Zhuojing Luo

Electrical regulation of Schwann cells using conductive polypyrrole/chitosan polymers

Electrical stimulation (ES) can dramatically enhance neurite outgrowth through conductive polymers and accelerate peripheral nerve regeneration in animal models of nerve injury. Therefore, conductive tissue engineering graft in combination with ES is a potential treatment for neural injuries. Conductive tissue engineering graft can be obtained by seeding Schwann cells on conductive scaffold. However, when ES is applied through the conductive scaffold, the impact of ES on Schwann cells has never been investigated. In this study, a biodegradable conductive composite made of conductive polypyrrole (PPy, 2.5%) and biodegradable chitosan (97.5%) was prepared in order to electrically stimulate Schwann cells. The tolerance of Schwann cells to ES was examined by a cell apoptosis assay. The growth of the cells was characterized using DAPI staining and a MTT assay. mRNA and protein levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in Schwann cells were assayed by RT-PCR and Western blotting, and the amount of NGF and BDNF secreted was determined by an ELISA assay. The results showed that the PPy/chitosan membranes supported cell adhesion, spreading, and proliferation with or without ES. Interestingly, ES applied through the PPy/chitosan composite dramatically enhanced the expression and secretion of NGF and BDNF when compared with control cells without ES. These findings highlight for the first time the possibility of enhancing nerve regeneration in conductive scaffolds through ES-increased neurotrophin secretion.

Tanshinone IIA Attenuates the Inflammatory Response and Apoptosis after Traumatic Injury of the Spinal Cord in Adult Rats

Background Spinal cord injury (SCI), including immediate mechanical injury and secondary injury, is associated with the inflammatory response, apoptosis and oxidative stress in response to traumatic injury. Tanshinone IIA (TIIA) is one of the major extracts obtained from Salvia miltiorrhiza BUNGE, which has anti-inflammatory and anti-apoptotic effects on many diseases. However, little is known about the effects of TIIA treatment on SCI. Therefore, the aim of the present study is to evaluate the pharmacological action of TIIA on secondary damage and the underlying mechanisms of experimental SCI in rats. Methodology/Principal Findings SCI was generated using a weight drop device on the dorsal spinal cord via a two-level T9-T11 laminectomy. SCI in rats resulted in severe trauma, characterized by locomotor disturbance, edema, neutrophil infiltration, the production of astrocytes and inflammatory mediators, apoptosis and oxidative stress. TIIA treatment (20 mg/kg, i.p.) after SCI induced significant effects: (1) improved motor function (Basso, Beattie and Bresnahan scores), (2) reduced the degree of tissue injury (histological score), neutrophil infiltration (myeloperoxidase activity) and the expression of astrocytes, (3) inhibited the activation of SCI-related pathways, such as NF-κB and MAPK signaling pathways, (4) decreased the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and iNOS, (5) reduced apoptosis (TUNEL staining, and Bcl-2 and caspase-3 expression) and (6) reversed the redox state imbalance. Conclusions/Significance The results clearly show that TIIA has a prominent protective effect against SCI through inhibiting the inflammatory response and apoptosis in the spinal cord tissue after SCI.

Electrical stimulation induces calcium-dependent release of NGF from cultured Schwann cells

Production of nerve growth factor (NGF) from Schwann cells (SCs) progressively declines in the distal stump, if axonal regeneration is staggered across the suture site after peripheral nerve injuries. This may be an important factor limiting the outcome of nerve injury repair. Thus far, extensive efforts are devoted to modulating NGF production in cultured SCs, but little has been achieved. In the present in vitro study, electrical stimulation (ES) was attempted to stimulate cultured SCs to release NGF. Our data showed that ES was capable of enhancing NGF release from cultured SCs. An electrical field (1 Hz, 5 V/cm) caused a 4.1‐fold increase in NGF release from cultured SCs. The ES‐induced NGF release is calcium dependent. Depletion of extracellular or/and intracellular calcium partially/ completely abolished the ES‐induced NGF release. Further pharmacological interventions showed that ES induces calcium influx through T‐type voltage‐gated calcium channels and mobilizes calcium from 1, 4, 5‐trisphosphate‐sensitive stores and caffeine/ryanodine‐sensitive stores, both of which contributed to the enhanced NGF release induced by ES. In addition, a calcium‐triggered exocytosis mechanism was involved in the ES‐induced NGF release from cultured SCs. These findings show the feasibility of using ES in stimulating SCs to release NGF, which holds great potential in promoting nerve regeneration by enhancing survival and outgrowth of damaged nerves, and is of great significance in nerve injury repair and neuronal tissue engineering.

