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.
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 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 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 develop a tractive spinal cord injury model in rats with a novel spinal distractor so as to supply the rel iable animal model for researching the pathological mechanism and rehabil itation treatment of tractive spinal cordinjury. Methods A novel spinal distractor was prepared based on previous study. Sixty adult Sprague Dawley rats (weighing 250-300 g) were randomly divided into 5 groups, 12 rats in each group. T12-L3 spinal structures in the rear area were exposed and then T13-L2 spinal cords were revealed via dual laminectomy and kept integrity. In group A, a novel spinal distractor was placed without distraction; in groups B, C, D, and E, the T12-L3 spines were tracted with a novel spinal distractor which put on transverses process of T12-L3 vertebrae. During the tractive period, the somatosensory evoked potential (SEP) was used to monitor spinal cord function. The SEP ampl itudes descended 50% and kept distracting for 5 minutes in group B and for 10 minutes in group C, and descended 70% and kept distracting for 5 minutes in group D and for 10 minutes in group E, respectively to establ ish the tractive spinal cord injury model of T11-L2. The improved combine behavioral score (ICBS) was recorded at 1 and 7 days after injury in 6 rats of each group. The T13-L2 spinal tissue specimens were harvested for the morphological observation by HE and Nissl’s staining and for neurons counting. Results In group A, the ICBS score was 0 at 1 and 7 days after operation, showing significant difference when compared with the scores of the other groups (P lt; 0.05). The ICBS scores of groups D and E were significantly higher than those of groups B and C (P lt; 0.05). Edema and hemorrhage were observed in spinal cord surface and normal morphological structures were destroyed at different extent in groups B, C, D, and E at 1 day. There were adherence and congestion between spinal cord surface and peripheral issue without luster at 7 days, and dura depression was observed at the injury section, especially in group E. Necrosis and dissolution occurred in some neurons, and Nissl body structure dissolved or disappeared in groups B, C, D, and E. The neuron counting gradually decreased in accordance with the aggravation of injury in groups B, C, D, and E, showing significant difference when compared with group A (P lt; 0.05). Significant differences in neuron counting were found among groups B, C, D, and E (P lt; 0.05). Conclusion The tractive spinal cord injury model in rats can be successfully establ ished with novel spinal distractor, and the model establ ished by SEP ampl itude descending 70% and keeping distracting for 10 minutes is more suitable for study in tractive spinal cord injury.
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 effect of methylprednisolone sodium succinate (MP) and mouse nerve growth factor (mNGF) for injection in treating acute spinal cord injury (ASCI) and cauda equina injury. Methods Between December 2004 and December 2007, 43 patients with ASCI and cauda equina injury were treated, including 33 males and 10 females with an average age of 43 years (range, 32-66 years). Injured vertebral columns were C2 in 1 case, C4 in 5 cases, C5 in 7cases, C6 in 3 cases, T8 in 1 case, T10 in 1 case, T11 in 2 cases, T12 in 3 cases, L1 in 9 cases, L2 in 5 cases, L3 in 3 cases, L4 in 1 case, and L5 in 2 cases. All the patients had sensory disturbance and motor dysfunction at admission. The Frankel scale was used for assessment of nerve function, 5 cases were rated as Grade A, 12 as Grade B, 22 as Grade C, and 4 as Grade D before operation. In 43 patients, 23 cases were treated with MP and mNGF (group A), 20 cases with MP only (group B). There was no significant difference in general data between 2 groups (P gt; 0.05). All the patients were admitted, received drug treatment within 8 hours of injury, and were given spinal canal decompression, bone transplantation, and internal fixation within 48 hours. The neurological function score systems of American Spinal Injury Association (ASIA) were used for neurological scores before treament, at 1 week and 2 years after treatment. The scores of the activity of daily l iving (ADL) were evaluated and compared. Results All the patients achieved heal ing of incision by first intention. Forty-three cases were followed up 24-61 months with an average of 30 months. Bone graft fusion was achieved after 6-17 months, 11 months on average with stable fixation. No death and compl ications of osteonecrosis and central obesity occurred. There was no significant difference in neurological function scores and ADL scores between 2 groups before treatment (P gt; 0.05); however, the neurological function scores and ADL scores at 1 week and 2 years after treatment were higher than those before treatment (P lt; 0.01) in 2 groups. Group A had higher neurological function scores and ADL scores than group B (P lt; 0.01). At 1 week and 2 years after treatment, the improvement rates of neurological function of group A (47.8%, 11/23 and 91.3%, 21/23) were significantly higher (P lt; 0.01) than those of group B (30.0%, 6/20 and 70.0%, 14/20). Conclusion MP and mNGF play an important role in improving the neurological function in patients with ASCI and cauda equina injury.
