Objective To explore the influence factors for the functional improvement after the fetal olfactory ensheathing cell (OEC) transplantation for chronic spinal cord injury(SCI). Methods The olfactory bulbs were harvested and trypsinized down to single fetal OEC. They were cultured for 12-17 days prepared for use. From November 2001 to December 2003, a total of 300 patients volunteered for the fetal OEC transplantation, among whom 222 suffered from complete chronicSCI and 78 suffered from incomplete chronic SCI. The procedures were performed on the patients with a disease course ranging from 6 months to 31 years (average 3.1 years) after their injuries. The fetal OEC was transplanted by the form of injections into the spinal cord at the upper and lower ends of the injury site. All the patients were assessed by the ASIA standard before the transplantation and 2-8 weeks after the transplantation. The influence factors including age, sex, duration after the injury, and injury degreesand levels were compared with those in the functional improvement after fetal OEC transplantation. Results The partially-improved neurological functions assessed by the ASIA standard were indicated by the motor scores increasing from 39.1±20.6 to 45.9±20.3 (Plt;0.001), the light touch scores from 51.7±24.9 to 63.4±23.0 (Plt;0.001), and the pin prick scores from 53.0±24.2 to 65.3±22.7(Plt;0.001). There was no significant difference in the functional improvement of the motor, light touch, and pin brick when compared with the age, sex, duration after theinjury, and the injury degrees and levels. The motor scores and light touch scores at the cervical level were higher than the scores at the thoracic level. Conclusion The fetal OEC transplantation can partially improve the neurological functions quickly in treatment of the chronic spinal cord injury. All the influence factors except the motor scores and light touch scores, which were higher at the cervical level than at thoracic level, have no impact on the functional improvement after the fetal OEC transplantation.
Objective To determine the safety of the fetal olfactory ensheathing cell(OEC) transplantation in patients with chronic spinal cord injury (SCI) by examination of the magnetic resonance imaging (MRI). Methods A prospective clinical study involving 16 patients with chronic SCI was designed to investigate the feasibility and biological safety of the fetal OEC transplantation in treatment of SCI. The olfactory bulbs from the 3-4-month-old aborted human fetuses following the strict ethical guidelines were harvested and trypsinized down to single fetal OEC. These cells were then cultured for 12-17 days and were prepared for a clinical use. From November 2001 to December 2002, 16 patients with chronic SCI were randomly enrolled. The patients suffered from SCI for1.5-8 years (average 4-3 years) after the injury. The suspension (50 μl) containing about 1×106 fetal OECs was transplanted by an injection into the patients’ spinal cords above and below the injury site. All the patients were assessed before thetransplantation and were followed up with MRI for 29-42 months (average 38 mon)after the transplantation. Results No cell-related adverse effects were observed in any patient during the followup period. The follow-up with MRI did not reveal any development of optic glial tumor, tumor-like mass, new hemorrhage,edema, expanding cyst, new cyst formation, infection or disruption of the neuralstructure in the transplant site of all the patients. Conclusion This is the first clinical study demonstrating the long-term safety of theOEC therapy for SCI. The results indicate that our protocol is feasible and safe in treatment of patients with chronic SCI within 38 months after the injury. Although the size of the samples for our study was not big enough, the positive results of the study have encouraged us to make a further research in this field.
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.
OBJECTIVE To study the early protective effects of basic fibroblast growth factor(bFGF) on the experimental acute spinal cord injury. METHODS Thirty-four SD rats were randomly divided into three groups, and were subjected to contusion of thoracolumbar spinal cord. A thin plastic tube was placed in subarachnoid space below the injury level for perfusion. The bFGF-treated rats were received 20 microliters bFGF(containing bFGF 100 U) at once, 30 min, 1, 2, 3, 4, 6, 12, 24 and 48 hours after injury, and an equal volume of normal saline was given to the control group at the same time. The injured spinal cord was detected by morphological observation and biochemical index after injury. RESULTS The degree of ionic disorder in bFGF-treated rats was significantly ameliorated and the contents of H2O were also markedly decreased. The morphological finding showed that the damages of gray and white matter in bFGF-treated rats were slighter than those of saline-treated rats. CONCLUSION bFGF has some protective effects on the secondary lesion of early spinal cord injury in rats.
In order to observe the role of genetically modified Schwann cell (SC) with pSVP0Mcat in the regeneration of injured spinal cord, the cells were implanted into the spinal cord. Ninety SD rats were used to establish a model of hemi-transection of spinal cord at the level of T8, and were divided into three groups, randomly, that is, pSVP0Mcat modified SC implantation (Group A), SC implantation (Group B) and without cell implantation as control (Group C). After three months the presence of axonal regeneration of the injured spinal cord was examined by means of horseradish peroxidase (HRP) retrograde labelling technique and stereography. The results indicated that HRP labelled cells in Group A and B could be found in the superior region of injured spinal cord and the brain stem such as the red nuclei and oculomotor nuclei. The density of ventral hom neurons of the spinal cord and the number of myelinated axons in 100 microns of the white matter was A gt; B gt; C group. In brief, the pSVP0Mcat modified SC intraspinal implantation could promote regeneration of the injured spinal cord.
