Rat Model Of Spinal Cord Injury Research Articles

Thermosensitive hydrogel-mediated sphere/fiber multi-dimensional composite nanotube with controlled release of NGF for improved spinal cord injury repair

Spinal cord injury is a serious disabling disease that not only causes physical defects, but also causes psychological problems for patients. In the current study, a novel multi-dimensional functional composite nanotube was prepared by simultaneouslyriveting nerve growth factor (NGF)-loaded PLGA microspheres and tissue-engineered cells (BMSCs) into the PLA fiber nanotube by a designed thermosensitive hydrogel to improve spinal cord injury (SCI) repair. The PLGA microspheres allow the protection and long-term controlled release of NGF to guarantee enough time and drug concentration to promote the growth and differentiation of cells into axons and promote axonal regeneration. Thermosensitive hydrogels are served not only to provide a tunable physiologically simulated microenvironment, but also as scaffolds to provide a suitable matrix for cells and drugs, and PLA nanotube ensures the seal of the released NGF in the nanotube to improve its drug bioavailability, as well as the supporting and guiding of the nerve growth. The physicochemical properties of the composite nanotube are investigated and the repair efficacy is evaluated according to the functional recovery, histological, immunohistochemistry, immunofluorescence examinations as well as the westernblot analysis. The administration of the composite nanotube in the SCI rat models obviously improves the regeneration of the neuron, axon, myelin sheath, neural stem cell and nissnei body but decreases the astrocytes formation, as well as enhances the integrity of the spinal cord tissue and the recovery of locomotor functions. This present work provides a future perspective for NGF application and a potential strategy for SCI repair.

MiR‑222-3p reduces neuronal cell apoptosis and alleviates spinal cord injury by inhibiting Bbc3 and Bim

Spinal cord injury (SCI) is a severe traumatic event, but without any established effective treatment because of the irreversible neuronal death. Here, we investigated the role of miR-222-3p in neuronal apoptosis following SCI. Rat SCI models and neuron hypoxia models were accordingly established. The Bbc3, Bim, Bcl-2, Bax, cleaved-caspase 3, cleaved-caspase 9, Cytochrome c, and miR-222-3p expression levels were examined by Western blotting and real-time reverse transcription polymerase chain reaction (RT-qPCR). The possible association between miR-222-3p and Bbc3/Bim was analyzed by dual-luciferase assay. The neuron viability was assessed by Cell Counting Kit-8 assay and Nissl's staining. Live cell staining was performed to detect the mitochondrial membrane potential and neuronal apoptosis. Rat locomotor function was assessed using the Basso–Beattie–Bresnahan scores. Cytochrome c was outflowed from the mitochondria after SCI or hypoxia treatment, and Bbc3, Bim, Bax, cleaved-caspase 9, and cleaved-caspase 3 were significantly upregulated, while Bcl-2 and miR-222-3p were decreased remarkably. Meanwhile, neuronal cell viability was significantly inhibited. Treatment of miR-222-3p significantly suppressed the Cytochrome c efflux and neuronal apoptosis and improved neuronal cell viability and motor function in SCI rats. Moreover, we found that Bbc3 and Bim were the direct targets of miR-222-3p. Overall, our data suggest that miR-222-3p could alleviate the mitochondrial pathway-mediated apoptosis and motor dysfunction in rats after SCI by targeting Bbc3 and Bim.

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.

Intrathecal minocycline does not block the adverse effects of repeated, intravenous morphine administration on recovery of function after SCI

