Objective To investigate the effect of quantitative semi-transected blade on the improvement of spinal cord semi-transected and lump defect model. Methods Forty-eight male Sprague Dawley rats (weighing 220-250 g) were divided into the experimental group (n=24) and control group (n=24). The spinal cord semi-transected and lump defect model was made by self-made quantitative semi-transected blade in the experimental group, and by ophthalmic scalpel in the controlgroup. Then, the complications were observed; the electrophysiological results were detected before modeling and at 21 days after modeling; the histological changes at margin of lump defect were observed at 6 hours, 5 days, and 28 days; Basso, Beattie, and Bresnahan (BBB) scores were detected at 1, 3, 5, 7, 14, 21, 28, 35, 42, 56, and 84 days after modeling. Results There was significant difference in the mortality between the experimental group (0) and the control group (26.67%) (P=0.028). Electrophysiological examination: there was no significant difference in latency and ampl itude of motor evoked potentials (MEP) and sensory evoked potentials (SEP) between 2 groups at preoperation (P gt; 0.05); at 21 days after operation, latencies of MEP and SEP increased and the amplitude decreased in the control group, showing significant differences when compared with those in the experimental group and the preoperative values (P lt; 0.05), but no significant difference was seen between preoperation and postoperation in the experimental group (P gt; 0.05). Histological examination: in the control group, small hematoma could be observed at normal side at 6 hours after modeling, increased spaces of spinal tissue and perineural invasion were observed at 5 days, and small cavity formed without normal motoneurons at 28 days in the margin of lump defect. In the experimental group, no small hematoma could be observed at 6 hours after modeling, no inreversible injury of neuron and small cavity were observed at 5 days, and normal motoneurons were observed without small cavity at 28 days in the margin of lump defect.BBB scores: except the scores between experimental group and control group at affected side (P gt; 0.05), there were significant differences between groups, and between normal side and affected side for intragroup (P lt; 0.05). Conclusion Semi-transected and lump defect model could be set up successfully by self-made quantitate semi-transected blade, procedure is repetitive and the model is stable. This model is an ideal model for semi-transected spinal cord injury.
Objective To discuss the clinical characteristics, mechanism, and treatment of odontoid fracture combined with lower cervical spinal cord injuries without fracture or dislocation. Methods According to the inclusion and exclusion criteria, 7 male patients aged 37-71 years (mean, 51.4 years), suffered from odontoid fractures combined with lower cervical spinal cord injuries without fracture or dislocation were analyzed retrospectively between June 2007 and October 2015. The trauma causes were traffic accidents in 2 cases, fall in 2 cases, and hit injury in 3 cases. The time from injury to admission was 2 hours to 3 days with an average of 9 hours. According to Anderson-Grauer classification of odontoid fracture, 1 case of type IIA, 3 cases of type IIB, 2 cases of type IIC, and 1 case of shallow type III were found. The cervical spinal cord injuries affected segments included C4, 5 in 1 case, C4–6 in 2 cases, and C5–7 in 4 cases. All the cervical spine had different degenerative changes: 2 of mild, 3 of moderate, and 2 of severe. The lower cervical spinal cord injury was assessed by Sub-axial Injury Classification (SLIC) with scoring of 4-6 (mean, 5.1). The visual analogue scale (VAS) score was used to evaluate the occipital neck pain with scoring of 7.8±1.0; the neurological function was assessed by American Spinal Injury Association (ASIA) as grade B in 1 case, grade C in 4 cases, and grade D in 2 cases; and Japanese Orthopedic Association score (JOA) was 9.2±3.9. For the odontoid fractures, 4 cases were fixed with anterior screw while the others were fixed with posterior atlantoaxial fixation and fusion. For the lower cervical spine, 4 cases were carried out with anterior cervical corpectomy and titanium fusion while the others with anterior cervical disecotomy and Cage fusion. Results The operation time was 178-252 minutes (mean, 210.2 minutes); the intraoperative blood loss was 60-140 mL (mean, 96.5 mL) and with no blood transfusion. All incisions healed primarily. All the patients were followed up 12-66 months (mean, 18 months). There was no direct surgical related complications during operation, and all bone grafting got a fusion at 6-9 months (mean, 7.7 months) after operation. There was no inter-fixation failure or loosening. At last follow-up, the VAS score declined to 1.7±0.7 and JOA score improved to 15.1±1.7, showing significant differences when compared with preoperative ones (t=18.064, P=0.000; t=–7.066, P=0.000). The neurological function of ASIA grade were also improved to grade D in 5 cases and grade E in 2 cases, showing significant difference (Z=–2.530, P=0.011). Conclusion Complex forces and degeneration of lower cervical spine were main reasons of odontoid fracture combined with lower cervical spinal cord injuries without fracture or dislocation. The type of odontoid fracture and neurological deficit status of lower cervical spinal cord were important to guide making strategy of one-stage operation with a satisfactory clinic outcome.
