Xiaosong Gu

Bone marrow mesenchymal stem cells promote cell proliferation and neurotrophic function of Schwann cells in vitro and in vivo.

The use of bone marrow-derived mesenchymal stem cells (MSCs) in nerve tissue engineering leads to an improved functional outcome of peripheral nerve repair. Schwann cells (SCs) are primary structural and functional cells in peripheral nervous system and play a crucial role in peripheral nerve regeneration. We hypothesize that MSCs promote peripheral nerve regeneration not only via their direct release of neurotrophic factors, but through indirect modulation of cellular behaviors of SCs. To test this hypothesis we investigated the influences of MSCs on proliferation of and neurotrophic factor expression by SCs using an in vitro co-culture model and an in vivo system of rat sciatic nerve regeneration. The data from cell viability assay and flow cytometry, bromodeoxyuridine/Hoechst 33342 double staining, immunocyto/histochemistry, RT-PCR and quantitative real-time RT-PCR, as well as Western blot analysis collectively confirmed the effects of MSCs on the biological characteristics of SCs, especially during the period of peripheral nerve regeneration. Our results help to elucidate the mechanisms by which MSCs function as a cell therapy agent in peripheral nerve repair.

Salidroside attenuates glutamate-induced apoptotic cell death in primary cultured hippocampal neurons of rats

Salidroside, a compound of natural origin, has displayed a broad spectrum of pharmacological properties. This study aimed to evaluate the inhibitory effects of salidroside on glutamate-induced cell death in a primary culture of rat hippocampal neurons as compared to brain-derived neurotrophic factor (BDNF), a usual positive control. MTT and LDH assays, together with Hoechst 33342 staining, terminal deoxynucleotidyl transferase dUTP-mediated nicked end labeling (TUNEL) assay and flow cytometric analysis using annexin-V and propidium (PI) label, indicated that salidroside pretreatment attenuated glutamate-induced apoptotic cell death in primary cultured hippocampal neurons, showing a dose-dependent pattern. Furthermore, caspase-3 activity assay and calcium measurements with Fluo 4-AM, respectively, revealed that salidroside pretreatment antagonized activation of caspase-3 and elevation of intracellular calcium level, both of which were induced by glutamate stimulation, thus adding to the understanding of how salidroside offered neuroprotection against glutamate excitotoxicity.

Chitosan/polyglycolic acid nerve grafts for axon regeneration from prolonged axotomized neurons to chronically denervated segments

Peripheral nerve regeneration for long-term delayed injuries is usually unsatisfied. Here we attempted to use a chitosan/polyglycolic acid (PGA) artificial nerve graft to bridge a long-term delayed 10-mm defect in SD rats based on the previous studies on the graft used for immediate repair of 30-mm-long dog sciatic nerve defects and for clinical treatment of a 35-mm-long median nerve defect at elbow of a human patient. In this study, for experimental groups, the rat sciatic nerve had been transected leaving a 10-mm defect, which was maintained for 3 or 6 months before implantation with the chitosan/PGA artificial nerve graft. The animals non-grafted or grafted with autograft served as negative or positive control group. In experiment groups, nerve regeneration with functional recovery was achieved as measured by electrophysiological and histological techniques, although differences in the quantity and the quality of the regenerated nerve were observed between the 3- and 6-month delayed subgroups. The results showed that: (1) a few denervated Schwann cells survived and sustained their ability to myelinate axons at least 6 months, and (2) the atrophic denervated muscle could be reinnervated by regenerated axons through new muscle-nerve connections. These observations provide the possibility of guiding regenerated axons from survived axotomized neurons to distal nerve stump by the chitosan/PGA artificial nerve graft.

Nerve conduits based on immobilization of nerve growth factor onto modified chitosan by using genipin as a crosslinking agent.

