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BACKGROUND: Vascular endothelial cells are pivotal in the pathophysiological progression following spinal cord injury (SCI). The UTX (Ubiquitously Transcribed Tetratripeptide Repeat on Chromosome X) serves as a significant regulator of endothelial cell phenotype. The manipulation of endogenous neural stem cells (NSCs) offers a compelling strategy for the amelioration of SCI. METHODS: Two mouse models were used to investigate SCI: NSCs lineage-traced mice and mice with conditional UTX knockout (UTX KO) in endothelial cells. To study the effects of UTX KO on neural differentiation, we harvested extracellular vesicles (EVs) from both UTX KO spinal cord microvascular endothelial cells (SCMECs) and negative control SCMECs. These EVs were then employed to modulate the differentiation trajectory of endogenous NSCs in the SCI model. RESULTS: In our NSCs lineage-traced mice model of SCI, a marked decrease in neurogenesis was observed post-injury. Notably, NSCs in UTX KO SCMECs mice showed enhanced neuronal differentiation compared to controls. RNA sequencing and western blot analyses revealed an upregulation of L1 cell adhesion molecule (L1CAM), a gene associated with neurogenesis, in UTX KO SCMECs and their secreted EVs. This aligns with the observed promotion of neurogenesis in UTX KO conditions. In vivo administration of L1CAM-rich EVs from UTX KO SCMECs (KO EVs) to the mice significantly enhanced neural differentiation. Similarly, in vitro exposure of NSCs to KO EVs resulted in increased activation of the Akt signaling pathway, further promoting neural differentiation. Conversely, inhibiting Akt phosphorylation or knocking down L1CAM negated the beneficial effects of KO EVs on NSC neuronal differentiation. CONCLUSIONS: In conclusion, our findings substantiate that EVs derived from UTX KO SCMECs can act as facilitators of neural differentiation following SCI. This study not only elucidates a novel mechanism but also opens new horizons for therapeutic interventions in the treatment of SCI. Video Abstract.
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Vesículas Extracelulares , Molécula L1 de Adesão de Célula Nervosa , Células-Tronco Neurais , Traumatismos da Medula Espinal , Animais , Camundongos , Diferenciação Celular , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Molécula L1 de Adesão de Célula Nervosa/farmacologia , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapiaRESUMO
Spinal cord injury (SCI) can prompt an immediate disruption to the blood-spinal cord barrier (BSCB). Restoring the integrity of this barrier is vital for the recovery of neurological function post-SCI. The UTX protein, a histone demethylase, has been shown in previous research to promote vascular regeneration and neurological recovery in mice with SCI. However, it is unclear whether UTX knockout could facilitate the recovery of the BSCB by reducing its permeability. In this study, we systematically studied BSCB disruption and permeability at different time points after SCI and found that conditional UTX deletion in endothelial cells (ECs) can reduce BSCB permeability, decrease inflammatory cell infiltration and ROS production, and improve neurological function recovery after SCI. Subsequently, we used RNA sequencing and ChIP-qPCR to confirm that conditional UTX knockout in ECs can down-regulate expression of myosin light chain kinase (MLCK), which specifically mediates myosin light chain (MLC) phosphorylation and is involved in actin contraction, cell retraction, and tight junctions (TJs) protein integrity. Moreover, we found that MLCK overexpression can increase the ratio of p-MLC/MLC, further break TJs, and exacerbate BSCB deterioration. Overall, our findings indicate that UTX knockout could inhibit the MLCK/p-MLC pathway, resulting in decreased BSCB permeability, and ultimately promoting neurological recovery in mice. These results suggest that UTX is a promising new target for treating SCI.
