Objective To investigate the effect of carboxymethylated chitosan (CMCS) on the proliferation, cell cycle, and secretion of neurotrophic factors in cultured Schwann cells (SCs). Methods SCs were obtained from sciatic nerves of 20 Sprague Dawley rats (3-5 days old; male or female; weighing, 25-30 g) and cultured in vitro, SCs were identified and purified by immunofluorescence against S-100. The cell counting kit 8 (CCK-8) assay was used to determine the proliferation of SCs. The SCs were divided into 4 groups: 50 μg/mL CMCS (group B), 100 μg/mL CMCS (group C), 200 μg/mL CMCS (group D), and the same amount of PBS (group A) were added. The flow cytometry was used to analyze the cell cycle of SCs; the real-time quantitative PCR and Western blot analysis were used to detect the levels of never growth factor (NGF) and ciliary neurotrophic factor (CNTF) in cultured SCs induced by CMCS. Results The purity of cultured SCs was more than 90% by immunofluorescence against S-100; the CCK-8 results indicated that CMCS in concentrations of 10-1 000 μg/mL could promote the proliferation of SCs, especially in concentrations of 200 and 500 μg/mL (P lt; 0.01), but no significant difference was found between 200 and 500 μg/mL (P gt; 0.05). CMCS at a concentration of 200 μg/mL for 24 hours induced the highest proliferation, showing significant difference when compared with that at 0 hour (P lt; 0.01). The percentage of cells in phase S and the proliferation index were significantly higher in groups B, C, and D than in group A (P lt; 0.05), in groups C and D than in group B (P lt; 0.05); and there was no significant difference between group C and group D (P gt; 0.05). Real-time quantitative PCR and Western blot results showed that the levels of NGF and CNTF in groups B, C, and D were significantly higher than those in group A (P lt; 0.05), especially in group D. Conclusion CMCS can stimulate the proliferation, and induce the synthesis of neurotrophic factors in cultured SCs.
Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) embedded in fibrin glue around chemical extracted acellular nerve allograft (CEANA) on the peripheral nerve regeneration. Methods Twenty-oneadult male C57 mice (weighing 25-30 g) and 15 adult male Balb/c mice (weighing 25-30 g) were selected. The sciatic nerves were harvested from the Balb/c mice to prepare CEANA. The BMSCs were isolated from 3 C57 mice and were cultured; BMSCs embedded in fibrin glue were cultured for 3, 7, 14, and 21 days. Then the supernatant was obtained and co-cultured with PC12 cells for 2 days to observe the PC12 cell growth in vitro. The other 18 C57 mice were used to establ ish the left sciatic nerve defect models of 10 mm and divided into 3 groups: autogenous nerve graft with fibrin glue (group A, n=6), CEANA graft with BMSCs (5 × 106) embedded in fibrin glue (group B, n=6), and CEANA graft with fibrin glue (group C, n=6). The right sciatic nerves were exposed as the controls. At 2, 4, 6, and 8 weeks, the mouse static sciatic index (SSI) was measured. The histomorphometric assessment of triceps surae muscles and nerve grafts were evaluated by Masson staining, toluidine blue staining, and transmission electron microscope (TEM) observationat 8 weeks after operation. Results BMSCs were uniform distribution in fibrin glue, which were spherical in shape, and the cells began to grow apophysis at 3 days. PC12 cells differentiated into neuron-l ike cells after addition supernatant co-cultured after 2 days. Incisions healed well in each group. At 2, 4, 6, and 8 weeks, the SSI increased gradually in 3 groups. SSI in group A was higher than that in groups B and C at 4, 6, and 8 weeks after operation (P lt; 0.05). SSI in group B was sl ightly higher than that in group C, but had no significant difference (P gt; 0.05). At 8 weeks, the wet weight recovery rate of triceps surae muscles and fibers number of myel inated nerve were better in group B than in group C, but worse in group B than in group A, showing significant differences (P lt; 0.05). The triceps surae muscle fibers area and myel in sheath thickness had significant differences between group B and group C (P lt; 0.01), but there was no significant difference between group A and group B (P gt; 0.05). Conclusion BMSCs embedded in fibrin glue around CEANA can improve functional recovery of peripheral nerve injury.
Objective To review new progress of related research of peri pheral nerve defect treatment with tissue engineering in recent years. Methods Domestic and internationl l iterature concerning peri pheral nerve defect treatment with tissue engineering was reviewed and analyzed. Results Releasing neurotrophic factors with sustained release technology included molecular biology techniques, poly (lactic-co-glycol ic acid) microspheres, and polyphosphate microspheres. The mixture of neurotrophic factors and ductus was implanted to the neural tube wall which could be degraded then releasing factors slowly. Seed cells which were the major source of active ingredients played an important role in the repair and reconstruction of tissue engineering products. The neural tube of Schwann cells made long nerve repair and the quality of nerve regeneration was improved. Nerve scaffold materials included natural and synthetic biodegradable materials. Tube structure usually was adopted for nerve scaffold, which performance would affect the nerve repair effects directly. Conclusion With the further research of tissue engineering, the treatment of peripheral nerve defects with tissue engineering has made significant progress.
