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.
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 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 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.
Objective To investigate the effects of lycium barbarum polysaccharide (LBP) on the formation of traumatic neuroma and pain after transection of sciatic nerve in rats. Methods Forty Sprague-Dawley (SD) rats, weighing 200-220 g, half male and half female, were allocated into 2 groups randomly: LBP group and control group (n=20 per group). The right sciatic nerves were transected and 2 cm sciatic nerve were removed in all rats of the 2 groups. LBP were intraperitoneally injected in a volum of 10 mg/(kg·d) in the LBP group, while the same volum normal sal ine (NS) in the control group for 28 days. The deficiency of toenail and toe were observed to estimate the autophagy of the operated l imb. Light microscope and transmission electron microscope were used to observe the formation of traumatic neuroma aftertransection of sciatic nerve. Results Autophagy was observed in 5 rats (25%) of LBP group and in 12 rats (60%) of controlgroup at 4 weeks, showing significant difference (P lt; 0.05). Neuroma formed in 8 rats (40%) of LBP group and in 16 rats(80%) of control group, showing significant difference (P lt; 0.05). The observation of l ight microscope showed that there were unorganized growth cells in the neuroma, infiltrated muscle cells, the regeneration of axons and ensheathing cells to form small patch and funicular structure in the control group, while in the LBP group there were less prol iferation of nerve fibers with a regular arrangement. Transmission electron microscope showed that there were lots of axons in nerve tumour, more fusoid fibroblasts, more collagen fiber, and hyperplasia and degenerated myel in sheath in the control group, while in the LBP group there were less myel in sheath in the proximal end of injuring nerves, less Schwann cells and fibroblasts, and sparsed collagen fibers. Conclusion LBP can inhibit autophagy and the formation of traumatic neuroma after transection of sciatic nerve in rats.
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 To investigate the effect of exogenous erythropoietin (EPO) on the denervated muscle atrophy. Methods Twenty-four SD male rats, weighting 200-220 g were made the models of denervated gastrocnemius muscle after sciatic nerves were transected under the piriform muscle at the right lower leg, and were randomly divided into two groups (n=12). rhEPO (2 500 U/kg) was injected daily into the denervated gastrocnemius muscle in EPO group, and normal sal ine was injected into the denervated gastrocnemius muscle in control group. To observe the general state of health of the experimental animal, the muscle wet weight, the muscle cell diameter, the cross section area, the protein amount, thepercentage of the apoptotic muscle cells, and the Na+-K+-ATPase and Ca2+-ATPase activities were measured 2 and 4 weeks after operation. Results All experimental animals were survived during experiment without cut infection, and all animals could walk with pull ing the right knee. At 4 weeks after operation, 7 cases showed ulcer in the right heel, inculding 5 in the control group and 2 in the EPO group. At 2 and 4 weeks after operation, the muscle wet weight in EPO group was (885.59 ± 112.35) and (697.62 ± 94.74) g, respectively; in control group, it was (760.63 ± 109.05) and (458.71 ± 58.76) g, respectively; indicating significant differences between two groups (P lt; 0.01). The protein amount in EPO group was (77.37 ± 5.24) and (66.37 ± 4.87) mg/mL, respectivly；in control group, it was (65.39 ± 4.97) and (54.62 ± 6.32) mg/mL；indicating significant differences between two groups (P lt; 0.01). At 2 and 4 weeks after operation, the myofibrillar shapes were nearly normal in EPO group while there were muscle fiber atrophy, some collapse and obviously hyperblastosis between muscle bundle. There were significant differences in the muscle cell diameter and the cross section between two groups (P lt; 0.01). However, the percentage of the apoptotic muscle cells was 11.80% ± 1.74% and 28.47% ± 1.81% in control group, respectively, which was significantly smaller than that in EPO group (21.48% ± 2.21% and 55.89% ± 2.88%, P lt; 0.01). At 2 and 4 weeks after operation, Na+-K+-ATPaseand Ca2+-ATPase activities in EPO group were higher than those in control group (P lt; 0.01). Conclusion EPO can delay the denervated muscle atrophy.
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 investigate whether the peri pheral administration of amitri ptyl ine and bupivacaine produces anti-hyperalgesic effect and to screen the neurotoxicological effect on sciatic nerve blockade in a rat model of neuropathic pain. Methods Twenty-four adult male SD rats [weighing (200 ± 20) g] were made the models of chronic constriction injury (CCI) and randomly divided into 3 groups (n=8) 5 days after operation: group A (amitriptyl ine), group B (bupivacaine) and group C (normal sal ine). 0.5 mL 0.5% amitriptyl ine, 0.5% bupivacaine or normal sal ine were given in group A, group B, and group C, respectively through implanted cannulas after 5, 7 and 9 days of CCI once a day for successive 3 days. The motor function was measured before administration and 1, 2, 4, 8, 12 and 24 hours after every administration. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured before administration and 1, 3, 5 and 7 days after the third administration. The operated sciatic nerve samples were obtained for neuropathological examination under l ight microscope. Results Twenty-four CCI rats were all survival without infection, palsy and catheter fall ing off. Compared with group C, the rats of group A and group B both produced significant ambulation deficits after every administration (P lt; 0.05). The ambulation deficits lasted 2 hours (group B) and 8 hours (group A) respectively. But the ambulation deficits of CCI rats were all reversible. The MWT and TWL of group A 1 and 3 days after the third administration increased when compared with those before administration and 5 and 7 days after the third administration, and when compared with group B and group C (P lt; 0.05). There was no significant difference (P gt; 0.05) in l ight microscopic neuropathological examination among three groups. Epineurial tissue and endoneurium tissue integrity, tidy arrangement of fibers, less inflammatory cell and no marked degeneration of myel inated fibers were observed. Conclusion Repeated sciatic nerve blockade with 0.5% amitriptyl ine has peripheral anti-hyperalgesic effects on neuropathic pain of rats. No morphological evidence of neurotoxicity in the sciatic nerve of rats is observed in 0.5% amitriptyl ine.