ARTICLE https://doi.org/10.1038/s41467-019-08446-0 OPEN Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury Victor Bellver-Landete1, Floriane Bretheau1, Benoit Mailhot1, Nicolas Vallières1, Martine Lessard1, Marie-Eve Janelle2, Nathalie Vernoux1, Marie-Ève Tremblay 1, Tobias Fuehrmann 3, Molly S. Shoichet3 & Steve Lacroix 1 1234567890():,; The role of microglia in spinal cord injury (SCI) remains poorly understood and is often confused with the response of macrophages. Here, we use specific transgenic mouse lines and depleting agents to understand the response of microglia after SCI. We find that microglia are highly dynamic and proliferate extensively during the first two weeks, accu- mulating around the lesion. There, activated microglia position themselves at the interface between infiltrating leukocytes and astrocytes, which proliferate and form a scar in response to microglia-derived factors, such as IGF-1. Depletion of microglia after SCI causes disruption of glial scar formation, enhances parenchymal immune infiltrates, reduces neuronal and oligodendrocyte survival, and impairs locomotor recovery. Conversely, increased microglial proliferation, induced by local M-CSF delivery, reduces lesion size and enhances functional recovery. Altogether, our results identify microglia as a key cellular component of the scar that develops after SCI to protect neural tissue. 1 Axe neurosciences du Centre de recherche du Centre hospitalier universitaire (CHU) de Québec–Université Laval et Département de médecine moléculaire de l’Université Laval, Québec, QC G1V 4G2, Canada. 2 Département de biologie-biotechnologie du Cégep de Lévis-Lauzon, Lévis, QC, Canada G6V 6Z9. 3 Department of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto, ON M5S 3E1, Canada. Correspondence and requests for materials should be addressed to S.L. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:518 | https://doi.org/10.1038/s41467-019-08446-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08446-0 icroglia is derived from primitive yolk sac progenitors 85.9 ± 4.6 microglia per mm2. Following a moderate contusive SCI, that arise during embryogenesis1–3. They are main- only 28.8 ± 1.9 microglia per mm2 were left at the lesion epicenter at M 4,5 tained after birth and into adulthood by self-renewal , 1 dpi, which corresponds to a 67% reduction in cell numbers. independently from bone marrow-derived hematopoietic stem Hardly any TdT+ microglia were observed in the lesion core at this cells (HSCs) and their differentiated progeny (e.g. monocyte- early time point, suggesting that they underwent rapid cell death. derived macrophages, MDMs)6,7. After a CNS injury, blood- Despite the fact that the impactor tip measures 1.25 mm of derived monocytes are massively recruited in the tissue where diameter, microglia were lost across several spinal cord segments they differentiate into macrophages and adopt many of the rostrocaudally. This microglial cell loss ranged from ~20% to 65% at markers and behaviors of microglia. These similarities have rostrocaudal distances up to 6 mm from the lesion epicenter complicated the development of efficient prediction tools to dis- (Fig. 1g, h), and was mediated in part through apoptosis (Fig. 1i–k). criminate between them. As a consequence, they are still referred At that time, residual microglia still expressed the purinergic to as microglia/macrophages in the neuroscience literature, and receptor P2ry12 (Supplementary Figure 3), a receptor implicated in accordingly, their individual roles remain to be clarified. microglia recruitment during the early acute phase of CNS injury15. Recent advances in genetic fate mapping and conditional gene Accordingly, we noticed a retraction of microglial processes as early targeting have allowed the study of the specific biology of as day 1. Expression levels of the lysosome-associated glycoprotein microglia in various experimental contexts, including spinal cord CD68, a marker of phagocytosis, remained low in TdT+ microglia injury (SCI)8. This, together with the newly developed strategies at 1dpi (Supplementary Figure 4). However, the situation changed to specifically eliminate microglia9, has moved forward knowl- at day 4, as we counted 119.1 ± 15.0 microglia per mm2 at the lesion edge about these cells substantially. For example, the application epicenter, which represents a four-fold increase in the number of of some of these advances to a mouse model of stroke has led to TdT+ microglia compared to day 1 (Fig. 1g). Microglia around the the discovery that microglia can protect neurons through the lesion epicenter exhibited a round morphology, downregulation of regulation of calcium levels10. In contrast, the