ABSTRACT
BACKGROUND: Spinal cord injury occurs frequently and its consequence is very severe. There is no effective method to rebuild the function of demylinated nerves. Transplantation of a kind of special glial cells in olfactory system of mammal attracts more attention.OBJECTIVE: To observe whether combined transplantation of human fetal olfactory ensheathing cells (human OECs) and rat embryonic spinal cord tissues (rat ECS) possesses synergistic effect in promoting axonal regeneration in the rats following spinal cord transection.transection.DESIGN: Open experiment.SETTING: Cell Room, Third People's Hospital of Wuxi City; Department of Neurology, First Hospital Affiliated to Soochow University; Department of Neurosurgery, Zhongda Hospital Affiliated to Central South University MATERIALS: This experiment was carried out in the cell laboratory of the Third People's Hospital of Wuxi from September 2002 to October 2004. ① Totally 36 adult female SD rats, of clean grade, were selected and randomly divided into 4 groups: human OECs group (n=10), rat ECS group (n=10), combined transplantation group (n=10) and sham-operation group (n=6). ② Fresh 12-week aborted human embryo was used for culture and purification of human OECs (Informed consent was obtained from the parturient). ③One SD rat at embryonic 14 days underwent caesarean operation, and fetal rat and fetal membrane were taken out together and used for preparing new embryonic spinal cord.METHODS: ①Rats of 4 groups were all created into hemisection cavity models. Gelatin sponge and complete culture medium of 8 μL were packed into the injured cavity of rats in the model group, and the same culture medium of 2 μL was injected at 1 mm above or below injure; Human OECs suspension of 8 μL was added to gelatin sponge in human fetal Human OECs group, and human OECs suspension of 2 μL was injected at 1 mm above and below injure; rat embryonic spinal cord tissue of rat ECS group was chipped into pieces, which were packed into the cavity,and gelatin sponge was spread on the injury part. Embryonic spinal cord with the same size was packed into the cavity of combined transplantation group, then 8 μL human OECs suspension was injected into cavity with micro sample injector, and gelatin sponge was spread on the injury part, and then cellular suspension of 2 μL was injected at 1 mm above and below the cavity, and muscular layer skin was sutured layer by layer. ②The rats of each group were performed ethological evaluation periodically. Combined with pathological observation, effect of human OECS and rat ECS on neuronal survival and regeneration was evaluated by performing horseradish peroxidase-tetramethyl benzidine tracer technique.MAIN OUTCOME MEASURES: ①In vitro culture and purification of human fetal human OECs. ② In vitro immunocytochemistrical analysis. ③BBB scoring of motor function of hindlimb of rats. ④ Immunohistochemical detection of implants and injured spinal cord repair⑤ Quantitative analysis on labeled neurons at the cortex and mesencephalic red nucleus ineach group with horseradish peroxidase-tetramethyl benzidine tracer technique.RESULTS: ① Most of human fetal OSCs presented double-polar spindle.Five to seven days after culture, OSCs weaved into net and a lot of mitosis phases were found. The cellular purity was 85%. ② The rate of P75 positive cells was (83±7)%. Glial fibrillary acidic protein was found in about (81±6)% of cells and Vimentin in (91±9)% of cells and the rate of Nestin positive was (77±5)%. ③Three to five days after operation, affected limb of rats of sham-operation group began to contract, the activity of hindlimb of intact side was limited a little. Fewer obvious contraction symptoms were found in the other 3 groups. From 2 weeks after operation,behavioral function recovered significantly fast in each group. BBB scores of combined transplanted group were significantly high than those of human OECs group, rat ECS group and sham-operation group [(6.2±1.13) vs.(5.0±1.15)vs.(3.9±0.88)vs.(3.3±1.03)scores,P ＜ 0.05]. ④In bipolar or multipolar cells, in which basic protein(+)granules were found, P75 and glial fibrillary acidic protein positive were found at the implanted part in the range of 2.0 to 5.0 mm of transplanted region in the human OECs group and combined transplantation group. A great many of small MAP2 positive neurons were found in the spinal defected focus in the rat ECS group and combined transplantation group. Nerve plexus positive fibers were observed in spinal defected region of human OECs group, rat ECS group and combined transplantation group to different extents, especially significantly in the combined transplantation group, but they were not found in the model group. ⑤ Horse radish peroxidase labeling was hardly found in neurons at the injured side of sham-operation group, while the number of labeled neurons at the cortex and nesencephalic red nucleus was significantly higher in the combined transplantation group than in the human OECs group and rat ECS group (P ＜ 0.05).CONCLUSION: Combined transplantation of OECs and ESC can obviously protect injured spinal cord, promote host spinal axonal regeneration and play s a complementary and synergetic effect in speeding up the functional recovery of rats.
ABSTRACT
BACKGROUND: One of the main causes of the failure of central nerve regeneration is that the microenvironment (lack of nerve growth factor, inhibitory factor produced by excretion and formation of glial scar) in the injured central nerves is not favorable for the regeneration of axons. Therefore, it is important to improve the microenvironment of injured area for the regeneration of axons. Recently, olfactory ensheathing cells (OECs) have been attracting much attention as a key method to treat central nervous injury.OBJECTIVE: To investigate the effect of OECs on traumatic brain injury (TBI) in rats and whether they can reduce neurological impairment after TBI.DESIGN: A randomized controlled experimental trial based on experimental animals.SETTING: Department of neurosurgery in a hospital affiliated to a university.MATERIALS: The experiment was conducted in the Central Laboratory of Department of Neurosurgery, Kaifeng Railway Hospital from April 2003 to August 2003. Altogether 100 healthy adult SD rats of either gender,weighting 250- 350 g, were randomly divided into four groups: normal group, TBI group, normal saline group and OEC group with 25 rats in each. Each group was further divided into five subgroups with 5 rats in each.INTERVENTIONS: The models of TBI in rats were established, and OECs were transplanted into brain tissues immediately after injury. The scores of nerve injury were assessed in the rats at day 1, day 4, week 1, week 2 and week 4. The distribution of OECs in brain tissues was observed after the rats were sacrificed.MAIN OUTCOME MEASURES: Neurological function recovery of rats and distribution of OECs in brain tissues.RESULTS: At week 2 and week 4 after operation, neurological severity scores (NSS) in OEC group significantly differed from those of TBI group and normal saline group. HE staining or immunohistochemistry of GFAP and p75 revealed that OECs could survive in the transplanted site and migrate toward the surrounding tissues. The total number of p75 positive cells in five coronal tissue slices of 6 μm thick was added up at different intervals. The results showed that the number of OECs was decreased with the passing time.CONCLUSION: OECs can survive in the transplanted site and migrate to the surrounding tissues when they are transplanted into the iujured brain tissues immediately after TBI. Giving OECs can reduce neurological and motor dysfunction induced by TBI.