Jeannette E Davies1 
, Christoph Pröschel2 , Ningzhe Zhang2 , Mark Noble2 , Margot Mayer-Pröschel2 and Stephen JA Davies1 
1Department of Neurosurgery, Anschutz Medical Campus, University of Colorado Denver, 12800 East 19th Ave, Aurora, CO 80045, USA
2Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
Journal of Biology 2008, 7:24doi:10.1186/jbiol85
The electronic version of this article is the complete one and can be found online at: http://jbiol.com/content/7/7/24
Abstract
Background
Two critical challenges in developing cell-transplantation therapies for injured or diseased tissues are to identify optimal cells and harmful side effects. This is of particular concern in the case of spinal cord injury, where recent studies have shown that transplanted neuroepithelial stem cells can generate pain syndromes.
Results
We have previously shown that astrocytes derived from glial-restricted precursor cells (GRPs) treated with bone morphogenetic protein-4 (BMP-4) can promote robust axon regeneration and functional recovery when transplanted into rat spinal cord injuries. In contrast, we now show that transplantation of GRP-derived astrocytes (GDAs) generated by exposure to the gp130 agonist ciliary neurotrophic factor (GDAsCNTF), the other major signaling pathway involved in astrogenesis, results in failure of axon regeneration and functional recovery. Moreover, transplantation of GDACNTF cells promoted the onset of mechanical allodynia and thermal hyperalgesia at 2 weeks after injury, an effect that persisted through 5 weeks post-injury. Delayed onset of similar neuropathic pain was also caused by transplantation of undifferentiated GRPs. In contrast, rats transplanted with GDAsBMP did not exhibit pain syndromes.
Conclusion
Our results show that not all astrocytes derived from embryonic precursors are equally beneficial for spinal cord repair and they provide the first identification of a differentiated neural cell type that can cause pain syndromes on transplantation into the damaged spinal cord, emphasizing the importance of evaluating the capacity of candidate cells to cause allodynia before initiating clinical trials. They also confirm the particular promise of GDAs treated with bone morphogenetic protein for spinal cord injury repair.
Background
Two critical challenges that must be addressed in the development of cell-based tissue repair strategies are the identification of optimal cell types and the identification of instances in which cell transplantation may create severe adverse side effects. The first problem is important because of the considerable resources that will be required to establish clinical efficacy of putative treatments. The second problem is perhaps of even greater importance, because adverse outcomes in clinical trials could seriously hinder the development of stem cell technology for tissue repair.
Diseases of the central nervous system (CNS) are of particular interest as candidates for clinical evaluation of cell transplantation therapies, with the treatment of spinal cord injury being one of the primary targets for early translation of laboratory efforts to clinical trials. A variety of cell types of both non-CNS and CNS origin, such as Schwann cells [1], olfactory ensheathing glia [2], marrow stromal cells [3,4] and oligodendrocyte progenitor cells [5], are being considered for clinical trial to treat spinal cord injuries. One of the most attractive reasons for considering the use of non-CNS cells such as Schwann cells, olfactory ensheathing cells and marrow stromal cells for CNS repair has been their relative ease of isolation compared to cells of CNS origin. However, continuing advances in stem cell technology are making the goal of utilizing CNS cell types to repair the injured CNS more readily attainable.
One new potential candidate for use in CNS repair is a population of astrocytes that is derived by treatment of glial progenitor cells (GRPs) of the embryonic spinal cord with bone morphogenetic protein (BMP) before transplantation. We call this astrocyte population GDAsBMP. The replacement of damaged neurons and oligodendrocytes in the injured or diseased spinal cord has been pursued by a number of laboratories (reviewed in [6]), but less attention has been given to the development of astrocyte replacement therapies, despite the fact that astrocytes account for the majority of cells in the adult CNS [7] and are critical to normal CNS function [8]. This relative lack of attention is probably due to the modest levels of axon regeneration and lack of functional recovery seen after transplantation into the injured CNS of astrocytes isolated from the immature cortex [9-12]. Factors such as contamination with microglia and undifferentiated progenitors, isolation from cortex rather than spinal cord, and a phenotype that is less supportive of axon growth (resulting from the prolonged in vitro growth required to generate postnatal astrocyte cultures) [13], may have rendered these glial cultures suboptimal for repairing the injured adult spinal cord.
