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Objectives: The goal of the study was to reveal the regulatory role of protein kinase R (PKR) in spinal cord injury (SCI), a devasting disorder of the neurological system, and to elucidate its potential mechanism., Methods: The established animal and cellular models of SCI were treated by the PKR inhibitor C12. Histological injury and tissue apoptosis were assessed via H&E staining and TUNEL assays, respectively. Basso-Beattie-Bresnahan (BBB) scoring as well as forelimb grip strength tests were employed to evaluate functional recovery. The production of ROS and cytokines were appraised via their related commercial kits. Western blot and immunofluorescence assay were used to examine protein expression. CCK-8 method was used to assay cell activity. Co-immunoprecipitation assay was conducted to measure the affinity of PKR with STAT1., Results: PKR expression was enhanced following SCI, and the PKR inhibitor C16 mitigated histological injury, cell apoptosis and water content in spinal cord, and improved function recovery following SCI. Meanwhile, C16 attenuated ER stress, pyroptosis, NLRP3 inflammasome and inflammation in mice with SCI and in BV-2 cells challenged with LPS. Additionally, PKR interacted with STAT1 in BV-2 cells, and STAT1 knockdown inhibited ER stress, pyroptosis and inflammation in BV-2 cells challenged with LPS. The protective role of C16 in BV-2 cells exposed to LPS were partly abolished by STAT1 overexpression., Conclusion: PKR inhibition might be a prospective effective approach to attenuating SCI and accelerating function recovery through modulating microglial pyroptosis and ER stress., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier Ltd.)

Materials and Methods: Locomotor outcomes in perforin-deficient (Pfp-/-) mice and wild-type littermate controls were measured after severe compression injury of the lower thoracic spinal cord up to six weeks after injury., Results: According to both the Basso mouse scale score and single frame motion analysis, motor recovery of Pfp-/- mice was similar to wild-type controls. Interestingly, immunohistochemical analysis of cell types at six weeks after injury showed enhanced cholinergic reinnervation of spinal motor neurons caudal to the lesion site and neurofilament-positive structures at the injury site in Pfp-/- mice, whereas numbers of microglia/macrophages and astrocytes were decreased compared with controls., Conclusions: We conclude that, although, loss of perforin does not change the locomotor outcome after injury, it beneficially affects diverse cellular features, such as number of axons, cholinergic synapses, astrocytes and microglia/macrophages at or caudal to the lesion site. Perforin's ability to contribute to Rag2's influence on locomotion was observed in mice doubly deficient in perforin and Rag2 which recovered better than Rag2-/- or Pfp-/- mice, suggesting that natural killer cells can cooperate with T- and B-cells in spinal cord injury.

As a serious trauma of the neurological system, spinal cord injury (SCI) results in permanent disability, gives rise to immediate vascular damage and a wide range of matters that induce the breakage of blood spinal cord barrier (BSCB). SCI activates the expression of MMP-2/9, which are considered to accelerate the disruption of BSCB. Recent research shows that Dl-3-n-butylphthalide (NBP) exerted protective effects on blood spinal cord barrier in animals after SCI, but the underlying molecular mechanism of NBP on the BSCB undergoing SCI is unknown. Here, our research show that NBP inhibited the expression of MMP-2/9, then improved the permeability of BSCB following SCI. After the T9 level of spinal cord performed with a moderate injury, NBP was managed by intragastric administration and further performed once a day. NBP remarkably improved the permeability of BSCB and junction proteins degration, then promoted locomotion recovery. The protective effect of NBP on BSCB destruction is related to the regulation of MMP-2/9 induced by SCI. Moreover, NBP obviously inhibited the MMP-2/9 expression and junction proteins degradation in microvascular endothelial cells. In conclusion, our results indicate that MMP-2/9 are relevant to the breakdown of BSCB, NBP impairs BSCB destruction through inhibiting MMP-2/9 and promotes functional recovery subjected to SCI. NBP is likely to become a new nominee as a therapeutic to treat SCI via a transigent BSCB., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Spinal cord injury (SCI) can cause chronic paralysis and incontinence and remains a major worldwide healthcare burden, with no regenerative treatment clinically available. Intraspinal transplantation of olfactory ensheathing cells (OECs) and injection of chondroitinase ABC (chABC) are both promising therapies but limited and unpredictable responses are seen, particularly in canine clinical trials. Sustained delivery of chABC presents a challenge due to its thermal instability; we hypothesised that transplantation of canine olfactory mucosal OECs genetically modified ex vivo by lentiviral transduction to express chABC (cOEC-chABC) would provide novel delivery of chABC and synergistic therapy. Rats were randomly divided into cOEC-chABC, cOEC, or vehicle transplanted groups and received transplant immediately after dorsal column crush corticospinal tract (CST) injury. Rehabilitation for forepaw reaching and blinded behavioural testing was conducted for 8 weeks. We show that cOEC-chABC transplanted animals recover greater forepaw reaching accuracy on Whishaw testing and more normal gait than cOEC transplanted or vehicle control rats. Increased CST axon sprouting cranial to the injury and serotonergic fibres caudal to the injury suggest a mechanism for recovery. We therefore demonstrate that cOECs can deliver sufficient chABC to drive modest functional improvement, and that this genetically engineered cellular and molecular approach is a feasible combination therapy for SCI., (Copyright © 2021. Published by Elsevier Inc.)

Long non-coding RNAs (lncRNAs) play important roles in various diseases, but the effect of lncRNA CASC9 on spinal cord injury (SCI) remains unclear. Therefore, the present study was conducted to explore the role of this lncRNA in SCI. SCI model was established by laminectomy in rats in vivo or induced by LPS in PC12 cells in vitro. Methylprednisolone (MP) was used for treatment in vivo. Spinal cord tissues were stained with H&E, and the oxidative stress- and inflammation-related factors were detected using their commercial kits. Cell apoptosis was determined using flow cytometry assay. Relative expression of corresponding genes was measured using qRT-PCR and western blotting. Luciferase reporter assay was used to verify binding site between CASC9 and miR-383-5p, as well as miR-383-5p and LDHA. The results showed that lncRNA CASC9 was downregulated and miR-383-5p was upregulated in SCI rats and LPS-induced PC12 cells. Severe histological injury and increased water content were also found in SCI rats. Increased levels of LDH, MDA, lactic acid, TNF-α, and IL-1β were found in SCI rats and LPS-induced PC12 cells. These changes could be reversed by MP treatment in vivo or overexpression of CASC9 in vitro. Besides, overexpression of CASC9 decreased cell apoptosis and protein expression of LDHA and increased protein expression of Nrf2 and HO-1 in LPS-induced PC12 cells. Furthermore, miR-383-5p was a direct target of CASC9 and was negatively regulated by CASC9. LDHA was a direct target of miR-383-5p and was negatively regulated by CASC9. In conclusion, lncRNA CASC9 exerted a protective role against oxidative stress, inflammation, and cell apoptosis in SCI, providing a novel therapeutic target or prognostic factor for SCI.

