Recovery Of Spinal Cord Injury In Rats Research Articles

Micro-RNA let-7a-5p Derived From Mesenchymal Stem Cell-Derived Extracellular Vesicles Promotes the Regrowth of Neurons in Spinal-Cord-Injured Rats by Targeting the HMGA2/SMAD2 Axis

Spinal cord injury (SCI) often causes neuronal and axonal damage, resulting in permanent neurological impairments. Mesenchymal stem cells (MSCs) and extracellular vesicles (EVs) are promising treatments for SCI. However, the underlying mechanisms remain unclear. Herein, we demonstrated that EVs from bone marrow-derived MSCs promoted the differentiation of neural stem cells (NSCs) into the neurons and outgrowth of neurites that are extending into astrocytic scars in SCI rats. Further study found that let-7a-5p exerted a similar biological effect as MSC-EVs in regulating the differentiation of NSCs and leading to neurological improvement in SCI rats. Moreover, these MSC-EV-induced effects were attenuated by let-7a-5p inhibitors/antagomirs. When investigating the mechanism, bioinformatics predictions combined with western blot and RT-PCR analyses showed that both MSC-EVs and let-7a-5p were able to downregulate the expression of SMAD2 by inhibiting HMGA2. In conclusion, MSC-EV-secreted let-7a-5p promoted the regrowth of neurons and improved neurological recovery in SCI rats by targeting the HMGA2/SMAD2 axis.

A study on the mechanism of PP2A in the recovery of SCI in rats through downregulation of MMP-9 via MAPK signaling pathway.

The aim of this study was to investigate the mechanism of action of protein phosphatase 2A (PP2A) in the recovery of spinal cord injury (SCI) in rats by downregulating matrix metalloproteinase 9 (MMP-9) via the mitogen-activated protein kinase (MAPK) signaling pathway. A model of SCI was first successfully established in rats. A total of three groups were set, including: sham operation group (A group), SCI group (B group) and PP2A group (C group). The Basso, Beattie and Bresnahan (BBB) motor function score and inclined plane test were adopted to evaluate the motor ability and limb muscle strength of rats in each group. The water content in spinal cord tissues was detected as well. Quantitative Polymerase Chain Reaction (qPCR) assay was performed to analyze the messenger ribonucleic acid (mRNA) expression levels of MAPK, MMP-2, and MMP-9 in spinal cord tissues. The expressions of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in each group of rats were determined via enzyme-linked immunosorbent assay (ELISA). Western blotting (WB) was employed to measure the protein expression levels of MAPK, MMP-2 and MMP-9 in each group of rats. Additionally, the apoptosis of nerve cells in spinal cord tissues was analyzed through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The BBB score was 8.8 points in C group at 5 d after operation, which was significantly different from that in B group (p<0.05). The slope in B and C groups was clearly lower than that in A group at each time point (p<0.001). Meanwhile, it was significantly higher in C group than that in B group at 5, 7 and 9 d (p<0.05). The edema rate rose notably in B group compared with A group (p<0.001). However, spinal cord edema was remarkably relieved after treatment with FRY720 (p<0.01), suggesting that PP2A agonist could treat SCI in rats. The levels of cytokines TNF-α, IL-1β and IL-6 were markedly higher in B group than those in A group (p<0.01). However, they were significantly reduced after treatment with PP2A agonist (p<0.01). In comparison with A group, B group exhibited remarkably decreased mRNA expression of MAPK and elevated mRNA expressions of MMP-2 and MMP-9 (p<0.01). However, C group exhibited an upregulated mRNA expression of MAPK (p<0.05), a downregulated mRNA expression of MMP-9 (p<0.01), and an undifferentiated mRNA expression of MMP-2 (p>0.05). Compared with B group, the protein expression level of MAPK significantly increased (p<0.05), while that of MMP-9 evidently decreased in C group (p<0.05). Besides, no statistically significant difference was observed in the protein expression level of MMP-2 between C group and B group (p>0.05). Compared with that in A group, the apoptosis rate significantly increased in B group (p<0.001). In addition, the apoptosis rate was significantly lower in C group than that in B group, showing a statistically significant difference (p<0.01). PP2A downregulates MMP-9 through the MAPK signaling pathway, thereby conducing to the recovery of SCI in rats.

