baylor College of Medicine, Houston, TX or the IACUC of University of Colorado Health Sciences Center, Denver, CO, or the IACUC of University of Rochester Medical Center, Rochester, NY.