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.
Objective To evaluate the effect of self-designed antirotation reduction internal fixator(ARRIF) on treating different spine segment fracture.Methods From August 1999 to March 2003, 76 patients(48 males and 28 females, aged from 22 to 59 with an average of 34.1) with thoracolumbar fracture were operatively treated by ARRIF. The follow-up period ranged from 6 to 21 months(15 months in average). Classification according to injury segment: flexion compression racture 27 cases, burst fracture 42 cases, flexion distraction injury 3 cases, flexion revolving type fracture dislocation 2 cases, shear force type dislocation 2 cases. Classification according Frankel’s grade:A grade 16 cases, B grade 15 cases, C grade 27 cases, D grade 10 cases, E grade 8 cases.Operation duration, volume of bleeding, incidence postoperation complication and effect of reduction fixation were observed. Results The operation duration of ARRIF was 1.2 h in average, and there was about 200 ml volume of bleeding during operation. The nerve function showed one Frankel’s grade improvement after operation were as follows:A grade 8 cases(50%), B grade 11 cases (73.3%), C grade20 cases(74.1%), D grade 3 cases (30%); 2 Frankel’s E cases have no nerve function changes.The nerve function damage have no aggravation in all the patients,the postoperation Cobb’s angle was averagely corrected 22°. The horizontal displacement of dislocation vertebrae was averagely corrected 28% in sagittal plane, the statistical analysis had significant variance(Plt;0.01).ARRIF had no complications of the breakage of screws and rods. Conclusion ARRIF proves to be a valid internal fixator in reducing and fixing different thoracic lumbar segment spine fracture.
Objective To explore the expression of nestin and glialfibrillary acidic protein (GFAP) at different time and sites after spinal cord injury in adult rats. Methods Seventy-two adult Sprague-Dawley rats, aging 8 weeks and weighing from 180 to 220 g, were randomly divided into 11 experimental groups(66, n=6) and 1 control group(n=6). In the experimental groups, the rat spinal cord injury models were established by aneurysm clip compression, and the expression and proliferation of nestin and GFAP at different time（1 day,3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,7 weeks and 8 weeks）and at different sites(injured site and adjacent site) were observed with toludine blue staining, immunofluorescent staining and the analytical system of photographs. In control group, the same site of the rat spinal cord was exposed without aneurysm clip compression. The same preparation and examination were done as the experimental groups. Results Toluidine blue staining results showed thatcontour of neurite and pericaryon were distinct and nucleus were deep blue in normal control rats. One day after injury, the number of big and medium-sized neuron decreased obviously; neurite was deep blue with clouding Nissl bodies and ellipse or triangular typed nucleus. In the normal control group, the expression of nestin was hardly seen except ependymal cells of central canal, and the low expression of GFAP was seen. In the experimental groups, the nestin and GFAP expressions increased obviously in the injured sites and adjacent sites 24 hours after injury, reached the peak value after 3-7 days and followed by gradual decrease. There were statistically significant differences in the nestin and GFAP expressions between theexperimental groups and the control group.Conclusion The aboveresults suggestthat spinal cord injury can induce the expression of nestin and GFAP. There is a positive correlation between nestin expression and the proliferation of the reactive astrocytes.
In order to study the prophylactic and therapeutic effect of methyl-prednisolone (MP) on traction injury of spinal cord, 48 rabbits were divided into four groups randomly. According to decreasing amount of the amplitude of P1-wave, 50% reduction lasted for 5 min and 10 min with MP as experimental group, and 50% 5 min and 10 min with NS as control, the changes of amplitudes were monitored by, and the function of the spinal cord was assessed. The amounts of MDA and SOD of the spinal cord tissue were determined and the pathomorphological changes of the spinal cord were observed. The results showed that in the experimental groups, the recovery of P1-wave was quicker, the Tarlov and Molt value were decreased, the density of gray matter of the anterior horn and the myelinated nerve fiber of white matter of 100 microns diameter were higher, the SOD and MDA was decreased and the degenerative and necrotic degree of neuron and nerve fiber were milder. Where in the control groups all the above items were just on the opposite. The conclusions list as follows: the application of MP before operation of spinal deformity might prevent traction injury of the spinal cord during operative correction of spinal deformity, and could also minimized the secondary damage to spinal cord from traction injury if MP was used in time. The action to MP were summarized as improving the microcirculation, inhibiting the hyperoxidation of lipid and accelerating the recovery of SCEP.
