A comparative analysis of titanium anatomic plate and titanium reconstructive plate for treatment of extra-articular fractures of the scapula (Miller types IIb, IIc, and IV).

BACKGROUND: Because of the irregular shape of the scapula and the different types of fractures, a standard internal fixation device is lacking in clinical practice, even though there are many options available. This study aimed to compare the therapeutic efficacy of titanium anatomic and reconstructive plates for extra-articular fractures of the scapula (Miller types IIb, IIc, and IV). METHODS: A retrospective study of 41 patients who underwent treatment for extra-articular fractures of the scapula between March 2017 and March 2020 was conducted. Patients were divided into 2 groups based on the fixation device: titanium anatomic plate group (20 patients) and titanium reconstructive plate group (21 patients). After follow-up for 12-18 months, the general characteristics, perioperative characteristics, postoperative follow-up findings, and imaging data of the 2 groups were compared. RESULTS: The surgical procedures were uneventful in both groups. The anatomic plate group significantly outperformed the reconstructive plate group with respect to surgical duration and intraoperative blood loss (P < .001). At 3-month postoperative follow-up, the Constant-Murley score (P = .026), shoulder flexion range of motion, and shoulder abduction range of motion in the anatomic plate group were all significantly better than those in the reconstructive plate group (P < .001). The postoperative Western Ontario Shoulder Instability scores of the 2 groups were similar. Imaging showed functional recovery and stable and reliable fixation in both groups. Time to bone union was similar in both groups (9.10 ± 1.25 weeks in anatomic plate group and 9.24 ± 1.41 weeks in reconstructive plate group, P = .742). No complications occurred in any patient, such as fixation failure, instability, or bone nonunion. CONCLUSION: Surgeons should favorably consider titanium anatomic plates for the treatment of extra-articular scapular fractures owing to their clear efficacy associated with a shorter surgical time, less intraoperative blood loss, better fixation, and rapid short-term functional recovery.

MiR-429 improved the hypoxia tolerance of human amniotic cells by targeting HIF-1α.

MicroRNA-429(miR-429) plays an important role in mesenchymal stem cells. Hypoxia-inducible factor 1α (HIF-1α) is a nuclear transcription factor that regulates the proliferation, apoptosis and tolerance to hypoxia of mesenchymal stem cells. HIF-1α is also a target gene of miR-429. We investigated whether miR-429 plays a role in hypoxia tolerance with HIF-1α in human amniotic mesenchymal stem cells (hAMSCs). The expression of miR-429 was increased by hypoxia in hAMSCs. miR-429 expression resulted in decreased HIF-1α protein level, but little effect on HIF-1α mRNA. While overexpression of HIF-1α increased the survival rate and exhibited anti-apoptosis effects in hAMSCs under hypoxia, co-expression of miR-429 reduced survival and increased apoptosis. However, miR-429 silencing with HIF-1α overexpression stimulated cell survival and reduced apoptosis. Co-expression of HIF-1α and miR-429 reduced VEGF and Bcl-2 proteins and increased Bax and C-Caspase-3 levels in hAMSCs under hypoxia compared with cells expressing only HIF-1α; cells with HIF-1α overexpression and miR-429 silencing showed the opposite effects. These results indicate that HIF-1α and angomiR-429 reciprocally antagonized each other, while HIF-1α and antagomiR-429 interacted with each other to regulate survival and apoptosis in hAMSCs under hypoxia. miR-429 increased VEGF and Bcl-2 protein levels and decreased Bax and cleaved Caspase-3 protein levels by promoting the synthesis of HIF-1α. These results indicate that miR-429 negatively regulates the survival and anti-apoptosis ability of hAMSCs by mediating HIF-1α expression and improves the ability of hAMSCs to tolerate hypoxia.

Overexpression of miR-100 inhibits growth of osteosarcoma through FGFR3.

