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 study the outcomes of nerve defect repair with the tissue engineered nerve, which is composed of the complex of SCs, 30% ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeable poly (D, L-lacitic acid) (PDLLA) catheters. Methods SCs were cultured and purified from the sciatic nerves of 1-day-old neonatal SD rats. The 1st passage cells were compounded with bFGF-PLGA sustained release microspheres andECM gel, and then were injected into permeable PDLLA catheters with PLGA microfilaments inside. In this way, the tissueengineered nerve was constructed. Sixty SD rats were included. The model of 15-mm sciatic nerve defects was made, and then the rats were randomly divided into 5 groups, with 12 rats in each. In group A, autograft was adopted. In group B, the blank PDLLA catheters with PBS inside were used. In group C, PDLLA catheters, with PLGA microfilaments and 30% ECM gel inside, were used. In group D, PDLLA catheters, with PLGA microfilaments, SCs and 30% ECM gel inside, were used. In group E, the tissue engineered nerve was appl ied. After the operation, observation was made for general conditions of the rats. The sciatic function index (SFI) analysis was performed at 12, 16, 20 and 24 weeks after the operation, respectively. Eelectrophysiological detection and histological observation were performed at 12 and 24 weeks after the operation, respectively. Results All rats survived to the end of the experiment. At 12 and 16 weeks after the operation, group E was significantly different from group B in SFI (P lt; 0.05). At 20 and 24 weeks after the operation, group E was significantly different from groups B and C in SFI (P lt; 0.05). At 12 weeks after the operation, electrophysiological detection showed nerve conduct velocity (NCV) of group E was bigger than that of groups B and C (P lt; 0.05), and compound ampl itude (AMP) as well as action potential area (AREA） of group E were bigger than those of groups B, C and D (P lt; 0.05). At 24 weeks after the operation, NCV, AMP and AREA of group E were bigger than those of groups B and C (Plt; 0.05). At 12 weeks after the operation, histological observation showed the area of regenerated nerves and the number of myel inated fibers in group E were significantly differents from those in groups A, B and C (Plt; 0.05). The density and diameter of myel inated fibers in group E were smaller than those in group A (Plt; 0.05), but bigger than those in groups B, C and D (P lt; 0.05). At 24 weeks after the operation, the area of regenerative nerves in group E is bigger than those in group B (P lt; 0.05); the number of myel inated fibers in group E was significantly different from those in groups A, B, C (P lt; 0.05); and the density and diameter of myel inated fibers in group E were bigger than those in groups B and C (Plt; 0.05). Conclusion The tissue engineered nerve with the complex of SCs, ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeables PDLLA catheters promote nerve regeneration and has similar effect to autograft in repair of nerve defects.
Objective To investigate the promotion effect of neurotropic reinnervation with chemically extracted acellular nerve allograft. Methods The sciatic nerves of 5 healthy adult SD rats, regardless of gender and weighing 270-300 g, were collected to prepare chemically extracted acellular nerve allograft. Eighteen healthy adult Wistar rats, regardless of genderand weighing 300-320 g, were made the model of left sciatic nerve defect (10 mm) and randomly divided into 2 groups: autograft (control group, n=9) and allograft group (experimental group, n=9). The defects were bridged by acellular nerve allograft in experimental group and by autograft by turning over the proximal and distal ends of the nerve in control group. At 3 months after transplantation, dorsal root ganglion (DRG) resection operation was performed in 6 rats of 2 groups. At 3 weeks after operation, the sural nerves were harvested to calculate the misdirection rate of nerve fibers with pathological staining and compute-assisted image analysis. Cholinesterase staining and carbonic anhydrase staining were performed in the sural nerve of 3 rats that did not undergo DRG resection at 3 months. Results The results of chol inesterase staining and carbonic anhydrase staining showed that experimental group had less brown granules and more black granules than control group. After DRG resection, count of myelinated nerve fiber were 4 257 ± 285 in the experimental group and 4 494 ± 310 in the control group significant (P lt; 0.05). The misdirection rate was 2.27% ± 0.28% and 7.65% ± 0.68% in the experimental group and in the control group respectively, showing significant difference (P lt; 0.05). Conclusion Chemically extracted acellular nerve allograft can not only act as a scaffold in the period of nerve defects repair, but markedly enhance the effects of neurotropic reinnervation.
