Required growth facilitators propel axon regeneration across complete spinal cord injury

Abstract

Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury1,2,3, but efficient reversal of this regrowth failure remains elusive4. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults—(i) neuron intrinsic growth capacity2,5,6,7,8,9, (ii) growth-supportive substrate10,11 and (iii) chemoattraction12,13—are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor16,17 delivered via spatially and temporally controlled release from biomaterial depots18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.

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Fig. 1: Stimulated and chemoattracted propriospinal axons regrow robustly across anatomically complete SCI lesions in mice receiving combined delivery of AAV-OIC plus FGF, EGF and GDNF in two sequentially placed hydrogel depots.
Fig. 2: Stimulated, supported and chemoattracted mouse propriospinal axons regrow through the proximal borders of astrocyte scars and lesion core stromal cells along laminin that has been upregulated by delivered growth factors, in spite of CSPG presence.
Fig. 3: Stimulated and chemoattracted mouse propriospinal axons regrow past astrocyte scar distal borders into grey matter and form synapse-like contacts with neurons.
Fig. 4: Stimulated and chemoattracted propriospinal axons regrow robustly and conduct electrophysiological signals across anatomically complete SCI lesions in rats after combined delivery of AAV-OIC plus FGF, EGF and GDNF in two sequentially placed hydrogel depots.

Data availability

Files of Source Data of individual values for all quantitative figures are provided with the paper. Raw images of dot blots are provided as Supplementary Fig. 1. RNA-seq data are available at the NCBI Gene Expression Omnibus under accession number GSE111529. Other data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by US National Institutes of Health (NS084030 to M.V.S., F32NS096858 to J.E.B., NS096294 to Z.H., and NS062691 to G.Cop.); Dr. Miriam and Sheldon G. Adelson Medical Foundation (M.V.S., Z.H., T.J.D. and G.Cop.); International Foundation for Research in Paraplegia (146 to M.A.A. and G.Cou.); ALARME Foundation (531066 to M.A.A. and G.Cou.); Association Song Taaba (M.A.A.); Craig H. Neilsen Foundation (381357 to T.M.O. and M.V.S); Consolidator Grant from the European Research Council [ERC-2015-CoG HOW2WALKAGAIN 682999] (G.Cou.); Paralyzed Veterans Foundation of America (3080 to J.E.B. and M.V.S.); Swiss National Science Foundation (323530-164220 to S.L.B and G.Cou.); Microscopy Core Resource of UCLA Broad Stem Cell Research Center; Microscopy Core Resource of the Wyss Center for Bio and Neuroengineering; and Wings for Life (M.V.S., J.E.B. and Z.H.).

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Nature thanks J. Fawcett, P. Letourneau and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Contributions

M.A.A., T.M.O., J.E.B., T.J.D., Z.H., G.Cou. and M.V.S. designed experiments; M.A.A., T.M.O., J.E.B., Y.A., S.L.B., A.M.B., N.D.J., A.R., A.L.W. and C.W. conducted experiments; M.A.A., T.M.O., Y.A., J.E.B., N.D.J., J.H.K., B.K., R.K., G.Cop. and M.V.S. analysed data. M.A.A., T.M.O., J.E.B., T.J.D., G.Cou. and M.V.S. prepared the manuscript.

Corresponding authors

Correspondence to Gregoire Courtine or Michael V. Sofroniew.

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Extended data figures and tables

Extended Data Fig. 1 Experimental models and timelines.

