Yansong Wang

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.

The effect of cigarette smoke exposure on spinal cord injury in rats.

In this study, we examined whether cigarette smoke has neuroprotective or toxic effects on spinal cord injury (SCI). Male Sprague-Dawley rats were included in the study and received either cigarette smoke exposure or fresh air exposure. Twenty-four hours after the last cigarette smoke or fresh air exposure, all rats were injured at thoracic level 12 (T12), using an established static compression model. Our data showed that the cigarette smoke group had higher water content; higher permeability of the blood-spinal cord barrier (BSCB); higher malondialdehyde (MDA) levels, aquaporin-4 (AQP4) and hypoxia-inducible factor 1-alpha (HIF-1α) protein expression, and mRNA levels; and lower glutathione (GSH) levels than the control group values at 12 h, 24 h, and 48 h after SCI. There was no significant difference in these between the cigarette smoke group and the control group at 0 h after SCI. The results of the Basso, Beattie, and Bresnahan (BBB) hindlimb locomotor rating scale showed that rats in the cigarette smoke group had greater dysfunction in hindlimb movement than did rats in control group from 2 to day 6 after SCI. The extent of recovery did not make any difference from day 7 to day 10 after SCI between the cigarette smoke group and the control group. These results suggested that cigarette smoke can reinforce the oxidative stress injury via HIF-1α and AQP4 in the early stage after SCI. It is possible that cigarette smoke exposure does not affect SCI recovery in the long term; however, it can aggravate the edema and deteriorate BSCB disruption via HIF-1α and AQP4 in the early stage after SCI. More studies will be essential to consider this hypothesis and elucidate the mechanisms involved.

Supplement moderate zinc as an effective treatment for spinal cord injury

Spinal cord repair is a challenging task that has puzzled clinical specialists and scientists for a long time. Zinc plays an important role in regulating the expression of brain-derived neurotrophic factor (BDNF) in nervous system, which can improve the pathological state of neurons and promote regeneration of injured neurons, reduce neuronal apoptosis. Our previous studies demonstrated that the serum zinc levels in SCI model group were significantly decreased and zinc concentrations in spinal cord were gradually increased in 24 h after SCI, which induces the up-regulation of zinc transporter 1 (ZnT-1). The mRNA levels of ZnT1 and BDNF were both increased after SCI, and there is a positive correlation between them. Excess zinc exposure has been proved to be a risk factor for neuron death in brain and spinal cord injuries, but supplement of the right amount of zinc may be useful in promoting the recovery of spinal cord function.

Curcumin improves the integrity of blood-spinal cord barrier after compressive spinal cord injury in rats.

Previous studies have shown that curcumin (Cur) can produce potent neuroprotective effects against damage due to spinal cord injury (SCI). However, whether Cur can preserve the function of the blood-spinal cord barrier (BSCB) is unclear. The present study was performed to investigate the mechanism underlying BSCB permeability changes, which were induced by treatment with Cur (75, 150, and 300 mg/kg, i.p.) after compressive SCI in rats. BSCB permeability was evaluated by Evans blue leakage. Motor recovery of rats with SCI was assessed using the Basso, Beattie, and Bresnahan scoring system every day until the 21st days post-injury. The protein levels of heme oxygenase-1 (HO-1), tight junction protein, and inflammatory factors were analyzed by western blots. The expression of the inflammatory factors tumor necrosis factor-α (TNF-α) and nuclear factor-kappaB (NF-κB) mRNA was determined with reverse transcription-polymerase chain reactions. Treatment with Cur (150 and 300 mg/kg) significantly reduced Evans blue leakage into the spinal cord tissue at 24h after SCI. Cur (150 mg/kg) significantly increased HO-1 protein expression. The levels of TNF-α and NF-κB mRNA and protein greatly increased at 24h after SCI, and this increase was significantly attenuated by Cur treatment. ZO-1 and occludin expression was upregulated by Cur (150 mg/kg) treatment after SCI, and this effect was blocked by the HO-1 inhibitor zinc protoporphyrin. Long-term effects of Cur on motor recovery after SCI were observed. Our results indicated that Cur can improve motor function after SCI, which could correlate with improvements in BSCB integrity.

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.

Supplement zinc as an effective treatment for spinal cord ischemia/reperfusion injury in rats.

