Discussion

This study provides several new discoveries related to the treatment of traumatic spinal cord injury by cell transplantation. We show for the first time that different types of astrocytes derived from the same population of embryonic glial precursor cells have markedly different effects on repair and functional recovery when transplanted into the injured adult spinal cord. Transplantation of GDAs^BMP promoted axon regeneration, neuroprotection and robust recovery of function. In sharp contrast, transplantation of GDAs^CNTF or of undifferentiated GRPs did not provide any of these beneficial effects (see Table 1). Moreover, transplantation of GDAs^CNTF or undifferentiated GRPs resulted in both mechanical allodynia and thermal hyperalgesia, problems that were not caused by transplantation of GDAs^BMP. Our study provides further evidence that astrocytes derived from BMP-treated GRPs are a particularly promising population of cells for CNS repair and provide the first identification of a specific glial cell type – GDAs^CNTF – that can induce pain-related syndromes following transplantation into the injured spinal cord.

Controlled differentiation of glial precursors and spinal cord repair

The remarkably consistent and robust support of endogenous axon regeneration, neuroprotection and functional recovery provided by transplantation of GDAs^BMP in our previous [14] and present studies and the equally consistent failure of GRP transplantation to provide these benefits clearly show that controlled differentiation of glial precursors prior to transplantation to acute spinal cord injuries can result in significantly better outcomes. This hypothesis is consistent with previous studies showing failures of transplanted GRPs to promote axon growth [31] or functional recovery after spinal cord injury unless combined with additional treatments. Genetic manipulation of GRP cells to express the multifunctional-neurotrophin D15A before transplantation [32], or their transplantation in combination with neuron-restricted precursors (NRPs) [33], resulted in some locomotor recovery, with both studies showing improvements of approximately 2.5 points on the Basso, Beattie, and Bresnahan (BBB) open-field locomotor test. However, even the NRP/GRP-treated rats that had shown improved BBB scores failed to show a statistically significant improvement after grid-walk analysis [33]. As the NRP/GRP transplants were carried out in animals with contusion spinal cord injuries, the outcomes cannot be directly compared with our current studies, but do indicate the importance of conducting future experiments to compare the effects of NRP/GRP versus GDA^BMP transplantation in promoting recovery from both transection and contusion spinal cord injuries.

A striking and somewhat unexpected result of our study is that the two populations of astrocytes derived by pre-differentiation of embryonic spinal cord GRPs by two classical astrogenesis signaling pathways had completely opposite effects on axon regeneration, neuroprotection, functional recovery and neuropathic pain after transplantation, clearly demonstrating that not all astrocytes that can be derived from glial precursors are beneficial for CNS repair. Previous studies have shown functional differences in astrocytes from different regions of the CNS in respect of promotion of neurogenesis, neurite outgrowth, or promotion of axonal versus dendritic specialization [34-37]. Our current study, however, is the first to demonstrate that astrocytes generated by exposing the same precursor cell to different signaling agents have markedly different effects when transplanted into acute spinal cord injuries.

Factors regulating GRP differentiation into beneficial astrocytes

In the light of our new findings, the question arises as to whether any astrocyte generated by exposure of precursor cells to BMP would be suitable for use in repair of spinal cord injuries. This may not be the case, however, as exposure of O-2A progenitors cells (a type of glial progenitor that arises later in development than GRP cells) to BMP generate astrocytes with a phenotype that appears to be like that of GDAs^CNTF [38], and BMP treatment of acute spinal cord injuries can promote scar formation [39]. These findings suggest that glial precursors isolated from later stages of neural development may not be able to generate beneficial GDA^BMP-like astrocytes in response to BMP.

Endogenous GDA^CNTF-like astrocytes in the injured CNS

It will be of great interest to determine whether the Olig2⁺/GFAP⁺ cells generated in spinal cord injuries (Figure 3c), cerebral cortex stab injuries [21] and in rodent models of experimental autoimmune encephalomyelitis [20] are astrocytes generated from endogenous O-2A progenitor cells, and thus represent the long-sought in vivo counterpart of the type-2 astrocytes generated from these progenitor cells in vitro [40]. Our findings that GDAs^CNTF can become Olig2-negative after transplantation shows that the phenotype of such cells can be labile in vivo, as has also been seen in studies of Olig2⁺ astrocytes during CNS development [41]. This raises the possibility that the search for endogenous GDA^CNTF-like cells may have to be conducted with a variety of markers, at multiple time points after injury. Nonetheless, the fact that astrocytes within adult CNS scar tissue share many characteristics with GDAs^CNTF, such as poor support of axon growth and expression of Olig2 and inhibitory CSPGs, supports the hypothesis that they are functionally similar.

GDAs and suppression of axon-growth-inhibitory scar formation

Our study sheds new light on the role of GDA-mediated suppression of glial scar formation in supporting axon regeneration across acute spinal cord injuries. Logic dictates that improved alignment of host tissue can increase the efficiency of axon growth into and out of a site of injury by creating a shorter, less tortuous path for axons to follow. Neurocan and NG2 are axon-growth-inhibitory CSPGs [42,43] that are upregulated at sites of spinal cord injury [18,44,45] and whose suppression has been shown to correlate with the ability of adult sensory axons to cross acute spinal cord injuries [46]. In light of our previous finding that transplantation of GDAs^BMP to acute dorsal column transection injuries resulted in a remarkable alignment of host astrocytes within injury margins and a transient suppression of neurocan and NG2 [14], we proposed that these effects played significant roles in promoting axon growth across GDA^BMP-bridged spinal cord injuries.