Traumatic spinal cord injury (SCI) enhances the activity of S-nitrosoglutathione reductase (GSNOR) and inhibits the mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity, resulting in prolonged and sustained pain and functional deficits. This study's objective was to test the hypotheses that GSNOR's specific inhibitor N6022 mitigates pain and improves functional recovery in a mouse model of SCI. Furthermore, the degree of recovery is enhanced and the rate of recovery is accelerated by an ALDH2 activator Alda-1. Using both wild-type and GSNOR - / - mice, the SCI model deployed for groups was contusion at the T9-T10 vertebral level. The enzymatic activity of GSNOR and ALDH2 was measured, and the expression of GSNOR and ALDH2 was determined by western blot analysis. Functional improvements in experimental animals were assessed with locomotor, sensorimotor, and pain-like behavior tests. Wild-type SCI animals had enhanced GSNOR activity and decreased ALDH2 activity, leading to neurovascular dysfunction, edema, and worsened functional outcomes, including locomotor deficits and pain. Compared to wild-type SCI mice, GSNOR - / - mice had better functional outcomes. Monotherapy with either GSNOR inhibition by N6022 or enhanced ALDH2 activity by Alda-1 correlated well with functional recovery and lessened pain. However, combination therapy provided synergistic pain-relieving effects and more significant functional recovery compared with monotherapy. Conclusively, dysregulations in GSNOR and ALDH2 are among the causative mechanisms of SCI injury. Either inhibiting GSNOR or activating ALDH2 ameliorates SCI. Combining the specific inhibitor of GSNOR (N6022) with the selective activator of ALDH2 (Alda-1) provides greater protection to the neurovascular unit and confers greater functional recovery. The study is novel, and the combination therapy (N6022 + Alda-1) possesses translational potential., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The pathological process of traumatic spinal cord injury (SCI) involves excessive activation of microglia leading to the overproduction of proinflammatory cytokines and causing neuronal injury. Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P), plays an important role in mediating inflammation, cell proliferation, survival, and immunity., Methods: We aim to investigate the mechanism and pathway of the Sphk1-mediated neuroinflammatory response in a rodent model of SCI. Sixty Sprague-Dawley rats were randomly assigned to sham surgery, SCI, or PF543 (a specific Sphk1 inhibitor) groups. Functional outcomes included blinded hindlimb locomotor rating and inclined plane test., Results: We discovered that Sphk1 is upregulated in injured spinal cord tissue of rats after SCI and is associated with production of S1P and subsequent NF-κB p65 activation. PF543 attenuated p65 activation, reduced inflammatory response, and relieved neuronal damage, leading to improved functional recovery. Western blot analysis confirmed that expression of S1P receptor 3 (S1PR3) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) are activated in microglia of SCI rats and mitigated by PF543. In vitro, we demonstrated that Bay11-7085 suppressed NF-κB p65 and inhibited amplification of the inflammation cascade by S1P, reducing the release of proinflammatory TNF-α. We further confirmed that phosphorylation of p38 MAPK and activation of NF-κB p65 is inhibited by PF543 and CAY10444. p38 MAPK phosphorylation and NF-κB p65 activation were enhanced by exogenous S1P and inhibited by the specific inhibitor SB204580, ultimately indicating that the S1P/S1PR3/p38 MAPK pathway contributes to the NF-κB p65 inflammatory response., Conclusion: Our results demonstrate a critical role of Sphk1 in the post-traumatic SCI inflammatory cascade and present the Sphk1/S1P/S1PR3 axis as a potential target for therapeutic intervention to control neuroinflammation, relieve neuronal damage, and improve functional outcomes in SCI.

Axonal demyelination is a consistent pathological characteristic of Spinal cord injury (SCI). Promoting differentiation of oligodendrocytes is of importance for remyelination. Conversion of reactive astrocytes with stem cell potential to oligodendrocytes is proposed as an innovative strategy for SCI repair. Neuregulin-1 (Nrg1) plays an essential role in the differentiation of oligodendrocytes. Therefore, it's a potential treatment for demyelination in SCI that using Nrg1 to drive reactive astrocytes toward oligodendrocyte lineage cells. In this study, tumor necrosis factor-α (TNF-α) was used to induce dedifferentiation of primary rat spinal cord astrocytes into reactive astrocytes and Nrg1 was used to induce astrocytes in vitro and in vivo. The results showed that astrocytes treated with TNF-α expressed immaturity markers CD44 and Musashi1 at mRNA and protein levels, indicating that TNF-α induced the stem cell state of astrocytes. Nrg1 induced reactive astrocytes to express oligodendrocyte markers PDGFR-α and O4 at mRNA and protein levels, indicating that Nrg1 directly converts reactive astrocytes toward oligodendrocyte lineage cells. Moreover, upregulation of PI3K-AKT-mTOR signaling activation in response to Nrg1 was observed. In rats with SCI, intrathecal treatment with Nrg1 converted reactive astrocytes to oligodendrocyte lineage cells, inhibited astrogliosis, promoted remyelination, protected axons and eventually improved BBB score. All the biological effects of Nrg1 were significantly reversed by the co-administration of Nrg1 and ErbB inhibitor, suggesting that Nrg1 functioned through the receptor ErbB. Our findings indicate that Nrg1 is sufficient to trans-differentiate reactive astrocytes to oligodendrocytes via the PI3K-AKT-mTOR signaling pathway and repair SCI. Delivery of Nrg1 for the remyelination processes could be a promising strategy for spinal cord repair., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

The inhibition of glycogen synthase kinase-3 (GSK-3) can induce neurogenesis, and the associated activation of Wnt/β-catenin signaling via GSK-3 inhibition may represent a means to promote motor function recovery following spinal cord injury (SCI) via increased astrocyte migration, reduced astrocyte apoptosis, and enhanced axonal growth. Herein, we assessed the effects of GSK-3 inhibition in vitro on the neurogenesis of ependymal stem/progenitor cells (epSPCs) resident in the mouse spinal cord and of human embryonic stem cell-derived neural progenitors (hESC-NPs) and human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) and in vivo on spinal cord tissue regeneration and motor activity after SCI. We report that the treatment of epSPCs and human pluripotent stem cell-derived neural progenitors (hPSC-NPs) with the GSK-3 inhibitor Ro3303544 activates β-catenin signaling and increases the expression of the bIII-tubulin neuronal marker; furthermore, the differentiation of Ro3303544-treated cells prompted an increase in the number of terminally differentiated neurons. Administration of a water-soluble, bioavailable form of this GSK-3 inhibitor (Ro3303544-Cl) in a severe SCI mouse model revealed the increased expression of bIII-tubulin in the injury epicenter. Treatment with Ro3303544-Cl increased survival of mature neuron types from the propriospinal tract (vGlut1, Parv) and raphe tract (5-HT), protein kinase C gamma-positive neurons, and GABAergic interneurons (GAD65/67) above the injury epicenter. Moreover, we observed higher numbers of newly born BrdU/DCX-positive neurons in Ro3303544-Cl-treated animal tissues, a reduced area delimited by astrocyte scar borders, and improved motor function. Based on this study, we believe that treating animals with epSPCs or hPSC-NPs in combination with Ro3303544-Cl deserves further investigation towards the development of a possible therapeutic strategy for SCI.

We investigate the anti-inflammatory effects of injectable hydrogel containing tauroursodeoxycholic acid (TUDCA) in a spinal cord injury (SCI) model. To this end, TUDCA-hydrogel (TC gel) is created by immersing the synthesized hydrogel in a TUDCA solution for 1 h. A mechanical SCI was imposed on rats, after which we injected the TC gel. After the SCI and injections, motor functions and lesions were significantly improved in the TC gel group compared with those in the saline group. The TC gel significantly decreased pro-inflammatory cytokine levels compared with the saline; TUDCA and glycol chitosan-oxidized hyaluronate were mixed at a ratio of 9:1 (CHA) gel independently. In addition, the TC gel significantly suppressed the phosphorylation of extracellular signal-regulated kinase (p-ERK) and c-Jun N-terminal kinase (p-JNK) in the mitogen-activated protein kinase (MAPK) pathway compared with the saline, TUDCA, and CHA gel independently. It also decreased tumor necrosis factor-α (TNF-α) and glial fibrillary acidic protein (GFAP), inflammatory marker, at the injured sites more than those in the saline, TUDCA, and CHA gel groups. In conclusion, the results of this study demonstrate the neuroinflammatory inhibition effects of TC gel in SCI and suggest that TC gel can be an alternative drug system for SCI cases.

The objective of this study was to explore the neuroprotective molecular mechanisms of erythropoietin (EPO) in rats following spinal cord injury (SCI). First, a standard SCI model was established. After drug or saline treatment was administered, locomotor function was evaluated in rats using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. H&E, Nissl, and TUNEL staining were performed to assess the ratio of cavities, number of motor neurons, and apoptotic cells in the damaged area. The relative protein and mRNA expressions were examined using western blot and qRT-PCR analyses, and the inflammatory markers, axon special protein, and neuromuscular junctions (NMJs) were detected by immunofluorescence. Both doses of EPO notably improved locomotor function, but high-dose EPO was more effective than low-dose EPO. Moreover, EPO reduced the cavity ratio, cell apoptosis, and motor neuron loss in the damaged area, but enhanced the autophagy level and extracellular-regulated protein kinase (ERK) activity. Treatment with an ERK inhibitor significantly prevented the effect of EPO on SCI, and an activator mimicked the benefits of EPO. Further investigation revealed that EPO promoted SCI-induced autophagy via the ERK signaling pathway. EPO activates autophagy to promote locomotor function recovery in rats with SCI via the ERK signaling pathway.