In contrast to the lack of effect of astrocyte transplantation in previous studies, GDAsBMP promote robust axon regeneration, neuroprotection and functional recovery after acute spinal cord injury [14]. The ability to generate specific subtypes of astrocytes from defined glial precursors provides a new platform for the development of astrocyte-based transplantation therapies for the injured adult CNS. Transplantation of GDAsBMP to acute transection injuries of adult rat spinal cord promoted first, a 39% efficiency of endogenous ascending dorsal column axon regeneration across sites of injury; second, protection of axotomized red nucleus neurons; third, a significant reduction of inhibitory scar formation; and fourth, a degree of behavioral recovery from dorsolateral funiculus injuries that enabled rats to generate an average score by 4 weeks after transplantation that was statistically indistinguishable from that obtained for uninjured animals on a stringent test of volitional foot placement [14]. Moreover, this strategy allows the rapid generation of astrocytes directly from embryonic precursor cells, thus eliminating the use of the prolonged in vitro purification procedures that result in a phenotype that is less supportive of axon growth [13].
Recent studies demonstrating the ability of transplanted neuroepithelial stem cells (NSCs) to cause pain syndromes in animals with spinal cord injury have, however, raised concerns that the astrocytes generated by transplanted stem or progenitor cells might cause adverse effects that outweigh any benefits. Two recent studies have shown that transplantation of NSCs into acute spinal cord injuries in rats promotes the onset of both mechanical allodynia (a painful response to normally non-painful touch stimuli) and thermal hyperalgesia (abnormal sensitivity to heat) [15,16]. These adverse side effects correlated with the differentiation of the transplanted NSCs into astrocytes, and were prevented by the suppression of astrocyte generation by overexpression of the transcription factor neurogenin-2 in the transplanted NSCs [15]. It was therefore very important to determine whether transplantation of astrocytes, or of precursor cells capable of generating astrocytes, would promote the onset of allodynia, or whether this is a problem unique to the transplantation of NSCs.
The study reported here was carried out to determine whether all astrocytes generated from GRPs [17] were equally able to promote repair of adult injured spinal cord. Two types of astrocytes can be generated from embryonic spinal GRPs – GDAsBMP and GDAsCNTF (astrocytes derived from the gp130 receptor agonist ciliary neurotrophic factor (CNTF)). We found that transplantation of these two types of astrocytes into acute spinal cord injuries (Figure 1) yielded significantly different outcomes. In contrast to GDAsBMP, we found that GDAsCNTF provided no benefit and, more importantly, transplantation of either GDAsCNTF or undifferentiated GRPs caused neuropathic pain. Our results also confirm earlier work [14] showing that transplantation of GDAsBMP generated by controlled pre-differentiation of GRPs can provide substantial benefits after spinal cord injury and that this pre-differentiation can avoid the problem of transplanted glial precursors themselves causing pain syndromes.
Figure 1. Schematic illustration of the adult rat models of spinal cord injury used in this study. (a) Dorsal view of rat brain and spinal cord. Dorsal column white matter on the right side was transected (shaded area) at the C1/C2 spinal level, and the ability of either BDA-labeled endogenous axons or axons from microtransplanted GFP-expressing adult sensory neurons (DRGs) to cross injuries bridged with GDAs or GRPs was assayed. (b) Horizontal and (c) sagittal views of the dorsal column white-matter pathways at the C1/C2 cervical vertebrae of the spinal cord. (b) Injections of GDAs or GRPs (black diamonds) suspended in medium were made directly into the centers of the injury sites as well as their rostral and caudal margins in the cervical spinal cord. (c) A discrete population of endogenous ascending axons within the cuneate and gracile white-matter pathways of dorsal columns was labeled by BDA injection at the C4/C5 spinal level (5 mm caudal to the injury site, shaded). Alternatively, microtransplants of GFP+ DRGs were injected 500 μm caudal to the injury site. (d) The right-side dorsolateral funiculus white matter containing descending axons of the rubrospinal tract was transected at the C3/C4 spinal level and GDAs or GRPs were transplanted as described for dorsal column injuries. CC, central canal; Cf, cuneate fasciculus; CST, corticospinal tract; DF, dorsolateral funiculus; Gf, gracile fasciculus; GM, gray matter; RN, red nucleus; RST, rubrospinal tract; T1, level of the first thoracic vertebra.
