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. 2016 Apr 14;532(7598):195-200.
doi: 10.1038/nature17623. Epub 2016 Mar 30.

Astrocyte scar formation aids central nervous system axon regeneration

Mark A Anderson  1 , Joshua E Burda  1 , Yilong Ren  1 , Yan Ao  1 , Timothy M O'Shea  1 , Riki Kawaguchi  2 , Giovanni Coppola  2 , Baljit S Khakh  3 , Timothy J Deming  4 , Michael V Sofroniew  1
Affiliations

Astrocyte scar formation aids central nervous system axon regeneration

Mark A Anderson et al. Nature. .

Abstract

Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended data figure 1
Extended data figure 1. SCI model schematic, locomotor behavioral effects, and AAV vector targeting specificity and effects
(a) Schematic of severe lateral crush SCI at thoracic level T10 that generates a large lesion core (LC) of non–neural tissue surrounded by an astrocyte scar (AS) and completely transects descending and ascending axons. (b) Open field hindlimb locomotor score at various times after SCI assessed using a 5 point scale where 5 is normal and 0 is no movement of any kind. No significant differences were observed among any of the experimental groups at any time point. n = 6 ns at all time points p>0.5 (ANOVA with Newman-Keuls post hoc analysis). (c) Horizontal sections through a severe SCI lesion of a representative tdT reporter mouse injected with an AAV vector with a minimal Gfap promoter regulating Cre (AAV2/5-GfaABC1D-Cre) into the lesion at two weeks after SCI and perfused at three weeks. tdT labeling demonstrates that this AAV2/5-GfaABC1D-Cre efficiently and specifically targets GFAP-positive astrocytes. In this mouse, the amount AAV2/5-GfaABC1D-Cre injected was intentionally titrated on the basis of previous trial and error to target primarily the astrocyte scar border in an approximately 500μm zone immediately abutting the SCI lesion core (LC). High magnification analysis of individual fluorescence channels stained for tdT plus various cell markers shows the specificity of Cre-activity targeting to cells expressing the astrocyte marker, GFAP, but not to cells expressing either the neuronal marker, NeuN, or the mature oligodendrocyte marker, GSTπ. AAV2/5-GfaABC1D-Cre was prepared using a previously described and well-characterized cloning strategy. (d) Open field hindlimb locomotor scores at various times after SCI. There was no difference in scores of control mice and loxP-DTR mice that received AAV2/5-GfaABC1D-Cre prior to injections of DTX. Five weeks after DTX injections, loxP-DTR mice that received AAV2/5-GfaABC1D-Cre exhibited a slightly, but significantly, lower locomotor score. Hindlimb locomotion was assessed using a 5 point scale where 5 is normal and 0 is no movement of any kind. n = 6 per group, * p<0.05 versus WT (ANOVA with Newman-Keuls). (e) GFAP immunohistochemistry of a sagittal section after ablation of a chronic astrocyte scar plus adjacent astrocytes. DTX was administered to a transgenic mouse expressing DTR targeted selectively to astrocytes around a severe SCI. In this case, the amount of AAV2/5-GfaABC1D-Cre injected was titrated to target not only primarily the astrocyte scar border but also adjacent astrocytes spread over approximately two mm on either side of the center (Cn) of the SCI lesion core (LC). Note the profound degeneration of neural tissue resulting from the selective ablation of the chronic astrocyte scar plus adjacent astrocytes after SCI.
Extended data figure 2
Extended data figure 2. Single channel CSPG and GFAP immunofluorescence and stained area quantification
(a) Individual fluorescence channels of CS56 and GFAP immunohistochemistry from horizontal sections of uninjured mice and at two weeks after severe SCI shown in main text figure 3b. Sections are taken from WT mice and mice with transgenic ablation (TK+GCV) or attenuation (STAT3-CKO) of astrocyte scar formation. (b) Example of black and white thresholding of single channels of immunofluorescence staining for image analysis to quantify (using NIH Image J software) the amount of CSPG or GFAP stained area in different tissue compartments in SCI lesions. Boxes denote areas quantified to obtain values for lesion core (LC) and grey (g) or white (w) matter in astrocyte scar (AS) or equivalent regions in uninjured tissue. Graphs show percent of areas stained for CSPG or GFAP determined using Image J. n = 4 WT, n = 6 TK+GCV and STAT3-CKO; # p<0.05 versus uninjured white matter, * p<0.05 versus uninjured grey matter in same experimental group (ANOVA with Newman-Keuls); ˆ p<0.05 versus equivalent anatomical region in WT (ANOVA with Newman-Keuls).