Osteosarcoma (OS) is a prevalent, fast growing cancer. Identification of molecular regulation of OS growth may result in development of a novel therapy. Previous studies have highlighted a role of microRNAs (miRNAs) in the regulation of the carcinogenesis of OS, whereas the underlying mechanisms are not completely understood. Moreover, a role of miR-100 in the growth control of OS is not clear. Here we reported significantly higher levels of fibroblast growth factor receptor 3 (FGFR3) and significantly lower levels of miR-100 in the OS specimen, compared to those in the paired normal bone tissues. Bioinformatics analysis and luciferase reporter assay suggest that miR-100 binds to the 3'UTR of FGFR3 mRNA to prevent its translation. To prove it, we modified miR-100 levels in OS cells. We found that overexpression of miR-100 in OS cells decreased FGFR3 protein levels, whereas inhibition of miR-100 increased FGFR3 protein levels, without affecting FGFR3 transcripts. Moreover, overexpression of miR-100 suppressed the OS growth in vitro and in vivo, while inhibition of miR-100 significantly increased OS growth. Taken together, our data demonstrate that miR-100 may inhibit the growth of OS through FGFR3.

Finite element analysis of titanium anatomic plate and titanium reconstructive plate for treatment of extra-articular fractures of the scapula.

BACKGROUND: Due to the lack of postoperative reporting outcomes and bio-mechanical studies, an optimal management of scapular fractures has not been well-established in clinical treatment, even though there are many options available. This study aimed to compare the stability of the new titanium anatomic and traditional titanium reconstructive plates for extra-articular scapular fractures through finite element analysis. METHODS: Two models of scapular assembly were constructed, including one anatomic plate (AP model) and one reconstructive plate (RP model). After meshing, material parameter, and boundary condition settings, we applied four loading conditions to simulate forces acting on the scapula and osteosynthesis material. To evaluate the bio-mechanical properties, the equivalent von Mises stress, equivalent elastic strain, and total deformation were investigated. RESULT: The stress and strain distribution of model AP has better performance than model RP, with more uniform and lower values. The maximum stress value of the scapula in model AP is smaller than that of the scapula in model RP (102.83 MPa vs. 166.71 MPa). The maximum stress of the anatomic plate is half that of the reconstructive plate (218.34 MPa vs. 416.01 MPa). The maximum strain of the scapula in model AP is smaller than that of the scapula in model RP (0.0071 vs. 0.0106). The maximum strain of the anatomic plate is half that of the reconstructive plate (0.0019 vs. 0.0037). The maximum displacement of each model is all at the acromion, with a similar value (2.2947 mm vs. 1.8308 mm). CONCLUSIONS: With sufficient bio-mechanical stability, the anatomic plate to support scapular fracture fragments was superior to that of the reconstructive plate.

Construction and validation of a prognostic marker and risk model for HCC ultrasound therapy combined with WGCNA identification.

Background: Hepatocellular carcinoma (HCC) is a malignant tumor with a highly aggressive and metastatic nature. Ultrasound remains a routine monitoring tool for screening, treatment and post-treatment recheck of HCC. Therefore, it is of great significance to explore the role of ultrasound therapy and related genes in prognosis prediction and clinical diagnosis and treatment of HCC. Methods: Gene co-expression networks were developed utilizing the R package WGCNA as per the expression profiles and clinical features of TCGA HCC samples, key modules were identified by the correlation coefficients between clinical features and modules, and hub genes of modules were determined as per the GS and MM values. Ultrasound treatment differential expression genes were identified using R package limma, and univariate Cox analysis was conducted on the intersection genes of ultrasound differential expression genes and hub genes of key HCC modules to screen the signatures linked with HCC prognosis and construct a risk model. The median risk score was used as the threshold point to classify tumor samples into high- and low-risk groups, and the R package IOBR was used to assess the proportion of immune cells in high- and low-risk groups, R package maftools to assess the genomic mutation differences in high- and low-risk groups, R package GSVA's ssgsea algorithm to assess the HALLMARK pathway enrichment analysis, and R package pRRophetic to analyze drug sensitivity in patients with HCC. Results: WGCNA analysis based on the expression profiles and clinical data of the TCGA LIHC cohort identified three key modules with two major clinical features associated with HCC. The intersection of ultrasound-related differential genes and module hub genes was selected for univariate Cox analysis to identify prognostic factors significantly associated with HCC, and a risk score model consisting of six signatures was finally developed to analyze the prognosis of individuals with HCC. The risk model showed strength in the training set, overall set, and external validation set. The percentage of immune cell infiltration, genomic mutations, pathway enrichment scores, and chemotherapy drug resistance were significantly different between high- and low-risk groups according to the risk scores. Expression of model genes correlated with tumor immune microenvironment and clinical tumor characteristics while generally differentially expressed in pan-cancer tumor and healthy samples. In the immunotherapy dataset, patients in the high-risk group had a worse prognosis with immunotherapy, indicating that subjects in the low-risk group are more responsive to immunotherapy. Conclusion: The 6-gene signature constructed by ultrasound treatment of HCC combined with WGCNA analysis can be used for prognosis prediction of HCC patients and may become a marker for immune response.