Opioids are among the most effective analgesics for the management of pain in the acute phase of a spinal cord injury (SCI), and approximately 80% of patients are treated with morphine in the first 24 h following SCI. We have found that morphine treatment in the first 7 days after SCI increases symptoms of pain at 42 days post-injury and undermines the recovery of locomotor function in a rodent model. Prior research has implicated microglia/macrophages in opioid-induced hyperalgesia and the development of neuropathic pain. We hypothesized that glial activation may also underlie the development of morphine-induced pain and cell death after SCI. Supporting this hypothesis, our previous studies found that intrathecal and intravenous morphine increase the number of activated microglia and macrophages present at the spinal lesion site, and that the adverse effects of intrathecal morphine can be blocked with intrathecal minocycline. Recognizing that the cellular expression of opioid receptors, and the intracellular signaling pathways engaged, can change with repeated administration of opioids, the current study tested whether minocycline was also protective with repeated intravenous morphine administration, more closely simulating clinical treatment. Using a rat model of SCI, we co-administered intravenous morphine and intrathecal minocycline for the first 7 days post injury and monitored sensory and locomotor recovery. Contrary to our hypothesis and previous findings with intrathecal morphine, we found that minocycline did not prevent the negative effects of morphine. Surprisingly, we also found that intrathecal minocycline alone is detrimental for locomotor recovery after SCI. Using ex vivo cell cultures, we investigated how minocycline and morphine altered microglia/macrophage function. Commensurate with published studies, we found that minocycline blocked the effects of morphine on the release of pro-inflammatory cytokines but, like morphine, it increased glial phagocytosis. While phagocytosis is critical for the removal of cellular and extracellular debris at the spinal injury site, increased phagocytosis after injury has been linked to the clearance of stressed but viable neurons and protracted inflammation. In sum, our data suggest that both morphine and minocycline alter the acute immune response, and reduce locomotor recovery after SCI.

Dual detection of spinal cord injury biomarkers in rat model using gold nanorod array substrate based on surface-enhanced Raman scattering

Tumor necrosis factor alpha (TNF­α) and glial fibrillary acidic protein (GFAP) are candidate biomarkers for early diagnosis and evaluation of spinal cord injury (SCI). In fact, their concentrations in serum, plasma, or cerebrospinal fluid are highly correlated with the SCI severity and neurological prognosis. Therefore, development of a rapid, ultrasensitive, and accurate technique for dual-detection of these important biomarkers is critical for SCI diagnosis, monitoring, and recovery assessment. We synthesized gold nanorods (AuNRs) with stable aspect ratio and fabricated an AuNR array parallelly aligned on a substrate using evaporation-induced self-assembly. The uniform morphology and neat arrangement of the AuNRs produce many hotspots that increase the sensitivity and reproducibility of the array. For dual detection of TNF-α and GFAP, the self-assembled AuNR array substrate was modified with anti-TNF­α and anti-GFAP. Then, special Raman probe molecules, specific antibodies, and AuNRs were combined to fabricate surface-enhanced Raman scattering (SERS) tags of AuNR@4-MPBA and AuNR@NBA modified with anti-TNF­α and anti-GFAP, respectively. Sandwich immunoassay using standard solutions of TNF­α and GFAP provided limits of detection of 0.023 and 0.018 pg/mL, respectively. The detector was evaluated on concentrations of TNF­α and GFAP in plasma of SCI rat models. Cross-validation against ELISA (enzyme-linked immunosorbent assay) indicated that the SERS sandwich immunoassay detector based on AuNR array substrate has a high dual-detection accuracy. Overall, the proposed detector provides high sensitivity, reproducibility, stability, and accuracy, and it correctly performs dual detection. The fabricated detector platform seems promising for detection of multiple molecules to achieve early and accurate disease diagnosis in clinical practice.

Implantation of adipose-derived mesenchymal stem cell sheets promotes axonal regeneration and restores bladder function after spinal cord injury