Protection by salidroside against bone loss via inhibition of oxidative stress and bone-resorbing mediators.

Oxidative stress is a pivotal pathogenic factor for bone loss in mouse model. Salidroside, a phenylpropanoid glycoside extracted from Rhodiola rosea L, exhibits potent antioxidative effects. In the present study, we used an in vitro oxidative stress model induced by hydrogen peroxide (H2O2) in MC3T3-E1 cells and a murine ovariectomized (OVX) osteoporosis model to investigate the protective effects of salidroside on bone loss and the related mechanisms. We demonstrated that salidroside caused a significant (P<0.05) elevation of cell survival, alkaline phosphatase (ALP) staining and activity, calcium deposition, and the transcriptional expression of Alp, Col1a1 and Osteocalcin (Ocn) in the presence of H2O2. Moreover, salidroside decreased the production of intracellular reactive oxygen species (ROS), and osteoclast differentiation inducing factors such as receptor activator of nuclear factor-kB ligand (RANKL) and IL-6 induced by H2O2. In vivo studies further demonstrated that salidroside supplementation for 3 months caused a decrease in malondialdehyde (MDA) and an increase in reduced glutathione (GSH) concentration in blood of ovariectomized mouse (P<0.05), it also improved trabecular bone microarchitecture and bone mineral density in the fourth lumbar vertebra and distal femur. Our study indicated that the protection provided by salidroside in alleviating bone loss was mediated, at least in part, via inhibition of the release of bone-resorbing mediators and oxidative damage to bone-forming cells, suggesting that salidroside can be used as an effective remedy in the treatment or prevention of osteoporosis.

Electrical Stimulation to Conductive Scaffold Promotes Axonal Regeneration and Remyelination in a Rat Model of Large Nerve Defect

Background Electrical stimulation (ES) has been shown to promote nerve regeneration when it was applied to the proximal nerve stump. However, the possible beneficial effect of establishing a local electrical environment between a large nerve defect on nerve regeneration has not been reported in previous studies. The present study attempted to establish a local electrical environment between a large nerve defect, and examined its effect on nerve regeneration and functional recovery. Methodology/Findings In the present study, a conductive scaffold was constructed and used to bridge a 15 mm sciatic nerve defect in rats, and intermittent ES (3 V, 20 Hz) was applied to the conductive scaffold to establish an electrical environment at the site of nerve defect. Nerve regeneration and functional recovery were examined after nerve injury repair and ES. We found that axonal regeneration and remyelination of the regenerated axons were significantly enhanced by ES which was applied to conductive scaffold. In addition, both motor and sensory functional recovery was significantly improved and muscle atrophy was partially reversed by ES localized at the conductive scaffold. Further investigations showed that the expression of S-100, BDNF (brain-derived neurotrophic factor), P0 and Par-3 was significantly up-regulated by ES at the conductive scaffold. Conclusions/Significance Establishing an electrical environment with ES localized at the conductive scaffold is capable of accelerating nerve regeneration and promoting functional recovery in a 15 mm nerve defect in rats. The findings provide new directions for exploring regenerative approaches to achieve better functional recovery in the treatment of large nerve defect.

Electrical Stimulation Accelerates Motor Functional Recovery in the Rat Model of 15-mm Sciatic Nerve Gap Bridged by Scaffolds With Longitudinally Oriented Microchannels

