Zhengxu Ye

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.

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.

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.

A synthetic oxygen carrier in fibrin matrices promotes sciatic nerve regeneration in rats

Tissue-engineering nerve conduits have been studied for a long time in bridging large nerve defects. However, the low oxygen availability within the nerve conduits, which results in death of migratory Schwann cells (SC) or loss of the newly formed tissues function, is still an obstacle for axonal regeneration. Thus, it was hypothesized that an oxygen-enriched conduit would enhance axonal regeneration and functional recovery in vivo. To address this issue, perfluorotributylamine (PFTBA) enriched fibrin hydrogel was prepared and injected into collagen-chitosan conduits. The conduit containing PFTBA-enriched fibrin hydrogel was then used to bridge a 12-mm sciatic nerve defect in rats. The control rats were bridged with collagen-chitosan conduits filled with fibrin matrices without PFTBA. It was found that axonal regeneration and functional recovery in the combined PFTBA group were significantly higher than those in the control group without PFTBA. Further investigations showed that the mRNA and protein levels of S-100, brain-derived neurotrophic factor and nerve growth factor were enhanced by PFTBA at 1 and 3weeks after surgery. However, the mRNA and protein levels of vascular endothelial growth factor were in a similar range between the combined PFTBA group and the control group without PFTBA. In addition, immunohistochemical results showed that the morphological appearances of regenerated nerve and survival of SC were enhanced by PFTBA at 4 and 12weeks after surgery. In conclusion, PFTBA-enriched nerve conduit is capable of enhancing axonal regeneration, which provides a new avenue for achieving better functional recovery in the treatment of nerve defect.

Morphological changes of the neural cells after blast injury of spinal cord and neuroprotective effects of sodium β-aescinate in rabbits

OBJECTIVE Explosive blast neurotrauma is becoming more and more common not only in the military population but also in civilian life due to the ever-present threat of terrorism and accidents. However, little attention has been offered to the studies associated with blast wave-induced spinal cord injury in the literatures. The purpose of this study is to report a rabbit model of explosive blast injury to the spinal cord, to investigate the histological changes, focusing especially on apoptosis, and to reveal whether beta-aescinate (SA) has the neuroprotective effects against the blast injury. METHODS Adult male New Zealand white rabbits were randomly divided into sham group, experimental group and SA group. All rabbits except the sham group were exposed to the detonation, produced by the blast tube containing 0.7 g cyclotrimethylene trinitramine, with the mean peak overpressure of 50.4 MP focused on the dorsal surface of T9-T10 level. After evaluation of the neurologic function, spinal cord of the rabbits was removed at 8 h, 1, 3, 7, 14 or 30 days and the H&E staining, EM examination, DNA gel electrophoresis and TUNEL were progressively performed. RESULTS The study demonstrated the occurrence of both necrosis and apoptosis at the lesion site. Moreover, the SA therapy could not only improve the neurologic outcomes (P<0.05) but also reduce the loss of motoneuron and TUNEL-positive rate (P<0.05). CONCLUSIONS In the rabbit model of explosive blast injury to the spinal cord, the coexistent apoptotic and necrotic changes in cells was confirmed and the SA had neuroprotective effects to the blast injury of the spinal cord in rabbits. This is the first report in which the histological characteristics and drug treatment of the blast injury to the spinal cord is demonstrated.

CaMKII-Mediated CREB Phosphorylation Is Involved in Ca2+-Induced BDNF mRNA Transcription and Neurite Outgrowth Promoted by Electrical Stimulation

Electrical stimulation (ES)-triggered up-regulation of brain-derived neurotrophic factor (BDNF) and neurite outgrowth in cultured rat postnatal dorsal root ganglion neurons (DRGNs) is calcium (Ca2+)-dependent. The effects of increased Ca2+ on BDNF up-regulation and neurite outgrowth remain unclear. We showed here that ES increased phosphorylation of the cAMP-response element binding protein (CREB). Blockade of Ca2+ suppressed CREB phosphorylation and neurite outgrowth. Down-regulation of phosphorylated (p)-CREB reduced BDNF transcription and neurite outgrowth triggered by ES. Furthermore, blockade of calmodulin-dependent protein kinase II (CaMKII) using the inhibitors KN93 or KN62 reduced p-CREB, and specific knockdown of the CaMKIIα or CaMKIIβ subunit was sufficient to suppress p-CREB. Recombinant BDNF or hyperforin reversed the effects of Ca2+ blockade and CaMKII knockdown. Taken together, these data establish a potential signaling pathway of Ca2+-CaMKII-CREB in neuronal activation. To our knowledge, this is the first report of the mechanisms of Ca2+-dependent BDNF transcription and neurite outgrowth triggered by ES. These findings might help further investigation of complex molecular signaling networks in ES-triggered nerve regeneration in vivo.

