Functional Recovery Of Spinal Cord Injury Rats Research Articles

Repeated trans-spinal magnetic stimulation promotes microglial phagocytosis of myelin debris after spinal cord injury through LRP-1

Spinal cord injury (SCI) is a serious trauma of the central nervous system. The clearance of myelin debris is a critical step in the functional recovery following spinal cord injury (SCI). Recent studies have begun to reveal critical roles for professional phagocytes in the central nervous system, microglia, and their receptors in the control of myelin debris in neurodegenerative disease. Repeated trans-spinal magnetic stimulation (rTSMS) has been demonstrated as a noninvasive SCI treatment that enhances tissue repair and functional recovery. In this study, we investigated the role and molecular mechanism of rTSMS on microglial phagocytosis of myelin debris in a rat SCI model. In our studies, we found that rTSMS significantly promoted the motor function recovery of SCI rats associated with the inhibition the neuroinflammation and glia scar formation. Immunofluorescence results further showed that the rTSMS promotes the clearance of myelin debris by microglia in vivo and in vitro. Additionally, receptor-associated protein (RAP), a Low-density lipoprotein receptor-related protein-1 (LRP-1) inhibitor, could cancel the accelerated microglial phagocytosis of myelin debris after rTSMS in vitro experiments. Simultaneously, Elisa's results and western blotting respectively showed that rTSMS significantly decreased the levels of soluble LRP-1(sLRP-1) and the LRP-1 splicing enzyme of ADAM17. In conclusion, rTSMS could promote the clearance of myelin debris by microglia through LRP-1 to improve the functional recovery of SCI rats.

Evaluation of benefits and risks of immunosuppressive drugs in biomaterial-based neural progenitor cell transplantation for spinal cord injury repair

Spinal cord injury (SCI) can disrupt neural connections and impair locomotion and sensation function. Currently, biomaterials containing neural progenitor cells (NPCs) are widely used to replace damaged tissue or provide support for neural regeneration following SCI. Immunosuppressive drugs (ISDs) are commonly utilized to ensure the survival of transplanted NPC in SCI. However, the potential benefits and risks of using ISDs in NPC transplantation with biomaterials for SCI repair has not been deeply studied. Herein, an orderly aligned collagen sponge scaffold (ACSS) combined with human embryonic spinal cord neural progenitor cells (hscNPCs) was transplanted into SCI rats, with or without ISDs. Results showed that ISDs suppressed cell-mediated immune and inflammatory responses, maintained early survival and differentiation capacity of transplanted cells, and promoted early functional recovery of SCI rats. However, ISDs did not influence endogenous nerve regeneration, angiogenesis, glial scarring or long-term functional recovery after ACSS and hscNPC transplantation. Additionally, ISDs can induce toxicity in multiple organ and impact the survival of animals after SCI. Thus, careful evaluation of the benefits and risks is necessary to maximize therapeutic efficiency when administrating ISD in combination with scaffold-loaded NPCs for SCI repair.

Microvascular endothelial cells derived from spinal cord promote spinal cord injury repair.

Neural regeneration after spinal cord injury (SCI) closely relates to the microvascular endothelial cell (MEC)-mediated neurovascular unit formation. However, the effects of central nerve system-derived MECs on neovascularization and neurogenesis, and potential signaling involved therein, are unclear. Here, we established a primary spinal cord-derived MECs (SCMECs) isolation with high cell yield and purity to describe the differences with brain-derived MECs (BMECs) and their therapeutic effects on SCI. Transcriptomics and proteomics revealed differentially expressed genes and proteins in SCMECs were involved in angiogenesis, immunity, metabolism, and cell adhesion molecular signaling was the only signaling pathway enriched of top 10 in differentially expressed genes and proteins KEGG analysis. SCMECs and BMECs could be induced angiogenesis by different stiffness stimulation of PEG hydrogels with elastic modulus 50-1650Pa for SCMECs and 50-300Pa for BMECs, respectively. Moreover, SCMECs and BMECs promoted spinal cord or brain-derived NSC (SNSC/BNSC) proliferation, migration, and differentiation at different levels. At certain dose, SCMECs in combination with the NeuroRegen scaffold, showed higher effectiveness in the promotion of vascular reconstruction. The potential underlying mechanism of this phenomenon may through VEGF/AKT/eNOS- signaling pathway, and consequently accelerated neuronal regeneration and functional recovery of SCI rats compared to BMECs. Our findings suggested a promising role of SCMECs in restoring vascularization and neural regeneration.

