Objective To investigate the synergistic effect of a combination of grafted olfactory ensheathing cells (OECs) from the olfactory bulbs and intrathecal injection of vascular endothel ial growth factor (VEGF) on repairing spinal cord injury, and to explore the neuroprotection on both neurons and nerve fibers. Methods OECs from neonatal rats were cultured, purified, and collected with 0.25% trypsin after 9 days. A total of 75 adult female Wistar rats (weighing 200-250 g) were randomly divided into 5 groups: group A was sham-surgery group receiving laminectomy; the spinal cord injury model was establ ished with weight-dropped apparatus in the rats of groups B, C, D, and E. Then group B was injected with 10 μL DMEM-F12 medium without serum at injury site on the 1 day and was intrathecally administrated with 10 μL sal ine solutiontwice a day during the following 1 week; group C was injected with 10 μL DMEM-F12 medium and 25 ng recombined ratVEGF165 (rrVEGF165); group D was injected with 10 μL DMEM-F12 medium containing 1 × 105 OECs and 10 μL sal ine solution; group E was injected with 10 μL DMEM-F12 medium containing 1 × 105 OECs and 25 ng rrVEGF165. The functional recovery of hindl imb was evaluated by the Basso-Beattie-Bresnahan (BBB) score at 1 day and each week from 1 to 8 weeks. The histological changes and the changes of ultrastructure were observed at 8 weeks after operation by HE and electron microscope, and the immunohistochemistry staining was used for p75 nerve growth factor receptor (p75NGFR), Caspase-3, and von Willebrand factor (vWF). Results The function of hindl imb recovered rapidly in group E; the BBB score reached the peak at 8 weeks, and it was significantly higher than those in other groups (P lt; 0.05). The histology and ultrastructure observation showed that nerve fibers and neurons were damaged seriously in group B, oderately in groups C and D, and sl ightly in group E. Numerous spared tissue between nerve stumps, fibers with regular myel ination, and neurons with l ittle vacuolar mitochondria were observed in group E. The immunohistochemistry staining revealed that Caspase-3 positive cells in groups B, C, D, and E were significantly more than that in group A (P lt; 0.05); more Caspase-3 positive cells were found in groups B and D than in groups C and E (P lt; 0.05), while no significant difference was found between groups C and E (P gt; 0.05). And more vessels per high field were examined in groups C and E than in groups A, B, and D (P lt; 0.05), while no significant difference was found between groups C and E (P gt; 0.05). The p75NGFR positive results showed the survival of OECs in groups D and E at 8 weeks after OECstransplantation. Conclusion Grafted OECs combined with intrathecal injection of VEGF has significant promotive effects on restoration of spinal cord injury in rats, can improve part function of nerve fibers, and shows neuroprotection on damaged cells and fibers, which have a synergistic effect.