Mice or rats received different combinations of procedures including adeno-associated virus (AAV) injections, complete crush SCI, injections of one or two depots of hydrogel containing different molecular cargoes and injections of biotinylated dextran amine (BDA) for axonal tract-tracing. AAV injections were made two weeks before SCI to allow time for molecular expression and were targeted at propriospinal neurons (PrSp) between one and two segments rostral to planned locations of SCI lesions. AAVs were used to deliver either potential axon-growth reactivating molecules, GFP to identify targeted neurons or RFP as an axonal tract-tracer. Complete crush SCI lesions were placed at the level of spinal segment T10. Two days after SCI, all animals were behaviourally evaluated for completeness of SCI and only animals with functionally complete SCI were included in subsequent experimental steps. Additional animals with complete SCI were evaluated without hydrogel injections (SCI only). a, Schematic and timeline of one-depot experiments. Two days after complete crush SCI, animals received hydrogel injections targeted to the centre of the non-neural lesion core. These depots (D1) contained different molecular cargos as listed in the schematic. Depots without cargo were referred to as ‘empty’. b, Schematic and timeline of two-depot experiments. Two days after complete crush SCI, animals received a D1 hydrogel injection into the centre of the non-neural lesion core to deliver the growth factors FGF + EGF + GDNF. Nine days after SCI, the animals received a second hydrogel injection (D2) targeted to spared neural tissue 1 to 2 mm caudal to the lesion centre to deliver GDNF to sequentially chemoattract propriospinal axons that had regrown into the lesion core. BDA injections for axonal tract-tracing were targeted at propriospinal neurons between one and two segments rostral to SCI lesions and were placed at the time of injecting either D1 (a) or D2 (b). Tissue was collected for evaluation at either two or four weeks after SCI. Electrophysiological evaluations were conducted at four weeks after SCI. For abbreviations, see Extended Data Table 1.

Extended Data Fig. 2 AAV targeting, axon tracing and axon quantification.

a, AAV targeting of green fluorescent protein (GFP) to propriospinal neurons. Multi-fluorescent, survey (left) and detail (right, boxed area) confocal images of horizontal section through mouse grey (gm) and white (wm) matter. Essentially all NeuN+ propriospinal neurons targeted with AAV express GFP. b, Multi-fluorescent, orthogonal 3D confocal images show that AAV-targeted propriospinal neurons express GDNFR. c, Multi-fluorescent, survey images show tract-tracing of propriospinal axons using biotinylated dextran amine (BDA) in tiled confocal scans of horizontal section from uninjured mouse. Hatched area indicates densely labelled location of BDA injections. d, e, Multiple channel fluorescent images compare BDA-labelled propriospinal axons and immunohistochemically stained serotonin (5HT) axons in mice after SCI + AAV-OIC + 1D + FGF + EGF + GDNF. d, Survey and orthogonal 3D confocal detail from an area proximal to the SCI lesion shows a complete lack of overlap of BDA-labelling and 5HT immunohistochemistry, indicating that BDA-tracing did not label 5HT axons of passage. e, Survey images of the same field examined with different filters show BDA-labelled propriospinal axons (bottom image) regrowing robustly past the astrocyte scar proximal border and through the non-neural lesion core; by contrast, 5HT axons (top and bottom image) did not regrow into or through the lesion core. f, Open field hindlimb locomotor score at various times after SCI assessed using a 6-point scale in which 5 is normal walking and 0 is no movement of any kind. Data are mean ± s.e.m., n = 6 mice per group. Source data

Extended Data Fig. 3 Procedures for quantification of BDA-labelled propriospinal axons after SCI.

a, Schematics show demarcation of SCI lesion centre (Cn) and evenly spaced lines beyond the lesion centre placed by image analysis software (Neurolucida, MicroBrightField) for quantification of axon intercepts in horizontal tissue sections of mice with SCI and one (D1) or two (D1 + D2) hydrogel depots. b, Multi-fluorescent, survey images show BDA-labelled axons and GFAP-labelled astrocytes that demarcate astrocyte scar proximal borders and distal borders around the non-neural lesion core after SCI. The hydrogel of the empty depot (left) was tagged with a blue fluorescent label for visualization. Note the essential absence of axons passing the astrocyte scar (AS) proximal border to reach the lesion centre (Cn) or beyond in the mouse with SCI plus empty depot (left), in contrast to the large number of axons that regrew through the lesion core and passed beyond the distal astrocyte scar border into spared grey matter in the mouse with full treatment of stimulatory AAV plus growth factors (right). GFAP staining shows that the SCI lesions are anatomically complete across the entire width of the spinal cord in both cases. Note that the second depot was placed at nine days after SCI, by which time the distal astrocyte scar border was essentially formed35. Note also that astrocytes do not migrate into the depots, potentially giving the mistaken impression of cavity formation when looking only at the GFAP channel alone. Nevertheless, examination of other fluorescence channels shows that depot sites clearly contain DAPI-stained stromal cells and BDA-positive axons. c, Large area survey images of BDA-labelled axons in composite mosaic scans of horizontal sections. In a control mouse (top) that received SCI plus empty depot, few axons reach the lesion centre, almost none pass beyond and no axons are present at 3 mm. In the treated mouse (middle) that received stimulatory AAV plus growth factors, many axons regrow through the lesion core and reach or pass 1.5 mm beyond the lesion centre, which is the equivalent length of a full thoracic spinal segment in mice46. Note also that there are no axons present at 3 mm, demonstrating that the SCI lesion was complete and that axons that are found past the lesion centre represent axon regrowth after SCI in response to the experimental manipulations. In an uninjured mouse (bottom), there are many labelled axons at the distance equivalent to 3 mm beyond the location of SCI in injured mice. d, Numbers of axon intercepts at lesion centres for all experimental groups. Data are mean ± s.e.m., dots in graphs show numbers and distribution of individual mice per group. NS, not significant versus SCI only; #P < 0.01, versus SCI only and not significant versus each other; **P < 0.01; ***P < 0.001, versus all other groups; one-way ANOVA with Bonferroni, F(12, 57) = 22.3. Source data