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 investigate the division, prol iferation and differentiation abil ities of nestin+/GFAP+cell after spinal cord injury and to identify whether it has the characteristic of neural stem cells (NSCs). Methods Twelvemale SD rats, aged 8 weeks and weighing 200-250 g, were randomized into 2 groups (n=6 per group): model group inwhich the spinal cord injury model was establ ished by aneurysm cl ip compression method, and control group in which no processing was conducted. At 5 days after model ing, T8 spinal cord segment of rats in each group were obtained and the gray and the white substance of spinal cord outside the ependymal region around central tube were isolated to prepare single cellsuspension. Serum-free NSCs culture medium was adopted to culture and serum NSCs culture medium was appl ied to induce differentiation. Immunohistochemistry detection and flow cytometry were appl ied to observe and analyze the type of cells and their capabil ity of division, prol iferation and differentiation. Results At 3-7 days after injury, the model group witnessed a plenty of nestin+/GFAP+ cells in the single cell suspension, while the control group witnessed few. Cell count of the model and the control group was 5.15 ± 0.71 and 1.12 ± 0.38, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Concerning cell cycle, the proportion of S-phase cell and prol iferation index of the model group (15.49% ± 3.04%, 15.88% ± 2.56%) were obviously higher than those of the control group (5.84% ± 0.28%, 6.47% ± 0.61%), indicating there were significant differences between two groups (P lt; 0.01). In the model group, primary cells gradually formed threedimensional cell clone spheres, which were small in size, smooth in margin, protruding in center and positive for nestin immunofluorescence staining, and large amounts of cell clone spheres were harvested after multi ple passages. While in the control group, no obvious cell clone spheres was observed in the primary and passage culture of single cell suspension. At 5 days after induced differentiation of cloned spheres in the model group, immunofluorescence staining showed there were a number of galactocerebroside (GaLC) -nestin+ cells; at 5-7 days, there were abundance of β-tubul in III-nestin+ and GFAP-nestin+ cells; and at 5-14 days, GaLC+ ol igodendrocyte, β-tubul in II+ neuron and GalC+ cell body and protruding were observed. Conclusion Nestin+/GFAP+ cells obtained by isolating the gray and the white substance of spinal cord outside the ependymal region around central tube after compressive spinal cord injury in adult rat has the abil ity of self-renewal and the potential of multi-polarization and may be a renewable source of NSCs in the central nervous system.
OBJECTIVE: To observe the effect of nerve growth factor (NGF) and nimodipine (NP) on fetal spinal cord graft in repair of injury of spinal cord. METHODS: A total of 144 adult Wistar rats were included in this study. All were made as the hemi-section cavity injury model at the lumbar enlargement and divided into three groups: fetal spinal cord graft (group Tr), fetal spinal cord graft with NGF (group TN), and fetal spinal cord graft with NGF and NP (group TNN). The intracellular concentration of free ionic calcium was measured at the 4th, 8th, and 24th hour, and superoxidase (SOD) and malondialdehyde (MDA) at 3rd, 6th, 12th, 24th and 72nd hour after operation. RESULTS: After spinal cord was injured, the concentration of MDA and intracellular concentration of free ionic calcium increased and reached to the peak at the 6th and 8th hour respectively, but SOD decreased and at 24th hour to its vale. The MDA was significantly lower in group TN than in group Tr, while the SOD was higher (P lt; 0.05). There was no significant difference on intracellular free ionic calcium concentration between group Tr and TN. The concentration of SOD of group TNN was the highest and the intracellular concentration of free ionic calcium was the lowest in the three groups (P lt; 0.05). The weekly mortality was 33%, 31%, 17% respectively in group Tr, TN and TNN. The mortality of group TNN was significantly lower than the other two groups (P lt; 0.01). CONCLUSION: Although the fetal spinal cord graft is an effective method to repair laboratory spinal cord injury, NGF and ND can interrupt secondary injury and increase survival rate of the host.
Objective To investigate a new composite matrix (BMSCs seeded on the denuded human amniotic membrane, BMSCs-DHAM) bridging the both stumps of spinal cord injury in rats to promote axon regeneration and improve motor function of hind l imbs. Methods The human amniotic membrane (HAM) was voluntarily donated by the healthy pregnant women after a caesarean section. The cells on the HAM were completely removed with a tryptic and mechanical approach to prepare DHAM. The BMSCs were separated and cultured from 4-week-old female rats (n=4), then the forth passage of BMSCs were labeled by PKH26 and seeded on DHAM (BMSCs-DHAM). The growing state of BMSCs was observed under themicroscopy. Moreover, 40 female rats (8-week-old, weighting 200-220 g) were made spinal cord injury models by transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). The both stumps were respectively wrapped by BMSCs- DHAM or simple DHAM in groups A and C, and the same dose of BMSCs or physiological sal ine were also respectively injected the central lesion in groups B and D. At 12 weeks after surgery, the functional recovery of the hindl imbs was evaluated by the BBB locomotor rating score, and other indexes were tested including cortical motion evoked potential (MEP), anterograde biopinylated dextan amine (BDA) tracing, and immunofluorescence of neurofilament protein 200 (NF-200). Results HE staining proved that the DHAM was devoid of cellular components by this way, and BMSCs grew well on the substrate under the microscopy. At 12 weeks after operation, the BBB score (12.50 ± 1.26) in group A was significantly higher than those of other groups (P lt; 0.05), and the recovery in latency (3.52 ± 2.45) ms and ampl itude (480.68 ± 18.41) μV of MEP was also obviously improved in group A (P lt; 0.05) when compared with other groups. In addition, anterograde BDA tracing revealed that the rate of the positive BDA axons 54.12% ± 3.30% under the lesion level in group A was higher than those of other groups (P lt; 0.05), and lots of the regeneration axons (positive NF-200) were found to grow into the spinal cord under the composite matrix in group A. Conclusion The BMSCs-DHAM composite matrix can improve hindl imb motor function to some extent after spinal cord injury. It will be widely appl ied as the matrix material in the future.
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.