ObjectiveTo investigate the expression pattern and significance of Sonic Hedgehog (Shh) signaling pathway by observing whether the Shh signaling pathway components express in the adult rat after spinal cord injury (SCI). MethodsSixty-four healthy male Sprague-Dawley rats were randomly divided into normal group (group A, 8 rats), sham group (group B, 8 rats), and SCI group (group C, 48 rats). In group A, the rats served as controls without any treatment; a decompressive laminectomy was performed on T7-9 levels without SCI in group B; and modified Allen's method was used to make SCI model in group C. Basso Beattie Bresnahan (BBB) scale was used to assess the hind limb motor function at 12 hours, 1 day, 3 days, 7 days, 14 days, and 21 days after SCI; the immunofluorescence staining, real-time PCR, and Western blot were performed to detect the mRNA and protein expression levels of Shh and Glioma-associated oncogene homolog-1 (Gli-1) in SCI zone. ResultsThe BBB score slowly increased with time in group C, but the scores at each time point in group C were significantly lower than those in group A and group B (P<0.05). The results of immunofluorescence staining showed that Shh and Gli-1 rapidly increased after SCI in astrocytes. Real-time PCR and Western blot showed that the relative expression levels of Shh and Gli-1 mRNA and protein were gradually increased in group C and reached a maximum at 7 days. In addition, the relative expression levels of Shh and Gli-1 mRNA and protein in group C were significantly higher than those in group A and group B (P<0.05). On the other hand, compared with group A, the expression of Gli-1 protein was reduced in the cytoplasm but increased in nucleus in group C. ConclusionAstrocytes synthesize and secrete Shh and Gli-1 signaling molecules after SCI, both Shh and Gli-1 significantly up-regulate and exhibit dynamic changes, which suggests Shh signaling pathway may be involved in nerve cell regeneration after SCI.
ObjectiveTo fabricate the bionic scaffolds of rat spinal cord by combining three dimensional (3D) printer and 3D software, so as to lay the foundation of theory and technology for the manufacture of scaffolds by using biomaterials. MethodsThree female Sprague Dawley rats were scanned by 7.0T MRI to obtain the shape and position data of the cross section and gray matter of T8 to T10 spinal cord. Combined with data of position and shape of nerve conduction beam, the relevant data were obtained via Getdata software. Then the 3D graphics were made and converted to stereolithography (STL) format by using SolidWorks software. Photosensitive resin was used as the materials of spinal cord scaffolds. The bionic scaffolds were fabricated by 3D printer. ResultsMRI showed that the section shape of T8 to T10 segments of the spinal cord were approximately oval with a relatively long sagittal diameter of (2.20±0.52) mm and short transverse diameter of (2.05±0.24) mm, and the data of nerve conduction bundle were featured in the STL format. The spinal cord bionic scaffolds of the target segments made by 3D printer were similar to the spinal cord of rat in the morphology and size, and the position of pores simulated normal nerve conduction of rat spinal cord. ConclusionSpinal cord scaffolds produced by 3D printer which have similar shape and size of normal rat spinal cord are more bionic, and the procedure is simple. This technology combined with biomaterials is also promising in spinal cord repairing after spinal cord injury.
Objective To investigate the possibility of constructing eukaryotic expression vector for human glial derived neurotrophic factor （hGDNF）, transfecting it to spinal cord tissue of rats so as to treat acute spinal cord injury. Methods The eukaryotic expression vector pcDNA3-hGDNF was constructed by recombinant DNA technique, transfected into glial cell and neuron of spinal cord by liposome DOTAP as experimental group. In control group, mixture of empty vector and liposome was injected. The mRNA and protein expressions of hGNDF were detected by RT-PCR and Western blot. Results After the recombinant eukaryotic expression vector for hGDNF was digested with Hind III and XbaⅠ, electrophoresis revealed 400 bp fragment for hGDNF gene and 5 400 bp fragment for pcDNA3 vector. In the transfected spinal cord tissue, the mRNA and protein expressions of hGDNF gene were detected with RT-PCR and Western blot. Conclusion The constructed eukaryotic expression vector pcDNA3hGDNF could be expressed in the transfected spinal cord tissue of rat, so it provide basis for gene therapy of acute spinal cord 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.