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Cadeias Leves de Miosina , Traumatismos da Medula Espinal , Animais , Camundongos , Ratos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Histona Desmetilases/genética , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Permeabilidade , Fosforilação , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
Macrophages polarized to the M2 subtype after spinal cord injury (SCI) are beneficial for promoting neurological recovery. The crosstalk between endothelial cells (ECs) and macrophages is crucial for the imbalance between proinflammatory and pro-resolving responses caused by macrophage heterogeneity; however, this crosstalk is strengthened post-SCI, leading to inflammatory cascades and second damage. As a powerful means to regulate gene expression, epigenetic regulation of the interaction between immune cells and ECs in SCI is still largely unknown. Our previous research demonstrated that the histone demethylase UTX deletion in ECs (UTX-/- ECs) promotes neurological recovery, while the precise mechanism is unrevealed. Here, we discovered that UTX-/- ECs polarize macrophages toward the M2 subtype post-SCI. Macrophage deficiency could block the neurological recovery caused by the knockdown of UTX. The exosomes from UTX-/- ECs mediate this crosstalk. In addition, we found UTX, H3K27, and miR-467b-3p/Sfmbt2 promoters forming a regulatory complex that upregulates the miR-467b-3p in UTX-/- ECs. And then, miR-467b-3p transfers to macrophages by exosomes and activates the PI3K/AKT/mTOR signaling by decreasing PTEN expression, finally polarizing macrophage to the M2 subtype. This study reveals a mechanism by epigenetic regulation of ECs-macrophages crosstalk and identifies potential targets, which may provide opportunities for treating SCI.
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MicroRNAs , Traumatismos da Medula Espinal , Humanos , Células Endoteliais/metabolismo , Epigênese Genética , Fosfatidilinositol 3-Quinases/metabolismo , Macrófagos/metabolismo , Traumatismos da Medula Espinal/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Exploring the three-dimensional (3D) morphology of neurons is essential to understanding spinal cord function and associated diseases comprehensively. However, 3D imaging of the neuronal network in the broad region of the spinal cord at cellular resolution remains a challenge in the field of neuroscience. In this study, to obtain high-resolution 3D imaging of a detailed neuronal network in the mass of the spinal cord, the combination of synchrotron radiation micro-computed tomography (SRµCT) and the Golgi-cox staining were used. We optimized the Golgi-Cox method (GCM) and developed a modified GCM (M-GCM), which improved background staining, reduced the number of artefacts, and diminished the impact of incomplete vasculature compared to the current GCM. Moreover, we achieved high-resolution 3D imaging of the detailed neuronal network in the spinal cord through the combination of SRµCT and M-GCM. Our results showed that the M-GCM increased the contrast between the neuronal structure and its surrounding extracellular matrix. Compared to the GCM, the M-GCM also diminished the impact of the artefacts and incomplete vasculature on the 3D image. Additionally, the 3D neuronal architecture was successfully quantified using a combination of SRµCT and M-GCM. The SRµCT was shown to be a valuable non-destructive tool for 3D visualization of the neuronal network in the broad 3D region of the spinal cord. Such a combinatorial method will, therefore, transform the presentation of Golgi staining from 2 to 3D, providing significant improvements in the 3D rendering of the neuronal network.
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Complexo de Golgi/química , Imageamento Tridimensional , Neurônios/citologia , Medula Espinal/citologia , Coloração e Rotulagem , Microtomografia por Raio-X , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , SíncrotronsRESUMO
The regeneration of the blood vessel system post spinal cord injury (SCI) is essential for the repair of neurological function. As a significant means to regulate gene expression, epigenetic regulation of angiogenesis in SCI is still largely unknown. Here, we found that Ubiquitously Transcribed tetratricopeptide repeat on chromosome X (UTX), the histone H3K27 demethylase, increased significantly in endothelial cells post SCI. Knockdown of UTX can promote the migration and tube formation of endothelial cells. The specific knockout of UTX in endothelial cells enhanced angiogenesis post SCI accompanied with improved neurological function. In addition, we found regulation of UTX expression can change the level of microRNA 24 (miR-24) in vitro. The physical binding of UTX to the promotor of miR-24 was indicated by chromatin immunoprecipitation (ChIP) assay. Meanwhile, methylation sequencing of endothelial cells demonstrated that UTX could significantly decrease the level of methylation in the miR-24 promotor. Furthermore, miR-24 significantly abolished the promoting effect of UTX deletion on angiogenesis in vitro and in vivo. Finally, we predicted the potential target mRNAs of miR-24 related to angiogenesis. We indicate that UTX deletion can epigenetically promote the vascular regeneration and functional recovery post SCI by forming a regulatory network with miR-24.