Enolase inhibition is a potential therapeutic strategy currently being investigated for treatment of spinal cord injury (SCI) as it reduces pro-inflammatory cytokines and chemokines, alters metabolic factors, and reduces gliosis in acute SCI. Herein, the role of enolase in SCI has been examined to better understand the effects of this enzyme on inflammation, metabolic hormones, glial cell activation, and neuroprotection under these shorter injury conditions. Immunohistochemical analyses of inflammatory markers vimentin, Cox-2, and caspase-1 indicated that enolase inhibition attenuated the elevated levels of inflammation seen following SCI. Iba1, GFAP, NFP, and CSPG staining indicated that enolase inhibition with prolonged administration of ENOblock reduced microglia/astrocyte activation and lead to enhanced neuroprotection in SCI. An analysis of metabolic hormones revealed that ENOblock treatment significantly upregulated plasma concentrations of peptide YY, glucagon-like peptide 1, glucose-dependent insulinotropic peptide, glucagon, and insulin hormones as compared to vehicle-treated controls (Mann-Whitney, p ≤ 0.05). ENOblock did not have a significant effect on plasma concentrations of pancreatic polypeptide. Interestingly, ENOblock treatment inhibited chondroitin sulfate proteoglycan (CSPG), which is produced by activated glia and serves to block regrowth of axons across the lesion site following injury. An increased level of NeuN and MBP with reduced caspase-1 was detected in SCI tissues after ENOblock treatment, suggesting preservation of myelin and induction of neuroprotection. ENOblock also induced improved motor function in SCI rats, indicating a role for enolase in modulating inflammatory and metabolic factors in SCI with important implications for clinical consideration., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Spinal cord injury (SCI) is a neurological disease commonly caused by traumatic events on spinal cords. MiRNA-92a-3p is reported to be down-regulated after SCI. Our study investigated the effects of up-regulated miR-92a-3p on SCI and the underlying mechanisms. SCI mice model was established to evaluate the functional recovery of hindlimbs of mice through open-field locomotion and scored by Basso, Beattie, and Bresnahan (BBB) locomotion scale. Apoptosis of spinal cord cells was determined by flow cytometry. The effects of miR-92a-3p on SCI were detected by intrathecally injecting miR-92a-3p agomiR (agomiR-92) into the mice prior to the establishment of SCI. Phosphatase and tensin homolog (PTEN) was predicted as a target of miR-29a-3p by TargetScan. We further assessed the effects of agomiR-92 or/and overexpressed PTEN on apoptosis rates and apoptotic protein expressions in SCI mice. Moreover, the activation of protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling was determined by Western blot. The results showed that compared with the sham-operated mice, SCI mice had much lower BBB scores, and theapoptosis rate of spinal cord cells was significantly increased. After SCI, the expression of miR-92a-3p was down-regulated, and increased expression of miR-92a-3p induced by agomiR-92 further significantly increased the BBB score and decreased apoptosis. PTEN was specifically targeted by miR-92a-3p. In addition, the phosphorylation levels of Akt and mTOR were up-regulated under the treatment of agomiR-92. Our data demonstrated that the neuroprotective effects of miR-92a-3p on spinal cord safter SCI were highly associated with the activation of the PTEN/AKT/mTOR pathway., (© 2020 The Author(s).)

Spinal cord injury (SCI) is a devastating disease. Strategies that enhance the intrinsic regenerative ability are very important for the recovery of SCI to radically prevent the occurrence of sensory disorders. Epidermal growth factor (EGF) showed a limited effect on the growth of primary sensory neuron neurites due to the degradation of phosphorylated-epidermal growth factor receptor (p-EGFR) in a manner dependent on Casitas B-lineage lymphoma (CBL) (an E3 ubiquitin-protein ligase). MiR-22-3p predicted from four databases could target CBL to inhibit the expression of CBL, increase p-EGFR levels and neurites length via STAT3/GAP43 pathway rather than Erk1/2 axis. EGF, EGFR, and miR-22-3p were downregulated sharply after injury. In vivo miR-22-3p Agomir application could regulate CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis, and restore spinal cord sensory conductive function. This study clarified the mechanism of the limited promotion effect of EGF on adult primary sensory neuron neurite and targeting miR-22-3p could be a novel strategy to treat sensory dysfunction after SCI., (© 2019 Wiley Periodicals, Inc.)

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. The aim of this study was to investigate the potential effect and mechanism of action of MGMT in strengthing neuronal apoptosis following SCI. To determine MGMT-mediated apoptosis in spinal cord injury, we performed western blot and analyzed the expression change of MGMT with different timepoints. Western blot analysis showed the upregulation of MGMT has a peak at 21 days in injured spinal cord tissues. Expression and location was observed in the neurons after SCI. Upregulation of p53, Bax, cleaved caspase3 and cleaved caspase9 and downregulation of Bcl2 were detected after SCI. Co-localization of cleaved caspase3 with MGMT indicated MGMT involved in apoptosis taking place after SCI. In addition, we carried out H 2 O 2 stimulation to further confirm MGMT played a role in neuron apoptosis process and activated p53 signaling pathway in vitro. Finally, based above data, we packaged lenti-associated virus inhibit MGMT expression and injected into rat spinal cords after SCI model was built. LV-MGMT not only reduces the neuron apoptosis, but also increases GAP43 expression and promotes hindlimbs locomotor function recovery. Taken together, the in vivo data and the in vitro observations prove MGMT-mediated apoptosis in the injured spinal cord., Competing Interests: Declaration of Competing Interest The authors report no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: This study aims to investigate the potential function of miR-325-3p in vascular integrity and inflammatory response following spinal cord injury (SCI)., Materials and Methods: The protein levels of ANG-1, ANG-2, and caspase-3 in HUVECs incubated with 0, 100, 200, 400, and 800 ng/ml NE for 24 h were determined. The regulatory effect of overexpressed miR-325-3p on the protein levels of ANG-1 and ANG-2 was determined by Western blot. The SCI model in SD rats was established by spinal injury at T10. Subsequently, the relative levels of miR-325-3p, ANG-1, and ANG-2 were determined in SCI rats and controls. Furthermore, SCI rats were administrated with miR-325-3p mimics or negative control and the relative levels of miR-325-3p, ANG-1, and ANG-2 were examined as well. At day 14, the protein levels of iNOS and GFAP in SCI rats and those overexpressing miR-325-3p were detected. BBB (Basso, Beattie, and Bresnahan) locomotor rating scale was applied for evaluating the locomotor function recovery at day 1, 3, 7, 14, 21, and 28 following SCI., Results: NE treatment in HUVECs downregulated ANG-1 and upregulated ANG-2 and caspase-3 in a dose-dependent manner. The overexpression of miR-325-3p upregulated NE-induced decreased the level of ANG-1 and downregulated NE-induced increased level of ANG-2. After the establishment of the SCI model in rats, the miR-325-3p level gradually decreased in SCI rats relative to controls in a time-dependent manner. ANG-1 level in SCI rats decreased to the lowest on the first day following SCI, and gradually increased at day 3, 5, and 7. ANG-2 level was firstly upregulated and achieved the peak on day 3, and then decreased at day 5 and 7. Moreover, SCI rats overexpressing miR-325-3p showed a higher level of ANG-1 and lower level of ANG-2 than those of SCI rats. Overexpression of miR-325-3p downregulated the protein levels of iNOS and GFAP in SCI rats. BBB scale showed elevated locomotor function recovery in SCI rats overexpressing miR-325-3p compared with SCI rats., Conclusions: MiR-325-3p protects the integrity of the vascular wall, reduces infiltration of inflammation, and improves locomotor function recovery at post-SCI.

Spinal cord injury (SCI) is terrible damage leading to the deficiencies and results in infinite inconvenience to sufferers. The effective treatment for SCI still meets a larger number of problems. Herein, the underlying molecular mechanism and novel therapy of SCI are urgently to investigate. Arachidonate 12-lipoxygenase (ALOX12) is widely expressed in various cell types and plays important role in modulating different cellular processes, such as platelet aggregation, cell migration and cancer cell proliferation. Nevertheless, the effects of ALOX12 on SCI are unclear. In the study, SCI model was established in wild type (WT) mice and ALOX12 knockout mice. First, ALOX12 expression was up-regulated in spinal cord tissues of WT mice after SCI. ALOX12-knockout mice exhibited improved behavior after SCI operation. Glial activation triggered by SCI was also alleviated in mice with the loss of ALOX12, as evidenced by the down-regulated expression of glial fibrillary acidic protein (GFAP) and Iba-1 in spinal cord samples. Further, SCI-induced inflammation was markedly prevented in ALOX12-knockout mice through blocking inhibitor of NF-κB α (IκBα)/nuclear factor-κB (NF-κB) pathway signaling. Additionally, reducing ALOX12 expression attenuated apoptosis in spinal cord tissues of SCI mice by decreasing Cyto-c, cleaved Caspase-3 and poly (ADP-ribose) polymerases (PARP) expression. The protective role of ALOX12-decrease against SCI was verified in LPS-incubated glial cells through repressing inflammatory response and apoptotic formation. Moreover, transgenic mice with ALOX12 over-expression showed accelerated SCI, associated with intensified inflammation and apoptosis. Based on these results, strategies for inhibiting ALOX12 could be used to prevent SCI development by repressing inflammation and apoptosis., (Copyright © 2019. Published by Elsevier Inc.)