Preparation of NIR-Responsive Gold Nanocages as Efficient Carrier for Controlling Release of EGCG in Anticancer Application.

Hepatocellular carcinoma (HCC) is a type of cancer that has a restricted therapy option. Epigallocatechin gallate (EGCG) is one of the main biologically active ingredients in tea. A large number of studies have shown that EGCG has preventive and therapeutic effects on various tumors. In addition, the development of near-infrared (NIR)-responsive nano-platforms has been attracting cancer treatment. In this work, we designed and synthesized a strategy of gold nanocages (AuNCs) as an efficient carrier for controlling release of EGCG for anti-tumor to achieve the synergistic functions of NIR-response and inhibited tumor cell proliferation. The diameter of AuNCs is about 50 nm and has a hollow porous (8 nm) structure. Thermal imaging-graphic studies proved that the AuNCs-EGCG obtained have photothermal response to laser irradiation under near-infrared light and still maintain light stability after multiple cycles of laser irradiation. The resulted AuNCs-EGCG reduced the proliferation rate of HepG2 cells to 50% at 48 h. Western blot analysis showed that NIR-responsive AuNCs-EGCG can promote the expression of HepG2 cell apoptosis-related proteins HSP70, Cytochrome C, Caspase-9, Caspase-3, and Bax, while the expression of Bcl-2 is inhibited. Cell confocal microscopy analysis proved that AuNCs-EGCG irradiated by NIR significantly upregulates Caspase-3 by nearly 2-fold and downregulates Bcl-2 by nearly 0.33-fold, which is beneficial to promote HepG2 cell apoptosis. This study provides useful information for the NIR-responsive AuNCs-EGCG as a new type of nanomedicine for HCC.

Mesoporous Bioactive Glass Scaffold Delivers Salvianolic Acid B to Promote Bone Regeneration in a Rat Cranial Defect Model.

BACKGROUND: Mesoporous Bioactive Glass (MBG) has been widely studied because of its excellent histocompatibility and degradability. However, due to the lack of good osteoinductive activity, the pure MBG scaffold is not effective in repairing large-scale bone defects. OBJECTIVE: To observe the repair effect of MBG scaffolds delivering Salvianolic acid B (SB) on critical bone defects in rats. METHODS: In this study, MBG scaffolds were used as delivery vehicle. SB, a small molecular active drug with good osteogenic differentiation ability, was loaded into the MBG scaffolds at low, medium and high doses. The effect of SB released from the MBG scaffolds on osteogenic differentiation of rat Bone Marrow Mesenchymal Stem Cells (rBMSCs) was investigated using alkaline phosphatase staining, alizarin red staining and real-time quantitative polymerase chain reaction. Moreover, 8 weeks after implantation of the scaffolds, the bone regeneration was evaluated by micro- CT, sequential fluorescence labeling and histological staining analysis. RESULTS: The in vitro results showed that different doses of SB had similar release rate from scaffolds and could be released from scaffolds continuously. The middle dose (MBG/MSB) and high dose (MBG/HSB) groups significantly promoted the osteogenic differentiation of rBMSCs when compared with a low dose (MBG/LSB) group. Moreover, SB produced significant increases in newly formed bone of calvarial bone defects in rats. CONCLUSION: It is concluded that the use of MBG scffolds delivering SB is an effective strategy for the treatment of bone defects.

Outcome of Humeral Shaft Fracture Treated with Intramedullary Nail and Plate Fixation.