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Células Endoteliais/citologia , Epigênese Genética , Deleção de Genes , Histona Desmetilases/fisiologia , Regeneração , Traumatismos da Medula Espinal/terapia , Animais , Movimento Celular , Proliferação de Células , Metilação de DNA , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Regiões Promotoras Genéticas , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologiaRESUMO
There has been increasing interest in using high-resolution micro-tomography to investigate the morphology of neurovascular networks in the central nervous system, which remain difficult to characterize due to their microscopic size as well as their delicate and complex 3D structure. Synchrotron radiation X-ray imaging, which has emerged as a cutting-edge imaging technology with a high spatial resolution, provides a novel platform for the non-destructive imaging of microvasculature networks at a sub-micrometre scale. When coupled with computed tomography, this technique allows the characterization of the 3D morphology of vasculature. The current review focuses on recent progress in developing synchrotron radiation methodology and its application in probing neurovascular networks, especially the pathological changes associated with vascular abnormalities in various model systems. Furthermore, this tool represents a powerful imaging modality that improves our understanding of the complex biological interactions between vascular function and neuronal activity in both physiological and pathological states.
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Sistema Nervoso Central/irrigação sanguínea , Microvasos/diagnóstico por imagem , Síncrotrons , Microtomografia por Raio-X/métodos , Animais , HumanosRESUMO
STUDY DESIGN: Experimental animal study. OBJECTIVES: To validate the anti-apoptosis effect of microRNA-21 in neurons after spinal cord injury (SCI) and explore the mechanism. SETTING: Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China. METHODS: In situ hybridization was used to detect the expression of miR-21 in spinal cord neurons (n = 24). In a rat contusion SCI model (n = 48), we upregulated the miR-21 level around the injured area using miR-21 lentiviral vectors and evaluated the therapeutic effect with histology and behavioural scores. In neuronal cells, oxygen-glucose deprivation (OGD) was exerted to imitate SCI, and we explored the biomechanism using molecular biology and a dual-luciferase reporter assay. RESULTS: miR-21 was expressed in spinal cord neurons and was found to improve neuronal survival and promote functional recovery in rat SCI models. The in vitro results in PC-12 cells revealed that the augmentation of endogenous miR-21 was able to reduce neuronal cell death after OGD. In addition, overexpression of miR-21 was able to reduce cellular apoptosis via decreasing PDCD4 protein levels, and caspase-3 activity was also influenced. Transfection of miR-21 into 293T cells was able to decrease luciferase activity in a reporter assay system, including the 3' untranslated region of PDCD4. CONCLUSIONS: miR-21 may have a protective role in neuronal apoptosis after SCI. PDCD4 may be a functional target gene involved in the miR-21-mediated anti-apoptotic effect through an miR-21/PDCD4/caspase-3 pathway.
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MicroRNAs/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Regulação para Cima/fisiologia , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Contagem de Células , Modelos Animais de Doenças , Hipóxia/patologia , Hipóxia/terapia , Masculino , MicroRNAs/genética , Neurônios Motores , Oxigênio/uso terapêutico , Células PC12 , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Transdução Genética , TransfecçãoRESUMO
Spinal cord injury (SCI) causes severe axon damage, usually leading to permanent paraparesis, which still lacks effective regenerative therapy. Recent studies have suggested that exosomes derived from neural stem cells (NSCs) may hold promise as attractive candidates for SCI treatment. Epidermal Growth Factor Receptor positive NSC (EGFR+NSC) is a subpopulation of endogenous NSCs, showing strong regenerative capability in central nervous system disease. In the current study, we isolated exosomes from the EGFR+NSCs (EGFR+NSCs-Exos) and discovered that local delivery of EGFR+NSCs-Exos can effectively promote neurite regrowth in the injury site of spinal cord-injured mice and improve their neurological function recovery. Using the miRNA-seq, we firstly characterized the microRNAs (miRNAs) cargo of EGFR+NSCs-Exos and identified miR-34a-5p which was highly enriched in EGFR+NSCs derived exosomes. We further interpreted that exosomal miR-34a-5p could be transferred to neurons and inhibit the HDAC6 expression by directly binding to its mRNA, contributing to microtubule stabilization and autophagy induction for aiding SCI repair. Overall, our research demonstrated a novel therapeutic approach to improving neurological functional recovery by using exosomes secreted from a subpopulation of endogenous NSCs and providing a precise cell-free treatment strategy for SCI repair.