Buyang Huanwu decoction improves neural recovery after spinal cord injury in rats through the mTOR signaling pathway and autophagy

Background: Spinal cord injury (SCI) refers to the interruption of the tracts inside the spinal cord caused by various factors. The repair of damaged axons has always been a difficult point in clinical treatment and neuroscience research. The treatment of SCI with Buyang huanwu decoction (BYHWD), a well-known recipe for invigorating Qi (a vital force forming part of any living entity in traditional Chinese culture) and promoting blood circulation, shows a good effect. Methods: The rubrospinal tract (RST) transection model in rats was established in this study and rats were administrated with low (BL), medium (BM), or high (BH) doses of BYHWD. Results: Compared with the SCI group, BL, BM moderately, and BH significantly improved the motor function of forelimbs and increased the number of red nucleus neurons in SCI rats. As for the possible molecular mechanism, BL, BM moderately, and BH significantly increased mTOR whereas decreased Beclin-1 and LC3 in the red nucleus. Conclusion: In conclusion, low, medium, and high doses of BYHWD could promote neural recovery in SCI rats through improving motor function and neuron survival in the red nucleus. The neuroprotective effects of BYHWD might be associated with affecting the mTOR signaling pathway and autophagy.

CircTYW1 serves as a sponge for microRNA-380 in accelerating neurological recovery following spinal cord injury via regulating FGF9

ABSTRACT As one of the most severe kinds of neurological damage, spinal cord injury (SCI) contributes to persistent motor dysfunction and involves a large repertoire of gene alterations. The participation of circular RNAs (circRNAs) in neurological recovery following SCI needs to be clarified. In the current work, we attempted to assess the function of hsa_circRNA_0003962/circTYW1 and its underlying mechanism in SCI. By accessing the GEO repository, the expression of circTYW1, microRNA-380 (miR-380), and FGF9 in SCI and sham-operated rats was evaluated. PC12 cells after oxygen-glucose deprivation (OGD) treatment were prepared to mimic the SCI model. circTYW1 and FGF9 were poorly expressed, whereas miR-380 was highly expressed in the spinal cord tissues of SCI rats. circTYW1 promoted neurological recovery in SCI rats and inhibited apoptosis in spinal cord tissues. In PC12 cells exposed to OGD, circTYW1 suppressed PC12 cell apoptosis; however, miR-380 overexpression reversed the protective effect of circTYW1 on PC12 cells. Also, circTYW1 promoted FGF9 expression through competitively binding to miR-380, which activated the ERK1/2 signaling. In summary, our results demonstrated that declines in circTYW1 prevented SCI rats from neurological recovery by regulating the miR-380/FGF9/ERK1/2 axis, which might provide new understanding for SCI treatment.

MicroRNA-145-Mediated KDM6A Downregulation Enhances Neural Repair after Spinal Cord Injury via the NOTCH2/Abcb1a Axis.

Spinal cord injury (SCI) causes a significant physical, emotional, social, and economic burden to millions of people. MicroRNAs are known players in the regulatory circuitry of the neural repair in SCI. However, most microRNAs remain uncharacterized. Here, we demonstrate the neuroprotection of microRNA-145 (miR-145) after SCI in vivo and in vitro. In silico analysis predicted the target gene KDM6A of miR-145. The rat SCI model was developed by weight drop, and lipopolysaccharide- (LPS-) induced PC12 cell inflammatory injury model was also established. We manipulated the expression of miR-145 and/or KDM6A both in vivo and in vitro to explain their roles in rat neurological functional recovery as well as PC12 cell activities and inflammation. Furthermore, we delineated the mechanistic involvement of NOTCH2 and Abcb1a in the neuroprotection of miR-145. According to the results, miR-145 was poorly expressed and KDM6A was highly expressed in the spinal cord tissue of the SCI rat model and LPS-induced PC12 cells. Overexpression of miR-145 protects PC12 cells from LPS-induced cell damage and expedites neurological functional recovery of SCI in rats. miR-145 was validated to target and downregulate the demethylase KDM6A expression, thus abrogating the expression of Abcb1a by promoting the methylation of NOTCH2. Additionally, in vivo findings verified that miR-145 expedites neuroprotection after SCI by regulating the KDM6A/NOTCH2/Abcb1a axis. Taken together, miR-145 confers neuroprotective effects and enhances neural repair after SCI through the KDM6A-mediated NOTCH2/Abcb1a axis.

