ObjectiveTo investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats.MethodsOne hundred and twenty male Sprague Dawley (SD) rats (weighing 220-250 g) were used for the study, 70 of them were randomly divided into sham-operation group and SCI group (n=35), the rats in SCI group were given SCI according to Allen’s method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen (PCNA) by Western blot assay. Another 20 SD rats were grouped by the same method as above (n=10). In these rats, a negative control lentiviral LV3NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other 30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3NC injected after SCI, and group C with MSK1 small interfering RNA (siRNA) lentivirus injected after SCI, with 10 rats each group. The Basso, Beatlie, Bresnahan (BBB) score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins.ResultsWestern blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from 8 hours after injury to the lowest level at 3 days after injury, and then gradually increased; the expression change of PCNA protein was opposite to MSK1. The expression of MSK1 in SCI group was significantly lower than that in the sham-operation group at 1, 2, 3, and 5 days after injury (P<0.05), and the expression of PCNA protein of SCI group was significantly higher than that of the sham-operation group at 8 hours and 1, 2, 3, 5, and 7 days after injury (P<0.05). The fluorescence expression of both the SCI group and the sham-operation group has be found and peaked at 7 days. There was a positive correlation between fluorescence intensity and time in 7 days after transfection. With the prolongation of postoperative time, the BBB scores of groups A, B, and C showed a gradually increasing trend. The BBB score of group C was significantly lower than those of groups A and B at 5, 7, and 14 days after treatment (P<0.05). After transfection for 7 days, Western blot results showed that the relative expression of MSK1 protein in group C was significantly lower than that in groups A and B (P<0.05); and the relative expression of PCNA protein was significantly higher than that in groups A and B (P<0.05). Immunofluorescence staining showed that MSK1 was expressed in the nuclei of the spinal cord and colocalized with green fluorescent protein, neuronal nuclei, and glial fibrillary acidic protein (GFAP). The relative expression area of MSK1 positive cells in group C was significantly higher than that in group B (P<0.05), and the relative expression areas of PCNA and GFAP positive cells were significantly lower than those in group B (P<0.05).ConclusionLentivirus-mediated MSK1 siRNA can effectively silence the expression of MSK1 in rat spinal cord tissue. MSK1 may play a critical role in the repair of SCI in rats by regulating the proliferation of glial cells.
Objective To investigate tissue engineered spinal cord which was constructed of bone marrow mesenchymal stem cells (BMSCs) seeded on the chitosan-alginate scaffolds bridging the both stumps of hemi-transection spinal cord injury (SCI) in rats to repair the acute SCI. Methods BMSCs were separated and cultured from adult male SD rat. Chitosan-alginate scaffold was produced via freeze drying, of which the structure was observed by scanning electron microscope (SEM) and the toxicity was determined through leaching l iquor test. Tissue engineered spinal cord was constructed by seeding second passage BMSCs on the chitosan-alginate scaffolds (1 × 106/mL) in vitro and its biocompatibil ity was observed under SEM at 1, 3, and 5 days. Moreover, 40 adult female SD rats were made SCI models by hemi-transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). Tissue engineered spinal cord or chitosan-alginate scaffolds or BMSCs were implanted in groups A, B, and C, respectively. Group D was blank control whose spinal dura mater was sutured directly. After 1, 2, 4, and 6 weeks of surgery, the functional recovery of the hindl imbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. Other indexes were tested by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing, HE staining and immunofluorescence staining after 6 weeks of surgery. Results Chitosan-alginate scaffold showed three-dimensional porous sponge structure under SEM. The cells adhered to and grew on the surface of scaffold, arranging in a directional manner after 3 days of co-culture. The cytotoxicity of chitosan-alginate scaffold was in grade 0-1. At 2, 4, and 6 weeks after operation, the BBB score was higher in group A than in other groups and was lower in group D than in other groups; showing significant differences (P lt; 0.05). At 4 and 6 weeks, the BBB score was higher in group B than in group C (P lt; 0.05). After 6 weeks of operation, WGA-HRP retrograde tracing indicated that there was no regenerated nerve fiber through the both stumps of SCI in each group. HE and immunofluorescence staining revealed that host spinal cord and tissue engineering spinal cord l inked much compactly, no scar tissue grew, and a large number of neurofilament 200 (NF-200) positive fibers and neuron specitic enolase (NSE) positive cells were detected in the lesioned area in group A. In group B, a small quantity of scar tissue intruded into non-degradative chitosan-alginate scaffold at the lesion area edge, and a few of NSE flourescence or NF-200 flourescence was observed at the junctional zone. The both stumps of SCI in group C or group D were filled with a large number of scar tissue, and NSE positive cells or NF-200 positive cells were not detected. Otherwise, there were obviously porosis at the SCI of group D. Conclusion The tissue engineered spinal cord constructed by multi-channel chitosan-alginate bioscaffolds and BMSCs would repair the acute SCI of rat. It would be widely appl ied as the matrix material in the future.