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Human neural progenitor cells (hNPCs) hold promise for treating spinal cord injury. Studies to date have focused on improving their regenerative potential and therapeutic effect. Equally important is ensuring successful delivery and engraftment of hNPCs at the injury site. Unfortunately, no current imaging solution for cell tracking is compatible with long-term monitoring in vivo. The objective of this study was to apply a novel bright-ferritin magnetic resonance imaging (MRI) mechanism to track hNPC transplants longitudinally and on demand in the rat spinal cord. We genetically modified hNPCs to stably overexpress human ferritin. Ferritin-overexpressing (FT) hNPCs labeled with 0.2 mM manganese provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, morphology, proliferation, and differentiation. In vivo, 2 M cells were injected into the cervical spinal cord of Rowett nude rats. MRI employed T1-weighted acquisitions and T1 mapping on a 3 T scanner. Conventional short-term cell tracking was performed using exogenous Mn labeling prior to cell transplantation, which displayed transient bright contrast on MRI 1 day after cell transplantation and disappeared after 1 week. In contrast, long-term cell tracking using bright-ferritin allowed on-demand signal recall upon Mn supplementation and precise visualization of the surviving hNPC graft. In fact, this new cell tracking technology identified 7 weeks post-transplantation as the timepoint by which substantial hNPC integration occurred. Spatial distribution of hNPCs on MRI matched that on histology. In summary, bright-ferritin provides the first demonstration of long-term, on-demand, high-resolution, and specific tracking of hNPCs in the rat spinal cord.
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Rastreamento de Células , Ferritinas , Imageamento por Ressonância Magnética , Células-Tronco Neurais , Ratos Nus , Medula Espinal , Animais , Imageamento por Ressonância Magnética/métodos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/transplante , Células-Tronco Neurais/metabolismo , Rastreamento de Células/métodos , Humanos , Ratos , Ferritinas/metabolismo , Medula Espinal/metabolismo , Medula Espinal/diagnóstico por imagem , Transplante de Células-Tronco/métodos , Diferenciação Celular , Traumatismos da Medula Espinal/terapiaRESUMO
Electrical neuromodulation as a palliative treatment has been increasingly used in the control of epilepsy. However, current neuromodulations commonly implement predetermined actuation strategies and lack the capability of self-adaptively adjusting stimulation inputs. In this work, rooted in optimal control theory, we propose a Koopman-MPC framework for real-time closed-loop electrical neuromodulation in epilepsy, which integrates i) a deep Koopman operator based dynamical model to predict the temporal evolution of epileptic electroencephalogram (EEG) with an approximate finite-dimensional linear dynamics and ii) a model predictive control (MPC) module to design optimal seizure suppression strategies. The Koopman operator based linear dynamical model is embedded in the latent state space of the autoencoder neural network, in which we can approximate and update the Koopman operator online. The linear dynamical property of the Koopman operator ensures the convexity of the optimization problem for subsequent MPC control. The proposed deep Koopman operator model shows greater predictive capability than the baseline models (e.g., vector autoregressive model, kernel based method and recurrent neural network (RNN)) in both synthetic and real epileptic EEG data. Moreover, compared with the RNN-MPC framework, our Koopman-MPC framework can suppress seizure dynamics with better computational efficiency in both the Jansen-Rit model and the Epileptor model. Koopman-MPC framework opens a new window for model-based closed-loop neuromodulation and sheds light on nonlinear neurodynamics and feedback control policies.
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Educação a Distância , Epilepsia , Humanos , Redes Neurais de Computação , Convulsões , RetroalimentaçãoRESUMO
After T5 (forging + aging) and different T6 (forging + solution + aging) heat treatments, the AZ80A Mg alloys exhibited microstructures with different fractions of continuous precipitate (CP) regions and discontinuous precipitate (DP) regions. The effects of the fractions of DP regions and CP regions on the tensile properties and fracture behaviors were investigated using microstructural characterizations and analysis. The results showed that increasing the fraction of DP regions enhanced the yield strength and tensile strength at room temperature. However, at the same high temperature, increasing the fractions of DP regions improved the elongation but deteriorated the tensile strength significantly. The different resultant tensile properties at different temperatures were caused by the different precipitation-strengthening effects in the CP and DP regions. The strengthening contribution of the DP regions was more effective at room temperature but became inferior to the effect brought about by the CP regions at high temperatures. Micro-cracks were usually initiated and propagated in the CP regions at room temperature. At high temperatures, however, micro-voids formed more easily in the DP regions, and the fracture path preferred to locate there.
