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Despite advances in promoting axonal regeneration after acute spinal cord injury (SCI), elicitation of bridging axon regeneration after chronic SCI remains a formidable challenge. We report that combinatorial therapies administered 6 weeks, and as long as 15 months, after SCI promote axonal regeneration into and beyond a mid-cervical lesion site. Provision of peripheral nerve conditioning lesions, grafts of marrow stromal cells and establishment of NT-3 gradients, supports bridging regeneration. Controls receiving partial components of the full combination fail to exhibit bridging. Notably, intraneuronal molecular mechanisms recruited by delayed therapies mirror those of acute injury, including activation of transcriptional activators and regeneration-associated genes. Collectively, these findings provide evidence that regeneration is achievable at unprecedented post-injury time points.
Injured axons fail to spontaneously regenerate in either acute or chronic stages of spinal cord injury (SCI), resulting in persistent functional loss. Knowledge gained over the last several years indicates that the failure of axonal regeneration after CNS injury is attributable at least in part to: 1) an absence of permissive substrates to support axonal attachment and extension through lesion sites (Bunge, 2001), 2) a lack of neurotrophic stimulation (Jones et al., 2001), 3) myelin-based (Filbin, 2003) and extracellular matrix inhibitors in the injured region (Fawcett, 2006), 4) partial deficiency in the intrinsic growth capacity of adult neurons (Neumann et al., 2002; Qiu et al., 2002), and 5) extensive secondary damage resulting from inflammatory mechanisms (Jones et al., 2005). Chronic SCI presents additional obstacles: 1) glial scars and inhibitory extracellular matrices become well-established around the lesion site (Busch and Silver, 2007), a mechanism that is thought to increase the refractory nature of the chronically injured spinal cord to regeneration; 2) chronically injured neurons downregulate regeneration-associated genes and become atrophic (Kwon et al., 2002); 3) injured axons exhibit varying degrees of retraction and retrograde degeneration (Pallini et al., 1988), and 4) progressive anterograde degeneration in white matter beyond lesion sites potentially generates a particularly refractory milieu for axonal extension (Houle and Tessler, 2003). Collectively, these mechanisms constitute formidable barriers to regeneration in the chronically injured state.
Recent studies indicate that experimental strategies targeting multiple mechanisms that limit adult CNS plasticity and regeneration may be capable of eliciting axonal regeneration into and beyond sites of CNS injury, when administered immediately after injury (Lu et al., 2004; Pearse et al., 2004; Houle et al., 2006). These mechanisms include trophic factor availability (Lu et al., 2004), the provision of permissive cellular or extracellular matrices within injury sites ( Lu et al., 2004; Pearse et al., 2004; Houle et al., 2006), and augmentation of the intrinsic growth state of the neuron by pre-conditioning lesions (pre-CL) or cAMP administration (Lu et al., 2004; Pearse et al., 2004). We hypothesized that combinatorial therapies administered at prolonged time points after SCI: 1) would exhibit persistent effects in eliciting the expression of regeneration-associated genes, 2) thereby enhance the intrinsic growth capacity of sensory neurons, and 3) when combined with delayed application of neurotrophic factors beyond the lesion site, would be sufficient to elicit bridging axonal regeneration. We now report successful bridging regeneration of adult CNS axons when treatment is initiated from 6 weeks to as long as 15 months after the original injury, generated by modification of both cell-extrinsic and cell-intrinsic growth mechanisms.
We initially examined whether combinatorial therapies would promote bridging sensory axonal regeneration when treatment was delayed 6 weeks after SCI. A total of 51 adult F344 rats underwent C3 spinal cord dorsal column wire knife lesions (Lu et al., 2004; Taylor et al., 2006). Six weeks later, combinatorial therapies were applied consisting of: 1) a peripheral CL (sciatic nerve crush bilaterally) to upregulate neuron-intrinsic regeneration-associated gene expression (Neumann and Woolf, 1999); 2) placement of a syngenic bone marrow stromal cell (MSC) graft mixed with 1µg NT-3 protein in the established C3 cystic lesion cavity, 1 week after CL (7 weeks after the initial SCI) (Lu et al., 2004);and 3) provision of a neurotrophic factor gradient by injection of lentivirus expressing neurotrophin-3 (NT-3) into dorsal column white matter (Taylor et al., 2006), 2.5mm rostral to the lesion site, 1 week after CL (7 weeks after the initial SCI, n=16 animals; Fig. 1A). In addition, 4 control groups were examined: a) 9 subjects received C3 lesions, CL 6 weeks later, and a cell graft in the lesion site an additional week later; neither NT-3 protein nor vector were administered; b) 9 subjects received C3 lesions, a CL 6 weeks later, then a cell graft mixed with 1µg NT-3 protein in the lesion site and lentivirus expressing the reporter gene Green Fluorescent Protein (GFP) into the dorsal white matter 2.5mm beyond the lesion an additional week later; NT-3 beyond the lesion was not provided; c) 8 subjects underwent C3 lesions, no CL, and delivery of 1µg NT-3 protein into a cell graft in the lesion site plus Lenti-NT-3 vector 7 weeks after the initial injury; and d) 9 subjects underwent C3 lesions and a cell graft 7 weeks after the initial injury. Of note, chronic scars surrounding the cystic lesion cavity were not resected in any case, to avoid damage to the surrounding spinal cord (Tuszynski et al., 2003); thus, bridging axonal regeneration in this paradigm would require growth into and beyond the peri-lesion scar. An additional 6 weeks later, or 13 weeks after the original C3 lesion, dorsal column sensory axons were traced transganglionically by injections of cholera toxin B (CTB) subunit into the sciatic nerve, and animals were sacrificed 3 days later.
