Abstract In case of sciatic nerve injury, there is degeneration of neuron in the corresponding segment of spinal cord. To study whether NGF could protect the dorsal root ganglia in this situation, the following experiments were performed: 72 SD mice were divided into 2 groups. In each mouse, the sciatic nerve was sectioned at the middle of the right thigh, and then,the proximal end of the sciatic nerve was inserted into a one ended silastic tube. The NGF 0.15ml (contain 2.5S NGF 0.15mg) was injected into the tubes of the experimental group, while a equal amount of normal saline was injected into the tubes of the control group. After 1, 3, 5, 9, 20 and 30 days, 6 mice of each groupwere sacrificed respectively, and 5th to 6th lumbar segments of the spinal cords were resected for examination. By histochemical study, the activity of fluoride resistant acid phosphatase (FRAP) of each animal was detected. The results showed: (1) Excision of the sciatic nerve led to decrease of FRAP activity, it suggested that the injury of sciatic nerve could damage the dorsal root ganglia; (2) The use of exogenous NGF could protect the FRAP activity. It was concluded that NGF played an important role in protecting the dorsal root ganglia in peripheral nerve injury, in vivo.
In order to understand the change of free radicals in the course of injury and regeneration of nerve, the sciatic nerve of Wistar rat was crushed to, prepare the model of nerve injury and measured the content of Malondialdehyde (MDA) and superoxide dismutase (SOD) of the nerve. Thirty rats were used in this study. The sciatic nerve on one side was crushed, the contralateral sciatic nerve was served as control. According to the time of assessment (2,4,6,11,21 days after crushing), the rats were divided into 5 groups. The MDA concentration of the controlwas 19.65±0.27 and that of the crushing groups at different time were 21.25±0.36, 21.98±0.35, 22.77±0.38, 23.73±0.13, 23.92±0.44, respectively (nmol/100mg pro, x±s), while the SOD concentration of the control was 119.18±0.58 and that of the crushing groups at different time were 144.85±1.70, 136.14±1.71, 130.58±0.57, 126.41±0.98, 122.36±0.79, respectively (ug/mg pro, x±s), In the experimental groups, all the MDA concentrations were markedly higher than that of the control Plt;0.01, t-test) and tended to increase with the time passing by. The SOD concentrations in the experimental groups were also higher than that of the control Plt;0.01, t-test) and tended to decrease with the time passing on. The study suggested that after crushing or ligation of the nerve, the free radicals would increase.
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 Peri pheral nerve injury is a common cl inical disease, to study the effects of the physical therapy on the regeneration of the injured sciatic nerve, and provide a reference for cl inical treatment. Methods Sixty-four female adult Wistar rats (weighing 252-365 g) were chosen and randomly divided into 4 groups (n=16): group A, group B, groupC, and group D. The experimental model of sciatic nerve defect was establ ished by crushing the right sciatic nerve in groups B, C, and D; group A served as the control group without crushing. At 2 days after injury, no treatment was given in group B, electrical stimulation in group C, and combined physical therapies (decimeter and infrared ray) in group D. At 0, 7, 14, and 30 days after treatment, the sciatic nerve function index (SFI) and the motor nerve conduction velocity (MNCV) were measured, and morphological and transmission electron microscopy (TEM) examinations were done; at 30 days after treatment, the morphological evaluation analysis of axons was performed. Results At 0 and 7 days after treatment, the SFI values of groups B, C, and D were significantly higher than that of group A (P lt; 0.05); at 14 and 30 days after treatment, the SFI value of group D decreased significantly, no significant difference was observed between group D and group A (P gt; 0.05) at 30 days; whereas the SFI values of groups B and C decreased, showing significant difference when compared with the value of group A (P lt; 0.05). At 0, 7, and 14 days after treatment, the MNCV values of groups B, C, and D were significantly lower than that of group A (P lt; 0.05), and there were significantly differences between group B and groups C, D (P lt; 0.05); at 14 days, the MNCV value of group D was significantly higher than that of group C (P lt; 0.05); and at 30 days, the MNCV values of groups B and C were significantly lower than that of group A (P lt; 0.05), but there was no significant difference between group D and group A (P gt; 0.05). At 0 and 7 days, only collagen and l i pid were observed by TEM; at 14 and 30 days, many Schwann cells and perineurial cells in regeneration axon were observed in groups B, C, and D, especially in group D. Automated image analysis of axons showed that there was no significant difference in the number of myelinated nerve fibers, axon diameter, and myelin sheath thickness between group D and group A (P gt; 0.05), and the number of myelinated nerve fibers and axon diameter of group D were significantly higher than those of groups B and C (P lt; 0.05). Conclusion Physical therapy can improve the regeneration of the injured sciatic nerve of rats.
