Zhibin Shi

Resveratrol improves neuron protection and functional recovery in rat model of spinal cord injury

Researches on the pathology of spinal cord injury (SCI) have been recently focused on oxidative radicals stress and inflammation associated neuronal apoptosis. Resveratrol, a natural phenolic compound, has been extensively studied and shown a wide variety of health beneficial effects, including prevention of cardiovascular diseases and cancer and neuroprotective activities. However, the study of its potential role in neuroprotection and underlying mechanism in SCI model has been limited. In this study, we investigated the effect of resveratrol on neurologic functions and histopathologic changes after SCI and the mechanism underlying its neuroprotective effects. First, neuronal function after SCI was evaluated with Basso Beattle Bresnahan locomotor rating scale (BBB) and the result showed that injured animals treated with resveratrol showed a significant increase in BBB scores. Further, histopathological alternations were evaluated with HE and Nissl staining, showing a restored neural morphology and an increase of the number of neurons after resveratrol administration. To explore the underlying mechanism, anti-oxidation effect of resveratrol was assessed by measuring superoxide dismutase (SOD) activity and malondialdehyde (MDA) level after SCI. Resveratrol treatment reversed the decrease of SOD activity and increase of MDA level caused by SCI, suggesting its anti-oxidation role in response to the injury. In addition, resveratrol treatment suppressed immunoreactivity and expression of inflammatory cytokines including IL-1β, IL-10, TNF-α, and myeloperoxidase (MPO) after SCI, suggesting an anti-inflammation effect of resveratrol. Finally, resveratrol treatment inhibited injury-induced apoptosis as assessed by electrical microscopy and TUNEL staining and affected the expression level of apoptosis-related gene Bax, Bcl-2 and caspase-3, which indicated its anti-apoptosis role after SCI. Our data suggest that resveratrol significantly promotes the recovery of rat dorsal neuronal function after SCI, and this effect is related to its characteristics of anti-oxidation, anti-inflammation and anti-apoptosis.

Tetramethylpyrazine protects spinal cord and reduces inflammation in a rat model of spinal cord ischemia-reperfusion injury

OBJECTIVE Inflammation, which is known to be detrimental to the neurologic outcome during the acute phase after an ischemic stroke, provides a potential target for preventive or therapeutic approach for spinal cord ischemia-reperfusion injury. Tetramethylpyrazine (TMP), a pure compound derived from Ligusticum chuanxiong, is widely used in the treatment of ischemic stroke. The present study aimed to gain a deeper insight into the mechanism underlying the anti-inflammatory effects of TMP on spinal cord ischemia-reperfusion injury. METHODS Spinal cord ischemia was induced in male Sprague-Dawley rats by balloon occlusion of the thoracic aorta. The experimental groups (n = 30 per group) included sham operation, control (receiving only normal saline), and TMP (30 mg/kg, 30 minutes before occlusion). Neurologic function was assessed by the Basso, Beattie, and Bresnahan (BBB) score at 1, 6, 12, 24, and 48 hours after reperfusion. Histologic changes were studied using Nissl staining. Infarct volume was analyzed using 2,3,5-triphenyltetrazolium chloride staining. Myeloperoxidase (MPO) activity was determined by using a rat MPO assay kit. Interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-10 and nuclear factor (NF)-κB were examined with immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Western blotting. RESULTS Compared with the control group, the TMP group showed significantly improved neurologic outcome (P < .05), decreased infarct volume (42.3% vs 17.4%), and alleviated neutrophil infiltration (0.35 vs 0.18 U/g). TMP treatment reduced the expressions of proinflammatory cytokines TNF-α (28.62 vs 15.23 pg/mg protein) and IL-1β (13.62 vs 8.24 pg/mg protein), upregulated the expression of anti-inflammatory cytokine IL-10 (18.35 vs 31.26 pg/mg protein), and inhibited the activation of NF-κB (2.78 vs 1.22) in ischemic spinal cord. CONCLUSIONS Treatment with TMP exerted a neuroprotective effect against spinal cord ischemia-reperfusion injury. The anti-inflammatory effect was believed to be one of the contributing mechanisms.

Osteogenesis of the construct combined BMSCs with β-TCP in rat

BACKGROUND The use of artificial bone graft substitutes has increased as the surgical applications widen and the availability of allograft bone decreases. The present study was to evaluate the construct combined bone marrow stromal cells (BMSCs) with beta-tricalcium phosphate (beta-TCP) as bone substitute implanted in rat dorsal muscles. METHODS To study the osteogenic capability in vivo, specimens were harvested on 1 week, 4 weeks and 8 weeks after implantation, and were analyzed by hematoxylin and eosin (HE) staining. The percentages of new bone formation for each implant type and implantation period were determined by histomorphometry. RESULTS After 1 week of implantation, new bone formation for both beta-TCP and BMSCs+beta-TCP group had no formed. After 4 weeks of implantation, the amount of bone formation was increased to 1.32 % in beta-TCP group and 6.35% in BMSCs+beta-TCP group. After 8 weeks of implantation, more bone was found in the BMSCs+beta-TCP group (21.58 %), while in the beta-TCP group bone formation was increased to 4.78%. Significant differences between the two groups have been observed. CONCLUSIONS Based on these results, we conclude that bone substitutes constructed by porous beta-TCP scaffold loaded with osteogenically induced BMSCs could promote newly formed bone.

Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and osteogenesis in rabbit femoral head osteonecrosis

PURPOSE Osteonecrosis of the femoral head may be a disease resulting from abnormal proliferation or differentiation of mesenchymal stem cells. The present investigation explored the novel strategy of hypoxia-preconditioned BMMSCs to reverse the impairment of osteonecrosis BMMSCs and enhance the therapeutic potential of hypoxia-treated BMMSC transplantation. METHODS BMMSCs from the anterior superior iliac spine region of osteonecrosis rabbit were cultured under 20% O2 or 2% O2 conditions. Normal BMMSCs were cultured under 20% O2 condition as control. Growth factors secreted were examined by enzyme-linked immunosorbent assay. 20% O2 or 2% O2 BMMSCs were injected into the femoral head of rabbits after core decompression. Cell viability and apoptosis were assessed in vitro, and TUNEL staining of the femoral head was analyzed after transplantation. Angiogenesis (capillary-like structure formation, CD31 immunohistochemical staining and ink infusion angiography) and osteogenesis (Alizarin red-S staining, micro-CT scanning and OCN immunohistochemical staining) tests were conducted as well. RESULTS 2% O2 exposure up-regulated growth factor secretion in BMMSCs. Apoptosis in 2% O2 group was lower when compared with that in 20% O2 osteonecrosis group. Cell viability in 2% O2 was significantly higher when compared with that in 20% O2 osteonecrosis group. Growth factor secretion, cell viability, apoptosis, capillary-like structure formation, Alizarin red-S staining, and ALP staining showed no difference between the 2% O2 BMMSC and normal BMMSC groups. Transplantation of 2% O2 versus 20% O2 mesenchymal stem cells after core decompression resulted in an increase in angiogenesis function and a decrease in local tissue apoptosis. Our study also found that osteogenesis function was improved after hypoxic stem cell transplantation. CONCLUSION Hypoxic preconditioning of BMMSCs is an effective means of reversing the impairment of osteonecrosis BMMSCs, promoting their regenerative capability and therapeutic potential for the treatment of osteonecrosis.

Design and numerical investigation of A HVDC vacuum switch based on artificial current zero

With the fast development of multi-terminal high-voltage direct-current (HVDC) transmission technology, there is an urgent demand for the HVDC interruption technique. In this paper, an interruption scheme for HVDC based on artificial current zero is proposed with its main circuit breaker consisting of modularized vacuum switch in series. A triggered sphere gap is adopted as the commutation switch to achieve bidirectional DC interruption. The interruption process, particularly, the most important two commutation processes, is simulated based on a circuit model. The residual current and its influence after current commutation, which might exist due to the weak arc extinguishing capacity of the sphere gap, are analyzed. It is proposed to use back-up circuit breakers to interrupt the residual current. Furthermore, the influence of the frequency of the countercurrent on the interruption process, particularly, on the integral of i2t in the triggered sphere gap due to the residual current, is discussed.

Investigation on DC interruption based on artificial current zero of vacuum switch

Direct current interruption utilizing vacuum switch based on artificial current zero is investigated. A high-speed vacuum switch, which is driven by electromagnetic repulsion force, is adopted as the main switch. The injection of high-frequency countercurrent is controlled by a triggered vacuum switch. The main experimental current is supplied by a LC circuit. The overvoltage generated during interruption is suppressed by metal oxide arrester. The interruption process is also simulated via a circuit model, with which the two important current commutation processes are analyzed, i.e., the current commutation from main switch to countercurrent branch, and the current commutation from countercurrent branch to arrester branch. Simulation results are compared with measured waveforms of voltage and current.

Prefabrication of Vascularized Porous Three-Dimensional Scaffold Induced from rhVEGF165: A Preliminary Study in Rats

Background/Aims: Several shortcomings have limited the routine use of autogenous vascularized bone graft. The present study investigates the prefabrication of vascularized scaffold with the desired shape and microarchitecture combined with recombinant human vascular endothelial growth factor 165 (rhVEGF165) to mimic autogenous vascularized bone graft. Methods: Eighty-five porous calcium phosphate cement scaffolds constructed by rapid prototyping technology were divided into four groups: group A [rhVEGF165-fibrin sealant (FS) scaffold], group B (hVEGF165 scaffold), group C (FS scaffold), and group D (scaffold alone). The release of rhVEGF165 from the scaffolds was examined in vitro. The vessel density, relative functionalized vessels, vessel diameter and relative vessel area were also measured. Results: The sustained release of hVEGF165 lasted 14 days in the absence of plasmin and 12 days in the presence of plasmin in group A and 10 days in group B. There was no statistical difference between groups A and B at 2 or 4 weeks in terms of vessel density, relative functionalized vessels, vessel diameter, and relative vessel area, as between groups C and D. However, the above parameters were greater in groups A and B than groups C and D. Conclusion: The scaffolds with the desired shape and microarchitecture combined with rhVEGF165 could shorten the time needed for the construction of prefabricated vascularized grafts and accelerate the maturation of the vessels.