BackgroundCell-based therapy using adipose-derived mesenchymal stem cells (ADSCs) is a promising treatment strategy for neurogenic bladder (NB) associated with spinal cord injury (SCI). However, therapeutic efficacy is low because of inefficient cell delivery. Cell sheets improve the efficacy of cell transplantation. Therefore, this study was conducted to investigate the therapeutic efficacy of transplanting ADSC sheets into an SCI rat model and focused on the function and pathological changes of the bladder.MethodsADSC sheets were prepared from adipose tissue of Sprague–Dawley (SD) rats using temperature-responsive cell culture dishes. Adult female SD rats were subjected to SCI by transection at the T10 level and administered ADSC sheets or gelatin sponge (the control group). Four and 8 weeks later, in vivo cystometrograms were obtained for voiding function assessment. Rats were sacrificed and the expression of various markers was analyzed in spinal and bladder tissues.ResultsThe number of β-tubulin III-positive axons in the ADSC sheet transplantation group was higher than that in the control group. Conversely, expression of glial fibrillary acidic protein in the ADSC sheet transplantation group was lower than that in the control group. Cystometry showed impairment of the voiding function after SCI, which was improved after ADSC sheet transplantation with increased high-frequency oscillation activity. Furthermore, ADSC sheet transplantation prevented disruption of the bladder urothelium in SCI rats, thereby maintaining the intact barrier. Compared with fibrosis of the bladder wall in the control group, the ADSC sheet transplantation group had normal morphology of the bladder wall and reduced tissue fibrosis as shown by downregulation of type 1 collagen. ADSC sheet transplantation also resulted in strong upregulation of contractile smooth muscle cell (SMC) markers (α-smooth muscle actin and smoothelin) and downregulation of synthetic SMC markers (MYH10 and RBP1).ConclusionADSC sheet transplantation significantly improved voiding function recovery in rats after SCI. ADSC sheet transplantation is a promising cell delivery and treatment option for NB related to SCI.

Cerium Oxide Nanoparticles Alleviate Neuropathic Pain by Modulating Macrophage Polarization in a Rat SCI Model.

ContextChronic neuropathic pain (NP) frequently occurs after spinal cord injury (SCI) but lacks effective therapeutic options in the clinic. Numerous evidence indicates the involvement of macrophages activation in the NP, and the modulation of macrophages is promising for NP treatment. In this study, we introduce Cerium oxide nanoparticles (CONPs) and aim to investigate whether it can relieve the NP by modulating macrophage polarization.MethodsCONPs were prepared using the hydrothermal method. In vitro, different concentrations of CONPs were used to cultivate macrophages (RAW 264.7). In vivo, the analgesic effect of CONPs was investigated in a contusive rat SCI model. Mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) were tested to evaluate pain behaviors. Immunofluorescence staining and real-time quantitative polymerase chain reaction were applied to assess macrophage phenotypes.ResultsThe synthesized CONPs were 6.8 ± 0.5 nm in size, presenting a cubic morphology. Live/dead staining showed that the relatively low concentrations of CONPs (less than 800 μg/mL) displayed good biocompatibility with macrophages. Intrathecal injection of CONPs could significantly increase the mechanical PWT and thermal PWL of SCI rats. Molecular experiments results showed the expression of M2 macrophage-related markers (CD206, Arg-1, IL-10) were significantly increased, while that of M1 macrophage-related markers (CD86, TNF-α, iNOS) were downregulated after CONPs treatment.ConclusionOur study suggests that CONPs can relive the NP following SCI by promoting M2 macrophages polarization, which provides a novel insight for the treatment of SCI induced NP.

Upregulation of TRPC6 inhibits astrocyte activation and proliferation after spinal cord injury in rats by suppressing AQP4 expression

AimsThis work investigates the effects and mechanisms of inhibiting TRPC6 (a non-selective cation channel) downregulation on rat astrocyte activation and proliferation following spinal cord injury (SCI) by suppressing AQP4 expression. We used HYP9 (TRPC6-specific agonist) and TGN-020 (AQP4-specific inhibitor) to explore the relationship between TRPC6 and AQP4 and their probable protective effects on SCI. Methods: In a rat SCI model, we randomly assigned female Sprague-Dawley rats into the following four groups: Sham, SCI, SCI+HYP9, and SCI+TGN-020. Western blotting and immunofluorescence staining were used to determine protein expression among groups following SCI. TUNEL and immunofluorescence staining were used to identify changes in the rate of apoptosis and the fraction of surviving neurons after SCI. The Basso-Beattie-Bresnahan open-field locomotor scale was used to identify changes in motor function after SCI. In vitro astrocyte scratch model, we first used the CCK8 assay to test the effects of varying doses of HYP9 or TGN-020 on astrocytes and then split the astrocytes into four groups: Con, Scratch, Scratch+HYP9, and Scratch+TGN-020. Western blotting and immunofluorescence were used to identify changes in the expression of target proteins. Results: In vivo and in vitro models, SCI dramatically decreased TRPC6 while considerably upregulating AQP4, glial fibrillary acidic protein (GFAP), and proliferating cell nuclear antigen (PCNA) expression. However, HYP9 or TGN-020 significantly suppressed activation of astrocytes, promoted neurons survival in the anterior horn of the spinal cords, and benefited the recovery of motor function in the hind limbs of rats following SCI. Interestingly, TRPC6 agonists dramatically suppressed AQP4 overexpression, indicating that the probable mechanism of HYP9 benefiting alleviation of SCI may be connected to AQP4 inhibition and astrocyte activation and proliferation reduction. Conclusion: we discovered for the first time that HYP9 inhibits astrocyte activation and proliferation by inhibiting AQP4 in SCI rats in vivo and in vitro models and that it preserves neuronal survival and functional recovery after SCI.