OBJECTIVE: To compare the results of the intramedullary nail (IMN) and compression plate fixation performed for humeral shaft fracture as to determine a better option out of the two. STUDY DESIGN: Comparative study. PLACE AND DURATION OF STUDY: First Affiliated Hospital of Jinzhou Medical University, Liaoning, PR China, from October 2016 to January 2019. METHODOLOGY: Patients treated with IMN (n=26) or plate fixation (n=30) for humeral shaft fracture were included in this study. Assessment was done in terms of perioperative parameters, complications, union time, and functional outcomes. Functional outcome were compared between the two groups at each follow-up (6 weeks, 3, 6, and 12 months) and between the subsequent follow-ups in both groups using the repeated measures ANOVA. RESULTS: Intraoperative blood loss, operative time, hospital stay, and union time were significantly lower in the IMN group. There was no significant difference in the functional outcomes when it was compared between the two groups at each follow-up. However, when it was compared between subsequent follow-ups, a significant improvement was observed in both groups. Increase incidence of individual complication and reoperation were established in the plating group, but without a significant difference. Yet, the overall complications rate was significantly higher in the plating group. CONCLUSION: IMN fixation led to a significant decrease in intraoperative blood loss, shorter operating time, hospital stay, union time, and a lower rate of overall complications. Thus, IMN may be a better choice of internal fixation as it also accelerates the patients' recovery, and increases their satisfaction.

Nuclear Heme Oxidase-1 Inhibits Endoplasmic Reticulum Stress-Mediated Apoptosis after Spinal Cord Injury.

The treatment goal for spinal cord injury (SCI) is to repair neurites and suppress cellular apoptosis. This study is to investigate the effects of nuclear heme oxidase-1 (HO-1) on the acute spinal cord injury and the related mechanisms. The rat model of the SCI was established. On day 7, before model establishment, the adenovirus vector carrying nuclear HO-1 (Ad-GFP-HO-1CΔ23) was injected into the animals into the tenth thoracic spine (T10) segment by the intrathecal injection. Starting from after the model establishment to day 28, the recovery of motor function was assessed by the Basso-Beattie-Bresnahan (BBB) scoring method. Immunofluorescence was performed to detect the expression patterns of nuclear and cytoplasmic proteins. HE and Nissl staining methods were used to evaluate the structural damage and the number of surviving neurons near the injured area. The TUNEL method was conducted to evaluate the apoptotic degree. Protein expression levels were detected with the Western blot analysis. The BBB assay scores in the nuclear HO-1 group were significantly higher than the blank and adenovirus control groups. Moreover, compared to the blank and adenovirus control groups, the neuronal apoptosis in the nuclear HO-1 group was significantly alleviated. Furthermore, the expression levels of the endoplasmic reticulum stress-related proteins, i.e., CHOP, GRP78, and caspase-12, were significantly decreased in the nuclear HO-1 group. Nuclear HO-1 significantly improves the SCI, promotes the functional recovery, inhibits the endoplasmic reticulum stress, and alleviates the apoptotic process after SCI.

[HO-1Cδ23 reduces the permeability of the blood-spinal barrier by upregulating miR-125a-5p after hypoxic injury].

Objective To detect the effects of microRNA 125a-5p (miR-125a-5p) in heme oxygenase 1 (HO-1) truncation (HO-1Cδ23) regulating the blood-spinal barrier (BSCB) after hypoxic injury. Methods HCMEC/D3 and astrocytes were co-cultured in TranswellTM chamber to establish BSCB model in vitro, and then prepared with cobalt chloride to establish BSCB hypoxic injury model. The experiment was divided into blank group, non-virus loading group, HO-1Cδ23 group (Lv-HO-1Cδ23), miRNA negative control group (Lv-HO-1Cδ23 combined with miR-NC treatment), miRNA mimic group (Lv-HO-1Cδ23 combined with miR-125a-5p mimics treatment) and miRNA inhibitor group (Lv-HO-1Cδ23 combined with miR-125a-5p inhibitor treatment). The expression levels of miR-125a-5p and zonula occludens-1 (ZO-1), occludin and vascular endothelial adhesion protein (VE-cadherin) were detected by reverse transcription PCR and real-time quantitative PCR. Western blot was used to detect the expressions of HO-1, ZO-1, occludin and VE-cadherin proteins. The permeability of BSCB in vitro was evaluated by the spillage rate of horseradish peroxidase (HRP). Results The success rate of HO-1Cδ23 transfection was about 70%. After transfection, the content of HO-1Cδ23 was significantly increased, and the expression of miR-125a-5p up-regulated compared with the blank group. Compared with the blank group, the mRNA and protein levels of ZO-1, occludin, and VE-cadherin in the HO-1Cδ23 group, the miRNA negative control group, and miRNA mimic group increased, but the HRP spillover rate decreased; on the contrary, the mRNA and protein levels of ZO-1 occludin and VE-cadherin in the miRNA inhibitor group decreased, while the HRP spillover rate increased significantly. Compared with the HO-1Cδ23 group, the mRNA and protein levels of ZO-1, occludin and VE-cadherin increased significantly in the miRNA mimic group, and the HRP spillover rate decreased significantly, while the mRNA and protein levels of ZO-1, occludin, and VE-cadherin as well as the HRP spillover rate in the miRNA inhibitor group showed the opposite trend. Conclusion Under hypoxic injury, HO-1Cδ23 may promote the transcription and translation of the genes involved in junction and adhesion and reduce the permeability of BSCB by up-regulating the expression of miR-125a-5p.