Our current study shows that transplanted GDAs^CNTF and GRP cells promote the suppression of neurocan and NG2 in host tissues to an extent comparable to that previously observed for GDAs^BMP at 4 days post-transplantation [14]; nevertheless, they completely fail to align host astrocytes or support the regeneration of ascending dorsal column axons across sites of injury. A suppression of CSPG expression combined with a failure of axon regeneration has also previously been shown after GRP transplantation into acute spinal cord injuries [31]. Although these results do not rule out the possibility that suppression of CSPGs in host tissues may play an important role in the ability of GDAs^BMP to promote axon growth across sites of injury, they do show that such suppression is not by itself sufficient to promote axon growth. It may be that despite being able to suppress expression of axon-growth-inhibitory CSPGs, GDAs^CNTF and GRPs transplanted in acute spinal cord injuries fail to actively support axon growth and/or express molecules themselves that actively inhibit it. These concepts are supported by the expression of neurocan and NG2 by transplanted GDAs^CNTF and GRPs, a result that was not observed for transplanted GDAs^BMP [14]. The low-level expression of axon-growth-inhibitory CSPGs by GDAs^BMP compared with GDAs^CNTF, both in vitro and within spinal cord injuries, is one potential mechanism that might account for the clear difference in the ability of these astrocytes to support axon growth.

Precursor-derived astrocytes, spinal cord injury and neuropathic pain

Some of the most important results of our experiments concern the ability of both GDAs^CNTF and GRP transplants to cause mechanical allodynia and thermal hyperalgesia. Two studies of NSC transplantation to acute traumatic spinal cord injury sites in adult rats showed similar degrees of both mechanical and thermal forelimb allodynia [15,16]. That suppressing the differentiation of the transplanted NSCs to astrocytes prevented the onset of allodynia [15] could be interpreted to mean that all astrocytes generated from precursor cells have the capacity to promote allodynia. It was therefore crucial to determine whether the onset of allodynia after spinal cord injury is a problem that applies generally to the transplantation of astrocytes and astrocyte precursors. Our finding that mechanical allodynia and thermal hyperalgesia are caused by transplantation of GRPs and GDAs^CNTF – but not of GDAs^BMP – shows that only specific types of astrocytes or glial precursors cause these adverse outcomes.

Aberrant sprouting of CGRP-positive c-fibers has been shown to correlate with the onset of neuropathic pain after spinal cord injury [30]. We found a doubling of the density of CGRP-positive c-fibers within lamina III of the dorsal horns of injured spinal cords receiving GDA^CNTF or GRP transplants, an effect that was also correlated with neuropathic pain after transplantation of neural precursor cells into acute spinal cord injuries [15,16]. Interestingly, a reduction in allodynia and sprouting of CGRP⁺ c-fibers has been observed after transplantation of mixed NRP/GRP populations to spinal cord injuries [33]; however, the effects of GRP transplantation alone on allodynia were not tested in that study. Whether this benefit of combined NRP/GRP transplantation reflects suppression of the generation of GDA^CNTF-like astrocytes at the site of injury is an interesting question for the future.

It has long been a concern that therapies designed to promote axon growth after spinal cord injury would result in sprouting of CGRP⁺ c-fibers and the induction of neuropathic pain. Our results show, however, that GDAs^BMP have the remarkable ability to promote functional recovery without inducing pain, and promote axon regeneration without promoting aberrant sprouting of CGRP⁺ c-fibers. These results also show for the first time that two different types of precursor-derived astrocytes can have markedly different effects on the growth of different types of sensory axons.

Glial activation and neuropathic pain after spinal cord injury

Glial activation within the injured adult spinal cord is thought to have an important role in the development and maintenance of neuropathic pain [47,48]. A current model of glial cell function in neuropathic pain hypothesizes that injury-activated microglia are critical for initiation of enhanced pain perception via activation of astrocytes, and that activated astrocytes and microglia are also involved in the maintenance of neuropathic pain after traumatic spinal cord injury [49]. It has been shown that increases in spinal cord astrocytic GFAP expression following peripheral nerve injury correlates with the development of neuropathic pain [50] and that specific activation of microglia and astrocytes in the adult rat spinal cord is sufficient to promote neuropathic pain [51]. Whether transplanted GDAs^BMP or GDAs^CNTF can alter the activation state of microglia, whether these cells respond differently to the presence of activated microglia in an acute spinal cord injury, or whether transplantation of GDAs^CNTF bypasses any requirement for activated microglia to initiate neuropathic pain, are all presently unknown and will be the subject of future investigations.