Objective To investigate the expression pattern of hypoxia-inducible factor 1α (HIF-1α) in experimental secondary spinal cord injury (SSCI) in rats and its potential effects on SSCI. Methods A total of 66 SD rats (female or male) with weight (250 ± 20) g were randomly divided into 3 groups: normal control group (group A, n=6), pseudo injury group (group B, n=6), and spinal cord injury (SCI) group (group C, n=54). In group A, no treatment was given as normal control. In groupB, only laminectomy was appl ied. In group C, laminectomy was appl ied and static compression model of SCI was built at T10 level. The expression of HIF-1α was measured with HE and immunohistochemical staining in groups A, B (1 hour after pseudo injury), and C (1, 3, 6, 12 hours and 1, 2, 3, 7, 14 days after SCI). Results All rats survived to the end of the experiment. HE staining showed that the spinal tissue of groups A and B were dense and the nucleus were round and big with l ight staining and clear nucleolus. The injured neuron at 1-12 hours after SCI of group C presented pyknosis and deep eosin staining. The swelling axon with bubbles and the disintegrated and disorganized medullary sheath in white matter appeared at 1-3 days after SCI. The hyperplasia of gl ial cells were obvious and gray matter cells were broken and apoptosis with cavities in injured spinal segment was observed at 7 and 14 days after SCI. Immunohistochemical staining showed that HIF-1α was poorly expressed in group A and increased a l ittle in group B. The positive expression in group C increased at 3 hours after SCI, which was found in spinal cord anterior horn neurons and a small amount of gangl ion cells. It reached peak at 1 day, maintained at a high level during 1-3 days and then decl ined. At 14 days, it appeared only in a small amount of gangl ion cells of white matter. There was no significant difference in the number of HIF-1α positive cells between groups A and B (t=1.325, P=0.137). The number of HIF-1α positive cells at each time point in group C was more than those in groups A and B (P lt; 0.05), and there were significant differences between all time points in group C (P lt; 0.05). Conclusion The expression of HIF-1α increases after SCI, it is related to the ischemia hypoxia after SSCI, and the expression pattern was correlated with the injury time.
Objective To determine the feasibility, safety, and efficacy of common pedicle screw placement under direct vision combined with dome shaped decompression via small incision for double segment thoracolumbar fracture with nerve injury. Methods A retrospective analysis was performed on the clinical data of 32 patients with double segment thoracolumbar fracture with nerve injury undergoing common pedicle screw placement under direct vision combined with dome shaped decompression via small incision between November 2011 and November 2015 (combined surgery group), and another 32 patients undergoing traditional open pedicle screw fixation surgery (traditional surgery group). There was no significant difference in gender, age, cause of injury, time of injury-to-surgery, injury segments and Frankel classification of neurological function between two groups (P>0.05). The length of soft tissue dissection, the operative time, the blood loss during surgery, the postoperative drainage, the visual analogue scale (VAS) of incision after surgery, and recovery of neurological function after surgery were evaluated. Results All cases were followed up 9 to 12 months (mean, 10.5 months) in combined surgery group, and 8 to 12 months (mean, 9.8 months) in traditional surgery group. The length of soft tissue dissection, the operative time, the blood loss during surgery, the postoperative drainage, and the postoperative VAS score in the combined surgery group were significantly better than those in the traditional surgery group (P<0.05). Dural rupture during surgery and pedicle screw pulling-out at 6 months after surgery occurred in 2 cases and 1 case of the combined surgery group; dural rupture during surgery occurred in 1 case of the traditional surgery group. The X-ray films showed good decompression, and fracture healing; A certain degree of neurological function recovery was achieved in two groups. Conclusion Common pedicle screw placement under direct vision combined with dome shaped decompression via small incision can significantly reduce iatrogenic trauma and provide good nerve decompression. Therefore, it is a safe, effective, and minimally invasive treatment method for double segment thoracolumbar fracture with neurological injury.
