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 study the effect of allogenic different cells injected into denervated muscles on nerve regeneration. Methods Thirty-six adult female SD rats, weighed 120-150 g, were divided into four groups randomly (n=9, each group). Left sciatic nerves were cut down on germfree conditions and given primary suture of epineurium. Different cells were injected into the muscles of calf at once after operation every seven days and in all four times (group A: 1 ml Schwann cells at concentration of 1×106/ml; group B: 1 ml mixed cells of Schwann cells and myoblast cells at concentration of 1×106/ml; group C: 1 ml extract from the culture medium of kidney endothelial cells; and group D: 1 ml culture medium without FCS as control ). After 3 months, the specimen was observed on macrobody and histology, and the densities of neurilemma cell and myoceptor were counted. Results The means of proximate neurilemma cells were 0.187 7±0.054 2 in group A, 0.155 1±0.032 1 in group B, 0.072 4±0.023 7 in group C, and 0.187 7±0.054 2 in group D. The densities of myoceptor were 6.000±0.866 in group A，9.000±2.291 in group B,12.780±1.394 in group C, 3.110±0.782 in group D. Conclusion Schwann cells, mixed cells of Schwann cells with myoblast cells, and the extract from kidney endothelial cells canall accelerate the nerve regeneration. And the effect of extract from the kidney endothelial cell is superior to that of Schwann cell and mixed cell.
OBJECTIVE: To evaluate the nerve regeneration after implantation of chitin tubes containing nerve growth factor(NGF) in the rabbit facial nerve. METHODS: Bilateral 8 mm defect of superior buccal divisions of the facial nerves were made in 16 New Zealand rabbits. Chitin tubes containing NGF were implanted into the gaps, and autologous nerves were implanted into the right gaps as control. The nerve regeneration was evaluated with electrophysiological and ultrastructural examination after 8 and 16 weeks of operation. RESULTS: Chitin tubes containing NGF successfully induced the nerve regeneration, regularly arranged myelinated and unmyelinated axons could be observed across the 8 mm gaps, and the myelin sheath was thick with clear lamellar structure at 8 weeks after operation, The regenerated nerve fibers increased and were more mature at 16 weeks after operation. There were no significant difference in electrical impulse conduction velocity through the neural regeneration between the experimental and control sides (P gt; 0.05). CONCLUSION: Chitin tubes containing NGF can provide optimal conditions for regeneration of rabbit facial nerve.
OBJECTIVE Following the delayed repair of peripheral nerve injury, the cell number of anterior horn of the spinal cord and its ultrastructural changes, motorneuron and its electrophysiological changes were investigated. METHODS In 16 rabbits the common peroneal nerves of both sides being transected one year later were divided into four groups randomly: the degeneration group and regeneration of 1, 3 and 5 months groups. Another 4 rabbits were used for control. All transected common peroneal nerves underwent epineural suture except for the degeneration group the electrophysiological examination was carried out at 1, 3 and 5 months postoperatively. Retrograde labelling of the anterior horn cells was demonstrated and the cells were observed under light and electronmicroscope. RESULTS 1. The number of labelled anterior horn cell in the spinal cord was 45% of the normal population after denervation for one year (P lt; 0.01). The number of labelled cells increased steadily from 48% to 57% and 68% of normal values at 1, 3 and 5 months following delayed nerve repair (P lt; 0.01). 2. The ultrastructure of the anterior horn cells of the recover gradually after repair. 3. With the progress of regeneration the latency become shortened, the conduction velocity was increased, the amplitude of action potential was increased. CONCLUSION Following delayed repair of injury of peripheral nerve, the morphology of anterior horn cells of spinal cord and electrophysiological display all revealed evidence of regeneration, thus the late repair of injury of peripheral nerve was valid.
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 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 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 study the biological activities ofthe nerve regeneration conditioned fluid (NRCF), especially to further separateand identify the protein bands of the relative molecular mass of (232-440)×103. Methods The silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve in 6 New Zealand white rabbits (weight, 1.8-2.5 kg). The proteins in NRCF were separated by the native-polycrylamide gel electrophoresis (Native-PAGE), the protein bands of the relative molecular mass of (232-440)×103 were analyzed by the Shotgun technique, liquid chromatography, and mass spectrometry. Results The Native-PAGE result showed that there was 1 protein band of the relative molecular mass over 669×103, (232-440)×103 and (140-232)×103,respectively, and 6 bands of the relative molecular mass of (67-140)×103.Besides, 54 proteins were identified with at least 2 distinct peptides in 1 protein band of the relative molecular mass of (232-440)×103, including 4 unnamed protein products, mainly at the isoelectric points of 5.5-8.0 and of the relative molecular mass of (10-40)×103. Based on their functions in the protein database, allthe identified proteins in this study were classified into the following 5 groups: conjugated protein (43%), transport protein (30%), enzyme (6%), signal transducer (4%), and molecular function-unknown protein (17%). At the subcellular localization of the identified proteins, there was mainly a secreted protein (63%), and the remaining proteins were localized in the membrane and cytoplasm. Conclusion Native-PAGE and the Shotgun technique can effectively separate and identify proteins from NRCF, and can identify the components of the protein band of the relative molecular mass of (232-440)×103 and provide basicinformation on the unnamed protein products in NRCF.
ObjectiveTo investigate the regularity of myelin degeneration and regeneration and the difference of axonal density between tibial nerve and common peroneal nerve after sciatic nerve injury repair in rhesue monkey. MethodsNine adult rhesue monkeys (male or female, weighing 3.5-4.5 kg) were selected to establish the model of rat sciatic nerve transaction injury. The tibial nerve and common peroneal nerve of 5 mm in length were harvested at 5 mm from injury site as controls in 3 monkeys; the distal tibial nerve and common peroneal nerve were repaired with 9-0 suture immediately in the other 6 monkeys. And the gross observation and neural electrophysiological examination were performed at 3 and 8 weeks after repair respectively. Then, distal tibial nerve and common peroneal nerve at anastomotic site were harvested to observe the myelin sheath changes, and to calculate the number of axon counts and axonal density by staining with Luxol Fast Blue. ResultsAtrophy of the lower limb muscle and various degrees of plantar ulcer were observed. Gross observation showed nerve enlargement at anastomosis site, the peripheral connective tissue hyperplasia, and obvious adhesion. The compound muscle action potential (CMAP) of tibial nerve and common peroneal nerve could not be detected at 3 weeks; the CMAP amplitude of common peroneal nerve was less than that of the tibial nerve at 8 weeks. Different degrees of axonal degeneration was shown in the tibial nerve and common peroneal nerve, especially in the common peroneal nerve. The average axonal density of common peroneal nerve was lower than that of tibial nerve at 3 weeks (13.2% vs. 44.5%) and at 8 weeks (10.3% vs. 35.3%) after repair. ConclusionThe regeneration of tibial nerve is better and faster than that of common peroneal nerve, and gastrocnemius muscle CMAP recovers quicker, and amplitude is higher, which is the reason of better recovery of tibial nerve.
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.