Adenovirus-delivered GFP-HO-1C[INCREMENT]23 attenuates blood-spinal cord barrier permeability after rat spinal cord contusion.

The blood-spinal cord barrier (BSCB) plays a key role in maintaining the microenvironment and is primarily composed of tight junction proteins and nonfenestrated capillary endothelial cells. After injury, BSCB damage results in increasing capillary permeability and release of inflammatory factors. Recent studies have reported that haem oxygenase-1 (HO-1) fragments lacking 23 amino acids at the C-terminus (HO-1C[INCREMENT]23) exert novel anti-inflammatory and antioxidative effects in vitro. However, no study has identified the role of HO-1C[INCREMENT]23 in vivo. We aimed to investigate the protective effects of HO-1C[INCREMENT]23 on the BSCB after spinal cord injury (SCI) in a rat model. Here, adenoviral HO-1C[INCREMENT]23 (Ad-GFP-HO-1C[INCREMENT]23) was intrathecally injected into the 10th thoracic spinal cord segment (T10) 7 days before SCI. In addition, nuclear and cytoplasmic extraction and immunofluorescence staining of HO-1 were used to examine the effect of Ad-GFP-HO-1C[INCREMENT]23 on HO-1 nuclear translocation. Evan's blue staining served as an index of capillary permeability and was detected by fluorescence microscopy at 633 nm. Western blotting was also performed to detect tight junction protein expression. The Basso, Beattie and Bresnahan score was used to evaluate kinematic functional recovery through the 28th day after SCI. In this study, the Ad-GFP-HO-1C[INCREMENT]23 group showed better kinematic functional recovery after SCI than the Ad-GFP and Vehicle groups, as well as smaller reductions in TJ proteins and capillary permeability compared with those in the Ad-GFP and Vehicle groups. These findings indicated that Ad-GFP-HO-1C[INCREMENT]23 might have a potential therapeutic effect that is mediated by its protection of BSCB integrity.

Regulation of autophagy by AMP-activated protein kinase/sirtuin 1 pathway reduces spinal cord neurons damage.

OBJECTIVES: AMP-activated protein kinase/sirtuin 1 (AMPK/SIRT1) signaling pathway has been proved to be involved in the regulation of autophagy in various models. The aim of this study was to evaluate the effect of AMPK/SIRT1 pathway on autophagy after spinal cord injury (SCI). MATERIALS AND METHODS: The SCI model was established in rats in vivo and the primary spinal cord neurons were subjected to mechanical injury (MI) in vitro. The apoptosis in spinal cord tissue and neurons was assessed by TUNEL staining and Hoechst 33342 staining, respectively. The autophagy-related proteins levels were detected by Western blot. The activation of AMPK/SIRT1 pathway was determined by Western blot and immunohistochemical staining. RESULTS: We found that the apoptosis of spinal cord tissue and cell damage of spinal cord neurons was obvious after the trauma. The ratio of LC3II/LC3I and level of p62 were first increased significantly and then decreased after the trauma in vivo and in vitro, indicating the defect in autophagy. The levels of p-AMPK and SIRT1 were increased obviously after the trauma in vivo and in vitro. Further activation of the AMPK/SIRT1 pathway by pretreatment with resveratrol, a confirmed activator of the AMPK/SIRT1 pathway, alleviated the cell damage and promoted the autophagy flux via downregulation of p62 in spinal cord neurons at 24 hr after MI. CONCLUSION: Our results demonstrate that regulation of autophagy by AMPK/SIRT1 pathway can restrain spinal cord neurons damage, which may be a potential intervention of SCI.

