Zinc deficiency impairs axonal regeneration and functional recovery after spinal cord injury by modulating macrophage polarization via NF-κB pathway.

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, axonal regeneration, 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 like phenotype and expression of pro-inflammatory cytokines. The inflammatory response exacerbated by zinc deficiency led to worsening motor function by inducing more apoptosis of oligodendrocytes and demyelination and inhibiting axonal regeneration in the lesion site compared to the 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 axonal regeneration and motor functional recovery after SCI by negatively regulating NF-κB activity and the subsequent inflammatory response in macrophages. Our findings suggest that zinc supplementation after SCI may be a novel therapeutic strategy for SCI.

Reduced Neuroinflammation Via Astrocytes and Neutrophils Promotes Regeneration After Spinal Cord Injury in Neonatal Mice.

Neonatal spinal cord injury (SCI) shows better functional outcomes than adult SCI. Although the regenerative capability in the neonatal spinal cord may have cues in the treatment of adult SCI, the mechanism underlying neonatal spinal cord regeneration after SCI is unclear. We previously reported age-dependent variation in the pathogenesis of inflammation after SCI. Therefore, we explored differences in the pathogenesis of inflammation after SCI between neonatal and adult mice and their effect on axon regeneration and functional outcome. We established two-day-old spinal cord crush mice as a model of neonatal SCI. Immunohistochemistry of the spinal cord revealed that the nuclear translocation of NF-κB, which promotes the expression of chemokines, was significantly lower in the astrocytes of neonates than in those of adults. Flow cytometry revealed that neonatal astrocytes secrete low levels of chemokines to recruit circulating neutrophils (e.g., Cxcl1 and Cxcl2) after SCI in comparison with adults. We also found that the expression of a chemokine receptor (CXCR2) and an adhesion molecule (ß2 integrin) quantified by flow cytometry was lower in neonatal circulating neutrophils than in adult neutrophils. Strikingly, these neonate-specific cellular properties seemed to be associated with no neutrophil infiltration into the injured spinal cord, followed by significantly lower expression of inflammatory cytokines (Il-1ß, Il-6 and TNF-α) after SCI in the spinal cords of neonates than in those of adults. At the same time, significantly fewer apoptotic neurons and greater axonal regeneration were observed in neonates in comparison with adults, which led to a marked recovery of locomotor function. This neonate-specific mechanism of inflammation regulation may have potential therapeutic applications in controlling inflammation after adult SCI.

Bone marrow-derived fibroblast migration via periostin causes irreversible arthrogenic contracture after joint immobilization.

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-GFPTG ; Postn-/- mice and chimeric mice with Col1a2-GFPTG ; tdTomatoTG bone marrow showed fibrosis in joint capsules caused by bone marrow-derived fibroblasts, and POSTN promoted the migration of bone marrow-derived fibroblasts, contributing to fibrosis and contracture. Conversely, POSTN-neutralizing antibody attenuated contracture exacerbation. Our findings identified POSTN as a key inducer of fibroblast migration that exacerbates capsule fibrosis, providing a potential therapeutic strategy for joint contracture.

Glial scar survives until the chronic phase by recruiting scar-forming astrocytes after spinal cord injury.

Spinal cord injury (SCI) causes reactive astrogliosis, the sequential phenotypic change of astrocytes in which naïve astrocytes (NAs) transform into reactive astrocytes (RAs) and subsequently become scar-forming astrocytes (SAs), resulting in glial scar formation around the lesion site and thereby limiting axonal regeneration and motor/sensory functional recovery. Inhibiting the transformation of RAs into SAs in the acute phase attenuates the reactive astrogliosis and promotes regeneration. However, whether or not SAs once formed can revert to RAs or SAs is unclear. We performed selective isolation of astrocytes from glial scars at different time points for a gene expression analysis and found that the expression of Sox9, an important transcriptional factor for glial cell differentiation, was significantly increased in chronic phase astrocytes (CAs) compared to SAs in the sub-acute phase. Furthermore, CAs showed a significantly lower expression of chondroitin sulfate proteoglycan (CSPG)-related genes than SAs. These results indicated that SAs changed their phenotypes according to the surrounding environment of the injured spinal cord over time. Even though the integrin-N-cadherin pathway is critical for glial scar formation, collagen-I-grown scar-forming astrocytes (Col-I-SAs) did not change their phenotype after depleting the effect of integrin or N-cadherin. In addition, we found that Col-I-SAs transplanted into a naïve spinal cord formed glial scar again by maintaining a high expression of genes involved in the integrin-N-cadherin pathway and a low expression of CSPG-related genes. Interestingly, the transplanted Col-I-SAs changed NAs into SAs, and anti-ß1-integrin antibody blocked the recruitment of SAs while reducing the volume of glial scar in the chronic phase. Our findings indicate that while the characteristics of glial scars change over time after SCI, SAs have a cell-autonomous function to form and maintain a glial scar, highlighting the basic mechanism underlying the persistence of glial scars after central nervous system injury until the chronic phase, which may be a therapeutic target.

