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 construct chemically extracted acellular nerve allograft (CEANA) with Schwann cells (SCs) from different tissues and to compare the effect of repairing peripheral nerve defect. Methods Bone marrow mesenchymal stem cells (BMSCs) and adi pose-derived stem cells (ADSCs) were isolated and cultured from 3 4-week-old SD mice with weighing 80-120 g. BMSCs and ADSCs were induced to differentiated MSC (dMSC) and differentiated ADSC (dADSC) in vitro.dMSC and dADSC were identified by p75 protein and gl ial fibrillary acidic protein (GFAP). SCs were isolated and culturedfrom 10 3-day-old SD mice with weighing 6-8 g. CEANA were made from bilateral sciatic nerves of 20 adult Wistar mice with weighing 200-250 g. Forty adult SD mice were made the model of left sciatic nerve defect (15 mm) and divided into 5 groups (n=8 per group) according to CEANA with different sources of SCs: autografting (group A), acellular grafting with SCs (5 × 105) (group B), acellular grafting with dMSCs (5 × 105) (group C), acellular grafting with dADSCs (5 × 105) (group D), and acellular grafting alone (group E). Motor and sensory nerve recovery was assessed by Von Frey and tension of the triceps surae muscle testing 12 weeks after operation. Then wet weight recovery ratio of triceps surae muscles was measured and histomorphometric assessment of nerve grafts was evaluated. Results BMSCs and ADSCs did not express antigens CD34 and CD45, and expressed antigen CD90. BMSCs and ADSC were differentiated into similar morphous of SCs and confirmed by the detection of SCs-specific cellsurface markers. The mean 50% withdrawal threshold in groups A, B, C, D, and E was (13.8 ± 2.3), (15.4 ± 6.5), (16.9 ± 5.3), (16.3 ± 3.5), and (20.0 ± 5.3) g, showing significant difference between group A and group E (P lt; 0.01). The recovery of tension of the triceps surae muscle in groups A, B, C, D, and E was 87.0% ± 9.7%, 70.0% ± 6.6%, 69.0% ± 6.7%, 65.0% ± 9.8%, and 45.0%± 12.1%, showing significant differences between groups A, B, C, D, and group E (P lt; 0.05). No inflammatory reactionexisted around nerve graft. The histological observation indicated that the number of myel inated nerve fiber and the myel in sheath thickness in group E were significantly smaller than that in groups B, C, and D (P lt; 0.01). The fiber diameter of group B was significantly bigger than that of groups C and D (P lt; 0.05) Conclusion CEANA supplementing with dADSC has similar repair effect in peripheral nerve defect to supplementing with dMSC or SCs. dADSC, as an ideal seeding cell in nerve tissue engineering, can be benefit for treatment of peripheral nerve injuries.
Objective To make a histological evaluation of poly(dextrogyr-levogyr)lactide acide-triiodothy-ronine (PDLLA-T3) in sciatic nerve defect of rat. Methods Ninety SD rats were evenly divided into 3 groups (autograft group A, PDLLA-T3 group B and PDLLA group C). Group D was control group. The left sciatic nerves were cut off by operation and 1 cm-nerve-defect was set up. The specimens were collected 2 weeks,1 month and 2 months after the operation respectively, simultaneously the right sciatic nerves were collected as normal control group D. HE stainning, electron microscope, S100 immunohistochemistry, and Bielschowsky staining were done in all the specimens, the quantity and quality of the regenerated nerves were observed, and all the results were processed by image analyzer.Results Two weeks after the operation,histological observation indicated that the materials in groups B and C were not completely degraded. Transmission electron microscopic observationshowed that the myelin sheath was not thick and it was about 0.5 μm in thickness. There was no significant difference among the 3 groups. One month after theoperation, histological observation indicated that in group A the regenerated nerves passed through the scaffold and in the new nerves there were regenerated blood vessels. The materials in groups B and C were not completely degraded. S-100 immunohistochemical observation and Bielschowsky staining showed that in groupB PDLLA-T3 repaired the defect successfully and the regenerated nerve myelinsheath was 1.81±0.19 μm in thickness. The effect in group B was better thanthat of groups A and C (P＞0.05). Two months after the operation, the materials in groups B and C were completely degraded. The quantity of the regeneratednerves in group B confirmed by S-100 immunohistochemical observation and Bielschowslcy staining was more than that in group C(P＜0.05) and close to that in group A. The regenerated nerve myelin sheath in group B was 2.15±0.27 μm in the thickness and was thicker than that in group C (P＜0.05)， but thinner than that in groups A and D (P＜0.05). Conclusion PDLLA-T3 can repair the defect of rat sciatic nerve with satisfactory quantity andquality of regenerated nerves.