Objective To transplant intravenously human brain-derived neurotrophic factor (hBDNF) genemodified bone marrow mesenchymal stem cells (BMSCs) marked with enhanced green fluorescent protein (EGFP) to injured spinal cord of adult rats, then to observe the viabil ity of the cells and the expressions of the gene in spinal cord, as well as theinfluence of neurological morphological repairing and functional reconstruction. Methods Ninety-six male SD rats weighing (250 ± 20) g were randomly divided into 4 groups: hBDNF-EGFP-BMSCs transplantation group (group A, n=24), Ad5-EGFPBMSCs transplantation group (group B, n=24), control group (group C, n=24), and sham operation group (group D, n=24). In groups A, B, and C, the spinal cord injury models were prepared according to the modified Allen method at the level of T10 segment, and after 3 days, 1 mL hBDNF-EGFP-BMSCs suspension, 1 mL Ad5-EGFP-BMSCs suspension and 1 mL 0.1 mol/L phosphate buffered sal ine (PBS) were injected into tail vein, respectively; in group D, the spinal cord was exposed without injury and injection. At 24 hours after injury and 1, 3, 5 weeks after intravenous transplantation, the structure and neurological function of rats were evaluated by the Basso-Beattie-Bresnahan (BBB) score, cortical somatosensory evoked potential (CSEP) and transmission electron microscope. The viabil ity and distribution of BMSCs in the spinal cord were observed by fluorescent inverted phase contrast microscope and the level of hBDNF protein expression in the spinal cord was observed and analyzed with Western blot. Meanwhile, the expressions of neurofilament 200 (NF-200) and synaptophysin I was analyzed with immunohi stochemistry. Results After intravenous transplantation, the neurological function was significantly improved in group A. The BBB scores and CSEP in group A were significantly higher than those in groups B and C (P lt; 0.05) at 3 and 5 weeks. The green fluorescence expressions were observed at the site of injured spinal cord in groups A and B at 1, 3, and 5 weeks. The hBDNF proteinexpression was detected after 1, 3, and 5 weeks of intravenous transplantation in group A, while it could not be detected in groups B, C, and D by Western blot. The expressions of NF-200 and synaptophysin I were ber and ber with transplanting time in groups A, B, and C. The expressions of NF-200 and synaptophysin I were best at 5 weeks, and the expressions in group A were ber than those in groups B and C (P lt; 0.05). And the expressions of NF-200 in groups A, B, and C were significantly ber than those in group D (P lt; 0.05), whereas the expressions of synaptophysin I in groups A, B, and C were significantly weaker than those in group D (P lt; 0.05). Ultramicrostructure of spinal cords in group A was almost normal. Conclusion Transplanted hBDNF-EGFP-BMSCs can survive and assemble at the injured area of spinal cord, and express hBDNF. Intravenous implantation of hBDNF-EGFP-BMSCs could promote the restoration of injured spinal cord and improve neurological functions.