Rho Kinase Inhibitor Y27632 Improves Recovery After Spinal Cord Injury by Shifting Astrocyte Phenotype and Morphology via the ROCK/NF-κB/C3 Pathway

Spinal cord injury (SCI) usually results in loss or reduction in motor and sensory functions. Despite extensive research, no available therapy can restore the lost functions after SCI. Reactive astrocytes play a pivotal role in SCI. Rho kinase inhibitors have also been shown to promote functional recovery of SCI. However, the role of Rho kinase inhibitors in reactive astrocytic phenotype switch within SCI remains largely unexplored. In this study, astrocytes were treated with proinflammatory cytokines and/or the Rho kinase inhibitor Y27632. Concomitantly the phenotype and morphology of astrocytes were examined. Meanwhile, the SCI model of SD rats was established, and nerve functions were evaluated following treatment with Y27632. Subsequently, the number of A1 astrocytes in the injured area was observed and analyzed. Eventually, the expression levels of nuclear factor kappa B (NF-κB), C3, and S100A10 were measured. The present study showed that the Rho kinase inhibitor Y27632 improved functional recovery of SCI and elevated the proliferation and migration abilities of the astrocytes. In addition, Y27632 treatment initiated the switch of astrocytes morphology from a flattened shape to a process-bearing shape and transformed the reactive astrocytes A1 phenotype to an A2 phenotype. More importantly, further investigation suggested that Y27632 was actively involved in promoting the functional recovery of SCI in rats by inhabiting the ROCK/NF-κB/C3 signaling pathway. Together, Rho kinase inhibitor Y27632 effectively promotes the functional recovery of SCI by shifting astrocyte phenotype and morphology. Furthermore, the pro-regeneration event is strongly associated with the ROCK/NF-κB/C3 signal pathway.

Injectable and fast gelling hyaluronate hydrogels with rapid self-healing ability for spinal cord injury repair

Self-healing natural hydrogels still suffer from some issues such as unfit stiffness, poor healing efficiency, and lack of biocompatibility and hydrolytic stability, although they have been used to treat spinal cord injury (SCI). Herein, we develop the injectable, self-healing hyaluronate hydrogels based on multiple dynamic covalent bonds. The hydrogels exhibit fast gelation and excellent self-healing capability as well as injectability, favoring in situ formation for the hydrogels in target sites and maintaining their structural stability. Furthermore, the hydrogels are compatible with neural stem cells and various tissues and possess proper stiffness similar to nervous tissue. Interestingly, the hydrogel can induce neural differentiation of neural stem cells. In vivo experiment further illustrates that the hydrogels promote angiogenesis and remyelination as well as neuron regeneration, leading to the significant locomotor recovery of the SCI model rats. This injectable self-healing hyaluronic acid-based hydrogel is a potential candidate for nerve repair.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Promotes Forelimb Functional Recovery after Cervical Spinal Cord Injury.