Objective To investgate the effects of neurotrophic factor 3 (NT-3) genes modified SC on facil itating nerve regeneration and protecting neuronal survival after the sciatic nerve transection in rats. Methods The double sciatic nerves were harvested from 3-day-old Wistar rats and the SCs were separated, cultured and purified with double enzyem digestion and adherent culture. The third generation purified SCs were used. The NT-3 cDNA gene was transfected into culturedSCs by using cationic l iposome. The NT-3 expression were identified by ELISA after 1, 2, 4 and 8 weeks. The plasmids expressing NT-3 genes were transfected into SCs with l ipofectamine. The purity of SCs were detecting before and after modified with NT-3. The nerve-grafting complexes were constructed by SCs (3 × 107/mL) modified NT-3, third generation SCs (3 × 107/mL), NT-3 gene, respectively. And the nerve-grafting complexes were combined with ECM gel and PLGA conduit. Forty-eight adult SD rats were made the models of the right sciatic nerve defect (10 mm). According to the nerve-grafting complexes which were repaired the sciatic nerve defects, the models were divided into 4 groups randomly (n=12): group A (ECM gel and PLGA conduits), group B (SC, ECM gel and PLGA conduits), group C (NT-3 gene, ECM gel and PLGA conduits) and group D (NT-3 modified SC, ECM gel and PLGA conduits). At 2, 4, 6, 8 and 12 weeks after operation, the nerve gross were observed. Electrophysiological examination, histological observation and transmission electron microscope observation were performed at 12 weeks after operation. Results The concentrations of NT-3 protein were 0.39 ± 0.25, 0.76 ± 0.22, 1.06 ± 0.38 and 1.61 ± 0.35 at 1, 2, 4 and 8 weeks after operation; showing statistically significant differences (P lt; 0.05). The purity of SCs was 94.7% ± 2.1% and 95.6% ± 2.5% before and after modified with NT-3, respectively; showing a statistically significant difference (P lt; 0.05). The feet of injury rats began inflammation and ulcer, which healed at 12 weeks in group D, followed by groups C and B, but which was serious in group A gradually. The observations of gross, sections under microscope and transmission electron microscope at 12 weeks showed the regeneration of defect nerve was best in group D, followed by groups C and B, and group A was worst. There were statistically significant differences (P lt; 0.05) in latent period, ampl itude, motor nerve conduction velocity, the number and thickness of axon, the diameter of nerve fiber, the percentage of the nerve tissue area between group A and groupsB, C, D, between groups B, C and group D at 12 weeks. At 12 weeks after operation, the transmission electron microscope showed observation the maturation of medullary sheath was best in group D, followed by groups C and B, and group A was worst. Conclusion The nerve-grafting complex of NT-3 genes modified SCs could repair injured nerve. The competence is superior to SCs and neurotrophic factors.
Objective To separate each protein band from the nerve regeneration conditioned fluid(NRCF）and to study whether there are somenew and unknown neurotrophic factors in the protein bands with a relative molecular mass of 220×103. Methods The silicone nerve regenerationchambers were formed in the sciatic nerve of the 25 New Zealand rabbits (weight,1.8-2.5 kg), and NRCF was taken from it at 1 week after operation. The Nativepolyacrylamide gel electrophoresis (Native-PAGE) was used for separating the proteins from NRCF and detecting the relative molecular mass. The Western blot and ELISA were used to observe whether the protein bands ［220×103 (Band a), (20-40)×103(Band c)］ of NRCF could combine with the antibody of the known antibody of neurotrophic factor (NTF):nerve growth factor(NGF), glial cell-derived neurotrophic factor(GDNF), brainderived neurotrophic factor(BDNF), neurotrophin 3(NT-3)， NT-4, ciliang neurotrophic factor(CNTF). Results Separated by Native-PAGE, NRCF mainly contained two protein bands:Band a had a relative molecular mass about 220×103, and Band c had a relative molecular mass about (20-40)×103. Band a could not combine with the antibodies of the NGF, BDNF, CNTF, and NT-3, but could combine with the antibody of NT-4.Band c could combine with the antibodies of NGF, BDNF, CNTF and NT-3, but could not combine with the antibodies of NT-4 and GDNF. Conclusion The protein bands with a relative molecular mass of 220×103 have ber neurotropic and neurotrophic effects than the protein bands with a relative molecular mass of (20-40)×103, which contains NGF,CNTF, etc. NT-4 just has a weak or no effect on the sympathetic neurone. This indicates that there is a new NTF in the protein bands with a relative molecular mass of 220×103, which only combines with the antibody of NT-4.