Objective: To investigate the use of surrogate plasma fatty acid analysis to provide further insights into the underlying adiposity and the development of metabolic syndrome in men with spinal cord injury (SCI)., Design: Case-control, cross-sectional study., Setting: Community-based individuals with spinal cord injury and healthy controls., Participants: Twenty men with SCI age, height and weight matched with 20 able-bodied controls., Outcome Measures: Lean tissue (LTM) and fat mass (FM) were determined using dual energy X-ray absorptiometry. Fasting blood samples were taken for analysis of fatty acids, adiponectin, insulin, glucose and leptin. Enzymatic indices were calculated using relevant fatty acids., Results: Total FM, leptin, stearoyl coenzyme A desaturase (SCD) Δ9 (SCD-16, 16:1/16:0, and SCD-18, 18:1/18:0) indices and Δ6 desaturase index were significantly higher (P < 0.05) in the SCI group than the controls. Significant differences between the groups was observed for several individual fatty acids. Correlational analysis revealed a different pattern between blood biomarkers and indices of SCDs, de novo lipogenesis and elongase. Associations between the desaturase and elongase indices and biomarkers in the controls followed those reported elsewhere for able bodied participants; the same associations were not observed in the SCI group., Conclusion: We have identified disturbances in fatty acid biosynthesis in SCI individuals likely associated with the development of adipose tissue below the lesion and a decrease in LTM. Loss of LTM may disturb the normal skeletal muscle-fatty acid metabolic processes leading to the disruption of metabolic homeostasis, previously identified in persons with SCI.

Aims: Usually, spinal cord injury (SCI) develops into a glial scar containing extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). Chondroitinase ABC (ChABC), from Proteus vulgaris degrading the glycosaminoglycan (GAG) side chains of CSPGs, offers the opportunity to improve the final outcome of SCI. However, ChABC usage is limited by its thermal instability, requiring protein structure modifications, consecutive injections at the lesion site, or implantation of infusion pumps., Methods: Aiming at more feasible strategy to preserve ChABC catalytic activity, we assessed various stabilizing agents in different solutions and demonstrated, via a spectrophotometric protocol, that the 2.5 mol/L Sucrose solution best stabilized ChABC as far as 14 days in vitro., Results: ChABC activity was improved in both stabilizing and diluted solutions at +37°C, that is, mimicking their usage in vivo. We also verified the safety of the proposed aqueous sucrose solution in terms of viability/cytotoxicity of mouse neural stem cells (NSCs) in both proliferating and differentiating conditions in vitro. Furthermore, we showed that a single intraspinal treatment with ChABC and sucrose reduced reactive gliosis at the injury site in chronic contusive SCI in rats and slightly enhanced their locomotor recovery., Conclusion: Usage of aqueous sucrose solutions may be a feasible strategy, in combination with rehabilitation, to ameliorate ChABC-based treatments to promote the regeneration of central nervous system injuries., (© 2018 John Wiley & Sons Ltd.)

Objective: To investigate the effect of microRNA-210 on the spinal cord injury (SCI) and its underlying mechanism., Materials and Methods: The mouse SCI model was established. Mice were randomly assigned into 4 groups, namely the sham operation group (sham group), surgery group (SCI group), surgery+NC group (SCI+NC group) and surgery+microRNA-210 overexpression group (SCI+microRNA-210 mimics group). The mRNA levels of microRNA-210 and the key genes in the JAK-STAT pathway of the four groups were detected by Real-Time Polymerase Chain Reaction (RT-PCR) at different time points. Protein levels of JAK2 and STAT3 in mice of the four groups were detected by Western blot. To investigate the role of microRNA-210 in SCI recovery, changes in the motor function of mice were detected., Results: Grip strengths of right and left forelimbs in mice from the sham group were temporarily decreased at the early stage after surgery, which were gradually recovered to the preoperative levels on the 3rd postoperative day. However, mice in SCI group were unable to complete the grip strength determination at the early stage after surgery. Mice in SCI group were capable of grasping on the 7th postoperative day. Besides, grip strengths of mice in SCI group were remarkably lower than those of sham group until the end-point (on the 50th day). Furthermore, mRNA levels of microRNA-210 in mice of SCI group were decreased in a time-dependent manner (p<0.05). Higher grip strengths were observed in mice of SCI+microRNA-210 mimics group in comparison with those of SCI group and SCI+NC group (p<0.05). In addition, Western blot showed that protein levels of JAK2 and STAT3 in mice of SCI group were increased in a time-dependent manner (p<0.05). Moreover, protein levels of JAK2, STAT3, and MCP-1 in mice of SCI+NC group were remarkably higher than those in the sham group and SCI+microRNA-210 mimics group (p<0.05)., Conclusions: MicroRNA-210 is down-regulated in SCI mice. Grip strengths of SCI mice can be recovered after microRNA-210 overexpression via inhibiting inflammatory response by the JAK-STAT pathway.

HIPK2 is considered to be a tumor suppressor. It also has been implicated in several functions such as apoptosis and inflammation that are linked to spinal cord injury (SCI). However, whether HIPK2 ameliorates the neurological pain of SCI remains unclear. Here, we investigated the effects of HIPK2 on neurological function, oxidative stress, levels of inflammatory cytokines and expression of Bcl-2/Bax in an SCI model. Firstly, we evaluated the therapeutic effects of HIPK2 on neurological pain in the SCI rat using the Basso, Beattie and Bresnahan scores and H & E staining. Overexpression of HIPK2 significantly elevated the levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), and reduced the mRNA expression of Nogo-A and RhoA in SCI rats. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays showed that overexpression of HIPK2 significantly reduced the number of apoptotic cells. Overexpression of HIPK2 also decreased expression of Bax and Caspase-3 and elevated expression of Bcl-2 in the SCI model, indicating that HIPK2 exhibited its protective activity by inhibiting SCI-induced apoptosis. Then, we measured the serum concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). We also determined the mRNA and protein levels of nuclear factor-κB p65 unit, tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β. HIPK2 overexpression reduced oxidative stress and the levels of inflammatory cytokines compared with SCI control animals. Additionally, acetylation of HIPK2 was reduced in SCI rats. Overexpression of HIPK2 could enhance autophagy by elevating the expression of Beclin-1 and LC3-II while autophagy is regarded as a beneficial regulator to improve spinal cord injury. Together, overexpression of HIPK2 improved contusive SCI induced pain by modulating oxidative stress, Bcl‑2 and Bax signaling, and inflammation, and also regulating autophagy., (Copyright © 2018. Published by Elsevier Masson SAS.)

Aim: Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury., Methods: Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied., Results: Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser 473 and Akt Thr 308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser 2448 phosphorylation, as well as FOXO1 Ser 256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser 235/236 phosphorylation, as well as 4E-BP1 Thr 37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury., Conclusion: Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

BACKGROUND The correlation between serum concentration of neuron specific enolase (NSE), S100B, and the prognosis of patients with acute spinal cord injury (ASCI) remains controversial. MATERIAL AND METHODS Sixty patients with confirmed diagnosis of ASCI were recruited for this study from February 2015 to January 2017. The serum level of NSE and S100B were dynamically measured: on the day of injury and for 2 weeks. The 60 cases were divided into Group A (1 or more than 1 ASIA grade improved at 6 months after the injury) and Group B (ASIA grades changed <1 at 6 months after the injury). The serum level of the 2 groups were compared at different time points. And the prognostic value of serum NSE and S100B as biomarkers in patients with ASCI were calculated by Bayes theorem. RESULTS The serum levels of NSE in Groups A and B on the 2nd day of injury reached a peak at 66.80±13.76 g/L and 98.87±20.12 μg/L, respectively, and then declined gradually. On the 14th day of injury, the serum levels of NSE in both groups were 21.23±8.45 and 39.32±16.31 μg/L, respectively, which were much lower than those on the 2nd day (P<0.05). The serum levels of S100B in Groups A and B rose after the injury and reached a peak on the 4th day of injury. Then, the levels declined gradually to 1.14±0.64 and 1.97±0.98 μg/L, respectively, 2 weeks after the injury. Serum levels of NSE and S100B were good biomarkers for predicting the prognosis of ASCI patients with the sensitivity of 74.35% and 71.79%, the specificity of 71.43% and 66.67%. The cutoff value for serum NSE and S100B were 29.07 μg/L and 1.67 μg/L respectively. The AUCs were 0.78 (95% CI: 0.66-0.89) and 0.76 (95% CI: 0.63-0.89) respectively for serum NSE and S100B. CONCLUSIONS Serum levels of NSE and S100B protein can reflect the degree of spinal cord injury and could be potential biomarkers for the prognosis of acute spinal cord injury.