The Role of Netrin-1 in Improving Functional Recovery through Autophagy Stimulation Following Spinal Cord Injury in Rats.

Our previous findings indicated that treatment with Netrin-1 could improve functional recovery through the stimulation of autophagy, by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway in rats following spinal cord injury (SCI). However, the underlying mechanisms were not elucidated. The purpose of this study was to investigate the underlying mechanisms by which Netrin-1 promotes autophagy and improves functional recovery after SCI. Following controlled SCI in Sprague-Dawley rats, we observed that the autophagic flux in neurons was impaired, as reflected by the accumulation of light chain 3-II (LC3-II)-positive and LC3-positive autophagosomes (APs), accompanied by the accumulation of the autophagic substrate, Sequestosome 1 (SQSTM1; also known as p62). Our results showed that treatment with Netrin-1 increases the levels of the lysosomal protease cathepsin D (CTSD) and lysosomal-associated membrane protein 1 (LAMP1), through the regulation of the nuclear localization of Transcription factor EB (TFEB) via the AMPK/mTOR signaling pathway. In addition, this enhancement of lysosomal biogenesis correlated strongly with the restoration of autophagic flux, inhibition of neural apoptosis and improved functional recovery. Suppression of lysosomal biogenesis via the inhibition of the nuclear translocation of TFEB by Compound C abolished this restoration of autophagic flux and the functional recovery effects of Netrin-1 following SCI. Taken together, these results indicate that Netrin-1 enhances lysosomal biogenesis by regulating the nuclear translocation of TFEB via the AMPK/mTOR signaling pathway. Furthermore, the enhancement of lysosomal biogenesis by Netrin-1 following SCI promotes autophagic flux and improves functional recovery in rats. Thus, the regulation of lysosomal biogenesis by modulating the nuclear localization of TFEB might be a novel approach for the treatment of SCI.

HMGB1/Advanced Glycation End Products (RAGE) does not aggravate inflammation but promote endogenous neural stem cells differentiation in spinal cord injury.

Receptor for advanced glycation end products (RAGE) signaling is involved in a series of cell functions after spinal cord injury (SCI). Our study aimed to elucidate the effects of RAGE signaling on the neuronal recovery after SCI. In vivo, rats were subjected to SCI with or without anti-RAGE antibodies micro-injected into the lesion epicenter. We detected Nestin/RAGE, SOX-2/RAGE and Nestin/MAP-2 after SCI by Western blot or immunofluorescence (IF). We found that neural stem cells (NSCs) co-expressed with RAGE were significantly activated after SCI, while stem cell markers Nestin and SOX-2 were reduced by RAGE blockade. We found that RAGE inhibition reduced nestin-positive NSCs expressing MAP-2, a mature neuron marker. RAGE blockade does not improve neurobehavior Basso, Beattie and Bresnahan (BBB) scores; however, it damaged survival of ventral neurons via Nissl staining. Through in vitro study, we found that recombinant HMGB1 administration does not lead to increased cytokines of TNF-α and IL-1ß, while anti-RAGE treatment reduced cytokines of TNF-α and IL-1ß induced by LPS via ELISA. Meanwhile, HMGB1 increased MAP-2 expression, which was blocked after anti-RAGE treatment. Hence, HMGB1/RAGE does not exacerbate neuronal inflammation but plays a role in promoting NSCs differentiating into mature neurons in the pathological process of SCI.

Netrin-1 Improves Functional Recovery through Autophagy Regulation by Activating the AMPK/mTOR Signaling Pathway in Rats with Spinal Cord Injury.

Autophagy is an process for the degradation of cytoplasmic aggregated proteins and damaged organelles and plays an important role in the development of SCI. In this study, we investigated the therapeutic effect of Netrin-1 and its potential mechanism for autophagy regulation after SCI. A rat model of SCI was established and used for analysis. Results showed that administration of Netrin-1 not only significantly enhanced the phosphorylation of AMP-activated protein kinase (AMPK) but also reduced the phosphorylation of mammalian target of rapamycin (mTOR) and P70S6K. In addition, the expression of Beclin-1 and the ratio of the light-chain 3B-II (LC3B-II)/LC3B-I in the injured spinal cord significantly increased in Netrin-1 group than those in SCI group. Moreover, the ratio of apoptotic neurons in the anterior horn of the spinal cord and the cavity area of spinal cord significantly decreased in Netrin-1 group compared with those in SCI group. In addition, Netrin-1 not only preserved motor neurons but also significantly improved motor fuction of injured rats. These results suggest that Netrin-1 improved functional recovery through autophagy stimulation by activating the AMPK/mTOR signaling pathway in rats with SCI. Thus, Netrin-1 treatment could be a novel therapeutic strategy for SCI.