Objective To investigate the feasibil ity of establ ishment of physiological micturition reflex arc by simultaneously reconstructing the sensory and the motorial nerve of atonic bladder after spinal cord injury. Methods Eight 1-year-old Beegle male canine were selected, weighing 7-12 kg. The left side was the experimental side, while the right side wasthe control side. Epidural microanastomosis of vertebral canal of the left L7 ventral root to S2 ventral root and L7 dorsal root to S2 dorsal root was performed to reconstruct the sensory and the motorial function of atomic bladder. The right side was used as a control without treatment. The new motor-to-motor, and sensory-to-sensory physiological bladder reflex pathway were establ ished after 12 months of axonal regeneration. Then S1-4 segmental spinal cord was destroyed for preparation of complete paraplegia. The electrophysiological examination and the bladder pressure were detected before and after paraplegia. The canine micturition was observed for 3 months after paraplegia. Nurohistological observation was performed after canine sacrifice. Results Of 8 canine, 7 canine survived. After paraplegia, canines displayed urinary incontinence and frequent micturition at first, nocturnal continence was achieved gradually without frequent micturition after 1 month. Urinary infection at different degrees occurred in 3 canines and was controlled after Norfloxacin was administered orally. The bladder pressure increased to (1.00 ± 0.13) kPa, (0.90 ± 0.12) kPa after trains of stimulation (300 mV, 0.3 ms, 20 Hz, 5 seconds) of S2 dorsal root at the experimental side before and after paraplegia respectively, showing no significant difference (P gt; 0.05). It increased to (1.90 ± 0.10) kPa after the same train of stimulation of S2 dorsal root at control side. There was significant difference between the experimental side and the control side (P lt; 0.01). Single stimulation (300 mV, 0.3 ms) of the S2 dorsal root at the experimental side resulted in evoked potentials recorded from the left S2 ventral root before and after paraplegia. Before and after paraplegia, the ampl itudes of the evoked potentials were (0.68 ± 0.11) mV and (0.60 ± 0.08) mV respectively, showing no significant difference (P gt; 0.05). It was (1.21 ± 0.13) mV while stimulating at the control side. There was significant difference between the experimental side and the control side (P lt; 0.01). Neurofibra of L7 dorsal and ventral root grew into S2 dorsal and ventral root on tissue sl ice under l ight microscope. Conclusion Reconstruction of the bladder physiological micturition reflex arc is feasible by anastomosis of sacral dorsal and ventral root below injured spinal plane with the suprasacral survival dorsal and ventral root above the plane respectively for restoration of atonic bladder after spinal cord injury.
Objective To investigate the expressions of heat shock protein 27 (HSP27), Bcl-2, and Bax proteins of the nerve cells after spinal cord ischemia/reperfusion injury (SCII) in rats and their relationship. Methods Seventy adult male Sprague Dawley rats (weighing, 200-220 g) were randomly divided into the sham operated group (sham group, n=35) and the SCII group (n=35). Only the left renal artery was exposed with no occlusion of the abdominal aorta in the rats of sham group. The left renal artery was exposed with occlusion of the abdominal aorta for 20 minutes in the rats of SCII group. At 4, 8, and 12 hours and at 1, 2, 3, and 5 days, reperfusion treatment was performed in 5 rats respectively, and then the spinal cord tissue was harvested to detect the expressions of HSP27, Bcl-2, and Bax protein of the nerve cells by using immunohistochemistry staining. Results The HSP27 began to express at 4 hours, reached the peak at 3 days, and decreased at 5 days in SCII group; significant differences were found between at 3 and 5 days and at the other time points (P lt; 0.05). The Bcl-2 expression increased at 4 hours, reached the peak at 1 day and maintained a high level at 2 days, and then gradually decreased; significant differences were found between at 1 and 2 days and at the other time points (P lt; 0.05). The Bax expression reached the peak at 12 hours and 3 days, and decreased at 5 days; significant differences were found between at 12 hours and 3 days and at the other time points (P lt; 0.05). A little expression of each protein was observed in sham group at different time points; the expressions of HSP27, Bcl-2, and Bax proteins in SCII group were significantly higher than those in sham group at different time points (P lt; 0.05). Conclusion There may be the time window of self repair after SCII. High expression of HSP27 has an obvious protective effect on the SCII in rat, by promoting the expression of the anti-apoptotic protein Bcl-2 and reducing the expression of the pro-apoptotic protein Bax so as to inhibit spinal cord cell apoptosis.