Objective To evaluate the feasibility and safety of percutaneous endoscopic technique in the treatment of intraspinal cement leakage after percutaneous vertebroplasty (PVP). Methods Between May 2014 and March 2016, 5 patients with lower limb pain and spinal cord injury caused by intraspinal cement leakage after PVP, were treated with percutaneous endoscopic spinal decompression. Of 5 cases, 3 were male and 2 were female, aged from 65 to 83 years (mean, 74.4 years). The course of disease was 10-30 days (mean, 16.2 days). Imageological examinations confirmed the levels of cement leakage at T 12, L 1 in 3 cases, and at L 1, 2 in 2 cases; bilateral sides were involved in 1 case and unilateral side in 4 cases. Two patients had lower limb pain, whose visual analogue scale (VAS) were 8 and 7; 3 patients had lower extremities weakness, whose Japanese Orthopedic Association (JOA) 29 scores were 18, 20, and 19. According to American Spinal Injury Association (ASIA) impairment scale, neural function was rated as grade E in 2 cases and grade D in 3 cases. Results The operation time was 55-119 minutes (mean, 85.6 minutes), and the blood loss was 30-80 mL (mean, 48 mL). CT scan and three-dimensional (3D) reconstruction at 1 day after operation showed that cement leakage was removed in all patients. Five cases were followed up 6-21 months (mean, 12 months). In 2 patients with lower limb pain, and VAS score was significantly decreased to 2 at last follow-up. In 3 patients with lower extremities weakness, the muscle strength was improved progressively, and the JOA29 scores at last follow-up were 21, 23, and 22. Conclusion Percutaneous endoscopic technique for intraspinal cement leakage after PVP is safe, effective, and feasible.
Objective Aminoguanidine (AG) can reduce brain edema and increase the recovery of neuron functions in surgical brain injury and stroke. To investigate the effect of AG on spinal cord injury (SCI) in rats and its mechanism. Methods A total of 150 adult male Sprague Dawley rats (weighing, 230-255 g) were divided into control group (group A, 25 rats without treatment), the sham-operated group (group B, 25 rats undergoing laminectomy), SCI group (group C, 25 SCI rats with injection of 5%DMSO), SCI + AG groups (groups D, E, and F, 25 SCI rats and AG injection of 75, 150, and 300 mg/kg, respectively). The optimal dosage of AG was screened by dry-wet weight method with the percentage of water content at 0, 12, 24, and 48 hours after injury. The blood-spinal cord barriar permeability was further detected by Evans blue (EB) method, aquaporins 4 (AQP4) mRNA expression by RT-PCR, AQP4 protein expression by immunohistochemistry and Western blot. Results AG injection at dosage of 150 mg/kg can significantly reduce edema of spinal cords at 12, 24, and 48 hours after SCI (P lt; 0.05), so 150 mg/kg was the optimal dosage. The EB content in group E was significantly lower than that in group C at 12, 24, and 48 hours after SCI, and the permeability of blood-spinal cord barrier was significantly decreased compared with group C (P lt; 0.05). The AQP4 mRNA expressions in groups B and E were significantly lower than that in group C at 12, 24, and 48 hours after SCI (P lt; 0.05). AQP4 protein expressions in groups B and E were significantly lower than that in group C at 24 and 48 hours after SCI (P lt; 0.05) by Western blot. Immunohistochemical staining revealed that AQP4 protein expression in group C was significantly higher than that in groups B and E (P lt; 0.05) at 48 hours after SCI, but no significant difference was found between group B and group E (P gt; 0.05). Conclusion AG injection at dosage of 150 mg/kg can induce spinal cord edema and injury in rats, which could be correlated with the down-regulation of AQP4 expression.
In order to investigate the clinical significance of electron-neurogram for evaluating the degree and prognosis of acute traumatic cervical spinal cord injury without fracture or dislocation, electron-neurogram and sensory evoked potential (SEP) of the upper limbs in 4 such cases were recorded from the 3rd to 30th day after the injury. The results showed SEP and MEP could be obtained from every nerve in both upper limbs, and continous monitoring of SEP and MEP could provide valuable data to judge the degree and prognosis of the injury in spinal cord.
