Objective To observe the result of reconstructing quadriceps femoris function in the paraplegia rats by using the 7th cervical nerve root (C7) transposition with autologous and allogeneic neural transplantation. Methods Twenty16-week-old SPF male Wistar rats were adopted to prepare frozen sciatic nerve. Thirty-six Wistar rats were divided into 2 groups (group A and group B, n=18). The left paraplegia model was establ ished with left spinal cord hemisection by the micro scissors under the operation microscope. After the model establ ishment, the homolateral autologous sciatic nerve was bridged with the femoral nerve root by the translocation of C7 in group A, while the allogeneic sciatic nerve was bridged with the femoral nerve root by the translocation of C7 in group B. At 16 weeks and 24 weeks after operation, 9 rats in each group were selected for the neuroelectric-physiological test and then the histomorphology of the nerves was observed under the microscope and the electron microscope. The fresh weight recovery rate of quadriceps femoris was calculated. Results At 16 and 24 weeks after operation, the nerve action-evoked potential (NAP) was (1.14 ± 0.07) mV and (1.21 ± 0.07) mV in group A, and (0.87 ± 0.06) mV and (0.99 ± 0.05) mV in group B; the nerve conduction velocity (NCV) was (17.34 ± 2.15) m/s and (19.00 ± 3.02) m/s in group A, and (11.23 ± 1.45) m/s and (12.54 ± 1.59) m/s in group B, respectively, indicating significant differences (P lt; 0.05) between 2 groups. At 16 and 24 weeks after operation, HE staining and Bielschowsky staining showed that group A had a large number of nerve fiber regeneration, with a regular arrange of axons; while group B had l ittle nerve fiber regeneration with a scattered arrange of axons. At 24 weeks after operation, images in TEM showed a large number of regeneration myel inated nerve fibers and a small number of unmyel inated nerve fibers through the transplanted nerve in two groups. At 16 weeks after operation, the number of myel inated nerve fibers in group A and group B was (438 ± 79) and (196 ± 31) / vision, the areas of myel inated nerve fiberswere (5 596.00 ± 583.94) and (4 022.63 ± 615.75) μm2 / vision; after 24 weeks, the number of myel inated nerve fibers in groups A and B were (642 ± 64) and (321 ± 75)/vision, the areas of myel inated nerve fibers were (6 689.50 ± 1 142.10) and ( 4 733.00 ± 982.22) μm2/vision, indicating significant differences between two groups (P lt; 0.05). There was no statistically significant difference (P gt; 0.05) in the wet weight recovery rate of quadriceps between group A and group B at 16 weeks (87.96% ± 4.93% vs. 86.47% ± 7.47%) and at 24 weeks after operation (90.10% ± 4.22% vs. 87.66% ± 3.14%). Conclusion C7 transposition combined with autograft and allograft of sciatic nerve can reconstruct the partial function of the quadriceps femoris in paraplegia rats. The effect of graft is better than that of graft obviously.
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.
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 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.
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.
ObjectiveTo construct recombinant adenovirus expressing nerve growth factor (NGF) and myelin associated glycoprotein (MAG) (Ad-NGF-MAG) and to investigate its effect on repair and regeneration of sciatic nerve injury in rats. MethodsNGF and MAG gene sequences were cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK293 cells, the recombinant Ad-NGF-MAG underwent sequence and identification. Thirty-two male Sprague Dawley rats were randomly divided into 4 groups (n=8): control group (normal control), adenovirus vector group (Ad group), Ad-NGF group, and Ad-NGF-MAG group. The sciatic nerve injury model was established by transection of the right sciatic nerve; then, the empty adenovirus vector, Ad-NGF, and Ad-NGF-MAG were injected into the gastrocnemius muscle of the affected limb at a dose of 1×108 PFU every other day for 3 times in Ad group, AdNGF group, and Ad-NGF-MAG group, respectively. The right sciatic nerve was exposed only, and then the incision was closed in the control group. The sciatic nerve function index (SFI) was measured, and neuro-electrophysiology was observed; mRNA and protein expressions of NGF and MAG were detected by RT-PCR and Western blot; and histological examination was performed at 31 days after operation. ResultsRecombinant adenovirus vectors of Ad-NGF and Ad-NGF-MAG were constructed successfully. All rats survived and incision healed by first intension. The SFI, nerve conduction velocity, evoked potential amplitude, and latent period of Ad-NGF-MAG group were significantly better than those of Ad group and Ad-NGF group (P < 0.05). MAG mRNA and protein expressions of Ad-NGF-MAG group were the highest in all the groups (P < 0.05). The expressions of NGF mRNA and protein increased in Ad-NGF group and AdNGF-MAG group when compared with control group and Ad group (P < 0.05). Histological examination showed that the nerve had good continuity in control group; nerve fibers disarranged in Ad group; neurons connections formed in some nerve fibers of Ad-NGF group, but nerve fibers arrange disorderly; and the growth of the nerve were ordered and wellstructured in Ad-NGF-MAG group. ConclusionAd-NGF-MAG can effectively promote the growth of the nerve and inhibit the form of abnormal branches, facilitating the repair of sciatic nerve injury in rats.
