Traumatic spinal cord injury (SCI) brings numerous inflammatory cells, including macrophages, from the circulating blood to lesions, but pathophysiological impact resulting from spatiotemporal dynamics of macrophages is unknown. Here, we show that macrophages centripetally migrate toward the lesion epicenter after infiltrating into the wide range of spinal cord, depending on the gradient of chemoattractant C5a. However, macrophages lacking interferon regulatory factor 8 (IRF8) cannot migrate toward the epicenter and remain widely scattered in the injured cord with profound axonal loss and little remyelination, resulting in a poor functional outcome after SCI. Time-lapse imaging and P2X/YRs blockade revealed that macrophage migration via IRF8 was caused by purinergic receptors involved in the C5a-directed migration. Conversely, pharmacological promotion of IRF8 activation facilitated macrophage centripetal movement, thereby improving the SCI recovery. Our findings reveal the importance of macrophage centripetal migration via IRF8, providing a novel therapeutic target for central nervous system injury.

Background Spinal cord injury (SCI) is a catastrophic trauma accompanied by intralesional bleeding and neuroinflammation. Recently, there is increasing interest in tranexamic acid (TXA), an anti-fibrinolytic drug, which can reduce the bleeding volume after physical trauma. However, the efficacy of TXA on the pathology of SCI remains unknown. Methods After producing a contusion SCI at the thoracic level of mice, TXA was intraperitoneally administered and the bleeding volume in the lesion area was quantified. Tissue damage was evaluated by immunohistochemical and gene expression analyses. Since heme is one of the degraded products of red blood cells (RBCs) and damage-associated molecular pattern molecules (DAMPs), we examined the influence of heme on the pathology of SCI. Functional recovery was assessed using the open field motor score, a foot print analysis, a grid walk test, and a novel kinematic analysis system. Statistical analyses were performed using Wilcoxon’s rank-sum test, Dunnett’s test, and an ANOVA with the Tukey-Kramer post-hoc test. Results After SCI, the intralesional bleeding volume was correlated with the heme content and the demyelinated area at the lesion site, which were significantly reduced by the administration of TXA. In the injured spinal cord, toll-like receptor 4 (TLR4), which is a DAMP receptor, was predominantly expressed in microglial cells. Heme stimulation increased TLR4 and tumor necrosis factor (TNF) expression levels in primary microglial cells in a dose-dependent manner. Similarly to the in vitro experiments, the injection of non-lysed RBCs had little pathological influence on the spinal cord, whereas the injection of lysed RBCs or heme solution significantly upregulated the TLR4 and TNF expression in microglial cells. In TXA-treated SCI mice, the decreased expressions of TLR4 and TNF were observed at the lesion sites, accompanied by a significant reduction in the number of apoptotic cells and better functional recovery in comparison to saline-treated control mice. Conclusion The administration of TXA ameliorated the intralesional cytotoxicity both by reducing the intralesional bleeding volume and preventing heme induction of the TLR4/TNF axis in the SCI lesion. Our findings suggest that TXA treatment may be a therapeutic option for acute-phase SCI. Electronic supplementary material The online version of this article (10.1186/s12974-019-1536-y) contains supplementary material, which is available to authorized users.

Background After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. Recently, we reported that anti-β1 integrin antibody (β1Ab) had a therapeutic effect on astrocytes by preventing the induction of glial scar formation. However, the cellular components within the glial scar are not only astrocytes but also microglia, and whether or not β1Ab treatment has any influence on microglia within the glial scar remains unclear. Methods To evaluate the effects of β1Ab treatment on microglia within the glial scar after SCI, we applied thoracic contusion SCI to C57BL/6N mice, administered β1Ab in the sub-acute phase, and analyzed the injured spinal cords with immunohistochemistry in the chronic phase. To examine the gene expression in microglia and glial scars, we selectively collected microglia with fluorescence-activated cell sorting and isolated the glial scars using laser-captured microdissection (LMD). To examine the interaction between microglia and astrocytes within the glial scar, we stimulated BV-2 microglia with conditioned medium of reactive astrocytes (RACM) in vitro, and the gene expression of TNFα (pro-inflammatory M1 marker) was analyzed via quantitative polymerase chain reaction. We also isolated both naïve astrocytes (NAs) and reactive astrocytes (RAs) with LMD and examined their expression of the ligands for β1 integrin receptors. Statistical analyses were performed using Wilcoxon’s rank-sum test. Results After performing β1Ab treatment, the microglia were scattered within the glial scar and the expression of TNFα in both the microglia and the glial scar were significantly suppressed after SCI. This in vivo alteration was attributed to fibronectin, a ligand of β1 integrin receptors. Furthermore, the microglial expression of TNFα was shown to be regulated by RACM as well as fibronectin in vitro. We also confirmed that fibronectin was secreted by RAs both in vitro and in vivo. These results highlighted the interaction mediated by fibronectin between RAs and microglia within the glial scar. Conclusion Microglial inflammation was enhanced by RAs via the fibronectin/β1 integrin pathway within the glial scar after SCI. Our results suggested that β1Ab administration had therapeutic potential for ameliorating both glial scar formation and persistent neuroinflammation in the chronic phase after SCI.

