Experimental Study On The Therapy Of Spinal Cord Contusion By Transplantation Of Olfactory Ensheating Cells

Objective:To explore a simple, practical and high performance method of cultivation olfactory ensheathing cells(OECs),to get ready for transplantation of olfactory ensheating cells into injuried spinal cord;To observe the effect of OECs on hindlimb movement in rats with spinal cord contusion;To detect the expression of brain-derived neurotrophic factor(BDNF)and myelin basic protein (MBP) in spinal cord contusion of rats in order to investigate the mechanism of repair spinal cord injury by transplantation of OECs.Methods:Under anatomical microscope,the OECs were dissociated from the first outer layer of the olfactory bulb of the green fluorescent protein (GFP) transgene adult rats which were 2.5 months old,then enzymatic digestion and culture in DMEM/F12 with 20%FCS were performed.The morphology of OECs was regularly observed and photographied under light and electronic microscope.At 10th day,the OECs were identified by the immunocytochemistry of S100 and NGFRp75,and its purity was calculated according to the positive rate,the cell ultrastructure revealed by electron microscope.There were total 28 adult female Sprague Dawlet rats, of which 6 rats received no operation were regarded as normal group(group C), another 22 rats contused by NYUâ…¡impact instrument were randomly divided inio two grous: group A (16 rats) and group B (6 rats).After the contusion, rats of group A were acutely transplanted with suspension of green fluorescent protein-Olfactory ensheathing cells (GFP-OECs), rats of group B were acutely transplanted with suspension of DMEM culture media. Rats of group A and B were tested by Basso-Beattie-Bresnahan locomotor rating scale (BBB) after postoperation at first day, 1th week, 2th week, 3th week and 4th week.The rats of groups were anesthetized by 3% pentobarbital sodium at 3th week after transplantation.The corresponding segmemts of spinal cords were took out from living experimental rats,and were detected expression of BDNF by RT-PCR .All groups rats were anesthetized by 3% pentobarbital sodium,infused and fixed with 4% paraform at 3th week after transplantation,the corresponding segmemts of injuried spinal cords were taken out from the experiment rats, then, those spinal cords were made into paraffin sections which were detected expression of BDNF by immunohistostaining.In addition,3 rats from group A was anesthetized by 3% pentobarbital sodium, infused and fixed with 4% paraform,the corresponding segmemts of injuried spinal cord were take out from the rat,and were made into frozen section to observe the transplantation GFP-OECs in injuried spinal cord.3 rats from group A were anesthetized by 3% pentobarbital sodium,infused and fixed with 4% paraform at 4th week after transplantation,the corresponding segmemts of injuried spinal cord were took out from the rat,and were made into frozen section,to detect expression of BDNF by immunohistostaining.3 rats from group A were anesthetized by 3% pentobarbital sodium,infused and fixed with 4% paraform at 4th week after transplantation, the corresponding segmemts of injuried spinal cords were take out from the rats,and were made into frozen section,to detect MBP expression in group A by immunofluorescence.Results:Under fluorescent microscope,strong green fluorescence GFP-OECs were observed after primary cultured for 7 days,which appeared three typical morphology:dipolar-like(spindle),three pole-like and flat round. After primary cells were cultured for 10 days,major cells were dipolar-like.More than 95% cultured cells were identified as OECs,which expressed both S100 and NGFR p75.Under electron microscope, GFP-OECs nucleis were irregular;there were rich rough endoplasmic reticulums,free ribosomes and mitochondrion in the cytoplasm;there were tuberculum mallei on cell surface.The BBB scores of transplantation groups were decreased to zero after the postoperative 1 day, the scores increased gradually. The BBB scores of group A were higher than group B during all postoperative period. After transplantation 21 days, rats of group B creeped with no weight support or plantar placement of the paw with no weight support, but, rats of group A creeped with weight support or plantar placement of the paw with weight support.All results showed that recovery of hind limb motor function in group A was better than in group B.3 weeks after transplantation, BDNF located in cytoplasm by immunohistochemistry observation, and was brow-yellow.The expression of BDNF was the strongest in group A and was the weakest in group C. The expression of BDNF mRNA in group A was the greatest .The difference was statistically significant (p<0.05).Frozen section of spinal cord of post-transplantation 4 weeks was observed by the fluorescence microscope,GFP-OECs located in the surrounding spinal cord injury cavity, making a strong green fluorescence. BDNF immunohistochemistry yellow masculine production was distributed the tissue which was near the GFP-OECs in spinal cord injury, and BDNF immunohistochemistry masculine production in the place of GFP-OECs was stronger, the results showed OECs secreted BDNF and enhanced the BDNF expression of contusion spinal cord which was near the OECs.4 weeks after transplantation, MBP immunofluorescence red masculine production of contusion spinal cord frozen section was followed with green fluorescence GFP-OECs.Conclusion:1. The good active and high purity OECs could be cultured successfully in vitro by our cell culture method.2. Green fluorescent protein from GFP-OECs was intensive,persistent and no side effect.GFP-OECs could be used as an ideal tool in study of repair spinal cord injury by transplantation of OECs.3. The hindlimb movement function of rats could be enhanced by OECs transplantation into contusion spinal cord.4. OECs transplantation promoted the expression of BDNF and MBP of spinal cord contusion to protect injuried nerves and form myelin. It was one of mechanism that OECs repair spinal cord injury.