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The microstructure, interfacial characteristics, and corrosion resistance of Fe-W-Mn-Al-B alloys in molten zinc at 520 °C have been investigated using scanning electron microscopy (SEM), X-ray diffractometry (XRD), and electron probe micro-analysis (EPMA). The experimental results indicate that the Fe-B alloy with 11 wt.% W, 7 wt.% Mn, and 4 wt.% Al addition displays a lamellar eutectic microstructure and excellent corrosion resistance to molten zinc. The toughness of M2B-type borides in the hyper-eutectic Fe-4.2B-11W-7Mn-4Al alloy can be more than doubled, reaching 10.5 MPa·m1/2, by adding Mn and Al. The corrosion layer of the Fe-3.5B-11W-7Mn-4Al alloy immersed in molten zinc at 520 °C comprises Fe3AlZnx, δ-FeZn10, ζ-FeZn13, and η-Zn. The lamellar borides provide the mechanical protection for α-(Fe, Mn, Al), and the thermal stability of borides improves as the fracture toughness of the borides increases, which jointly contribute to the improvement of the corrosion resistance to the molten zinc.
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The m6A methylation is reported to function in multiple physiological and pathological processes. However, the functional relevance of m6A modification to post-spinal cord injured (SCI) damage is not yet clear. In the present study, methylated RNA immunoprecipitation combined with microarray analysis showed that the global RNA m6A levels were decreased following SCI. Then, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses were conducted to demonstrate the potential function of differential m6A-tagged transcripts and the altered transcripts with differential m6A levels. In addition, we found that the m6A "writer," METTL3, significantly decreased after SCI in mice. The immunostaining validated that the expression of METTL3 mainly changed in GFAP or Iba-1+ cells. Together, this study shows the alteration of m6A modification following SCI in mice, which might contribute to the pathophysiology of the spinal cord after trauma.
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Dimensionality reduction is crucial for the visualization and interpretation of the high-dimensional single-cell RNA sequencing (scRNA-seq) data. However, preserving topological structure among cells to low dimensional space remains a challenge. Here, we present the single-cell graph autoencoder (scGAE), a dimensionality reduction method that preserves topological structure in scRNA-seq data. scGAE builds a cell graph and uses a multitask-oriented graph autoencoder to preserve topological structure information and feature information in scRNA-seq data simultaneously. We further extended scGAE for scRNA-seq data visualization, clustering, and trajectory inference. Analyses of simulated data showed that scGAE accurately reconstructs developmental trajectory and separates discrete cell clusters under different scenarios, outperforming recently developed deep learning methods. Furthermore, implementation of scGAE on empirical data showed scGAE provided novel insights into cell developmental lineages and preserved inter-cluster distances.
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Visualização de Dados , RNA-Seq/métodos , Análise de Célula Única/métodos , Mineração de Dados/métodos , Processamento Eletrônico de Dados/métodos , Análise de Sequência de RNA/métodosRESUMO
Traumatic spinal cord injury (SCI) is a devastating disease of the central nervous system with long-term disability and high mortality worldwide. Revascularization following SCI provides nutritional supports to rebuild and maintain the homeostasis of neuronal networks, and the subsequent promotion of angiogenesis is beneficial for functional recovery. Oxidative stress drastically produced following SCI has been contributed to endothelial dysfunction and the limited endogenous repair of microvasculature. Recently, exosomes, being regarded as potential therapeutic candidates for many kinds of diseases, have attracted great attentions due to its high bioavailability, safety, and stability. Microglia have been reported to exhibit proangiogenic function and guide the forming of vasculature during tissue repair. However, the specific role of microglia-derived exosomes (MG-Exos) played in SCI is still largely unknown. In the present study, we aimed to evaluate whether MG-Exos could protect spinal cord microvascular endothelial cells (SCMECs) against the toxic effects of oxidative stress, thus promote SCMECs' survival and function. We also investigated the protective effects of MG-Exos in the mouse model of SCI to verify their capability. Our results demonstrated that MG-Exo treatment significantly decreased the level of oxidative stress (ROS), as well as did the protein levels of NOX2 when bEnd.3 cells were exposed to H2O2-induced oxidative stress in vitro and in vivo. Functional assays showed that MG-Exos could improve the survival and the ability of tube formation and migration in H2O2-induced bEnd.3 in vitro. Moreover, MG-Exos exhibited the positive effects on vascular regeneration and cell proliferation, as well as functional recovery, in the mouse model of SCI. Mechanically, the keap1/Nrf2/HO-1 signaling pathway was also investigated in order to unveil its molecular mechanism, and the results showed that MG-Exos could increase the protein levels of Nrf2 and HO-1 via inhibiting the keap1; they also triggered the expression of its downstream antioxidative-related genes, such as NQo1, Gclc, Cat, and Gsx1. Our findings indicated that MG-Exos exerted an antioxidant effect and positively modulated vascular regeneration and neurological functional recovery post-SCI by activating keap1/Nrf2/HO-1 signaling.