Locomotor function after spinal cord injury (SCI) is critical for assessing recovery. Currently, available means to improve locomotor function include surgery, physical therapy rehabilitation and exoskeleton. Stem cell therapy with neural progenitor cells (NPCs) transplantation is a promising reparative strategy. Along this line, patient-specific induced pluripotent stem cells (iPSCs) are a remarkable autologous cell source, which offer many advantages including: great potential to generate isografts avoiding immunosuppression; the availability of a variety of somatic cells without ethical controversy related to embryo use; and vast differentiation. In this current work, to realize the therapeutic potential of iPSC-NPCs for the treatment of SCI, we transplanted purified iPSCs-derived NPCs into a cervical contusion SCI rat model. Our results showed that the iPSC-NPCs were able to survive and differentiate into both neurons and astrocytes and, importantly, improve forelimb locomotor function as assessed by the grooming task and horizontal ladder test. Purified iPSC-NPCs represent a promising cell type that could be further tested and developed into a clinically useful cell source for targeted cell therapy for cervical SCI patients.

Modulation of Neuroinflammation via Selective Nanoparticle‐Mediated Drug Delivery to Activated Microglia/Macrophages in Spinal Cord Injury

AbstractInflammation after spinal cord injury (SCI) is characterized by immune cell invasion and activation, combined with inflammatory mediator release that worsens outcomes following primary trauma. Effective therapies targeting neuroinflammation remain an unmet need, and modulation of the injury microenvironment to induce a comprehensive pro‐regenerative response is an attractive therapeutic approach. Given its crucial role in cell stress and inflammation after SCI, the potential of pharmacologically targeting MAPK‐activated protein kinase‐2 (MK2) to modulate the response of microglia/macrophages after injury is focused. Nanoparticles (NPs) containing an MK2 inhibitor for specific targeting of microglia/macrophages is developed. NPs selectively target and modulate activated microglia/macrophages in vitro and in a rat model of SCI. NPs in the acute injury setting reduce the pro‐inflammatory cytokine IL‐6 and increase the anti‐inflammatory cytokine IL‐10. Importantly, NPs have a significant effect on microglia/macrophage distribution and accumulation, leading to ≈65% reduction of immune cells around the injury. Last, microglia/macrophage populations with activated morphology are significantly reduced compared to resting or ramified cells around the lesion site. This strategy exhibits potential therapeutic efficiency and specificity for local, pharmacologic manipulation of activated microglia/macrophages, and is a versatile tool to manage acute inflammation and glia plasticity after central nervous system trauma.

Transplantation of NEP1-40 and NT-3 Gene-Co-Transduced Neural Stem Cells Improves Function and Neurogenesis after Spinal Cord Injury in a Rat Model.

Spinal cord injury (SCI) generally results in necrosis, scarring, cavitation, and a release of inhibitory molecules of the nervous system, which lead to disruption of neurotransmission and impede nerve fiber regeneration. This study was intended to evaluate the therapeutic efficacy rates of the transplantation of NEP1-40- and NT-3 gene-co-transduced neural stem cells (NSCs) in a rat model of SCI. Ninety Sprague-Dawley rats were subdivided randomly into six groups: sham-operated, SCI model, SCI + NSCs-NC, SCI + NEP1-40-NSCs, SCI + NT-3-NSCs, and SCI + NEP1-40/NT-3-NSCs. Motor function at different time points was evaluated using the Basso, Beattie, and Bresnahan locomotor activity scoring system (BBB). At 8 weeks post-transplantation, histological analysis, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, immunofluorescent assay, immunocytochemical staining, and cholera toxin subunit B (CTB) retrograde tracing were performed. BBB scores of the co-transduction group significantly surpassed those of other transplantation groups and of the SCI-model group after 2 weeks post-transplantation. The apoptotic rate of neurocytes was significantly lower in the co-transduction group than in other experimental groups. Expression of NF-200, MBP, and ChAT was significantly higher in the SCI + NEP1-40/NT-3-NSCs group than in other transplantation groups, whereas the expression of GFAP and GAD67 was the second lowest after the sham-operated group. CTB retrograde tracing showed that CTB-positive neural fibers on the caudal side of the hemisected site were more numerous in the SCI + NEP1-40/NT-3-NSCs group than in other experimental groups. Transplantation of NEP1-40- and NT-3-gene-co-transduced NSCs can modify the protein expression following acute SCI and promote neuron formation and axonal regeneration, thus having a neuroprotective effect. Furthermore, this effect surpasses that of transplantation of single-gene-transduced NSCs. Transplantation of NEP1-40- and NT-3-gene-co-transduced NSCs is effective at the neural recovery of the rat model of SCI and may be a novel strategy for clinical treatment of SCI.