Extended data figure 3
Extended data figure 3. Specificity of HA-targeting to astrocytes and enrichment of HA-IP for astrocyte specific RNA transcripts
(a) Individual fluorescence channels of immunohistochemistry for transgenically-targeted hemagglutinin (HA) plus various cell markers showing the specificity of HA targeting to cells expressing the astrocyte marker, GFAP, and not to cells expressing either the neuronal marker, NeuN, or the mature oligodendrocyte marker, GSTπ, in uninjured grey and white matter and in astrocyte scar at 2 weeks after SCI. (b) CNS cell type-specific gene transcript enrichment of ribosome-associated mRNA (ramRNA) isolated from WT uninjured spinal cord by HA immune-precipitation (HA-IP). Differential expression analysis by RNA-sequencing (RNA-Seq) indicates significant enrichment (red) for astrocyte-specific gene transcripts, and de-enrichment (green) for gene transcripts enriched in other CNS cell types, FDR<0.1. A log2 scale is used so that positive and negative differences are directly comparable. The mean numerical enrichment of three quintessential astrocyte genes, Gfap, Aldh1l1 and Aqp4, is 25 fold greater in HA samples than in flow through samples. (c) Gene transcript enrichment of HA-IP ramRNA relative to P7 mouse primary cortical astrocytes. Of the 200 most highly expressed genes previously described for post-natal mouse cortical astrocytes, 71.5% (red line) are at least 4-fold enriched (blue line) in HA-IP ramRNA isolated from uninjured spinal cord relative to flow through RNA from non-astrocyte cells. (d) Pearson correlation plots of total normalized RNA-Seq reads from individual biological replicates for each treatment condition. Correlation coloring indicates little (white) to high (red) similarity. n = 4 for uninjured controls and wild type SCI (SCI-WT); n = 3 for STAT3-CKO SCI (SCI-STAT3). FDR<0.1 for differential expression and enrichment analysis. Raw and normalized data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE76097 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE76097).
Extended data figure 4
Extended data figure 4. Comparison of genomic data from astrocytes and non-astrocyte cells from WT and STAT3-CKO mice after SCI
(a) Heat maps depicting all significantly differentially expressed genes (DEG), as determined by RNA-Seq, for WT and STAT3-CKO astrocytes and non-astrocytes from independent biological replicates two weeks after SCI relative to uninjured WT control. Red upregulated, green downregulated. (b) Total numbers and Venn diagrams of significant DEGs in WT and STAT3-CKO astrocytes and non-astrocytes two weeks after SCI relative to uninjured control. Red and green numerical values indicate significantly upregulated and downregulated genes, respectively. (c) Comparison of altered gene expression in our SCI-reactive astrocytes and previously reported forebrain stroke-reactive astrocytes. Of the 200 most highly elevated genes in forebrain astrocytes 1 week following stroke, 58.5% (red line) are also significantly elevated in astrocytes after SCI, relative to uninjured. (d) Comparison of expression by WT SCI and STAT3-CKO SCI reactive astrocytes of a selected cross-section of genes that are highly regulated after SCI by WT reactive astrocytes. Many of the regulated genes exhibit changes that are expected and implicated in WT reactive astrogliosis mechanisms and roles, and some of the changes appear to be newly identified in this context. Note that many of the genes are not regulated or exhibit attenuated changes in STAT3-CKO SCI astrocytes. n = 4 for uninjured and WT SCI; n = 3 for STAT3-CKO SCI (SCI-STAT3). FDR<0.1 for differential expression and enrichment analysis.