Notably, axonal bridging beyond the lesion occurred only in animals that received combinatorial treatment with CLs, MSC grafts in the lesion cavity, and lenti-NT-3 injections (Fig. 1, ,2).2). 10-of-16 animals in the full treatment group exhibited axonal bridging beyond the lesion site; in the 6 remaining animals, lenti-NT-3 failed to diffuse from the rostral injection site to the host/graft interface (Suppl. Fig. 1), and no axonal bridging was observed in these subjects. In subjects with complete vector diffusion from the injection site to the graft in the lesion cavity, axons extended in several instances the full 2.5mm distance beyond the lesion to the rostral site of lenti-NT-3 injection. Further, growth occurred through host white matter undergoing Wallerian degeneration. The morphology of extending axons was not typical of intact axons: circuitous trajectories and frequent swellings of axons were evident (Fig. 1, ,2).2). Lesions were complete, confirmed by absence of CTB label in the nucleus gracilis of all subjects (Suppl. Fig. 1). Thus, regeneration of axons into and beyond the lesion site in combination-treated animals was not an artifact of axon sparing.
Axon quantification beyond the lesion revealed significantly greater regeneration in combination-treated subjects compared to all other groups (χ2 p<0.03; Dunn posthoc with Bonferroni correction, p<0.005; Fig. 2I). Similarly, the maximum distance of axonal regeneration was significantly greater in animals receiving combination treatment (χ2 p<0.01; Dunn posthoc with Bonferroni correction, p<0.005;Fig. 2J). Among controls, axon growth beyond the lesion was detectable only among subjects that received MSC grafts plus CLs, and only for very short distances of 100µm or less (Fig. 2I). Unlike findings in models of acute SCI (Taylor et al., 2006), treatment with MSC grafts plus lenti-NT-3, in the absence of a CL, failed to elicit detectable axon extension for even short distances beyond the lesion (Fig. 2I). On the other hand, CLs with or without NT3 protein in the graft significantly increased penetration of sensory axons into MSC grafts in the lesion cavity, relative to non-CL controls (ANOVA p<0.0001; post-hoc Fisher’s p<0.05; Fig. 2K).
Next, we determined whether the same combinatorial therapies would promote bridging axonal regeneration when administered 15 months after SCI. 27 adult Fischer 344 rats underwent C3 dorsal column lesions as described above. 15 months later, subjects underwent bilateral CLs of the sciatic nerve. One week later, the chronic lesion site was re-exposed, and syngenic MSCs were grafted into the chronic lesion cavity and lenti--NT-3 was injected 2.5mm rostral to the lesion site (n=11 animals). In addition to this “full treatment” group, the following controls were examined. a) 4 subjects received MSC grafts, no CL, and injection of lenti-GFP 2.5mm rostral to the lesion (i.e., no NT-3 gradient); b) 6 subjects received MSC grafts, no CL, and injection of lenti-NT-3 2.5mm rostral to the lesion; and c) 6 subjects received MSC grafts, CLs, and lenti-GFP 2.5mm rostral to the lesion.
