Objective Neuron purification is essential to procedure of various nerve cell experimental research, however, at present there is few reports on the effect of various factors on neural axons during purification. To find out a simple method of neuron purification, and to investigate the influence factors of corresponding purification culture in dorsal root gangl ion (DRG) tissue culture on β3-tubul in positive axon. Methods The DRGs were obtained from the 3 days neonatal SD rat microscopically and were made into cell suspension. Then, the amount of attached DRG neurons and non neuronal cells in poly-D-lysine (PDL) group, PDL/Laminin (PDL/LN) group and collagen-I (Col I) group was observed from 10 to 100 minutes. Then, the extension and arborization of β3-tubul in positive axons were observed after 72 hours completely randomised DRG tissue culture for the research of the influences among culture substrates (PDL, PDL/LN, and Col I), FBS (0, 5%, and 10%), 5 fluorouracil (5-Fu, 0, 20, and 40 μmol/L), and cytrarabine (Ara-C, 0, 10, and 20 μmol/L). Results Adherent cells were observed instantly after inoculation by inverted phase contrast microscope and inverted fluoresence microscope; after cell suspension was removed, adherent growth of DRGn cells and non-DRGn cells were still seen. In PDL group, the amount of NSE negative cells was significantly higher than that of NSE positive cells at 10 and 30 minutes (P lt; 0.05); the amount of NSE positive cells was significantly higher than that of NSE negative cells at 80, 90 and 100 minutes (P lt; 0.05). In PDL/LN gruop, there was no significant difference (P gt; 0.05) in the amount of NSE negative cells and NSE positive cells at 10, 20, 30, 40, and 50 minutes; the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 60, 70, 80, 90, 100 minutes. In Col I group, the amount of NSE negative cells was higher than that of NSE positive cells at 10-40 minutes, but showing no significant difference (P gt; 0.05); the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 70-100 minutes. At 72 hours after DRG tissue culture, the best result of β3-tubul in positive axon extension and arborization was obtained when the substrate level was PDL/LN, and the average length of PDL/LN level was significantly larger than that of other two substrates (P lt; 0.05). The highest number of β3-tubul in positive axon distal end was obtained at 5% concentration level of FBS (P lt; 0.05), but showing no significant differences in β3-tubul in positive axon length among three levels (P gt; 0.05). Both the most of β3-tubul in positive axon distal ends and the longest β3-tubul in positive axon average length were obtained at 0 μmol/L concentration level of 5-Fu, showing significant differences between 0 μmol/L level and 20, 40 μmol/L levels (P lt; 0.05). A similar result of β3-tubul in positive axon distal end was got at the 0 μmol/L level and 10 μmol/L level of Ara-C, which was significantly higher than that of 20 μmol/L level (Plt; 0.05). Conclusion? A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 minutes. When DRG neuron culture, neuron special medium, PDL/LN substrate and 10 μmol/L Ara-C are recommended in β3-tubul in positive axon research.
Objective To study the cl inical effects of modified Galveston technology in the treatment of lumbosacral tuberculosis. Methods From January 2001 to May 2008, 19 patients with lumbosacral tuberculosis were treated, including13 males and 6 females aged 21-58 years old (average 38 years old). The course of disease was 8-22 months. The tuberculosis was at the L4-S1 level in 3 cases, the L5, S1 level in 10 cases, the L5-S2 level in 5 cases, and the S1, 2 level in 1 case. Seven cases were compl icated with neural symptom of the lower l imbs, 3 cases of them were grade C and 4 cases were grade D according to the Frankel scale of nerve function. The preoperative JOA score of lower back pain was 5-22 (average 19). Six cases were compl icated with il iac abscess, 3 cases with psoas abscess, 3 cases with sacroil iac joint tuberculosis, and 2 cases with pulmonary tuberculosis. For 12 patients, the operation of modified Galveston internal fixation via the posterior approach, focus debridement via vertebral canal, and interbody fusion with autogeneous il iac bone fragment grafting was performed; for 7 cases, the operation of modified Galveston internal fixation via the posterior approach, vertebral lamina fusion with autogeneous il iac bone fragment grafting, and anterior focus debridement was performed. Results The incision of 18 cases was healed by first intention, and 1 case had sinus 3 weeks after operation and healed 3 months after operation. Nineteen patients were followed up for 12-82 months (average 21 months). There was no recurrence of the local tuberculosis, and the common toxic symptom of tuberculosis disappeared 6-12 months after operation. All the patients achieved bony fusion 4-6 months postoperatively, and 3 patients with sacroil iac joint tuberculosis achieved sacroil iac joint fusion. For those 7 patients with combinations of the neural symptomof the lower l imbs, the symptoms disappeared and their Frankel scales were improved to grade E. The JOA score of low back pain at the final follow-up was 22-29 (average 26). There was a significant difference between preoperation and postoperation (P lt; 0.05). Conclusion The modified Galveston technology is helpful to reconstruct the stabil ity of lumbosacral vertebrae, improve bony fusion rate, reduce the postoperative in-bed time.