ObjectiveTo study the effects of astaxanthin on the apoptosis after spinal cord injury in rats.MethodsOne hundred and forty-four healthy adult Sprague Dawley rats were divided into experimental group, control group, and sham group according to the random number table (n=48). In the control group and the experimental group, the modified Allen’s method was used to make the spinal cord injury model; in the sham group, only the lamina was cut without damaging the spinal cord. At immediate after operation, the rats in the experimental group were given intragastric administration of astaxanthin (75 mg/kg) twice a day; and the rats in the control group and the sham group were given equal amount of olive oil by gavage twice a day. BBB score was used to assess the motor function at 1 day and 1, 2, 3, and 4 weeks after operation. The malondialdehyde (MDA) content was determined by the thiobarbituric acid method at 24 hours after operation; and the activity of superoxide dismutase (SOD) was determined by the xanthine oxidase method. Apoptosis index (AI) was determined by TUNEL method at 6, 24, and 48 hours after operation. At 48 hours after operation, the water content of spinal cord was measured by dry-wet weight method, the lesion ratio of spinal cord was calculated, the ultrastructure of the spinal cord was observed by transmission electron microscopy, and ultrastructure scoring was performed using the Kaptanoglu score method.ResultsThe BBB score in the control group and the experimental group was significantly lower than that in the sham group at each postoperative time point (P<0.05); and the BBB score in the experimental group were significantly higher than that in the control group at 1-4 weeks postoperatively (P<0.05). The MDA content in the control group and the experimental group was significantly higher than that in the sham group at 24 hours after operation, and in the experimental group was significantly lower than in the control group (P<0.05). The SOD activity in the control group and the experimental group was significantly lower than that in the sham group, and in the experimental group was significantly higher than in the control group (P<0.05). At each time point postoperatively, the AI in the control group and the experimental group was significantly higher than that in the sham group, and in the experimental group was significantly lower than in the control group (P<0.05). At 48 hours after operation, the water content of spinal cord, the lesion ratio of spinal cord, and the ultrastructure score in the control group and the experimental group were significantly higher than those in the sham group, and in the experimental group were significantly lower than in the control group (P<0.05).ConclusionAstaxanthin can inhibit the lipid peroxidation, reduce the apoptosis, reduce the spinal cord edema, reduce the spinal cord lesion, reduce the histopathological damage after spinal cord injury, and improve the motor function of rats with spinal cord injury, and protect the spinal cord tissue, showing an obvious neuroprotective effect.
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.
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 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.
Objective To investigate the effects of removing microglia from spinal cord on nerve repair and functional recovery after spinal cord injury (SCI) in mice. MethodsThirty-nine 6-week-old female C57BL/6 mice were randomly divided into control group (n=12), SCI group (n=12), and PLX3397+SCI group (n=15). The PLX3397+SCI group received continuous feeding of PLX3397, a colony-stimulating factor 1 receptor inhibitor, while the other two groups were fed a standard diet. After 14 days, both the SCI group and the PLX3397+SCI group were tested for ionized calcium binding adapter molecule 1 (Iba1) to confirm that the PLX3397+SCI group had completely depleted the spinal cord microglia. The SCI model was then prepared by clamping the spinal cord in both the SCI group and the PLX3397+SCI group, while the control group underwent laminectomy. Preoperatively and at 1, 3, 7, 14, 21, and 28 days postoperatively, the Basso Mouse Scale (BMS) was used to assess the hind limb function of mice in each group. At 28 days, a footprint test was conducted to observe the gait of the mice. After SCI, spinal cord tissue from the injury site was taken, and Iba1 immunofluorescence staining was performed at 7 days to observe the aggregation and proliferation of microglia in the spinal cord. HE staining was used to observe the formation of glial scars at the injury site at 28 days; glial fibrillary acidic protein (GFAP) immunofluorescence staining was applied to astrocytes to assess the extent of the injured area; neuronal nuclei antigen (NeuN) immunofluorescence staining was used to evaluate neuronal survival. And 5-hydroxytryptamine (5-HT) immunofluorescence staining was performed to assess axonal survival at 60 days. Results All mice survived until the end of the experiment. Immunofluorescence staining revealed that the microglia in the spinal cord of the PLX3397+SCI group decreased by more than 95% compared to the control group after 14 days of continuous feeding with PLX3397 (P<0.05). Compared to the control group, the BMS scores in the PLX3397+SCI group and the SCI group significantly decreased at different time points after SCI (P<0.05). Moreover, the PLX3397+SCI group showed a further decrease in BMS scores compared to the SCI group, and exhibited a dragging gait. The differences between the two groups were significant at 14, 21, and 28 days (P<0.05). HE staining at 28 days revealed that the SCI group had formed a well-defined and dense gliotic scar, while the PLX3397+SCI group also developed a gliotic scar, but with a more blurred and loose boundary. Immunofluorescence staining revealed that the number of microglia near the injury center at 7 days increased in the SCI group than in the control group, but the difference between groups was not significant (P>0.05). In contrast, the PLX3397+SCI group showed a significant reduction in microglia compared to both the control and SCI groups (P<0.05). At 28 days after SCI, the area of spinal cord injury in the PLX3397+SCI group was significantly larger than that in SCI group (P<0.05); the surviving neurons significantly reduced compared with the control group and SCI group (P<0.05). The axonal necrosis and retraction at 60 days after SCI were more obvious. ConclusionThe removal of microglia in the spinal cord aggravate the tissue damage after SCI and affecte the recovery of motor function in mice, suggesting that microglia played a neuroprotective role in SCI.