Glycine and N-Acetylcysteine (GlyNAC) Combined with Body Weight Support Treadmill Training Improved Spinal Cord and Skeletal Muscle Structure and Function in Rats with Spinal Cord Injury.

Skeletal muscle atrophy is a frequent complication after spinal cord injury (SCI) and can influence the recovery of motor function and metabolism in affected patients. Delaying skeletal muscle atrophy can promote functional recovery in SCI rats. In the present study, we investigated whether a combination of body weight support treadmill training (BWSTT) and glycine and N-acetylcysteine (GlyNAC) could exert neuroprotective effects, promote motor function recovery, and delay skeletal muscle atrophy in rats with SCI, and we assessed the therapeutic effects of the double intervention from both a structural and functional viewpoint. We found that, after SCI, rats given GlyNAC alone showed an improvement in Basso-Beattie-Bresnahan (BBB) scores, gait symmetry, and results in the open field test, indicative of improved motor function, while GlyNAC combined with BWSTT was more effective than either treatment alone at ameliorating voluntary motor function in injured rats. Meanwhile, the results of the skeletal muscle myofiber cross-sectional area (CSA), hindlimb grip strength, and acetylcholinesterase (AChE) immunostaining analysis demonstrated that GlyNAC improved the structure and function of the skeletal muscle in rats with SCI and delayed the atrophication of skeletal muscle.

MiR-145a-5p/Plexin-A2 promotes the migration of OECs and transplantation of miR-145a-5p engineered OECs enhances the functional recovery in rats with SCI

BackgroundOlfactory ensheathing cells (OECs) serve as a bridge by migrating at the site of spinal cord injury (SCI) to facilitate the repair of the neural structure and neural function. However, OEC migration at the injury site not only faces the complex and disordered internal environment but also is closely associated with the migration ability of OECs. MethodsWe extracted OECs from the olfactory bulb of SD rats aged <7 days old. We verified the micro ribonucleic acid (miR)-145a-5p expression level in the gene chip after SCI and OEC transplantation using quantitative reverse transcription (qRT)-polymerase chain reaction (PCR). The possible target gene Plexin-A2 of miR-145a-5p was screened using bioinformatics and was verified using dual-luciferase reporter assay, Western blot, and qRT-PCR. The effect of miR-145a-5p/plexin-A2 on OEC migration ability was verified by wound healing assay, Transwell cell migration assay, and immunohistochemistry. Nerve repair was observed at the injured site of the spinal cord after OEC transplantation using tissue immunofluorescence and magnetic resonance imaging, diffusion tensor imaging, and the Basso–Beattie–Bresnahan locomotor rating scale were further used for imaging and functional evaluation. ResultsmiR-145a-5p expression in the injured spinal cord tissue after SCI considerably decreased, while Plexin-A2 expression significantly increased. OEC transplantation can reverse miR-145a-5p and Plexin-A2 expression after SCI. miR-145a-5p overexpression enhanced the intrinsic migration ability of OECs. As a target gene of miR-145a-5p, Plexin-A2 hinders OEC migration. OEC transplantation overexpressing miR-145a-5p after SCI can increase miR-145a-5p levels in the spinal cord, reduce Plexin-A2 expression in the OECs and the spinal cord tissue, and promote OEC migration and distribution at the injured site. OEC transplantation overexpressing miR-145a-5p can promote the repair of neural morphology and neural function. ConclusionsOur study demonstrated that miR-145a-5p could promote OEC migration by down-regulating the target gene Plexin-A2, and transplantation of miR-145a-5p engineered OECs was beneficial to enhance neural structural and functional recovery in SCI rats.

