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 probe the possibility of direct transfer of exogenous gene into peripheral nerve and its following expression in vivo. METHODS The PCMV beta plasmid containing cytomegalovirus (CMV) promoter and Escherichia Coli (E. Coli), beta-Galactosidease (beta-Gal) structural gene (lacZ gene) was constructed and injected into the rabbit sciatic nerve. The control group was injected PBS solution. The injected nerves were sampled and tested by beta-Gal enzyme activity assay of the 5-bromo-4-chloro-3-indolyl-beta-D-galactoside and beta-Gal histochemical stain. RESULTS In the control group, no beta-Gal enzyme activity was detected in the different stages after operation, and beta-Gal histochemical stains showed positive. In the experimental group, enzyme activity could be detected from 2 days to 30 days after operation, and the histochemical stains showed negative. CONCLUSION The exogenous gene can be transferred into peripheral nerve and expressed with bioactivity, thus the gene therapy to accelerate the recovery of nerve is practical.
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.
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.
Objective To realize the visualization of three-dimensional microstructure of rabbit sciatic nerve bundles by micro-CT and three-dimensional visualization software Mimics17.0. Methods The sciatic nerve tissues from 6 New Zealand rabbits were divided into 2 groups (n=3), and the sciatic nerve tissues were stained by 1% (group A) and 5% (group B) Lugol solution respectively. After staining for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 hours, the imaging changes of specimens were observed by light microscope and micro-CT. The clear micro-CT images were exported to the Mimics software to complete the visualization of three-dimensional microstructure of rabbit sciatic nerve according to three-dimensional reconstruction tool. Results The clear three-dimensional microstructure images could be observed in group A at 2.5 hours after staining and in group B at 1.5 hours after staining by light microscope and micro-CT. The sciatic nerve of New Zealand rabbits were divides into 3 bundles and each of them was relatively fixed. There was no obvious crossing or mergers between each bundle. The cross-sectional area of each bundle was (0.425±0.013), (0.038±0.007), and (0.242±0.026) mm2 respectively. The digital model could clearly reflect the microstructure of the sciatic nerve at all cross sections. Conclusion The internal structure of New Zealand rabbits sciatic nerve can be clearly reflected by micro-CT scanning. It provides a reliable method for establishing a nerve microstructure database with large amount specimens.
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.
ObjectiveTo study the inducting differentiation effect of the sciatic nerve extracts on rabbit adipose-derived stem cells (ADSCs) in vitro. MethodsThe ADSCs were isolated from 2 healthy 4-month-old New Zealand rabbits (weighing, 2.0-2.5 kg) and cultured to passage 3, which were pretreated with 10 ng/mL basic fibroblast growth factor (bFGF) for 24 hours before induction. Then the induction media containing the extracts of normal sciatic nerve (group B) and injured sciatic nerve at 3, 7, and 14 days (group C, group D, and group E) were used, and D-Hank was used in group A as blank control group. The morphological changes of the cells were observed. At 7 days of induction, the gene expressions of neuron-specific enolase (NSE), nestin (NES), and S-100 were detected by real-time fluorescent quantitative PCR. The S-100 protein expression was tested by immunocytochemical staining. ResultsAt 4 days after induction, some ADSCs of groups C, D, and E showed the morphology of Schwann-like cells or neuron-like cells, the change of group D was more obvious; and the ADSCs of group A and B had no obvious change, which were still spindle. The S-100 immunocytochemical staining showed positive expression in groups C, D, and E (more obvious in group D) and negative expression in groups A and B. The gene expression of S-100 displayed time-dependent increases in groups C and D, which was significantly higher than that of groups A, B, and E (P<0.05), but no significant difference was found between groups C and D (P>0.05). The gene expression of NSE showed the same tendency to S-100, which reached the peak in group D; the gene expression of NSE in groups D and E was significantly higher than that of groups A, B, and C (P<0.05), and groups D and E showed significant difference (P<0.05). However, the gene expression of Nestin showed no significant difference among different groups (P>0.05). ConclusionThe ADSCs can be induced to differentiate into Schwann-like cells or neuron-like cells with sciatic nerve extracts; and the early stage (3-7 days) after injury is the best time for stem cell transplantation.
ObjectiveTo observe the effect of Mongolian medicine fumigation combined with sciatic nerve and rectal probe electrical stimulation on muscle spasticity of spinal cord injury.MethodsBetween January 2012 and January 2018, a total of 65 patients with muscle spasticity after spinal cord injury were randomly divided into two group: the observation group (32 cases) and the control group (33 cases). The patients in the observation group were treated with Mongolian medicine (Wu Wei Gan Lu-Decoction) fumigation combined with sciatic nerve and rectal probe electrical stimulation, while the patients in the control group were treated with medicine, physical therapy, and exercise therapy. Both two groups were treated for 8 weeks. The patients were scored with Ashworth Score, American Spinal Injury Association (ASIA) score, and Barthel Index before and after treatment.ResultsThe pre-treatment ASIA scores (light touch sensation, pain sensation, and muscle strength) and Barthel Index of the two groups were not statistically significant (P>0.05). The post-treatment ASIA scores and Barthel Index of both groups performed significantly better than the pre-treatment levels (P<0.05). The post-treatment ASIA muscle strength item was 58.55±10.83 in the observation group and 50.69±11.32 in the control group (P<0.05). The post-treatment Barthel Index was 74.22±11.53 in the observation group and 68.46±9.92 in the control group (P<0.05). The effective rate in the observation group was significantly better than that in the control group (84.4% vs. 60.6%, P<0.05). Conclusion Mongolian medicine fumigation combined with sciatic nerve and rectal probe electric stimulation could improve the muscle spasticity of spinal cord injury and patients’ ability of daily life effectively.
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.