Objective To evaluate an effect of the vascularendothelial growth factor (VEGF) geneactivated matrix (GAM) on repair of the sciatic nerve defect in rats. Methods The peripheral nerve extracellular matrix（ECM） was harvested by the chemical extraction from 30 SD rats. The VEGF-GAM comprised of ECM and the plasmids encoding VEGF. Thirty adult Wistar rats were made as a model of the asciatic nerve defect and were randomly divided into the following 3 groups(n=10): Group A (VEGF-GAM conduits), Group B (ECM conduits),and Group C (autografts). At 12 weeks, the rats from each groupwere subjected to an inspection for the walking tract analysis and electrophysiological and histomorphological studies.Results The VEGF DNA could be retained in GAM, promoting the transgene expressing in the sciatic nerve, and more importantly, in the axotomized neurons in the spinal cord for 12 weeks. The motor neuron recovery rate in Group A (79.13％±2.53％) was similar to that in Group C (75.26％±4.48％, Pgt;0.05), but significantly better than that in Group B (56.09％±1.89％, Plt;0.01). The number of the regenerationaxons in the distal sciatic nerve in Group A (13 463±794/mm2) was significantly lower than that in Group C (16 809±680/mm2, Plt; 0.01), but significantly higher than that in Group B (10 260±1 117/mm2,Plt;0.01). The motor nerve conduction velocity in Group A (16.44±1.65 m/s) was significantly lowerthan that in Group C (23.79±2.75 m/s, Plt;0.01), but significantly higherthan that in Group B (12.8 ±1.42 m/s, Plt;0.01). The recovery rate of thegastrocnemius muscle wet weight in Group A (71.40％±3.05%) was significantlylower than that in Group C (87.00％±1.87％,Plt;0.01), but significantly higher than that in Group B (50.00％±4.90％, Plt;0.01). The sciatic nerve function index in Group A （39.37%±4.81%） was significantly lower 〖KG6〗than that in Group C (26.27%±2.71%, Plt;0.01), but significantly higher than that in Group B (4693％±296％, Plt;0.01). Conclusion The results indicate that VEGF-GAM as a bridge can promote the functional recovery of the defected sciatic nerve in rats, but the effect is not so good as that by autografts.
OBJECTIVE To investigate the effects of basic fibroblast growth factor(bFGF) on repairing transected sciatic nerves in rats. METHODS The animal models of the transected sciatic nerve of 40 SD rats were established, which divided into 4 groups: normal saline (NS) group, nerve growth factor (NGF) group, bFGF group and normal control group. The epineurium of the transected sciatic nerve was sutured under microscope, then bFGF or NGF was dropped into local sites and injected intramuscularly once a day for 30 days after operation. Functional repair for the transected sciatic nerves was studied by nerve conductive velocity (NCV) and sciatic nerve function index (SFI). RESULTS As a criterion, the level of the normal control group was regarded as zero, SFI of NS group, NGF group and bFGF group were -114.30 +/- 10.34, -70.50 +/- 11.01, -50.45 +/- 7.82 respectively at 1 month after operation, and they were -54.96 +/- 16.46, -35.21 +/- 10.80, -27.53 +/- 11.23 respectively in 3 months after operation. NCV of bFGF group was significantly faster than NS group and NGF group. CONCLUSION bFGF can significantly promote the functional repair of injured peripheral nerve, and its effects are better than NGF.
ObjectiveTo observe the changes of microstructure of rats'sciatic nerves with non-freezing cold injury after treated with dexamethasone. MethodsTwelve male Wistar rats were randomly divided into cooling group and treating group.Unilateral sciatic nerves of the rats in the cooling group received cooling treatment with 3-5℃ for 2 hours;while unilateral sciatic nerves of the rats in the treating group received cooling treatment with 3-5℃ for 2 hours and underwent the celiac injection with dexamethasone in addition.The other sciatic nerves were exposed,as the control.The bilateral sciatic nerves of rats in each group were harvested after 24 hours.The microstructure of nerves was examined under the light microscope and electron microscope. ResultsLight microscopic examination revealed extensive myelinated fibre degeneration in form of giant empty axons or shrunken dark axons on the first day after cooled.And the endoneurial capillary lumen was narrowed because of swollen endothelial cells.After the treatment,myelinated fibre degeneration was still similar to that before the treatment,but the endoneurial capillary lumen and endothelial cells were normal.By electron microscopy,sciatic nerves showed extensive myelinated fiber degeneration,and swollen endothelial cells.But unmyelinated fibers and tight junction were preserved on the first days after cooled.Aggregated red cells and platelet thrombus were not found.After the treatment,myelinated fibre degeneration was still similar to that before the treatment.Unmyelinated fibers and tight junction were preserved.The endoneurial capillary lumen and endothelial cells were normal. ConclusionAfter the treatment,the damage of endoneurial capillary had improved,but myelinated fiber degeneration was similar to that before the treatment.It suggested that dexamethasone may only improve the vascular system in non-freezing cold injury of sciatic nerve.
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.