Baicalein-functionalized collagen scaffolds direct neuronal differentiation toward enhancing spinal cord injury repair.

Spinal cord injury (SCI) repair remains a major challenge in clinics. Though neural stem cells (NSCs) have shown great potentials in SCI treatment, their applications were hampered since they primarily differentiate into astrocytes rather than neurons in the injured area, indicating a high demand for effective strategies to direct neuronal differentiation. Baicalein is a clinical drug with multiple pharmacological activities, while its effects on NSCs have rarely been reported. In the current work, inspired by a similarity of the metabolic reprogramming required in neuronal differentiation and that involved in chemoresistance reversal of cancer cells induced by baicalein, we studied the role of baicalein in NSC differentiation and discovered its promotion effects on neuronal differentiation. Based on this observation, baicalein-functionalized collagen scaffolds (BFCSs) were developed and applied for SCI treatment. The BFCSs released the payload in a sustained way and possessed comparable physical properties to the commonly used collagen. Both in vitro studies with primary NSCs and in vivo studies in SCI rats showed that the BFCSs containing a low amount of baicalein can facilitate not only neurogenesis and axon extension, but also reduce astrocyte production and glial scar formation. More importantly, the BFCS implantation led to improvement in the motor functional recovery of SCI rats. Thus, the BFCSs provided a potential strategy to induce neuronal differentiation towards facilitating SCI repair, as well as for the treatment of other central nervous system injuries.

MiR-30b-5p attenuates the inflammatory response and facilitates the functional recovery of spinal cord injury by targeting the NEFL/mTOR pathway.

Neurofilament light chain (NEFL) has been identified as a biomarker for spinal cord injury (SCI), but its effect and underlying mechanism in SCI remain unclear. SCI rat models were established for in vivo studies. Lipopolysaccharide (LPS)-induced cell models were used for in vitro studies. The protein and mRNA expression levels of genes were evaluated by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The pathological changes in rats after SCI were subjected to histological examinations. The interaction of NEFL and upstream miRNAs was explored using dual-luciferase reporter gene assays. NEFL was highly expressed in SCI rat spinal cord tissues and LPS-stimulated PC12 cells. NEFL silencing showed an inhibitory effect on the morphological changes of SCI rats and the secretion of inflammatory factors and facilitated functional recovery of SCI rats. MiR-30b-5p was demonstrated to target NEFL and negatively regulate NEFL mRNA and protein levels. Downregulation of miR-30b-5p in SCI cell and rat models was demonstrated. MiR-30b-5p alleviated the inflammatory response in SCI rat models and LPS-stimulated PC12 cells and promoted functional recovery in rats by targeting NEFL. NEFL activated mTOR signaling. MiR-30b-5p inactivated mTOR signaling by negatively regulating NEFL. MiR-30b-5p alleviated the inflammatory response and facilitated the functional recovery of SCI rats by targeting NEFL to inactivate the mTOR pathway.

Inhibition of IL1R1 or CASP4 attenuates spinal cord injury through ameliorating NLRP3 inflammasome-induced pyroptosis.

Spinal cord injury (SCI) is a devastating trauma characterized by serious neuroinflammation and permanent neurological dysfunction. However, the molecular mechanism of SCI remains unclear, and few effective medical therapies are available at present. In this study, multiple bioinformatics methods were used to screen out novel targets for SCI, and the mechanism of these candidates during the progression of neuroinflammation as well as the therapeutic effects were both verified in a rat model of traumatic SCI. As a result, CASP4, IGSF6 and IL1R1 were identified as the potential diagnostic and therapeutic targets for SCI by computational analysis, which were enriched in NF-κB and IL6-JAK-STATA3 signaling pathways. In the injured spinal cord, these three signatures were up-regulated and closely correlated with NLRP3 inflammasome formation and gasdermin D (GSDMD) -induced pyroptosis. Intrathecal injection of inhibitors of IL1R1 or CASP4 improved the functional recovery of SCI rats and decreased the expression of these targets and inflammasome component proteins, such as NLRP3 and GSDMD. This treatment also inhibited the pp65 activation into the nucleus and apoptosis progression. In conclusion, our findings of the three targets shed new light on the pathogenesis of SCI, and the use of immunosuppressive agents targeting these proteins exerted anti-inflammatory effects against spinal cord inflammation by inhibiting NF-kB and NLRP3 inflammasome activation, thus blocking GSDMD -induced pyroptosis and immune activation.