Extended data figure 5
Extended data figure 5. Immunohistochemistry of specific CSPGs
(a) Absence of aggrecan (ACAN) production by scar-forming astrocytes. Images show individual fluorescence channels of ACAN and GFAP immunohistochemistry from horizontal sections two weeks after severe SCI in a representative WT mouse. Boxes denote areas of astrocyte scar (AS) or uninjured tissue (Uninj) shown at higher magnification. Note that ACAN (i) is heavily present in the perineuronal nets that surround neurons in uninjured tissue, (ii) is almost absent from AS and lesion core (LC), and (iii) is not detectably produced by newly generated scar-forming astrocytes (arrows). (b) Brevican (BCAN) production by scar-forming astrocytes and non-astrocyte cells. Images show individual fluorescence channels of BCAN and GFAP immunohistochemistry from horizontal sections two weeks after severe SCI, in WT mice and mice with transgenic ablation (TK+GCV) or attenuation (STAT3-CKO) of astrocyte scar formation. Note that BCAN is produced both by GFAP-positive scar-forming astrocytes (arrowheads) and by non-astrocyte cells (arrows).
Extended data figure 6
Extended data figure 6. Immunohistochemistry of specific CSPGs
(a) Neurocan (NCAN) production by scar-forming astrocytes and non-astrocyte cells. Images show individual fluorescence channels of NCAN and GFAP immunohistochemistry from horizontal sections two weeks after severe SCI, in a representative WT mouse. Box denotes area of lesion core (LC) and astrocyte scar (AS) shown at higher magnification. Note that NCAN is produced both by GFAP-positive scar-forming astrocytes and by non-astrocyte cells (arrows) in the lesion core (LC). (b) NG2 (CSPG4) production by newly proliferated scar-forming astrocytes. Images show individual channels and various combinations of immunofluorescence staining for NG2, GFAP, tdT, BrdU (proliferation marker) and DAPI showing astrocytes in a mature SCI scar. The images are representative of findings from tdT-reporter mice injected with AAV2/5-GfaABC1D-Cre vector into multiple sites of the uninjured spinal cord to label mature astrocytes. Three weeks after AAV2/5-GfaABC1D-Cre injection, the mice received a severe SCI and were administered BrdU from days 2–7 after SCI. The mice were perfused after two weeks after SCI. Images comparing individual fluorescence channels show that astrocytes labeled 1 and 3: (i) incorporated BrdU and thus are newly proliferated after SCI, (ii) express tdT reporter, (iii) express GFAP, the prototypical marker of reactive and scar-forming astrocytes, and (iv) express NG2 both intracellularly and along their cell surfaces. In contrast, astrocyte number 2 is also BrdU-labeled and expresses both tdT and GFAP, but does not appear to express detectable levels of NG2. (c) CSPG5 (Neuroglycan C) production by scar-forming astrocytes. Images show individual channels and various combinations of immunofluorescence staining for CSPG5 or GFAP. Note that CSPG5 is present within and along the processes of GFAP-positive scar-forming astrocytes (arrows).
Extended data figure 7
Extended data figure 7. Specificity and effects of treatments to stimulate AST axon regrowth after SCI
(a) BDNF and NT3 treatment does not alter the appearance or density of astrocyte scars in WT or STAT3-CKO mice. Images show horizontal sections of mice at two weeks after SCI or after SCI followed by delayed injection of hydrogel only (as a control) or hydrogel releasing NT3 and BDNF. Top images show GFAP immunofluorescence; boxed area denotes size of areas taken from multiple locations in the astrocyte scar (AS) for GFAP area quantification shown in graph. n = 5 mice per group, ns p>0.05 (ANOVA with Newman-Keuls). Bottom images show brightfield immunohistochemistry simultaneously of GFAP+TK to stain both astrocyte cell processes (GFAP) and cell bodies (TK) in mGFAP-TK transgenic mice for quantification of astrocyte cell numbers shown in graph. For these experiments the transgene derived TK is used as a reporter protein that efficiently labels astrocyte cell bodies and thereby improves cell quantification and the mice were not given GCV. n = 4 mice per group, * p<0.05 versus uninjured (ANOVA with Newman-Keuls); ns p>0.05 (ANOVA with Newman-Keuls). (b) AST axon regrowth through scar-forming astrocytes and CSPGs in SCI lesions. Images show individual channels and various combinations of immunofluorescence staining for CTB, GFAP and CS56 to detect total CSPGs from a WT mouse after SCI followed by delayed injection of a hydrogel depot releasing NT3 and BDNF, shown as multichannel image in main text figure 5e1. Arrows denote robust regrowth of many AST axons along, through and past scar-forming astrocytes into and through the lesion core. Note that the stimulated axons are regrowing through CSPG containing areas in the astrocyte scar and lesion core. Boxed area is shown at higher magnification in Extended data figure 8. (c) Graph shows numbers of AST axons at various distances past the proximal border of the astrocyte scar under different conditions. n = 5 per group. * p<0.001 significant difference SCI+CL+BDNF+NT3 versus all other groups (ANOVA with post-hoc Newman-Keuls).
