ObjectiveTo establish an efficient method of isolating and culturing high activity and high purity of Schwann cells, and to identify the cells at the levels of transcription and translation. MethodsThe sciatic nerves harvested from a 4-week-old Sprague Dawley rat were digested in the collagenase I for 15 minutes after dissecting, and then the explants were planted in culture flask directly. The cells were cultured and passaged in vitro, the growth state and morphological changes of the cells were observed under inverted phase contrast microscope. MTT assay was used to test the proliferation of cells and the cells growth curve was drawn. RT-PCR and immunohistochemistry staining were used to detect S100 and glial fibrillary acidic protein (GFAP) at the levels of transcription and translation, respectively. The purity of cells was caculated under microscope. ResultsAfter the digestion of collagenase I, fibroblast-like cells appeared around explants within 24 hours, with slender cell body and weak refraction. After tissues were transferred to another culture flask, a large number of dipolar or tripolar cells were seen after 48 hours, with slender ecphyma, plump cell body, and b refraction, and the cells formed colonies within 72 hours. The cells were covered with the bottom of culture flask within 48-72 hours after passaging at a ratio of 1∶2, and spiral colonies appeared. Cells showed vigorous growth and full cytoplasm after many passages. MTT assay results showed that the cells at passage 3 entered the logarithmic growth phase on the 3rd day, reached the plateau phase on the 7th day with cell proliferation, and the growth curve was “S” shape. RT-PCR results showed that the cells expressed S100 gene and GFAP gene, and immunohistochemistry staining showed that most of the cells were positively stained, indicating that the majority of cells expressing S100 protein and GFAP protein. The purity of Schwann cells was 98.37% ± 0.30%. ConclusionHigh activity and high purity of Schwann cells can be acquired rapidly by single-enzyme digestion and explant-culture method.
OBJECTIVE To analysis the clinical characters of gluteal sciatic nerve injuries and investigate the treatment options. METHODS From October 1962 to June 1997, 190 patients with gluteal sciatic nerve injuries were adopted in this retrospective study. In these cases, the sciatic nerve injuries were caused by injection in 164 patients(86.32%), stab injury in 14 patients, pelvic fracture and hip dislocation in 11 patients, and contusion injury in 1 patient. Among them, 15 cases were treated by conservative method and the other 175 cases were operated. According to the observation during the operations, the injuries were occurred at the region of gluteal muscle in 146 cases, at the region of piriform muscle in 26 cases, and at the region of pelvic cavity in 3 cases. Then neurolysis was performed in 160 cases, epineurial neurorrhaphy in 12 cases and nerve grafting in 2 cases, and nerve exploration but no repair in 1 case. Late stage functional reconstruction of the foot and ankle was carried out in 23 cases. RESULTS One hundred and fifty-one patients were followed up 8.5 years in average. The occurrence of excellent and good nerve recovery was 56.95% and the occurrence of excellent and good functional reconstruction of late stage was 78.26%. CONCLUSION The gluteal sciatic nerve injury has since been challenging because of the tremendous difficulty in treatment and the poor outcome. The injury situation at the different region was closely related to the regional anatomy. According to this study, it is advised that the surgical treatment should be carried out actively. Neurolysis should be performed as soon as possible in the cases of injection injury. Epineurial neurorrhaphy should be performed in the cases of nerve rupture. In case of the gluteal sciatic nerve injury which caused by pelvic fracture or hip dislocation, the reduction and decompression is suggested in the early stage, and exploration and nerve repair is indicated in the late stage. The functional reconstruction of foot and ankle should be carried out in the late stage for the improvement of the limb function.
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 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 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.
OBJECTIVE: To investigate the mechanism, diagnosis, and treatment of common fibular nerve compression syndrome secondary to sciatic nerve injury. METHODS: Based on the clinical manifestation and Tinel’s sign at fibular tunnel, 5 cases of common fibular nerve secondary compression following sciatic nerve injury were identified and treated by decompression and release of fibular tunnel. All 5 cases were followed up for 13-37 months, 25 months in average, and were evaluated in dorsal flexion strength of ankle. RESULTS: The dorsal flexion strength of ankle in 4 cases increased from 0-I degrees to III-V degrees, and did not recover in 1 case. CONCLUSION: Fibular tunnel is commonly liable to fibular nerve compression after sciatic nerve injury. Once the diagnosis is established, either immediate decompression and release of the entrapped nerve should be done or simultaneous release of fibular tunnel is recommended when the sciatic nerve is repaired.
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.
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.
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.
