Objective To explore the effect of controlled release of nerve growth factor (NGF) on peripheral nerve defect repaire by acellular nerve graft. Methods The microspheres of NGF were prepared with drug microsphere technologyand fixed with the fibrin glue to make the compl icated controlled release NGF. Twenty healthy male SD rats weighing 280-300 g were adopted to prepare acellular xenogenous nerve, 52 male Wistar rats weighing 250-300 g were adopted to prepare the 10 mm defect model of left sciatic nerve. and thereafter were randomly divided into 4 groups: autograft group(group A), acellular nerve allograft combined with the double controlled release NGF (group B), acellular nerve allograft (group C) and acellular nerve allograft combined with fibrin glue (group D). Without any operation, the right sciatic nerve was regarded as control group. General observation was conducted after operation. The nerve axon regeneration length was measured 2 weeks after operation. The effects of peripheral nerve regeneration were evaluated by neural electrophysiology, the recovery rate of triceps surae muscular tension and weight and histological assessment 16 weeks after operation. Results All the animals survived till the end of experiment. The length of nerve regeneration was measured at 2 weeks after transplantation. The regeneration nerve of group A was longer than that of other groups (P lt; 0.05), group B longer than groups C and D (P lt; 0.05), and there were no difference between group C and group D (P gt; 0.05). At 16 weeks after operation, the recovery rates of nerve conduction velocity of groups A and B (73.37% ± 7.82% and 70.39% ± 8.45%) were larger than that of groups C and D (53.51% ± 6.31% and 55.28% ± 5.37%) (P lt; 0.05). The recovery rates of the triceps surae muscular tension in group A (85.33% ± 5.59%) were larger than that in groups B, C and D (69.79% ± 5.31%, 64.46% ± 8.49％ and 63.35% ± 6.40%) (P lt; 0.05). There were no significant differences among groups B, C and D (P gt; 0.05). The recovery rates of the triceps surae weight in group A (62.54% ± 8.25%) werelarger than that in groups B, C and D (53.73% ± 4.56%, 46.37% ± 5.68% and 45.78% ± 7.14%, P lt; 0.05). There was significant difference between group B and groups C, D (P lt; 0.05) and no significant differences between group C and group D (P gt; 0.05). The histological observation indicated that axon number and myel in thickness in group B were larger than those in group C and group D (P lt; 0.05). The axonal diameter in group B was significantly less than that in group A (P lt; 0.05). Conclusion Acellular nerve graft combined with the controlled release NGF is a satisfactory alternative to repair the peripheral nerve defect.
Basing on the experimental results, 48 nerve defects (with the length of 3-4 cm in 21 cases, 4.1-5cm in 25 cases and 6cm in 2 cases) were repaired clinically by using vaseularized nerve sheath canal with living Schwann s cells, 87.5 percent of them obtained good results. The advantages were: (1) The neural sheath had rich blood supply with resultant less scar from its healing; (2) The living Schwann s cells would secrete somatomedin to promote the reproduction of neural tissues; and (3) The useless neurofib...
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.
Schwanns cells were obtained from the distal end of the sciatic nerve following Wallerian degeneration of SD rats. These cells were cultured with the anteriorhorn neuron of spinal cord of 14dayold SD rat fetus. The two kinds of cells were separated by a slice. Through the microscope, the dendrites and the morphology changes at the 24th, 48th, 72th, and 96 th hour after culture were observed. It was demonstrated that the Schwanns cells played the role of maintaining the survival of neuron and promoting the growth of dendrites. It was said that the Schwanns cells could secrete neurotrophic factor which made the body enlarged and caused the dendrites enlonged to several times of the body.
A comparative study of four methods of laryngeal muscle reinnervation in dogs is presented. Twenty-eight cases were divided into four groups to undergo main branch and branch of ansa cervicalis nerve anastomosis, and nerves implantation an neuromuscular pedicles transfer respectively for restoration of vocal cord adduction on left sides. The results showed that the four procedures seemed to induce effective reinnervation of adductor muscles. But the main branch of ansa cervicalis nerve suture was superior to the other methods among which little difference was noted in the functional recovery, electrophysiological activity and muscle strength. It demonstrated that main branch of ansa nerve suture was the best procedure for treatment of unilateral vocal cord paralysis among the four methods.
Objective Inducing human amniotic membrane mesenchymal stem cells (hAMSCs) to Schwann cells-like cells (SCs-like cells) in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps. Methods hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR (qPCR). The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by ELISA in the supernatant of the 3rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups (n=25). The 3rd generation of hAMSCs (1×106 cells) in the proliferation period of culturing for 6 days, the SCs-like cells (1×106 cells), and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody (NF-01) by immunofluorescence staining. Results The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining, Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100, p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group (P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group (P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation. Conclusion The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.