Role of Transcription Factor Nrf2 in Pyroptosis in Spinal Cord Injury by Regulating GSDMD.

Spinal cord injury (SCI) is a prevalent disease that debilitates millions of people. Nuclear factor E2-related factor 2 (Nrf2) is an important regulator of SCI. The current study sought to elaborate on the effects of Nrf2 on gasdermin D (GSDMD)-mediated microglia pyroptosis to repair SCI. The SCI rat model was established via the percussion of the T10 spinal cord and in vitro SCI model was established on BV-2 cells via lipopolysaccharide (LPS)/adenosine triphosphate (ATP) treatment. Nrf2 expression in SCI rats and BV-2 cells was overexpressed via pcDNA3.1-Nrf2 injection. Functional assays were carried out to evaluate SCI rat pathological injury, BV-2 cell viability, the release of lactate dehydrogenase (LDH), and pyroptotic factors. The binding relations of Nrf2 and microRNA (miR)-146a and miR-146a and GSDMD were verified. BV-2 pyroptosis was analyzed after the combined experiment of miR-146a-inhibitor and pcDNA3.1-GSDMD. Our experiments revealed that Nrf2 was downregulated in SCI, and Nrf2 overexpression relieved SCI pathological injury, promoted BV-2 cell viability, inhibited the release of LDH, and repressed pyroptosis. Mechanically, Nrf2 bound to the miR-146a promoter and promoted miR-146a expression, and miR-146a targeted GSDMD transcription. Rescue experiments revealed that miR-146a knockdown or GSDMD overexpression annulled the inhibitory function of Nrf2 overexpression in LPS/ATP-induced microglia pyroptosis. Overall, our findings initially highlighted that Nrf2 inhibited GSDMD-mediated microglia pyroptosis and accelerated SCI repair by repressing miR-146a.

Acute Magnetic Resonance Imaging Predictors of Chronic Motor Function and Tissue Sparing in Rat Cervical Spinal Cord Injury.

Predicting functional outcomes from spinal cord injury (SCI) at the acute setting is important for patient management. This work investigated the relationship of early magnetic resonance imaging (MRI) biomarkers in a rat model of cervical contusion SCI with long-term functional outcome and tissue sparing. Forty rats with contusion injury at C5 at either the spinal cord midline (bilateral) or over the lateral cord (unilateral) were examined using in vivo multi-modal quantitative MRI at 1 day post-injury. The extent of T2-weighted hyperintensity reflecting edema was greater in the bilateral model compared with the unilateral injury. Diffusion tensor imaging (DTI) exhibited microscopic damage in similar regions of the cord as reductions in fractional anisotropy (FA) and mean diffusivity (MD), but DTI parameter maps were also confounded by the presence of vasogenic edema that locally increased FA and MD. In comparison, filtered diffusion-weighted imaging (fDWI) more clearly delineated the location of acute axonal damage without effects of vasogenic edema. Pairwise correlation analysis revealed that 28-day motor functional outcomes were most strongly associated with the extent of edema (R = -0.69). Principal component analysis identified close associations of motor functional score with tissue sparing, the extent of edema, lesion area, and injury type (unilateral or bilateral). Among the diffusion MRI parameters, lesion areas measured with fDWI had the strongest association with functional outcome (R = -0.41). Voxelwise correlation analysis identified a locus of white matter damage associated with function in the dorsal white matter, although this was likely driven by variance across the two injury patterns (unilateral and bilateral injury). Nonetheless, correlation with motor function within the damaged region found in the voxelwise analysis outperformed morphological lesion area measurement as a predictor of chronic function. Collectively, this study characterized anatomical and diffusion MRI signatures of acute SCI at cervical spine and their association with chronic functional outcomes and histological results.