A reactive oxygen species-responsive hydrogel encapsulated with bone marrow derived stem cells promotes repair and regeneration of spinal cord injury.

Spinal cord injury (SCI) is an overwhelming and incurable disabling event accompanied by complicated inflammation-related pathological processes, such as excessive reactive oxygen species (ROS) produced by the infiltrated inflammatory immune cells and released to the extracellular microenvironment, leading to the widespread apoptosis of the neuron cells, glial and oligodendroctyes. In this study, a thioketal-containing and ROS-scavenging hydrogel was prepared for encapsulation of the bone marrow derived mesenchymal stem cells (BMSCs), which promoted the neurogenesis and axon regeneration by scavenging the overproduced ROS and re-building a regenerative microenvironment. The hydrogel could effectively encapsulate BMSCs, and played a remarkable neuroprotective role in vivo by reducing the production of endogenous ROS, attenuating ROS-mediated oxidative damage and downregulating the inflammatory cytokines such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), resulting in a reduced cell apoptosis in the spinal cord tissue. The BMSCs-encapsulated ROS-scavenging hydrogel also reduced the scar formation, and improved the neurogenesis of the spinal cord tissue, and thus distinctly enhanced the motor functional recovery of SCI rats. Our work provides a combinational strategy against ROS-mediated oxidative stress, with potential applications not only in SCI, but also in other central nervous system diseases with similar pathological conditions.

Atractylenolide III ameliorates spinal cord injury in rats by modulating microglial/macrophage polarization.

BackgroundInflammatory reactions induced by spinal cord injury (SCI) are essential for recovery after SCI. Atractylenolide III (ATL‐III) is a natural monomeric herbal bioactive compound that is mainly derived in Atractylodes macrocephala Koidz and has anti‐inflammatory and neuroprotective effects.ObjectiveHere, we speculated that ATL‐III may ameliorate SCI by modulating microglial/macrophage polarization. In the present research, we focused on investigating the role of ATL‐III on SCI in rats and explored the potential mechanism.MethodsThe protective and anti‐inflammatory effects of ATL‐III on neuronal cells were examined in a rat SCI model and lipopolysaccharide (LPS)‐stimulated BV2 microglial line. The spinal cord lesion area, myelin integrity, and surviving neurons were assessed by specific staining. Locomotor function was evaluated by the Basso, Beattie, and Bresnahan (BBB) scale, grid walk test, and footprint test. The activation and polarization of microglia/macrophages were assessed by immunohistofluorescence and flow cytometry. The expression of corresponding inflammatory factors from M1/M2 and the activation of relevant signaling pathways were assessed by Western blotting.ResultsATL‐III effectively improved histological and functional recovery in SCI rats. Furthermore, ATL‐III promoted the transformation of M1 into M2 and attenuated the activation of microglia/macrophages, further suppressing the expression of corresponding inflammatory mediators. This effect may be partly mediated by inhibition of neuroinflammation through the NF‐κB, JNK MAPK, p38 MAPK, and Akt pathways.ConclusionThis study reveals a novel effect of ATL‐III in the regulation of microglial/macrophage polarization and provides initial evidence that ATL‐III has potential therapeutic benefits in SCI rats.

