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.

The Posterolaterally Oriented and Laterally Downward Sloping Facet Joint Is a Risk Factor for Degenerative Cervical Spondylolisthesis and Myelopathy.

Introduction: Facet joints are anatomical structures that are known to be crucial for determining spinal biomechanical motion; however, the potential relationship between facet orientation and the development of cervical spondylolisthesis remains unclear. Thus, in this study, we aimed to explore the relationship between facet orientation and cervical spondylolisthesis as well as myelopathy. Methods: Facet orientation in the cervical spine was investigated using computed tomography in 103 patients with cervical myelopathy, and facet inclination was measured on axial, coronal, and sagittal reconstructed images. Patients were divided into anterolisthesis, retrolisthesis, and no spondylolisthesis groups at each intervertebral level (C2/3-C6/7 levels). Results: Facet joints in the anterolisthesis and retrolisthesis groups tended to slope posterolaterally and downward laterally compared with those in the no spondylolisthesis group at C3/4, C4/5, and C5/6 levels (P<0.001). Conclusions: The posterolaterally oriented and laterally downward sloping facet at C3/4 and C4/5 levels may be a risk factor for the development of cervical spondylolisthesis as well as symptomatic myelopathy.

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.

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.