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After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism by which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3−/− mice, which showed enhanced astrocyte migration, and bone marrow from IRF8−/− mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8−/ bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism in which migrating macrophages attracted astrocytes and affected the pathophysiology and outcome after SCI.

Joint contracture causes distressing permanent mobility disorder due to trauma, arthritis, and aging, with no effective treatment available. A principal and irreversible cause of joint contracture has been regarded as the development of joint capsule fibrosis. However, the molecular mechanisms underlying contracture remain unclear. We established a mouse model of knee joint contracture, revealing that fibrosis in joint capsules causes irreversible contracture. RNA-sequencing of contracture capsules demonstrated a marked enrichment of the genes involved in the extracellular region, particularly periostin (Postn). Three-dimensional magnetic resonance imaging and immunohistological analysis of contracture patients revealed posterior joint capsule thickening with abundant type I collagen (Col1a2) and POSTN in humans. Col1a2-GFP TG ; Postn −/− mice and chimeric mice with Col1a2-GFP TG ; tdTomato TG bone marrow showed fibrosis in joint capsules caused by bone marrow-derived fibroblasts, and POSTN promoted the

Background Spinal cord injury (SCI) is a devastating disease that results in permanent paralysis. Currently, there is no effective treatment for SCI, and it is important to identify factors that can provide therapeutic intervention during the course of the disease. Zinc, an essential trace element, has attracted attention as a regulator of inflammatory responses. In this study, we investigated the effect of zinc status on the SCI pathology and whether or not zinc could be a potential therapeutic target. Methods We created experimental mouse models with three different serum zinc concentration by changing the zinc content of the diet. After inducing contusion injury to the spinal cord of three mouse models, we assessed inflammation, apoptosis, demyelination and the number of nuclear translocations of NF-κB in macrophages by using qPCR and immunostaining. In addition, macrophages in the injured spinal cord of these mouse models were isolated by flow cytometry, and their intracellular zinc concentration level and gene expression were examined. Functional recovery was assessed using the open field motor score, a foot print analysis, and a grid walk test. Statistical analysis was performed using Wilcoxon rank-sum test and ANOVA with the Tukey-Kramer test. Results In macrophages after SCI, zinc deficiency promoted nuclear translocation of NF-κB, polarization to pro-inflammatory phenotype and expression of pro-inflammatory cytokines. The inflammatory response exacerbated by zinc deficiency induced more apoptosis of oligodendrocytes and demyelination at the lesion site, leading to worsened motor function compared to normal zinc condition. Furthermore, zinc supplementation after SCI attenuated these zinc-deficiency-induced series of responses and improved motor function. Conclusion We demonstrated that zinc affected motor functional recovery after SCI by negatively regulating NF-κB activity and the subsequent inflammatory response. Our findings suggest that zinc supplementation after SCI may be a novel therapeutic strategy for SCI.

In crush syndrome, massive muscle breakdown resulting from ischemia–reperfusion muscle injury can be a life-threatening condition that requires urgent treatment. Blood reperfusion into the ischemic muscle triggers an immediate inflammatory response, and neutrophils are the first to infiltrate and exacerbate the muscle damage. Since free zinc ion play a critical role in the immune system and the function of neutrophils is impaired by zinc depletion, we hypothesized that the administration of a zinc chelator would be effective for suppressing the inflammatory reaction at the site of ischemia–reperfusion injury and for improving of the pathology of crush syndrome. A crush syndrome model was created by using a rubber tourniquet to compress the bilateral hind limbs of mice at 8 weeks. A zinc chelator N , N , N ′, N ′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) was administered immediately after reperfusion in order to assess the anti-inflammatory effect of the chelator for neutrophils. Histopathological evaluation showed significantly less muscle breakdown and fewer neutrophil infiltration in TPEN administration group compared with control group. In addition, the expression levels of inflammatory cytokine and chemokine such as IL-6, TNFα, CXCL1, CXCL2, CXCR2, CCL2 in ischemia–reperfusion injured muscle were significantly suppressed with TPEN treatment. Less dilatation of renal tubules in histological evaluation in renal tissue and significantly better survival rate were demonstrated in TPEN treatment for ischemia–reperfusion injury in crush syndrome. The findings of our study suggest that zinc chelators contributed to the resolution of exacerbation of the inflammatory response and attenuation of muscle breakdown in the acute phase after crush syndrome. In addition, our strategy of attenuation of the acute inflammatory reaction by zinc chelators may provide a promising therapeutic strategy not only for crush syndrome, but also for other diseases driven by inflammatory reactions.