Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted precursors have opposite effects on recovery and allodynia after spinal cord injury

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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. © 2008 Davies et al.; licensee BioMed Central Ltd.

Figures

  • 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.
  • 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) antiphosphacan. 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.
  • 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) Highmagnification 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 postinjury/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.
  • 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 GDAtransplanted/dorsolateral funiculus injured rats versus the controlinjured 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.

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Davies, J. E., Pröschel, C., Zhang, N., Noble, M., Mayer-Pröschel, M., & Davies, S. J. A. (2008). Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted precursors have opposite effects on recovery and allodynia after spinal cord injury. Journal of Biology, 7(7). https://doi.org/10.1186/jbiol85

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