PTEN-silencing combined with ChABC-overexpression in adipose-derived stem cells promotes functional recovery of spinal cord injury in rats

The efficiency of cell therapy after spinal cord injury (SCI) depend on the survival of transplanted cells. However, sterile microenvironment and glial scar hyperplasia extremely reduce their numbers. Our previous study found overexpression of ChABC gene is positively correlated to migration ability. Expression of PTEN gene is closely associated with proliferation. However, whether manipulation of PTEN and ChABC on adipose-derived mesenchymal stem cells (ADSCs) promote motor recovery is unknown. This study aimed to promote hindlimb function recovery in SCI rats by enhancing proliferation and migration ability of ADSCs, transiently silencing expression of PTEN following overexpression of ChABC (double-gene modified ADSCs, DG-ADSCs). After PTEN silencing, we observed strong proliferation and accelerated G1-S transition in DG-ADSCs using CCK8 assay and flow cytometry. In addition, we demonstrated that migration numbers of DG-ADSCs were higher than control group using Transwell assay. The protein and mRNA levels of MAP2 and βⅢ-tubulin in DG-ADSCs were increased compared with ADSCs. These results were further confirmed in SCI rats. Increased survival cells and reduction of glial scars were quantitatively analyzed in DG-ADSCs groups, which is definitely correlated to function recovery. Recovery of motor function was observed in DG-ADSCs treatment rats using BBB score, which emphasized that improved viability of transplanted cells and reduction of glial scars were an effective strategy for enhancing recovery of neurological function after SCI.

The role of hepatocyte growth factor in mesenchymal stem cell-induced recovery in spinal cord injured rats

BackgroundMesenchymal stem cells (MSCs) have become a promising treatment for spinal cord injury (SCI) due to the fact that they provide a favorable environment. Treatment using MSCs results in a better neurological functional improvement through the promotion of nerve cell regeneration and the modulation of inflammation. Many studies have highlighted that the beneficial effects of MSCs are more likely associated with their secreted factors. However, the identity of the factor that plays a key role in the MSC-induced neurological functional recovery following SCI as well as its molecular mechanism still remains unclear.MethodsA conditioned medium (collected from the MSCs) and hepatocyte growth factor (HGF) were used to test the effects on the differentiation of neural stem cells (NSCS) in the presence of BMP4 with or without a c-Met antibody. In SCI rats, Western blot, ELISA, immunohistochemistry, and hematoxylin-eosin staining were used to investigate the biological effects of MSC-conditioned medium and HGF on nerve cell regeneration and inflammation with or without the pre-treatment using a c-Met antibody. In addition, the possible molecular mechanism (cross-talk between HGF/c-Met and the BMP/Smad 1/5/8 signaling pathway) was also detected by Western blot both in vivo and in vitro.ResultsThe conditioned medium from bone marrow-derived MSCs (BMSCs) was able to promote the NSC differentiation into neurons in vitro and the neurite outgrowth in the scar boundary of SCI rats by inhibiting the BMP/Smad signaling pathway as well as reduces the secondary damage through the modulation of the inflammatory process. The supplementation of HGF showed similar biological effects to those of BMSC-CM, whereas a functional blocking of the c-Met antibody or HGF knockdown in BMSCs significantly reversed the functional improvement mediated by the BMSC-CM.ConclusionsThe MSC-associated biological effects on the recovery of SCI rats mainly depend on the secretion of HGF.

