Objective To establish the artificial bladder reflex arc by the normal body reflex pathway above the horizon of spinal cord injury to reinnervate the flaccid bladder and restore bladder micturition function. Methods An intradural microanastomosis was performed on the L6 ventral root tothe S2 ventral root. After axonal regeneration,the “patellar ligament-spinal cord center-bladder” reflex pathway was reestablished. A longterm function of the reflex arc was observed in the nerve electrophysiological experiment, detrusor electromyography experiment, and urodynamic testing 8 months after anastomosis. Results Trains of the stimuli(200 μV,5 ms) in the left L6 dorsal root and the nerve at the anastomosizedsite resulted in motor evoked potential from the disal to the anastomosized site before and after the spinal cord was destroyed horizontally between S1 and S4 segment levels in 2 Beegle dogs.The figure and amplitude of the evoked potential were similar to those of the control and general stability which showed anoninterventional wave. The urodynamic test revealed a rapid increase of the bladder pressure and a minor increase in the abdominal pressure. This showed that the bladder detrusor mainly resulted in the pressure increase.The bladder pressure increased to 60% of the normal on average compared with the controls when resulted in the left L6 dorsal root and the nerve anastomosized site were stinulated. Conclusion The long-term observation by the nerveelectrophysiological experiment, detrusor electromyography experiment, and urodynamic test indicate that the new artificial reflex arc can be established successfully. The somatic motor axons can regenerate into the parasympathetic endoneurial tubes of the autonomic nerve.
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.
The capacity of embryonic spinal cord tissue in the repair of injured structure of spinal cord has been noted for years. In order to investigate the embryonic spinal cord graft in the repair of motor function of injured spinal cord, the embryonic spinal cord tissue was transplanted to the hemisection cavity in spinal cord in adult rat. One hundred adult Wistar Rats were used to simulate the hemisectional injury of spinal cord by drilling 2-3 mm cavity in lumbar enlargement. Sixty rats were treated with rat embryonic spinal cord tissue grafting while the other forty were chosen as control. The outcome was evaluated according the combined behavioural score (CBS) and motor evoked potential (MEP) in the 1, 2, 4 and 12 weeks. The grafting group was superior to the control as assessed by CBS (P lt; 0.05), especially within 4 weeks. (P lt; 0.01). The restoration of the latent peak of early wave(P1, N1) was better in the grafting group, too. This suggested that embryonic spinal cord graft could improve the recovery of motor function of injured spinal cord in adult rat. The effect of the embryonic spinal cord tissue graft might be concerned with its secretion of several kinds of neurotrophic factors, nerve growth factor, nerve transmitted factor, or adjustment of hormone.
Objective To investigate the effects of Neuritin on the regeneration of the neural axons after acute spinal cord injury (SCI) in rats. Methods The model of acute SCI at T10 was establ ished in 54 adult healthy Wistar rats (half males and half females) weighing 250-300 g by using the improved Allen’s weight-drop method. The rats were randomly dividedinto 3 groups. 100 μL (6 μg) Neuritin and His protein was injected into group A (n=24) and group B (n=24), respectively,through subarachnoid catheter. Six rats from each group were killed 3, 7, 14, and 28 days after injury to receive Basso, Beattie and Bresnahan (BBB) locomotor rating scal ing, HE staining observation, and immunohistochemistry staining observation for neurofilament 200 (NF-200) and growth associated protein 43 (GAP-43). Group C (n=6) served as sham-operated group receiving laminectomy without spinal injury and with an empty catheter in the subarachnoid space and received the above observations 7 days after injury. Results BBB scale: after operation, the scale of groups A and B was increased over time; group A was significantly higher than group B from 14 days (P lt; 0.05); group C was higher than groups A and B at different time points after operation (P lt; 0.05). HE staining: in group C, the injured spinal tissue was normal after operation; from 7 days after operation, group A presented deeper-stained nissl body, less physal iferous cells, and more nerve synapses when compared with group B. NF-200 and GAP-43 immunohistochemistry observation: in group C, there was just l ittle positive expression; while in groups A and B, positive expression of NF-200 and GAP-43 was evident in the spinal cord from 7 days after operation. Mean density integral absorbency (IA) value of NF-200 and GAP-43: group A was higher than group B at each time point (P lt; 0.05) and group C was lower than groups A and B at each time point (P lt; 0.05). Conclusion Local application of exogenous Neuritin can promote the axonal regeneration after acute SCI in rats and the recovery of the locomotion function of hind-limbs in rats.
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.
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.