To introduce a micturition alert device dedicated to neurogenic bladders. Methods The design and mechanism of the micturition alert device were explained, the effectiveness was tested in a cranine experiment. Results The micturition alert device consisted of a permanent magnet sutured on the anterior bladder wall and a warning unit sutured on theinferior abdominal wall. The warning unit was assembled with a compass-l ike switch, a power supply, a buzzer and a power switch. Bladder volume determined the position of the magnet which determined the magnetic field at the point of the warning unit. The change of magnetic field was read by the warning unit. With increasing bladder volume from initial state to 200 mL in 8 dogs, the magnet moved cranially 32.8 mm averagely (from 31.3 mm to 34.1 mm) and the hand of warning unit turned 52° (from 47° to 57°). The value of the warning unit was correlated positively to the bladder volume (r =1.0, P lt; 0.01）. If the desired bladder volume was determined as 150 mL to activate the warning unit to alarm in advance, the fullness of bladder was 147.6 mL averagely from135 mL to 160 mL, with an error less than 15 mL (10%). Conclusion The micturition alert device including a warning unit and permanent magnet could monitor bladder volume continuously and alarm in time for the patients with loss of micturition desire. It is simple, easily-made, cheap and conveniently used. It is worth of further study.
Objective To discuss operative strategies of posterior deformity vertebra resection and instrumentation fixation in the treatment of congenital scol iosis or kyphoscol iosis in child and adolescent patients, and to evaluate the surgicalresults. Methods From May 2003 to December 2007, 28 patients with congenital scol iosis or kyphoscol iosis were treatedwith one stage posterior deformity vertebra resection. There were 11 males and 17 females with an average age of 9.6 years (1.5-17.0 years). The locations were thoracic vertebra in 13 cases, thoracolumbar vertebra in 10 cases, and lumbar vertebra in 5 cases. All the patients underwent one stage posterior deformity vertebra resection, fusion and correction with pedicle instrumentation. According to different types of deformities, the patients underwent three different surgeries: hemivertebra resection (13 patients), hemivertebra resection combined contralateral unsegmental resection (7 patients), and total vertebral column resection (8 patients). Based on short or long segmental pedicle instrumentation, deformities were corrected and fixed, in 7 patients with short segmental fixation (group A), in 13 patients with long segmental fixation with hemivertebra resection or combined contralateral unsegmental resection (group B), and in 8 patients with long segmental fixation with total vertebral column resection (group C). The operative duration and the volume of blood loss were recorded, and the correction rate was calculated through measurement of Cobb angles of scol iosis and kyphosis before and after operation. Results The operation time of groups A, B, and C was (98 ± 17), (234 ± 42), and (383 ± 67) minutes, respectively, and the blood loss during operation was (330 ± 66), (1 540 ± 120), and (4 760 ± 135) mL, respectively; showing significant differences among three groups (P lt; 0.05). All patients achieved one-stage heal ing of incision. No deep infection, respiratory failure or deep vein thrombosis occurred. One patient had the signs of ischemical reperfusion injury of spinal cord 6 hours after operation and recovered after 2 weeks of relative therapy in group C; no neurological compl ication occurred in other patients. The mean follow-up period was 32.8 months (24-72 months). Intervertebral rigid fusion was identified from radiological data 6 months after operation according to contiguous callus crossed intervertebral gap and maintenance of correction results. No instrumentation failure occurred. There were significant differences in the Cobb angle between before and after operations (P lt; 0.01). There were significant differences in the corrective rate of scol iosis between groups A, B and group C (P lt; 0.05). Meanwhile, there were significant differences in the corrective rate of kyphosis between groups A, C and group B (P lt; 0.05). Conclusion One-stage posterior deformity vertebra resection has a good capabil ity of correcting congenital scol iosis or kyphoscol iosis on coronal and sagittal plane rel ied on removal deformity origin. It is important to select appropriated strategies on deformity resection and segmental fixation according to different ages and deformity situations of patient.
OBJECTIVE: To investigate a animal model of spinal cord injury in different degrees of impact. METHODS: A new weight-drop device was designed with the character of controlled degree of impact and time. After thirty-five rats underwent different degrees of impact, their motor function and pathological changes were observed. RESULTS: In control group, the rats could walk after reviving, and the micro-structure of spinal cord was normal. With 0.5 mm depth of impact, the rats also could walk, and the micro-structure of spinal cord did not change obviously. With 0.8 mm depth of impact, the rats could walk after several days of injury and only slight damage could be found in spinal cord. When the impact depth increased to 1.0 or 1.5 mm, the rats were paralyzed completely and could not walk after four weeks of injury. Severe injury was observed in spinal cord. CONCLUSION: This animal model of spinal cord injury is based on different degrees of impact. It has stable and repetitive characters for the research on spinal cord injury.
