| BackgroundsSpinal cord injury (SCI) is a long-lasting, debilitating condition that affects 250,000 people each year in the United States, thus is the researching focus of scientists all over the world. Drugs, neurotrophic factors, tissue and cells graft and gene therapy are main approaches to promote the spinal cord recovery after injury. Because of the continual capacity of myelination, olfactory ensheathing cells(OECs) are preferred by numerous researchers. The ischemia and secondary changes after injury initiate events that deteriorate local conditions of survival and aggravate neural deficiency. Thus, researchers turn to improve blood circulation and alleviate secondary injury.Vascular endothelial growth factor (VEGF), has been proved up regulated and may relate to function recovery after injury.AimsTo explore a simple and pragmatic method to obtain sufficient olfactory ensheathing cells from neonatal rat. To re-assess the effect of transplantation of OECs originating from the olfactory bulbs and intrathecal administration of VEGF to repair spinal cord injury and the synergistic effect of a combination of two approaches to protect both neurons and nerve fibers.Methods The neonatal female Wistar rats (2-3 d old) were used in this study. The outer two layers of the olfactory bulb, the olfactory nerve and glomerular layers were dissected and retained. The tissues were minced with a eye scissors, triturated using mechanical blow and trypsinized (0.25% w/v trypsin).The single cell suspension of OECs was obtained. After using different attachment rates, DMEM/F12 culture solution including 20% fetal bovine serum and 10ug/ml of cytarabine was used to culture olfactory ensheathing cells for 48 h. Then OECs were cultured in DMEM/F12 solution with 25ng/ml NGF and 1% of FBS. The conditions and growth degree of OECs were observed and immunocytochemistry was used to estimate the purity of the cells at the 9th culture day·And the transplanted cells were collected at the density of 1 x107cells/ml.A total of 75 animals were randomly divided into 5 groups:the control group(CON group), spinal cord injury group (SCI group), vascular endothelial growth factor group (VEGF group), olfactory ensheathing cells group(OECs group) and combinative treated group (VEGF+OECs group). BBB scale was obtained each week. Animals were deeply anesthetized (pentobarbital) 8 weeks postoperatively, intracardially perfused. Spinal cords corresponding with T7-L1 were carefully dissected.Histological changes were determined by hematoxyhn and eosin(HE), the situations of apoptosis and angiogenesis were evaluated by immunohistochemistry staining against caspase-3 and vWF. Transmission electron microscopy (TEM) was used to observe the ultrastructure of fifteen animals (3 uninjured and 3 injured for each group, transplanted for 8 weeks)Continuous values are reported as x±s. All datas were analyzed using SPSS 16.0. Statistical comparisons between groups involved ANOVA of repeated measurement datas. Other datas were analyzed by t test. P<0.05 was considered statistically significant.ResultsOECs were positive against NGFRp75 by immunocytochemical staining. They appeared to be bipolar or tripolar cells and their processes formed a network in vitro. The purity of OECs in good conditions reached about 95% on 7 d and 90% on 9d.Behaviors of rats gained 21 scores before operation. Control group presented the same results after operation. Spontaneous functional recovery was observed in injured animals post operation, especially in former 4 weeks. During the latter 4 weeks, VEGF group and OECs group were better than SCI group.Combinative treated group obtained the highest behavior scores.Histological examination and ultrastructure observation indicated the fact that SCI group possessed the lest spared tissue and seriously damaged nerve fibers and neurons. VEGF group had moderated injured nerve fibers and neurons, In OECs group, numerous vacuolar mitochondria were observed in astrocytes. Combinative treated group showed slightly damaged fibers and neurons, a few of ventral nerve fibers connecting two stumps and numerous myelinated fibers nerve fibers including microfilaments, microtubules and more lightly damaged mitochondria.Immunohistochemical staining against caspase-3 showed that the numbers of CON group, SCI group, VEGF group, OECs group and VEGF+OECs group were (8.33±5.43),(25.89±10.75),(14.62±5.60),(23.47±8.33),(13.38±6.33)/HPF respectively. The amount of last 4 groups were more than that of CON group (P<0.05). SCI group and OECs group possessed more caspase-3 positive cells than VEGF group and VEGF+OECs group respectively (P<0.01)The number of CON group, SCI group, VEGF group, OECs group and VEGF+OECs group against vWF were (13.87±2.59),(11.25±3.19),(14.42±4.05),(11.94±3.77),(16.50±6.25)/HPF respectively. Statistical differences were found comparing VEGF group and VEGF+OECs group with SCI group and OECs group respectively.Conclusions1,The method of using cytarabine combined with the technique of using NGF can culture and purify OECs simply and effectively.2,Transplantation of OECs has promotive effects on restoration of spinal cord injury in rat partly, it can improve part function of nerve fibers and show neuroprotection on cells and fibers.3,Administration of VEGF has limited promotive effects on restoration of spinal cord injury partly,it can improve part function of nerve fibers and show neuroprotection on damaged cells and fibers.4,Combinative treatments, OECs graft and intrathecally administrated VEGF,can significantly improve function and show neuroprotection on damaged cells and fibers, which have a significant synergistic effect.SignificancesIn this study, OECs originating from the olfactory bulbs were cultured and purified by the means of using cytarabine combined with NGF and the model of spinal cord injury were established by weight-drop apparatus. Then OECs were grafted into injury site and VEGF were administrated intrathecally. The study aims to re-assess the effect of transplantation of OECs and VEGF to repair spinal cord injury and the synergistic effect of both approaches to repair protect both neurons and nerve fibers. |
PDF Full Text Request