To explore the advantage and indication of combined anterior and posterior surgeries for lumbarsacral junction tuberculosis. Methods Eleven cases of the lumbarsacral junction tuberculosis were treated with combined anterior (radical debridement and autograft) and posterior (instrumentation and fusion) surgeries in one stage between January 2002 and December 2006. There were 9 males and 2 females with the age of 20-56 years old. The courseof disease was 4 to 15 months, 6 months on average. The lessons were located at L5, S1 in 7 patients, at L4,5, S1 in 2 patients and at L5, S2 in 2 patients. The involved vertebral bodies were at 2 segments in 7 patients; and 3 segments in 5 patients. The preoperative kyphosis was 5 to 8° with an average 9°. The sinus was associated in 3 patients, 3 patients had radiculopathy; 4 had paeumonophthisis and 9 had abscess. Results The followed-up period was from 6 months to 3 years, 18 months on average. According to Chen score, among the 11 cases, there were excellent in 9, good in 2. All incisions were healed up primarily. After operation, spinal fusion was achieved in 10 cases within 5 months to 7 months, 6 months on average, and pseudoarthrosis in 1 case was found by the CT examination. The postoperative kyphosis was 0 to 4° with the mean of 2° and the radiculopathy in 3 cases all got nerve function recovery. Conclusion Lumbarsacral junction tuberculosis treated with this surgical technique can achieve a high satisfactory rate with restoring the spinal stabil ity, arresting the disease early, providing early fusion, correcting the kyphosis and preventing progression of kyphosis particularly if lumbosacral spine tuberculosis is associated with sinus or preoperative diagnosis cannot exclude suppurative spondyl itis.
Objective To investigate the therapeutic effects of transplanting allogeneic marrow mesenchymal stem cells (MSCs) via subarachnoid space on spinal cord injury(SCI) and the T cell subpopulation. Methods Density gradient centrifugation was used to isolate and expand MSCs from bone marrow of 10 six-week-old SD rats. The SCI model was produced by weightbeating from 60 eight-week-old female SD rats. Forty survival SCI rats,which BBB scores were zero, were divided randomly into 2 groups:experimental group(group A) and control group(group B). In addition, 20 normal eightweekold SD ratswere used as blank group (group C). In group A, 1 ml cells suspention containing MSCs(the 6th generation, 2×106/ml) was injected via subarachnoid space. Ingroup B, equal volume of L-DMEM was injected in the same way. The BBB score was obtained after 1st,2nd and 3rd weeks of injection. At the same time,T cell subpopulation was detected by flow cytometry. Results The BBB score in group A was better than that in group B, but fewer than that in group C in the 3rd week. CD4+T cells in group A were less than those in groups B and C in the 1st, 2nd, and 3rd weeks. CD8+T cells in group A were less than those in groups B and C in the 2nd and 3rd weeks. The ratio of CD4+/CD8+T cells in group A was less than those in groups B and C in the 1st week. Above differences showed statistically significant difference(P＜0.05). However, there were no statistically significant differences in the ratio of CD4+/CD8+T cells between group A and groups B, C in the 2nd and 3rd weeks (P＞0.05). Conclusion The above results suggest that allogeneic MSCs transplantation via subarachnoid space is beneficial to SCI to some extend, do not result in rejection in vivo. Furthermore, it can lead to immunosuppression in short time. So, it provides clues to apply MSCs to treat SCI and other diseases.
目的:探讨后路内固定治疗脊柱结核的必要性及适应证。方法:2002年1月~2008年12月采用后路器械固定、融合结合前路彻底病灶清除、植骨治疗脊柱结核17例。病变位于胸椎3例,胸腰段2例,腰椎4例,腰骶椎8例;累及2个节段7例,3个节段7例,4个节段3例;有3例伴窦道形成;9例伴不同程度的脊髓和(或)神经根受压症状;术前后凸成角10°~72°,平均31°。所有患者均一期手术。结果:术后随访6个月~5年,平均3.1年,术后切口均Ⅰ期愈合,后凸成角7°~58°,平均16°,椎间植骨平均在5个月融合,植骨融合率95.6%,优良率达89.6%,无一例复发。结论:后路器械固定结合前路彻底病灶清除、植骨治疗脊柱结核主要适用于多个节段受累、腰骶段及伴窦道者,利于恢复脊柱的稳定性、提高植骨融合率、纠正和预防后凸畸形。
ObjectiveTo explore the pathogenesis of cervical spondylosis combined with cervicogenic vertigo, and to investigate the clinical results of anterior discectomy and fusion in treating the disease. MethodsA retrospective study was performed on 83 patients with cervical spondylosis myelopathy (n=60, 72%) or radiculopathy (n=23, 37.3%) accompanied by sympathetic symptoms such as dizziness between March 2008 and November 2012. The disease involved single segment in 29 cases, double segment in 50 cases, and triple in 4 cases. All the segments involved were treated with anterior discectomy and fusion. Vertigo alleviation was observed before surgery, 3 days after surgery, and during the final follow-up. Neurological status was evaluated by Japanese Orthopedic Association (JOA) score system and sympathetic symptoms were evaluated with vertigo symptom and function scoring system. ResultsThe average follow-up was 21 months (ranging from 12 to 30 months). Significant difference was observed between sympathetic symptom scores and JOA scores before surgery and 3 days after surgery or at the final follow-up (P<0.05), but no significant difference was found between the scores 3 days after surgery and during the final follow-up (P>0.05). ConclusionThe surgical effect for cervicogenic vertigo is often accompanied by the relief of spinal cord and nerve roots symptoms. Surgery is effective for cervical spondylosis combined with cervicogenic vertigo.