Extended data figure 8
Extended data figure 8. AST axon regrowth through scar-forming astrocytes and CSPGs in SCI lesions
(a,b) Images show individual channels and various combinations of immunofluorescence staining for CTB, GFAP and CS56 to detect total CSPGs from a WT mouse after SCI followed by delayed injection of a hydrogel depot releasing NT3 and BDNF, shown as multichannel images in main text Figures 5e2 and 5e3. Arrows in (a) denote robust regrowth of many AST axons along, through and past scar-forming astrocytes into and through the lesion core; note that the stimulated axons are regrowing through CSPG containing areas in the astrocyte scar and lesion core. (b) High magnification orthogonal images of axons in three visual planes. Arrows in (b) denote AST regrowing axons tracking along CSPG-positive and GFAP-negative structures. Arrowheads in (b) denote AST axons tracking along GFAP-positive and CSPG-positive astrocyte processes, passing from one astrocyte process to another.
Extended data figure 9
Extended data figure 9. AST axon regrowth in SCI lesions is dependent on laminin
(a–g) Tract-tracing for of AST axons using CTB and laminin immunohistochemistry. (a–c) Same fields imaged for CTB alone (a1–c1), or CTB plus laminin (a2–c2). (a,d) Intact gracile cuneate tract (GCT). (b) SCI only. (c,f) SCI plus conditioning lesion (CL) plus hydrogel with growth factors. (e) SCI plus conditioning lesion. (g) SCI plus conditioning lesion plus hydrogel with growth factors and anti-CD29. (d–g) High magnification orthogonal images of axons in three visual planes. Arrows indicate regrowing axons in direct contact with laminin. Arrowheads indicate axons not in direct contact with laminin in the intact GCT (d) or with anti-CD29 treatment (g). Note the difference in appearance of axons in the intact gracile cuneate tract (GCT), which are independent of laminin, compared with regrowing axons in lesion core (LC), which track along laminin. (h) Axon length per tissue volume in intact GCT or in SCI lesions under different conditions. (Intact GCT values were not included in ANOVA comparison of other 3 groups.) (i) Percent of AST axon length in direct contact with laminin under different conditions. n = 5 per group. * p<0.001 (ANOVA with post-hoc Newman-Keuls); ns non-significant (ANOVA with post-hoc Newman-Keuls).
Figure 1
Figure 1. Preventing astrocyte scar formation does not lead to spontaneous regrowth of CST, AST, or 5HT axons after SCI
(a) Experiment summary schematic. Horizontal view of lesion core (LC) and astrocyte scar (AS) after SCI. Intercepts of CST, AST or 5HT axons with lines drawn at various distances from lesion center (Cn) were counted and expressed as a percent of axons 3mm proximal. (b,c,e,f) Dotted lines demarcate Cn and 500μm on either side. (b,d) Area occupied by GFAP-positive scar-forming astrocytes within 500μm on either side of Cn at 2 or 8 weeks after SCI. n = 6; (c, e) Arrows depict most caudal CST axons (BDA-tracing) or most rostral AST axons (CTB-tracing). (f) Arrows in top images depict most caudally penetrating 5HT axons, boxed areas are shown below. In WT mice, 5HT axons are surrounded by AS (arrowheads). In TK+GCV and STAT3-CKO mice, many 5HT axons are not in contact with AS (arrows), but have not regrown. (g–i) Numbers of CST (g), AST (h) and 5HT (i) axons at various distances from the SCI lesion center (Cn) as a percent of the number of axons present 3mm proximal. n = 6 CST; n = 5 AST and 5HT; * p<0.05 versus WT (ANOVA with Newman-Keuls).