Salvianolic acid A ameliorates the integrity of blood-spinal cord barrier via miR-101/Cul3/Nrf2/HO-1 signaling pathway.

Salvianolic acid A (Sal A), a bioactive compound isolated from the Chinese medicinal herb Danshen, is used for the prevention and treatment of cardiovascular diseases. However, the protective function of Sal A on preserving the role of blood-spinal cord barrier (BSCB) after spinal cord injury (SCI) is unclear. The present study investigated the effects and mechanisms of Sal A (2.5, 5, 10mg/kg, i.p.) on BSCB permeability at different time-points after compressive SCI in rats. Compared to the SCI group, treatment with Sal A decreased the content of the Evans blue in the spinal cord tissue at 24h post-SCI. The expression levels of tight junction proteins and HO-1 were remarkably increased, and that of p-caveolin-1 protein was greatly decreased after SCI Sal A. The effect of Sal A on the expression level of ZO-1, occluding, and p-caveolin-1 after SCI was blocked by the HO-1 inhibitor, zinc protoporphyrin IX (ZnPP). Also, Sal A inhibited the level of apoptosis-related proteins and improved the motor function until 21days after SCI. In addition, Sal A significantly increased the expression of microRNA-101 (miR-101) in the RBMECs under hypoxia. AntagomiR-101 markedly increased the RBMECs permeability and the expression of the Cul3 protein by targeting with 3'-UTR of its mRNA. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1 was significantly increased after agomiR-101 treatment. Therefore, Sal A could improve the recovery of neurological function after SCI, which could be correlated with the repair of BSCB integrity by the miR-101/Cul3/Nrf2/HO-1 signaling pathway.

Mdivi-1 Inhibits Astrocyte Activation and Astroglial Scar Formation and Enhances Axonal Regeneration after Spinal Cord Injury in Rats.

After spinal cord injury (SCI), astrocytes become hypertrophic, and proliferative, forming a dense network of astroglial processes at the site of the lesion. This constitutes a physical and biochemical barrier to axonal regeneration. Mitochondrial fission regulates cell cycle progression; inhibiting the cell cycle of astrocytes can reduce expression levels of axon growth-inhibitory molecules as well as astroglial scar formation after SCI. We therefore investigated how an inhibitor of mitochondrial fission, Mdivi-1, would affect astrocyte proliferation, astroglial scar formation, and axonal regeneration following SCI in rats. Western blot and immunofluorescent double-labeling showed that Mdivi-1 markedly reduced the expression of the astrocyte marker glial fibrillary acidic protein (GFAP), and a cell proliferation marker, proliferating cell nuclear antigen, in astrocytes 3 days after SCI. Moreover, Mdivi-1 decreased the expression of GFAP and neurocan, a chondroitin sulfate proteoglycan. Notably, immunofluorescent labeling and Nissl staining showed that Mdivi-1 elevated the production of growth-associated protein-43 and increased neuronal survival at 4 weeks after SCI. Finally, hematoxylin-eosin staining, and behavioral evaluation of motor function indicated that Mdivi-1 also reduced cavity formation and improved motor function 4 weeks after SCI. Our results confirm that Mdivi-1 promotes motor function after SCI, and indicate that inhibiting mitochondrial fission using Mdivi-1 can inhibit astrocyte activation and astroglial scar formation and contribute to axonal regeneration after SCI in rats.

A disintegrin and metalloprotease 17 promotes microglial cell survival via epidermal growth factor receptor signalling following spinal cord injury.