Objective To investigate the time l imit of repairing old sciatic nerve defect in rats and observe the repair effect of autogenous nerve transplantation on old sciatic nerve defect in rats. Methods Thirty-six SD rats of clean grade wererandomized into 6 groups (n=6 per group). The animal model of nerve defect was made by transecting left sciatic nerve at the mid-thigh level. For groups A1, B1 and C1, defects were repaired by the contralateral autogenous nerve transplantation 1, 3 or 6 months after nerve damage and for the control groups of A2, B2 and C2, defects were not repaired. After operation, the gait, toe skin and leg muscle were examined weekly. Three months after autograft, a combination of electrophysiology examination, fluoro gold (FG) retrograde tracing and histological assessment including l ight microscopy, TEM was util ized to investigate the nerve functional recovery. Results Lameness and foot skin ulcers were observed in each group after nerve damage. At 2 months after autograft, such denervation symptoms were only improved in groups A1 and B1. At 3 months after autograft, the motor conduction velocity was (21.84 ± 6.74), (20.02 ± 4.17) and (16.09 ± 8.21) m/s in groups A1, B1 and C1, respectively, showing no statistically significant difference between them (P gt; 0.05). The ampl itude of compound muscle action potential (CAMP) was (12.68 ± 4.38), (9.20 ± 3.43) and (1.22 ± 0.39) mV in groups A1, B1 and C1, respectively, indicating significant differences between groups A1, B1 and group C1 (P lt; 0.05). No CAMP was evident in groups A2, B2 and C2. FG retrograde tracing conducted 3 months after autograft showed that the positive cells were most common in group A1 with big soma, mild in group B1 and lest in group C1 with smallest soma. Gastrocnemius Masson staining showed that the fiber morphology of gastrocnemius in groups A1 and B1 was close to normal, while the rest 4 groups had an obvious atrophy of muscle fiber. The fiber cross-section area was (340.73 ± 118.46), (299.88 ± 119.75), (54.33 ± 53.43), (78.60 ± 51.38), (65.62 ± 25.36), and (40.93 ± 28.22) μm2 in groups A1, B1, C1, A2, B2 and C2, respectively, indicating a significant difference between groups A1, B1 and groups C1, A2, B2 (P lt; 0.05). Neurohistology observation showed that more regenerated nerve fibers were observed in group A1 and B1, but less in group C1. The myel in sheath was thick in groups A1 and B1, while it was thin in group C1. Only SCs and hyperplastic collagen fiber were found in groups A2, B2 and C2. Conclusion Autogenous nerve transplantation is capable of repairing 1- and 3- month sciatic nerve defect to some degree in rat, but repair effect is not obvious on 6-month sciatic nerve defect in rats.
Objective To explore a new method for the pre-degeneration of peripheral nerve in vitro for obtaining many effective Schwann cells so as to provide a large number of seed cells for the research and application of tissue engineered nerves. Methods The bone marrow derived cells (BMDCs) from transgenic green fluorescent protein C57BL/6 mouse and the sciatic nerve segments from the C57BL/6 mouse were co-cultured to prepare the pre-degeneration of sciatic nerve in vitro (experimental group, group A), and only sciatic nerve was cultured (control group, group B). At 7 days after culture, whether BMDCs can permeate into the sciatic nerve in vitro for pre-degeneration was observed by gross and immunohistofluorescence staining. And then Schwann cells were obtained from the sciatic nerves by enzymic digestion and cultured. The cell number was counted, and then the purity of primary Schwann cells was determined using immunohistofluorescence staining and flow cytometer analysis. Results At 7 days after pre-degeneration, gross observation showed that enlargement was observed at nerve stumps, and neuroma-like structure formed; the group A was more obvious than group B. Immunohistofluorescence staining showed many BMDCs permeated into the nerve segments, with positive F4/80 staining in group A. After culture, the yield of Schwann cells was (5.59 ± 0.19) × 104 /mg in group A and (3.20 ± 0.21) × 104/mg in group B, showing significant difference (t=2.14, P=0.03). At 48 hours after inoculation, the cells had blue bipolar or tripolar cell nuclei with small size and red soma by immunohistofluorescence staining; fibroblasts were flat polygonal with clear nucleus and nucleolus, showing negative p75NTR staining; and there were few of fibroblasts in group A. The purity of Schwann cells was 88.4% ± 5.8% in group A and 76.1% ± 3.7% in group B, showing significant difference (t=2.38, P=0.04). And the flow cytometer analysis showed that the purity was 89.6% in group A and 74.9% in group B. Conclusion BMDCs can promote the pre-degeneration of peripheral nerve in vitro, and it is a new method to effectively obtain Schwann cells for tissue engineered nerve.