Figure 2
Figure 2. No spontaneous regrowth of CST, AST or 5HT axons after deleting chronic astrocyte scars
(a) Experiment summary schematic. (b) Selective targeting of tdT reporter to GFAP-positive scar-forming astrocytes in 500μm zone occupied by astrocyte scar (see Extended Data Figure 1c). (c) DTR-DTX mediated ablation of chronic astrocyte scar after severe SCI. Dotted lines indicate Cn and 500μm on either side normally occupied by scar-forming astrocytes in WT mice. Boxes show locations of d–f in adjacent sections. (d) CST axons are found only among GFAP-positive astrocytes proximal to ablated scar. (e) AST axons at margins of large area depleted of scar but have not regrown (arrows). (f) 5HT axons are within area depleted of scar but have not regrown (arrows). (g–i) Numbers of CST (F), AST (G), or 5HT (H) axons at various distances from SCI lesion centers as a percent of the number of axons present at 3mm proximal. n = 6 CST; n = 5 AST and 5HT; * p<0.05 versus WT (ANOVA with Newman-Keuls).
Figure 3
Figure 3. CSPG production by non-astrocyte cells in SCI lesions after ablation or attenuation of astrocyte scars
(a) Dot blots and quantitation of total CSPG detected by CS56 antibody relative to total protein (Ponceau). n = 4; * p<0.05 versus uninjured (ANOVA with Newman-Keuls). (b) CS56 and GFAP immunohistochemistry (see Extended Data Fig. 2). (c) Details of boxed areas in b showing CSPG production by GFAP-negative cells.
Figure 4
Figure 4. Genomic dissection of astrocytes and non-astrocyte cells in SCI lesions
(a) Schematic of tissue harvested for RNA-sequencing. (b) Transgenically-targeted HA-tagged ribosome clusters (arrows) among GFAP filaments in scar-forming astrocytes (see Extended Data Fig. 3a). (c) Unsupervised hierarchical clustering dendrogram based on Pearson correlations shows that transcriptome profiles of STAT3-CKO SCI astrocytes cluster nearer to (and are more similar to) those of uninjured as compared to WT SCI astrocytes. (d) Numbers and heat map of significantly differentially expressed genes (DEGs) in WT astrocytes after SCI and the comparative differential expression profile of that specific cohort of genes in STAT3-CKO SCI astrocytes. Differential expression relative to uninjured, FDR<0.1. (e) Histogram of astrocyte and non-astrocyte expression of axon inhibitory or permissive molecules after SCI shown as mean FPKM. * significant difference astrocytes versus non-astrocytes, FDR<0.1. Numbers show Log2 fold significant differences. Red upregulated, green downregulated, ns non-significant.
Figure. 5
Figure. 5. Robust regrowth of AST axons can be stimulated after WT SCI and is significantly attenuated by preventing astrocyte scar formation
(a1–c1) AST axons (CTB-tracing) plus GFAP immunohistochemistry. (a2–c2) AST axons alone. (a) WT mouse, SCI and hydrogel only (no growth factors). Arrowhead denotes most rostrally penetrating axons that do not pass beyond AS. (b) WT mouse, SCI plus conditioning lesions (CL) and hydrogel depot (D) with NT3+BDNF. Arrows denote robust regrowth of AST axons past AS into LC and along, but not into, the depot that releases NT3+BDNF but provides no adhesive matrix. (c) STAT3-CKO mouse, SCI plus CL and NT3+BDNF depot. Arrows denote regrowth of AST axons into LC. (d) Experiment summary schematic. (e–f) WT mice. (e1–e3) AST plus GFAP and CSPG (CS56) immunohistochemistry. Box in e1 is shown in e2. (e1 and e2) Arrows denote robust regrowth of stimulated AST axons past AS into LC through CSPG. (e3) Regrowing AST axons track along CSPG-positive GFAP-negative structures (arrows) or along CSPG-positive GFAP-positive astrocyte processes (arrowheads) (See Extended Data Figures 7,8). (f,g) AST axons plus laminin immunohistochemistry. (f) Arrows denote regrowing stimulated AST axons tracking along laminin. (g) Arrowheads denote stimulated AST axons exposed to anti-CD29 antibody and failing to maintain contact with laminin. (h,i) Numbers of AST axons at SCI Cn (h) or on either side (i) expressed as percent of axons 3mm proximal. n = 5; * p<0.05 versus all other groups, ˆ p<0.05 versus all groups except STAT3-CKO (ANOVA with Newman-Keuls).
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