【 Abstract】 Objective To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40(NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. Methods The DNA fragment ofNEP1-40 coding sequence was ampl ified by PCR with designed primer from the cDNA l ibrary including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40， and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene del ivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. Results The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation， and NEP1-40 protein expression was detected in 293T cells by Western blot. Conclusion The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP1-40.
Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.
Objective Bone marrow mesenchymal stem cells (BMSCs), as replacement cells of Schwann cells, can increase the effect of peripheral nerve repair. However, it has not yet reached any agreement to add the appropriate number of seeded cells in nerve scaffold. To investigate the effect of different number of BMSCs on the growth of rat dorsal root gangl ia(DRG). Methods Three 4-week-old Sprague Dawley (SD) rats (weighing 80-100 g) were selected to isolate BMSCs, whichwere cultured in vitro. Three 1- to 2-day-old SD rats (weighing 4-6 g) were selected to prepare DRG. BMSCs at passage 3 were used to prepare BMSCs-fibrin glue complex. According to different number of BMSCs at passage 3 in fibrin glue, experiment was divided into group A (1 × 103), group B (1 × 104), group C (1 × 105), and group D (0, blank control), and BMSCs were cocultured with rat DRG. The axon length of DRG, Schwann cell migration distance, and axon area index were quantitatively evaluated by morphology, neurofilament 200, and Schwann cells S-100 immunofluorescence staining after cultured for 48 hours. Results Some long cell processes formed in BMSCs at 48 hours; migration of Schwann cells and axons growth from the DRG were observed, growing in every direction. BMSCs in fibrin glue had the biological activity and could effect DRG growth. The axon length of DRG and Schwann cell migration distance in groups A, B, and C were significantly greater than those in group D (P lt; 0.05). The axon length of DRG and Schwann cell migration distance in group C were significantly less than those in group B (P lt; 0.05), but there was no significant difference between group A and group C, and between group A and group B (P gt; 0.05). The axon area index in groups A and B was significantly greater than that in group D (P lt; 0.05), but there was no significant difference between group C and group D (P gt; 0.05); there was no significant difference in groups A, B, and C (P gt; 0.05). Conclusion In vitro study on DRG culture experiments is an ideal objective neural model of nerve regeneration. The effect of different number of BMSCs in fibrin glue on the growth of DRG has dose-effect relationship. It can provide a theoretical basis for the appropriate choice of the BMSCs number for tissue engineered nerve.
Objective To investigate the effects of chitosan/polyvinyl alcohol (PVA) nerve conduits for repairing radial nerve defect in Macaques. Methods Twelve adult Macaques weighing 3.26-5.35 kg were made the models of radial nerve defect (2 cm in length) and were randomly divided into 3 groups according to nerve grafting, with 4 Macaques in each group. Chitosan/PVA nerve conduit, non-graft, and autografts were implanted in the defects in groups A, B, and C, respectively. And the right radial nerves were used as normal control. At 8 months postoperatively, the general observation,electrophysiological methods, and histological examination were performed. Results At 8 months postoperatively, theregenerated nerve bridged the radial nerve defect in group A, but no obvious adhesion was observed between the tube and the peripheral tissue. The regenerated nerve had not bridged the sciatic nerve defect in group B. The adhesions between the implanted nerve and the peri pheral tissue were significant in group C. Compound muscle action potentials (CMAP) were detected in group A and group C, and no CMAP in group B. Peak ampl itude showed a significantly higher value in normal control than in groups A and C (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). Nerve conduction velocity and latency were better in normal control than in groups A and C, and in group C than in group A, all showing significant differences (Plt; 0.05). The density of myl inated fibers in groups A and C was significantly lower than that in normal control (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). The diameter and the myel in sheath thickness of the myl inated fibers in normal control were significantly higher than those in groups A and C, and in group C than in group A, all showing significant differences (P lt; 0.05). Conclusion The chitosan/PVA nerve conduits can promote the peripheral nerve regeneration, and may promise alternative to nerve autograft for repairing peripheral nerve defects.
Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) embedded in fibrin glue around chemical extracted acellular nerve allograft (CEANA) on the peripheral nerve regeneration. Methods Twenty-oneadult male C57 mice (weighing 25-30 g) and 15 adult male Balb/c mice (weighing 25-30 g) were selected. The sciatic nerves were harvested from the Balb/c mice to prepare CEANA. The BMSCs were isolated from 3 C57 mice and were cultured; BMSCs embedded in fibrin glue were cultured for 3, 7, 14, and 21 days. Then the supernatant was obtained and co-cultured with PC12 cells for 2 days to observe the PC12 cell growth in vitro. The other 18 C57 mice were used to establ ish the left sciatic nerve defect models of 10 mm and divided into 3 groups: autogenous nerve graft with fibrin glue (group A, n=6), CEANA graft with BMSCs (5 × 106) embedded in fibrin glue (group B, n=6), and CEANA graft with fibrin glue (group C, n=6). The right sciatic nerves were exposed as the controls. At 2, 4, 6, and 8 weeks, the mouse static sciatic index (SSI) was measured. The histomorphometric assessment of triceps surae muscles and nerve grafts were evaluated by Masson staining, toluidine blue staining, and transmission electron microscope (TEM) observationat 8 weeks after operation. Results BMSCs were uniform distribution in fibrin glue, which were spherical in shape, and the cells began to grow apophysis at 3 days. PC12 cells differentiated into neuron-l ike cells after addition supernatant co-cultured after 2 days. Incisions healed well in each group. At 2, 4, 6, and 8 weeks, the SSI increased gradually in 3 groups. SSI in group A was higher than that in groups B and C at 4, 6, and 8 weeks after operation (P lt; 0.05). SSI in group B was sl ightly higher than that in group C, but had no significant difference (P gt; 0.05). At 8 weeks, the wet weight recovery rate of triceps surae muscles and fibers number of myel inated nerve were better in group B than in group C, but worse in group B than in group A, showing significant differences (P lt; 0.05). The triceps surae muscle fibers area and myel in sheath thickness had significant differences between group B and group C (P lt; 0.01), but there was no significant difference between group A and group B (P gt; 0.05). Conclusion BMSCs embedded in fibrin glue around CEANA can improve functional recovery of peripheral nerve injury.
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 influence of nerve growth factor (NGF) on neuroal regeneration of somatovisceral heterogenic reinnervation using a rat phrenic-to-vagus anastomosis model. Methods Forty male SD rats, aging 3 months and weighing 200 g, were selected and randomly divided into 3 groups. In group A (n=10, control group), phrenic and vagusnerves were exposed and no neurorraphy was performed. In group B (n=15) and group C (n=15), both nerves were transected and proximal stump of phrenic nevers were microsurgically anastomosed to the distal stump of vagus nerves. Postoperatively, group C was intraperitoneally injected with NGF (20 μg/kg·d), while groups A and B were given matching sal ine solution. Twelve weeks later, cardiac function was examined under electrical stimulation of the regenerated nerve. Light and electron microscopies were used to examine the heterogenic regenerated nerve, and the passing rate of axon and thickness of myel in sheath were calculated. Results Under electrical never stimulation in groups A, B, and C, the decreases of blood pressure were (20.12 ± 2.57), (10.63 ± 2.44), and (14.18 ± 2.93) mmHg (1 mmHg=0.133 kPa), respectively; and the decreases of heart rate were (66.77 ± 9.96), (33.44 ± 11.82), and (43.27 ± 11.02)/minutes, respectively. In group B, the decrease ampl itudes of blood pressure and heart rate were 52.83% and50.08% of group A, respectively. Blood pressure and heart rate in group C also decreased dramatically; the decrease ampl itudes of blood pressure and heart rate in group C were 70.48% and 64.80% of group A. There were significant differences in the decrease ampl itudes of blood pressure and heart rate (P lt; 0.05) between group B and group C. Morphological observation showed that heterogenic nerve fibers had the structure of matured myel in sheath and their axons could regenerate into the vagus nerve. In group B and group C, the passing rates of axon were 66.83% ± 4.46% and 81.63% ± 3.56%, respectively; and the thicknesses of myel in sheath were (0.25 ± 0.10) μm and (0.46 ± 0.08) μm, respectively; showing significant differences (P lt; 0.05) between group B and group C. Conclusion Heterogenic nerve is primarily a somatic motor nerve; NGF can promote the axons of heterogenic nerve to regenerate into the parasympathetic nerve.
Objective To investigate the differences in biomechanical properties between fresh and chemically extracted acellular peri pheral nerve. Methods Thirty-six sciatic nerves were harvested from 18 adult male Wistar rats of 3 months old and randomly assigned into 3 groups (n=12 per group): normal control group (group A), the nerve segments received no treatment; Sondell method group (group B), the nerve segments were chemically extracted with the detergents of Triton X-100 and sodium deoxycholate; and improved method group (group C), chemically extracted acellular treatment of nerve was done with the detergents of Triton X-200, Sulfobetaine-10 (SB-10), and SB-16. After the acellularization, the structural changes of nerves in each group were observed by HE staining and field emission scanning electron microscope,then the biomechanical properties of nerves were tested using mechanical apparatus (Endura TEC ELF 3200). Results HE staining and field emission scanning electron microscope showed that the effect of acellularization of group C was similar to that of group B, but the effects of demyel ination and integrity of nerve fiber tube of group C were better than those of group B; the structure of broken nerves was more chaotic than before biomechanical test. The biomechanical test showed that the ultimate load, ultimate stress, ultimate strain, mechanical work to fracture in group A were the largest, the next was group C, the least was group B; the tenacity and elastic modulus in group C were the largest, the next was group B, the least was group A; but the differences were not significant (P gt; 0.05). Conclusion Compared with Sondell method, the nerve treated by improved method is more appropriate for use in vivo.
