Abstract
Spinal cord injury in mammals is thought to trigger scar formation with little regeneration of axons1,2,3,4. Here we show that a crush injury to the spinal cord in neonatal mice leads to scar-free healing that permits the growth of long projecting axons through the lesion. Depletion of microglia in neonatal mice disrupts this healing process and stalls the regrowth of axons, suggesting that microglia are critical for orchestrating the injury response. Using single-cell RNA sequencing and functional analyses, we find that neonatal microglia are transiently activated and have at least two key roles in scar-free healing. First, they transiently secrete fibronectin and its binding proteins to form bridges of extracellular matrix that ligate the severed ends of the spinal cord. Second, neonatal—but not adult—microglia express several extracellular and intracellular peptidase inhibitors, as well as other molecules that are involved in resolving inflammation. We transplanted either neonatal microglia or adult microglia treated with peptidase inhibitors into spinal cord lesions of adult mice, and found that both types of microglia significantly improved healing and axon regrowth. Together, our results reveal the cellular and molecular basis of the nearly complete recovery of neonatal mice after spinal cord injury, and suggest strategies that could be used to facilitate scar-free healing in the adult mammalian nervous system.
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Data availability
RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) (www.ncbi.nlm.nih.gov/geo) under the accession number GSE150871. Source data are provided with this paper.
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Acknowledgements
We thank J. Sanes and I. Benhar for discussion and S. Hegarty, J. Sanes, B. Stevens, F. Wang, K. H. Wang, C. Winter and C. Woolf for critically reading the manuscript. This study was supported by NIH R01 grants NS096294 and NS110850 (to Z.H.); NIH-NINDS R35NS111582 and a Ray W. Poppleton endowment (to P.G.P.); grants from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to Z.H., R.K., V.S. and D.H.G.); the Craig H. Neilsen Foundation (to Y.L.); and a NIH training grant T32NS007473 (to A.M.).The Intellectual and Developmental Disabilities Research Center (IDDRC) and viral cores supported by the grants NIH HD018655 and P30EY012196 were used for this study.
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Y.L., X.H., P.G.P. and Z.H. conceived the project; Y.L., X.H., Q.W., A.M., B.C., Z.S., H.M., S.Z., J.Z. and A.J. performed the experiments and discussed the results; R.K., Y.Z., Z.Y., V.S. and D.H.G. participated in data analysis; Y.L., X.H. and Z.H. prepared the manuscript with inputs from all authors.
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Z.H. is an advisor of SpineX. The remaining authors declare no competing interests.
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Peer review information Nature thanks Elizabeth Bradbury, Jerry Silver and Binhai Zheng for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data figures and tables
Extended Data Fig. 1 Age-dependent decline in serotonergic axon regrowth and wound healing.
a, Representative images of spinal cord sagittal sections showing 5HT-labelled axons from sham, P7 or P20 mice at two weeks after inury. Scale bar, 500 μm. b, Representative images of spinal cord sections from sham, P7 or P20 mice at two weeks after injury, stained with antibodies against collagen I, CD68, P2Y12 or CD31. Scale bar, 250 μm. c, Representative images of spinal sagittal sections at four weeks after sham control or P2 injury, with CST axons labelled with AAV-ChR2-mCherry. Red stars indicate the lesion site. Scale bar, 500 μm. d, Representative images of sagittal spinal cord sections at two weeks after injury, stained with antibodies against laminin, CSPG and GFAP. Scale bar, 250 μm. e, Representative images of sagittal spinal cord sections at two weeks after P20 injury, stained with antibodies against laminin, GFAP and 5-HT. Scale bar, 250 μm. All experiments shown were independently repeated three times with similar results.
Extended Data Fig. 2 Distinct microglia and macrophage responses after crush injury to neonatal or adult spinal cord.
a, Images of spinal cord sections stained with antibodies against CD68 and P2Y12 from mice at 3 dpi, 7 dpi or 14 dpi. Higher-magnification images showing P2Y12+ cells were co-labelled with CD68 at 3 dpi. Cells with highly ramified morphology at 7 dpi and 14 dpi can be seen around lesion sites. Scale bar, 100 μm. b, Higher-magnification images from Fig. 2a showing that CD68+ cells and fibronectin matrix form bridges between gaps at 3 dpi. Scale bar, 50 μm. c, Immunolabelling for CD68 and P2Y12 in adult mice at 3 dpi, 7 dpi or 14 dpi, showing CD68+ cells that lack P2Y12 expression. Scale bar, 200 μm. All experiments shown were independently repeated three times with similar results.
Extended Data Fig. 3 Histological assessments of bridges formed after neonatal spinal cord injury.
a, Higher-magnification images of sections of the spinal cord bridge area stained with antibodies against fibronectin, GFAP, P2Y12 and collagen I, or with DAPI (blue), at 3 dpi in P2 injury. Scale bar, 50 μm. b, Representative images of spinal sections of Cx3cr1GFP mice immunolabelled with caspase-3 showing cells around the lesion sites at 3 dpi in P2 injury. Scale bar, 200 μm. All experiments shown were independently repeated three times with similar results.