Extended Data Fig. 4 BDA tract-tracing of propriospinal axons after SCI and different treatment conditions.

Survey images show tiled mosaic scans of horizontal sections from representative mice of all experimental conditions. Experimental treatment conditions are listed in the upper left of each scan. Mouse identification numbers are given in the upper right. Scans are oriented with their lesion centres aligned along the dashed lines so that axon growth to or past this point can be easily compared. Axon regrowth was quantified by counting axon intercepts with lines drawn through lesion centres and at regular intervals beyond by using image analysis software.

Extended Data Fig. 5 Stimulated, supported and chemoattracted mouse propriospinal axons regrow through the lesion core in contact with various substrate molecules, including putatively inhibitory CSPGs.

a, Left and left middle, multi-fluorescent, detail images show BDA-labelled axons regrowing along and among surfaces decorated with fibronectin or collagen. Right middle, orthogonal 3D confocal images of the outlined areas show direct contact between BDA-labelled axons and fibronectin or collagen. Right, quantification of fibronectin or collagen levels and dot blots. Data are mean ± s.e.m. of density, n = 4 mice per group. *P < 0.01, one-way ANOVA with Bonferroni, F(3, 12) = 13.0 for fibronectin dot blot and F(3, 12) = 10.2 for collagen dot blot). b, Left and middle, multi-fluorescent, detail images show BDA-labelled axons regrowing along and among surfaces decorated with brevican (BCAN). Right, orthogonal 3D confocal image of the outlined area shows direct contact between BDA-labelled axons and BCAN. c, Multi-fluorescent, orthogonal 3D confocal images show BDA-labelled axons regrowing along and in direct contact with surfaces decorated with both CSPG4 and laminin (arrows). Source data

Extended Data Fig. 6 BDA tract-tracing of propriospinal axon regrowth after SCI along and among different cell types.

a, Multiple channel fluorescent images show the same BDA-labelled axon transitioning from contact with GFAP-positive astrocytes in proximal scar border to contact with CD13+ stromal cells in the lesion core. Numbers and arrows indicate the same locations in images of different combinations of fluorescent markers: 1, axons in contact with astrocyte processes; 2, axons in contact with both astrocyte process and stromal cell; 3, axons in contact with stromal cell. b, Multiple channel fluorescent images show axons regrowing along, and following the trajectory of, stromal cells (S) while circumventing clusters of inflammatory cells (Inf) in the lesion core. c, Multiple channel fluorescent images show axons (A) regrowing along the trajectory of blood vessels (bv) in contact with stromal cells that are present on endothelia (E) positive for platelet endothelial cell adhesion molecule (PECAM). d, Multiple channel fluorescent images show BDA-labelled propriospinal axons and cells expressing the combinatorial Schwann cell markers, p75 and SOX10, in the lesion core. Some stromal cells expressing only SOX10 but not p75 are also visible. Numbers and arrows indicate the same locations in images of different combinations of fluorescent markers: 1, axons in partial contact with cells expressing Schwann cell markers; 2, axons not in detectable contact with Schwann cells. Note that some axons are partially in contact with, and partially not in contact with, Schwann cells in lesion core.