Objective: To investigate the effects of hyperbaric oxygen preconditioning (HBO-PC) on neuronal apoptosis, Ca2+ concentration, and Caspases expression after spinal cord injury (SCI) in rats., Materials and Methods: A total of 36 rats were randomly divided into control group (CON group), hyperbaric oxygen preconditioning group (HBO-PC group) and spinal cord injury group (SCI group), with 12 rats in each group. Rats in group HBO-PC were given HBO-PC intervention before modeling. SCI model was established by modified Allen method in group HBO-PC and group SCI. Basso-Beattie-Bresnahan (BBB) locomotor rating scale and motor evoked potential (MEP) examination were used to assess the neurological function. The expression of apoptosis gene caspase (3, 7, 8, 12) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The concentration of Ca2+ in spinal cord tissue of each group was detected., Results: CON group, HBO-PC group, and SCI group were gradually diminishing in BBB score and potential value and amplitude of MEP, respectively. The differences between groups were statistically significant (p<0.05). The expressions of Caspase-3 and 7, 8 and 12 mRNA in SCI group were significantly higher than those in CON group and HBO-PC group, respectively (p<0.05). There was no significant difference between CON group and HBO-PC group (p>0.05). The concentrations of Ca2+ in the CON group, HBO-PC group and SCI group were gradually increased; differences between groups were statistically significant (p<0.05)., Conclusions: HBO-PC can reduce the loss of motor function of SCI rats, which may inhibit the activation of endoplasmic reticulum pathway of neural apoptosis, and reduce the calcium overload through inhibiting the expressions of pro-apoptotic proteins (Caspase-3/7/8/12), thus reducing the cell apoptosis and protecting neurons.

Necroptosis, a regulated necrosis pathway mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), is induced following spinal cord injury (SCI) and thought to contribute to neuronal and glial cell death. However, mechanisms leading to activation of necroptosis after SCI remain unclear. We have previously shown that autophagy, a catabolic pathway facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner, is inhibited following SCI in rats. Our current data confirm that inhibition of autophagy also occurs after thoracic contusive SCI in the mouse model, as indicated by accumulation of both the autophagosome marker, LC3-II and autophagy cargo protein, p62/SQSTM1. This was most pronounced in the ventral horn neurons and was caused by rapid inhibition of lysosomal function after SCI. Interestingly, RIPK1, RIPK3, and the necroptosis effector protein MLKL also rapidly accumulated after SCI and localized to neurons with disrupted autophagy, suggesting that these events may be related. To determine if lysosomal dysfunction could contribute to induction of necroptosis, we treated PC12 cells and primary rat cortical neurons with lysosomal inhibitors. This led to rapid accumulation of RIPK1 and RIPK3, confirming that they are normally degraded by the lysosomal pathway. In PC12 cells lysosomal inhibition also sensitized cells to necroptosis induced by tumor necrosis factor α (TNFα) and caspase inhibitor. Imaging studies confirmed that RIPK1 partially localized to lysosomes in both untreated and lysosomal inhibitor treated cells. Similarly, we detected presence of RIPK1, RIPK3 and MLKL in both cytosol and at lysosomes after SCI in vivo. Furthermore, stimulation of autophagy and lysosomal function with rapamycin treatment led to decreased accumulation of RIPK1 and attenuated cell death after SCI. These data suggest that lysosomal dysfunction after SCI may contribute to both inhibition of autophagy and sensitize cells to necroptosis by promoting RIPK1 and RIPK3 accumulation.

Spinal cord injury (SCI) is a type of long-term disability with a high morbidity rate in clinical settings for which there is no effective clinical treatment to date. Usually, lithium is used as a popular mood stabilizer. Recently, growing evidence has shown that lithium has clear neuroprotective effects after SCI, and the administration of lithium can effectively improve locomotor recovery. However, the exact neuroprotective mechanism of lithium is still not understood. Glycogen synthase kinase-3 beta (GSK3β) is a serine/threonine kinase that plays an important role in the neuroprotective effects of lithium both in vivo and in vitro. In this study, we discovered that lithium inhibits GSK3β activity through two different signaling pathways in spinal cord neurons. In the acute phase, lithium inhibited GSK3β activity by stimulating phosphorylation of AKT; in the chronic phase, we first discovered that lithium additionally upregulated the expression of Na + , K + -ATPase α1 (NKA α1), which had an inhibitory effect on GSK3β activity by inducing the expression of glucocorticoid inducible kinase 1 (SGK1). SGK1 is well known as a regulator of the GSK3β/β-catenin signaling pathway. Moreover, the suppressed activity of GSK3β increased the level of β-catenin in the cytoplasm, which gave rise to the translocation of the freely stabilized β-catenin to the nucleus. In addition, the accumulation of β-catenin in the nucleus had the benefits of neuronal survival. Hopefully our findings from this study are beneficial in revealing the neuroprotective mechanism of lithium and in offering novel targets for the development of new SCI therapeutic drugs.

Reactive astrocytes influence post-injury recovery, repair, and pathogenesis of the mammalian CNS. Much of the regulation of astrocyte reactivity, however, remains to be understood. Using genetic loss and gain-of-function analyses in vivo, we show that the conserved MAP3K13 (also known as leucine zipper-bearing kinase [LZK]) promotes astrocyte reactivity and glial scar formation after CNS injury. Inducible LZK gene deletion in astrocytes of adult mice reduced astrogliosis and impaired glial scar formation, resulting in increased lesion size after spinal cord injury. Conversely, LZK overexpression in astrocytes enhanced astrogliosis and reduced lesion size. Remarkably, in the absence of injury, LZK overexpression alone induced widespread astrogliosis in the CNS and upregulated astrogliosis activators pSTAT3 and SOX9. The identification of LZK as a critical cell-intrinsic regulator of astrocyte reactivity expands our understanding of the multicellular response to CNS injury and disease, with broad translational implications for neural repair., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-β1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.

After spinal cord injury (SCI), tight junction (TJ) protein degradation increases permeability and disrupts the blood-spinal cord barrier (BSCB). The BSCB is primarily formed of endothelial cell, which forms a specialized tight seal due to the presence of TJs. BSCB disruption after SCI allows neutrophil infiltration. Matrix metalloproteinase (MMP)-8 is believed to be mainly expressed by neutrophils and is quickly released upon neutrophil activation. Here, we determined whether MMP-8 is involved in the TJ protein degradation in endothelial cells and also determined its role in the neuroinflammation after SCI. MMP-8 recombinant protein treatment increases the TNF-α expression and decreased the TJ (occludin and zonula occludens-1) protein expression in the endothelial cells. Likewise, specific MMP-8 inhibitor (MMP-8I) significantly prevented the TNF-α-induced decrease in the expression of TJ protein in endothelial cells. Furthermore, MMP-8 expression was significantly increased 1 and 3 days after moderate compression (35 g for 5 min at T10 level) SCI, whereas TJ protein levels decreased as determined qRT-PCR, western blotting, and immunohistochemistry. MMP-8 was inhibited directly using a MMP-8I (5 mg/kg) and indirectly by reducing neutrophil infiltration with sivelestat sodium (50 mg/kg) or using the antioxidant N-acetyl-L-cysteine (100 mg/kg). The MMP-8I significantly decreased TNF-α expression, IL-6, and iNOS expression and increased TJ protein expression after SCI. In addition, MMP-8I significantly lessens the amount of Evans blue dye extravasation observed after injury. Thus, our result suggests that MMP-8 plays an imperative role in inflammation and degradation of TJ proteins. Increased MMP-8 expression was associated with the early inflammatory phase of SCI. Inhibiting MMP-8 significantly attenuated SCI-induced inflammation, BSCB breakdown, and cell injury.

Chondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of growth in the adult central nervous system. Use of the enzyme chondroitinase ABC I (ChABC I) as a strategy to reduce CSPG inhibition in experimental models of spinal cord injury has led to observations of its remarkable capacity for repair. More importantly, ChABC therapy has been demonstrated to promote significant recovery of function to spinal injured animals. Despite this incomparable function of ChABC I, its clinical application has been limited because of its thermal instability as reported in the literature. In a recent study by Nazari-Robati et al., thermal stability of ChABC I was improved by protein engineering using site-directed mutagenesis method. Here, in this study, molecular dynamics simulations were used to take a closer look into the phenomenon leading to the experimentally observed thermal stability improvement followed by the corresponding site-directed mutagenesis. We concluded that the mutations induce local flexibility along with a re-conformation into the native structure which consequently increase the protein thermal stability.

Hexokinase-3 (HK3) is a member of hexokinase family, which can catalyze the first step of glucose metabolism. It can increase ATP levels, reduce the production of reactive oxygen species, increase mitochondrial biogenesis, protect mitochondrial membrane potential and play an antioxidant role. However, the change of its expression in spinal cord after injury is still unknown. In this study, we investigated the spatio-temporal expression of HK3 in the spinal cords by using a spinal cord injury (SCI) model in adult female Sprague-Dawley rats. Quantitative reverse transcription-PCR and western blot analysis revealed that HK3 could be detected in sham-opened spinal cords. After SCI, it gradually increased, reached a peak at 7 days post-injury (dpi), and then gradually decreased with the prolonging of injury time, but still maintained at a higher level for up to 28 dpi (the longest time evaluated in this study). Immunofluorescence staining showed that HK3 was found in GFAP + , β-tubulin III + and IBA-1 + cells in sham-opened spinal cords. After SCI, in addition to the above-mentioned cells, it could also be found in CD45 + and CD68 + cells. These results demonstrate that HK3 is mainly expressed in astrocytes, neurons and microglia in normal spinal cords, and could rapidly increase in infiltrated leukocytes, activated microglia/macrophages and astrocytes after SCI. These data suggest that HK3 may be involved in the pathologic process of SCI by promoting glucose metabolism., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Cytosolic Phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of Spinal Cord Injury (SCI). The expression and activation of Cpla2 are significantly higher in SCI, leading to neuronal death in spinal cord tissue. Novel strategies are needed to substantially reverse the effect of cPLA2 activation; one such strategy is inhibiting cPLA2 by jamming its lipid binding C2 domain., Objective: To develop a much needed strategy to treat SCI, we used a Computer Aided Drug Design (CADD) method to discover novel cPLA2 inhibitors., Methods: we used a natural chemiome database for virtual screening, from which we selected the compounds exhibiting the greatest drug-likeliness properties for molecular docking simulation analysis., Results: We studied the interaction of lead compounds at the atomic level; the results yielded a cPLA2 inhibitor of natural origin with the potential for ameliorating secondary tissue damage and promoting recovery of function after SCI. The top compound, lead 4exibited a binding energy of -10.02 Kcal/mol and formed three hydrogen bonds with the lipid binding C2 domain of the cPLA2 protein. An evaluation of cell cytotoxicity revealed an IC50 for lead4 of 134.2 ± 6.8 µM. An in-vitro analysis of lead4 is indicated anti-apoptotic activity via a decrease in caspase-3 expression., Conclusion: We used the CADD method to make a novel lead discovery for the treatment of SCI using compounds of natural origin. The selected natural compounds are non-toxic promising drugs against cPLA2 protein, allowing us to limits our focus on single compound for future in-vitro and invivo testing., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The focus of this article is to highlight novel inhibitors and current examples where the use of selective small-molecule inhibitors has been critical in defining the roles of matrix metalloproteinases (MMPs) in disease. Selective small-molecule inhibitors are surgical chemical tools that can inhibit the targeted enzyme; they are the method of choice to ascertain the roles of MMPs and complement studies with knockout animals. This strategy can identify targets for therapeutic development as exemplified by the use of selective small-molecule MMP inhibitors in diabetic wound healing, spinal cord injury, stroke, traumatic brain injury, cancer metastasis, and viral infection. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Spinal cord ischemia is a serious injury that threatens human health and life. Furthermore, it was widely accepted that miR-136 was mediated in the spinal injury, while whether it regulated neurocytes apoptosis in I/R-induced spinal cord injury remains unclear., Methods: Spinal cord ischemia injury (SCII) rats were induced by clamping the nontraumatic vascular clip on the abdominal aorta. Real-time PCR was conducted to determine the mRNA expression, and western blot was carried out to measure protein expression. TUNEL assay was used to measure cell apoptosis., Results: MiR-136 was up-regulated, while Tissue Inhibitor of Metalloproteinases-3 (TIMP3) was down-regulated in both SCII rats and hypoxic neurocytes. MiR-136 overexpression protected neurocytes against injury that induced by hypoxia. TIMP3 was the target gene of miR-136. Hypoxia supplementation decreased the expression of miR-136, promoted TIMP3 expression, and urged cell apoptosis, cells transfected with miR-136 mimic reversed the effect that induced by hypoxia, while cells co-transfected with pcDNA-TIMP3 abolished the results that induced by overexpressed miR-136., Conclusion: MiR-136 regulated neurocytes apoptosis of SCII by mediating TIMP3., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Understanding how the transcription factor signal transducer and activator of transcription-3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in Nestin - Cre mice, we show that reduction of PTEN rescues glial scar formation in Nestin-Stat3 +/- mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation., (© 2017 Renault-Mihara et al.)

The mammalian target of rapamycin (mTOR) positively regulates axon growth in the mammalian central nervous system (CNS). Although axon regeneration and functional recovery from CNS injuries are typically limited, knockdown or deletion of PTEN, a negative regulator of mTOR, increases mTOR activity and induces robust axon growth and regeneration. It has been suggested that inhibition of S6 kinase 1 (S6K1, gene symbol: RPS6KB1), a prominent mTOR target, would blunt mTOR's positive effect on axon growth. In contrast to this expectation, we demonstrate that inhibition of S6K1 in CNS neurons promotes neurite outgrowth in vitro by twofold to threefold. Biochemical analysis revealed that an mTOR-dependent induction of PI3K signaling is involved in mediating this effect of S6K1 inhibition. Importantly, treating female mice in vivo with PF-4708671, a selective S6K1 inhibitor, stimulated corticospinal tract regeneration across a dorsal spinal hemisection between the cervical 5 and 6 cord segments (C5/C6), increasing axon counts for at least 3 mm beyond the injury site at 8 weeks after injury. Concomitantly, treatment with PF-4708671 produced significant locomotor recovery. Pharmacological targeting of S6K1 may therefore constitute an attractive strategy for promoting axon regeneration following CNS injury, especially given that S6K1 inhibitors are being assessed in clinical trials for nononcological indications. SIGNIFICANCE STATEMENT Despite mTOR's well-established function in promoting axon regeneration, the role of its downstream target, S6 kinase 1 (S6K1), has been unclear. We used cellular assays with primary neurons to demonstrate that S6K1 is a negative regulator of neurite outgrowth, and a spinal cord injury model to show that it is a viable pharmacological target for inducing axon regeneration. We provide mechanistic evidence that S6K1's negative feedback to PI3K signaling is involved in axon growth inhibition, and show that phosphorylation of S6K1 is a more appropriate regeneration indicator than is S6 phosphorylation., Competing Interests: The authors declare no competing financial interests., (Copyright © 2017 the authors 0270-6474/17/377079-17$15.00/0.)

Neuronal cell death following spinal cord injury (SCI) is an important contributor to neurological deficits. The purpose of our work was to delineate the function of mammalian sterile 20-like kinase 1 (Mst1), a pro-apoptotic kinase and key mediator of apoptotic signaling, in the pathogenesis of an experimental mouse model of SCI. Male mice received a mid-thoracic spinal contusion injury, and it was found that phosphorylation of Mst1 at the injured site was enhanced significantly following SCI. Furthermore, when compared to the wild-type controls, Mst1-deficient mice displayed improved locomotor function by increased Basso mouse scale score. Deletion of Mst1 in mice attenuated loss of motor neurons and suppressed microglial and glial activation following SCI. Deletion of Mst1 in mice reduced apoptosis via suppressing cytochrome c release and caspase-3 activation following SCI. Deletion of Mst1 attenuated mitochondrial dysfunction and increased ATP formation following SCI. Deletion of Mst1 in mice inhibited local inflammation following SCI, evidenced by reduced activities of myeloperoxidase and protein levels of TNF-α, IL-1β, and IL-6. In conclusion, the present study demonstrated that deletion of Mst1 attenuated neuronal loss and improved locomotor function in a mouse model of SCI, via preserving mitochondrial function, attenuating mitochondria-mediated apoptotic pathway, and suppressing inflammation, at least in part.