MiR-212-3p improves rat functional recovery and inhibits neurocyte apoptosis in spinal cord injury models via PTEN downregulation-mediated activation of AKT/mTOR pathway

BackgroundMultiple cellular and molecular changes are involved in the etiology of spinal cord injury (SCI) and the recovery from SCI. Accumulating studies showed aberrant expression of microRNAs (miRNAs) after SCI. Here, we established in vivo and in vitro models to analyze the role of miR-212-3p in SCI. MethodsAn in vivo model of SCI was established in Sprague-Dawley rats. SCI-induced histopathological changes of the spinal cord were observed by hematoxylin-eosin staining. Functional recovery of rats with SCI was evaluated using the Basso-Beattie-and-Bresnahan scale. PC12 cells were stimulated by lipopolysaccharide (LPS) to establish SCI model of neuronal apoptosis in vitro. Dual-luciferase reporter assay was performed to validate the potential target of miR-212-3p predicted by TargetScan 7.2. MTT assay and flow cytometry were carried out to measure the viability and apoptosis of PC12 cell, respectively. The expressions of miR-212-3p, PTEN, phosphorylated (p)-AKT, AKT, p-mTOR, mTOR, Cleaved caspase-3 and BCl-2 in spinal cord tissues and PC12 cells were analyzed by qRT-PCR or Western blot. ResultsIn the spinal cord of rats with SCI, the expressions of miR-212-3p, p-AKT, p-mTOR and BCl-2 were downregulated, whereas those of PTEN and Cleaved caspase-3 were upregulated. BBB scores were low, and there were histopathological changes, which were all reversed after the injection of agomiR-212-3p. MiR-212-3p directly targeted PTEN. Upregulated miR-212-3p in LPS-injured PC12 cells suppressed apoptosis, downregulated the expressions of PTEN and Cleaved caspase-3, promoted viability and upregulated the expressions of p-AKT, p-mTOR and BCl-2, which were all reversed by overexpressed PTEN. ConclusionMiR-212-3p improved functional recovery of SCI rats and inhibited LPS-induced neurocyte apoptosis by targeting PTEN to activate AKT/mTOR pathway.

Conotoxin loaded dextran microgel particles alleviate effects of spinal cord injury by inhibiting neuronal excitotoxicity

Spinal cord injury (SCI) is a severe refractory disabling condition accompanied by complicated pathological processes, such as excessive glutamate release in the extracellular environment and massive influx of Ca2+ into cells. Dextran can absorb extracellular glutamate and Ca2+ to attenuate persistent excitatory processes that are detrimental to normal cells. Calcium channel inhibitors reduce the influx of Ca2+, thereby relieving subsequent cytotoxicity such as mitochondria damage. Here, a bifunctional microgel was designed for delivering GVIA, an N-type calcium ion channel blocker, to achieve a combinational therapy of simultaneously reducing the Ca2+ and glutamate concentrations in extracellular environment and inhibiting excess Ca2+ influx. The obtained dextran microgels could effectively encapsulate GVIA peptide, which displayed sustained release. Microgels loaded with GVIA (DexGVIA) played an excellent neuroprotective role by relieving the influx of Ca2+in vitro. DexGVIA also exhibited promising therapeutic effect in vivo via reducing the lesion cavity, saving neurons and gliacytes from the injury, and improving continuity and integrity of the spinal cord, thus distinctly accelerated motor functional recovery of SCI rats. Our work provided a combinational strategy against excitotoxicity, with potential applications not only in SCI, but also in other pathologically similar conditions of the central nervous system.

Mash-1 modified neural stem cells transplantation promotes neural stem cells differentiation into neurons to further improve locomotor functional recovery in spinal cord injury rats.

Spinal cord injury (SCI) leads to severe motor and sensory dysfunctions. Neural stem cells (NSCs) transplantation therapy plays a positive role in functional recovery after SCI, but the effectiveness of this therapy is limited by inadequate differentiation ability of transplanted NSCs. Mammalian achaete-scute homologue-1 (Mash-1) has been reported to improve differentiation of NSCs. Thus, this study modified NSCs with Mash-1 to repair SCI. NSCs isolated from rat embryo hippocampus were cultured and identified in vitro and further transfected with the lentiviral vectors (Lv-Mash-1). After establishing a SCI rat model, the rats were transplanted with Mash-1 modified NSCs, the histopathological changes of rat spinal cord were detected by hematoxylin-eosin (HE) staining, and the locomotor activity of rats was evaluated with the Basso, Beattie and Bresnahan (BBB) scale. The NSCs cultured in vitro or extracted from SCI rat spinal cord were identified by immunofluorescence (IF). Mash-1, β3-Tubulin, and NeuN expressions in those cells were determined by Western blotting and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). NSCs isolated from rat embryo hippocampus were Nestin- and NeuN-positive. NSC transplantation modified by Mash-1 increased BBB score of SCI rats and promoted recovery in lesion site of SCI rats. Mash-1 overexpression also promoted β3-Tubulin and NeuN expressions in NSCs cultured in vitro or extracted from spinal cord of SCI rats. Mash-1 overexpression promoted NSC differentiation into neurons, and further improved locomotor functional recovery of SCI rats.

