Experimental Study On Recovery Of Adult Rat With Hemisection Of Spinal Cord Injury By Transplantation Of Olfactory Ensheathing Cells

Objective:To investigate a simple and inexpensive method of primary culture and purification of olfactory ensheathing cells (OECs) from the adult rat olfactory bulbs; To observe the effect of OECs graft on hindlimb movement in rats with hemisection of spinal cord injury(hSCI); To detect the expression of Sema3A, NP-1 and Versican in spinal cord ventral horn in rats with hSCI in order to explore the mechanism of repairing spinal cord injury with transplantation of OECs.Methods:The adult female Sprague-Dawley rats (2.5 months old) were used in this study. The outer two layers of the olfactory bulb, the olfactory nerve fibrasa 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. OECs were planted in culture flask at a density of 1×10⁶ cells/mL, and cultured in DMEM/F12 nutrient with 20% foetal calf serum, and purified by a method of combination of the different rates of attachment and the Arabinosylcytosine (AraC) inhibition. The cell suspension was transferred twice, when it was incubated at 12th hours and 24th hours. In order to kill the olfactory nerve fibroblasts, the AraC was added in supernatant (5×10^-5mol/L ) at the 7th culture day, and the inhibition was quenched by renewing the all medium in 36 hours.The morphological changes of cultured OECs were observed. The OECs in culture were identified by glial fibrillary acidic protein (GFAP) and nerve growth factor receptor p75 (NGFRp75) immunohistochemistry (avidin biotin-peroxidase complex technique, ABC) at the 14th culture day. The purity of the positive cells was calculated.At the 14th culture day, the transplanted cells were collected at the density of 1×10⁷ cells/mL, meanwhile, the supernatant was collected.A total of 35 female adult healthy Sprague-Dawley rats were used. 5 rats were normal group without treatment. 30 rats were randomly divided into five groups and acutely transplanted after hSCI with suspension of purified OECs , medium from cultured purified OECs , suspension of impurified OECs, medium from cultured impurified OECs and DMEM/F 12 culture fluid in group A, B, C, D ,E respectively.Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale and Inclined Plan (IP) Experiment were tested during 6 postoperative weeks (9 time points: dayl, day3, day5, weekl, week2, week3, week4, week5, week6).The normal and transplanted rats were infused and fixed with paraform (4% w/v) at 6th postoperative week. Their left triceps surae and T₁₁～T₁₂ segment of spinal cord were dissected and retained.The skeletal muscle samples were processed with paraffin sectioning, 5μm thickness, and hematoxylin-eosin staining. The cross section area and diameter of muscle fibers were measured with image analysis system (Image-pro plus 5.0.2). The spinal cord samples were made into 30μm thickness frozen sections, then stained with specific Sema3A (1:1600), NP-1 (1 : 400) and Versican (1:1200) antibody immunohistochemistry ABC technique. At the same time, blank control and serum replacement group were examined. The positive neuron were observed and calculated, the mean optical density (OD) of immunoreactivity were measured with image analysis system in spinal cord ventral horn.The One-way ANOVA and Student-Newman-Keuls mothod were used to test intergroup differences.Results:The cultured OECs were presented three main morphological types: multipolar, bipolar and fiat, most of cells were bipolar and multipolar. For GFAP positive cells, the plasma and processes were stained while the nuclei were not, but for NGFRp75 positive cells the nuclei were stained more deeply than the plasma and processes.～91% cultured' cell was GFAP⁺ and～90% was NGFRp75⁺.These positive cells were OECs.The BBB scores of transplanted groups were decreased to zero at the 1st postoperative day but normolity was 21. The scores were increased gradually and achieved～6 during 2 weeks. After 3th postoperative week, the scores of group A compared to other transplanted groups were significantly increased(P＜0.05).At the 1st postoperative day, the scores of IP experiment of transplanted groups were decreased to～12.5°, the normal score was～85°. The scores were increased gradually. The scores of all groups had no significant difference during 3 weeks. After 4th postoperative week, the scores of group A compared to other transplanted groups were significantly increased(P＜0.05)At 6th postoperative week, the cross section area and diameter of skeletal muscle fibers in left triceps surae were lesser than normal group. The decreasing extent of group E was the greatest, that of group A was the least. The difference was significant (P＜0.05).In spinal cord ventral horn, for all of the Sema3A, NP-1 and Versican positive motoneurons, the plasma were stained while the nuclei were not. The number of positive motoneuron and mean OD of transplanted groups compared with the normal group were increased. The increasing extent of groups E was the greatest, that of group A was the least among transplanted groups .The difference was significant(P＜0.05).Conclusion:1. The high purity OECs could be cultured successfully in vitro by the combination of the different rates of attachment and the AraC inhibition methods. This method was simple and inexpensive.2. The hindlimb movement functional recovery was promoted by transplantation of OECs in rats with hemisection of spinal cord injury.3. The atrophy of triceps surae was relieved by transplantation of OECs in rats with hemisection of spinal cord injury.4. The expression of Sema3A, NP-1 and Versicaln were down-regulated in spinal cord of rats with hemisection of spinal cord injury after transplantation of OECs. It's maybe one of the possible reasons that injuried spinal cord was repaired by transplantation of OECs.