Study On Repair Of Adult Rat Spinal Cord Injury By Transplants Of Olfactory Ensheathing Cells

Spinal cord injury (SCI) is a common disease in clinic, and the serious spinal cord injury is easily happened to adult people, furthermore, these are rising in our country year after year. It easily leads to crippledom and seriously damages to people's health, especially serious spinal cord injury. It not only makes huge loss to individual and society, but also becomes an important factor of shorting people's longevity, and there is not effective method for this disease by now. At present, the strategies for this disease aim at: to prevent and reduce secondly injury, and help to recove nervous function and adjust regeneration of central nerve system. Nearly years, the methods of treatment spinal cord injury have made advanced rapidly development with the techniques of molecular biology. The experiments show that embryo spinal cord transplantation, cells (stem cells) transplantation, gene transfer cells transplantation and gene therapy have achieved part functional recovery of spinal cord injury. These are available effective methods for this disease, especially olfactory ensheathing cells transplantation treating spinal cord injury that has made preliminary success. So we believe the method of transplantation olfactory ensheathing cells to treat spinal cord injury maybe become capable of the best one of solving this century difficult question.This paper studied systematically on related questions to olfactory ensheathing cells transplantation treating SCI as follows: olfactory ensheathing cells culture; investigating an effective method of purification for establishing primary cultures ofolfactory ensheathing cells (OECs) from the adult rat olfactory bulb, especially from olfactory mucosa, and investigate the morphological changes of the cultured OECs; detecting the glial cell line-derived neurotrophic factor (GDNF) in olfactory ensheathing cells supernatant and studing the relationship that concentration of GDNF in cultured OECs supernatant was changed with time, then providing the theoretic support for optimal time to transplant OECs. This paper also studied the curative effect of repairing spinal cord injury with transplantation of olfactory bulb tissue block, cell suspension, olfactory ensheathing cells (olfactory bulb and olfactory mucisa), olfactory ensheathing cells in combination with Methylprednisolone, olfactory mucosa and olfactory mucosa in combination with Methylprednisolone in rat, for the sake of theoretic support of repairing SCI with transplantation of OECs for clinic application.Part I: Olfactory ensheathing cells culture 1. Primary culture and morphologic study of olfactory ensheathing cells fromthe adult rat olfactory bulbsObjective To investigate an effective method of purification for establishing primary cultures of olfactory ensheathing cells (OECs) from the adult rat olfactory bulb and investigate the morphological changes of the cultured OECs. Methods OECs were harvested from olfactory bulbs and cultured, purified by the method of combining both the different rates of attachment among the various cells types and the Ara-c inhibition. The morphological changes of cultured OECs were observed at different stages and identified by both nerve growth factor receptor p75 (NGFRp75) and glial flbrillary acidic protein (GFAP) immunohistochemistry. Results The cultured OECs that presented three morphological types: fusiform, process-bearing multipolar, fried egg-like were high purity and their processes weaved into net. Conclusion The method of purification for OECs through combining both the different rates of attachment among the various cells types and the Ara-c inhibition is simple, inexpensive and practical.2. Primary culture and morphology of olfactory ensheathing cells from theadult rat olfactory mucosaObjective To investigate an effective method of purification for establishing primary cultures of olfactory ensheathing cells (OECs) from the adult rat nasal olfactory mucosa and investigate the morphological changes of the cultured OECs. Methods The nasal olfactory mucosa was harvested from the posterior part of the nasal septum and cultivated, purified by the method of combining the different rates of attachment among the various cells types, mechanical curettage and the Ara-c inhibition. The morphological changes of cultured OECs were observed at different stages and identified by both nerve growth factor receptor p75 (NGFRp75) and glial fibrillar acidic protein (GFAP) immunohistochemistry. Results The cultured mucosa cells presented mainly four morphological types: fusiform cell, bipolar cell, nest cell and giant cell, and part of these bipolar or fusiform cell were positive for NGFRp75 and GFAP, and a few giant cells were positive for above two antibodies. Conclusion The method of purification for OECs through combining the different rates of attachment among the various cells types, mechanical curettage and the Ara-c inhibition was able to culture and purify OECs.Part II : Detection of glial cell line-derived neurotrophic factor in culturedolfactory ensheathing cells supernatantObjective To study the relationship that concentration of glial cell line-derived neurotrophic factor (GDNF) in cultured olfactory ensheathing cells(OECs) supernatant was changed with time, then provide the theoretic support for optimal time to transplant OECs. Methods OECs were harvested from olfactory bulbs and olfactory mucosa, and cultured, purified by the method of the different rates of attachment among the various cells types. The OECs were cultured for 21 days and harvested the samples every 3 days. The GDNF in the OECs supernatant