Extended Data Fig. 4 Infiltrated CCR2+ monocytes and macrophages were eliminated after neonatal but not adult spinal cord injury.
a, b, Representative images of sagittal sections of injured spinal cord of Ccr2RFP mice at 3 or 14 dpi in P2 injury (a) or adult injury (b). Sections were immunostained for CD68 and RFP (for Ccr2RFP). Scale bar, 250 μm. All experiments shown were independently repeated three times with similar results.
Extended Data Fig. 5 Microglia depletion impairs wound healing and axon regrowth after neonatal spinal cord injury.
a, Left, representative P2Y12-stained spinal cord images showing PLX3397-mediated depletion of microglia cells. Right, quantification of microglia depletion in spinal cord treated with PLX3397 or vehicle at 0, 7 or 14 dpi (n = 3, 5 and 5 for 0, 7 and 14 dpi, respectively). Student’s t-test (two-tailed, unpaired), ***P < 0.0001. Data are mean ± s.e.m. Scale bar, 250 μm. b, Representative images of P2Y12-stained spinal cord sections from control (Csf1rlfl/fl) and Csf1r-knockout (Cx3crcre;Csf1rfl/fl) mice showing an approximately 70% reduction of microglia throughout the spinal cord in the mutant mice (n = 5 per group). Student’s t-test (two-tailed, unpaired), ***P = 0.0004. Data are mean ± s.e.m. Scale bar, 250 μm. c, Representative images of sagittal spinal sections taken at 14 days after P2 injury and immunostained with antibodies against 5-HT, GFAP, laminin, CSPG or CD31. Scale bar, 200 μm. d, Higher-magnification images from c, showing 5-HT+ axons and GFAP+ astrocytes in the lesion site. Scale bar, 50 μm.
Extended Data Fig. 6 Isolation and scRNA-seq results for immune cells after neonatal spinal cord injury.
a, FACS plots showing selection of CD11b+ and CD45+ cells from dissociated cells from neonatal spinal cord. b, t-SNE plot showing 14 clusters and population annotations. c, d, Relative proportions of microglia among total cells (c) and dividing microglia among microglia cells (d). e, Left, table showing the percentage of each cluster and their signature genes. Right, heat map depicting the top 30 differentially expressed genes for each of the 14 clusters.
Extended Data Fig. 7 Feature plots showing examples of differentially expressed genes in different clusters.
Gene expression of P2ry12, Tmem119, Siglech, Fn1, Igf1, Spp1, Ms4a7, Thbs1, Anxa1, Cstb, Serpinb6a and Stfa1 in different microglia clusters.
Extended Data Fig. 8 Comparison of changes in gene expression in proliferative-region-associated microglia, disease-associated microglia and MG3.
Dot plots of gene expression correlation between proliferative-region-associated microglia (PAM) and disease-associated microglia (DAM) (normalized to homeostatic microglia) and MG3 (normalized to MG0), showing different sets of upregulated and downregulated genes (n = 13,755 and 14,423 genes for PAM and DAM, respectively).
Extended Data Fig. 9 Network analysis and further characterization of MG3 differentially expressed genes.
a, Diagram depicting correlated gene modules that underlie cluster identities of MG3 microglia. b, c, Expression of genes associated with endopeptidase inhibitor activity (b) and the ECM (c), in adult microglia at 0, 3 and 5 dpi, using bulk RNA-seq (n = 3 per group). One-way ANOVA followed by post-hoc Bonferroni correction. *P = 0.03 (Lgals3), 0.04 (Lgals1), 0.01 (Ecm1), 0.01(Pf4); **P = 0.003 (Fn1), 0.0013 (Pf4). Data are mean ± s.e.m.
Extended Data Fig. 10 Microglia isolation and transplantation.
a, Representative images (left) and quantification (right) of isolated microglia (P2Y12+) from neonatal or adult Cx3cr1GFP mice (3 h after isolation, n = 400 cells examined over three independent experiments). Data are mean ± s.e.m. Scale bar, 50 μm. b, Representative images of spinal cord sections showing the activation of transplanted microglia in the adult lesion at two days after grafting. Scale bar, 250 μm. All experiments shown in b were independently repeated three times with similar results. c, Representative images of spinal cord sections showing the GFAP and CSPG staining in the adult lesion at 14 days after grafting. Quantification results are shown in Fig. 5d. Scale bar, 250 μm.
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Li, Y., He, X., Kawaguchi, R. et al. Microglia-organized scar-free spinal cord repair in neonatal mice. Nature 587, 613–618 (2020). https://doi.org/10.1038/s41586-020-2795-6
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DOI: https://doi.org/10.1038/s41586-020-2795-6
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