Objective To investigate the feasibility of selenium-methylselenocysteine (SMC) to promote peripheral nerve regeneration and its mechanism of action. Methods Rat Schwann cells RSC96 cells were randomly divided into 5 groups, which were group A (without any treatment, control group), group B (adding 100 μmol/L H2O2), group C (adding 100 μmol/L H2O2+100 μmol/L SMC), group D (adding 100 μmol/L H2O2+200 μmol/L SMC), group E (adding 100 μmol/L H2O2+400 μmol/L SMC); the effect of SMC on cell proliferation was detected by MTT method, and the level of oxidative stress was detected by immunofluorescence for free radicals [reactive oxygen species (ROS)] after determining the appropriate dose group. Thirty-six 4-week-old male Sprague Dawley rats were randomly divided into 3 groups, namely, the sham operation group (Sham group), the sciatic nerve injury group (PNI group), and the SMC treatment group (SMC group), with 12 rats in each group; the rats in the PNI group were fed with food and water normally after modelling operation, and the rats in the SMC group were added 0.75 mg/kg SMC to the drinking water every day. At 4 weeks after operation, the sciatic nerves of rats in each group were sampled for neuroelectrophysiological detection of highest potential of compound muscle action potential (CMAP). The levels of inflammatory factors [interleukin 17 (IL-17), IL-6, IL-10 and oxidative stress factors catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA)] were detected by ELISA assay. The luxol fast blue (LFB) staining was used to observe the myelin density, fluorescence intensity of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) was observed by immunofluorescence staining, and myelin morphology was observed by transmission electron microscopy with measurement of axon diameter. Western blot was used to detect the protein expressions of p38 mitogen-activated protein kinases (p38MAPK), phosphorylated p38MAPK (p-p38MAPK), heme oxygenase 1 (HO-1), and nuclear factor erythroid 2-related factor 2 (Nrf2). ResultsMTT assay showed that the addition of SMC significantly promoted the proliferation of RSC96 cells, and the low concentration could achieve an effective effect, so the treatment method of group C was selected for the subsequent experiments; ROS immunofluorescence test showed that group B showed a significant increase in the intensity of ROS fluorescence compared with that of group A, and group C showed a significant decrease in the intensity of ROS fluorescence compared with that of group B (P<0.05). Neuroelectrophysiological tests showed that the highest potential of CMAP in SMC group was significantly higher than that in PNI and Sham groups (P<0.05). ELISA assay showed that the levels of IL-6, IL-17, and MDA in PNI group were significantly higher than those in Sham group, and the levels of IL-10, SOD, and CAT were significantly lower; the levels of IL-6, IL-17, and MDA in SMC group were significantly lower than those in PNI group, and the levels of IL-10, SOD, and CAT were significantly higher (P<0.05). LFB staining and transmission electron microscopy showed that the myelin density and the diameter of axons in the SMC group were significantly higher than those of the PNI group and the Sham group (P<0.05). Immunofluorescence staining showed that the fluorescence intensity of GFAP and MBP in the SMC group were significantly stronger than those in the PNI group and Sham group (P<0.05). Western blot showed that the relative expressions of Nrf2 and HO-1 proteins in the SMC group were significantly higher than those in the PNI group and Sham group, and the ratio of p-p38MAPK/p38MAPK proteins was significantly higher in the PNI group than that in the SMC group and Sham group (P<0.05). Conclusion SMC may inhibit oxidative stress and inflammation after nerve injury by up-regulating the Nrf2/HO-1 pathway, and then inhibit the phosphorylation of p38MAPK pathway to promote the proliferation of Schwann cells, which ultimately promotes the formation of myelin sheaths and accelerates the regeneration of peripheral nerves.
OBJECTIVE To observe the degeneration and regeneration of the Meissner’s corpuscles after implanted sensory nerve into the denervated monkey’s fingers under electron microscope. METHODS The two finger nerves of the monkey’s fingers were denervated. Afterwards, one finger nerve was cut off, and the other was reimplanted into the denervated finger. After 1, 3, 5, 8 and 12 months, the finger skin was cut off and observed under electron microscope. RESULTS The degenerative changes of nerve ending in Meissner’s corpuscles were observed after 1 month of denervation, and the basic structure of the corpuscles had no obvious changes. After 3 months, the axons of corpuscles were disappeared, and the volume of corpuscles was shrunk. The basic structure of nerves was disappeared, and the lemmocyte and neurolemma plate were changed after 5 months. The collagen fibrils in the corpuscles were gradually increased in 8 months, the endoneurial structure and interneurial matrix were completely disappeared and replaced by collagen fibrils in 12 months. After 3 months of nerve implantation, unmyelinated nerve fibers were appeared and grew into the corpuscles. A part of corpuscles innervated in 5 months. Most of corpuscles innervated and myelinated nerve fibers were observed in 8 months. And in 12 months, corpuscles innervated to normal level. CONCLUSION The implantative sensory nerve by means of reinnervating the original corpuscles and regenerating new corpuscles could innervate the degenerative Meissner’s corpuscles.
