Objective To clone the genes of nogo-66 and NEP1-40 from spinal cord of rat and to realize the expression of its protein in vitro. Methods The nogo-66 and NEP1-40 genes were cloned from the spinal cord of juvenil rat by use of RT-PCR techniques, and the objective genes were bonded to T vector through gene coupled action, recombinant plasmid were sequencing, and the genes were cloned into PQE30-GST vector, then the recombinant plasmids were induced by isopropylthiogalactoside(IPTG) to express the proteins. The two proteins were purified by Ni-column and detected by using Westernblot test. Results The Nogo-66 and NEP1-40 genes were successfully cloned from rat, which were 215 bp and 137 bp for each one when add the enzyme site. No gene mutations were detected in the two genes after sequencing. The expression plasmids were cut by the two enzyme (BamH Ⅰ and Hind Ⅲ), the target bands were seen on the results of electrophoresis. The expression plasmids were induced by IPTG and got the purified GST fusion protein nogo-66 and NEP1-40, which relative molecular weight were 33.2×103 and 30.3×103 respectively. The results of Westernblot test confirmed that the antigenicity of the two proteins was precise. Conclusion Nogo-66 and NEP1-40 proteins can be expressed in a high efficiency in vitro using genetic engineering, so it provides a good basis for further research on its function and vaccine for spinal injury.
Objective To investigate the method of cultivation and the feature of differentiation of spinal cordderived stem cells in vitro.Methods The neural stemcells from spinal cord of 15 days fetal rats were harvested and cultivated in aserumfree limited medium. The stem cells were induced to differentiate and theresults were identified by cellular immunohistochemistry. Results Lots of stem cells were obtained from the spinal cord of fetal rats and the sphere of stemcells was formed about 10 days. Neural stem cells can give rise to mature neurons and astrocytes.Conclusion Epidermal growth factor/basic fibroblast growth factor serum-free limited medium can promote the proliferation activity ofthe stem cells. Spinal cord-derived stem cells can differentiate into glial cells and neurons.
Objective To explore the influence of dexmedetomidine on wake-up test during spinal orthopaedic surgery. Methods All 80 patients taking spinal orthopaedic surgery were randomly divided into the trial group and the control group, with 40 cases in each group. The endotracheal intubation anesthesia was adopted in both groups with same anesthesia induction. Additionally, dexmedetomidine 0.8 μg/ (kg·h) was infused within 10 min in the trial group before anesthesia induction, and then another 0.5 μg/ (kg·h) was also infused from the intraoperation to suture of incision. For the control group, the same amount of normal saline was infused, and all the narcotics were stopped pumping 15 min before the wake-up test, but then were continued pumping after the wake-up test. Finally, the following indexes were analyzed: wake-up time, wake-up quality, hemodynamic changes at the time of 15 min before wake-up (T1), recovery of spontaneous breathing (T2), wake-up (T3) and 15 min after wake-up (T4), dosage of narcotics, and the incidence of adverse events. Results There was no significant difference in the operation time before wake-up between the two groups (P=0.07). For the trial group, the dosage of sevoflurane (P=0.03) and sufentanil (P=0.00) used before wake-up was significantly lower, the wake-up time (P=0.04) and bleeding amount during wake-up (P=0.00) were significantly less, the wake-up quality (P=0.03) was significantly higher, the blood pressure (P=0.00) and heart rate (P=0.00) when wake-up were significantly lower, and the incidence of adverse events (P=0.04) was significantly lower, compared with the control group. Conclusion Dexmedetomidine adopted in spinal orthopaedic surgery can significantly improve patient’s wake-up quality, shorten wake-up time, reduce bleeding amount when wake-up and adverse events after wake-up, and maintain the hemodynamic stability, so it has better protective effects.
