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Understanding the sequence of cellular responses and their contributions to pathomorphogical changes in spinal white matter injuries is a prerequisite for developing efficient therapeutic strategies for spinal cord injury (SCI) as well as neurodegenerative and inflammatory diseases of the spinal cord such as amyotrophic lateral sclerosis and multiple sclerosis. We have developed several types of surgical procedures suitable for acute one-time and chronic recurrent in vivo multiphoton microscopy of spinal white matter [1]. Sophisticated surgical procedures were combined with transgenic mouse technology to image spinal tissue labeled with up to four fluorescent proteins (FPs) in axons, astrocytes, microglia, and blood vessels. To clearly separate the simultaneously excited FPs, spectral unmixing including iterative procedures was performed after imaging the diversely labeled spinal white matter with a custom-made 4-channel two-photon laser-scanning microscope. In our longitudinal multicellular studies of injured spinal white matter, we imaged axonal dynamics and invasion of microglia and astrocytes for a time course of over 200 days after SCI. Our methods offer ideal platforms for investigating acute and chronic cellular dynamics, cell-cell interactions, and metabolite fluctuations in health and disease as well as pharmacological manipulations in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) remains a worldwide challenge due to limited treatment strategies. Repetitive trans-spinal magnetic stimulation (rTSMS) is among the most cutting-edge treatments for SCI. However, the mechanism underlying rTSMS on functional recovery is still unclear. In this study, 8-week-old C57BL/6J female mice were used to design SCI models followed by treatment with monotherapy (1 Hz rTSMS or LY364947) or combination therapy (rTSMS + LY364947). Our results showed obvious functional recovery after monotherapies compared to untreated mice. Immunofluorescence results demonstrated that rTSMS and LY364947 modulate the lesion scar by decreasing fibrosis and GFAP and possess the effect on neural protection. In addition, rTSMS suppressed inflammation and the activation of TGFβ1/Smad2/3 signaling pathway, as evidenced by markedly reduced TGF-βRⅠ, Smad2/3, and p-Smad2/3 compared with untreated mice. Overall, it was confirmed that 1 Hz rTSMS promotes SCI recovery by suppressing the TGFβ1/Smad2/3 signaling, revealing a novel pathological mechanism of 1 Hz rTSMS intervention, and may provide potential targets for clinical treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Spinal cord injury induces significant cellular stress responses. The Heat Shock Protein 90 (HSP90) plays a pivotal role as a molecular chaperone and is crucial for protein folding, stabilization, and cellular signaling pathways. Despite its important function in stress adaptation, the specific expression patterns and functional roles of HSP90 after nerve injury remain unclear. This study aimed to elucidate the expression dynamics and functional implications of HSP90 following central nervous system (CNS) injury. Using western blotting and immunohistochemical analyses, we observed upregulation of HSP90 expression in spinal cord tissues and within injured neurons in a spinal cord contusion injury model. Additionally, HSP90 was found to enhance neurite outgrowth in primary cortical neurons cultured in vitro. Furthermore, in a glutamate-induced neuronal injury model, the expression of HSP90 was up-regulated, and overexpression of HSP90 promoted neurite re-growth in damaged neurons. Overall, our findings highlight the critical involvement of HSP90 in the neural response to injury and offer valuable insights into potential therapeutic strategies for CNS repair., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Axonal regeneration is restricted in adults and causes irreversible motor dysfunction following spinal cord injury (SCI). In contrast, neonates have prominent regenerative potential and can restore their neural function. Although the distinct cellular responses in neonates have been studied, how they contribute to neural recovery remains unclear. To assess whether the secreted molecules in neonatal SCI can enhance neural regeneration, we re-analyzed the previously performed single-nucleus RNA-seq (snRNA-seq) and focused on Asporin and Cd109, the highly expressed genes in the injured neonatal spinal cord. In the present study, we showed that both these molecules were expressed in the injured spinal cords of adults and neonates. We treated the cortical neurons with recombinant Asporin or CD109 to observe their direct effects on neurons in vitro. We demonstrated that these molecules enhance neurite outgrowth in neurons. However, these molecules did not enhance re-growth of severed axons. Our results suggest that Asporin and CD109 influence neurites at the lesion site, rather than promoting axon regeneration, to restore neural function in neonates after SCI., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background Context: Elucidating the mechanism of neuropathic pain (NeP) is crucial as it can result in motor dysfunction and negatively impact quality of life in patients with spinal cord injury (SCI). Although it has been reported that cyclooxygenase 2 (COX2) is involved in NeP in rat models of peripheral nerve injury and that COX2 inhibitors can alleviate NeP, these mechanisms after SCI have not been fully investigated., Purpose: The purpose is to investigate whether the thoracic SCI affects the expression of mRNAs for COX1 and COX2 in the lumbar spinal cord, and the effect of COX2 inhibitor on its behavior., Study Design: Male Sprague-Dawley (SD) rats underwent thoracic (T10) spinal cord contusion injury using an Infinite Horizon (IH) impactor device. SCI rats received COX2 inhibitors (50 μg/day) on days 5 and 6 after SCI., Methods: Male SD rats underwent T10 laminectomy under mixed anesthesia, and IH impactors were applied to the same site to create a rat SCI model. Rats that underwent only laminectomy were designated as sham. Lumbar spinal cord at the L4-5 level was harvested at 3, 5, 7, 14, and 28 days after SCI, and COX2 and COX1 were quantified by reverse-transcription PCR (RT-PCR). COX2 expression, expression site, and expression time were determined by immunohistochemistry (IHC) and in situ hybridization histochemistry (ISHH) at the same time points. The expression site and time of COX2 expression were also examined at the same time point by ISHH. On 5th and 6th day after SCI, saline and COX2 inhibitor (50 μg/day) were administered into the subarachnoid space as a single dose, and the two groups were compared in terms of mechanical withdrawal latency using the dynamic plantar esthesiometer, which is an automated von Frey-type system., Results: COX2 was significantly increased at 5 and 7 days after SCI, but no significant difference in COX1 was observed after SCI by RT-PCR. ISHH targeting COX2 showed clear expression of COX2 in spinal cord vascular endothelial cells at 5 and 7 days after SCI. COX2 expression was almost abolished at day 14 and 28. Behavioral experiments showed that pain was significantly improved from day 2 after COX2 inhibitor administration compared to the saline group, with improvement up to day 14 after SCI, but no significant difference was observed after day 21., Conclusions: The present findings suggest that thoracic SCI increased COX2 in vascular endothelial cells in the lumbar spinal cord and that the administration of COX2 inhibitor significantly alleviated mechanical hypersensitivity of the hind-paw following the thoracic SCI. Therefore, endothelial cell derived COX2 in the lumbar spinal cord may be involved in the induction of neuropathic pain in the SCI model rats., Clinical Significance: The findings in the present study regarding the induction of endothelial COX2 and the effect of its inhibitor on the mechanical hypersensitivity suggest that endothelial cell-derived COX2 is one of the focuses for the treatment for neuropathic pain in the acute phase of SCI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) is a devastating trauma of the central nervous system, with high levels of morbidity, disability, and mortality. To explore the underlying mechanism of SCI, we analyzed the proteome and phosphoproteome of rats at one week after SCI. We identified 465 up-regulated and 129 down-regulated differentially expressed proteins (DEPs), as well as 184 up-regulated and 40 down-regulated differentially expressed phosphoproteins (DEPPs). Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, we identified the biological characteristics of these proteins from the perspectives of cell component, biological process, and molecular function. We also found a lot of enriched functional pathways such as GABAergic synapse pathway, ErbB signaling pathway, tight junction, adherens junction. The integrated analysis of proteomics and phosphoproteomics yielded 22 differently expressed co-identified proteins of DEPs and DEPPs, which revealed strongly correlative patterns. These findings may help clarify the potential mechanisms of trauma and repair in SCI and may guide the development of novel treatments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Spinal cord injury (SCI) is a destructive and disabling nerve injury from which complete recovery has not yet been achieved due to complex pathology. Casein kinase II (CK2) is a pleiotropic serine/threonine protein kinase that plays an essential role in the nervous system. This study aimed to investigate the role of CK2 in SCI to understand the pathogenesis of SCI and explore new therapeutic methods. The SCI rat model of C5 unilateral clamp was established by modified clamp method in male adult SD rats. Then, CK2 inhibitor DMAT was used to treat SCI rats, and the behaviour, pathological changes in the spinal cord and microglial polarization were analysed. Additionally, the effects of DMAT on the polarization and autophagy of microglial BV-2 cells were investigated in vitro, and the effects of BV-2 polarization on spinal cord neuronal cells were analysed by Transwell coculture. Results showed that DMAT significantly increased the BBB score, improved histopathological injury, decreased the expression of inflammatory cytokines, and promoted M2 polarization of microglia in SCI rats. In vitro experiments further confirmed that DMAT could promote the polarization of BV-2 to the M2 type, promote autophagy, and reverse the LPS-induced decline in cell viability and increase in apoptosis of neuronal cells. The use of 3-MA confirmed that autophagy plays an important role in DMAT promoting M2 polarization of BV-2 to improve neuronal cell viability. In conclusion, CK2 inhibitor DMAT improved SCI by inducing anti-inflammatory polarization of microglia through autophagy and is a potential therapeutic target for SCI., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Spasticity is a common complication in patients with spinal cord injury (SCI) and adversely affects patients' quality of life. Little is known about the distribution of the serotonin 1F receptor (5-HT1FR) in the spinal cord, especially in relation to the spasticity caused by SCI. Adult male Wistar rats were divided into a sham-operation group and spinalized group. SCI-induced spasticity was caused by spinal transection at the second sacral segment. The spinal cord below the transection was obtained at the end of the experiment. The expression and distribution of 5-HT1FR in the spinal cord were analyzed. The results showed that the expression of 5-HT1FR (mRNA and protein) exhibited the same downward trend after spinal transection and reached the lowest expression level at 2 and 5 days, respectively. The expression of 5-HT1FR (mRNA and protein) thereafter gradually approached the levels in the sham-operation group after 60 days. Immunostaining suggested that 5-HT1FR showed particularly strong expression in the ventral horn (VH) region. The time course of 5-HT1FR mRNA downregulation is positively correlated with the development of tail spasticity after sacral spinal cord transection. There may be a connection between 5-HT1FR and the occurrence of spasticity, but elucidation of the specific mechanism needs further experimental verification., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The role of CD93 in inflammatory response has been reported in multiple previous studies. However, the underlying mechanism of CD93 in microglial activation and migration during neuroinflammation post spinal cord injury (SCI) remains elusive. In the current study, we performed western blot, qRT-PCR, immunofluorescence analyses Transwell assay, and ELISA to determine the expression change and in-depth molecular mechanism of CD93 in microglia post inflammatory initiation. We found that CD93 expression was increased in microglia after SCI in vivo or lipopolysaccharide (LPS) stimuli in vitro. Additionally, CD93 interacted with TAK1 to inhibit NF-κB activation, thus attenuating inflammation and migration of microglia after treatment with LPS. These findings indicate that CD93 might participate in microglia-induced neuroinflammation development post SCI, suggesting that CD93 is a promising target for neuroimmunological regulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: To investigate the effects of treadmill training on lung injury and HMGB1/TLR4/NF-κB after spinal cord injury (SCI) in rats., Methods: A total of 108 female SD rats were randomly divided into three groups: sham operation group, SCI brake group, and SCI exercise group. The rats in the SCI exercise group began treadmill training on the 3rd day after the operation. The rats in the SCI brake group underwent braking treatment. The lung tissues were obtained on the 3rd, 7th, and 14th days after exercise. Locomotor functional recovery was determined using the BBB scores and inclined plane test. Respiratory function was determined via abdominal aortic blood gas analysis. HE staining was used to detect pathological changes in rat lung tissue. RNA sequencing was used to identify differentially expressed genes at different phases in each group of lung tissues. HMGB1, TLR4, and NF-κB in lung tissue were detected using immunohistochemistry and immunofluorescence. Detection of HMGB1 levels in serum, spinal cord tissues and lung tissues by ELISA. HMGB1, TLR4, NF-κB, IL-1β, IL-6, TNF-α mRNA, and protein expression levels were detected via qRT PCR and western blot., Results: Motor and respiratory functions significantly decreased after SCI (P < 0.05). However, locomotion and respiratory functions were significantly improved after treadmill training intervention (P < 0.05). HE staining showed that interstitial thickening, inflammatory cells, and erythrocyte infiltration occurred in lung tissue of rats after SCI (P < 0.05). Moreover, inflammatory reaction in lung tissue was significantly reduced after treadmill training intervention (P < 0.05). A total of 428 differentially expressed mRNAs [(|log2(FC)| > 2, P < 0.05)] were identified in the intersection of the three groups. KEGG analysis identified five enriched signal pathways, including NF-kappa B. ELISA results showed that treadmill training could significantly reduce the levels of HMGB1 in serum, spinal cord tissue and lung tissue that were elevated after SCI (P < 0.05). Immunohistochemistry, immunofluorescence, qRT PCR, and Western blot showed that HMGB1, TLR4, IL-1β, IL-6, TNF-α, and NF-κB expressions were significantly up-regulated at the 3rd, 7th and 14th days after SCI, compared with the sham group. Besides, inflammatory cytokines were significantly lower in the SCI exercise group than in the SCI brake group at all time points after intervention (P < 0.05)., Conclusion: Treadmill training alleviates lung tissue inflammation and promotes recovery of motor and respiratory functions by inhibiting the HMGB1/TLR4/NF-κB signaling pathway after SCI in rats., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: To study the protective effect of tauroursodeoxycholic acid (TUDCA) on the spinal cord nerve cells (SCN) of SD rats and to explore the protective mechanism of TUDCA against mechanical injury of the SCN., Material and Methods: The SCN of SD rats were cultured in vitro, and a mechanical injury models of 1 mm, 3 mm, and 5 mm SCN were established. The cell survival rate was determined using the MTT assay to determine the optimal degree and time of injury. Different concentrations (0.5, to 20 mmol/L) of TUDCA were used to detect SCN cell survival rate after mechanical injury. MTT assay was used to determine the optimal TUDCA intervention dose. SCN autophagy in different experimental groups were observed by electron microscopy after the best degree of mechanical injury, time of injury, and TUDCA concentration. Beclin-1 and LC3 II/I expressions were detected by western blotting and immunohistochemistry., Results: Survival rate of SCN was close to 50% when the injury interval was 3 mm and the injury time was 24 h, significantly different from those of each group (P < 0.05). At 3 mm injury interval and 24 h injury time, SCN survival rate was approximately 80% when TUDCA concentration was 4 mmol/L, which was significantly different from those of each group (P < 0.05). Cell morphology of the normal control group was complete, with few autophagy lysosomes. Compared with the normal control group, the number of autophagic lysosomes in the mechanical injury group increased, and cell damage was more severe. Compared with the mechanical injury group, the number of autophagy lysosomes in the mechanical injury + TUDCA intervention group increased significantly, and cell damage was less severe. Further, compared with the normal control group, beclin-1 and lc3ii / I expressions in the mechanical injury group were significantly higher (P < 0.05); compared with the mechanical injury group, beclin-1 and lc3ii / I expressions in the mechanical injury + TUDCA intervention group were significantly higher (P < 0.05)., Conclusion: TUDCA can protect SCN from mechanical injury in vitro, which may be related to the enhancement of the expression of autophagy-related protein beclin-1 and LC3 II/I., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The pathophysiology following spinal cord injury (SCI) progresses from its lesion epicenter resulting in cellular and systemic changes acutely, sub-acutely and chronically. The symptoms of the SCI depend upon the severity of the injury and its location in the spinal cord. However, there is lack of studies that have longitudinally assessed acute through chronic in vivo changes following SCI. In this combinatorial study we fill this gap by evaluating acute to chronic effects of moderate SCI in rats. We have used fluorodeoxyglucose (FDG) imaging with positron emission tomography (PET) as a marker to assess glucose metabolism, motor function, and immunohistochemistry to examine changes following moderate SCI. Our results demonstrate decreased FDG uptake at the injury site chronically at days 28 and 90 post injury compared to baseline. This alteration in glucose uptake was not restricted to the lesion site, showing depressed FDG uptake in non-injured areas (cervical spinal cord and cerebellum). The alteration in glucose uptake was correlated with reductions in neuronal cell viability and increases in glial cell activation at 90 days at the lesion site, as well as chronic impairments in motor function. These data demonstrate the chronic effects of SCI on glucose metabolism both within the lesion and distally within the spinal cord and brain., (Published by Elsevier B.V.)