Objective To review the advances in repair of spinal cord injury by transplantation of marrow mesenchymal stem cells(MSCs). Methods The related articles in recent years were extensively reviewed,the biological characteristic of MSCs,the experimental and clinical studies on repair of spinal cord injury by transplantation of MSCs,the machanisms of immigration and therapy and the problems were discussed and analysed. Results The experimental and clinical studies demonstrated that the great advances was made in repair of spinal cord injury by transplantation of MSCs. After transplantation, MSCs could immigrate to the position of spinal cord injury, and differentiate into nervelike cells and secrete neurotrophic factors.So it could promote repair of injuryed spinal cord and recovery of neurologicalfunction. Conclusion Transplantation of MSCs was one of effective ways in repair of spinal cord injury, but many problems remain to be resolved.
Objective To explore the effect of spinal neural progenitor transplantation to the cervical spinal on treating brachial plexus injury with the reimplantation of the avulsed spinal roots. Methods Thebrachial plexusavulsed injury model was made on 54 rats and they were evenly divided into 3 groups: fresh group, chronic group, control group. The spinal neural progenitor was cultured and identified. Then 10 μl（1×105/μl）cells were labelled with BrdUand transplanted into the fresh group (15 rats survived, being model for 1 week) and the chronic group (14 rats survived, being model for 2 months). No cell was transplanted into the control group. Two months after the transplantation, therecovery of function of the injured limb was evaluated. Electrophysiologic study and immunohistochemical study of the injured limb were made. Results Spinal neural progenitors were isolated from the spine and became neural sphere. The neural spheres were differentiated into neurons and astrocytes. Fourteen rats out of 15 in the fresh group were recovered, 7 rats out of 14 in the chronic groupwere recovered, and 5 rats out of 12 in the control group were recovered. Immunohistochemical study indicated that the transplanted progenitors in fresh group survived and differentiated into the neural cells, and the transplanted progenitors in chronic group existed and did not differentiate well. Conclusion Transplanted spinal neural progenitors can promote the recovery of the brachial plexus injury with the reimplantation of the avulsed spinal root.
ObjectiveTo explore the biological functions of Kip1 ubiquitylation-promoting complex 2 (KPC2) in the repair process of spinal cord injury (SCI) by studying the expression and cellular localization of KPC2 in rat SCI models. MethodsFifty-six adult Sprague-Dawley rats were randomly divided into 2 groups: in the control group (n=7), simple T9 laminectomy was performed;in the experimental group (n=49), the SCI model was established at T9, 7 rats were used to detect follow indexs at 6 hours, 12 hours, 1 day, 3 days, 5 days, 7 days, and 14 days after SCI. Western blot analysis was used to detect the protein expressions of P27kip1, KPC2, CyclinA and proliferating cell nuclear antigen (PCNA) after SCI. Immunohistochemistry was used to observed the cellular localization of KPC2 after SCI, double-labeling immunofluorescence staining to observe the co-localization of KPC2 with neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and PCNA. in vitro astrocytes proliferation model was used to further validate these results, Western blot to detect KPC2, P27kip1, and PCNA expressions. The interaction of P27kip1, KPC1, and KPC2 in cell proliferation was analyzed by co-immunoprecipitation. ResultsThe Western blot analysis showed a significant down-regulation of P27kip1 and a concomitant up-regulation of KPC2, CyclinA, and PCNA after SCI. Immunohistochemistry staining revealed a wide distribution of KPC2 positive signals in the gray matter and white matter of the spinal cord. The number of KPC2 positive cells in the experimental group was significantly higher than that in the control group (t=10.982, P=0.000). Double-labeling immunofluorescence staining revealed the number of KPC2/NeuN co-expression cells in the gray matter of spinal cord was (0.43±0.53)/visual field in the control group and (0.57±0.53)/visual field in the experimental group, showing no significant difference (t=0.548, P=0.604);in the white matter of spinal cord, the number of KPC2/PCNA co-expression cells was (3.86±0.90)/visual field in the control group and (0.71±0.49)/visual field in the experimental group, showing significant difference (t=7.778, P=0.000). And then, the number of KPC2/PCNA co-expression cells were (0.57±0.53)/visual field in the control group and (5.57±1.13)/visual field in the experimental group, showing significant difference (t=8.101, P=0.000). Concomitantly, there was a similar kinetic in proliferating astrocytes in vitro. The Western blot analysis showed a significant down-regulation of P27kip1 and a concomitant up-regulation of KPC2 and PCNA after serum stimulated. Co-immunoprecipitation demonstrated increased interactions between P27kip1, KPC1, and KPC2 after stimulation. ConclusionThe up-regulated expression of KPC2 after SCI is related to the down-regulation of P27kip1, this event may be involved in the proliferation of astrocytes after SCI.