Results
Characterization of GDAs in vitro
GRPs exposed to BMP-4 generate astrocytes (GDAsBMP) with a flat, type-1 antigenic phenotype that express glial fibrillary acidic protein (GFAP) and do not label with the A2B5 antibody [14]. In contrast, GRPs grown in the presence of the gp130 receptor agonist CNTF generate GFAP+ astrocytes (GDAsCNTF) with processes that are labeled by A2B5 [17]. In seeking to use GDAs for repairing the injured spinal cord, it is critical to know whether the favorable properties of GDAsBMP are solely a reflection of the embryonic age and/or identity of the glial precursor cell from which they are derived, or whether it is necessary to generate a very specific population of astrocytes from these precursor cells to promote repair.
To answer this question, we first characterized GDAsBMP and GDAsCNTF in vitro and found that GDAsCNTF had properties suggesting they would be less suitable than GDAsBMP for repairing the injured adult CNS. Compared with GDAsBMP, GDAsCNTF express elevated levels of the axon-growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) NG2 (Figure 2a,b) and phosphacan (Figure 2c,d), both of which are also expressed at high levels in glial scar tissue [18]. We also found that GDAsBMP and GDAsCNTF cells differed in their regulation of the transcriptional regulator Olig2 in vitro (Figure 3a,b). In agreement with previous observations of the effects of BMP on Olig2 expression in cortical neural progenitors [19], GRPs exposed to BMP-4 downregulated Olig2 expression (Figure 3a). In contrast, GDAsCNTF had high levels of Olig2 in their nuclei (Figure 3b). Several recent studies have reported the natural generation of cells that coexpress Olig2 and GFAP in vivo after injury to the brain [20,21]. Although those studies described cytoplasmic rather than nuclear localization of Olig2, our examination of control injured spinal cords at 8 days revealed the presence of endogenous GFAP+ cells with nuclear localization of Olig2 (Figure 3c).
Figure 2. GDAsBMP, GDAsCNTF and GRPs express different levels of NG2 and phosphacan in vitro. GRPs were induced to differentiate into astrocytes by exposure to BMP or CNTF. Relative levels of expression of NG2 and phosphacan proteins were determined by quantitative Western blot and immunocytochemical analysis. (a,c) Western blot analysis of whole-cell lysates demonstrates that GDAsCNTF express higher levels of (a) NG2 and (c) phosphacan. The graph shows fold change in protein levels for GDAs compared to GRPs. Error bars represent 1 standard deviation (SD). *p < 0.05. (b,d) Immunofluorescent labeling of cells using (b) anti-NG2 antibodies and (d) anti-phosphacan. Scale bars 50 μm.
Figure 3. Differential expression of Olig2 protein by different astrocyte populations. (a) GDAsBMP do not express Olig2. (b) In sharp contrast, GDAsCNTF are uniformly immunopositive for Olig2 in vitro. (c) A subset of endogenous GFAP+ astrocytes in the margins of untreated dorsal column spinal cord injuries is also Olig2-immunoreactive. Survival, 8 days post-injury. Note the nuclear localization of Olig2 in GDAsCNTF in vitro and in reactive, endogenous GFAP+ astrocytes in vivo. Scale bars: (a,b) 50 μm; (c) 25 μm.
Characterization of transplanted GDAsCNTF in vivo
Transplanted GDAsCNTF exhibited good survival and were able to completely span sites of injury (Figures 4, 5, 6, 7). We found that transplanted GDAsCNTF displayed phenotypes markedly different from those previously observed for transplanted GDAsBMP. The majority of GDAsCNTF retained their GFAP immunoreactivity after transplantation to acute spinal cord injury, particularly for those cells adjacent to injury margins (Figure 4a). Subsets of intra-injury GDAsCNTF also displayed immunoreactivity for the axon-growth-inhibitory proteoglycan neurocan at 4 and 8 days post-injury (Figure 4b) and the majority of GDAsCNTF had retained their in vitro immunoreactivity for NG2 (Figure 5). In contrast, our previous studies showed that GDAsBMP did not retain GFAP immunoreactivity after transplantation to identical acute spinal cord injuries [14]. More importantly, transplanted GDAsBMP within the center of the injured site remained negative for neurocan and NG2 immunoreactivity at 8 days after transplantation [14].