Cerebrospinal fluid-contacting neurons affect the expression of endogenous neural progenitor cells and the recovery of neural function after spinal cord injury

Objective To explore the relationship between cerebrospinal fluid-contacting neurons (CSF-cNs) and endogenous neural progenitor cells (ENPCs) and whether CSF-cNs are involved in nerve repair after spinal cord injury (SCI). Methods Cholera toxin B-horseradish peroxidase complex (CB-HRP) and cholera toxin B conjugated with saporin (CB-SAP) were injected into the lateral ventricles of spinal cord injured rats to mark and destroy the CSF-cNs. Then the rats in the experimental group were injured by SCI. Observe the content and co-expression of CSF-cNs and ENPCs in rats of each group, and observe the recovery of motor function after SCI in each group. Results After the destruction of CSF-cNs, the number of ENPCs decreased significantly in the long term after the surgery, and the recovery of motor function also deteriorated as compared to the group with intact CSF-cNs. Meanwhile some cells in the spinal cord express both the biological marker of CSF-cNs and ENPCs. Conclusion This study shows that the population of ENPCs and motor function recovery in SCI rats declined after the destruction of CSF-cNs, suggesting that CSF-cNs affect the ENPCs population and may be involved in the recovery of neural function after SCI.

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

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

Excess administration of miR-340-5p ameliorates spinal cord injury-induced neuroinflammation and apoptosis by modulating the P38-MAPK signaling pathway

Spinal cord injury (SCI) is a destructive polyneuropathy that can result in loss of sensorimotor function and sphincter dysfunction, and even death in critical situations. MicroRNAs (miRs) are a series of non-coding RNA molecules that are involved in transcriptional regulation. Previous studies have demonstrated that modulation of multiple miRs is involved in neurological recovery after SCI. However, the functions of miR-340-5p in SCI remain uncertain. Therefore, we probed the therapeutic effect and mechanism of miR-340-5p in microglia in vitro and in vivo in SCI rats. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were employed to examine the alterations in miR-340-5p and P38 levels in SCI rats. miR-340-5p targets in microglia were ascertained using luciferase reporter assays, immunofluorescence analyses, and western blotting. We also established an SCI model and administered miR-340-5p. The effects of miR-340-5p on the amelioration of inflammation, oxidative stress, and apoptosis following SCI were assessed using immunofluorescence, immunohistochemistry, and histological analyses. Finally, locomotor function recovery was determined using the Basso, Beattie, Bresnahan rating scale. In our study, the expression profiles and luciferase assay results clarified that P38 was a target of miR-340-5p, which was associated with activation of the P38-MAPK signaling pathway. Elevation of miR-340-5p decreased P38 expression, subsequently inhibiting the inflammatory reaction. SCI-induced secondary neuroinflammation was relieved under miR-340-5p treatment. Moreover, by controlling neuroinflammation, the increased levels of miR-340-5p might counter oxidative stress and reduce the degree of apoptosis. We also observed decreasing gliosis and glial scar formation and increasing neurotrophin expression at the chronic stage of SCI. Together, these potential effects of miR-340-5p treatment ultimately improved locomotor function recovery in SCI rats.

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

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

ASK1 phosphorylation regulates astrocytic reactive gliosis in vitro and in vivo

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.

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

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

MiR-325-3p promotes locomotor function recovery in rats with spinal cord injury via inhibiting the expression of neutrophil elastase.

This study aims to investigate the potential function of miR-325-3p in vascular integrity and inflammatory response following spinal cord injury (SCI). 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. 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. MiR-325-3p protects the integrity of the vascular wall, reduces infiltration of inflammation, and improves locomotor function recovery at post-SCI.

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

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

PPAR-γ Activation Exerts an Anti-inflammatory Effect by Suppressing the NLRP3 Inflammasome in Spinal Cord-Derived Neurons.