Macrophage-dominated inflammation by the activation of Toll-like receptor (TLR) pathway leads to neurological disruption after spinal cord injury (SCI). Regulator of G-protein signaling 1 (RGS1) is reported to be a regulator in inflammation. The present study thus purposes to identify the unknown role of RGS1 mediating TLR on inflammation post SCI. A mouse model of traumatic SCI was established by a mechanical trauma at T10. The mice underwent SCI and a macrophage line activated by lipopolysaccharide (LPS) were treated with shRNA-RGS1 to elucidate the role of RGS1 in inflammatory progression. The inflammatory factors were measured, and the degree of histology and function protection were determined. The expression levels of RGS1, myeloid differentiation primary response protein 88 (Myd88), (TIR-domain-containing adaptor inducing interferon-β (TRIF), p38, metalloproteinase (MMP)-2, and MMP-9 were determined. RGS1 was robustly increased both in LPS-activated macrophage and SCI mice. The TLR signaling pathway-induced inflammation was suppressed by RGS1 knockdown. shRNA-mediated silence of RGS1 was exhibited a prominent decrease in TNF-α, IL-1β and IL-6 via TLR/TRIF/ nuclear factor kappa-B (NF-κB) axis. Depletion of RGS1 also inhibited MMP-induced tissue degradation via MAPK-p38 pathway in SCI mice. Moreover, suppression of RGS1 improved spinal cord histology and function recovery. These findings suggest that RGS1 regulates inflammation and tissue disruption via TLR/TRIF/NF-κB signaling pathway in mice with SCI, thereby explaining a novel target that regulates macrophage inflammation post SCI., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Growing evidence has shown that microRNAs (miRNAs) play crucial roles in the physiopathology of spinal cord injury (SCI). Recent studies have confirmed that miR-338-5p regulates myelination, suggesting a potential role in the treatment of SCI. However, the molecular mechanism of miR-338-5p on SCI is still unknown. Recently, exosomes have emerged as an ideal vector to deliver therapeutic molecules such as miRNAs. Here, we explored the effects of miR-338-5p-overexpressing exosomes derived from bone marrow-derived mesenchymal stromal cells (BMSCs) on SCI. In vivo, a model of contusion SCI in rats was established, and we observed that overexpression of miR-338-5p in exosomes profoundly increased the expression levels of neurofilament protein-M and growth-associated protein-43 and decreased those of myelin-associated glycoprotein and glial fibrillary acidic protein, which provided neuroprotective effects after acute SCI. In an in vitro study, we found that overexpression of miR-338-5p in exosomes repressed cell apoptosis following H 2 O 2 -induced oxidative stress injury in PC12 cells. Additionally, we confirmed that cannabinoid receptor 1 (Cnr1) was the target gene of miR-338-5p by dual-luciferase reporter assays and that Rap1 was the downstream gene by the KEGG pathway analysis. We found that miR-338-5p increased cAMP accumulation as a consequence of downregulated expression of the target gene Cnr1, and then, Rap1 was activated by cAMP. Eventually, the activation of the PI3K/Akt pathway attenuated cell apoptosis and promoted neuronal survival by cAMP-mediated Rap1 activation. In brief, these findings showed that exosomes overexpressing miR-338-5p were a promising treatment strategy for SCI., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Molecule's mechanism of action interacting with CasL 1 (MICAL1) in spinal cord injury (SCI) is unclear. This study aimed to detect the function of MICAL1 in SCI. Western blot was used to analyze the change of MICAL1 in vivo. Immunofluorescence staining was used to detect the location of MICAL1 expression. Oligodendrocyte cells were treated with H 2 O 2 to induce oxidative injury. Subsequently, siRNA transfection was performed to decrease MICAL1 expression in oligodendrocyte cells. Then, the effects of MICAL1 on oxidative stress, apoptosis, and autophagy were assessed. We found that silencing of MICAL1 could significantly reduce the levels of the nuclear factor erythroid 2-related factor 2 (Nrf2), increase the expression of pro-apoptotic factors (Bax and C-caspase 3), decrease the levels of anti-apoptotic factor (Bcl-2) and pro-autophagy factors (Beclin1 and LC3B). Therefore, MICAL1 is a potential target gene for SCI clinical therapy., (Copyright © 2021. Published by Elsevier B.V.)

Objective: To verify the effect of metformin on spinal cord injury (SCI) through Wnt/β-catenin signaling pathway., Background: SCI 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., Methods: Rats 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., Results: Metformin(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., Conclusion: We 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Little is known about the effect of wnt-3a on motor nerve function and its specific molecular mechanisms after spinal cord injury (SCI). This study demonstrates that the downregulated expression levels of caspases-3, caspases-9 and chondroitin sulfate proteoglycan (CSPG) proteins and number of proportion of transferase UTP nick end labeling (TUNEL)-positive neurons by wnt-3a treatment. Then, Nissl and hematoxylin-eosin (HE) staining showed that wnt-3a significantly reduced the loss of spinal anterior horn motor neurons and promoted repair of injured spinal cord tissues after SCI. The above factors constructed a favorable microenvironment for the recovery of motor nerve function after SCI. To elucidate the molecular mechanism of neuroprotection of wnt-3a on SCI, the study showed that the expression levels of Beclin-1 and light chain (LC)3-II/I in spinal cord neurons were significantly improved by wnt-3a after SCI in vitro and vivo experiments, while the effect of wnt-3a was inhibited after mechanistic target of rapamycin (mTOR) signaling pathway being activated by MHY-1485. Besides, the level of p70S6K phosphorylation was inhibited by wnt-3a treatment, on the contrary, the level of p70S6K protein was elevated by wnt-3a, indicating that wnt-3a significantly activated neuronal autophagy by inhibiting mTOR signaling pathway after SCI. To further verify the correlation between neuroprotection of wnt-3a and autophagy, we found that after the rats and spinal cord neurons were combined treatment with wnt-3a and MHY-1485, the neuroprotection of wnt-3a on SCI was significantly inhibited. This study is the first to report that wnt-3a improves functional recovery through autophagy activation via inhibiting the mTOR signaling pathway after SCI., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) causes loss of locomotor function and chronic neuropathic pain (NeP). Hematogenous macrophages and activated microglia are key monocytic lineage cell types in the response to SCI, and each has M1- and M2-phenotypes. To understand the roles of these cells in neuronal regeneration and chronic NeP after SCI, differences in distribution and phenotypes of activated microglia and infiltrated macrophages after SCI were examined at the injured site and the lumbar enlargement, as a remote region. Chimeric mice were used for differentiating activated microglia from hematogenous macrophages. The prevalences of activated microglia and infiltrating macrophages increased at day 14 after SCI, at the time of most severe pain hypersensitivity, with mainly M1-type hematogenous macrophages at the injured site and M2-type activated microglia at the lumbar enlargement. Peak expression of TNF-α, an M1-induced cytokine, occurred on day 4 post-SCI at the injured site, but not until day 14 at the lumbar enlargement. Expression of IL-4, a M2-induced cytokine, peaked at 4 days after SCI at both sites. These results suggest different roles of activated microglia and hematogenous macrophages, including both phenotypes of each cell, in neuronal regeneration and chronic NeP after SCI at the injured site and lumbar enlargement. The prevalence of the M1 over the M2 phenotype at the injured site until the subacute phase after SCI