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 analyze the therapeutic effect of olfactory ensheathing cells (OECs) transplantation for central nervous system diseases. Methods Between November 2001 and January 2008, 1 255 participants with central nervous system diseases were enrolled in this cl inical study for fetal OECs transplantation. There were 928 males and 327 femalesaged 1.2-87 (mean 40) years. The course of disease was (4.52 ± 4.67) years. Among them, 656 participants suffered from chronic spinal cord injury (SCI), 457 amyotrophic lateral sclerosis (ALS), 68 cerebral palsy (CP), 20 multiple sclerosis (MS), 11 the sequelae of stoke, 10 ataxia, and 33 residual diseases. The participants came from 71 countries or regions. Accidentally abortional fetal olfactory bulbs were donated voluntarily and were cultured for 2 weeks, then were transplanted. Results One thousand one hundred and twenty-eight cases were followed up for 2-8 weeks (mean 4 weeks) to obtain integrated data. Among them, the neurological functional amel ioration was noticed in 994 participants with the overall short-term improvement rate of 88.12%. Seventy-six patients experienced the various perioperative compl ications with the incidence rate of 6.74%. One hundred and twenty patients with SCI received over 1 year follow-up. And according to ASIA assessment, motor scores increased from (39.82 ± 20.25) to (44.55 ± 18.99) points, l ight touch scores from (51.56 ± 25.89) to (59.81 ± 27.72) points, pain scores from (50.36 ± 27.44) to (57.09 ± 28.51) points for foreign patients (P lt; 0.05); motor scores increased from (40.52 ± 20.80) to (46.45 ± 20.35) points, l ight touch scores from (55.64 ± 26.32) to (68.64 ± 25.89) points, pain scores from (57.05 ± 26.00) to (66.13 ± 24.29) points for good rehabil itation Chinese patients (overall P lt; 0.05); motor scores from (37.03 ± 18.52) to (38.03 ± 18.50 points (P lt;0.05), l ight touch scores from (45.88 ± 22.56) to (46.63 ± 23.09) points (P gt; 0.05), pain scores from (45.25 ± 23.68) to (45.28 ± 23.63) points (P gt; 0.05) for poor rehabil itation Chinese patients. Compared foreign patients and good rehabil itation Chinese patients with poor rehabil itation Chinese patients, difference in score change was remarkable (P lt; 0.05). One hundred and six cases of ALS, 32 CP, 8 MS, 7 ataxia, and 2 stroke sequelae were followed up for 3-48, 3-36, 2-20, 7-17, 6 and 24 months, One hundred and six cases of respectively. Majority of them (113/155, 72.9%）were benefited from OECs transplantation. Conclusion OECs transplantation into brain and spinal cord is feasible and safe . The therapeutic strategy is valuable treatment for such central nervous system diseases such as chronic SCI, ALS, CP and stroke sequelae and can improve the patients’ neurological functions and/or decrease the progressive deterioration.
Objective To investigate the synergetic effect and possibil ity of repairing spinal cord injury (SCI) by transplantation of olfactory ensheathing cells (OECs) and chondroitinase ABC (ChABC) in adult rats. Methods Three adult male SD rats were used to isolated olfactory bulb and primarily cultured OECs. In the 8th or 9th day, OECs were transplanted, the concentration of cells was modulated to 1 × 105/μL. Fifty-four SD rats were made the models of T8 spinal cord crush injury and divided into 4 groups. In group A (control, n=36), injured site was not treated; in groups B, C and D (n=6), OECs, ChABC and OECs+ChABC were injected into injured site, respectively. At 1, 2, 3, 7 and 14 days after injury, the BBB score system was used to evaluate the motion function. At 0, 1, 2, 3, 7, 14 days in group A and at 14 days in groups B, C, D after injury, the maximal transverse diameter and gross area of necrosis were evaluated on HE stained sections. The immunofluorescence double label ing staining for gl ial-fibrillary acidic protein (GFAP)/CS56, GFAP/growth associated protein 43(GAP-43) and GFAP/neurofilament 160(NF160) was carried out to evaluate the regeneration of nerve fiber. Results At 14 days after injury, there were significant difference in the BBB scores between group A and groups B, C, D (P lt; 0.05), and between groups B, C and group D (P lt; 0.05), HE staining showed that the formation of cavity was observed in each group at 14 days after injury. There were significant difference in the maximal transverse diameter and gross area of necrosis between groups B, C, D and group A (P lt; 0.01), and between groups B, C and group D (P lt; 0.01). The immunofluorescence staining indicated that expression of GFAP were more intense in group A than in other groups, and the cavity of the lesion site was apparent, but it was moderate in groups B and C. The expression of GAP-43 was more intense in group D than in groups B and C. The expression of NF160 was more intense in group D. Conclusion Transplantation strategy of OECs combined with ChABC was effective in the repair of SCI in some extent.