ObjectiveTo explore the surgical indication and summarize the experiences of anterior cervical discectomy and fusion (ACDF) for the treatment of cervical spondylosis. MethodsSeventy-five cases of cervical spondylosis were treated with ACDF from January 2010 to October 2013, including 34 cases of cervical spondylotic radiculopathy and 41 cases of cervical spondylotic myelopathy. The pre/post-operative Japanese Orthopedic Association (JOA) score and imaging data were observed. ResultsThirty-six patients were followed up for 6 to 25 months, with an average of 16 months. The mean JOA score before surgery was 10.67±2.66, and 3 months later, the score was 13.47±2.06. Six months later the score was 14.11±1.56, and after 12 months the score was 14.97±1.78. No spinal cord injury or esophagus, trachea injury occurred, and there was no superior laryngeal nerve and recurrent laryngeal nerve injury during the operation. Eight cases of postoperative dysphagia obviously decreased within 3-5 days, 6 cases decreased within 5-7 days, and 10 cases decreased within 3 weeks. And all 36 patients recovered within one month. There was no titanium mesh subsidence, displacement or titanium plate failure after operation. ConclusionACDF is suitable for the decompression of spinal cord or nerve root which is compressed by the degenerated intervertebral disc, especially without rigid kyphosis, ossification of the posterior longitudinal ligament extending across multiple segments, and the compression located at the level of intervertebral disc. The appropriate approach can achieve a stable efficacy, less interference on the stability of the spine, and the incision heals fast. It is a kind of classic anterior cervical operation for it can reduce the complication effectively.
Objective To explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid (HA) by electrospinning. Methods The three-dimensional porous nano-scaffolds were prepared by electrospinning techniques with collagen type II and HA (8 ∶ 1, W ∶ W), which was dissolved in mixed solvent of 3-trifluoroethanol and water (1 ∶ 1, V ∶ V). The morphology were observed by light microscope and scanning electron microscope (SEM). And the porosity, water absorption rate, contact angle, and degradation rate were detected. Chondrocytes were harvested from 1-week-old Japanese white rabbit, which was disgested by 0.25% trypsin 30 minutes and 1% collagenase overlight. The passage 2 chondrocytes were seeded on the nano-scaffold. The cell adhesion and proliferation were evaluated by cell counting kit 8 (CCK-8). The cell-scaffold composites were cultured for 2 weeks in vitro, and the biological morphology and extracelluar matrix (ECM) secretion were observed by histological analysis. Results The optimal electrospinning condition of nano-scaffold was 10% electrospinning solution concentration, 10 cm receiver distance, 5 mL/ h spinning injection speed. The scaffold had uniform diameter and good porosity through the light microscope and SEM. The diameter was 300-600 nm, and the porosity was 89.5% ± 25.0%. The contact angle was (35.6 ± 3.4)°, and the water absorption was 1 120% ± 34% at 24 hours, which indicated excellent hydrophilicity. The degradation rate was 42.24% ± 1.51% at 48 days. CCK-8 results showed that the adhesive rate of cells with scaffold was 169.14% ± 11.26% at 12 hours, and the cell survival rate was 126.03% ± 4.54% at 7 days. The histological and immunohistochemical staining results showed that the chondrocytes could grow well on the scaffold and secreted ECM. And the similar cartilage lacuma structure could be found at 2 weeks after co-culture, which suggested that hyaline cartilage formed. Conclusion The collage type II and HA complex three-dimensional nano-scaffold has good physicochemical properties and excellent biocompatibility, so it can be used as a tissue engineered cartilage scaffold.
Object ive To summa r i z e the advanc ement of cytoske l e ton and axon outgrowth of neuron. Methods The recent l iterature concerning cytoskeleton and axon outgrowth of neuron was reviewed and summarized. Results The actin filaments and microtubules in neuron were highly polarized and dynamic structures confined to the ti ps of axons and the reci procal interactions between these two major cytoskeletal polymers was also dynamic. Attractive or a repulsive cue whose final common path of action was the growth cone cytoskeleton mediated the growth of axons of neuron by intracellular signaling cascades. Regulating the actin filament and microtubule dynamics as well as their interactions in growth cones played a key role in neurite outgrowth and axon guidance. Rho-GTPases and glycogen synthase kinase 3β (GSK-3β), the two major intracellular signal ing pathways had emerged in recent years as candidates for regulating the dynamics of actin filaments and microtubules. Conclusion The axon outgrowth and guidance depend on well-coordinated cytoskeletal and reciprocal interaction dynamics which also mediate axon regeneration after spinal cord injury. Regulating activity of Rho-GTPases and GSK- 3β simultaneously may acts as key role to regulate the dynamics of cytoskeletal and to determine axon outgrowth.