Extended Data Fig. 7 Comparison of genomic data from astrocytes and non-astrocyte cells from mice with or without FGF + EGF after SCI.

a, Heat maps showing significantly differentially expressed genes (DEGs) derived by RNA-seq of mRNAs from spinal cord tissue of mice treated with SCI + 1D + FGF + EGF (D + GF), and the expression of these genes in mice treated with SCI + 1D empty (D-Em), at two weeks after SCI. Data are shown for mRNAs derived selectively from astrocytes or from all other cell types (non-astrocytes), isolated as previously described19. Red, upregulated; green, downregulated; relative to SCI only. n = 3 mice per group; FDR < 0.1 for differential expression. b, Total numbers of significant DEGs in astrocytes and non-astrocytes from mice as shown in the heat map in a. Red and green numerical values indicate significantly upregulated and downregulated genes, respectively. Relative to SCI only, over 900 astrocyte genes and over 300 non-astrocyte genes were significantly up- or downregulated in mice after 12 days of growth factor treatment, which were not significantly altered by treatment with empty depots. c, Top five networks of genes significantly altered by SCI + 1D + FGF + EGF that were not altered by SCI + 1D empty after SCI relative to SCI only, as identified by unbiased analysis (Ingenuity). Full RNA-seq data are available at NCBI Gene Expression Omnibus under accession GSE111529.

Extended Data Fig. 8 RFP tract-tracing of propriospinal axons after SCI and different treatment conditions in rats.

a, b, Large area survey images of RFP-labelled axons in composite mosaic scans of horizontal sections. Tracer-injection sites are denoted by RFP-PrSp. a, Multiple channel fluorescent images showing BDA-labelled axons and GFAP-labelled astrocytes that demarcate astrocyte scar proximal borders and distal borders around the non-neural lesion core after SCI. GFAP-staining shows that SCI lesions were anatomically complete across the entire width of the spinal cord, with large lesion cores in a control rat (left), and in a rat treated with stimulatory AAV plus growth factors (right). In the control rat, few axons reach the lesion centre or beyond. In the treated rat, many axons regrow through the lesion core and reach or pass 3 mm beyond the lesion centre, which is the equivalent length of a full thoracic spinal segment in rats47. b, Completeness of SCI lesions was confirmed in all rats used in qualitative and quantitative evaluations by confirming that no axons were present at 5 mm or more past lesion centres, as shown here for control rats (top) and treated rats (middle), whereas in uninjured rats, abundant labelled axons are present at an equivalent distance past the RFP-injection site.

Extended Data Fig. 9 Growth factor induction of laminin, comparison of propriospinal and serotonin axons, and locomotor evaluations of rats after SCI without and with treatments.

a, b, Survey images show laminin 1 IHC in tiled mosaic scans of horizontal sections from representative rats. b, Top, mean ± s.e.m. quantification of laminin IHC in rats as per cent area per linear µm3, (n = 4 rats per group, dark coloured lines = means, lighter coloured shaded areas = s.e.m., colours indicate experimental groups as shown in graph below). Bottom, total laminin in rats summarized as mean ± s.e.m. area under the curve as calculated from graph above. (ns non-significant, **P < 0.005, one-way ANOVA/Bonferroni, F (2, 9) = 15.04). c, Multiple channel fluorescent images show RFP-labelled propriospinal axons and immunohistochemically stained serotonin (5HT) axons in rats after SCI + AAV-OIC + 2D + FGF + EGF + GDNF. The two survey images on the left show the same field with different filters. Note in the survey images on the left, and in the higher magnification image in centre, that RFP-labelled propriospinal axons regrow robustly past the astrocyte scar proximal border and through the non-neural lesion core. By contrast, 5HT axons did not regrow into or through the lesion core. The image on the right shows an orthogonal 3D confocal detail from an area proximal to the SCI lesion, demonstrating a complete lack of overlap of RFP labelling and 5HT immunohistochemistry, indicating that RFP tracing did not label 5HT axons of passage. d, Open field hindlimb locomotor score at various times after SCI in rats assessed using a 6-point scale in which 5 is normal walking and 0 is no movement of any kind. Data are mean ± s.e.m., n = 6 per rats group. Source data

Extended Data Table 1 Abbreviations used in the text and figures

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Anderson, M.A., O’Shea, T.M., Burda, J.E. et al. Required growth facilitators propel axon regeneration across complete spinal cord injury. Nature 561, 396–400 (2018). https://doi.org/10.1038/s41586-018-0467-6

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Keywords

  • Propriospinal Axons
  • Propriospinal Neurons
  • Lesion Core
  • Scar Border
  • High Pressure Tube

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