Objective To investigate the biocompatibil ity of silk fibroin nanofibers scaffold with olfactory ensheathing cells (OECs) and to provide an ideal tissue engineered scaffold for the repair of spinal cord injury (SCI). Methods Silk fibroin nanofibers were prepared using electrospinning techniques and were observed by scanning electron microscope (SEM). Freshly isolated OECs from SD rats purified by the modified differential adherent velocity method were cultured. The cells at passage 1 (1 × 104 cells/cm2) were seeded on the poly-l-lysine (control group) and the silk fibroin nanofibers (experimental group) coated coversl ips in Petri dish. At desired time points, the morphological features, growth,and adhesion of the cells were observed using phase contrast inverted microscopy. The OECs were identified by the nerve growth factor receptor p75 (NGFR p75) immunofluorescence staining. The viabil ity of OECs was examined by l ive/dead assay. The prol iferation of OECs was examined by MTT assay. The cytotoxicity of the nanofibers was evaluated. Results The SEM micrographs showed that the nanofibers had a smooth surface with sol id voids among the fibers, interconnecting a porous network, constituted a fibriform three dimensional structure and the average diameter of the fibers was about (260 ± 84) nm. The morphology of OECs on the experimental group was similar to the cell morphology on the control group, the cells distributed along the fibers, and the directions of the cell protrusions were in the same as that of the fibers. Fluorescence microscopy showed that the purity of OECs was 74.21% ± 2.48% in the experimental group and 79.05% ± 2.52% in the control group 5 days after culture. There was no significant difference on cell purity between two groups (P gt; 0.05). The OECs in the experimental group stained positive for NGFR p75 compared to the control group, indicating that the cells in the experimental group still maintained the OECs characteristic phenotype. Live/dead staining showed that high viabil ity was observed in both groups 3 days after culture. There was no significant difference on cell viabil ity between two groups. The prol iferation activity at 1, 3, 5, 7, and 10 days was examined by MTT assay. The absorbency values of the control group and the experimental group had significant differences 3 and 5 days after culture (P lt; 0.05). The relative growth rates were 95.11%, 90.35%, 92.63%, 94.12%, and 94.81%. The cytotoxicity of the material was grade 1 and nonvenomous according to GB/T 16886 standard. Conclusion Silk fibroin nanofibers scaffold has good compatibility with OECs and is a promising tissue engineered scaffold for the repair of SCI.
Objective To evaluate proton MR spectroscopy (1H-MRS) for detection of the motor cortex and adjacent brain in amyotrophic lateralsclerosis (ALS) patients with apparent upper motor neuron involvement after olfactory ensheathing cells(OECs) transplantation. Methods From December 2004 to February 2005, 7 patients with clinically definite ALS who could safely undergo MRS were admitted into the perspective study. The neurological status, ALS functional rating scale (ALSFRS), EMG, and 1H-MRS taken before and 2 weeks after operationswere carefully analyzed. The NAA/Cr and Cho/Cr ratios were measured in the cerebral peduncle,genu and posterior limb of the internal capsule, corona radiata and precentral gyrus. Results The ALSFRS in 2 cases mproved obviously whose ALSFRS increased from 30 to 33 and from 29 to 34 respectively. And 5 cases remained stable 2 weeks after OECs transplantation. Statistical analyses for all seven cases showed both theNAA/Cr and Cho/Cr ratios decreased, but in the two cases with ALSFRS improvement the NAA/Cr increased in the certain anatomic position which confirmed the neurological and EMG findings. Conclusion The proton MR spectroscopy is a suitablenoninvasive measure for ALS evaluation. The preliminary study suggests that twoof the seven ALS cases improved apparently shortterm after OECs transplantation. More patients are required for the clinical study and longer followup duration is needed for future research.