Figure 4. GDAsCNTF express GFAP and neurocan after transplantation into spinal cord injuries. (a) Intra-injury GDAsCNTF are uniformly GFAP+ within acute dorsal column injuries. Note the co-localization (yellow) of human placental alkaline phosphatase (hPAP, red) with GFAP (green). GDAsCNTF have also failed to align host astrocytic processes within injury margins. Survival, 8 days post-injury/transplantation. (b) High-magnification confocal image of neurocan immunoreactivity at the injury margin and within a GDACNTF-transplanted injury site at 8 days after injury/transplantation. Note that some GDAsCNTF are immunoreactive for neurocan (green). In contrast, intra-injury transplanted GDAsBMP (not shown) do not express GFAP or neurocan, and can align host astrocytic processes within injury margins [14]. Scale bars 100 μm.
Figure 5. NG2 immunoreactivity in GDACNTF-transplanted dorsal column injuries. (a) Transplanted hPAP+ GDAsCNTF (arrowheads) at 8 days post injury/transplantation. (b) The same slide stained for NG2 (green) showing that the transplanted cells (arrowheads) show immunoreactivity for NG2. (c) Co-localization (yellow) of NG2 and hPAP immunoreactivity in regions containing higher densities of GDAsCNTF (arrowheads). In general, regions of the injury site that contained higher densities of hPAP+ GDAsCNTF had a higher density of NG2 immunoreactivity. Scale bars 50 μm.
Figure 6. Transplanted GDAsCNTF express neurocan and NG2, but suppress host expression of these two CSPGs at 4 days post-injury/transplantation. (a) At 4 days after injury, control dorsal column injury margins express dense neurocan immunoreactivity (green) mainly associated with GFAP- processes. Note the absence of neurocan immunoreactivity in the injury center (to the left). (b,c) While neurocan immunoreactivity in host white matter was markedly lower and mainly associated with astrocyte cell bodies, many intra-injury GDAsCNTF within injury centers displayed neurocan immunoreativity. (d) NG2 immunoreactivity in control injuries is high in both injury centers and margins. (e,f) Although overall levels of NG2 immunoreactivity were reduced within injury centers and margins of GDACNTF-transplanted injury sites compared to untreated control injuries (compare (d) and (f)), levels of NG2 immunoreactivity were still higher than that previously observed for identical dorsal column injuries transplanted with GDAsBMP [14]. Scale bars 200 μm.
Figure 7. Failure of axons to regenerate across GDACNTF or GRP transplanted dorsal column injuries. (a) Biotinylated dextran amine (BDA)-labeled endogenous, ascending dorsal column axons (green) fail to cross GDACNTF-transplanted injury sites and instead form dystrophic endings within caudal injury margins. While a few axons sprout towards the injury center, BDA+ axons are rarely detected beyond the injury/transplantation site at 8 days post-injury/transplantation. Scale bar 200 μm. (b) In contrast, transplanted GDAsBMP support extensive axon growth across dorsal column injuries at 8 days after injury/transplantation. Scale bar 200 μm. (c) Quantification of numbers of regenerating BDA+ axons in GDA- or GRP-transplanted dorsal column white matter at 8 days after injury and transplantation. BDA-labeled axons were counted in every third sagittally oriented section within the injury center and at points 0.5 mm, 1.5 mm and 5 mm rostral to the injury site and within the dorsal column nuclei (DCN). Note that 55% of BDA+ axons reached the centers of GDABMP-transplanted injuries, and 36% to 0.5 mm beyond the injury site. After GDACNTF or GRP transplantation, however, only 7% and 5.3% of BDA+ axons, respectively, were observed within injury centers, with only 4.6% and 4.2% of the axons observed at 0.5 mm beyond the injury site. No BDA+ axons were detected beyond 1.5 mm rostral to the injury site in GDACNTF- or GRP-transplanted spinal cords. Error bars represent 1 SD.