Incorporation of nerve growth factor (NGF) into a nerve conduit can improve peripheral nerve regeneration. Here, genipin, a natural and low toxic agent, was used to crosslink chitosan, a natural polysaccharide, and concurrently to immobilize NGF onto modified chitosan, followed by fabrication of chitosan (CS)-genipin (GP)-NGF nerve conduits. MTT test showed that the cell viability of Schwann cells cultured in the conduit extract was not significantly different from that in plain medium. The neurite outgrowth measurement and immunocytochemistry with anti-growth-associated protein-43 and anti-neurofilament indicated that NGF released from CS-GP-NGF nerve conduits retained the bioactivity of stimulating neuronal differentiation of PC12 cells. Fracture strength measurements and vitamin B12 release analysis confirmed that CS-GP-NGF nerve conduits possessed good mechanical properties and adequate permeability. We also investigated the in vitro release kinetics of NGF from CS-GP-NGF nerve conduits by ELISA. The continuous release profile of NGF, within a 60-day time span, consisted of an initial burst that was controlled by a concentration gradient-driven diffusion, followed by a zero-order release that was controlled by a degradation of chitosan matrix. Collectively, CS-GP-NGF nerve conduits had an integrated system for continuous release of NGF, thus holding promise for peripheral nerve repair applications.

Evaluation on in vitro biocompatibility of silk fibroin-based biomaterials with primarily cultured hippocampal neurons.

Silk fibroin-based biomaterials have recently found increasing applications in the tissue-engineering field including the generation of artificial nerve guides for peripheral nerve repair. The aim of this study was to investigate the suitability of silk fibroin as a candidate biomaterial for central nervous system (CNS) therapy. We found that substrates made up of silk fibroin fibers supported the survival and growth of the attached hippocampal neurons by using morphological observation. We also cultured the hippocampal neurons in silk fibroin extract for different times, and observed no significant difference occurring in their morphology, cell viability for these cultured hippocampal neurons as compared to those cultured in plain neuronal culture medium. Moreover, immunocytochemistry, RT-PCR, and Western blot analysis revealed that no significant difference was found in mRNA or protein levels of the growth-associated molecules, such as brain-derived neurotrophic factor, growth-associated protein-43, neurofilament, nerve growth factor, and nerve growth factor-receptor P75, between the hippocampal neurons cultured in the silk fibroin extract and in plain neuronal culture medium. Taken together, all the results demonstrate that silk fibroin has good biocompatibility with primarily cultured hippocampal neurons without any significant cytotoxic effects on their cell phenotype and functions, suggesting a potential possible use of silk fibroin for preparing the tissue-engineered nerve guides or drug delivery vehicles to treat CNS injuries or diseases.

The protective effects of Achyranthes bidentata polypeptides against NMDA-induced cell apoptosis in cultured hippocampal neurons through differential modulation of NR2A- and NR2B-containing NMDA receptors.

Achyranthes bidentata Blume is a commonly prescribed Chinese medicinal herb with a variety of pharmaceutical properties. From its aqueous extract we have separated important constituents, referred to as A. bidentata polypeptides (ABPP). In this study, the neuroprotective effect of ABPP against N-methyl-d-aspartate (NMDA)-induced cell apoptosis was investigated in cultured rat hippocampal neurons. The results of MTT assay, Hoechst/PI double staining and DNA ladder detection indicated that ABPP significantly attenuated, in a concentration-dependent manner, apoptotic cell damage induced by exposure of cultured hippocampal neurons to NMDA (100 μM) for 30 min. The intracellular calcium measurement with fluo-3/AM revealed that ABPP antagonized the excess intracellular calcium triggered by NMDA. Furthermore, in addition to inhibiting the action of NR2B-containing NMDA receptors, ABPP can enhance the function of NR2A-containing NMDA receptors. Our data might suggest that ABPP may also prove to be a potential neuroprotective therapy owing to its differential modulation of NR2A- and NR2B-containing NMDA receptors.