Small extracellular vesicles derived from mesenchymal stem cell facilitate functional recovery in spinal cord injury by activating neural stem cells via the ERK1/2 pathway

Spinal cord injury (SCI) causes severe neurological dysfunction leading to a devastating disease of the central nervous system that is associated with high rates of disability and mortality. Small extracellular vesicles (sEVs) derived from human umbilical cord mesenchymal stem cells (hucMSC-sEVs) have been explored as a promising strategy for treating SCI. In this study, we investigated the therapeutic effects of the intralesional administration of hucMSC-sEVs after SCI and determined the potential mechanisms of successful repair by hucMSC-sEVs. In vivo, we established the rat model of SCI. The Basso, Beattie, Bresnahan (BBB) scores showed that hucMSC-sEVs dramatically promoted the recovery of spinal cord function. The results of the hematoxylin–eosin (HE) staining, Enzyme-Linked Immunosorbent Assay (ELISA), and immunohistochemistry showed that hucMSC-sEVs inhibited inflammation and the activation of glia, and promoted neurogenesis. Furthermore, we studied the effect of hucMSC-sEVs on neural stem cells(NSCs) in vitro. We found that hucMSC-sEVs did not improve the migration ability of NSCs, but promoted NSCs to proliferate and differentiate via the ERK1/2 signaling pathway. Collectively, these findings suggested that hucMSC-sEVs promoted the functional recovery of SCI by activating neural stem cells via the ERK1/2 pathway and may provide a new perspective and therapeutic strategy for the clinical application of hucMSC-sEVs in SCI treatment.

A Localized Materials-Based Strategy to Non-Virally Deliver Chondroitinase ABC mRNA Improves Hindlimb Function in a Rat Spinal Cord Injury Model.

Spinal cord injury often results in devastating consequences for those afflicted, with very few therapeutic options. A central element of spinal cord injuries is astrogliosis, which forms a glial scar that inhibits neuronal regeneration post-injury. Chondroitinase ABC (ChABC) is an enzyme capable of degrading chondroitin sulfate proteoglycan (CSPG), the predominant extracellular matrix component of the glial scar. However, poor protein stability remains a challenge in its therapeutic use. Messenger RNA (mRNA) delivery is an emerging gene therapy technology for in vivo production of difficult-to-produce therapeutic proteins. Here, mineral-coated microparticles as an efficient, non-viral mRNA delivery vehicles to produce exogenous ChABC in situ within a spinal cord lesion are used. ChABC production reduces the deposition of CSPGs in an in vitro model of astrogliosis, and direct injection of these microparticles within a glial scar forces local overexpression of ChABC and improves recovery of motor function seven weeks post-injury.

Release of O-GlcNAc transferase inhibitor promotes neuronal differentiation of neural stem cells in 3D bioprinted supramolecular hydrogel scaffold for spinal cord injury repair

Precise fabrication of biomimetic three-dimensional (3D) structure and effective neuronal differentiation under the pathological environment are the key to neural stem cell (NSC)-based spinal cord injury (SCI) therapy. In this study, we have developed a spinal cord-like bioprinted scaffold loading with OSMI-4, a small molecule O-GlcNAc transferase (OGT) inhibitor, to induce and guide the neuron differentiation of NSCs for efficient SCI repair. To achieve this, we developed a supramolecular bioink (SM bioink) consisting of methacrylated gelatin and acrylated β-cyclodextrins to load NSCs and OSMI-4. This bioink showed fast gelation and stable mechanical properties, facilitating bioprinting of functional neural scaffolds. Moreover, the weak host-guest cross-linking of the SM scaffolds significantly improved the cell-matrix interaction for the infiltration and migration of NSCs. What's more, the sustained delivery of OSMI-4 remarkably enhanced the intrinsic neuronal differentiation of the encapsulated NSCs in vitro by inhibiting Notch signaling pathway. In vivo experiment further revealed that the functional bioprinted scaffolds promoted the neuronal regeneration and axonal growth, leading to significant locomotor recovery of the SCI model rats. Together, the NSC-laden bioprinted SM scaffolds in combination with sustained release of the therapeutic agent OSMI-4 largely induced neuronal differentiation of NSCs and thus leading to efficient SCI repair. Statement of significanceEfficient neuronal differentiation of neural stem cells (NSCs) under the complex pathological microenvironment of spinal cord injury (SCI) is a major challenge of neural regeneration. By the use of a supramolecular bioink, we bioprinted a spinal cord-like scaffold loaded with NSCs and a small molecule drug OSMI-4 to significantly induce neuronal differentiation of NSCs for efficient SCI repair in vivo. The scaffolds with spinal cord-like structure can support the interaction and neuronal differentiation of NSCs by providing a dynamic matrix and a source of molecular release of OSMI-4. The influences of OSMI-4 on NSCs and its molecular mechanism were investigated for the first time in this study. Altogether, three-dimensional bioprinting fabrication of NSC- and small molecule drug-laden biomimetic construct may represent a promising therapeutic strategy for SCI repair.