Effects of GDAsCNTF and GRPs on scar formation
Transplanted GDAsCNTF and GDAsBMP also had substantially different effects on the reactivity of host astrocytes at sites of injury. We previously showed that transplantation of GDAsBMP suppressed the gliotic response of host astrocytes within injury margins and promoted a remarkable linearization of their processes [14]. Transplantation of GDAsCNTF, in contrast, did not suppress astrogliosis, nor did these cells align host astrocytes in injury margins. Instead, the margins of GDACNTF-transplanted injury sites contain a meshwork of misaligned, hypertrophic GFAP+ astrocytic processes (Figure 4a), similar to that observed in both control untreated injuries and the margins of GRP-transplanted injuries [14]. GDACNTF and GRP transplantation did, however, result in a suppression of neurocan and NG2 expression by host tissue at sites of injury at 4 days post-injury, an effect we previously observed following transplantation of GDAsBMP [14]. At 4 days after injury, the margins of control, untreated injuries displayed a high density of neurocan immunoreactivity (Figure 6a) associated with numerous fine GFAP- processes that we previously showed to be associated with NG2+ glia [18]. In contrast, at 4 days after injury and transplantation of GDAsCNTF (Figure 6b,c) or GRPs (Additional data file 1), neurocan immunoreactivity within injury margins was mainly associated with the cell bodies of GFAP+ host white-matter astrocytes, a pattern of expression similar to that observed for neurocan at 2 days after injury in untreated control animals [18]. However, by 8 days after injury and GDACNTF transplantation, neurocan and NG2 immunoreactivity at sites of injury was similar in intensity and distribution to that seen in untreated control injuries (Figures 4b and 5b). Thus, like GDAsBMP transplanted to acute spinal cord injuries, GRPs and GDAsCNTF had promoted transient suppression of axon-growth-inhibitory CSPGs by host tissues; but unlike GDAsBMP, neither GDAsCNTF nor GRPs [14] suppressed astrogliosis or aligned host astrocytes within injury margins.
GDAsCNTF do not support axon regeneration in vivo
We next examined the ability of GDAsCNTF to promote axon regeneration in vivo, both of endogenous ascending dorsal column axons and of axons emanating from transplanted adult dorsal root ganglion (DRG) neurons. For analysis of endogenous axon regeneration, a discrete population of ascending axons aligned with the injury site was traced with a single injection of biotinylated dextran amine (BDA) at a distance 6 mm caudal to GDACNTF-, GDABMP-, or GRP-transplanted or control transection injuries of the right-hand dorsal column cuneate and gracile white-matter pathways. This minimized the labeling of spared axons. Previous studies have shown that around 30–40% of ascending dorsal column axons projecting to the dorsal column nuclei arise from postsynaptic dorsal column neurons in spinal lamina IV and that 25% of ascending dorsal column axons are also propriospinal in origin [22,23]. It has been shown that only 15% of primary afferents of DRG neurons entering the spinal cord at lumbar levels reach the cervical spinal cord and that most leave dorsal column white matter within two to three segments of entering [24]. Therefore, our en passage labeling of dorsal column axons at the cervical level would have included significant proportions of axons from both CNS spinal neurons and DRG neurons. To further test the ability of transplanted GDAsCNTF to support axon growth across an acute spinal cord injury in a model that eliminates the possibility of axon sparing, we examined their ability to support the growth of adult sensory axons across identical stab injuries in an adult DRG neuron/GDA transplant spinal cord injury model [14]. In these experiments, a separate series of animals received microtransplants of adult mouse sensory neurons labeled with green fluorescent protein (GFP) acutely into dorsal column white matter at a distance of 400–500 μm caudal to GDACNTF-transplanted injuries (Figure 1c).
Transplantation of either GRPs or GDAsCNTF to acute dorsal column transection injuries failed to improve the regeneration of endogenous ascending dorsal column axons above that observed in untreated injuries (Figure 7). There was also a complete failure of axons grown from adjacent microtransplanted adult mouse DRG neurons expressing enhanced green fluorescent protein (EGFP) to cross GDACNTF-transplanted injuries (Additional data file 2). In both experimental models, the majority of axons instead formed dystrophic endings within the caudal injury margins of GDACNTF-transplanted injuries, (Figure 7a and Additional data file 2a), an axon morphology well known as the hallmark of failure of axon regeneration in CNS injury [25,26]. Quantitative analysis of the efficiency of ascending dorsal column axon regeneration in GDACNTF- and GRP-transplanted rats at 8 days after transplantation/injury showed that only 7% (SD ± 2.0) and 5.3% (SD ± 3.0), respectively, of BDA-labeled axons within white matter 0.5 mm caudal to injury sites had reached injury centers; 6.2% (SD ± 3.5) and 4.7% (SD ± 3.9) of axons had extended 0.5 mm beyond injury sites into distal white matter, with 4.6% (SD ± 2.3) and 4.2% (SD ± 3.6) reaching 1.5 mm beyond injury sites (Figure 7c). No BDA-labeled axons were detected beyond 1.5 mm in distal white matter or within the dorsal column nuclei of both GDACNTF- and GRP-transplanted rats (Figure 7c). All the percentages of BDA-labeled axons within injury sites and at all points beyond were not statistically different from those quantified for BDA-labeled endogenous ascending dorsal column axons in identical control, untransplanted injuries [14] (ANOVA, p > 0.05).