was detected by the method of enzyme-linked immunosorbent assay (ELISA). Results The concentration of GDNF in OECs supernatant of olfactory bulb and olfactory mucosa rised gradually with cultured time, but the neat content was very low. Conclusion The cultured OECs can secret GDNF and the best time to transplant is at 14 days after cell culture.Part m : Study on Repair of adult rat spinal cord injury by transplants of olfactory ensheathing cells and administration of a high dose ofMethylprednisoloneObjective To investigate the synergetic effect and possibility of spinal cord injury repaired by transplantation of olfactory ensheathing cells and methylprednisolone in adult rat. Methods Wistar rats with T10 spinal cord hemisection were divided into 4 groups: olfactory ensheathing cells group (OEC), olfactory ensheathing cells in combination with mthylprednisolone group (OEC+MP), methylprednisolone group (MP) and control group (CG) respectively. The functional recovery of spinal cord injury was observed with combined behavioral score (CBS) and pathological anatomy at different phases. The tissue sections were made at 5 weeks and 10 weeks postoperatively to observe the axon regeneration and the survival of olfactory ensheathing cells. Results The CBS showed that rats treated with OEC and OEC+MP had more improvement than that of MP and control group at series of phases after operation 3 weeks (p<0.01), but had not significant difference between OEC group and OEC+MP group (p>0.05). The pathological section showed same results as CBS. Conclusion The method of treating spinal cord injury by transplants of olfactory ensheathing cells and administration of a high dose of Methylprednisolone had good curative effect, but had not significant synergetic effect.Part IV: Study on repair of spinal cord injury by transplants of olfactory bulb tissue block or cell suspension in ratObjective To investigate the effect and possibility of spinal cord injury repair by transplantation of olfactory bulb tissue block or cell suspension in rat. Methods Wistar rats with T10 spinal cord hemisection were divided into 3 groups: tissue block group, cell suspension and control group, each group transplanted olfactory bulb tissue block(TB), olfactory bulb cell suspension(CS) and DMEM respectively. The functional recovery of spinal cord injury was observed with CBS scores at different phases. The tissue sections were made at 2 weeks and 10 weeks postoperatively to observe the axon regeneration and the survival of olfactory ensheathing cells. Results The experimentshowed that rats treated with TB and CS had more improvement than control group. The effect between TB group and CS group had not significant difference. Conclusion The method of treating spinal cord injury by transplanted TB or CS not only simplified operation but also had good curative effect. Therefore, this method is worth spreading and application in clinic.PartV: Study on Repair of adult rat spinal cord injury by transplants of olfactory mucosa and administration of a high dose ofMethylprednisoloneObjective To investigate the effect and possibility of spinal cord injury repaired by transplantation of olfactory mucosa, and the synergetic effect with methylprednisolone in adult rat. Methods Wistar rats with T10 spinal cord hemisection were divided into 4 groups: olfactory mucosa group(OM), olfactory mucosa in combination with mthylprednisolone group(OM+MP), methylprednisolone group(MP) and control group(CG) respectively. The functional recovery of spinal cord injury was observed with combined behavioral score (CBS) and pathological anatomy at different phases. The tissue sections were made at 5 weeks and 10 weeks postoperatively to observe the axon regeneration and the survival of olfactory ensheathing cells. Results The CBS showed that rats treated with OM and OM+MP had more improvement than that of MP and control group at series of phases after operation 3 weeks (p<0.01), but had not significant difference between OM group and OM+MP group (p>0.05). The pathological section showed same results as CBS. Conclusion The method of treating spinal cord injury by transplants of olfactory mucosa had good curative effect, but had not significant synergetic effect with administration of a high dose of Methylprednisolone at same time.PartVI: Study on repair of spinal cord injury by transplants of olfactoryensheathing cells derived from olfactory mucosa in ratObjective To investigate the effect and possibility of spinal cord injury repaired by transplantation of olfactory ensheathing cells derived from olfactory mucosa in adult rat.Methods Wistar rats with T10 spinal cord hemisection were divided into 4 groups: olfactory ensheathing cells derived from olfactory mucosa group(OM-OEC), olfactory ensheathing cells derived from olfactory bulb group(OB-OEC), methylprednisolone group(MP) and control group(CG) respectively. The functional recovery of spinal cord injury was observed with combined behavioral score (CBS) and pathological anatomy at different phases. The tissue sections were made at 10 weeks postoperatively to observe the axon regeneration and the survival of olfactory ensheathing cells. Results The CBS showed that rats treated with OM-OEC and OB-OEC had more improvement than that of MP and control group at series of phases after operation 3 weeks (p<0.01), but had not significant difference between group OM-OEC and OB-OEC group (p>0.05). The pathological section showed better results of two OEC groups than latter two groups. Conclusion The method of treating spinal cord injury by transplants of OM-OEC had good curative effect, just as that of OB-OEC, providing academic foundation for clinic practice .