elimination of P2ry12 and a strong upregulation of CD68 (Supplementary microglia in mouse models of Alzheimer’s disease and Tau Figures 3 and 4), which points to a potential increase in their pathology reduced disease progression11,12. Thus, depending on phagocytic activity starting around 4 dpi. The number of microglia the context, microglia may exert diverging roles. Whether these continued to increase at the lesion epicenter over time, reaching up cells are beneficial or deleterious after SCI remains unexplored. to 1204.61 ± 137.8 cells/mm2 at 14 dpi. Nearly all microglia Here, we took advantage of Cx3cr1creER mice13, a mouse line observed in these areas were TdT+ CD68hi P2Y12neg (Supplemen- that allows with an adequate regimen of tamoxifen to label tary Figures 3 and 4). A similar trend was seen in the surrounding microglia while excluding nearly all MDMs. Our results show that tissue (400–800 µm) of the lesion epicenter up to 35 dpi, after which microglia are highly dynamic and proliferate extensively during the total number of microglia (i.e. 673.91 ± 62.4 cells/mm2 at the the first week post-SCI. Notably, we reveal that microglia form a lesion epicenter) started to decrease compared to day 14 (Fig. 1g). dense cellular interface at the border of the lesion between Interestingly, TdT+ microglia started to gradually increase their reactive astrocytes and infiltrating MDMs, which we hereafter expression of P2ry12 and decrease their expression of CD68 from refer to as the “microglial scar”. Microglia depletion experiments day 14 up to day 35 (Supplementary Figures 3 and 4), suggesting a using PLX5622, a CSF1R inhibitor that crosses the blood–spinal partial return to homeostasis. In sum, our data indicate that cord barrier (BSCB), demonstrated that the absence of microglia microglia are rapidly recruited around the site of SCI, where they in the context of SCI disrupts the organization of the astrocytic accumulate extensively during the subacute phase and adopt an scar, reduces the number of neurons and oligodendrocytes at the activated state that is eventually partially resolved during the site of injury, and impairs functional recovery. The timing of the intermediate/chronic phases. beneficial effects of microglia was estimated to be during the first week post-SCI. Accordingly, CNS delivery of M-CSF during that Microglia proliferate extensively during the subacute phase of critical period boosted microglial proliferation and enhanced SCI. Under normal circumstances, the adult microglial popula- locomotor recovery. In light of these data, we conclude that tion remains stable in the brain throughout life by coupled cell microglia are an important component of the protective scar that death and cell proliferation4, but little is known about its dynamic forms after SCI. in the spinal cord and how it reacts following SCI. Here, we report that 0.6 ± 0.1 microglia/mm2 (0.58% of total) are pro- creER Results liferating in the normal thoracic spinal cord of Cx3cr1 ::R26- TdT mice, as revealed by the co-expression of TdT and the cell Microglia rapidly accumulate around the site of SCI. To dis- proliferation marker Ki67. One day after SCI, no significant tinguish microglia from MDMs, we took advantage of changes were observed in terms of microglial cell proliferation Cx3cr1creER::R26-TdT mice, and the slow turnover of microglia4,5. compared to the uninjured spinal cord (Fig. 1l, m). Strikingly, Mice received tamoxifen treatment one month before SCI to about 50% of microglia at the lesion epicenter expressed Ki67 at 4 activate the inducible Cre for recombination of TdT floxed + dpi. As shown in Supplementary Movie 1, proliferating TdT (Supplementary Figure 1a). As expected from our previous + + Ki67 microglia were round-shaped. The peak proliferation of work14, nearly all (99.6 ± 0.2%) CD11b cells in the spinal cord microglia, in terms of absolute numbers, was observed at day 7 parenchyma expressed TdT (Supplementary Figure 1b, c). In + (Fig. 1l, n–p). At 14 and 35 dpi, only a few (2–6%) microglia were contrast, only a few CD11b cells in the blood, spleen and bone + still expressing Ki67, suggesting that microglial proliferation was marrow were TdT , with average colocalization percentages of mostly inhibited by then. Together, these results show that 3.8 ± 1.7%, 6.7 ± 1.6%, and 2.4 ± 0.2%, respectively (Supplemen- microglia are highly dynamic after SCI, not only through their tary Figure 1d–f). Thus, inducible Cx3cr1creER::R26-TdT mice are recruitment and activation, but also through their ability to a good tool to study microglia in SCI. rapidly proliferate and surround the site of SCI during the sub- To understand the dynamics of the microglial response after