Tumour necrosis factor-α (TNF-α) converting enzyme (TACE), also termed a disintegrin and metalloprotease 17 (ADAM17), is involved in multiple cell signalling pathways. Through the secretion of epidermal growth factor receptor (EGFR) ligands, ADAM17 can activate the EGFR and is involved in various downstream signalling pathways. The present study aimed to investigate whether ADAM17­induced EGFR transactivation is involved in microglial cell survival following spinal cord injury (SCI). Reverse transcription quantitative polymerase chain reaction and western blot analysis revealed that ADAM17 was overexpressed in a mouse model following SCI. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay demonstrated that the viability of human microglia and oligodendrocytes were significantly reduced in a time- and dose-dependent manner following treatment with the ADAM17 antagonist, TNF protease inhibitor 2. Hoechst 33258 staining and flow cytometric analysis revealed that inhibiting ADAM17 increased the rate of cellular apoptosis in neuronal and glial cell cultures, which was accompanied by increased cleavage of caspase-3. Western blot analysis demonstrated that inhibiting ADAM17 resulted in a reduction in the phosphorylation of the EGFR signalling pathway components and thereby impaired functional recovery, inhibited cell viability and prompted microglial apoptosis following SCI. Pre-treatment with the EGFR inhibitor, AG1478, rescued the ADAM17­mediated proliferation of microglial cells. These data demonstrated that ADAM17 contributed to microglial cell survival, predominantly by EGFR signalling, following SCI.

Combining Bone Marrow Stromal Cells with Green Tea Polyphenols Attenuates the Blood-Spinal Cord Barrier Permeability in Rats with Compression Spinal Cord Injury.

This study was performed to investigate the effect of bone marrow stromal cells (BMSCs) combined with green tea polyphenols (GTPs) on the blood-spinal cord barrier (BSCB) permeability after spinal cord injury (SCI) in the rat model. In the model of SCI rats, we found that the water content and the BSCB permeability were decreased by BMSCs and GTPs treatment, and their combination had a synergistic effect. Further, the motor function of rats was also greatly improved by BMSCs and GTPs administration. After treated by the combination of BMSCs and GTPs, SCI rats showed the up-regulated expression of tight junction (TJ) associated proteins claudin-5, occludin and ZO-1 by Western blot, which was more remarkable than that in the single treatment. The increased expression levels of claudin-5, occludin, and ZO-1 were the most obvious in the spinal cord microvessels using immunohistochemistry assay. This led to the conclusion that the combination of BMSCs and GTPs could decrease the BSCB permeability by up-regulating protein expression levels of claudin-5, occludin, and ZO-1. In addition, after BMSCs and GTPs administration, the results of Western blot and enzyme-linked immunosorbent assay (ELISA) revealed a significant decrease in protein expression level and the activation of nuclear factor-кB (NF-кB) p65. Our results indicated that combination of BMSCs and GTPs could improve motor function after SCI, which might be correlated with improvements in BSCB integrity, and that NF-кB might be involved in the modulating process.

Mitochondrial fusion and fission after spinal sacord injury in rats.

Responsible for orchestrating cellular energy production, mitochondria are central to the maintenance of life and the gatekeepers of cell death. Its morphology is dynamic and controlled by continual and balanced fission and fusion events. In this study, we analyzed the mitochondrial dynamics and functions after spinal cord injury in rats and further to discuss the mechanisms of the mitochondria regulated cell injury during SCI. Using adult rat spinal cord injury model, it was found that the absolute number of mitochondria per area was significantly less and the individual mitochondrial cross-sectional area was significantly greater in the neurons of rats in SCI group than in the sham-operated group at 3h and 6h after SCI, and the reverse pattern at 12h and 24h after SCI. The results from Western blot and RT-PCR assays showed that the protein and mRNA levels of mitochondrial fusion-related genes (Mfn1 and Mfn2) decreased and fission-related genes (Drp1 and Fis1) increased at 3h and 6h after SCI. At 12h and 24h after SCI the reverse pattern of Mfn1, Mfn2, Drp1 and Fis1 expression was found. Taken together the results of the present study showed the mitochondrial tendency of elongation and fusion in the injured spinal cord at 3h and 6h after SCI, and the tendency of mitochondrial fission at 12h and 24h after SCI in our SCI models of rat. These findings have important implications for our understanding of the mechanisms of mitochondrial dynamics and functions after SCI injury. And mitochondrial fusion may potentially be used as a target for improving spinal cord function in the first 6h after SCI. Mitochondrial fusion may be inhibited at 12-24h after SCI for improving functional outcomes following SCI.