may be partially responsible for the lack of functional recovery and chronic NeP after SCI. Activation of M2-type microglia at the lumbar enlargement in response to inflammatory cytokines from the injured site might be important in chronic below-level pain. These findings are useful for establishment of a therapeutic target for prevention of motor deterioration and NeP in the time-dependent response to SCI., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) is a traumatic condition of the central nervous system (CNS) that can cause paralysis of the limbs. The molecular mechanisms of neural repair following SCI remain unclear and no effective treatment for SCI currently exists, since drugs have difficulty crossing the blood-brain barrier (BBB). The present study aimed to investigate whether exosomes could be used as specific carriers of resveratrol for induction of neuronal autophagy both in vitro and in vivo for the treatment of SCI. The results indicate that exosomes are able to enhance the solubility of resveratrol and enhance penetration of the drug through the BBB, thereby increasing its concentration in the CNS. Exosomes derived from resveratrol-treated primary microglia (Exo + Res) assisted the rehabilitation of paralyzed limbs in rats. Restoration of neural function following SCI was mediated through increased induction of autophagy and inhibition of apoptosis of neurons both in vitro and in vivo via activation of the PI3K signaling pathway. The mechanism of action of Exo + Res may be associated with the PI3K inhibitor 3-methyladenine (3-MA) in primary spinal neurons. The results suggest that Exo + Res are highly effective at crossing the BBB with good stability, suggesting they have potential for enhancing targeted drug delivery and the recovery of neuronal function in SCI therapy, likely associated with the induction of autophagy and inhibition of apoptosis via the PI3K signaling pathway., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Autophagy is a intracellular biological process that controls the homeostasis of nutrition deprivation and starvation and has been associated with the development of traumatic diseases. Zinc, an important chemical element involved in life activities, has improved nerve recovery effects through intraperitoneal injection. The purpose of this study was to probe the possible modulation of autophagy and apoptosis from the injured spinal cord and neurons by zinc administration. It was shown that zinc significantly induced the level of Beclin1 and LC3B by activating adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway. In addition, zinc suppressed apoptosis in the injured spinal cord. Taken together, these findings suggested that zinc through promoting neurons autophagy and inhibiting apoptosis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) is a worldwide problem and transplantation of neural progenitor cells (NPCs) represents a promising treatment strategy. Urine derived induced pluripotent stem cells (UiPSCs) which enable the generation of patient-specific NPCs, provide an invaluable source of autologous cells for future therapeutic applications after SCI. However, the fate and potential contribution of transplanted human UiPSCs-derived NPCs (UiPSC-NPCs) into injured spinal cords remain largely unknown. In this study, using a rat contusive SCI model, we evaluated the survival, migration and differentiation of UiPSC-NPCs after transplantation at subacute phase. Transplanted cells survived and migrated from the site of grafting towards the lesion epicenter. More than 25 % cells survived over 4 weeks post transplantation, with a few of them differentiated into neurons and astrocytes. Cytokine and chemokine levels within the injured spinal cord tissues were measured using multiplex immunoassays to evaluate the immune response. Pro-inflammatory factors and chemokines were significantly decreased at 3 days after UiPSC-NPCs transplantation. At 7 days post transplantation, a lower level of pro-inflammatory factor IFN-γ and a higher level of pro-inflammatory IL-2 were found in UiPSC-NPCs group than in the control. Transplantation of UiPSC-NPCs showed little effect on microglia activation at the lesion epicenter. However, the number of microglia cells at 4 mm rostral to the injury site was significantly decreased. The size of lesion cavity was reduced after transplantation of UiPSC-NPCs. In conclusions, the UiPSC-NPCs transplanted at the subacute phase of SCI showed a beneficial effect on tissue repairing., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Shh/Gli1 signaling plays important roles in development of spinal cord. How it is involved in spinal cord injury (SCI) remains unclear. In this study, we explored the roles of Shh/Gli1 signaling in SCI by using Shh signaling reporter Gli1 lz mice and Gli1 mutant Gli1 lz/lz mice. For detecting the Shh/Gli1 signaling after SCI, X-gal staining and double-immunostaining of Shh/PDGFR-β, Shh/GFAP and LacZ/GFAP was conducted at 3 days post injury (dpi) on Gli1 lz mice. To investigate the effects of Gli1 mutation on pathological changes after SCI, astrocytic proliferation and the content of intra-parenchymal Evans Blue were evaluated at 7dpi in wild-type and Gli1 lz/lz mice. Furthermore, locomotor recovery was assessed by BMS scoring at 1, 3, 5 and 7dpi. The results of X-gal staining and immunohistochemistry showed that Shh/Gli1 signaling was mainly activated in reactive astrocytes after SCI. The 5-bromo-2-deoxyuridine (BrdU) incorporation assay showed that mutation of Gli1 did not affect the proliferation of astrocytes. However, the leakage of Evans Blue was significantly increased in the injured cord of Gli1 lz/lz mice compared to wild-type mice. In addition, locomotor recovery was significantly impaired in the Gli1 lz/lz mice. The findings demonstrated that Shh/Gli1 signaling could be induced in reactive astrocytes by SCI, and plays important role in permeability of blood-spinal cord barrier (BSCB) and locomotor recovery after SCI., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Spinal cord injury (SCI) is one of the most serious neurological disorders and is characterized by high morbidity and disability. Unfortunately, there is a lack of effective treatment. Recently, the micro RNA, miR-384-5p, was reported to play a significant role in cell survival in response to different insults., Methods: In vitro model of traumatic neuronal injury was induced by application of a sharp sterile blade to generate cuts in PC12 cells, and in vivo SCI was produced by applying vascular clips (force of 15 g) to the dura via T9-T10 laminectomy, and then, the role of miR-384-5p in the development of SCI was investigated., Results: Dual-luciferase reporter assays confirmed that miR-384-5p regulates the gene expression of Beclin-1, an important promoter of autophagy. Quantitative polymerase chain reaction and western blot analyses revealed that treatment with miR-384-5p decreased mRNA and protein expression of Beclin-1 in the mechanically injured PC12 cells. In rats with spinal cord compression injuries, miR-384-5p expression was significantly decreased. Treatment with miR-384-5p increased spinal cord neuron survival and promoted locomotor function recovery in rats. Further study revealed that miR-384-5p administration decreased immunofluorescent labeling of Beclin-1 in spinal cord tissues and reduced autophagosome formation in neurons, as shown by transmission electron microscopy. These results indicated that miR-384-5p promotes recovery of rats with SCI by suppressing autophagy via direct targeting of Beclin-1. Moreover, miR-384-5p also inhibited the activation of endoplasmic reticulum (ER) stress by decreasing GRP78 expression in both in vitro and in vivo models., Conclusions: This study for the first time demonstrates that the protective role of miR-384-5p in the process of SCI is associated with simultaneous suppression of autophagy and ER stress and miR-384-5p could be a promising candidate for SCI therapeutics., Competing Interests: Declaration of Competing Interest None of the authors has any conflict of interest to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) triggers pronounced inflammatory responses that are accompanied by neuronal disruption and functional deficits. SCI treatment remains an unmet clinical need. Emerging evidence suggests that riluzole may exert a neuroprotective effect due to its anti-inflammatory properties. However, details of the underlying mechanisms remain poorly defined. The polarization of microglial/macrophages has an important role in neuroinflammation. Here, we examined whether riluzole can exert a neuroprotective effect after acute SCI, and whether this effect is associated with changes in microglia/macrophages polarization. Riluzole (4 mg/kg) or vehicle were injected intraperitoneally (i.p.) in female rats immediately following SCI and repeated for 7 consecutive days (b.i.d.). Compared with vehicle treatment, riluzole-treated SCI rats showed significant higher locomotor scores (Basso, Beattie, and Bresnahan score, Inclined Plane test score, n = 18/group). Riluzole-treated rats also developed smaller spinal cavities, showed higher levels of myelin basic protein (MBP) and neurofilament (NF)200 immunoreactivities, and lower levels of proinflammatory cytokines in the spinal cord at 7 days post-SCI. Immunofluorescence study revealed more CD206 + cells and less iNOS + cells in the injured spinal cord of riluzole-treated SCI rats, as compared to vehicle control. Using real-time PCR, we found that riluzole upregulated the mRNA levels of M2 markers, but downregulated that of M1 markers, as compared to the vehicle treatment. Current findings suggest that systemic administration of riluzole after acute SCI facilitated motor function recovery and inhibited inflammatory responses, which may be associated with polarization of M2 microglia/macrophages., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Spinal cord injury (SCI) induced by trauma is a devasting neurological consequences. Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, has be shown to have neuroprotective effects in several neurodegenerative diseases. However, the potential benefits of liraglutide as well as the underlying mechanisms for its therapeutic benefit for SCI are unclear. We aimed to investigate the therapeutic benefits and decipher the potential signaling pathways of liraglutide in spinal contusion injury. A SD rat model with controlled spinal contusion was established for this study. Behavioral tests and histological examinations were performed to assess the neuroprotective benefits. Several autophagy markers were measured by western blot analysis and immunofluorescence staining, including LC3B, Beclin-1 and p62. In addition, the AMPK-FOXO3 signaling pathway was investigated. Our results demonstrated that liraglutide treatment strongly enhanced motor function recovery and alleviated the degree of necrosis and loss of motor neurons in the spinal cord tissue after contusion. Autophagic responses were activated by liraglutide. LC3B-II/LC3B-I and Beclin-1 expression was enhanced while p62 expression was reduced. In addition, the levels of p-AMPK/AMPK, FOXO3 and p-FOXO3 (phospho S253) were notably up-regulated by liraglutide. These effects were partly reversed by Compound C, an AMPK inhibitor. In summary, our results demonstrated that liraglutide was therapeutically beneficial in treating spinal contusion injury and its underlying mechanism was through the activation of autophagic responses through the AMPK-FOXO3 signaling pathway., Competing Interests: Declaration of Competing Interest All authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Apoptosis signal-regulating kinase 1 (ASK1) may play a pivotal role in reactive gliosis. To assess the role of ASK1 in trauma-induced reactive gliosis, we examined the phosphorylation of ASK1 and the expression of glial fibrillary acidic protein (GFAP) and vimentin after scratch injury in cultured astrocytes and spinal cord injury (SCI) in rats. Enhanced phosphorylation of ASK1 was detected during reactive gliosis both in vitro and in vivo, and P38 MAPK relayed the signal from phosphorylated ASK1 to the activation of astrocytes. Immunoprecipitation analyses suggested that 14-3-3 was dissociated from ASK1 during astrocyte activation. Finally, treatment with thioredoxin reduced ASK1 phosphorylation and reactive gliosis and promoted hindlimb locomotion recovery in SCI rats. These results indicated that ASK1 may play an important role in mechanical-injury-induced reactive gliosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) is a devastating neuropathological condition. Long noncoding RNA X-inactive specific transcript (XIST) is an acknowledged cancer-related gene and participates in the development of SCI. However, role of XIST in SCI remains to be well revealed. Expression of XIST, miRNA-27a-3p (miR-27a) and smad ubiquitination regulatory factor 1 (Smurf1) was detected using RT-qPCR and western blotting. Cell apoptosis and inflammatory injury were assessed by sulforhodamine B (SRB) assay, flow cytometry, western blotting and enzyme-linked immunosorbent assay. The relationship among miR-27a, XIST and Smurf1 was confirmed by dual-luciferase reporter assay, RNA immunoprecipitation and RNA pull-down assay. As a result, we observed higher level of XIST and Smurf1, but lower level of miR-27a in SCI rats and lipopolysaccharide (LPS)-induced primary microglial cells. in vitro, LPS induced SCI microglia cells as described by decreased cell viability and B cell lymphoma 2 (Bcl-2) expression, and increased cell apoptosis rate, Bax and cleaved caspase 3 levels, and tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) secretions. in vivo, a T10 laminectomy caused SCI rats as evidenced by decreased Basso-Beattie-Bresnahan Locomotor Rating Scale (BBB) score and induced expression of Bax, cleaved caspase 3, TNF-α and IL-6. However, silencing of XIST could mitigate the apoptosis and inflammatory injury in LPS-induced microglia and SCI rats. Mechanically, miR-27a interacted with XIST and Smurf1 via target binding. Either miR-27a downregulation or Smurf1 overexpression partially reversed the role of XIST deletion in LPS-treated microglial cells. Collectively, knockdown of XIST could alleviate the apoptosis and inflammatory injury of SCI models in vitro and in vivo through directly modulating miR-27a/Smurf1 axis., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Backgrounds/aims: Brain-derived neurotrophic factor (BDNF) plays a primary role in the maturation, proliferation, and differentiation of neuronal cells, can induce bone-marrow-derived mesenchymal stem cells (MSCs) to differentiate into nerve cells. This study aims to explore whether regulation of BDNF through microRNAs (miRNAs) in MSCs may further enhance the therapeutic effect on spinal cord injury (SCI)., Methods: Bioinformatics analyses were done to predict miRNAs that target BDNF in MSCs. Dual-luciferase reporter gene assays were performed to verify the target relationship between microRNA and BDNF. We examined the mRNA and protein levels of BDNF in MSCs by RT-qPCR and Western blot, respectively. CCK 8 assay was chosen to assess cell viability. MSCs were transduced with miR-10a-5p-ASO, which were transplanted into rats that underwent SCI. The tissue integrity percentage, cavity volume, and Basso-Beattie-Bresnahan (BBB) scale were assessed. Neurofilament (NF) was detected using immunohistochemistry. Histological features of spinal cord tissues examined following HE staining., Results: MiR-10a-5p inhibited protein translation of BDNF, through binding to the 3'-UTR of the BDNF. MSCs transduced with MiR-10a-5p-ASO further increased the tissue integrity percentage, decreased cavity volume, and enhanced the recovery of BBB score in SCI model rats, compared to control MSCs., Conclusion: Upregulation of BDNF by miR-10a-5p suppression in MSCs further improve the therapeutic potential of MSCs in treating SCI in rats., (Copyright © 2019. Published by Elsevier B.V.)