The human natural killer cell antigen-1 (HNK-1) is functionally important in development, synaptic activity, and regeneration after injury in the nervous system of several mammalian species. It contains a sulfated glucuronic acid which is carried by neural adhesion molecules and expressed in nonmammalian species, including zebrafish, which, as opposed to mammals, spontaneously regenerate after injury in the adult. To evaluate HNK-1's role in recovery of function after spinal cord injury (SCI) of adult zebrafish, we assessed the effects of the two HNK-1 synthesizing enzymes, glucuronyl transferase and HNK-1 sulfotransferase. Expression of these two enzymes was increased at the messenger RNA (mRNA) level 11 days after injury in the brainstem nuclei that are capable of regrowth of severed axons, namely, the nucleus of medial longitudinal fascicle and intermediate reticular formation, but not at earlier time points after SCI. mRNA levels of glucuronyl transferase and sulfotransferase were increased in neurons, not only of these nuclei but also in the spinal cord caudal to the injury site at 11 days. Mauthner neurons which are not capable of regeneration did not show increased levels of enzyme mRNAs after injury. Reducing protein levels of the enzymes by application of anti-sense morpholinos resulted in reduction of locomotor recovery for glucuronyl transferase, but not for HNK-1 sulfotransferase. The combined results indicate that HNK-1 is upregulated in expression only in those neurons that are intrinsically capable of regeneration and contributes to regeneration after spinal cord injury in adult zebrafish in the absence of its sulfate moiety.

Objective: To observe the effect of electroacupuncture (EA) stimulation at "Dazhui" (GV 14) and "Mingmen" (GV 4) of the Governor Vessel at different time-points on spinal cord neuronal apoptosis and the expression of c-Jun N-terminal kinases (JNK) protein in spinal cord injury (SCI) rats, so as to reveal its mechanism underlying improving SCI., Methods: A total of 108 male SD rats were randomly divided into normal control, SCI model and EA groups which were further divided into 1, 3 and 7 d subgroups (12 rats/subgroup, 6 rats in each subgroup for TUNEL or Western blot, separately). SCI model was established by using the modified Allen's method. EA was applied to GV 14 and GV 4 for 20 min, once daily, for 1, 3 and 7 days, respectively. Basso-Beattie-Bresnahan (BBB) scale was adopted to assess the locomotor function of rats, the TUNEL method was used to examine neuronal apoptosis of injuried spinal cord, and the expression of phosphorylated (p)-c-Jun protein of T 9 -T 11 spinal cord was detected by using Western blot., Results: After modeling, the BBB scores of SCI rats on day 1, 3 and 7 were signi-ficantly decreased ( P <0.01), while the numbers of apoptotic neuronal cells and the expression levels of p-c-Jun protein in the spinal cord were considerably increased at the 3 time-points in the model group ( P <0.01, P <0.05). Following EA intervention, the decreased BBB scores on day 3 and 7, and the increased numbers of apoptotic neuronal cells on day 1, 3 and 7 and the up-regulated expression levels of p-c-Jun protein on day 3 and 7 were obviously suppressed ( P <0.05, P <0.01)., Conclusions: EA intervention can improve the locomotor function of SCI rats, which Feb be related to its effects in reducing neuronal apoptosis and down-regulating p-c-Jun protein in the injuried spinal cord.

Functional state of the neuromotor system after administration of a nonspecific NO synthase blocker L-NAME was studied on the model of experimental contusion of the spinal cord. Electron paramagnetic resonance measurements of NO production in the damaged segment of the spinal cord were performed for estimation of the dynamics of intensity of NO production during traumatic disease of the spinal cord and selection of optimal period for L-NAME administration. The status of the neuromotor system was evaluated by stimulation electromyography. Treatment with L-NAME during the acute period of traumatic injury to the spinal cord sharply reduced the intensity of evoked motor responses and more pronounced increase in excitability of peripheral motor structures. The results suggest that NO system is a factor of regulation of the stress-induced and adaptive responses of the body at the early stage of spinal cord injury.

After traumatic damage of the brain or spinal cord, many surviving neurons are disconnected, and recovery of function is limited by poor axon regeneration. Recent data have suggested that poly ADP-ribosylation plays a role in limiting axonal regrowth such that inhibition of poly (ADP-ribose) polymerase (PARP) may have therapeutic efficacy for neurological recovery after trauma. Here, we tested systemic administration of the PARP inhibitor, veliparib, and showed effective suppression of PARylation in the mouse CNS. After optic nerve crush injury or dorsal hemisection of the thoracic spinal cord in mice, treatment with veliparib at doses with pharmacodynamic action had no benefit for axonal regeneration or functional recovery. We considered whether PARP gene family specificity might play a role. In vitro mouse cerebral cortex axon regeneration experiments revealed that short hairpin RNA (shRNA)-mediated suppression of PARP1 promoted axonal regeneration, whereas suppression of other PARP isoforms either had no effect or decreased regeneration. Therefore, we examined recovery from neurological trauma in mice lacking PARP1. No increase of axonal regeneration was observed in Parp1 -/- mice after optic nerve crush injury or dorsal hemisection of the thoracic spinal cord, and there was no improvement in motor function recovery. Thus, comprehensive in vivo analysis reveals no indication that clinical PARP inhibitors will on their own provide benefit for recovery from CNS trauma., Competing Interests: S.M.S. is a co-founder of Axerion Therapeutics seeking to develop NgR1-based therapeutics.

Study designExperimental study.ObjectiveTo determine the effects of electroacupuncture (EA) at Zusanli (ST36) on colonic motility and neuronal nitric oxide synthase (nNOS) expression in rats with neurogenic bowel dysfunction (NBD) after spinal cord injury (SCI).SettingSecond School of Clinical Medical, Nanjing University of Chinese Medicine, Jiangsu, China.MethodsWe divided 30 adult Sprague-Dawley rats into a sham group (10 rats), a model group (SCI alone, 10 rats) and a EA group (SCI+EA at ST36, 10 rats). Defecation time was recorded as the time from activated carbon administration (on day 15) to evacuation of the first black stool. Immunohistochemical, real-time PCR and western blot analyses were performed to assess changes in nNOS-immunoreactive cells, and nNOS messenger RNA (mRNA) and protein, respectively, after 14 experimental days.ResultsDefecation time was lower in the EA group than in the model group (P<0.01). On immunohistochemical analysis, nNOS was localized in the myenteric plexus of the colon. The number of nNOS-immunoreactive cells and the intensity of nNOS staining were greater in the model group than in the sham group and lesser in the EA group than in the model group. Consistent with the immunohistochemical findings, nNOS mRNA and protein expression was higher in the model group than in the sham group and lower in the EA group than in the model group (P<0.05 for both).ConclusionIncreased colonic nNOS expression can induce/aggravate NBD in SCI rats. EA at ST36 ameliorated NBD, possibly by downregulating colonic nNOS expression.

Matrix metalloproteinase (MMP)-2 knockout (KO) mice show impaired neurological recovery after spinal cord injury (SCI), suggesting that this proteinase is critical to recovery processes. However, this finding in the KO has been confounded by a compensatory increase in MMP-9. We synthesized the thiirane mechanism-based inhibitor ND-378 and document that it is a potent (nanomolar) and selective slow-binding inhibitor of MMP-2 that does not inhibit the closely related MMP-9 and MMP-14. ND-378 crosses the blood-spinal cord barrier, achieving therapeutic concentrations in the injured spinal cord. Spinal-cord injured mice treated with ND-378 showed no change in long-term neurological outcomes, suggesting that MMP-2 is not a key determinant of locomotor recovery.