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Células Endoteliais/metabolismo , Exossomos/metabolismo , Microglia/metabolismo , Estresse Oxidativo/imunologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Humanos , CamundongosRESUMO
Vascularization following spinal cord injury (SCI) provides trophic support for rebuilding up and maintaining the homeostasis of neuronal networks, and the promotion of angiogenesis is beneficial for functional recovery after SCI. M2 macrophages have been reported to exhibit powerful pro-angiogenic functions during tissue repair. Exosomes are important paracrine mediators of their parent cells and play critical roles in tissue regeneration. However, the role of M2 macrophage-derived exosomes (M2-Exos) in SCI is still largely unknown. In the present study, we determined that M2-Exos could augment the angiogenic activities of spinal cord microvascular endothelial cells (SCMECs) in vitro. Hydrogel-mediated sustained release of M2-Exos significantly promoted vascular regeneration and functional recovery in mice after SCI. Furthermore, proteomics analysis showed that ubiquitin thioesterase otulin (OTULIN) protein was highly enriched in M2-Exos. Functional assays demonstrated that OTULIN protein was required for the M2-Exos-induced pro-angiogenic effects in SCMECs, as well as positive effects on vascular regeneration, cell proliferation, and functional recovery in the mouse model of SCI. Mechanically, OTULIN from M2-Exos could activate the Wnt/ß-catenin signaling by increasing the protein level of ß-catenin via inhibiting its ubiquitination and trigger the expression of angiogenesis-related genes that are reported to be the downstream targets of Wnt/ß-catenin signaling. Inhibition of the Wnt/ß-catenin signaling by ICG001 markedly attenuated the pro-angiogenic activities of M2-Exos in vitro/vivo. Our findings indicate that M2-Exos positively modulate vascular regeneration and neurological functional recovery after SCI by activating Wnt/ß-catenin signaling through the transfer of OTULIN protein. STATEMENT OF SIGNIFICANCE: M2 macrophages have been identified to promote vascular regeneration, cell proliferation and tissue growth after spinal cord injury (SCI), which is beneficial to the functional recovery. Exosomes are essential paracrine mediators involved in cell-to-cell communication and play important roles in tissue regeneration. In the present study, we revealed that M2 macrophages-derived exosomes (M2-Exos) could promote functional recovery post SCI by targeting angiogenesis. We demonstrated for the first time that OTULIN protein from M2-Exos mediated the angiogenic effects through activating Wnt/ß-catenin signaling and triggering the expression of angiogenic-related genes in spinal cord microvascular endothelial cells (SCMECs). The hydrogel-M2-Exos sustained released system provides potential therapeutic clues of local cell-free interventions for the treatment of SCI.
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Regeneração , Traumatismos da Medula Espinal , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Apoptose , Células Endoteliais , Macrófagos , Camundongos , Traumatismos da Medula Espinal/terapiaRESUMO
Spinal cord injury (SCI) is a catastrophic event mainly involving neuronal apoptosis and axonal disruption, and it causes severe motor and sensory deficits. Due to the complicated pathological process of SCI, there is currently still a lack of effective treatment for SCI. Microglia, a type of immune cell residing in the central nervous system (CNS), need to respond to various stimuli to protect neuronal cells from death. It was also reported that microRNAs (miRNAs) had been identified in microglia-derived exosomes that can be taken up by neurons. However, the kinds of miRNAs in exosome cargo derived from microglia and the underlying mechanisms by which they contribute to neuroprotection after SCI remain unknown. In the present study, a contusive SCI mouse model and in vitro experiments were applied to explore the therapeutic effects of microglia-derived exosomes on neuronal apoptosis, axonal regrowth, and functional recovery after SCI. Then, miRNA analysis, rescue experiments, and luciferase activity assays for target genes were performed to confirm the role and underlying mechanism of microglia-derived exosomal miRNAs in SCI. We revealed that microglia-derived exosomes could promote neurological functional recovery by suppressing neuronal apoptosis and promoting axonal regrowth both in vivo and in vitro. MicroRNA-151-3p is abundant in microglia-derived exosomes and is necessary for mediating the neuroprotective effect of microglia-derived exosomes for SCI repair. Luciferase activity assays reported that P53 was the target gene for miR-151-3p and that p53/p21/CDK1 signaling cascades may be involved in the modulation of neuronal apoptosis and axonal regrowth by microglia-derived exosomal microRNA-151-3p. In conclusion, our data demonstrated that microglia-derived exosomes (microglia-Exos) might be a promising, cell-free approach for the treatment of SCI. MicroRNA-151-3p is the key molecule in microglia-derived exosomes that mediates the neuroprotective effects of SCI treatments.