Of note, regeneration beyond the lesion was observed in full combination treatment animals when treatment was initiated 15 months after the original lesion (Fig. 3). 5-of-11 animals in the full treatment group exhibited axonal bridging beyond the lesion site; in 2 out of the 6 remaining animals, lenti-NT-3 vector failed to diffuse from the rostral injection site to the host/graft interface (Suppl. Fig. 1); in 1 out of the 6 remaining animals, the graft did not survive. CTB immunolabeling demonstrated dorsal column sensory axons first penetrating, and then emerging from, the graft/host interface and extending into rostral host white matter. Axons exhibited varicose morphology with frequent swellings, and circuitous trajectories, unlike spared axons (Fig. 3, ,4).4). Further indicating that these were not spared axons, medullary sectioning confirmed absence of CTB labeling (Suppl. Fig. 1). Triple labeling for CTB, GFP, and GFAP demonstrated that regenerating axons passed through the GFAP immunoreactive border within the lesion site, and beyond into host white matter. Notably, axons regenerating in host white matter were invariably associated with regions of lenti-NT-3 expression, indicated by co-association of axons with the reporter gene GFP (Fig. 4A–F). As noted above, the peri-lesion scar was not resected following the original lesion to avoid further damage to the spinal cord. Control groups lacking the combination treatment did not exhibit axonal bridging (Fig. 4G–I).
Quantification of axon number beyond the lesion revealed significantly greater regeneration in combination-treated subjects compared to all other groups (χ2 p<0.003; Dunn posthoc with Bonferroni correction, p<0.008; Fig. 4J). Similarly, the maximum distance of axonal regeneration was significantly greater in animals receiving combinatorial treatment (χ2 p<0.003; post-hoc p<0.008; Fig. 4K). Groups treated with CLs alone, or lenti-NT-3 injections alone, exhibited few sensory axons regenerating into grafts and no regeneration beyond the lesion (Fig. 4J–L). Thus, axons emerged from the graft and regenerated beyond only when treatments modified both the lesioned environment and the intrinsic state of neuronal activation.
To investigate intrinsic neuronal mechanisms related to enhanced axonal regeneration at prolonged times after SCI, we examined the expression of neuronal growth programs. This is particularly important as a previous study reported that CLs must be applied before SCI to promote regeneration (Neumann et al., 1999). We performed both in vitro assays and gene array analyses. For in vitro studies, adult rats underwent C3 dorsal column lesions, and 6 weeks later sciatic nerves were crushed. One week later, sensory neurons of lumbar dorsal root ganglia (DRG) L4-6 were cultured on myelin substrates for 48 hr. Neurite outgrowth was compared to sensory neurons obtained from: 1) intact subjects, 2) animals that underwent C3 lesions 6 weeks earlier, without subsequent CLs, and 3) animals that underwent CLs only, 1 week prior to explant. As previously described, sensory neurons from animals that underwent CL only (no central lesion) exhibited longer neurites compared to neurons from intact rats (p<0.0001 ANOVA; p<0.0001 post-hoc Fisher’s; Fig. 5E) (Smith and Skene, 1997). Sensory neurons from animals that underwent CLs 6 weeks after a C3 lesion also exhibited enhanced neurite outgrowth compared to neurons from intact rats (p<0.0001 post-hoc Fisher’s; Fig. 5E) that was equal in degree to animals that underwent CLs alone (Fig. 5E). In contrast, central lesions alone, without a CL, did not enhance outgrowth (p=0.54 post-hoc Fisher’s). Thus, growth of injured sensory neurons can be enhanced by CLs applied even 6 weeks after central injury.
Next, we examined expression of the regeneration-associated genes GAP43 and c-Jun in DRG neurons (Raivich et al., 2004). A 4-fold increase in the number of neurons labeled for GAP43 and c-Jun protein was observed when CLs were applied 6 weeks after C3 injury compared to intact DRGs (p<0.01 ANOVA; p<0.01 post-hoc Fisher’s), equal to increases observed when CLs were applied without a central lesion (“pre-CL”) (Fig. 5G–P). C3 lesions alone, without CL, did not increase GAP-43 or c-Jun. cAMP levels in DRGs, measured 1 week after placement of CLs, were also elevated equally when CLs were placed 6 weeks after a central lesion or as “pre-CLs” (Fig. 5F).