Objective To determine whether transplanting olfactory ensheathing cells (OECs) is effective in controlling or reversing the deterioration caused by amyotrophic lateral sclerosis (ALS). Methods Between February 2003 and April 2006, 327 patients (241 males and 86 females) with probable or definite ALS (diagnosed according to the El Escorial criteria) received the OECs transplantation. Their ages ranged from 20 to 84 years (51.6±11.1 years). The duration of symptoms before surgical treatment was 4-8 months to 13 years (2.9±2.0 years). OECs were cultured and injected into pathological regions of the spinal cord and/or bilateral corona radiata of the brain; the patients were divided into three groups, group A (cord only,n=29), group B (cord and brain,n=6), and group C (brain only,n=292) based on the transplant sites. Results The patient’s neurological function was assessed both before and at 4 weeks after transplantation by using the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS) of the ALS CNTF Treatment Study (ACTS). The scores were increased from 17.2±8.6 preoperation to 20.1±9.7 postoperation in group A (P＜0.05),from 24.2±6.8to 25.7±6.6 (P＞0.05) in group B, and from 20.3±8.6 to 22.0±9.4 (P＜0.001) in group C.There were no significant difference in increased ALSFRS scores among the threegroups (P＞0.05). The total improvement rate of neurological function was 77.1% (252/327). The result of electromyographic examination showed that spontaneouspotential diminished and/or disappeared, the amplitude of the motor unit actionpotential decreased remarkably and the numbers of motor unit action potential greatly increased in 261 cases (79.8%). Sixteen patients (4.9%) experienced thevarious complications including headache, shortterm fever, seizure attack, central nerve system infection, pneumonia, respiratory failure, urinary tract infection, heart failure, and possible pulmonary embolism; of them, there were 4 deaths(1.2%). Conclusion These preliminary results suggest that the OECs transplantation is effective in controlling or reversing the physiological deterioration caused by ALS.
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.
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 study the effect of olfactory ensheathingcells(OECs) transplantation on protecting spinal cord and neurons after peripheral nerve injury. Methods Fifty-five SD rats were randomly divided into blank group (n=5), experimental group (n=25) and control group (n=25). The right sciatic nerves of all the rats were transected. The proximal end was embedded in muscle and treated with OECs (experimental group) and DMEM (control group). No treatment was given to the blank group. The rats were sacrificed 1, 2, 3, 7, and 14 days after the transplantation, the related neurons were observed with histological and TUNEL methods. Results After sciatic nerves were transected, death of neurons occurred in spinal cord and ganglion. One, 2, 3 days after treatment, the neuron survival rate in experimental group was 98.4％±6.5%，97.6％±6.5%，95.2％±6.7% respectively. The neuron survival rate in control group was 97.8％±6.7%，97.4％±6.4%，94.3％±6.8% 1, 2, and 3 days after treatment respectively. There was no significant difference between experimental group and control group. Seven and 14 days after treatment, the neuron survival rate in experimental group was 92.4％±8.9%，87.7％±9.4% respectively. The neuron survival rate in control group was 87.4％±8.6%，83.4％±8.5% 7 and 14 days after treatment respectively. There was significant difference between experimental group and control group. On 1st and 2nd day, no apoptosis was seen in spinal cord anterior horn of the rats in both experimental group and control group. On 3rd, 7th, and 14th day, the apoptosis index of spinal cord anterior horn motoneuron in experimental rats were lower（1.2±0.8，1.4±0.6，4.1±1.3） than that in the control group（2.1±1.1，3.1±1.1，6.1±1.8）（Plt;0.05）. One, 2, and 3 days after the operation, no ganglion neurons apoptosis was observed in all rats. On 7th day the apoptosis index of ganglion neurons in experimental group（2.10±0.32）were lower than thatin control group （4.40±0.56）（Plt;0.05）. On 14th day there was no significant difference in the apoptosis index of ganglion neurons between experimental group （4.30±1.80）and control group（6.70±2.50）(P＜0.05). Conclusion Apoptosis of neurons occur after peripheral nerve injury in spinal cord and ganglion. OECs transplantation is effective in preventing apoptosis.