Aims: The present study investigated the therapeutic efficacy of asiatic acid (AA) on spinal cord injury (SCI) as well as the underlying mechanisms., Main Methods: Sprague-Dawley rats (n=150) were randomly assigned to five groups: sham, SCI, SCI+methylprednisolone (30mg/kg), SCI+AA (30mg/kg), and SCI+AA (75mg/kg). Motor function, histological changes, neutrophil infiltration, proinflammatory cytokine production, and oxidative stress as well as nuclear factor erythroid 2-related factor (Nrf)2, heme oxygenase (HO)-1, and nucleotide-binding domain-like receptor protein (NLRP)3 levels were evaluated., Key Findings: AA treatment increased Basso, Beattie and Bresnahan scores and inclined plane test scores that were reduced by SCI. In addition, AA suppressed myeloperoxidase activity and reduced the levels of interleukin-1β, -18, and -6 and tumor necrosis factor-α as well as reactive oxygen species (ROS), H2O2, and malondialdehyde levels while increasing superoxide dismutase activity and glutathione production. AA treatment results in the upregulation in Nrf2/HO-1 levels and downregulation of NLRP3 inflammasome protein expression in SC tissue., Significance: AA protects against SCI via suppression of inflammation and oxidative stress. The underlying mechanism likely involves activation of Nrf2 and HO-1 and inhibition of ROS and the NLRP3 inflammasome pathway. AA has therapeutic potential for SCI treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: To explore the associations between two endoplasmic reticulum (ER) stress proteins, protein disulfide isomerase (PDI), binding immunoglobulin protein (BIP), and the development and progression of pressure ulcers (PUs) in spinal cord injury (SCI) paraplegia patients., Methods: ELISA kits were used to measure the levels of serum PDI and BIP in 67 SCI paraplegia patients with PUs and 61 SCI paraplegia patients without PUs. The associations between PDI and BIP, PU formation, PU staging, and pressure ulcer scale for healing (PUSH) score were analyzed., Results: The patients in the PU group had higher levels of PDI and BIP than those in the non-PU group (both p < 0.05). Furthermore, the levels of PDI were positively correlated with those of BIP (r = 0.707, p < 0.0001). There were significant differences in the PDI and BIP levels among the different stages of PU (all p < 0.05). As the PU stages progressed, the levels of PDI and BIP first increased, then decreased, and finally peaked at stage III of the PUs. The PUSH scores significantly declined 7 days after debridement for the PU stage II (p < 0.01) but showed no significant difference between stages III and IV at 7 days after debridement (p > 0.05). The PUSH scores also decreased at 28 days after debridement for stages II, III, and IV (all p < 0.01). Higher PUSH scores indicated a longer time of debridement accompanied by a longer wound surface healing time (p < 0.05)., Conclusion: ER stress proteins may be involved in the process of PU formation and healing; moreover, the levels of PDI and BIP were also associated with the severity of the PUs. Finally, we found that the PUSH scores can be used as a reference to evaluate PU severity and healing.

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.)

Study Design: Ninety-six male adult CD-1 mice were randomly divided into sham, spinal cord injury (SCI) + vehicle, and SCI + IPA-3 groups. Expression of matrix metalloproteinase (MMP)-2 and MMP-9, production of tumor necrosis factors (TNF)-α and interleukin (IL)-1β, tissue edema, blood-spinal cord barrier penetrability, neural cell apoptosis, and neurological function recovery were measured., Objective: The aim of the study was to evaluate the effect of specific inhibition of p21-activated kinase 1 (PAK1) by IPA-3 on SCI and the underlying mechanisms thereof., Summary of Background Data: SCI is a devastating clinical condition that may result in long-lasting and deteriorating functional deficits. The major goal of SCI treatment is to limit the development of secondary injury. IPA-3, a PAK1 inhibitor, exhibited neuroprotection against secondary damage after traumatic brain injury and subarachnoid hemorrhage (SAH)., Methods: MMP-2, MMP-9, and cleaved caspase-3 expression were assessed by Western blot. Inflammatory cytokines TNF-α and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The blood-spinal cord barrier disruption was measured by water content and Evans blue extravasation of the spinal cord. Neuronal apoptosis was evaluated by Nissl staining and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay. The locomotor behavior of hind limb was evaluated by Basso Mouse Scale (BMS) at 1, 3, 7, 14, and 28 days post-injury., Results: Compared with SCI + vehicle mice, IPA-3 treatment showed decreased p-PAK1, MMP-2, MMP-9, cleaved caspase-3, TNF-α, and IL-1β expression. Moreover, inhibition of PAK1 by IPA-3 reduced spinal cord water content and Evans blue extravasation, increased neuronal survival, and reduced TUNEL-positive cells at 24 hours after SCI. Furthermore, IPA-3 improved spinal cord functional recovery 7 days after SCI., Conclusion: Inhibition of PAK1 by IPA-3 promoted recovery of neurological function, possibly by downregulating the expression of MMP-2, MMP-9, TNF-α, and IL-1β. Our data suggest that PAK1 may be a potential therapeutic target in patients with SCI., Level of Evidence: 1.

Mitogen-activated protein kinases (MAPKs) have been implicated in central nervous system injuries, yet the roles within neurodegeneration following spinal cord injury (SCI) still remain partially elucidated. We aimed to investigate the changes in expression of the three MAPKs following SCI and the role of spinal c-jun-NH2-terminal kinase (JNK) in motor impairment following the lesion. SCI induced at the T9 level resulted in enhanced expression of phosphorylated MAPKs shortly after trauma. SCI increased spinal cord myeloperoxidase levels, indicating a local neutrophil infiltration, and elevated the number of spinal apoptotic cells. Intrathecal administration of a specific inhibitor of JNK phosphorylation, SP600125, given at 1 and 4h after SCI, reduced the p-JNK expression, the number of spinal apoptotic cells and many of the histological signs of spinal injury. Notably, restoration of locomotor performance was clearly ameliorated by SP600125 treatment. Altogether, the results demonstrate that SCI induces activation of spinal MAPKs and that JNK plays a major role in mediating the deleterious consequences of spinal injury, not only at the spinal level, but also those regarding locomotor function. Therefore, inhibition of JNK activation in the spinal cord shortly after trauma might constitute a feasible therapeutic strategy for the functional recovery from SCI., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

BACKGROUND We investigated whether an intrathecal transplantation of mesenchymal stem cells (MSCs) activates extracellular adjusting protein kinase1 and 2(ERK1/2) in the spinal cords of rats following an ischemia-reperfusion injury, resulting in improved spinal cord function and inhibition of apoptosis. MATERIAL AND METHODS We observed the relationship between the activation of ERK1/2 in the rat spinal cord and intrathecal transplantation of MSCs, as well as the effect of U0126, a MEK1/2 (upstream protein of ERK1/2) inhibitor, on a spinal cord ischemia-reperfusion injury model in rats using Basso Beattie Bresnahan (BBB) scoring, somatosensory evoked potentials (SSEPs), immunohistochemistry, and Western blot analysis. RESULTS After transplantation of MSCs, the lower limb motor function score increased, and the incubation period of SSEPs and amplitude were improved. Moreover, following transplantation of MSCs, Bcl2 expression increased, whereas Bax expression decreased after reperfusion. Transplantation of MSCs significantly enhanced pERK1/2 expression in the spinal cord, as well as pERK1/2 in immunoreactive cells located in the grey matter of the L4/5 levels of the spinal cord, following ischemia reperfusion injury in rats. The effective dose of U0126 required to inhibit pERK1/2 expression was 200 µg/kg. Bcl-2 decreased and the level of Bax expression increased in the spinal cord after ischemia reperfusion injury, and the protective effects of MSCs were attenuated. CONCLUSIONS Our findings suggest that intrathecal transplantation of MSCs activates ERK1/2 in the spinal cord following ischemia reperfusion injury, partially improves spinal cord function, and inhibits apoptosis in rats.

Multiple players are involved in motor and sensory dysfunctions after spinal cord injury (SCI). Therefore, therapeutic approaches targeting these various players in the damage cascade hold considerable promise for the treatment of traumatic spinal cord injury. Soluble epoxide hydrolase (sEH) is an endogenous key enzyme in the metabolic conversion and degradation of P450 eicosanoids called epoxyeicosatrienoic acids (EETs). sEH inhibition has been shown to provide neuroprotective effects upon multiple elements of neurovascular unit under cerebral ischemia. However, its role in the pathological process after SCI remains unclear. In this study, we tested the hypothesis that sEH inhibition may have therapeutic effects in preventing secondary damage in rats after traumatic SCI. sEH was widely expressed in spinal cord tissue, mainly confined to astrocytes, and neurons. Administration of sEH inhibitor AUDA significantly suppressed local inflammatory responses as indicated by the reduced microglia activation and IL-1 β expression, as well as the decreased infiltration of neutrophils and T lymphocytes. Meanwhile, reactive astrogliosis was remarkably attenuated. Furthermore, treatment of AUDA improved angiogenesis, inhibited neuron cells apoptosis, alleviated demyelination and formation of cavity and improved motor recovery. Together, these results provide the first in vivo evidence that sEH inhibition could exert multiple targets protective effects after SCI in rats. sEH may thereby serve as a promising multi-mechanism therapeutic target for the treatment of SCI.