Objective To compare their competence of olfactory epithel ial gl iacytes, olfactory globular nerve layer (OGNL) gl iacytes and SC in repair nerve defect of sciatic nerve, and select the best gl iacytes for repair of peri pheral nerve defect. Methods Olfactory epithel ial gl iacytes, OGNL gl iacytes and SC were extracted from 20 female Wistar rats aged 2-3 months and cultured in vitro for 2 weeks, then purified and condensed for transplantation. Eighty adult female Wistar rats were randomized into groups A, B, C and D (n=20). The left sciatic nerves were excised 25 mm axons and retained epineuriumlumen anastomosed to proximal ends. The culture mediums, SC, OGNL gl iacytes, and olfactory epithel ial gl iacytes weretransplanted into the epineurium lumen of groups A, B, C and D, respectively. Three months postoperatively, the injured sciatic nerve regeneration was evaluated by methods of macroscopic observation, photomicroscope, transmission electron microscope, retro-marked fluorescence transportation distance, the gl ial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) were assayed by immunofluorescence, and the myel in basic protein (MBP) and neurofilament (NF) protein were assayed by ELISA. Results The scores of ankle joint were (3.325 ± 0.963), (4.200 ± 1.005), (5.143 ± 0.635) and (5.950 ± 0.154) in groups A, B, C and D, respectively; showing statistically significant difference between groups (P lt; 0.05). The obse vations of gross, sections under microscope and transmission electron microscope showed the regeneration of defect nerve was best in group D, followed by group C, and group B was superior to group A. The transportation distance of retro-marked fluorescence was longest in group D, followed by group C, and group B was superior to group A. The concentrations of GFAP and NGF were largest in group D, followed by group C, and group B was superior to group A. The MBP concentrations were (9.817 ± 3.267), (12.347 ± 3.091), (14.937 ± 2.075) and (22.757 ± 0.871) ng/mL in groups A, B, C and D, respectively; showing statistically significant difference between other groups (P﹤0.05) except between group A and group B (P gt; 0.05). And the NF concentrations were (13.869 ± 5.677), (18.498 ± 3.889), (23.443 ± 2.260) and (27.610 ± 1.125) ng/mL in groups A, B, C and D, respectively; showing statistically significant difference between groups (P﹤0.05). Conclusion Olfactory epithel ial gl iacytes, OGNL gl iacytes and SC transplantation could repair injured nerve. The competence of olfactory epithel iums is superior to the OGNL gl iacytes andSC, and the OGNL gl iacytes is better than SC.
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 establ ish the methods to get high activity, high purity, and adequate Schwann cells (SCs), and to provide sufficient seed cells for the peripheral nerve repair. Methods Six 5-day-old, male or female, Sprague Dawley rats were selected and the sciatic nerve (control group) and dorsal root gangl ion (DRG) (ex perimental group) were harvested.Then the sciatic nerves and DRG were digested by co-enzyme and dispersed by medium containing serum to isolate SCs. Freshlyisolated SCs from rats were cultured, purified and subcultured. The 1st generation of SCs were chosen to draw the growth curve of SCs by the counting method and to detect the prol iferation of SCs by MTT assay at 8 days of culture, the purity of SCs by immunocytochemistry of anti-S-100 and the brain-derived neurotrophic factor (BDNF) concentration by ELISA. Results A total of 36-43 DRGs could be obtained in each rat. The number of obtained single SC in experimental group [(7.5 ± 0.6）× 106] was significantly higher than that in control group [(3.5 ± 0.4）× 106 ] (t=13.175, P=0.000). SCs reached logarithm prol iferation phase at 3 days. With time, the cell number and the prol iferation absorbance (A) value of 2 groups all showed upward trend. The number and A value of experimental group were significantly higher than those of control group (P lt; 0.05). The SCs purity of experimental group (92.08% ± 3.45%) was significantly higher than that of control group (77.50% ± 3.57%) (t=6.689, P=0.001).The concentrations of BDNF at 3 days and 5 days in experimental group were significantly higher than those of control group (P lt; 0.05). Conclusion The sufficient amount, high purity, and viabil ity of SCs from DRGs can meet the needs of studies on peripheral nerve repairment.
Objective To discuss the effect of sciatic never repair at different angles on the neural regeneration in rats. Methods Seventy-two male Sprague Dawley rats were randomly divided into groups A, B, C, and D with 18 rats in each group. The right sciatic nerve was transected at 30, 45, 60, and 90° in groups A, B, C, and D, respectively, and then was repaired. The morphologic assessment of nerve regeneration was performed by gross observation, the wet weight recovery rateof gastrocnemius, histological and ultrastructural observations at 1, 2, and 3 months after operation. Results Three months later, the wet weight recovery rate of gastrocnemius, motor nerve conduction velocity and action potential of sciatic nerve, axonal diameter, medullary sheath thickness, and medullated nerve fiber counting in groups A and B were significantly better than those in groups C and D (P lt; 0.01); but no significant difference was found between group A and group B (P gt; 0.05), and between group C and group D (P gt; 0.05). Conclusion End-to-end neurorrhaphy at 30-45° can effectively promote the sciatic nerve regeneration in rats.