The failure of both GDAsCNTF and GRPs (see also [14]) to support axon regeneration is in stark contrast to the ability of transplanted GDAsBMP to promote regeneration of endogenous dorsal column axons across spinal cord injuries. In GDABMP-transplanted animals, 55% (SD ± 8.0) of labeled axons extended to the injury center, 36.5% (SD ± 11.0) extended to 0.5 mm beyond the injury site, and 30.4% (SD ± 9.2) had extended to 1.5 mm beyond the injury site (Figure 7c). Furthermore, 12.6% (SD ± 9.0) of labeled axons were detected within white matter at 5 mm beyond the injury site, and 2.1% (SD ± 1.4) were observed within the dorsal column nuclei (Figure 7c). This is consistent with our previous finding that intra-injury transplants of GDABMP cells promote regeneration of 60% (SD ± 11.0) of labeled endogenous ascending dorsal column axons into the center of injury sites, and more than two-thirds of these axons were within white matter beyond the injury site by 8 days after transplantation/injury [14].
Failure of GDAsCNTF to promote locomotor functional recovery after spinal cord injury
To make a direct comparison of the ability of GDAsCNTF and GDAsBMP to promote functional recovery following dorsolateral funiculus transection injuries to the spinal cord, an analysis of grid-walk performance for GDACNTF- and GDABMP-transplanted rats versus rats injected with control medium was carried out at times ranging from 3 to 28 days after injury/transplantation. Transection of the dorsolateral funiculus severs descending supraspinal axons and results in chronic deficits in both fore-and hindlimb motor function [27] that can be detected by the grid-walk behavioral test [28]. We have previously shown that transplantation of GDAsBMP into acute dorsolateral funiculus injuries resulted in robust improvements in grid-walk locomotor function compared to media-injected control injured animals at all time points ranging from 3 to 28 days post-injury. In contrast, transplantation of undifferentiated GRPs failed to improve scores to greater than those observed for control injured animals [14].
Animals that received GDACNTF transplants or injections of medium alone (controls) made an average of 6.2 (SD ± 0.5) and 6.0 (SD ± 0.3) mistakes, respectively, at 3 days after injury/transplantation and showed no statistically significant improvement at any later time point, with an average of 5.2 (SD ± 0.3) and 5.0 (SD ± 0.9) mistakes at 28 days post-injury (Figure 8). Thus, despite receiving transplants of astrocytes derived from embryonic GRP cells, GDACNTF-transplanted rats did not show any recovery of locomotor function when compared with controls. In contrast, at 3 days after injury/transplantation, animals receiving GDAsBMP were already making an average of 4.5 (SD ± 0.3) mistakes; that is, significantly fewer than GDACNTF-transplanted or control animals (Figure 8). Consistent with our previous report [14], animals receiving GDABMP transplants in the current study continued to improve significantly between 3 and 28 days after injury (two-way repeated measures ANOVA, p < 0.05). At 28 days after injury, GDABMP-treated rats made an average of just 1.7 (SD ± 0.3) mistakes on the grid walk apparatus (Figure 8), a score that was statistically indistinguishable from their pre-injury baseline scores.
Figure 8. Grid-walk analysis of locomotor recovery. Graph showing the average number of missed steps per experimental group from 1 day before injury (baseline pre-injury) to 28 days after injury for all GDA-transplanted/dorsolateral funiculus injured rats versus the control-injured animals. GDABMP-transplanted animals (green) performed significantly better than GDACNTF-transplanted animals and injured control animals at all post-injury time points (p < 0.05). Note that the performance of GDACNTF-transplanted animals was not different from untreated control injured rats at all time points (two-way repeated measures ANOVA, *p < 0.05). N = 9 rats per group.