Zinc chelator treatment in crush syndrome model mice attenuates ischemia-reperfusion-induced muscle injury due to suppressing of neutrophil infiltration.

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.

Microglial inflammation after chronic spinal cord injury is enhanced by reactive astrocytes via the fibronectin/ß1 integrin pathway.

BACKGROUND: After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. Recently, we reported that anti-ß1 integrin antibody (ß1Ab) 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 ß1Ab treatment has any influence on microglia within the glial scar remains unclear. METHODS: To evaluate the effects of ß1Ab treatment on microglia within the glial scar after SCI, we applied thoracic contusion SCI to C57BL/6N mice, administered ß1Ab 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 ß1 integrin receptors. Statistical analyses were performed using Wilcoxon's rank-sum test. RESULTS: After performing ß1Ab 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 ß1 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/ß1 integrin pathway within the glial scar after SCI. Our results suggested that ß1Ab administration had therapeutic potential for ameliorating both glial scar formation and persistent neuroinflammation in the chronic phase after SCI.

Tranexamic acid reduces heme cytotoxicity via the TLR4/TNF axis and ameliorates functional recovery after spinal cord 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.

Macrophage centripetal migration drives spontaneous healing process after spinal cord injury.

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.

The acute phase serum zinc concentration is a reliable biomarker for predicting the functional outcome after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a devastating disorder for which the accurate prediction of the functional prognosis is urgently needed. Due to the lack of reliable prediction methods, the acute evaluation of SCI severity and therapeutic intervention efficacy is extremely difficult, presenting major obstacles to the development of acute SCI treatment. We herein report a novel method for accurately predicting the functional prognosis using the acute-phase serum zinc concentration after SCI. METHODS: We produced experimental animal SCI models with different prognoses and examined the relationship among the SCI severity, functional outcome, and acute-phase serum zinc concentration. We also examined whether we could predict the functional prognosis by evaluating the serum zinc concentration within 72 h after SCI in a human prospective study. FINDINGS: In a mouse model, the acute serum zinc concentrations decreased in proportion to SCI severity and the serum zinc concentrations at 12 h after SCI accurately predicted the functional prognosis. We clarified the mechanism underlying this serum zinc proportional decrease, showing that activated monocytes took up zinc from blood-serum and then infiltrated the lesion area in a severity-dependent manner. A non-linear regression analysis of 38 SCI patients showed that the serum zinc concentrations in the acute-phase accurately predicted the long-term functional outcome (R2 = 0·84) more accurately than any other previously reported acute-phase biomarkers. INTERPRETATION: The acute-phase serum zinc concentration could be a useful biomarker for predicting the functional prognosis. This simple method will allow for more objective clinical trials and the development of patient-tailored treatment for SCI.

Pathological changes of distal motor neurons after complete spinal cord injury.

Traumatic spinal cord injury (SCI) causes serious disruption of neuronal circuits that leads to motor functional deficits. Regeneration of disrupted circuits back to their original target is necessary for the restoration of function after SCI, but the pathophysiological condition of the caudal spinal cord has not been sufficiently studied. Here we investigated the histological and biological changes in the distal part of the injured spinal cord, using a mice model of complete thoracic SCI in the chronic stage (3 months after injury). Atrophic changes were widely observed in the injured spinal cord both rostral and caudal to the lesion, but the decrease in area was mainly in the white matter in the rostral spinal cord while both the white and gray matter decreased in the caudal spinal cord. The number of the motor neurons was maintained in the chronic phase of injury, but the number of presynaptic boutons decreased in the lumbar motor neurons caudal to the lesion. Using laser microdissection, to investigate gene expressions in motor neurons caudal to the lesion, we observed a decrease in the expressions of neuronal activity markers. However, we found that the synaptogenic potential of postsynapse molecules was maintained in the motor neurons after SCI with the expression of acetylcholine-related molecules actually higher after SCI. Collectively, our results show that the potential of synaptogenesis is maintained in the motor neurons caudal to the lesion, even though presynaptic input is decreased. Although researches into SCI concentrate their effort on the lesion epicenter, our findings suggest that the area caudal to the lesion could be an original therapeutic target for the chronically injured spinal cord.

Periostin Promotes Fibroblast Migration and Inhibits Muscle Repair After Skeletal Muscle Injury.