Myeloid cells are important effector cells in the injured spinal cord tissue. Microglia and monocyte-derived macrophages serve important functions in the injured spinal cord, and their distinctive roles can now be studied more efficiently with the help of reporter mice and cell specific markers that were described in recent years. Focusing on microglia, this review discusses the microglial response to injury, microglia specific effects and the interaction between microglia and other cell types in the injured spinal cord., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Study Design: Animal study., Objectives: The aim of this study is to investigate the influence of inflammasomes in the injured spinal cord of a rat spinal cord injury model., Setting: University laboratory in Kanagawa, Japan., Methods: A thoracic contusion spinal cord injury (SCI) was induced in female Sprague Dawley rats using an IH-impactor to create a moderate injury group (LI) and a severe injury group (HI). Using a sham group as a control, the injured spinal cords were removed at several time points after injury to evaluate the levels of inflammasome component proteins in the injured spinal cord by immunohistochemistry and Western Blot., Results: Western blot analyses revealed that the expression of inflammasome component proteins leucine-rich repeat protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-2 significantly increased in the SCI animals compared to the sham animals. Thioredoxin interacting protein (TXNIP), which is a protein induced by ER stress that activates the NLRP3 inflammasome, was also significantly higher in the SCI animals. Immunohistochemistry revealed significantly higher expression of NLRP3, ASC, and Caspase-2 in oligodendrocyte progenitor cells (OPCs) of the SCI groups compared to astrocytes of the SCI groups and OPCs of the sham group., Conclusions: Inflammasome component protein expression increases after SCI in association with increased ER stress. OPCs had significantly higher levels of inflammasome proteins compared to astrocytes, which may be associated with the high rates of OPC cell death after SCI., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) results in pronounced focal tissue damage with subsequent formation of a glial scar that blocks axon regeneration and regrowth. Cellular changes and the composition of the extracellular matrix in regions distal from the injured area remain poorly characterized. In the present study, in the spinal cord distal to the damaged area (perilesion perimeter) there were minimal gross histological changes, but there were pronounced alterations in the extracellular proteoglycans even at 30 days after SCI. These abnormalities coincided with the appearance of reactive astrocytes and a reduction in main astrocytic glutamate transporter 1. Proteoglycan levels exhibited different kinetics and changes after SCI in areas near neuronal cell bodies and in areas distal from them. The results of the study suggest that SCI induces widespread changes in the spinal cord that may be responsible for neuronal dysfunction far from the damaged area and further aggravation of the SCI., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Receptor for advanced glycation end product (RAGE) is involved in neuronal inflammation, cell cycle and differentiation. However, the role of RAGE in autophagy in the process of spinal cord injury (SCI) is yet unknown. The present study investigated the effect of RAGE blockade on autophagy in SCI. A rat Allen SCI model was established and the animals were micro-injected with rabbit RAGE neutralizing antibody or rabbit polyclonal Ig G immediately after the injury. The oligodendrocytes(OLs) marker, 2', 3'-cyclic nucleotide 3'-phosphodiesterase(CNPase) and autophagy-related marker microtubule associated protein light chain 3B(LC3B) were evaluated by Western blot. Furthermore, myelin basic protein (MBP) and LC3B double staining were observed in the SCI via immunofluorescence. The results showed that RAGE blockade reduced the expression of CNPase, promoted LC3B-II/I and p62 expression after SCI. In addition, the MBP/LC3B double positive oligodendrocytes-expressing LC3B was up-regulated by RAGE blockade. Moreover, RAGE blockade attenuated the neuronal survival at ventral horn after SCI. The present study revealed the role of RAGE in maintaining oligodendrocyte autophagy to promote neuronal regeneration post-SCI., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Neuronal death and organization degeneration can happen inordinately after spinal cord injury (SCI), which lead to nerve dysfunction. We aimed to determine whether local application of a cell permeable calpain I inhibitor (MDL28170) can promote SCI recovery by increasing neuronal cell viability. MDL28170-loaded polycaprolactone (PCL) film was fabricated. Scanning electron microscopy showed the surface of PCL film was smooth with holes (diameter at μM level). The PCL film was non-toxic, biological compatibility, and had good neuron adhension and slow release characteristic. MDL28170 increased VSC4.1 motor neurons' viability under tunicamycin (an endoplasmic reticulum stress) induced injury. In a traumatic SCI rat model, MDL28170-loaded PCL film reduced the area of lesion cavity, and promoted recovery of locomotor behavior. Moreover, the expression of GAP-43 was upregulated after MDL28170-loaded PCL film treatment. Thus, our findings demonstrated that localized delivery of MDL28170 could promote SCI recovery by inhibiting endoplasmic reticulum stress, preserving survival of the motor neurons, which may point out a promising therapeutic target for treating SCI patient., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aging results in increased activation of inflammatory glial cells and decreased neuronal viability following spinal cord injury (SCI). Metabolism and transport of glucose is also decreased with age, although the influence of age on glucose transporter (GLUT) expression or glucose uptake in SCI is currently unknown. We therefore performed [ 18 F]Fluorodeoxyglucose (FDG) PET imaging of young (3 month) and middle-aged (12 month) rats. Glucose uptake in middle-aged rats was decreased compared to young rats at baseline, followed by increased uptake 14 days post contusion SCI. qRT-PCR and protein analysis revealed an association between 14 day glucose uptake and 14 day post-injury inflammation. Further, gene expression analysis of neuron-specific GLUT3 and non-specific GLUT4 (present on glial cells) revealed an inverse relationship between GLUT3/4 gene expression and glucose uptake patterns. Protein expression revealed increased GLUT3 in 3 month rats only, consistent with age related decreases in glucose uptake, and increased GLUT4 in 12 month rats only, consistent with age related increases in inflammatory activity and glucose uptake. Inconsistencies between gene and protein suggest an influence of age-related impairment of translation and/or protein degradation. Overall, our findings show that age alters glucose uptake and GLUT3/4 expression profiles before and after SCI, which may be dependent on level of inflammatory response, and may suggest a therapeutic avenue in addressing glucose uptake in the aging population., (Published by Elsevier B.V.)

Nerve growth factor (NGF) is reportedly involved in the changes in C-fiber bladder afferent pathways that induce detrusor overactivity (DO) following spinal cord injury (SCI). This study examined the roles of NGF in TRP channel expression in bladder afferent neurons in mice with SCI using laser-capture microdissection (LCM) methods. Spinal intact (SI) and SCI mice were divided into 3 groups: (1) SI with vehicle treatment; (2) SCI with vehicle treatment; and (3) SCI with anti-NGF antibody. Two weeks after SCI, an osmotic pump was placed subcutaneously into the back of the mice and vehicle or anti-NGF antibody was administered at a rate of 10 μg/kg per hour for two weeks. Four weeks after SCI, the L6 dorsal root ganglia (DRG) were removed. Expression of the TRPV1, TRPC1, TRPC3, and TRPC6 genes was analyzed using real-time polymerase chain reaction (PCR) following LCM of the bladder afferent neurons, which were labeled by Fast Blue injected into the bladder wall 1 week prior to tissue removal. The mRNA expression of TRPV1 was found to be higher in vehicle-treated SCI mice than in SI mice. The expression level of TRPC3 and TRPC6 in vehicle-treated SCI mice was lower than in SI mice. However, in SCI mice treated with anti-NGF antibody, the mRNA expression of TRPV1 was lower, and the mRNA levels of TRPC3 and TRPC6 were higher than in vehicle-SCI mice. These results suggest that the NGF-dependent changes in specific TRP channel genes, such as TRPV1, TRPC3, and TRPC6, could be involved in SCI-induced afferent hyperexcitability and DO., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Oxidative stress and inflammation are two key secondary pathological mechanisms following spinal cord injury (SCI). Ketogenic diet (KD) and its metabolite β-hydroxybutyrate have been found to exhibit anti-oxidative and anti-inflammatory properties both in rats with SCI and in healthy rats; however, the underlying mechanisms are not yet fully understood. We investigated the effects of KD on the suppression of oxidative stress and inflammation, activation of nuclear factor-E2 related factor 2 (Nrf2), and inhibition of the nuclear factor-κB (NF-κB) signaling pathway in rats with SCI. We assessed functional recovery and evaluated the status of oxidative stress and inflammation using tests of superoxide dismutase and myeloperoxidase activity. We further assessed the presence of the proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interferon γ (IFN-γ) by ELISA. Western blotting was used to detect Nrf2 and NF-κB pathway-associated proteins in spinal cord tissue. Finally, we measured the levels of the NF-κB downstream genes TNF-α, IL-1β, and IFN-γ by western blotting and real-time quantitative PCR. Following SCI, KD improved functional recovery, attenuated oxidative stress and inflammation, and induced Nrf2 activation. In addition, KD suppressed the NF-κB pathway and the expression of TNF-α, IL-1β, and IFN-γ. Together, these findings provide new insight into the underlying regulatory mechanisms of KD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ebselen is a fat-soluble small molecule and organic selenium compound that regulates the activity of glutathione peroxidase to alleviate mitochondrial oxidative stress and improve mitochondrial function. In the present study, we aimed to investigate the effects of ebselen on mitochondrial oxidative stress response, mitochondrial apotosis, and motor behaviors after spinal cord injury (SCI). We found that ebselen significantly increased the BBB score in motor behavior, thus suggesting a rescue effect of ebselen on motor function after SCI in rats. Meanwhile, we revealed that ebselen can increase glutathione (GSH) content as well as superoxide dismutase (SOD) and catalase (CAT) activities after SCI-this suggests ebselen has an antioxidant effect. Furthermore, the ATP content and Na + -K + -ATPase activity in mitochondria were increased by ebselen after SCI, while the mitochondrial membrane potential (MMP) was decreased by ebselen. The Cytochrome C and Smac release from mitochondria were reduced by ebselen after SCI, thus indicating improved membrane permeability by ebselen. Moreover, the alterations in caspase-3, Bax and Bcl-2 protein expression, as well as the proportion of cell apoptosis were improved by ebselen treatment, which together suggested that ebselen has an inhibitory effect on mitochondrial apotosis pathways after SCI. Taken together, our results suggest that ebselen can inhibit secondary damage caused by spinal cord injury. Indeed it plays a neuroprotective role in spinal cord injury perhaps by improving mitochondrial function and inhibiting the mitochondrial apoptosis pathway., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rationale: Spinal cord injury (SCI) may induce significant respiratory muscle weakness and paralysis, which in turn may cause a patient to require ventilator support. Central nervous system alterations can also exacerbate local inflammatory responses with immune cell infiltration leading to additional risk of inflammation at the injury site. Although mechanical ventilation is the traditional treatment for respiratory insufficiency, evidence has shown that it may directly affect distant organs through systemic inflammation., Objectives: This study aimed to better understand the impact of invasive mechanical ventilation on local spinal cord inflammatory responses following cervical or thoracic SCI., Methods: Five groups of female Sprague-Dawley rats were anesthetised for 24 h. Three groups received mechanical ventilation: seven rats without SCI, seven rats with cervical injury (C4-C5), and seven rats with thoracic injury (T10); whereas, two groups were non-ventilated: six rats without SCI; and six rats with thoracic injury (T10). Changes in inflammatory responses were determined in the spinal cord tissues collected at the local site of injury. Cytokines were measured using ELISA., Main Results: SCI induced local pro-inflammatory cytokine IL-6 expression for all groups. Mechanical ventilation also had effects on pro-inflammatory cytokines and independently increased TNF-α and decreased IL-1β levels in the spinal cords of anesthetized rats., Conclusion: These data provide the first evidence that mechanical ventilation contributes to local inflammation after SCI and in the absence of direct tissue injury., (Copyright © 2018. Published by Elsevier B.V.)