Persistent inflammation disrupts functional recovery after spinal cord injury (SCI). Peroxisome proliferator-activated receptor gamma (PPAR-γ) activation promotes functional recovery in SCI rats by inhibiting inflammatory cascades and increasing neuronal survival. We sought to clarify the relationship between PPAR-γ activation and NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inflammasome suppression, and the role of NF-κB in activating the NLRP3 inflammasome in neurons. In SCI rats, we found that rosiglitazone (PPAR-γ agonist) inhibited the expression of caspase-1. In in vitro neurons, G3335 (PPAR-γ antagonist) reversed the rosiglitazone-induced inhibition of caspase-1, interleukin 1 (IL-1β), and interleukin 6 (IL-6). Rosiglitazone inhibited the expression of NLRP3, caspase-1, IL-1β, and IL-6. However, the activator of NLRP3 could counteract this inhibition induced by PPAR-γ activation. NF-κB did not participate in the process of rosiglitazone-induced inhibition of NLRP3. Consistent with our in vitro results, we verified that locomotor recovery of SCI rats in vivo was regulated via PPAR-γ, NLRP3, and NF-κB. These results suggest that PPAR-γ activation exerts an anti-inflammatory effect by suppressing the NLRP3 inflammasome—but not NF-κB—in neurons and that PPAR-γ activation is a promising therapeutic target for SCI.

Mitochondrial Transfer from Bone Marrow Mesenchymal Stem Cells to Motor Neurons in Spinal Cord Injury Rats via Gap Junction.

Recent studies have demonstrated that bone marrow mesenchymal stem cells (BMSCs) protect the injured neurons of spinal cord injury (SCI) from apoptosis while the underlying mechanism of the protective effect of BMSCs remains unclear. In this study, we found the transfer of mitochondria from BMSCs to injured motor neurons and detected the functional improvement after transplanting.Methods: Primary rat BMSCs were co-cultured with oxygen-glucose deprivation (OGD) injured VSC4.1 motor neurons or primary cortical neurons. FACS analysis was used to detect the transfer of mitochondria from BMSCs to neurons. The bioenergetics profiling of neurons was detected by Extracellular Flux Analysis. Cell viability and apoptosis were also measured. BMSCs and isolated mitochondria were transplanted into SCI rats. TdT-mediated dUTP nick end labelling staining was used to detect apoptotic neurons in the ventral horn. Immunohistochemistry and Western blotting were used to measure protein expression. Re-myelination was examined by transmission electron microscope. BBB scores were used to assess locomotor function.Results: MitoTracker-Red labelled mitochondria of BMSCs could be transferred to the OGD injured neurons. The gap junction intercellular communication (GJIC) potentiator retinoid acid increased the quantity of mitochondria transfer from BMSCs to neurons, while GJIC inhibitor 18β glycyrrhetinic acid decreased mitochondria transfer. Internalization of mitochondria improved the bioenergetics profile, decreased apoptosis and promoted cell survival in post-OGD motor neurons. Furthermore, both transplantation of mitochondria and BMSCs to the injured spinal cord improved locomotor functional recovery in SCI rats.Conclusions: To our knowledge, this is the first evidence that BMSCs protect against SCI through GJIC to transfer mitochondrial to the injured neurons. Our findings suggested a new therapy strategy of mitochondria transfer for the patients with SCI.

Upregulation of UBAP2L in Bone Marrow Mesenchymal Stem Cells Promotes Functional Recovery in Rats with Spinal Cord Injury.