Therapeutic effect of metformin on inflammation and apoptosis after spinal cord injury in rats through the Wnt/β-catenin signaling pathway

ObjectiveTo verify the effect of metformin on spinal cord injury (SCI) through Wnt/β-catenin signaling pathway. BackgroundSCI is a serious traumatic disease of the central nervous system. Wnt/β-catenin signaling pathway plays important roles in SCI. Metformin has been reported to exert neuroprotective effects in the central nervous system. Whether metformin could improve SCI through Wnt/β-catenin signaling pathway remains unclear. MethodsRats were divided into sham group, SCI group, SCI + metformin group, metformin + XAV939 group (XAV939 is an effective inhibitor of the Wnt/β-catenin signaling pathway), and methylprednisolone group. BBB scores were used to detect motor function recovery at different time points (0, 1, 3, 7, 14, 21, and 28 days) in SCI rats. Western blot analysis, immunofluorescence, TUNEL, HE and Nissl staining were used to observe the morphological characteristics of spinal cord tissue and the expression of inflammation and apoptosis in spinal cord neurons. ResultsMetformin(50 mg/kg) promoted motor functional recovery in rats after SCI, increased the expressions of β-catenin and brain derived neurotrophic factor (BDNF), inhibited neuron apoptosis and inflammatory response, and improved the recovery of pathological morphology at the injury site by activating the Wnt/β-catenin signaling pathway. ConclusionWe found a possible mechanism that metformin could reduce inflammation and apoptosis, and promote functional recovery of SCI rats through activating Wnt/β-catenin signaling pathway.

Overexpression of Rictor in the injured spinal cord promotes functional recovery in a rat model of spinal cord injury.

Rictor is an essential component that directly activates the mammalian target of rapamycin (mTOR) activity, which contributes to the intrinsic axon growth capacity of adult sensory neurons after injury. However, whether its action also applies to regeneration after spinal cord injury (SCI) remains unknown. In this study, rats were given spinal cord contusion at the T9-10 level to establish the SCI model and were subsequently treated with intraspinal cord injection of a Rictor overexpression lentiviral vector to locally upregulate the Rictor expression in the injured spinal cord. Thereafter, we investigated the therapeutic effects of Rictor overexpression in the injured spinal cords of SCI rats. Rictor overexpression not only significantly attenuated the acute inflammatory response and cell death after SCI but also markedly increased the shift in macrophages around the lesion from the M1 to M2 phenotype compared to those of the control lentiviral vector injection-treated group. Furthermore, Rictor overexpression dramatically increased neurogenesis in the lesion epicenter, subsequently promoting the tissue repair and functional recovery in SCI rats. Interestingly, the mechanism underlying the beneficial effects of Rictor overexpression on SCI may be associated with the Rictor overexpression playing a role in the anti-inflammatory response and driving macrophage polarization toward the M2 phenotype, which benefits resident neuronal and oligodendrocyte survival. Our findings demonstrate that Rictor is an effective target that affects the generation of molecules that inhibit spinal cord regeneration. In conclusion, localized Rictor overexpression represents a promising potential strategy for the repair of SCI.