BACKGROUND Electrical stimulation (ES) can enhance the regenerative capacity of axotomized motor and sensory neurons. However, the impact of ES on axonal regeneration and functional recovery has not been investigated in an animal model of a lengthy peripheral nerve defect. OBJECTIVE To determine whether ES accelerates axonal regeneration and functional recovery of a 15-mm sciatic nerve defect in rats. METHODS A 15-mm excision of the sciatic nerve was bridged with a chitosan scaffold with longitudinally or randomly oriented pores or with autologous grafting of the segment. In half of the animals with chitosan grafts, the proximal nerve stump was electrically stimulated for 1 hour at 20 Hz immediately after the nerve repair with the scaffolds. Axonal regeneration was investigated by retrograde labeling and morphometric analysis. The rate of motor functional recovery was evaluated by electrical nerve stimulation, behavioral tests of stepping, and histological appearance of the target muscles. RESULTS Axonal regeneration and motor functional recovery were improved by ES in animals that received longitudinal pore grafts as compared with others. The maximal number of axons that regenerated across the longitudinal graft was achieved 2 to 4 weeks earlier in rats with ES. In addition, the latency of compound muscle action potentials (CMAPs), the peak amplitude of CMAPs, and nerve conduction velocity were improved by ES. Stepping indices were better, with less atrophy of target muscle in ES rats managed with longitudinal pores. CONCLUSION Brief ES may accelerate axonal regeneration and motor recovery after focal peripheral nerve transection when repaired with optimally tissue-engineered grafts.Background. Electrical stimulation (ES) can enhance the regenerative capacity of axotomized motor and sensory neurons. However, the impact of ES on axonal regeneration and functional recovery has not been investigated in an animal model of a lengthy peripheral nerve defect. Objective. To determine whether ES accelerates axonal regeneration and functional recovery of a 15-mm sciatic nerve defect in rats. Methods. A 15-mm excision of the sciatic nerve was bridged with a chitosan scaffold with longitudinally or randomly oriented pores or with autologous grafting of the segment. In half of the animals with chitosan grafts, the proximal nerve stump was electrically stimulated for 1 hour at 20 Hz immediately after the nerve repair with the scaffolds. Axonal regeneration was investigated by retrograde labeling and morphometric analysis. The rate of motor functional recovery was evaluated by electrical nerve stimulation, behavioral tests of stepping, and histological appearance of the target muscles. Results. Axonal regeneration and motor functional recovery were improved by ES in animals that received longitudinal pore grafts as compared with others. The maximal number of axons that regenerated across the longitudinal graft was achieved 2 to 4 weeks earlier in rats with ES. In addition, the latency of compound muscle action potentials (CMAPs), the peak amplitude of CMAPs, and nerve conduction velocity were improved by ES. Stepping indices were better, with less atrophy of target muscle in ES rats managed with longitudinal pores. Conclusion. Brief ES may accelerate axonal regeneration and motor recovery after focal peripheral nerve transection when repaired with optimally tissue-engineered grafts.

Rapid repair and regeneration of damaged rabbit sciatic nerves by tissue-engineered scaffold made from nano-silver and collagen type I

A tissue-engineered scaffold with nano-silver and collagen type I was constructed and investigated for its ability to adsorb laminin and the usefulness in the repair and regeneration of damaged peripheral nerves in animals. The nano-silver scaffold displayed ideal microtubule structure under electronic microscope; even distribution of the nano-silver particles was also seen with energy spectrometry. After immersion in a laminin solution, the laminin-attached scaffolds were implanted into rabbits to repair a 10-mm injury of the sciatic nerve. At 30 days post-implantation, regeneration of the damaged nerve was evaluated by transmission electron microscopy, electrophysiological examination and fluoro-gold (FG) retrograde labelling. Compared with the control collagen-scaffold without nano-silver, the nano-silver-containing scaffold showed a higher rate of laminin adsorption, regenerated a nerve with a thicker myelin sheath and improved the nerve conduction velocity and nerve potential amplitude. FG retrograde labelled the newly grown axons in the spinal cord cortex anterior horn and the dorsal root ganglion. These results demonstrate the superior functionality of the nano-silver-collagen scaffold in the adsorption to laminin and subsequent regeneration of damaged peripheral nerves.

Rapid repair of rat sciatic nerve injury using a nanosilver-embedded collagen scaffold coated with laminin and fibronectin

