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.
Objective To investigate the effect of olfactory ensheathing cell culture medium (OECCM) on the growth of spinal cord neurons and its protective effect on the injured neurons by H2O2, and to disscuss the probable protective mechanisms of olfactory ensheathing cells (OECs). Methods The primary olfactory ensheathing cells (OECs) were isolated from olfactory bulb of adult SD rat, and OECCM were prepared. The morphology of OECs was observed by inverted phase contrast microscope, identified by rabbit-antiratlow-affinity nerve growth factor p75 (NGFRp75), and its purity were calculated.Primary spinal cord neurons were cultured from 15 to 17 days pregnant SD rats, and injury model of neurons were prepared by H2O2. OECCM and control culture medium were added into the normal spinal neurons (groups A, B). OECCM and control culture medium were added into the injured spinal neurons by H2O2 (groups C, D). In groups A and C, 200 μL of control culture medium was used; in groups B and D, 100 μL of control culture medium and 100 μL of OECCM were used. Then the growth index such as average diameter of neuron body, the number and length of neuron axons were measured. The viabil ities of normal and injured neurons were assessed by MTT. Results OECs showed bipolar or tripolar after 6-9 days of culture. Primary spinal cord neurons were round and bigger, and neuron axons grew significantly and showed bipolar after 5-7 days of culture. The immunocytochemisty of OECs by NGFRp75 showed that membrane were stained. The degree of purity was more than 90%. Primary spinal cord neurons grew well after 6-9 days of culture, and compared with group A, neurons of group B grew b, whose cell density and diameter were bigger. The average diameter of neuron body, the number and length of neuron axons were (33.38 ± 6.80) D/μm, (1.67 ± 0.80), and (91.19 ± 62.64) L/μm in group A, and (37.39 ± 7.28) D/μm, (1.76 ± 0.82), and (121.33 ± 81.13) L/μm in group B; showing statistically significant differences (P lt; 0.05). The absorbency (A) value of neurons was 0.402 0 ± 0.586 9 in group A and 0.466 0 ± 0.479 0 in group B; showing statistically significant difference (P lt; 0.01). After 2 hours of injury by H2O2, the cell density of spinal cord neurons decreased, and neuron axons shortened. The A value of injured neurons was 0.149 0 ± 0.030 0 in group C and 0.184 0 ± 0.052 0 in group D, showing statistically significant difference (P lt; 0.01). Conclusion The results above suggest that OECCM could improve the growth of spinal cord neurons and protectthe injured neurons. The neurotrophic factors that OECs secrete play an important role in the treatment of spinal cord injury.
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 explore the construction of a canine model of vascularized allogeneic spinal cord transplantation (vASCT) and preliminarily evaluate its therapeutic efficacy for spinal cord injury (SCI). Methods Sixteen female Beagle dogs aged 8-12 months were randomly selected, with 8 dogs serving as donors for the harvesting of spinal cord tissue with a vascular pedicle [dorsal intercostal artery (DIA) at the T10 level and accompanying vein]. The remaining 8 dogs underwent a 1.5-cm-length spinal cord defect at the T10 level, followed by transplantation of the donor spinal cord tissue for repair. Polyethylene glycol (PEG) was applied to both ends to spinal cord graft; then, using a random number table method, the dogs were divided into an experimental group (n=4) and a control group (n=4). The experimental group received immunosuppressive intervention with oral tacrolimus [0.1 mg/(kg∙d)] postoperatively, while the control group received no treatment. The operation time and ischemia-reperfusion time of two groups were recorded. The recovery of hind limb function was estimated by Olby score within 2 months after operation; the motor evoked potentials (MEP) was measured through neuroelectrophysiological examination, and the spinal cord integrity was observed through MRI. ResultsThere was no significant difference in the operation time and ischemia-reperfusion time between the two groups (P>0.05). All dogs survived until the completion of the experiment. Within 2 months after operation, all dogs in the control group failed to regain the movement function of hind limbs, and Olby scores were all 0. In the experimental group, the movement and weight-bearing, as well as walking abilities of the hind limbs gradually recovered, and the Olby scores also showed a gradually increasing trend. There was a significant difference between the two groups from 3 to 8 weeks after operation (P<0.05). Neuroelectrophysiological examination indicated that the electrical signals of the experimental group passed through the transplanted area, and the latency was shortened compared to that at 1 month after operation (P<0.05), showing continuous improvement, but the amplitude did not show significant improvement (P>0.05). The control group was unable to detect any MEP changes after operation. MRI examination showed that the transplanted spinal cord in the experimental group survived and had good continuity with normal spinal cord tissue, while no relevant change was observed in the control group. ConclusionThe vASCT model of dogs was successfully constructed. This surgical procedure can restore the continuity of the spinal cord. The combination of tacrolimus anti-immunity is a key factor for the success of transplantation.