Objective To compare single cell suspension of neural stem cells (NSCs) with neurospheres transplantation for spinal cord injury (SCI) so as to explore the therapeutic effectiveness of two NSCs transplantation methods for SCI. Methods The NSCs were isolated from the spinal cord of adult Sprague Dawley (SD) rats, purified and cultured. At passage 3, the cells were identified by Hoechst33342, Nestin staining, and gl ial fibrillary acidic protein staining for differentiated cells. Sixty adult SD rats (weighing 230-250 g) were made the SCI models at T10 level with modified Allen method and randomlydivided into 3 groups (20 rats in each). The injury sites were treated by injecting 5 μL sal ine (group A), 5 μL single cellssuspensions of NSCs at passage 3 (group B), and 5 μL neurospheres cell suspensions at passage 3 (group C). At preoperation and 3, 7, 14, 21, and 28 days after operation, the locomotor functions of each group were assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale. HE staining was applied to observe the morphology of spinal cord. Subsequently immunofluorescence staining was used to observe microtubule-associated protein 2 (MAP-2). Results The cells cultured were NSCs by morphological observation and immunofluorescence staining. After 3 days of modeling surgery, BBB score significantly decreased when compared with preoperative score, and there was no significant difference among 3 groups at 3 and 7 days (P gt; 0.05). BBB score increased in different degrees with time; at 14, 21, and 28 days, BBB score of groups B and C was better than that of group A, and group C was better than group B, showing significant differences (P lt; 0.05). HE staining showed that spinal cord structure of group C was more clear than that of groups A and B, and had less scar. There was no significant difference in the number of MAP-2 positive cells among 3 groups at 3 and 7 days (P gt; 0.05). At 14, 21, and 28 days, the number of MAP-2 positive cells of groups B and C was significantly more than that of group A, and group C was more than group B, showing significant differences (P lt; 0.05). Conclusion Transplantation of neurospheres suspension compared with single cell can significantly promote NSCsto differentiate into neurons and is conducive to recover the lower extremity function after SCI.
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.
To investigate the protective effect of propofol on ischemia/reperfusion induced spinal cord injury in rabbits and its influence on excitatory amino acid (EAA). Methods Sixty New Zealand white rabbits weighing 2.0-2.5 kg, half males and half females, were selected. The infrarenal circumaortic clamping model was used. And 6 mL/kg different fluids were continuously infused through a catheter into the aorta distal to the clamping site at a speed of 12 mL/(kg?h) during the 30 minutes ischemia period. According to the different infusing l iquids, the rabbits were randomized into 6 groups(n=10 per group): group A, normal sal ine; group B, 10% intral ipid; group C, propofol 30 mg/kg; group D, propofol 40 mg/kg; group E, propofol 50 mg/kg; group F, propofol 60 mg/kg. At 0, 6, 24, and 48 hours after reperfusion, neurologic outcomes were scored on a Tarlov scale system. At 48 hours after reperfusion, the number of normal neurons in the anterior spinal cord was counted, and concentration of EAA in the lumbar spinal cord was measured by high performance l iquid chromatography. Results The neuroethological score was better in groups C, D, E and F than that of groups A and B (P lt; 0.05), the score of group E was the highest (P lt; 0.05), and there was no significant difference between group A and group B (P gt; 0.05). The number of normal neurons in the anterior spinal cord of groups C, D, E and F was greater than that of groups A and B (P lt; 0.05), and group E was greater than groups C, D and F (P lt; 0.05). The concentration of EAA in groups A, B, C, D, E and F was greater than that of normal tissue, the group E was the lowest (P lt; 0.05), the groups A and B were the highest (P lt; 0.05), and there was no significant difference between group A and group B (P gt; 0.05). Concentrations of glutamate and aspartic acid were negatively correlated to normal neuron numbers in the anterior spinal cord and neuroethological scores 48 hours after reperfusion, and the corresponding correlation coefficient was — 0.613, — 0.536, — 0.874 and — 0.813, respectively (P lt; 0.01). Conclusion Propofol can significantly inhibit the accumulation of EAA in spinal cord and provide a protective effect against the ischemia/reperfusion injury induced spinal cord in rabbits.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