Objective To observe the effects of neural stem cells(NSCs) transplantation on the glial cell line-derived neurotrophic factor (GDNF) and growth associated protein 43(GAP-43) after the spinal cord injury(SCI), and to investigate the mechanism of repairing the SCI by NSCs transplantation. Methods The neural stem cells from the hippocampus of rats’ embryo were cultured and identified by immunocytochemistry. The SCI model was made by the modified Allen device. Sixty adult Wistar rats were randomly divided into three groups: spinal cord injury was treated with transplantation of NSCs (group A, n=24), with DMEM solution(group B, n=24) and normal control group without being injured(group C, n=12). Seven days after the operation of SCI, the NSCs were transplanted into the injured site. Then GAP-43 and GDNF expressions were tested by RT-PCR and immunohistochemistry. Results Compared with group B, the GDNF mRNA expression of group A increased by 23.3％ on the 1st day, by 26.8% on the 3rd day and by 32.7% on the 7th day; the GAP-43 mRNA expression increased by 19.5% on the 1st day, 21.6% on the 3rd day and 23.1% on the 7th day. There were statistically significant differences(Plt;0.05). Conclusion The transplantation of NSCs can change the microenvironment injured site and promote the regeneration of axon by enhancing the expressions of GDNF mRNA and GAP-43 mRNA. It is one of the mechanisms of repairing the SCI by NSCs transplantation.
Objective To make a mouse model of traumatic spinal cord injury (SCI) by Allen’s weight dropping (WD),which might be helpful for further research on the mechanism of SCI. Methods A total of 180 healthy female mice, weighing 17 - 23 g (20 g on average), were randomized into 4 groups (n=45 per group): the experimental groups of A, B and C and the control group of D. Experimental groups were distinguished by the amount of weight or the height from which the weight was dropped onto an impounder resting on the dura (2.0 × 2.5 g·cm, 2.5 × 3.0 g·cm, 3.0 × 5.0 g·cm). In group D, neural scute was opened only and spinal cord was exposed without SCI. The recovery of the lower extremity was observed at various time points (0,6 and 12 hours, 1 and 3 days, 1, 2, 4 and 8 weeks) by using the Basso mouse scale (BMS) scoring system, motor evoked potentials (MEP) and histological observation. Results MEP displayed that the incubation period of N1 wave was extended in group B after 6 hours and in group C after 12 hours. As time passed by, the incubation periods of N1 wave in group A, group B and group C began to shorten. The incubation period in group A was close to normal at 4 weeks (2.40 ± 0.12) ms, and there was no significant difference compared with group D (P gt; 0.05). The incubation period in group B was close to normal at 8 weeks (2.96 ± 0.15) ms, and there was no significant difference compared with group D (P gt; 0.05). The incubation period in group C was still relatively high at 8 weeks (3.76 ± 0.13) ms, and there was a significant difference compared with group D (P﹤0.05). Both hind l imbs of all mice were paralytic instantly after SCI, the score of main BMS was 0 point; the score of main BMS was close to 0 at the first 3 days after SCI, the score of main BMS of group A was 8.00 ± 0.13 and group B was 7.50 ± 0.31 at 8 weeks;the score of main BMS of group A was 5.45 ± 0.12 at 1 week and group B was 5.45 ± 0.15 at 2 weeks which were significant difference compared with group D (P﹤0.05).There were significant differences among groups A, B and C after 1 week of SCI (P lt; 0.05), and group C was lower than the others(P﹤0.01). The score of adjuvant BMS of group A was 10.12 ± 0.76 at 2 weeks and group B was 9.85 ± 0.55 at 8 weeks which was no significant difference compared with the group D at the same time (P gt; 0.05). Histological observation showed hemorrhage, cellular edema, inflammatory cell infiltration, nerve cell swell and solution of Nissl body 12 hours after SCI in group C. As time passed by, the number of nerve cells decreased, the gl ial cell prol iferated and Nissl body vanished. There was much gl ial cell prol iferation and cavitation 2 weeks after SCI in group C. The nerve cell decrease and cavitation in group B was sl ighter than that in group C, and group A was the sl ightest. In group D, there was no obvious change of the number of cells during the observation apart from sl ight edema in early period. Conclusion The mouse model precisely reflects the pathological and physiological features and law of change after different degrees of SCI, and can be used as a standard of mouse model of traumatic SCI by Allen’s WD.
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.