Transplantation of sh-miR-199a-5p-Modified Olfactory Ensheathing Cells Promotes the Functional Recovery in Rats with Contusive Spinal Cord Injury

MicroRNAs (miRNAs) function as gene expression switches, and participate in diverse pathophysiological processes of spinal cord injury (SCI). Olfactory ensheathing cells (OECs) can alleviate pathological injury and facilitate functional recovery after SCI. However, the mechanisms by which OECs restore function are not well understood. This study aims to determine whether silencing miR-199a-5p would enhance the beneficial effects of the OECs. In this study, we measured miR-199a-5p levels in rat spinal cords with and without injury, with and without OEC transplants. Then, we transfected OECs with the sh-miR-199a-5p lentiviral vector to reduce miR-199a-5p expression and determined the effects of these OECs in SCI rats by Basso–Beattie–Bresnahan (BBB) locomotor scores, diffusion tensor imaging (DTI), and histological methods. We used western blotting to measure protein levels of Slit1, Robo2, and srGAP2. Finally, we used the dual-luciferase reporter assay to assess the relationship between miR-199-5p and Slit1, Robo2, and srGAP2 expression. We found that SCI significantly increased miR-199a-5p levels (P < 0.05), and OEC transplants significantly reduced miR-199a-5p expression (P < 0.05). Knockdown of miR-199a-5p in OECs had a better therapeutic effect on SCI rats, indicated by higher BBB scores and fractional anisotropy values on DTI, as well as histological findings. Reducing miR-199a-5p levels in transplanted OECs markedly increased spinal cord protein levels of Slit1, Robo2, and srGAP2. Our results demonstrated that transplantation of sh-miR-199a-5p-modified OECs promoted functional recovery in SCI rats, suggesting that miR-199a-5p knockdown was more beneficial to the therapeutic effects of OEC transplants. These findings provided new insights into miRNAs-mediated therapeutic mechanisms of OECs, which helps us to develop therapeutic strategies based on miRNAs and optimize cell therapy for SCI.

Salvianolic acid B activates Wnt/β-catenin signaling following spinal cord injury.

Neural cell apoptosis serves a key role in spinal cord injury (SCI), which is a threat to human health. The present study aimed to evaluate the neuroprotective mechanism of salvianolic acid B (Sal B) in a spinal cord injury (SCI) rat model. Basso, Beattie, and Bresnahan scores demonstrated that Sal B treatment significantly increased locomotor functional recovery in SCI rats compared with SCI model rats between 3 and 8 weeks. Nissl staining demonstrated that Sal B enhanced motor neuron survival and decreased lesion size after SCI. Reverse transcription-quantitative PCR analysis demonstrated that Sal B treatment significantly enhanced the mRNA levels of lymphoid enhancer biding factor-1 and HNF1 homeobox A. In addition, Sal B treatment enhanced the expression of β-catenin. Western blot analysis determined that Sal B treatment significantly decreased the expression of pro-apoptosis proteins, including Bax, cleaved caspase-3 and −9, in spinal cord tissues after SCI but enhanced the expression of Bcl-2, an anti-apoptotic protein. Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining demonstrated that, compared with the SCI group, Sal B treatment decreased the number of TUNEL-positive neurons. In summary, the present study produced novel data demonstrating the neuroprotective effect of Sal B on SCI with the mechanism likely primarily mediated via the Wnt/β-catenin signaling pathway. The present findings may be of potential therapeutic value for future SCI treatments.

MiR-21 derived from the exosomes of MSCs regulates the death and differentiation of neurons in patients with spinal cord injury.

In this study, we aimed to investigate the therapeutic effect of miR-21 in the treatment of spinal cord injury (SCI) as well as its underlying molecular mechanisms. Real-time PCR and western blot were performed to measure the expression of miR-21, PTEN, and PDCD4 in SCI rats. Locomotion recovery assessment, Nissl staining, IHC assay, and TUNEL assay were utilized to observe the therapeutic effect of miR-21 in the treatment of SCI. Bioinformatics analysis and luciferase assay were conducted to establish the signaling pathway of miR-21, PTEN, and PDCD4. The regulatory relationships between miR-21 and PTEN/PDCD4 were further validated by real-time PCR, western blot, MTT assay, and flow cytometry. Compared with sham-operated rats, SCI rats showed decreased expression of miR-21 along with increased expression of PTEN/PDCD4. Exosomes were equally distributed in MSCs transfected with miR-21, PTEN siRNA, or scramble controls. The exosomes isolated from the supernatant of cultured MSCs could improve the functional recovery of SCI rats by reducing SCI-induced neuron loss. In addition, miR-21 was shown to inhibit the expression of PTEN/PDCD4 and suppress neuron cell death. Moreover, PTEN and PDCD4 were validated as virtual targets of miR-21. In addition, the miR-21/PTEN/PDCD4 signaling pathway was shown to enhance cell viability and suppress cell death in vivo. The exosomes collected from the supernatant of transfected MSCs contained miR-21, which could improve the functional recovery of SCI rats and suppress cell death both in vivo and in vitro via the miR-21/PTEN/PDCD4 signaling pathway.