BACKGROUND: Skeletal muscle injury (SMI) can cause physical disability due to insufficient recovery of the muscle. The development of muscle fibrosis after SMI has been widely regarded as a principal cause of this failure to recover. Periostin (Postn) exacerbates tissue fibrosis in various organs. We investigated whether Postn is involved in the pathophysiology after SMI. METHODS: Partial laceration injuries of the gastrocnemius were created in wild-type (WT) and Postn knockout (Postn) mice. We examined the expression of the Postn gene before and after SMI. Regeneration and fibrosis of skeletal muscle were evaluated by histological analyses, and recovery of muscle strength was measured by physiological testing. Immunohistochemistry was used to examine the number and proliferative potential of infiltrating fibroblasts in injured muscle. A trans-well migration assay was used to assess the migration capability of fibroblasts. Control immunoglobulin G (IgG) or Postn-neutralizing antibody (Postn-nAb) was injected into injured muscle at 7 and 14 days after injury (dpi). We evaluated the effects of Postn-nAb on muscle repair after SMI. RESULTS: The expression of Postn was dramatically upregulated after SMI. Compared with WT mice, Postn mice had improved muscle recovery and attenuated fibrosis as well as a significantly reduced number of infiltrating fibroblasts. The proliferative potential of these fibroblasts in WT and Postn mice was comparable at 14 dpi; however, the migration capability of fibroblasts was significantly enhanced in the presence of Postn (mean, 258%; 95% confidence interval, 183% to 334%). Moreover, the administration of Postn-nAb inhibited fibroblast infiltration and promoted muscle repair after SMI. CONCLUSIONS: Postn exacerbates fibrotic scar formation through the promotion of fibroblast migration into injured muscle after SMI. Treatment with Postn-nAb is effective for attenuating fibrosis and improving muscle recovery after SMI. CLINICAL RELEVANCE: Our findings may provide a potential therapeutic strategy to enhance muscle repair and functional recovery after SMI.

Macrophage Infiltration Is a Causative Factor for Ligamentum Flavum Hypertrophy through the Activation of Collagen Production in Fibroblasts.

Ligamentum flavum (LF) hypertrophy causes lumbar spinal canal stenosis, leading to leg pain and disability in activities of daily living in elderly individuals. Although previous studies have been performed on LF hypertrophy, its pathomechanisms have not been fully elucidated. In this study, we demonstrated that infiltrating macrophages were a causative factor for LF hypertrophy. Induction of macrophages into the mouse LF by applying a microinjury resulted in LF hypertrophy along with collagen accumulation and fibroblasts proliferation at the injured site, which were very similar to the characteristics observed in the severely hypertrophied LF of human. However, we found that macrophage depletion by injecting clodronate-containing liposomes counteracted LF hypertrophy even with microinjury. For identification of fibroblasts in the LF, we used collagen type I α2 linked to green fluorescent protein transgenic mice and selectively isolated green fluorescent protein-positive fibroblasts from the microinjured LF using laser microdissection. A quantitative RT-PCR on laser microdissection samples revealed that the gene expression of collagen markedly increased in the fibroblasts at the injured site with infiltrating macrophages compared with the uninjured location. These results suggested that macrophage infiltration was crucial for LF hypertrophy by stimulating collagen production in fibroblasts, providing better understanding of the pathophysiology of LF hypertrophy.

Interaction of reactive astrocytes with type I collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury.

Central nervous system (CNS) injury transforms naive astrocytes into reactive astrocytes, which eventually become scar-forming astrocytes that can impair axonal regeneration and functional recovery. This sequential phenotypic change, known as reactive astrogliosis, has long been considered unidirectional and irreversible. However, we report here that reactive astrocytes isolated from injured spinal cord reverted in retrograde to naive astrocytes when transplanted into a naive spinal cord, whereas they formed astrocytic scars when transplanted into injured spinal cord, indicating the environment-dependent plasticity of reactive astrogliosis. We also found that type I collagen was highly expressed in the spinal cord during the scar-forming phase and induced astrocytic scar formation via the integrin-N-cadherin pathway. In a mouse model of spinal cord injury, pharmacological blockade of reactive astrocyte-type I collagen interaction prevented astrocytic scar formation, thereby leading to improved axonal regrowth and better functional outcomes. Our findings reveal environmental cues regulating astrocytic fate decisions, thereby providing a potential therapeutic target for CNS injury.

Periostin Promotes Scar Formation through the Interaction between Pericytes and Infiltrating Monocytes/Macrophages after Spinal Cord Injury.

Scar formation is a prominent pathological feature of traumatic central nervous system (CNS) injury, which has long been implicated as a major impediment to the CNS regeneration. However, the factors affecting such scar formation remain to be elucidated. We herein demonstrate that the extracellular matrix protein periostin (POSTN) is a key player in scar formation after traumatic spinal cord injury (SCI). Using high-throughput RNA sequencing data sets, we found that the genes involved in the extracellular region, such as POSTN, were significantly expressed in the injured spinal cord. The expression of POSTN peaked at 7 days after SCI, predominantly in the scar-forming pericytes. Notably, we found that genetic deletion of POSTN in mice reduced scar formation at the lesion site by suppressing the proliferation of the pericytes. Conversely, we found that recombinant POSTN promoted the migration capacity of the monocytes/macrophages and increased the expression of tumor necrosis factor-α from the monocytes/macrophages in vitro, which facilitated the proliferation of pericytes. Furthermore, we revealed that the pharmacological blockade of POSTN suppressed scar formation and improved the long-term functional outcome after SCI. Our findings suggest a potential mechanism whereby POSTN regulates the scar formation after SCI and provide significant evidence that POSTN is a promising therapeutic target for CNS injury.