Objective To investigate the effect of curcumin on calcitionin gene related peptide (CGRP) expression after spinal cord injury (SCI) in rats. Methods A total of 200 rats, weighing 250-300 g, were randomly divided into 4 groups (n=50): sham-operation group, normal saline (NS) group, low-dose curcumin group (30 mg/kg), and high-dose curcumin group (100 mg/kg). In sham-operation group, only vertebral lamina excision was performed without SCI; the SCI model was established in the other 3 groups. At immediate after modeling, 30 mg/kg and 100 mg/kg curcumin were injected intraperitoneally in 2 curcumin groups, equivalent NS was given in NS group (30 mg/kg), but no treatment in sham-operation group. At 1, 3, 7, 14, and 21 days after operation, the motor neural function was evaluated by the inclined plane test and Basso-Beattie-Bresnahan (BBB) scores; immunohistochemical staining and Western blot assay were used to observe CGRP expression. Results BBB score and inclined plane test score of NS group, low-dose curcumin group, and high-dose curcumin group were significantly lower than those of sham-operation group at each time point (P lt; 0.05). BBB score of low-dose curcumin group and high-dose curcumin group was significantly higher than that of NS group at 3, 7, 14, and 21 days after SCI (P lt; 0.05), and the score of high-dose group was significantly higher than that of low-dose curcumin group at 7, 14, and 21 days after SCI (P lt; 0.05). Inclined plane test score of low-dose curcumin group and high-dose curcumin group was significantly higher than that of NS group at 7, 14, and 21 days after SCI (P lt; 0.05), and the score of high-dose curcumin group was significantly higher than that of low-dose curcumin group at 7, 14, and 21 days after SCI (P lt; 0.05). Immunohistochemical staining results showed that the CGRP positive cells of sham-operation group was significantly more than those of the other 3 groups, and the CGRP positive cells of high-dose curcumin group were significantly more than those of low-dose curcumin group at each time point (P lt; 0.05); the CGRP positive cells of low- and high-dose curcumin groups were significantly more than those of NS group at 3, 7, 14, and 21 days after SCI (P lt; 0.05). Western blot assay results showed that the CGRP protein expressed at each time point after SCI in sham-operation group; the CGRP protein expression gradually decrease with time passing in NS group; but the CGRP protein expression gradually increased with time passing in low- and high-dose curcumin groups, and reached the peak at 14 days, then maintained a high level. Conclusion After SCI in rats, 30 mg/kg curcumin can improve rats’ motor function, and 100 mg/kg curcumin effect is more obvious, especially in promoting the expression of CGRP. That may be the mechanism of protection of the nervous system.
OBJECTIVE: To observe the effect of nerve growth factor (NGF) and nimodipine (NP) on fetal spinal cord graft in repair of injury of spinal cord. METHODS: A total of 144 adult Wistar rats were included in this study. All were made as the hemi-section cavity injury model at the lumbar enlargement and divided into three groups: fetal spinal cord graft (group Tr), fetal spinal cord graft with NGF (group TN), and fetal spinal cord graft with NGF and NP (group TNN). The intracellular concentration of free ionic calcium was measured at the 4th, 8th, and 24th hour, and superoxidase (SOD) and malondialdehyde (MDA) at 3rd, 6th, 12th, 24th and 72nd hour after operation. RESULTS: After spinal cord was injured, the concentration of MDA and intracellular concentration of free ionic calcium increased and reached to the peak at the 6th and 8th hour respectively, but SOD decreased and at 24th hour to its vale. The MDA was significantly lower in group TN than in group Tr, while the SOD was higher (P lt; 0.05). There was no significant difference on intracellular free ionic calcium concentration between group Tr and TN. The concentration of SOD of group TNN was the highest and the intracellular concentration of free ionic calcium was the lowest in the three groups (P lt; 0.05). The weekly mortality was 33%, 31%, 17% respectively in group Tr, TN and TNN. The mortality of group TNN was significantly lower than the other two groups (P lt; 0.01). CONCLUSION: Although the fetal spinal cord graft is an effective method to repair laboratory spinal cord injury, NGF and ND can interrupt secondary injury and increase survival rate of the host.
Objective To review the in vivo imaging research progress of two-photon microscopy (TPM) in spinal cord. Methods The recent literature concerning in vivo two-photon imaging of axon, microglia, and calcium in transgenic mice spinal cord was extensively consulted and reviewed. Results In vivo two-photon imaging of spinal cord provide dynamic information about axonal degeneration and regeneration, microglial accumulation, and calcium influx after spinal cord injury. Conclusion TPM in vivo imaging study on spinal cord will provide theoretical foundation for pathophysiologic process of spinal cord injury.