Gene therapy develops very rapidly during the resent years. Great prospects have been demonstrated from basic study and clinic test. However, the gene therapy in CNS is still in stage of laboratory. The research status and prospects of gene therapy in spinal cord injury (SCI) were introduced. The basic principle is to transplant certain cells genetically modified with NTFs to the site of the injuried spinal cord, then NTFs are expressed in vivo and stimulate axon regrowing. Virus vectors are usually used for gene transfer because of their high rate of transfection, and the receptor cells include fibroblast, myoblast, etc. Nowadays, gene therapy in SCI is studied in many laboratories and the problems include: 1. The ideal components of transfer gene. 2. The choice of carrier. 3. Immune reaction, and prolonged survival and persistent expression of the receptor cells in the spinal cord. If these problems could be solved, the gene therapy would become the key method in the therapy of SCI.
【Abstract】 Objective To review the progress in the treatment of spinal cord injury (SCI) by graft of neuralstem cells (NSCs) or bone marrow mesenchymal stem cells (BMSCs) as well as immune characteristics of two stemcells. Methods Different kinds of documents were widely collected, and then immunologic characteristics of NSCs andBMSCs were summarized. The therapy of SCI by stem cell transplantation was reviewed. Additionally, some problems intreatment were analyzed. Results Experimental study showed that graft of NSCs and BMSCs can promote the functionalrecovery of the injured spinal cord in animals. Due to immunologic properties of two stem cells, rejection reaction oftransplantation could produce a harmful effect on SCI treatment. Conclusion Transplantation of NSCs or BMSCs might bean effective measure for SCI treatment, but immunologic rejection reaction must be considered.
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 Adenosine tri phosphate (ATP) can promote the repair of spinal cord injury (SCI). To investigate the effect of ATP combined with bone marrow mesenchymal stem cells (BMSCs) transplantation on SCI, and to evaluate the synergistic action of ATP and BMSCs in the repair of SCI and the feasibil ity of the combined transplantation in the treatment of SCI. Methods BMSCs were isolated from the marrow of the tibia and the femur of a male SD rat (weighing 120 g), the 3rd generation BMSCs were labeled with BrdU, then BMSCs suspension of 5.0 × 107 cell/mL were prepared. Fortyeightadult female SD rats (weighing 240-260 g) were made SCI models at T12 levels according to the improved Allen’s method, and were randomly divided into 4 groups (groups A, B, C, and D, n=12). In group A, ATP (40 mg/kg) and BMSCs (6 μL) were injected to the central point and the other 2 points which were 1 mm from the each side of head and tail of the injured spinal cord; after blending the BMSCs suspension, the cells amount was about 3.0 × 105. In groups B, C, and D, the BMSCs suspension (6 μL), ATP (40 mg/kg), and PBS (40 mg/kg) were injected to the points by the same method as group A, respectively. The general conditions of the rats were observed after operation. The nerve function of low extremities was evaluated using the improved Tarlov scale and the Rivil in incl ined plane test at 1, 3, 7, 14, 21, and 28 days after operation. At 28 days after operation, the reparative effect of SCI was observed using histological and immunohistochemical staining. Results One rat of group A, 2 of group B, 2 of group C, and 3 of group D died of infection and anorexic, the others survived to the end of the experiment. Paralysis symptom in low extremities occurred in all rats after operation and was improved at 2-3 weeks postoperatively, the improvement of group A was the best, groups B and C were better, group D was the worst. There was no significant difference in the Tarlov scale and the Rivil in incl ined plane test among 4 groups at 1 and 3 days after operation and between groups B and C at 7, 14, 21, and 28 days after operation (P gt; 0.05), but there were significant differences among other groups at 7, 14, 21, and 28 days after operation (P lt; 0.05). At 28 days after operation, HE staining demonstrated that the injured region in group A was finely restored, without obvious scar tissue and cavity, and there existed clear stem cell differentiation characters; there was small amount of scar tissue and cavity in the injury site of groups B and C; and there was great deal of scar tissue in the injury site of group D, in which there were numerous inflammatory cells and fibroblasts infiltration and bigger cavity. Immunohistochemical staining showed that BrdU-positive BMSCs were seen in groups A and B, and positive cells of group A was significantly more than that of group B (P lt; 0.05). The expressions of neruofilament protein 200 and gl ial fibrillary acidic protein in group A were significantly higher than those in groups B, C, and D, and groups B and C were significantly higher than group D (P lt; 0.05). Conclusion ATP has protective effects on injured spinal cord, a combination of ATP and BMSCs can synergistically promote the reparation of SCI.