Study On Olfactory Ensheathing Cells Subarachnoid Transplantation For Treatment Of Spinal Cord Injury

Spinal cord injury (SCI) is a common clinical illnesses, which can lead the patients to high rate of disability and great suffering . After spinal cord injury, nerve regeneration and functional recovery has become a unsolvable medical problem. In recent years,olfactory ensheathing cells (OECs) with its unique biological characteristics and the promotion of axon regeneration of spinal cord injured is considered the best therapy for SCI in cell transplantation. Currently, the main methods of Olfactory ensheathing cell transplantation are SCI location injection and venous transplantation. But now there is not relevant research reports about Olfactory ensheathing cells subarachnoid transplantation for treatment of spinal cord injuryObjective:To investigate the effect and the possible mechanisms of olfactory ensheathing cells subarachnoid transplantation and provide a simple and easy transplantation for the clinical treatment of acute spinal cord injury; at the same time using a variety of transplant dose to observe the best dose of the transplanted cells.Materials and methods:1. culture, purification, identification and count of Olfactory ensheathing cells:Taked olfactory bulb of healthy Wistar rat, two months, irrespective of gender, weight 200-250g, used the pure culture methods of Nash differential speed adhesion and inhibition of Ara-c combination to perform primary culture, purification, identification, enumeration afer digesting, and marked nuclear with dual-benzylidene methylamine.2. Preparation and transplantation in the treatment group of Spinal cord hit injury model:80 healthy adult Wistar rats, used modified Allen hit model. The rats were randomly divided into olfactory ensheathing cells (OECs) subarachnoid transplantation with high-dose group (group A), subarachnoid transplantation with mid-dose group (group B), subarachnoid transplantation with lower-dose group (group C), local intramedullary transplantation group (group D), subarachnoid transplantation with DMEM/F12 medium(group E).3. The evaluation of recovery of spinal cord injury:Observed and scored motor function of animal models in 1 day, 1, 2, 3, 4, 8 and 10 weeks after transplantation by modified CBS score. Observed the general morphology of spinal cord specimens, the survival and distribution of labeled cells through fluorescent microscopy through frozen section,the regeneration of axons through HE staining and silver staining, the survival and distribution of labeled cells by immunohistochemical staining in 1 week, 4 weeks, 10 weeks after transplantation.Results:The pure culture methods of Nash differential speed adhesion and inhibition of Ara-c combination could develop high purity olfactory ensheathing cells. The purity was more than 70% and count was 1×108个/ml on 10th day,which meet the test.The cell bodies showed three forms: spindle, flat round and multi-shaped protrusions, NGFRP75 and GFAP staining was positive.The motor function of rat hindlimb could recover in varying degrees in the period 1-10 weeks after transplantation. 3 weeks later, the score of group A, B and D compared with group C, group E showed significant difference (P <0.05); but the efficacy of group A and B compared with group D was similar, not statistically significant difference (P> 0.05); group A was better than B, but not statistically significant (P> 0.05); the efficacy of group C and group E was not significant difference (P> 0.05).Obseved by HE staining and argyrophilic staining: group A, B and D had more regenerated axons, and more nerve fibers passed through the injury area, and the regeneration axon of group D was better than that of group group A and B; group C, E had only few scattered axonal regeneration, no continuity of nerve fibers passed through the injury area. Immunohistochemistry and fluorescence microscope could be seen the number of survival and distributed of olfactory ensheathing cells in the damage zone of group A ,B, D in 4 weeks after operation and group D was more than that of group A and B.Conclusion:1. The purified culture method of Nash differential speed adhesion could be attained a higher purity of olfactory ensheathing cells, and better to meet the practical application.2. The effect of OEC subarachnoid transplantation for treatment of SCI was similar to the local transplantation. It might got good curative effect that the number of OEC should reach more than 2×105.The transplanted olfactory ensheathing cells could survive and migrate in the SCI areas.3. olfactory ensheathing cells subarachnoid transplantation operation was simple, easy and avoided secondary injury of reoperation and postoperative complications, and could be repeated treatment, and had better clinical practice and value.