Post-translational modifications of cellular proteins with ubiquitin or ubiquitin-like proteins regulate many cellular processes, such as cell proliferation, differentiation, apoptosis, signal transduction, intercellular immune recognition, inflammatory response, stress response, and DNA repair. Nice4/UBAP2L is an important member in the family of ubiquitin-like proteins, and its biological function remains unknown. This study aimed to investigate the effect of UBAP2L on spinal cord injury (SCI). At first, rat bone marrow mesenchymal stem cells (BMSCs) were infected with adeno-associated virus to induce over-expression of Nice4. Subsequently, the infected BMSCs were transplanted into rats suffering from semi-sectioned SCI. The results showed that the over-expression of Nice4 significantly promoted the proliferation and differentiation of BMSCs. In addition, the transplantation of infected BMSCs into the injured area of SCI rats improved the function repair of SCI. Importantly, the immunohistochemical and hematoxylin-eosin staining and RT-PCR results showed that the number of neuronal cells, oligodendrocytes, and astrocytes was significantly increased in the injured area, along with significantly upregulated expression of cyclin D1 and p38 mitogen-activated protein kinase (MAPK). Meanwhile, the expression of caspase 3 protein was significantly down-regulated. In conclusion, the over-expression of Nice4 gene can promote the functional recovery in SCI rats by promoting cell proliferation and inhibiting apoptosis. The results of this study indicate an alternative option for the clinical treatment of SCI.

Intravenous infusion of adipose-derived stem/stromal cells improves functional recovery of rats with spinal cord injury

Background aimsAdipose tissue has therapeutic potential for spinal cord injury (SCI) because it contains multipotent cells known as adipose-derived stem/stromal cells (ASCs). In this study, we attempted intravenous ASC transplantation in rats with SCI to examine the effect on functional recovery. MethodsASCs (2.5 × 106) were intravenously infused into SCI rats, after which hindlimb motor function was evaluated. Distribution of transplanted ASCs was investigated and growth factor/cytokine levels were determined. ResultsIntravenous transplantation of ASCs promoted the functional recovery in SCI rats and reduced the area of spinal cord cavitation. A distribution study revealed that ASCs gradually accumulated at the site of injury, but long-term survival of these cells was not achieved. Levels of growth factors increased only slightly in the spinal cord after ASC transplantation. Unexpectedly, cytokine-induced neutrophil chemoattractant (CINC)-1 showed a transient but substantial increase in the spinal cord tissue and blood of the ASC group. CINC-1 was secreted by ASCs in vitro, and the sponge implantation assay showed that CINC-1 and ASCs induced angiogenesis. CINC-1 promoted functional recovery in SCI rats, which was similar to the ASCs. Expression of glial cell line-derived neurotrophic factor was greater in the ASC group than in the CINC-1 group, although both promoted extracellular signal-regulated kinase (ERK)1/2 phosphorylation; Akt phosphorylation was enhanced in the spinal cord after ASC transplantation. ConclusionsOur findings indicated that intravenously transplanted ASCs gradually accumulated in the injured spinal cord, where cytokines such as CINC-1 activated ERK1/2 and Akt, leading to functional recovery.

Gastrodin ameliorates spinal cord injury via antioxidant and anti-inflammatory effects.

Spinal cord injury (SCI) is one of the most severe traumatic injuries that results in dysfunction of limbs and trunk below the damaged section. Recent studies have shown that gastrodin (GAS) could improve the recovery of SCI. In the current study, we aimed to examine the possible mechanism underlying the effect of GAS on recovery of SCI in rats. In rats with SCI, GAS improved locomotor functions and decreased permeability of blood-spinal cord barrier, as illustrated by increase of Basso-Beattie-Bresnahan scores and decrease of Evans blue leakage. In addition, GAS inhibited inflammation, as evidenced by decrease of proinflammatory cytokines, including tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) in rats following SCI. Moreover, increase of TBARS content and decrease of glutathione (GSH) content and superoxide dismutase (SOD) activities in SCI rats were inhibited by GAS. Furthermore, GAS enhanced mRNA expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), catalytic subunit of γ-glutamylcysteine ligase (GCLc) and modified subunit of γ-glutamylcysteine ligase (GCLm). The data suggested that GAS may promote the recovery of SCI through the enhancement of Nrf2-GCLc/GCLm signaling pathway, and subsequent improvement of oxidative stress and inflammation, resulting in decrease of permeability of BSCB and improved recovery of locomotor function in rats with SCI. The results have provided novel insights into GAS-related therapy of SCI and associated neurodegenerative diseases.