Objective To review the advances of functional electrical stimulation(FES) in treatment of peripheral nerve injuries. Methods By index of recent literature, the measures of stimulation, the mechanisms of FES and unsolved problems were evaluated and analyzed. Results Great advances have been made in the treatment of peripheral nerve injuries. It can not only enhance the regeneration of injured peripheral nerve, but also prevent muscular atrophy. Conclusion FES is an effective treatment for peripheral nerve injuries.
Objective To investigate the effect of tetramethylpyrazine (TMP) with a certain concentration added to vitrification solution on peripheral nerve allografts regeneration. Methods Forty-eight healthy clean SD male rats were selected as donors, and 96 healthy clean Wistar male rats as recipients, all rats being 3 months old and weighing 200-250 g. The sciatic nerves segments of 15 mm were removed from the donors, then randomly divided into 4 groups according to vitrificationsolution containing TMP. No TMP was used in group A as the control group; 100 mg/L, 200 mg/L and 400 mg/L TMP were used in group B, group C and group D, respectively. Then them were cryo-preserved at — 196 ℃ for 3 weeks. Nerve defect of 10 mm in length was made in the sciatic nerves of recipients. After rewarming, the allografts were transplanted to the corresponding rats. The gross appearance, the morphological and electrophysiological changes, the image analysis of axons and motor end-plate were detected at 4, 8, 12 and 16 weeks. Results All rates survived to the end of the experiment. The adhesion and edema of allografts in group A and group B were obvious 4 weeks after operation; then adhesion and edema was obvious in group A and were improved in the other groups 8 weeks after operation. Adhesion was observed in groups A and B; no adhesion was observed in groups C and D at 12 weeks. The number of regeneration nerve, the latent, the ampl itude, the nerve conduction velocity, the medullary sheath/μm2, the medullary sheath density/μm2 and the image analysis of axons and motor end-plate in groups A and B were significantly lower than those in groups C and D (P lt; 0.01); and there were no significant differences between groups C and D (P gt; 0.05). The observation of transmission electron microscope showed that medullated nerve fibers and myel in sheath of groups C and D were thicker than groups A and B, layers of groups C and D were clear. Conclusion The vitrification solution with 200 mg/L tetramethylpyrazine has protective effect on regeneration of peripheral nerve allografts．
OBJECTIVE To understand the biological activities of the nerve regeneration conditioned fluid (NRCF). METHODS Nerve regeneration chamber was made by using silicone tube bridging distal and proximal ends of severed SD rat’s sciatic nerve. The biological activities of the proteins in NRCF, which were separated by natural polyacrylamide gel electrophoresis (PAGE), were analysed by being cocultured with excised neonatal dorsal root ganglia (DRG). RESULTS Eight separated protein bands of NRCF were observed between 67-669 ku in molecular weight, and the protein bands between 232-440 ku showed b neurotrophic and chemotactic function. CONCLUSION NRCF has the promoting effects on nerve regeneration.
Objective To develop a technique that can directly demonstrate collateral sprouting of intact nerve fibers at endtoside neurorrhaphy site. Methods Five Wistar adult rats were used in this study. The common peroneal nerves at one side were sectioned at the level of knee joint, and their distal ends were sutured to the tibial nerves after removal of a 1 mm-diameter window in the epineurium. Three months after the operation, the nerve segments at neurorrhaphy site and the normal tibial nerves at the contralateral site were harvested. The specimens were fixed in 10% formaldehyde and postfixed in 1% osmium tetroxide, thenmacerated in glycerol. Single fiber was teased out in pure glycerol under an operative microscope, then transferred to a slide and observed under light microscope. The nerve segments at neurorrhaphy site and distal peroneal nerves were alsoharvested for histological evaluation. Results At the neurorrhaphy site, small nerve fibers sprouted from a donor nerve fiber near node’s of Ranvier. While such phenomena were not found in normal tibial nerve. From the longitudinal sectionof neurorrhaphy site, bundles of nerve fibers ranged from tibial nerve to peroneal nerve were observed. Lots of regenerative nerve fibers emerged in distal peroneal nerve. Conclusion The phenomena of collateral sprouting at end-to-side neurorrhaphy site can be demonstrated directly by nerve fiber micro-tease technique.
Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.