Objective To explore the therapeutic effect of basic fibroblast growth factor (bFGF) on spinal cord injury (SCI) in rats and the influence of Notch/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods A total of 40 10-week-old male Sprague Dawley (SD) rats were selected to establish T10-segment SCI model by a free falling object. Among them, 32 successful models were randomly divided into model group and bFGF group, with 16 in each group. Another 16 SD rats were selected as sham-operation group, with only T10 processes, dura mater, and spinal cord exposed. After modeling, the rats in bFGF group were intraperitoneally injected with 100 μg/kg bFGF (once a day for 28 days), and the rats in model group and sham-operation group were injected with normal saline in the same way. The survival of rats in each group were observed after modeling. Basso-Beattie-Bresnahan (BBB) scores were performed before modeling and at immediate, 14 days, and 28 days after modeling to evaluate the functional recovery of hind limbs. Then, the spinal cord tissue at the site of injury was taken at 28 days and stained with HE, Nissl, and propidium iodide (PI) to observe the pathological changes, neuronal survival (number of Nissl bodies) and apoptosis (number of PI red stained cells) of the spinal cord tissue; immunohistochemical staining and ELISA were used to detect the levels of astrocyte activation markers [glial fibrillary acidic protein (GFAP)] and inflammatory factors [interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ)] in tissues, respectively. Western blot was used to detect the expressions of Notch/STAT3 signaling pathway related proteins [Notch, STAT3, phosphoryl-STAT3 (p-STAT3), bone morphogenetic protein 2 (BMP-2)] in tissues. Results All rats survived until the experiment was completed. At immediate after modeling, the BBB scores in model group and bFGF group significantly decreased when compared to sham-operation group (P<0.05). At 14 and 28 days after modeling, the BBB scores in model group significantly decreased when compared to sham-operation group (P<0.05); the bFGF group showed an increase compared to model group (P<0.05). Compared with before modeling, the BBB scores of model group and bFGF group decreased at immediate after modeling, and gradually increased at 14 and 28 days, the differences between different time points were significant (P<0.05). The structure of spinal cord tissue in sham-operation group was normal; in model group, there were more necrotic lesions in the spinal cord tissue and fewer Nissl bodies with normal structures; the number of necrotic lesions in the spinal cord tissue of the bFGF group significantly reduced compared to the model group, and some normally structured Nissl bodies were visible. Compared with sham-operation group, the number of Nissl bodies in spinal cord tissue significantly decreased, the number of PI red stained cells, GFAP, IL-1β, TNF-α, IFN-γ, Notch, p-STAT3 /STAT3, BMP-2 protein expression levels significantly increased in model group (P<0.05). The above indexes in bFGF group significantly improved when compared with model group (P<0.05). Conclusion bFGF can improve motor function and pathological injury repair of spinal cord tissue in SCI rats, improve neuronal survival, and inhibit neuronal apoptosis, excessive activation of astrocytes in spinal cord tissue and inflammatory response, the mechanism of which may be related to the decreased activity of Notch/STAT3 signaling pathway.
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.
ObjectiveTo evaluate whether long frozen elephant trunk (FET) increases the risk of spinal cord injury in patients with acute type A aortic dissection.MethodsFrom 2018 to 2019, 172 patients with acute type A aortic dissection were treated in Guangdong Provincial People’s Hospital. They were divided into two groups according to the length of FET: patients treated with stents of 100 mm in length were enrolled into a short FET group, and those with stents of 150 mm in length into a long FET group. There were 124 patients in the short FET group, including 108 (87.1%) males and 16 (12.9%) females with a mean age of 51.8±7.9 years. There were 48 patients in the long FET group, including 44 (91.7%) males and 4 (8.3%) females with a mean age of 50.6±9.7 years. The clinical data and prognosis of the patients were analyzed.ResultsThe mean distal stent graft was at the level of T 8.5±0.7 in the long FET group, and at the level of T 6.8±0.6 in the short FET group (P=0.001). Sixteen patients died after operation in the two groups, including 13 (10.5%) in the short FET group and 3 (6.2%) in the long FET group (P=0.561). There were 7 patients of spinal cord injury in the two groups, including 6 (4.8%) in the short FET group and 1 (2.2%) in the long FET group (P=0.675). There was no statistical difference in other complications between the two groups. The follow-up time was 16.7 (1-30) months. During the follow-up, 2 patients died in the long FET group and 5 died in the short FET group. No new spinal cord injury or distal reintervention occurred during the follow-up.ConclusionLong FET does not increase the incidence of spinal cord injury in patients with acute type A aortic dissection.