Objective To explore the effects of human urine-derived stem cells (hUSCs) and hUSCs combined with chondroitinase ABC (chABC) on the expressions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the spinal cord injury (SCI) of rats, and to investigate the underlying mechanism. Methods hUSCs were cultured from human urine, and their phenotypes were detected by flow cytometry. The SCI model of rats were made via Allen method. Sixty Sprague Dawley rats were divided into 5 groups (n=12): the sham operation group (group A), SCI group (group B), SCI+hUSCs group (group C), SCI+chABC group (group D), and SCI+hUSCs+chABC group (group E). Basso, Beattie, Bresnahan (BBB) score was used to measure the lower extremity motor function of rats in each group at 10, 20, and 30 days after operation. Real-time fluorescent quantitative PCR was used to detect the relative mRNA expressions of NGF and BDNF at 30 days. Meanwhile, the protein expression of NGF and BDNF were confirmed by immunohistochemistry staining. The relative protein expressions of Bax and Bcl-2 were detected by Western blot. Results The hUSCs were identified to have multipotential differentiation potential. At 10, 20, and 30 days, BBB score was significantly lower in group B than in groups A, C, D, and E, in groups C, D, and E than in group A, in groups C and D than in group E (P<0.05). Real-time fluorescent quantitative PCR and immunohistochemistry staining demonstrated that the expressions of NGF and BDNF were significantly lower in group B than in groups A, C, D, and E, in groups C, D, and E than in group A, in groups C and D than in group E (P<0.05); but there was no significant difference between groups C and D (P>0.05). Western blot results indicated that the protein expression of Bax was significantly higher in group B than in groups A, C, D, and E, in groups C, D, and E than in group A, in groups C and D than in group E (P<0.05). Meanwhile, the protein expression of Bcl-2 was significantly lower in group B than in groups A, C, D, and E, in groups C, D, and E than in group A, in groups C and D than in group E (P<0.05). Conclusion hUSCs can protect SCI and this positive effect can be enhanced by chABC; this neuro-protective effect may depend on promoting the expressions of NGF and BDNF, and suppressing the neuronal apoptosis.
ObjectiveTo systematically profile and characterize the circular RNA (circRNA) and microRNA (miRNA) expression pattern in the lesion epicenter of spinal tissues after traumatic spinal cord injury (TSCI) and predict the structure and potential functions of the regulatory network.MethodsForty-eight adult male C57BL/6 mice (weighing, 18-22 g) were randomly divided into the TSCI (n=24) and sham (n=24) groups. Mice in the TSCI group underwent T8-10 vertebral laminectomy and Allen’s weight-drop spinal cord injury. Mice in the sham group underwent the same laminectomy without TSCI. The spinal tissues were harvested after 3 days. Some tissues were stained with HE staining to observe the structure. The others were used for sequencing. The RNA-Seq, gene ontology (GO) analysis, and circRNA-miRNA network analyses (TargetScan and miRanda) were used to profile the expression and regulation patterns of network of mice models after TSCI.ResultsHE staining showed the severe damage to the spinal cord in TSCI group compared with sham group. A total of 17 440 circRNAs and 1 228 miRNAs were identified. The host gene of significant differentially expressed circRNA enriched in the cytoplasm, associated with positive regulation of transcription and protein phosphorylation. mmu-miR-21-5p was the most significant differentially expressed miRNA after TSCI, and circRNA6730 was predicted to be its targeted circRNA. Then a potential regulatory circRNA-miRNA network was constructed.ConclusionThe significant differentially expressed circRNAs and miRNAs may play important roles after TSCI. A targeted interaction network with mmu-miR-21-5p at the core of circRNA6730 could provide basis of pathophysiological mechanism, as well as help guide therapeutic strategies for TSCI.
Objective To investigate the effects of 17β-estradiol on the cell apoptosis after chronic spinal cord injury in ovariectomized rats. Methods A total of 90 female Wistar rats (weighing, 220-250 g) received removal of bilateral ovaries. After 2 weeks, the rats were randomly divided into 3 groups (n=30): sham-operation group (group A); chronic gradual spinal cord injury model and 17β-estradiol treatment group (group B); and chronic gradual spinal cord injury model and normal saline treatment group (group C). Rats of group A only received removal of spinous process at T10. Rats of groups B and C were made the models of chronic gradual spinal cord injury, and then 17β-estradiol (100 μg/kg, twice a week) and normal saline were given by peritoneal injection, respectively. The cell apoptosis and positive cells of Caspase-3 were examined by the TUNEL methods and Caspase-3 immunohistochemical staining at 1, 3, 7, 14, 28, and 60 days after modeling; and the neurological function was evaluated by Tarlov scale and inclined plane test scoring. Results At 14, 28, and 60 days after modeling, Tarlov scale and inclined plane test scores of group B were significantly better than those of group C (P lt; 0.05), but were significantly lower than those of group A (P lt; 0.05). At 28 days after modeling, HE staining showed that the edema of spinal gray matter and the neurons, the proliferation of glial cells and astrocytes, and less pathologic change were observed in group B; and the pathological changes in group B were mitigated than in group C. At 60 days after modeling, edema of spinal gray matter and the neurons was significantly ameliorated in group B. At 14, 28, and 60 days after modeling, the rate of Caspase-3 positive cells in group B was significantly lower than in group C (P lt; 0.05), but was significantly higher than in group A (P lt; 0.05). At 7, 14, 28, and 60 days after modeling, the cell apoptotic rate was significantly lower in group B than in group C (P lt; 0.05), but was significantly higher than in group A (P lt; 0.05). Conclusion 17β-estradiol can reduce the numbers of apoptotic cells and promote the nerve function recovery after chronic spinal cord injury of rats.