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 observe and evaluate the expression and significance of Nogo66 receptor (NgR) mRNA in adult ratsprime;optic nerve. Methods Optic and sciatic nerves of 8 adult rats were used to make the sections, which were divided into 3 groups: optic-nerve experimental group, sciatic-nerve control group, and optic-nerve negative control group. In situ hybridization was used to observe the expression of NgR mRNA in optic nerve and sciatic nerve. Results The expression of NgR mRNA in the 8 rats was positive in optic nerve and negative in sciatic nerve. The positive signals were arranged along the long axis of optic nerve. Conclusion The expression of NgR mRNA is positive in optic nerve while negative in sciatic nerve in adult rats, which suggests that the positive expression and distribution of NgR may be related to the poor regenerate ability of optic nerves. (Chin J Ocul Fundus Dis, 2005,21:246-248)
Objective Bone marrow mesenchymal stem cells (BMSCs), as replacement cells of Schwann cells, can increase the effect of peripheral nerve repair. However, it has not yet reached any agreement to add the appropriate number of seeded cells in nerve scaffold. To investigate the effect of different number of BMSCs on the growth of rat dorsal root gangl ia(DRG). Methods Three 4-week-old Sprague Dawley (SD) rats (weighing 80-100 g) were selected to isolate BMSCs, whichwere cultured in vitro. Three 1- to 2-day-old SD rats (weighing 4-6 g) were selected to prepare DRG. BMSCs at passage 3 were used to prepare BMSCs-fibrin glue complex. According to different number of BMSCs at passage 3 in fibrin glue, experiment was divided into group A (1 × 103), group B (1 × 104), group C (1 × 105), and group D (0, blank control), and BMSCs were cocultured with rat DRG. The axon length of DRG, Schwann cell migration distance, and axon area index were quantitatively evaluated by morphology, neurofilament 200, and Schwann cells S-100 immunofluorescence staining after cultured for 48 hours. Results Some long cell processes formed in BMSCs at 48 hours; migration of Schwann cells and axons growth from the DRG were observed, growing in every direction. BMSCs in fibrin glue had the biological activity and could effect DRG growth. The axon length of DRG and Schwann cell migration distance in groups A, B, and C were significantly greater than those in group D (P lt; 0.05). The axon length of DRG and Schwann cell migration distance in group C were significantly less than those in group B (P lt; 0.05), but there was no significant difference between group A and group C, and between group A and group B (P gt; 0.05). The axon area index in groups A and B was significantly greater than that in group D (P lt; 0.05), but there was no significant difference between group C and group D (P gt; 0.05); there was no significant difference in groups A, B, and C (P gt; 0.05). Conclusion In vitro study on DRG culture experiments is an ideal objective neural model of nerve regeneration. The effect of different number of BMSCs in fibrin glue on the growth of DRG has dose-effect relationship. It can provide a theoretical basis for the appropriate choice of the BMSCs number for tissue engineered nerve.
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.
Objective To investigate the influence of nerve growth factor (NGF) on neuroal regeneration of somatovisceral heterogenic reinnervation using a rat phrenic-to-vagus anastomosis model. Methods Forty male SD rats, aging 3 months and weighing 200 g, were selected and randomly divided into 3 groups. In group A (n=10, control group), phrenic and vagusnerves were exposed and no neurorraphy was performed. In group B (n=15) and group C (n=15), both nerves were transected and proximal stump of phrenic nevers were microsurgically anastomosed to the distal stump of vagus nerves. Postoperatively, group C was intraperitoneally injected with NGF (20 μg/kg·d), while groups A and B were given matching sal ine solution. Twelve weeks later, cardiac function was examined under electrical stimulation of the regenerated nerve. Light and electron microscopies were used to examine the heterogenic regenerated nerve, and the passing rate of axon and thickness of myel in sheath were calculated. Results Under electrical never stimulation in groups A, B, and C, the decreases of blood pressure were (20.12 ± 2.57), (10.63 ± 2.44), and (14.18 ± 2.93) mmHg (1 mmHg=0.133 kPa), respectively; and the decreases of heart rate were (66.77 ± 9.96), (33.44 ± 11.82), and (43.27 ± 11.02)/minutes, respectively. In group B, the decrease ampl itudes of blood pressure and heart rate were 52.83% and50.08% of group A, respectively. Blood pressure and heart rate in group C also decreased dramatically; the decrease ampl itudes of blood pressure and heart rate in group C were 70.48% and 64.80% of group A. There were significant differences in the decrease ampl itudes of blood pressure and heart rate (P lt; 0.05) between group B and group C. Morphological observation showed that heterogenic nerve fibers had the structure of matured myel in sheath and their axons could regenerate into the vagus nerve. In group B and group C, the passing rates of axon were 66.83% ± 4.46% and 81.63% ± 3.56%, respectively; and the thicknesses of myel in sheath were (0.25 ± 0.10) μm and (0.46 ± 0.08) μm, respectively; showing significant differences (P lt; 0.05) between group B and group C. Conclusion Heterogenic nerve is primarily a somatic motor nerve; NGF can promote the axons of heterogenic nerve to regenerate into the parasympathetic nerve.
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 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.