OBJECTIVE Brain-derived neurotrophic factor (BDNF) plays a key role in the pathophysiology process and therapy of spinal cord injury (SCI). Accordingly, zinc regulates the expression of BDNF and its receptor in the central nervous system, the mechanism of which is still unknown. The present study investigates whether supplement zinc could reduce neurological damage in a rat model, with spinal cord ischemia-reperfusion (I/R) injury and how the effect of zinc transporter 1(ZnT-1) was involved. METHODS 100 Sprague-Dawley male rats were randomly and evenly divided into four groups. They were subjected to spinal cord ischemia by clamping the abdominal aorta for 45 min. Rats in the zinc-deficient dietary model group (ZD), zinc-adequate dietary model group (ZA), and zinc-high dietary model group (ZH) were given free access to purified diet, containing 5, 30, or 180 mg Zn/kg. Sham operation rats were subjected to laparotomy without clamping of the aorta and were fed by ZA diet (30 mg Zn/kg). Neurological function was scored by Tarlovs score. The spinal cord segments (L5) were harvested for histological examination, auto-metallographic (AMG) analysis, myeloperoxidase (MPO) activity analysis, expression of ZnT-1 and BDNF. RESULTS The rats in the ZH group have shown the higher neurological scores, slighter histological changes and the attenuated MPO activity, compared with those in the ZD and ZA groups at the four observation time points (p<0.05). The AMG staining density in the ZH group was significantly higher than that of ZD group in 14 days later after the operation. Compared with other groups, ZH groups expression of Zn-T1 and BDNF were significantly increased, and was positively correlated with the same time points after surgery (Spearman rho=0.403, p=0.0152.) CONCLUSION These findings suggest that zinc supplement can significantly reduce the spinal cord I/R injury in rats. The mechanism may be related with restraining the MPO activity and increasing of ZnT-1, which promoted the synthesis and release of BDNF.

The Correlation Among the Dynamic Change of Zn2+, ZnT-1, and Brain-Derived Neurotrophic Factor After Acute Spinal Cord Injury in Rats

Zinc plays an important role in regulating the expression of brain-derived neurotrophic factor (BDNF) and its receptor in nervous system, but the correlation among Zn2+, zinc transporter, and BDNF in spinal cord injuries (SCI) is not fully understood. The purpose of this study was to investigate the expression of Zn2+, zinc transporter 1 (ZnT-1), and BDNF, as well as their correlation in spinal cord-injured rats. One hundred Wistar male rats were divided into two groups: sham-operated group (as control group) and model group. Spinal cord injury was induced in model groups by hemisection of T9 on the left side. Compared with the control group, the serum zinc levels in SCI model group were significantly decreased after surgery, but zinc concentrations in spinal cord were increased gradually. The mRNA levels of ZnT-1 and BDNF were significantly increased in SCI model group, and there is a positive correlation between them (Spearman rho = 0.381, P = 0.0204). The correlation found between BDNF and ZnT-1 allows us to speculate that these two factors may be physiologically co-regulated, which may provide an idea for the treatment of SCI.

Silencing of PHLPP1 promotes neuronal apoptosis and inhibits functional recovery after spinal cord injury in mice

Aim: Spinal cord injury (SCI) causes increased apoptosis of neurons, leading to irreversible dysfunction of the spinal cord. In this study, we investigated the effects of the progression of SCI and potential regulation of apoptosis after the Pleckstrin homology (PH) domain and leucine rich repeat protein phosphatase 1 (PHLPP1) gene was silenced. Main methods: Spinal cord injection, and neuronal transfection with a recombinant adenovirus vector encoding small interfering RNA (siRNA) against PHLPP1 (AdsiPHLPP1) successfully silenced PHLPP1. These created in vivo and in vitro PHLPP1‐silenced models, respectively, resulting in stable expression of the transgene in neurons. Key findings: The results showed that silencing of PHLPP1 evidently reduced levels of the nuclear factor erythroid 2‐related factor 2 (Nrf2) after SCI. Western blot analysis revealed that the mice injected with AdsiPHLPP1 showed increased the expression of pro‐apoptotic factors (Bax and cleaved‐caspase 3), and reduced levels of neurotrophic (BDNF) and anti‐apoptotic (Bcl‐2) factors, both in vivo and in vitro. The motor function of AdsiPHLPP1‐injected mice was restored more slowly than that of wild type (WT) mice. In addition, the number of motor neurons surviving in the anterior horn of the spinal cord was also reduced after SCI. Significance: Our results confirm that silencing of PHLPP1 promotes neuronal apoptosis and inhibits functional recovery after injury in vivo and in vitro. Consequently, PHLPP1 represents a potential therapeutic target gene for the clinical treatment of SCI.

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.