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Interrupted axons that fail to regenerate mainly cause poor recovery after spinal cord injury (SCI). How neurons epigenetically respond to injury determines the intrinsic growth ability of axons. However, the mechanism underlying epigenetic regulation of axonal regeneration post-SCI remains largely unknown. In this study, we elucidated the role of the epigenetic regulatory network involving ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX)/microRNA-24 (miR-24)/NeuroD1 in axonal regeneration and functional recovery in mice following SCI. Our results showed that UTX was significantly increased post-SCI and repressed axonal regeneration in vitro. However, downregulation of UTX remarkably promoted axonal regeneration. Furthermore, miR-24 was increased post-SCI and positively regulated by UTX. miR-24 also inhibited axonal regeneration. Chromatin immunoprecipitation (ChIP) indicated that UTX binds to the miR-24 promoter and regulates miR-24 expression. Genome sequencing and bioinformatics analysis suggested that NeuroD1 is a potential downstream target of UTX/miR-24. A dual-luciferase reporter assay indicated that miR-24 binds to NeuroD1; moreover, it represses axonal regeneration by negatively regulating the expression of NeuroD1 via modulation of microtubule stability. UTX deletion in vivo prominently promoted axonal regeneration and improved functional recovery post-SCI, and silencing NeuroD1 restored UTX function. Our findings indicate that UTX could be a potential target in SCI.
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The complex pathology of chronic thoracic spinal cord compression involves vascular and neuroarchitectural repair processes that are still largely unknown. In this study, we used synchrotron radiation microtomography (SRµCT) to quantitatively characterize the 3D temporal-spatial changes in the vascular and neuronal network after chronic thoracic spinal cord compression in order to obtain further insights into the pathogenesis of this disease and to elucidate its underlying mechanisms. Direct 3D characterization of the spinal cord microvasculature and neural microstructure of the thoracic spinal cord was successfully reconstructed. The significant reduction in vasculature and degeneration of neurons in the thoracic spinal cord visualized via SRµCT after chronic compression were consistent with the changes detected by immunofluorescence staining. The 3D morphological measurements revealed significant reductions of neurovascular parameters in the thoracic spinal cord after 1 month of compression and became even worse after 6 months without relief of compression. In addition, the distinct 3D morphological twist and the decrease in branches of the central sulcal artery after chronic compression vividly displayed that these could be the potential triggers leading to blood flow reduction and neural deficits of the thoracic spinal cord. Our findings propose a novel methodology for the 3D analysis of neurovascular repair in chronic spinal cord compression, both qualitatively and quantitatively. The results indicated that compression simultaneously caused vascular dysfunction and neuronal network impairment, which should be acknowledged as concurrent events after chronic thoracic spinal cord injury. Combining neuroprotection with vasoprotection may provide promising therapeutic targets for chronic thoracic spinal cord compression.
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Neuropathic pain (NP) is among the most intractable comorbidities of spinal cord injury. Dysregulation of non-coding RNAs has also been implicated in the development of neuropathic pain. Here, we identified a novel lncRNA, PKIA-AS1, by using lncRNA array analysis in spinal cord tissue of spinal nerve ligation (SNL) model rats, and investigated the role of PKIA-AS1 in SNL-mediated neuropathic pain. We observed that PKIA-AS1 was significantly upregulated in SNL model rats and that PKIA-AS1 knockdown attenuated neuropathic pain progression. Alternatively, overexpression of PKIA-AS1 was sufficient to induce neuropathic pain-like symptoms in uninjured rats. We also found that PKIA-AS1 mediated SNL-induced neuropathic pain by directly regulating the expression and function of CDK6, which is essential for the initiation and maintenance of neuroinflammation and neuropathic pain. Therefore, our study identifies PKIA-AS1 as a novel therapeutic target for neuroinflammation related neuropathic pain.