Effects of Mechanical Force on Cytoskeleton Structure and Calpain-Induced Apoptosis in Rat Dorsal Root Ganglion Neurons In Vitro

Background A sudden mechanical insult to the spinal cord is usually caused by changing pressure on the surface of the spinal cord. Most of these insults are mechanical force injuries, and their mechanism of injury to the spinal cord is largely unknown. Methods Using a compression-driven instrument to simulate mechanical force, we applied mechanical pressure of 0.5 MPa to rat dorsal root ganglion (DRG) neurons for 10 min to investigate cytoskeletal alterations and calpain-induced apoptosis after the mechanical force injury. Results The results indicated that mechanical forces affect the structure of the cytoskeleton and cell viability, induce early apoptosis, and affect the cell cycle of DRG neurons. In addition, the calpain inhibitor PD150606 reduced cytoskeletal degradation and the rate of apoptosis after mechanical force injury. Conclusion Thus, calpain may play an important role in DRG neurons in the regulation of apoptosis and cytoskeletal alterations induced by mechanical force. Moreover, cytoskeletal alterations may be substantially involved in the mechanotransduction process in DRG neurons after mechanical injury and may be induced by activated calpain. To our knowledge, this is the first report to demonstrate a relationship between cytoskeletal degradation and apoptosis in DRG neurons.

Mechanical compression insults induce nanoscale changes of membrane-skeleton arrangement which could cause apoptosis and necrosis in dorsal root ganglion neurons

In a primary spinal cord injury, the amount of mechanical compression insult that the neurons experience is one of the most critical factors in determining the extent of the injury. The ultrastructural changes that neurons undergo when subjected to mechanical compression are largely unknown. In the present study, using a compression-driven instrument that can simulate mechanical compression insult, we applied mechanical compression stimulation at 0.3, 0.5, and 0.7 MPa to dorsal root ganglion (DRG) neurons for 10 min. Combined with atomic force microscopy, we investigated nanoscale changes in the membrane-skeleton, cytoskeleton alterations, and apoptosis induced by mechanical compression injury. The results indicated that mechanical compression injury leads to rearrangement of the membrane-skeleton compared with the control group. In addition, mechanical compression stimulation induced apoptosis and necrosis and also changed the distribution of the cytoskeleton in DRG neurons. Thus, the membrane-skeleton may play an important role in the response to mechanical insults in DRG neurons. Moreover, sudden insults caused by high mechanical compression, which is most likely conducted by the membrane-skeleton, may induce necrosis, apoptosis, and cytoskeletal alterations. Graphical Abstract Mechanical compression can cause rearrangement of the membrane skeleton and cytoskeleton and different biological responses of DRG neurons.

Effects of Ginkgo Biloba Extract EGb-761 on Neuropathic Pain in Mice: Involvement of Opioid System

Neuropathic pain is considered as one of the most difficult types of pain to manage with conventional analgesics. EGb‐761 is extracted from leaves of Ginkgo biloba and has analgesia and anti‐inflammatory properties. This study aimed to examine the effect of EGb‐761 on chronic constriction injury (CCI)‐induced neuropathic pain behaviors, including thermal hyperalgesia and mechanical allodynia, and to explore the possible mechanisms underlying this action. To this end, CCI mice were intraperitoneally injected with EGb‐761 (10, 20, 40, and 80 mg/kg), and thermal hyperalgesia, mechanical allodynia, cytokines, and mu‐opioid receptor expression were measured. Results showed that EGb‐761 attenuated thermal hyperalgesia and mechanical allodynia dose‐dependently and the best delivery time window was from day 7 to day 14 after CCI. Additionally, EGb‐761 treatment significantly decreased pro‐inflammatory cytokines and enhanced mu opioid receptor (MOR) expression in the sciatic nerve. Moreover, the opioid antagonist naloxone prevented the effect of EGb‐761 on thermal hyperalgesia and mechanical allodynia but did not influence the effect of EGb‐761 on inflammatory cytokines. In conclusion, this study suggests that the potential of EGb‐761 as a new analgesic for neuropathic pain treatment, and opioid system may be involved in the EGb‐761‐induced attenuation of thermal hyperalgesia and mechanical allodynia. Copyright