OBJECTIVE To establish an artificial bladder reflex arc in canines to reinnervate the neuropathic bladder and restore bladder function after spinal cord injury. It involves a somatic reflex arc with a modified efferent branch which passes the somatic motor impulses to the bladder and initiates autonomic bladder detrusor contraction. METHODS Intradural microanastomosis of the right L5 ventral root to S2 ventral root was performed to maintain the right L5 dorsal root intact. After axonal regeneration, the new patellar ligament-spinal cord center-bladder artificial bladder reflex pathway was established, and micturition was induced by knocking the patellar ligament. The early and final function of the reflex arc was observed by electrophysiological examinations, bladder pressure tests and detrusor electromyograms(EMG) at 6 months and 18 months postoperatively. RESULTS Single stimuli (115 mV, 1.0 ms) of the right L5 dorsal root resulted in evoked potentials recorded from the right S2 ventral root distal to the anastomosis site before and after the spinal cord was transected horizontally at the T10 segment level in all 6 canines. Bladder contraction was very quickly initiated by trains of stimuli(1,000 mV, 10 Hz, 2 s) of the right L5 dorsal root and bladder pressures increased rapidly to 65% of normal, and bladder contraction induced by knocking the right patellar ligament was increased to 51% of normal through the new reflex arc in 4 canines after 6 months of operation. Bladder pressures were increased by the same stimuli to average 84% of normal and to 62% of normal by knocking the patellar ligament in 2 canines after 18 months of operation. Stimuli(3.8 mA, 1.0 Hz) of the right L5 dorsal root and femoral nerve resulted in EMG similar to normal EMG could be recorded from the detrusor in 2 canines after 18 months postoperatively. CONCLUSION The somatic motor axons can be regenerated into the parasympathetic endoneurial tubes of autonomic nerve. Using the survived somatic reflex under the horizon of spinal cord injury to reconstruct the bladder autonomic reflex arc by intradural microanastomosis of ventral root is practical in the canine model and may have a potential of clinical application.
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.
ObjectiveTo investigate the effect of acute non-isovolemic hemodilution (ANIH) on spinal surgery. MethodsFrom January 2012 to July 2013, 80 patients scheduled for spinal surgery were enrolled. The patients were randomized into four groups, with 20 in each group. Patients in group A were infused with Ringer's injection and polygeline for fluid loss, physiological needs and blood loss. In group B, the patients were infused with acute hypervolemic hemodilution. In group C, patients were infused with acute nomovolemic hemodilution. In group D, patients were infused with ANIH. The hemodynamics and arterial blood gas indexes were detected in all the patients. ResultsThe mean blood pressure in group A and C was significantly more stable than that in group B and D. The central vein pressure in group B and D was significantly higher than that in group A and C after hemodilution (P<0.05), while the hematocrit in group B and D was significantly lower than that in group A and C (P<0.05). Allogeneic blood transfusion was performed in group A and B, while it was not performed in group C and D. ConclusionANIH can reduce the volume of blood loss during spinal surgery, and it is safe.
OBJECTIVE: To investigate the etiology, pathological mechanism and treatment of cervical fracture-dislocation without spinal cord injury. METHODS: Nine patients with cervical fracture-dislocation without spinal cord injury were male and aged 22 to 63 years. Based on the clinical symptoms and roentgenographic changes, the injury mechanism was analyzed; and the pathological characteristics and treatment principle were put forward. RESULTS: Anterior reduction was employed in all 9 cases. Eight cases were reduced completely while 1 case was reduced partially. After following up 1 to 3 years, 7 cases recovered completely and the other 2 cases relieved their symptoms obviously. No nervous symptoms aggravated during the following-up period. CONCLUSION: Fracture-dislocation of the cervical spine without spinal cord injury has special pathological mechanism. The surgical intervention is needed for solid fixation and complete decompression without any delayed neurosymptoms.
OBJECTIVE: To investigate the protective effect of tumor necrosis factor-alpha(TNF-alpha) on spinal motor neurons after peripheral nerve injury. METHODS: Twenty Wistar rats were divided into two groups, the right sciatic nerves of 20 Wistar rats were transected, the proximal stumps were inserted into a single blind silicone tube. 16 microliters of normal saline(NS) and TNF-alpha(30 U/ml) were injected into the silicone tubes. After 2 weeks, the 4th, 5th lumbar spinal cord were taken for examination. Enzyme histochemical technique and image analysis were used to show acetylcholinesterase(AChE) and nitric oxide synthase(NOS) activity of spinal motor neurons. RESULTS: The number of AChE and NOS staining neurons were 8.65 +/- 1.98 and 5.92 +/- 1.36 in the experimental group and 6.37 +/- 1.42 and 8.67 +/- 1.45 in the control group respectively, there were significant difference between the two groups(P lt; 0.01). CONCLUSION: It suggests that TNF-alpha has protective effect on motor neurons after peripheral nerve 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.