Next, we performed gene array analysis using Affymetrix whole-genome chips in 4 groups of adult rats: Group 1 DRGs were harvested 1week after CL (n=9 animals, pooling mRNA from 3 subjects, providing 3 arrays per group); Group 2 DRGs were harvested 7 weeks after C3 spinal cord lesion and 1 week after CL (i.e., CLs were placed 6 weeks after SCI; n=9 animals, 3 arrays); Group 3 DRGs were harvested 1 week after C3 lesion (no CL; n=9, 3 arrays); and Group 4 DRGs were harvested 7 weeks after C3 lesion (no CL; n=9, degradation of pooled mRNA in one group resulted in 2 arrays from 6 animals in this group). mRNA was also obtained from DRGs of 12 intact animals (4 arrays). Using a significance criterion of 5% false discovery rate (FDR) and a log ratio greater than 0.2 (15%), we found 5,609 differentially expressed probes in subjects that underwent CLs (Group 1) compared to intact animals: of these, 2,936 probes were upregulated and 2,673 were downregulated (Fig. 5Q). In marked contrast, no probes changed at this level of significance in samples from animals with C3 lesions only (no CL, Groups 3 and 4). Thus, peripheral but not central lesions led to extensive and long-lasting changes in gene expression. Notably, CLs performed 6 weeks after C3 lesions also resulted in extensive changes in gene expression (6,092 differentially expressed probes), and these changes overlapped to an exceptionally high degree with animals that underwent pre-CLs. Indeed, 4,244 probes, corresponding to 76% of all probes differentially expressed in animals that received pre-CLs, were also differentially expressed in animals that underwent “post-CLs”. Probes for GAP-43 and c-Jun were elevated in subjects with pre- and post-CLs (+85−92% and +227−201% elevations, respectively, relative to controls), providing independent confirmation of probe data. Functional annotation identified, among others, modulation of transcriptional networks centered around GAP-43 and c-Jun in both groups with CLs, whereas a C3 lesion had little effect on these pathways (Suppl. Fig. 2).
These findings indicate that bridging axonal regeneration in the adult CNS can be induced when experimental treatments are administered at unprecedented delays of more than a year after SCI. Regeneration after established injury requires modification of both the intrinsic growth state of the neuron, achieved by CLs, and modification of the non-permissive injury environment, accomplished by cell grafting and placement of growth factor gradients beyond the lesion site. Manipulation of the intrinsic neuronal growth state alone, or the environment alone, are insufficient to support axonal bridging beyond the lesion. Delayed conditioning of the injured neuron elicits modulation of broads set of genes remarkably similar in profile to CLs placed before central injury, suggesting extensive recruitment of intrinsic molecular mechanisms that contribute to axonal regeneration. Thus, neuron-intrinsic mechanisms and the injured environment can both be modified at extended delays after injury to successfully elicit axonal bridging into, and beyond, sites of SCI.
Few studies have examined axonal regeneration when therapies are administered at delays after injury, and most of these have examined relatively brief delays of 1–8 weeks (Houle and Tessler, 2003). Further, most delay treatment paradigms resect the “chronic scar” (Coumans et al., 2001; Jin et al., 2000), risking spinal cord re-injury (Tuszynski et al., 2003). Of these studies, only one to our knowledge has reported axonal growth beyond the lesion, using a combination of a fetal graft and growth factors applied to a re-lesioned spinal cord 4 weeks after the original injury (Coumans et al., 2001). The latter study reported regeneration through both gray matter and inhibitory white matter below the lesion resulting purely from cell-extrinsic experimental manipulations within the lesion site; however, examples of specific axons leaving the graft site were not provided. In contrast, axonal bridging in the present study is observed when experimental therapies are provided at exceptionally prolonged delays after the original injury, and only when both the intrinsic neuronal growth state and the surrounding, inhibitory environment are modified, consistent with findings in acute injury models (Lu et al., 2004). Re-lesioning the spinal cord was not necessary to achieve axonal growth either into or beyond the chronic lesion site in this study, enhancing clinical relevance: re-lesioning the spinal cord risks further deterioration of function, particularly in cervically injured subjects that are critically dependent of neural systems spared immediately above the lesion site to support residual function.
We examined a long-projecting axonal system, the dorsal column sensory tract, as a model system because the projection is well defined anatomically, it normally fails to regenerate after central injury, and lesion completeness is readily verifiable by examination of the gracilar target of this projection (Lu et al., 2004; Taylor et al., 2006). Five observations suggest that axons observed beyond the lesion actually regenerated, and were not simply spared. First, the morphology of axons extending beyond the lesion site was circuitous and their course was nonlinear, unlike intact axons (Fig. 1, ,3).3). Second, CTB-labeled axons emerged across the lesion-host interface at different dorsoventral levels of the graft, not simply the most ventral or dorsal aspects of the graft/lesion site where spared axons might be located (Fig. 1–Fig 4). Third, CTB-labeled axons were not observed ventral to the lesion site or in lateral, unlesioned portions of the spinal cord. Fourth, the lesion site was devoid of GFAP immunoreactivity, which would otherwise be present in strands of spared spinal cord parenchyma (Fig. 4). Fifth, sectioning of the medulla through the entire extent of the nucleus gracilis revealed an absence of CTB-labeled axons in all lesioned subjects, indicating that lesions were complete, whereas dense labeling was readily detected in unlesioned subjects (Suppl. Fig. 1). The latter observation also indicates that axons did not regenerate over extended distances to re-enter the denervated nucleus.