AIM Scaffold with micro-channels has shown great promise in facilitating axonal regeneration after peripheral nerve injury. Significant research has focused on mimicking, in terms of composition and function, the ability of the basement membrane of Schwann cells to both promote and guide axonal regeneration. We aim to investigate the ability of a tissue-engineered scaffold with nanosilver and collagen to adsorb laminin and fibronectin, and the usefulness of this scaffold for repairing and regenerating a 10-mm peripheral nerve gap in rats. METHODS In this study, nanosilver-embedded collagen scaffolds were prepared and coated with laminin (LN) or LN plus fibronectin (FN). Scanning electron microscopy of the transverse and longitudinal sections of the scaffold revealed axially oriented microtubules ranging from 20 to 80 µm in diameter, and the internal surface of microtubules was found to be evenly coated with LN and FN. Energy dispersive spectrometry also confirmed an even distribution of nanosilver particles within the scaffold. To test its effectiveness in restoring neuronal connection, the scaffold was used in order to bridge 10 mm gaps in the severed sciatic nerve of rats. The rats were divided into an experimental group (receiving scaffold coated with LN and FN), a control group (receiving scaffold coated with LN only) and an autologous graft group. The functional recovery 40 days after surgery was examined by electrophysiology and sciatic nerve functional index (SFI) evaluation. FluoroGold™ (FG) retrograde tracing, toluidine blue staining and transmission electron microscopy were also used to examine the regenerated nerve fibers and to establish their myelination status. RESULTS The experimental group displayed partially restored nerve function. The recovery was comparable to the effect of autologous nerve graft and was better than that observed in the control group. A better functional recovery correlated with more FG-labeled neurons, higher density of toluidine blue stained nerve fibers and thicker myelin sheath. CONCLUSION Our results demonstrated that nanosilver-embedded collagen scaffolds with LN and FN coating is effective in aiding axonal regeneration, and recovery is comparable to the effect of an autologous nerve graft.

Electrical regulation of olfactory ensheathing cells using conductive polypyrrole/chitosan polymers.

Electrical stimulation (ES) applied to a conductive nerve graft holds the great potential to improve nerve regeneration and functional recovery in the treatment of lengthy nerve defects. A conductive nerve graft can be obtained by a combination of conductive nerve scaffold and olfactory ensheathing cells (OECs), which are known to enhance axonal regeneration and to produce myelin after transplantation. However, when ES is applied through the conductive graft, the impact of ES on OECs has never been investigated. In this study, a biodegradable conductive composite made of conductive polypyrrole (PPy, 2.5%) and biodegradable chitosan (97.5%) was prepared in order to electrically stimulate OECs. The tolerance of OECs to ES was examined by a cell apoptosis assay. The growth of the cells was characterized using DAPI staining and a CCK-8 assay. The mRNA and protein levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neural cell adhesion molecule (N-CAM), vascular endothelial growth factor (VEGF) and neurite outgrowth inhibitor-A (NOGO-A) in OECs were assayed by RT-PCR and Western blotting, and the amount of BDNF, NGF, N-CAM, VEGF and NOGO-A secreted was determined by an ELISA assay. The results showed that the PPy/chitosan membranes supported cell adhesion, spreading, and proliferation with or without ES. Interestingly, ES applied through the PPy/chitosan composite dramatically enhanced the expression and secretion of BDNF, NGF, N-CAM and VEGF, but decreased the expression and secretion of NOGO-A when compared with control cells without ES. These findings highlight the possibility of enhancing nerve regeneration in conductive scaffolds through ES increased neurotrophin secretion in OECs.

Oral administration of quercetin inhibits bone loss in rat model of diabetic osteopenia.

Diabetic osteopenia can result in an increased incidence of bone fracture and a delay in fracture healing. Quercetin, one of the most widely distributed flavonoids in plants, possesses antioxidant property and beneficial effect on osteoporosis in ovariectomized mice. All these properties make quercetin a potential candidate for controlling the development of diabetic osteopenia. Therefore, the present study was designed to investigate the putative beneficial effect of quercetin on diabetic osteopenia in rats. Diabetes mellitus was induced by streptozotocin. The diabetic rats received daily oral administration of quercetin (5mg/kg, 30 mg/kg and 50mg/kg) for 8 weeks, which was started at 4 weeks after streptozotocin injection. Quercetin at 5mg/kg showed little effect on diabetic osteopenia, while quercetin at 30 mg/kg and 50mg/kg could increase the decreased serum osteocalcin, serum alkaline phosphatase activity, and urinary deoxypyridinoline in diabetic rats. In addition, quercetin (30 mg/kg and 50mg/kg) could partially reverse the decreased biomechanical quality and the impaired micro-architecture of the femurs in diabetic rats. Histomorphometric analysis showed that both decreased bone formation and resorption were observed in diabetic rats, which was partially restored by quercetin (30 mg/kg and 50mg/kg). Further investigations showed that quercetin significantly lowered the oxidative DNA damage level, up-regulated the total serum antioxidant capability and the activity of serum antioxidants in diabetic rats. All those findings indicate the beneficial effect of quercetin on diabetic osteopenia in rats, and raise the possibility of developing quercetin as potential drugs or an ingredient in diet for controlling diabetic osteopenia.