Ryanodine receptors (RyRs) are highly conductive intracellular Ca 2+ release channels and are widely expressed in many tissues, including the central nervous system. RyRs have been implicated in intracellular Ca 2+ overload which can drive secondary damage following traumatic injury to the spinal cord (SCI), but the spatiotemporal expression of the three isoforms of RyRs (RyR1-3) after SCI remains unknown. Here, we analyzed the gene and protein expression of RyR isoforms in the murine lumbar dorsal root ganglion (DRG) and the spinal cord lesion site at 1, 2 and 7 d after a mild contusion SCI. Quantitative RT PCR analysis revealed that RyR3 was significantly increased in lumbar DRGs and at the lesion site at 1 and 2 d post contusion compared to sham (laminectomy only) controls. Additionally, RyR2 expression was increased at 1 d post injury within the lesion site. RyR2 and -3 protein expression was localized to lumbar DRG neurons and their spinal projections within the lesion site acutely after SCI. In contrast, RyR1 expression within the DRG and lesion site remained unaltered following trauma. Our study shows that SCI initiates acute differential expression of RyR isoforms in DRG and spinal cord., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Many studies have indicated that autophagy and apoptosis play an important role in the pathogenesis of spinal cord injury. In recent years, research on autophagy-related signal transduction pathways has demonstrated that the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is closely associated with the initiation of autophagy. However, the mechanism of the pathological relationship between this signaling pathway and apoptosis in spinal cord injury is unclear. In this study, we used an in vitro model of spinal cord injury to observe the mechanism of the PI3K/Akt/mTOR signaling pathway and the apoptosis of neurons via the mitochondrial pathway. Mitochondrial pathway apoptosis-related proteins were detected by western blot. Akt and mTOR phosphorylation levels peaked 4h after mechanical damage and then decreased. Following administration of the PI3K-specific inhibitor LY294002, the phosphorylation levels of Akt and mTOR decreased, and the ratio of autophagy-specific protein microtubule-associated protein 1 light chain (LC3)II/I was higher in the inhibitor-treated injury group than in the simple-injury group. TUNEL staining was used to detect apoptosis, and apoptosis was significantly reduced after the inhibition of the PI3K/Akt/mTOR signaling pathway. In summary, the PI3K/Akt/mTOR signaling pathway is involved in the apoptosis of neurons after mechanical injury and can induce apoptosis through the mitochondrial pathway., (Copyright © 2017. Published by Elsevier B.V.)

Injured neurons in the adult mammalian central nervous system (CNS) have a very limited capacity for axonal regeneration and neurite outgrowth. This inability to grow new axons or to regrow injured axons is due to the presence of molecules that inhibit axonal growth, and age related changes in the neuron's innate growth capabilities. Available levels of cAMP are thought to have an important role in linking both of these factors. Elevated levels of cAMP in the developing nervous system are important for the guidance and stability of growth cones. As the nervous system matures, cAMP levels decline and the growth promoting effects of cAMP diminish. It has frequently been demonstrated that increasing neuronal cAMP can enhance neurite growth and regeneration. Some methods used to increase cAMP include administration of cAMP agonists, conditioning lesions, or electrical stimulation. Furthermore, it has been proposed that multiple stages of cAMP induced growth exist, one directly caused by its downstream effector Protein Kinase A (PKA) and one caused by the eventual upregulation of gene transcription. Although the role cAMP in promoting axon growth is well accepted, the downstream pathways that mediate cAMP-mediated axonal growth are less clear. This is partly because various key studies that explored the link between PKA and axonal outgrowth relied on the PKA inhibitors KT5720 and H89. More recent studies have shown that both of these drugs are less specific than initially thought and can inhibit a number of other signalling molecules including the Exchange Protein Activated by cAMP (EPAC). Consequently, it has recently been shown that a number of intracellular signalling pathways previously attributed to PKA can now be attributed solely to activation of EPAC specific pathways, or the simultaneous co-activation of PKA and EPAC specific pathways. These new studies open the door to new potential treatments for repairing the injured spinal cord., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Recovery from injuries to the central nervous system, including spinal cord injury, is constrained in part by the intrinsically low ability of many CNS neurons to mount an effective regenerative growth response. To improve outcomes, it is essential to understand and ultimately reverse these neuron-intrinsic constraints. Genetic manipulation of key transcription factors (TFs), which act to orchestrate production of multiple regeneration-associated genes, has emerged as a promising strategy. It is likely that no single TF will be sufficient to fully restore neuron-intrinsic growth potential, and that multiple, functionally interacting factors will be needed. An extensive literature, mostly from non-neural cell types, has identified potential mechanisms by which TFs can functionally synergize. Here we examine four potential mechanisms of TF/TF interaction; physical interaction, transcriptional cross-regulation, signaling-based cross regulation, and co-occupancy of regulatory DNA. For each mechanism, we consider how existing knowledge can be used to guide the discovery and effective use of TF combinations in the context of regenerative neuroscience. This mechanistic insight into TF interactions is needed to accelerate the design of effective TF-based interventions to relieve neuron-intrinsic constraints to regeneration and to foster recovery from CNS injury., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

There is no treatment for people with spinal cord injury that leads to significant functional improvements. The extracellular matrix is an intricate, 3-dimensional, structural framework that defines the environment for cells in the central nervous system. The components of extracellular matrix have signaling and regulatory roles in the fate and function of neuronal and non-neuronal cells in the central nervous system. This review discusses the therapeutic potential of extracellular matrix components for spinal cord repair., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

After injury, the adult mammalian central nervous system (CNS) lacks long-distance axon regeneration. This review discusses the similarities and differences of sensory and motor neurons, seeking to understand how to achieve functional sensory and motor regeneration. As these two types of neurons respond differently to axotomy, growth environment and treatment, the future challenge will be on how to achieve full recovery in a way that allows regeneration of both types of fibres simultaneously., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Cell-based therapy is a promising strategy to alleviate neuropathic pain caused by spinal cord injury (SCI). We transplanted olfactory ensheathing cells (OECs) into SCI rats with neuropathic pain and quantitatively detected the sensory nerve function. The expression levels of P2X4 receptor (P2X4R), 200kD neurofilament heavy polypeptide (NF200), and glial fiber acidic protein (GFAP) were measured by immunofluorescence and Western blot analyses. Results showed that NF200 expression significantly increased, GFAP expression decreased, and sensory nerve function improved. In addition, OEC transplantation inhibited the overexpression of P2X4R, which plays an important role in neuropathic pain. Thus, OEC is a candidate target for the treatment of sensory functional loss and P2X4R-mediated neuropathic pain caused by SCI., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We recently showed that the RNA regulator, HuR, is translocated to the cytoplasm in astrocytes in the acute phase of spinal cord injury (SCI), consistent with its activation. HuR positively modulates expression of many pro-inflammatory factors, including IL-1β, TNF-α, and MMP-12, which are present at high levels in the early phase of SCI and exacerbate tissue damage. Knockdown of HuR in astrocytes blunts expression of these factors in an in vitro stretch injury model of CNS trauma. In this report, we further investigate the impact of HuR in early SCI using a mouse model in which human HuR is transgenically expressed in astrocytes. At 24h following a mid-thoracic contusion injury, transgenic HuR translocated to the cytoplasm of astrocytes, similar to endogenous HuR, and consistent with its activation. Compared to littermate controls, the transgenic mice showed a global increase in astrocyte activation at the level of injury and a concomitant increase in vascular permeability. There was a significant decrease in neuronal survival at this time interval, but no differences in white matter sparing. Long term behavioral assessments showed no difference in motor recovery. In summary, transgenic expression of HuR in astrocytes accentuated neuronal injury and other secondary features of SCI including increased vascular permeability and astrocyte activation. These findings underscore HuR as a potential therapeutic target in early SCI., (Published by Elsevier B.V.)