Objective To explore an effect of the artificial nerve graft wrapped in the pedicled greater omentum on the early revascularization and an effectof the increased blood supply to the artificial nerve graft on the nerve regeneration. Methods Seventy-five rabbits were randomized into 3 groups, in which there were 2 experimental groups where the rabbits were made to abridge respectively with the artificial nerve grafts wrapped in the pedicled greater omentum (Group A) and with the artificial nerve grafts only (Group B), and the control group where the rabbits were abridged with the autologous nerve (Group C).On the 3rd, 7th and 14th days after operation, the evans blue bound to albumin (EBA) was injected into the vessels in all the grafts to show their revascularization. Twelve weeks after operation the nerve regeneration was evaluated with theelectrophysiological and histological observations on the serial sections, and was evaluated also with the transmission electron microscope. Results The artificial nerve grafts wrapped in the pedicled greater omentum in Group A and the autologous nerve grafts in Group C showed a beginning of revascularization on the3rd day after operation, and the revascularization was increased on the 7th and14th days. Compared with Groups A and C, the artificial nerve grafts in Group Bshowed a delayed revascularization on the7th day after operation. At 12 weeks after operation, there were no significant differences in the motor never conduction velocity, density of the regenerated myelinated nerve fibers, myelin sheath thickness, and diameter between Group A and Group C(Pgt;0.05). However, both Group A and Group C were superior to Group B in the above variables, with significant differences(Plt;0.05). Conclusion Utilization of the pedicled greater omentum to wrapthe artificialnerve grafts can promote an early revascularization of the artificial nerve graft and an early nerve regeneration of the artificial nerve graft because of an enhanced blood supply to the nerve graft.
Objective To investigate the effect of extract of ginkgo biloba leaves (EGb50) on the prol iferation of SCs cultured in vitro. Methods The SCs were isolated from 3-day-old SD rats’ sciatic nerves by the method of enzyme gradationdigestion (n=20) and the purified 2nd passage of SCs were divided into 2 groups: the experimental group, in which SCs were cultured in FBS-DMEM medium with EGb50 (terminal concentration: 50 μg/mL); the control group, in which SCs were cultured in the FBS-DMEM medium without EGb50. The absorbance (A) value was detected by the 2, 3-bis- (2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazol ium-5-carboxanil ide (XTT) method 1, 3, 5, 7 and 9 days after culture, then the growth curves was drawn. Cell cycle was detected by flow cytometry (FCM). Disintegration per minute (DPM) of SCs was detected by the method of 3H-thymine nucleoside (3H-TdR) 2 and 3 days after culture and nerve growth factor (NGF) synthesis in SCs culture media was detected by ELISA method. Results Most SCs were spindle-shaped with a purity above 90%. XTT detection showed that A value of SCs in the control group was gradually increased 3 days after culture, reached the peak 5 days after culture and gradually decreased from then; the A value in the experimental group experienced the similar changes, but it was higher than that in the control group at each time point (P lt; 0.01). 3H-TdR showed that the DPM of the experimental group was 1 961.78 ± 231.13 and 4 601.51 ± 605.08 at 2 and 3 days after culture, while for the control group, the A value was 1 347.15 ± 121.57 and 3 740.42 ± 158.73 at the same time point, indicating a significant difference between two groups (P lt; 0.01). FCM observation indicated that the SCs prol iferation index of the experimental group and the control group was 18.6% ± 3.2% and 9.7% ± 2.9%, indicating a significant difference between two groups (P lt; 0.01). ELISA observation showed that the NGF concentration in the experimental and the control group was (0.065 6 ± 0.003 9) ng/mL and (0.038 6 ± 0.003 6) ng/mL, indicating a significant difference (P lt; 0.01). Conclusion EGb50 is capable of enhancing the prol iferation of SCs cultured in vitro, which may be one of the important mechanisms to promote peripheral nerve regeneration.
OBJECTIVE: To explore the possibility to bridge peripheral nerve defects by xenogeneic acellular nerve basal lamina scaffolds. METHODS: Thirty SD rats were randomly divided into 5 groups; in each group, the left sciatic nerves were bridged respectively by predegenerated or fresh xenogeneic acellular nerve basal lamina scaffolds, autogenous nerve grafting, fresh xenogeneic nerve grafting or without bridging. Two kinds of acellular nerve basal lamina scaffolds, extracted by 3% Triton X-100 and 4% deoxycholate sodium from either fresh rabbit tibial nerves or predegenerated ones for 2 weeks, were transplanted to bridge 15 mm rat sciatic nerve gaps. Six months after the grafting, the recovery of function was evaluated by gait analysis, pinch test, morphological and morphometric analysis. RESULTS: The sciatic nerve function indexes (SFI) were -30.7% +/- 6.8% in rats treated with xenogeneic acellular nerve, -36.2% +/- 9.7% with xenogeneic predegenerated acellular nerve, and -33.9% +/- 11.3% with autograft respectively (P gt; 0.05). The number of regenerative myelinated axons, diameter of myelinated fibers and thickness of myelin sheath in acellular xenograft were satisfactory when compared with that in autograft. Regenerated microfascicles distributed in the center of degenerated and acellular nerve group. The regenerated nerve fibers had normal morphological and structural characters under transmission electron microscope. The number and diameter of myelinated fibers in degenerated accellular nerve group was similar to that of autograft group (P gt; 0.05). Whereas the thickness of myelin sheath in degenerated accellular nerve group was significantly less than that of autograft group (P lt; 0.05). CONCLUSION: The above results indicate that xenogeneic acellular nerve basal lamina scaffolds extracted by chemical procedure can be successfully used to repair nerve defects without any immunosuppressants.
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.