Combinational protective therapy for spinal cord injury medicated by sialic acid-driven and polyethylene glycol based micelles

Spinal cord injury (SCI) leads to immediate disruption of neuronal membranes and loss of neurons, followed by extensive secondary injury process. Treatment of SCI still remains a tremendous challenge clinically. Minocycline could target comprehensive secondary injury via anti-inflammatory, anti-oxidant and anti-apoptotic mechanisms. Polyethylene glycol (PEG), a known sealing agent, is able to seal the damaged cell membranes and reduce calcium influx, thereby exerting neuroprotective capacity. Here, an E-selectin-targeting sialic acid - polyethylene glycol – poly (lactic-co-glycolic acid) (SAPP) copolymer was designed for delivering hydrophobic minocycline to achieve combinational therapy of SCI. The obtained SAPP copolymer could self-assemble into micelles with critical micelle concentration being of 13.40 μg/mL, and effectively encapsulate hydrophobic minocycline. The prepared drug-loaded micelles (SAPPM) displayed sustained drug release over 72 h, which could stop microglia activation and exhibited excellent neuroprotective capacity in vitro. The SAPP micelles were efficiently accumulated in the lesion site of SCI rats via the specific binding between sialic acid and E-selectin. Due to the targeting distribution and combinational effect between PEG and minocycline, SAPPM could obviously reduce the area of lesion cavity, and realize more survival of axons and myelin sheaths from the injury, thus distinctly improving hindlimb functional recovery of SCI rats and conferring superior therapeutic effect in coparison with other groups. Our work presented an effective and safe strategy for SCI targeting therapy. Besides, neuroprotective capacity of PEG deserves further investigation on other central nervous system diseases.

Astragoloside IV Loaded Polycaprolactone Membrane Repairs Blood Spinal Cord Barrier and Recovers Spinal Cord Function in Traumatic Spinal Cord Injury.

Traumatic Spinal Cord Injury (SCI) is a serious challenge of CNS which may oftenly result in permanent paralysis and disability. The disruption of blood spinal cord barrier (BSCB) is a major step in the secondary injury of SCI. Until recently there are no restorative therapies for traumatic SCI. BSCB is considered to be a therapeutic target for SCI, however few biomaterials have been developed to restore the disrupted BSCB in SCI. In this study, an AST-PCL membrane was fabricated with a steady release of Astragoloside IV (AST) and its effects on BSCB repair as well as the functional recovery of SCI were evaluated. Firstly, this study demonstrated that AST-PCL (polycaprolactone) degradation media protects endothelial cells from apoptosis by down-regulating the expression of cleaved Caspase 3 and decreasing the ratio of Bax/Bcl-2, it also attenuates the stress fiber formation in vitro. Secondly, the rat model of traumatic SCI showed that AST-PCL treatment inhibits the disruption of BSCB permeability, as detected by MRI, Evan's Blue extravasation and water content. Thirdly, this study found that AST-PCL up-regulates the level of tight junction proteins including Occludin, Claudin5 and ZO-1. Furthermore, it is also demonstrated that AST-PCL treatment down-regulates Matrix metalloproteinase-9 secretion and diminishes neutrophil infiltration. Finally, this study found that AST-PCL treatment could significantly inhibit apoptosis, decrease tissue damage and improve functional recovery in SCI rats. Taken together, this study shows that AST-PCL might be an efficient biomaterial for BSCB repair and a potential drug therapy for SCI.