Exosomes from microRNA 146a overexpressed bone marrow mesenchymal stem cells protect against spinal cord injury in rats

BackgroundThis study aimed to investigate the effect of microRNA 146a (miR-146a) overexpressed bone marrow mesenchymal stem cells (BMMSCs) exosomes on spinal cord injury (SCI) recovery. MethodsRat BMMSCs were isolated and transfected with miR-146a mimic (miR-mimic) and control mimic (NC-mimic), after which their exosomes were isolated. Afterward, SCI rat models were constructed, then treated with phosphate buffer saline (PBS), NC BMMSCs exosomes (separated from the culture medium of BMMSCs with NC-mimic), and miR-146a overexpressed BMMSCs exosomes (isolated from the culture medium of BMMSCs with miR-mimic), respectively; additionally, rats underwent sham surgery were treated with PBS as controls. ResultsMiR-146a was upregulated in BMMSCs, and BMMSCs derived exosomes post miR-mimic transfection. Then in SCI rats, BMMSCs exosomes elevated the Basso, Beattie, and Bresnahan (BBB) score and reduced hematoxylin&eosin-reflected spinal cord tissue injury. In addition, BMMSCs exosomes did not affect TUNEL positive cells rate while increased NeuN(+) cells/field in spinal cord tissue from SCI rats. As for inflammation, BMMSCs exosomes repressed pro-inflammatory cytokine expressions, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, in spinal cord tissue from SCI rats. Furthermore, miR-146a overexpressed BMMSCs exosomes presented with notably better effects regarding elevating BBB score in SCI rats and reducing tissue injury, neuron apoptosis, and inflammation while enhancing neuron viability in spinal cord tissue from SCI rats. ConclusionsMiR-146a overexpressed BMMSCs exosomes enhance locomotor capacity and neuron viability while reducing neuron apoptosis and spinal cord tissue inflammation in SCI rats.

Longitudinal electrophysiological changes after mesenchymal stem cell transplantation in a spinal cord injury rat model.

Transcranial electrically stimulated motor-evoked potentials (tcMEPs) are widely used to evaluate motor function in humans and animals. However, the relationship between tcMEPs and the recovery of paralysis remains unclear. We previously reported that transplantation of mesenchymal stem cells to a spinal cord injury (SCI) rat model resulted in various degrees of recovery from paraplegia. As a continuation of this work, in the present study, we aimed to establish the longitudinal electrophysiological changes in this SCI rat model after mesenchymal stem cell transplantation. SCI rats were established using the weight-drop method. The model rats were transvenously transplanted with two types of mesenchymal stem cells (MSCs), one derived from rat cranial bones and the other from the bone marrow of the femur and tibia bone, 24 h after SCI. A phosphate-buffered saline (PBS) group that received only PBS was also created for comparison. The degree of paralysis was evaluated over 28 days using the Basso-Beattie-Bresnahan (BBB) scale and inclined plane task score. Extended tcMEPs were recorded using a previously reported bone-thinning technique, and the longitudinal electrophysiological changes in tcMEPs were investigated. In addition, the relationship between the time course of recovery from paralysis and reappearance of tcMEPs was revealed. The appearance of the tcMEP waveform was earlier in MSC-transplanted rats than in PBS-administered rats (earliest date was 7 days after SCI). The MEP waveforms also appeared at approximately the same level on the BBB scale (average score, 11 points). Ultimately, this study can help enhance our understanding of the relationship between neural regeneration and tcMEP recording. Further application of tcMEP in regenerative medicine research is expected.