Objective To explore the factors to affect severity of hyperextension injury of the cervical spinal cord (HEICSC). Methods Forty-five patients with HEICSC, 35 males and 10 females, aged 27-67 years old (mean 48.2 years old), were retrospectively analyzed. The disease course was 30 minutes to 16 days. According to modified Frankel grading, there were 6 cases of grade A, 8 cases of grade B, 16 cases of grade C and 15 cases of grade D. Spinal cord injuries (SCI) segments were determined according to SCI plane and high signal change (HSC) in spinal cord on MR images. The whole or large part of HSC segments were supposed to be main injured spinal cord segments (MISCSs) and the staccato or patchy HSC ones were supposed to be common injured spinal cord segments (CISCSs). When the external force acting on head or face suffered was larger, the force produced during high-speed movement or forehead and/or face had severe contused and/or) lacerated wound, the force was defined severe traumatic strength, whereas the reverse was true for sl ight traumatic strength. According to signal magnitude of the cervical discs on T2-weighted MR images, degeneration of cervical discs and cervical vertebras were classified into 5 grades: grade 0-4. Cervical spinal stenosis were graded to 5 grades according to the width of anterior or posterior cerebrospinal fluid layer to spinal cord on T2-weighted MR images and compressed degree of spinal cord on T1-weighted MR images. The influence of traumatic strength, cervical spinal degeneration or cervical spinal stenosis on SCI were explored. Results Among the 45 cases, 12 cases were caused by sl ight traumatic strength, 33 cases were caused by severe one. The cervical spinal cord was injuried more sl ightly and the patients were older in the sl ight traumatic strength cases than in the severe ones (P lt; 0.05). The number of MISCSs were 45 in 40 cases and the 25 segments were located at C3, 4 level. The number of CISCSs were 39 in 21 cases. All the cervical vertebraes of the 45 patients had degenerated. The most were in grade 3 in 22 patients and the severest degenerative segments were mostly located in C5,6 discs in 35 ones. The number of the MISCSs in different degenerative grades of discs was 0 in grade 0, 9 in grade 1, 20 in grade 2, 14 in grade 3, and 2 in grade 4. The ratios of the segment number of injuried spinal cord to the segment number of spinal stenosis in every grade of stenosis were 1/62 in grade 0, 2/11 in grade 1, 27/52 in grade 2, 33/33 in grade 3, 21/22 in grade 4. Conclusion Three main factors including the magnitude of traumatic strength, the degree of instabil ity of cervical vertebrae and the degree of cervical stenosis contribute to development and progress of HEICSC.
OBJECTIVE: To explore the potential possibility of synaptic connection and 3D adhesion between fetal spinal cord cell suspension (FSCS) and host, and to observe the synapses developing process of FSCS transplantation. METHODS: Spinal cord injury model produced in 42 Wistar rats on T7 by use of modified Allen’s impact method (10 g x 5 cm); 3 days after injury, 20 microliters FSCS with a density of 1 x 10(5)/microliter prepared from E14 rat were injected into the epicenter of the traumatized cavity. Animals were sacrificed after 2, 4, 6, 8, 10 and 12 weeks of transplantation, the graft survival, its differentiation and integration with the host were observed by light and electronmicroscopic study as well as immunohistochemical assay (NF, GFAP, CGRP, 5-HT). RESULTS: In the transplantation area, the neuroblasts stretched out the terminal endings 4 weeks after implantation, followed by the presenting of the pre- and postsynaptic membrane. After 8 weeks, the dense or developed projections were observed in the pre- and postsynaptic membrane; the synaptic cleft filled with the high electron dense substance. All the spherical clear vesicles, granular vesicles, elliptical vesicles and flattened-f type vesicles were seen under the electronmicroscope. After 10 weeks, the axosomatic, dendrosomatic, dendro-dendritic, axo-axonic, dendro-axonic synapses coexisted. Light microscopy showed that the graft cell grew gradually. Immunohistochemical assay showed that NF, 5-HT, CGRP and GFAP positive fibers were in the graft. Synapses, gliafibers and blood brain barrier integrated each other. CONCLUSION: (1) The transplanted FSCS can develop mature synapses with miscellaneous synaptic vesicles in the acute injured spinal cord, host injury cavity wall may induce the FSCS into 3D adhesion. (2) Co-existence of different type of synapse and the immunohistochemistry findings indicate the possibility of synaptic connection between FSCS and host.
OBJECTIVE Following the delayed repair of peripheral nerve injury, the cell number of anterior horn of the spinal cord and its ultrastructural changes, motorneuron and its electrophysiological changes were investigated. METHODS In 16 rabbits the common peroneal nerves of both sides being transected one year later were divided into four groups randomly: the degeneration group and regeneration of 1, 3 and 5 months groups. Another 4 rabbits were used for control. All transected common peroneal nerves underwent epineural suture except for the degeneration group the electrophysiological examination was carried out at 1, 3 and 5 months postoperatively. Retrograde labelling of the anterior horn cells was demonstrated and the cells were observed under light and electronmicroscope. RESULTS 1. The number of labelled anterior horn cell in the spinal cord was 45% of the normal population after denervation for one year (P lt; 0.01). The number of labelled cells increased steadily from 48% to 57% and 68% of normal values at 1, 3 and 5 months following delayed nerve repair (P lt; 0.01). 2. The ultrastructure of the anterior horn cells of the recover gradually after repair. 3. With the progress of regeneration the latency become shortened, the conduction velocity was increased, the amplitude of action potential was increased. CONCLUSION Following delayed repair of injury of peripheral nerve, the morphology of anterior horn cells of spinal cord and electrophysiological display all revealed evidence of regeneration, thus the late repair of injury of peripheral nerve was valid.