Neuropathic pain, glial scar formation and gp130 receptor activation

The results reported here may also prove relevant to a better understanding of the role of gp130 agonists in promoting glial scar formation and neuropathic pain syndromes. In addition to its interactions with CNTF, the gp130 protein is a shared receptor for several related cytokines, including leukemia inhibitory factor (LIF) and interleukin-6 (IL-6, which has tertiary structure homology with CNTF) [52]. In some contexts, these agents may have beneficial effects on the injured nervous system, such as promoting oligodendrocyte generation and survival as well as neuronal protection [53-59]. Our results show, however, that exposure of glial precursors to the gp130 agonist CNTF results in the generation of astrocytes that are poorly supportive of axon growth and promote pain when transplanted into spinal cord injuries. CNTF, LIF and IL-6 are known to be upregulated at sites of spinal cord injury [60-65] and it is possible that some or all of these factors may also drive the differentiation of local endogenous glial precursors to a GDA^CNTF-like phenotype (Olig2⁺/GFAP⁺) that contributes to the formation of axon-growth-inhibitory scar tissue. Our finding of endogenous Olig2⁺/GFAP⁺ astrocytes within the margins of control untreated spinal cord injuries lends at least preliminary support to this hypothesis. Recent experiments showing that blocking of the gp130 receptor suppressed scar formation and improved functional recovery after spinal cord injury [66], and that specific inhibition of CNTF induction of astrocyte differentiation within transplanted fetal tissue improved axon growth across acute spinal cord injuries [67], also support this hypothesis. Future experiments will determine whether blocking CNTF or gp130 receptor activity in spinal injuries or after transplantation of undifferentiated glial precursors will increase axon growth across the injury and suppress the onset of neuropathic pain.

Conclusion

The results reported here lend significant new support to our hypothesis that pre-differentiation of glial precursor cells into a specific population of astrocytes such as GDAs^BMP before transplantation into spinal cord injuries results in significantly better outcomes, and they also provide further evidence that GDAs^BMP are a particularly promising cell type for promoting CNS repair. We have also provided the first identification of a specific glial cell type – GDAs^CNTF – that is capable of inducing pain-related syndromes following its transplantation into the injured spinal cord. This clearly demonstrates that not all astrocytes that can be derived from embryonic glial precursors have beneficial effects in spinal injuries. As gp130 agonists are of broad interest as inducers of astrocyte reactivity after injury to the CNS, our present findings are of particular relevance to the future study of gp130 agonists and glial precursors in CNS scar formation and onset of allodynia. The generation of a pain syndrome is one of the most adverse outcomes that could result from cell transplantation therapy for spinal cord injury [68-71], rivaled only by loss of remaining function or increased mortality. Our findings demonstrate that a better understanding of the origins and functional properties of different subpopulations of astrocytes is required if we are to safely utilize CNS stem or progenitor cell transplantation for treating the injured or diseased adult CNS.

Materials and methods

Isolation of GRPs and generation of GDAs

A2B5⁺ 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 cells/cm² 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 – GDAs^BMP (A2B5^-/GFAP⁺) and GDAs^CNTF (A2B5⁺/GFAP⁺), 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 D_C 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 GDA^CNTF cell suspensions included a small number (average 1.3%) of A2B5⁺/GFAP^- cells. To ensure that GDA^BMP 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 2 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 1a–c), the right-side dorsal column was unilaterally transected between cervical vertebrae 1 and 2 using a 30-gauge needle as a blade (see also [18,25,46]). 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 1d). 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 GDA^CNTF, GDA^BMP 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].

A total of 6 μl of GDA^CNTF, GDA^BMP 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 1b). All dorsal column injury experiments were conducted in the absence of immunosuppressants. Transplants of either GDAs^BMP, GDAs^CNTF 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 [25,46,74]. 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 1c).

Histology

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 1c). 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-, GDA^BMP- and GDA^CNTF-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 > 0.05).

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 GDA^BMP- and GDA^CNTF-transplanted versus untreated injured controls, trained rats were randomly assigned to one of three groups: RST injury + GDA^BMP + cyclosporine (n = 9); RST injury + GDA^CNTF + 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.

Sensory testing

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 GDA^BMP-treated, GDA^CNTF-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 GDA^CNTF, GDA^BMP, 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.

Additional data files

Additional data file 1 is a figure showing that transplanted GRP cells express neurocan and NG2, but suppress host expression of these molecules at 4 days post transplantation to dorsal column injuries. Additional data file 2 is a figure showing failure of axons to regenerate across GDACNTF-transplanted injuries. Additional data file 3 is a figure showing neuroprotection of injured red nucleus neurons.

Acknowledgements

This work was supported by funding from the Christopher and Dana Reeve Foundation, NIH RO1-NS046442, NIH RO1-NS42820, the New York State Department of Health Spinal Injury Research Program grants CO19772, CO20942 and CO16889, the New York State Center of Research Excellence for Spinal Cord Injury and the Lone Star Paralysis Foundation. Private donations from members of the spinal cord injury community also played a major role in supporting this work. The 1F6 anti-neurocan and 3F8 anti-phosphacan antibody was obtained from the Developmental Hybridoma Bank developed under the auspices of the NICHID and maintained by the University of Iowa, Department of Biological Sciences.

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