Protective effects and mechanism of Panax Notoginseng saponins on oxidative stress-induced damage and apoptosis of rabbit bone marrow stromal cells

ObjectiveTo investigate the effects and possible mechanism of Panax Notoginseng saponins (PNS) on oxidative stress-induced damage and apoptosis in bone marrow stromal cells (BMSCs).MethodsBMSCs were isolated and cultured from 2-month-old New Zealand rabbits by the density gradient centrifugation combined with adherent method. The third passage cells were used for subsequent experiments. Oxidative stress was induced in cultured BMSCs by H2O2 (0.1 mmol/L). BMSCs were pretreated with 25–200 μg/mL PNS for 4 h before H2O2 treatment. Proliferation of BMSCs was observed using MTT assay. Alkaline phosphatase (ALP) activity, as an index of early osteoblastic differentiation, was determined with an ALP assay kit. Flow cytometry was used to observe the apoptosis of BMSCs by staining with annexinV-FITC/propidium iodide. Oxidative stress level was examined by reactive oxygen species (ROS) assay. The protein expressions of Bax, Bcl-2 and Caspase-3 in BMSCs were analyzed by Western blotting.ResultsPNS had different concentration-dependent effects on proliferation and osteoblast differentiation of BMSCs induced by H2O2. A PNS concentration of 100 μg/mL was determined as the optimal effective concentration. PNS markedly attenuated H2O2-induced apoptosis rate from 41.91% to 14.67% (P<0.01). PNS significantly decreased ROS level induced by H2O2 (P<0.01). Furthermore, pretreatment with PNS significantly reversed H2O2-induced inhibition of Bcl-2 expression and augmentation of Bax and Caspase-3 expression (P<0.01).ConclusionPNS had a protective effect on oxidative stress-induced damage and apoptosis in cultured rabbit BMSCs through scavenging ROS and regulating the Bcl-2/Bax pathway.

Association of toll-like receptor 4 signaling pathway with steroid-induced femoral head osteonecrosis in rats

Osteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramuscularly with 20 mg/kg methylprednisolone (MP) for 8 weeks, twice per week. The animals were sacrificed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups (n=24 each). Rats in the control group (n=12) were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase (TRAP) in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-κB p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR (RT-PCR) and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentration of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in control rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased significantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.SummaryOsteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramuscularly with 20 mg/kg methylprednisolone (MP) for 8 weeks, twice per week. The animals were sacrificed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups (n=24 each). Rats in the control group (n=12) were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase (TRAP) in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-κB p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR (RT-PCR) and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentration of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in control rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased significantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.

rhVEGF165 delivered in a porous β-tricalcium phosphate scaffold accelerates bridging of critical-sized defects in rabbit radii

Segmental bone defects are a common obstacle in major orthopedic procedures, and the treatment of these defects remains a challenging clinical problem. Bone tissue engineering has been attracting much attention in recent years. We evaluated the ability of the specific combination of 3 microg rhVEGF(165) with a novel porous beta-tricalcium phosphate (beta-TCP) scaffold coated with fibrin sealant (FS) to facilitate bone regeneration. Unilateral 15-mm long critical-sized defects were prepared in the radial diaphysis of rabbits and treated with rhVEGF(165)/FS/scaffold or FS/scaffold. Healing of the defects was assessed at 4, 8, and 12 weeks, radiologically, histologically, and biomechanically. The results of the study demonstrated that the critical-sized defects in the midshaft of the rabbit radius, treated with rhVEGF(165) incorporated in porous beta-TCP scaffold by FS, can be completely bridged by cortical bone in 12 weeks. The bone marrow space was also reformed histologically and radiologically at 12 weeks postsurgery in the rhVEGF(165)-treated group. Furthermore, biomechanical examination demonstrated that the segmental bone defects were not only radiologically and histologically repaired but were also mechanically repaired. Interestingly, none of the defects was completely repaired at 12 weeks following treatment with FS/scaffold without rhVEGF(165). A solution-driven process is likely the predominant mechanism of accelerating biodegradation of the beta-TCP scaffold in the presence of rhVEGF(165); furthermore, cell-mediated phagocytosis also contributes to biodegradation of the biomaterials.