Objective To investigate the early change of brain-derived neurotrophic factor (BDNF) in denervated red and white muscles and the regeneration of nerves innervating the muscles and to discuss the effect of the target organs on regeneration of the injured nerves.Methods Forty Wistar rats were divided into 5 groups. The sciatic nerves in 4 groups were sheared to make the models of the denervated muscles and the other one as control group. The amount of BDNF in muscles was measured with immunohistochemistry 1 day, 3 days, 7 days and 14 days after injury. The models of the regeneration of the nerves were made in another 15 rats whose sciatic nerves were disconnected with forceps. The nerve conduction velocity and electromyogram were tested with neuroelectrophysiology7 days and 14 days after injury. Results The expression of BDNF in soleus increased significantly on the 1st day, the 3rd day and the 7th day (P＜0.01); theexpression ingastrocnemius was lower, but there was no significant difference(P＞0.05) on the 1st day, the 3rd day,the 7th day and the 14th day when compared with control group. After 14 days of injury in the nerves innervating GAS and SOL, the nerve conduction velocities and the amplitudes of wave M recovered to (36.60±7.40)% and (19.9±6.4)% of normal value, and (42.50±3.50)% and (13.7±4.0)% of normal value respectively; there were no significant differences between the two muscles(P＞0.05).Conclusion There is- difference in BDNF amount between the denervated red and white muscles, but the recovery of the two kinds of the motornerves is similar,and the neurotrophism of denervated muscles was determined by all kinds of neurotrophic factors.
OBJECTIVE: To clarify the character of nerve growth factor (NGF) in the denervated red and white muscles and the relationship between the amount of NGF and sensitive neurotrophism of denervated red and white muscles. METHODS: The model of the denervated gastrocnemius and soleus was made by clipping the sciatic nerve of Wister rats. The immunohistochemistry was taken to measure the amount of NGF in muscles, and the neurotrophism of extracts of muscles was tested with culture of dorsal root ganglions at the 1st, 3rd, 7th and 14th days after injury. RESULTS: The amount of NGF in denervated gastrocnemius and soleus decreased, especially in soleus. The neruotrophism of the extracts of the two kinds of denervated muscles did not decrease; on the contrary, it increased after a week after injury. CONCLUSION: The injury of peripheral nerves causes the amount of NGF in the target tissues to increase, but the change is different between the denervated muscles; the neurotrophism of the extracts of musclesis determined by all kinds of neurotrophic factors, and can not be explained by a single factor.
OBJECTIVE: To investigate the variation of neurotrophic factors expression in spinal cord and muscle after root avulsion of brachial plexus. METHODS: Forty-eight Wistar rats were involved in this study and according to the observing time in 1st day, 1st week, 4th week, 8th week, and 12th week after avulsion, and the control, were divided into 6 groups. By immunohistochemical and hybridization in situ assays, the expression of nerve growth factor (NGF) on muscle, basic fibroblast growth factor(bFGF) and its mRNA on the neurons of corresponding spinal cord was detected. Computer image analysis system was used to calculate the result. RESULTS: After the root avulsion of brachial plexus occurred, expression of NGF increased and reached to the peak at the 1st day. It subsided subsequently but was still higher than normal control until the 12th week. While expression of bFGF and its mRNA increased in the neurons of spinal cord and reached to the peak at the 1st week. Then it dropped down and at the 12th week it turned lower than normal control. CONCLUSION: After root avulsion of brachial plexus, neurotrophic factors expression increase on target muscle and neurons of corresponding spinal cord. It maybe the autoregulation and may protect neuron and improve nerve regeneration.
Gene therapy develops very rapidly during the resent years. Great prospects have been demonstrated from basic study and clinic test. However, the gene therapy in CNS is still in stage of laboratory. The research status and prospects of gene therapy in spinal cord injury (SCI) were introduced. The basic principle is to transplant certain cells genetically modified with NTFs to the site of the injuried spinal cord, then NTFs are expressed in vivo and stimulate axon regrowing. Virus vectors are usually used for gene transfer because of their high rate of transfection, and the receptor cells include fibroblast, myoblast, etc. Nowadays, gene therapy in SCI is studied in many laboratories and the problems include: 1. The ideal components of transfer gene. 2. The choice of carrier. 3. Immune reaction, and prolonged survival and persistent expression of the receptor cells in the spinal cord. If these problems could be solved, the gene therapy would become the key method in the therapy of SCI.
Schwanns cell (SC) was isolated from sciatic nerve of adult rat with Wallerine degeneration. After culture, SC-serum free culture media (SCSFCM) was obtained. By ultrafiltration with PM-10 Amicon Membrane, electrophoresis with DiscPAGE,and electrical wash-out with Biotrap apparatus, D-band protein was isolated from the SC-SFCM. The D-band protein in the concentration of 25ng/ml could affect the survival of the spinal anterior horn neuron in vitro, prominently and itsactivity was not changed after being frozen. The molecular weight of the protein ranged from 43 to 67 Kd. The D-band protein might be a neurotrophic substancedifferent from the known SCderived neurotrophic factors (NTF). Its concentration with biological activity was high enough to be detected. The advantages of MTT in assessment of NTF activity were also discussed.