Transplantation of GDAsCNTF or GRPs fails to suppress atrophy of red nucleus neurons
Transection of axons of the rubrospinal tract (RST) in the dorsolateral funiculus of the spinal cord causes atrophy of significant numbers of red nucleus neurons, a process that begins approximately 1 week after spinal cord injury [29]. In the absence of intervention, the number of neurons with a cell-body diameter greater than 20 μm in the injured left-side red nucleus in control, untreated RST-injured animals fell to 52% (SD ± 4.2%) of the values in the uninjured right-side nucleus at 5 weeks after injury (Figure 9).
Figure 9. Neuroprotection of red nucleus neurons. Injured left-side red nuclei contained an average of 52% of the neurons counted in uninjured right-side red nuclei at 5 weeks after transection of the right-side rubrospinal tract. The numbers of neurons in the injured left-side red nuclei of GRP- and GDACNTF-transplanted animals were no different from controls, and contained an average of 55% and 51%, respectively, of the neurons counted in the uninjured right-side nuclei. In contrast, the number of neurons in the injured left-side red nuclei of GDABMP-transplanted animals was 81% of the total number of neurons in uninjured right-side nuclei. *p < 0.01. Error bars represent 1 SD.
Consistent with our previous findings [14], animals that had received intra-spinal cord injury transplants of GDAsBMP (Figure 9) once again showed a significant suppression of red nucleus neuron atrophy with 82% (SD ± 6.1) of neurons in the injured left-side red nucleus having cell body diameters greater than 20 μm when normalized to the uninjured right-side nucleus (Figure 9). In contrast, transplantation of GDAsCNTF or undifferentiated GRPs into identical dorsolateral funiculus injuries completely failed to suppress neuron atrophy in the injured left-side red nucleus (Figure 9; see also Additional data file 3). Counts of neurons with a cell-body diameter greater than 20 μm in the injured left-side red nucleus in GDACNTF- or GRP-treated animals were only 51% (SD ± 8.7%) and 55% (SD ± 8.0%), respectively, of the values in uninjured right-side red nucleus at 5 weeks after injury and did not differ statistically from untreated injured animals (ANOVA, p < 0.05). Thus, despite the fact that GDAsBMP and GDAsCNTF are both astrocytes derived from the same embryonic precursor cells, they do not share the same ability to rescue red-nucleus neuronal populations from atrophy.
GDAsCNTF or GRPs, but not GDAsBMP, induce mechanical allodynia and thermal hyperalgesia when transplanted into sites of spinal cord injury
To test whether transplantation of GDAsBMP, GDAsCNTF and GRPs might promote the induction of mechanical allodynia and thermal hyperalgesia in acute spinal cord injuries, initial experiments were carried out to test for increases in mechanical and thermal sensitivity in control rats receiving injections of medium into transection injuries of the right-side dorsolateral funiculus at 2, 3, 4, and 5 weeks after injury. Importantly, compared with pre-injury scores, injured medium-injected control rats did not show statistically significant increases in gram force withdrawal thresholds for right-side forepaws in response to application of graded Von Frey filaments at all time points after injury and transplantation (Figure 10a). Similarly, analysis of paw-withdrawal response latencies to an experimental heat source pre- and post-injury revealed no statistically significant induction of thermal hyperalgesia in injured controls at all time points post-injury (Figure 10b). These results enable a direct comparison of the effects of intra-injury transplantation of GDAsBMP, GDAsCNTF or GRPs on the induction of mechanical allodynia and thermal hyperalgesia in rats with identical dorsolateral funiculus transection injuries.
Figure 10. Von Frey filament and hot-plate analysis of mechanical and thermal allodynia. (a) Withdrawal threshold of the right front paw to a mechanical stimulus (force in grams). Measurements were made on GRP- or GDA-transplanted and injured control animals at 2, 3, 4 and 5 weeks after dorsolateral funiculus injury/transplantation. (b) Latency (in seconds) to paw withdrawal from a heat source. Note that injury alone and GDABMP transplantation do not induce statistically significant mechanical or thermal allodynia at any time point. However, the mechanical threshold and latency to withdrawal from a heat source are significantly lower in GDACNTF- and GRP-transplanted rats beginning at 2 and 3 weeks, respectively, post-injury/transplantation. Asterisks denote a statistical difference from time-matched control animals (two-way repeated measures, ANOVA, p < 0.05). Error bars represent 1 SD.