Objective To construct chemically extracted acellular nerve allograft (CEANA) with Schwann cells (SCs) from different tissues and to compare the effect of repairing peripheral nerve defect. Methods Bone marrow mesenchymal stem cells (BMSCs) and adi pose-derived stem cells (ADSCs) were isolated and cultured from 3 4-week-old SD mice with weighing 80-120 g. BMSCs and ADSCs were induced to differentiated MSC (dMSC) and differentiated ADSC (dADSC) in vitro.dMSC and dADSC were identified by p75 protein and gl ial fibrillary acidic protein (GFAP). SCs were isolated and culturedfrom 10 3-day-old SD mice with weighing 6-8 g. CEANA were made from bilateral sciatic nerves of 20 adult Wistar mice with weighing 200-250 g. Forty adult SD mice were made the model of left sciatic nerve defect (15 mm) and divided into 5 groups (n=8 per group) according to CEANA with different sources of SCs: autografting (group A), acellular grafting with SCs (5 × 105) (group B), acellular grafting with dMSCs (5 × 105) (group C), acellular grafting with dADSCs (5 × 105) (group D), and acellular grafting alone (group E). Motor and sensory nerve recovery was assessed by Von Frey and tension of the triceps surae muscle testing 12 weeks after operation. Then wet weight recovery ratio of triceps surae muscles was measured and histomorphometric assessment of nerve grafts was evaluated. Results BMSCs and ADSCs did not express antigens CD34 and CD45, and expressed antigen CD90. BMSCs and ADSC were differentiated into similar morphous of SCs and confirmed by the detection of SCs-specific cellsurface markers. The mean 50% withdrawal threshold in groups A, B, C, D, and E was (13.8 ± 2.3), (15.4 ± 6.5), (16.9 ± 5.3), (16.3 ± 3.5), and (20.0 ± 5.3) g, showing significant difference between group A and group E (P lt; 0.01). The recovery of tension of the triceps surae muscle in groups A, B, C, D, and E was 87.0% ± 9.7%, 70.0% ± 6.6%, 69.0% ± 6.7%, 65.0% ± 9.8%, and 45.0%± 12.1%, showing significant differences between groups A, B, C, D, and group E (P lt; 0.05). No inflammatory reactionexisted around nerve graft. The histological observation indicated that the number of myel inated nerve fiber and the myel in sheath thickness in group E were significantly smaller than that in groups B, C, and D (P lt; 0.01). The fiber diameter of group B was significantly bigger than that of groups C and D (P lt; 0.05) Conclusion CEANA supplementing with dADSC has similar repair effect in peripheral nerve defect to supplementing with dMSC or SCs. dADSC, as an ideal seeding cell in nerve tissue engineering, can be benefit for treatment of peripheral nerve injuries.
Objective To investigate the promotion effect of neurotropic reinnervation with chemically extracted acellular nerve allograft. Methods The sciatic nerves of 5 healthy adult SD rats, regardless of gender and weighing 270-300 g, were collected to prepare chemically extracted acellular nerve allograft. Eighteen healthy adult Wistar rats, regardless of genderand weighing 300-320 g, were made the model of left sciatic nerve defect (10 mm) and randomly divided into 2 groups: autograft (control group, n=9) and allograft group (experimental group, n=9). The defects were bridged by acellular nerve allograft in experimental group and by autograft by turning over the proximal and distal ends of the nerve in control group. At 3 months after transplantation, dorsal root ganglion (DRG) resection operation was performed in 6 rats of 2 groups. At 3 weeks after operation, the sural nerves were harvested to calculate the misdirection rate of nerve fibers with pathological staining and compute-assisted image analysis. Cholinesterase staining and carbonic anhydrase staining were performed in the sural nerve of 3 rats that did not undergo DRG resection at 3 months. Results The results of chol inesterase staining and carbonic anhydrase staining showed that experimental group had less brown granules and more black granules than control group. After DRG resection, count of myelinated nerve fiber were 4 257 ± 285 in the experimental group and 4 494 ± 310 in the control group significant (P lt; 0.05). The misdirection rate was 2.27% ± 0.28% and 7.65% ± 0.68% in the experimental group and in the control group respectively, showing significant difference (P lt; 0.05). Conclusion Chemically extracted acellular nerve allograft can not only act as a scaffold in the period of nerve defects repair, but markedly enhance the effects of neurotropic reinnervation.