Oxidative damage induced-mitochondrial dysfunction and apoptosis has been widely studied in spinal cord injury (SCI). Lycopene, a polyunsaturated hydrocarbon, has the highest antioxidant capacity compared to the other carotenoids. However, the role of lycopene in SCI is unknown. In the present study, we evaluated the antioxidant effects of lycopene on mitochondrial dysfunction and apoptosis following T10 contusion SCI in rats. The rats were randomized into 5 groups: the sham group, the SCI group and the SCI pre-treated with lycopene (5, 10, or 20mg/kg) group. The SCI group showed increased malondialdehyde (MDA) content, decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) ability, which indicated that SCI could induce oxidative damage. What's more, the SCI group showed decreased mRNA expression of cytochrome b and mitochondrial transcription factor A (Tfam), and decreased mitochondrial membrane potential (ΔYm), which indicated that SCI could induce mitochondrial dysfunction. Besides, the SCI group showed decreased protein expression of bcl-2 and mitochondrial cytochrome C, increased protein expression of cytosolic cytochrome C, cleaved caspase-9, cleaved caspase-3 and bax, and increased TUNEL-positive cell numbers, which indicated that SCI could induce cell apoptosis. Fortunately, the lycopene treatment significantly ameliorated oxidative damage, mitochondrial dysfunction and cell apoptosis via the reversion of those parameters described above in the dose of lycopene of 10 and 20mg/kg. In addition, lycopene significantly ameliorated the hind limb motor disturbances in the SCI+lyco10 group and the SCI+lyco20 group compared with the SCI group. These results suggested that lycopene administration could improve total antioxidant status and might have neuroprotective effects on SCI., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Suppression of the gamma-aminobutyric acid (GABA)ergic activity of the zona incerta (ZI) reportedly plays a role in neuropathic pain after spinal cord injury (SCI). A reduction in GABAergic signaling in the ZI of a thoracic hemisection-SCI rat model has been suggested, but not clearly demonstrated. Accordingly, our objective was to investigate whether GABAergic signals influence SCI-induced neuropathic pain., Methods: In vivo, we recorded and compared single-unit, neuronal activity between hemisection-SCI and sham-operated rat models. Furthermore, we analyzed neuronal activity in both models following treatment with either a GABAA receptor agonist (muscimol) or antagonist (bicuculline)., Results: Rats that underwent hemisection SCI exhibited reduced hindpaw withdrawal thresholds, latencies, and decreased ZI neuronal activity compared with sham-operated controls. Importantly, muscimol treatment increased, whereas bicuculline decreased, the firing rates of the ZI neurons. The muscimol treated, hemisection-SCI rats also exhibited increased hindpaw withdrawal thresholds and latencies., Conclusions: These data provide evidence that neuropathic pain after SCI is caused by decreased GABAergic signaling in the ZI. Furthermore, our data demonstrate that infusion of a GABAergic drug into the ZI could restore its inhibitory action and improve neuropathic pain behaviors., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Neuropathic pain is a frequent complication of spinal cord injury (SCI), still refractory to conventional treatment. The presence and biological activity of steroidogenic regulatory proteins and enzymes in the spinal cord suggests that neurosteroids locally generated could modulate pain messages. In this study we explored temporal changes in the spinal expression of the 18kDa translocator protein TSPO, the steroidogenic acute regulatory protein (StAr) and the steroidogenic enzyme 5α-reductase (5α-RI/II) in an experimental model of central chronic pain. Male Sprague-Dawley rats were subjected to a SCI and sacrificed at different time points (1, 14 or 28days). The development of mechanical and cold allodynia was assessed. Injured animals showed an early increase in the mRNA levels of TSPO and 5α-RII, whereas in the chronic phase a significant decrease in the expression of 5α-RI and 5α-RII was observed, coinciding with the presence of allodynic behaviors. Furthermore, since we have shown that progesterone (PG) administration may offer a promising perspective in pain modulation, we also evaluated the expression of steroidogenic proteins and enzymes in injured animals receiving daily injections of the steroid. PG-treated did not develop allodynia and showed a marked increase in the mRNA levels of TSPO, StAR, 5α-RI and 5α-RII 28days after injury. Our results suggest that in the acute phase after SCI, the increased expression of TSPO and 5α-RII may represent a protective endogenous response against tissue injury, which is not maintained in the chronic allodynic phase. PG may favor local steroidogenesis and the production of its reduced metabolites, which could contribute to the antiallodynic effects observed after PG treatment., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Spinal cord injury (SCI) results in an acute reduction in neuronal and glial cell viability, disruption in axonal tract integrity, and prolonged increases in glial activity and inflammation, all of which can influence regional metabolism and glucose utilization. To date, the understanding of glucose uptake and utilization in the injured spinal cord is limited. Positron emission tomography (PET)-based measurements of glucose uptake may therefore serve as a novel biomarker for SCI. This study aimed to determine the acute and sub-acute glucose uptake pattern after SCI to determine its potential as a novel non-invasive tool for injury assessment and to begin to understand the glucose uptake pattern following acute SCI. Briefly, adult male Sprague-Dawley rats were subjected to moderate contusion SCI, confirmed by locomotor function and histology. PET imaging with [(18)F] Fluorodeoxyglucose (FDG) was performed prior to injury and at 6 and 24h and 15days post-injury (dpi). FDG-PET imaging revealed significantly depressed glucose uptake at 6h post-injury at the lesion epicenter that returned to sham/naïve levels at 24h and 15 dpi after moderate injury. FDG uptake at 15 dpi was likely influenced by a combination of elevated glial presence and reduced neuronal viability. These results show that moderate SCI results in acute depression in glucose uptake followed by an increase in glucose uptake that may be related to neuroinflammation. This acute and sub-acute uptake, which is dependent on cellular responses, may represent a therapeutic target., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)