Unlike animals that received GDAsCNTF or GRPs, GDABMP-transplanted animals did not show a statistically significant increase in sensitivity to mechanical or heat stimuli at any times (2, 3 and 4 weeks) up to 5 weeks post-injury (Figure 10a,b) compared to pre-injury responses (two-way repeated measures ANOVA, p > 0.05). GDACNTF-transplanted animals showed a significant increase in sensitivity to both mechanical and heat stimuli by 2 weeks after injury, an effect that persisted to 5 weeks after injury, the last time point tested (Figure 10a,b). Animals that received intra-injury transplants of undifferentiated GRPs also developed increased sensitivity to both mechanical and heat stimuli, although with a delayed time course. GRP-transplanted animals began to show mechanical allodynia and thermal hyperalgesia at 3 weeks post injury and transplantation, effects that persisted to 5 weeks post-injury (Figure 10a,b).
GDAsCNTF and GRPs promote sprouting of CGRP c-fibers after spinal cord injury
Previous studies have shown a correlation between sprouting of calcitonin-gene-related peptide (CGRP) immunoreactive nociceptive c-fibers within lamina III of the dorsal horn and the development of neuropathic pain after spinal cord injury [30]. To assay for this, we carried out a comparative quantitative analysis at 5 weeks post-injury of the density of CGRP immunoreactivity in lamina III of the dorsal horn at spinal level C6 ipsilateral to injury sites in media-injected injured controls, and GDABMP-, GDACNTF- or GRP-transplanted animals. Notably, the GDABMP-transplanted animals showed no statistically significant change in the density of CGRP-positive c-fibers compared with the control injured animals (Figure 11). This result correlated with the absence of statistically significant mechanical allodynia and thermal hyperalgesia in GDABMP-treated animals compared with uninjured controls.
Figure 11. Aberrant CGRP+ c-fiber sprouting into lamina III of GDACNTF- or GRP-transplanted spinal cords that have received dorsolateral funiculus transection injuries. The density of pixels within images of lamina III of the right-side dorsal horn caudal to the injury and transplantation site in GDA- or GRP-transplanted, or injury-only control animals is presented as the average percentage of CGRP+ pixels per total pixels (area) of lamina III. (a) Averages of 5.7% and 6.2% of the total pixels in lamina III were CGRP+ in GDACNTF- and GRP-transplanted spinal cords, respectively. In contrast, only 2.2% and 3.4% of lamina III pixels were CGRP+ in GDABMP-transplanted and injury-only spinal cords. The asterisk indicates significant difference from both control injury-only and GDABMP-transplanted groups (two-way repeated measures ANOVA, p < 0.05). Error bars represent 1 SD. (b-e) Sample images of sections labeled with anti-CGRP antibodies from rats transplanted at the spinal C6 level: (b) control, (c) GDABMP, (d) GDACNTF and (e) GRP. Area enclosed with a dashed line in (b-e) indicates lamina III. Note the increased density of CGRP+ immunoreactivity within lamina III of the dorsal horn of (d) GDACNTF- and (e) GRP-treated spinal cords compared to (b) control injured and (e) GDABMP-treated spinal cords. Scale bar 200 μm.
In contrast, significant increases in the density of CGRP immunoreactivity were found in animals that received intra-injury transplants of GDAsCNTF or GRPs. Comparison of the density of CGRP immunoreactivity in GDACNTF-, GRP- and GDABMP-transplanted cords revealed 2.6- and 2.9-fold increases for GDACNTF- and GRP-treated cords, respectively, above levels in GDABMP-transplanted cords at 5 weeks post-injury (Figure 11).
These results demonstrate that transplantation of GDAsCNTF or GRPs, but not of GDAsBMP, into spinal cord injuries induces both mechanical allodynia and thermal hyper-algesia, effects that correlated with the relative densities of CGRP immunoreactive c-fibers in dorsal horn lamina III. Collectively, these results show that pain syndromes are not a necessary consequence of astrocyte transplantation or of the generation of astrocytes from transplanted precursor cells at sites of spinal cord injury, but instead indicate that the generation of specific subtypes of astrocyte, such as GDAsCNTF, is responsible for this adverse effect.
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 GDAsBMP promoted axon regeneration, neuroprotection and robust recovery of function. In sharp contrast, transplantation of GDAsCNTF or of undifferentiated GRPs did not provide any of these beneficial effects (see Table
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