Effect of chitooligosaccharide on neuronal differentiation of PC-12 cells

Chitosan is now being widely used biomaterial in the tissue engineering field, and has great potential as a candidate material for preparing nerve guidance conduits due to its various favorable properties, especially that of good nerve cell affinity. Chitosan can be degraded in vivo into chitooligosaccharide. We have investigated the in vitro effects of chitooligosaccharide on neuronal differentiation of PC‐12 cells to see what effects chitooligosaccharide have on certain functions in the regenerating neurons. The morphologic observation and assessment using the specific reagent of tetrazolium salt WST‐8 indicated that neurite outgrowths from PC‐12 cells and the viability of PC‐12 cells were enhanced by treatment of chitooligosaccharide. The real‐time quantitative RT‐PCR and Western blot analysis revealed showed that chitooligosaccharide could upregulate the expression of neurofilament‐H mRNA or protein and N‐cadherin protein in PC‐12 cells. The maximum effect of 0.1 mg/ml chitooligosaccharide was obtained after 2 week culture. All the data suggest that chitooligosaccharide possesses good nerve cell affinity by supporting nerve cell adhesion and promoting neuronal differentiation and neurite outgrowth.

The promotion of peripheral nerve regeneration by chitooligosaccharides in the rat nerve crush injury model

Chitooligosaccharides (COSs), the biodegradation product of chitosan, have shown many biological functions. In this study, we examined the possible benefits of treatment with COSs (M.W. 800) on regeneration of rat crushed sciatic nerves. The rats with sciatic nerve crush injury were administered intraperitoneally daily with 3 or 6mg/kg body weight of COSs over a 3-week period. During and at the end of COSs treatment, a series of functional and histological examinations, including the measurement of withdrawal reflex latency (WRL) values, walking track analysis, electrophysiological assessments, morphometric analysis of gastrocnemius muscle, as well as immunohistochemistry and electromicroscopy to regenerated sciatic nerves, were performed to evaluate the therapeutic outcomes of COSs. The experimental data demonstrated that COSs promoted peripheral nerve regeneration with the desired functional recovery in the rat sciatic nerve crush injury model. This study raises a possibility of developing COSs as a potential neuroprotective agent for peripheral nerve repair applications.

Surgical repair of a 30 mm long human median nerve defect in the distal forearm by implantation of a chitosan–PGA nerve guidance conduit

This paper describes a clinical case study in which a chitosan/polyglycolic acid nerve guidance conduit (chitosan–PGA NGC) was utilized to repair a 30 mm long median nerve defect in the right distal forearm of a 55 year‐old male patient. Thirty‐six months after the nerve repair, the palm abduction of the thumb and the thumb–index digital opposition recovered, facilitating the patient to accomplish fine activities, such as handling chopsticks. Static two‐point discrimination measured 14, 9 and 9 mm in the thumb, index and middle fingers of the right hand. Reproducible compound muscle action potentials were recorded on the right abductor pollicis. The ninhydrin test, a classical method for assessing sympathetic nerve function, showed partial recovery of the perspiration function of the injured thumb, index and middle fingers. This repair case suggested a possible strategy for the clinical reconstruction of extended defects in human peripheral nerve trunks by the implantation of chitosan–PGA NGCs. Copyright

Chitooligosaccharides protect cultured hippocampal neurons against glutamate-induced neurotoxicity

Chitooligosaccharides (COSs), the biodegradation product of chitosan, have demonstrated a diverse array of biological activities. Here we report the protective effect of COSs (M.W. 800) against glutamate-induced neurotoxicity in cultured hippocampal neurons. The cell viability assessments, together with Hoechst 33342 staining and flow cytometry for cell apoptosis analysis, indicated that glutamate (125 microM)-induced cell apoptosis in cultured hippocampal neurons was attenuated in a concentration-dependent manner by COSs pretreatment. After measurement with Fluo 4-AM, COSs were found to depress glutamate-induced elevation in intracellular calcium concentration ([Ca(2+)](c)). The enzymatic assay indicated that COSs antagonized glutamate-evoked activation of caspase-3. These results collectively suggest that COSs prevent cultured hippocampal neurons from glutamate-induced cell damage by interfering with an increase in [Ca(2+)](c) and inhibiting caspase-3 activity.