Having established that bridging axonal regeneration is feasible in the sensory model at remarkably extended delays after injury, findings can be adapted to descending motor projections of the spinal cord. While pools of axons recruitable for regeneration persist at prolonged time points post-injury, the number of sensory axons regenerating beyond the lesion site is lower when therapy is applied 15-months post-injury compared to 6 weeks post-injury, and the distance that axons regenerate beyond the lesion is also reduced after prolonged treatment times (Fig. 2, ,4).4). For these reasons, attempts to achieve chronic functional recovery should likely target cervical level lesions, because potential neuronal targets of regenerating axons can be contacted immediately below the lesion site. If reinnervation of denervated motor neuron pools even one level below a lesion were achieved, the impact on quality of life in spinal cord-injured subjects could be substantial, potentially providing movement of a wrist or hand that could support greater independence. Incremental improvements in function are a more tractable goal for human translation, and may be achievable with rational, safe therapies that target neuron-intrinsic and environmental mechanisms.
Female Fisher 344 rats (n=182) weighing 160–200g were experimental subjects. C3 dorsal columns were completely transected bilaterally using a tungsten wire knife (Lu et al., 2004). Peripheral CLs were made by firm compression of the exposed nerve at mid-thigh level using jeweler’s forceps for 15sec._ MSCs were isolated as described previously (Azizi et al., 1998). The chronic lesion site was re-exposed and 2µl (75,000 cells/µl) of passage 4 MSCs, mixed with fibrin glue, were grafted into the lesion cavity. Lenti-NT-3 vector and Lenti-GFP vector were generated as previously described (Taylor et al., 2006) and 2.5µl were injected 2.5mm rostral to the lesion site in dorsal column white matter (titer 100 µg/ml p24, ~1×108 Infectious Units/ml). Dorsal-column sensory axons were labeled transganglionically by CTB injection into the sciatic nerve proximal to the CL site (2µl 1% solution) 3 days before perfusion. Animals were perfused with 4%PFA and subjected to CTB, GFP and GFAP immunocytochemistry on 30µm-thick spinal cord sections.
Axon number was quantified in 1-in-6 sections within and beyond the lesion site. Lesion margins were determined using GFAP immunoreactivity (Taylor et al., 2006), and axons crossing a vertical line placed in sagittal sections within the graft midpoint, and 0, 50, 100, 250, 500, 1000, 2000 and 2500 µm rostral to the rostral lesion border, were counted. Total axon number/subject was calculated by multiplying the counted number by 6. The longest distance of regenerating axons from the rostral lesion border was also measured. Observers were blinded to group identity.
Dissected adult L4-6 DRGs were digested for 1h in 0.25% collagenase type XI, triturated, and resuspended on 35mm cell culture dishes coated with myelin (18 µg/ml/ per well). Cells were fixed 48h later with 4%PFA and labeled for NF200. Longest neurite length/cell was measured from a minimum of 150 cells/animal (n=4 DRGs/group). 35μm sections of L4-6 adult DRG were immunolabeled for GAP43 and c-Jun and counterstained with Hoechst 33342. %GAP43- or c-Jun-labeled neurons was quantified as described, n=3 DRGs/group (Qiu et al., 2005).
L4-6 DRGs were dissected, homogenized and subjected to cAMP ELISA (Assay designs), n=7 DRGs/group.
L4-6 DRGs were dissected, frozen at −80°C, and RNA extracted. RNA samples were reverse transcribed and labeled per manufacturer’s instructions, and hybridized to Affymetrix high-density oligonucleotide GeneChip Rat Genome 230 2.0 Arrays (Affymetrix).
All experiments and analyses were conducted under blinded conditions. Quantification of axon number beyond lesion sites and axonal length was assessed by Kruskall Wallis followed by Dunn post-hoc and Bonferroni adjustment. In all other quantifications, multiple group comparisons were made by ANOVA, with post-hoc Fisher’s. A significance criterion of p<0.05 was used. Data presented as mean ± SEM.
We thank Maya Culbertson, Lori Graham and Fuying Gao for experimental assistance. Funded by NIH (R01 NS09881and NS054883), the Veterans Administration, International Spinal Research Trust, the Bernard and Anne Spitzer Charitable Trust and the Dr. Miriam and Sheldon G Adelson Medical Research Foundation.
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