Experimental Study On The Repair Of Rat Spinal Cord Injury By Transplantation Of Olfactory Ensheathing Cells And NGF

Objective: To investigate the restoration of the motor functions, improvement of the electrophysiological recordings, alteration of the pathomorphology and the relation to each other in rat spinal cord injury(SCI) after olfactory ensheathing cells(OECs) and nerve growth factor(NGF) transplantation. To explore if there is synergistic effect between OECs and NGF and study possible mechanism in order to provide the theory data for the repair of the acute SCI.Methods: 1.Primary culture and purification of OECs: A adult Wistar rat was sacrificed by dislocation of cervical joint, which body weight was 200-250g, then it's olfactory bulb was taken into D-Hanks solution. Capsule of bulb was removed, then washed by D-Hanks solution two times. The outest two layers of bulb was acquired by forceps, cutted into pieces of 1 mm3, being digested with 0.25% trpsin for 20 minutes, cell suspension was centrifuged for 5 minutes with 800rpm, then being discarded upper solution and put DMEM into it which contains 10% FBS. The tissue was blowed with dropping tube, filterd and centrifuged for 5 minutes with 800rpm, then upper solution was discarded. The cell suspension was gathered and cultured in culture flask which was put in incubator (37℃, 5 % CO₂) for 24 hours. Cell suspension was absorbed into new culture flask which contains DF12(DMEM:F12 1:1 FBS 10% penicillin 100 IU/L) and polylysine. Ara-C(5-10mmol/L) was put into it after 7 days for 48 hours, then Forskolin (2μM) and BPE (20μg/ml) in order to culture better. Solution was substituted each 2 days until 14 days. Cells were Observed regularly andphoto of cells was taken. OECs were gathered by direct digestion before transplantation, concentration of cell was 1X 107/ml. Part of OECs were marked by Hochest.2. The fabrication of the model of the SCI: The spinal cord at rat T10 level was exposed after the spinous processes and lamina of vertebra were taken away carefully, then right spinal cord was resected using double blade according to the posteromedian blood vessel to get a gap of 3 mm in the spinal cord. At last the semi-resected model of the SCI was attained.3. Grouping and treatment: 40 wistar rats were grouped in Group A* IK C^ D randomly after their spinal cords were semi-resected. 10 H 1 NS was injected into the gap of the spinal cord in the right in group A, 10 y 1(30 v g) NGF in group B, 10 u 1 OECs in group C, which 2 n 1 were injected into spinal cord 1 mm away from gap and 8 n 1 in gap, Both OECs and NGF were injected into spinal cord gap in group D, two of each group were marked by Hochest. Animals were behaviorally tested for 8 weeks to observe the motor function improvement using the Basso, Beattie, Bresnahan locomotor rating scale, somatosensory evoked potential(SEP) of left were observed every week, but SEP of right only preoperative and postoperative immediately.The rats were anesthetized with 2% sodium pentobarbital and fixed in platform. SEP were recorded by stimulating the posterior tibial nerve at the left ankle. A recording needle electrode was placed in the scalp between two ears. A reference needle electrode was inserted into the scalp of forehead. A ground electrode was placed into the right forelimb. SEP was measured for each wave. The latency of SEP was measured from the onset of the stimulus to the peak, which indicated the conduction function of sensing system. The amplitude of a positive peak was measured from the positive peak immediately preceding negative peak, indicating the mount of neuron which had electrophysiological function.The tissue sections were dong 8 weeks postoperatively, which were observed by fluorescence microscope and electron microscope, stained by hematoxylin and eosin(HE) and immunohistochemistry staining in order to observe the axon regeneration and the survival of OECs .The data were analyzed by ANOVA, t-test with statistical software spss 12.0. P-value less than 0.05 was considered statistically significant.Resu Its: 1. The BBB scale of Group C and D rats increased from 3th week to 7th week obvoiusly, then became slowly in last two week, final score was above 15. The BBB scale of Group A and B rats didn't increased obviously. Though BBB scale of Group B was higher than Group A, there was not significant difference between them. There was not sinnificant difference between GroupA,B,C,D in 1th week, P>0.05. GroupC and D were significant higher than Group A and B(P<0.01), but there was not significant difference between Group C and D in 2th week (P>0.05). From 3th week to 8th week /There was significant difference between Group C and Group A^ B(P<0.01). the same significant difference between Group D and C(P<0.01). It indicated that motor function of Group D improved best.2. The SEP wave of Group C> D appeared from 3th week and its latent period became shorter and potential amplitude increased gradually, which suggested the recovery of electrophysiological functions of spinal cord. The wave of Group B appeared from 3th week, Group A from 4th week. Its latent period became longer and amplitude lowered obviously, there were not obvious change during 8 weeks. There was significant difference between Group C and Group As B(P<0.05), the same outcome between Group D and C(P<0.01). There was not significant difference between Group A and B(P>0.05), except that latent period of Group B was significant shorter than Group A in 8th week.3. The OECs had been being active in Group C and D 8 weeks after transplantation, fluorescence particles which were Olfactory ensheathing cells nuclei were found in tissue section of Group C and D when observed by ultraviolet light of 365 nm wave length, There was negative result in Group A and B. It indicated that OECs had been survived for 8 weeks. OECs of Group D were much more than Group C significantly. There were many cells which were positive to P7:>NGFR and had yellow endochylema in Group C> D. There rarely were positive OECs in spinal cord 1 cm away from injury gap. There were more degeneration and necrosis of OECs in Group C than Group D. The regeneraton axons both appeared in group C and D,which had same direction with longitudinal axis of spinal cord and went through injury gap, myelin sheath layers became tight. Few axons regenerated in Group A % B, which had a disorderly direction and din't go through the injury gap, Myelin sheath loosen badly. The average number of axons were counted by picture analysis system in each group, Group A: 109.0+31.8, Group B: 112.7+41.6, Group C: 362.6 + 64.8, GroupD: 607.2 + 77.8. The data were analyzed by ANOVA and LSD, There was not significant difference between Group A and B, P>0.05, Group C was significantly more than Group A> B, P<0.01, Group D was significantly more than Group A> B^ C,P<0.01. The quality and quantity of axons in Group C> D were better than Group A and B signifcantly, among of which, Group D was the best.Cone I us i on: 1 .NGF can promote functional improvement slightly after SCI, but there is not significant difference, axons cannot go through spinal cord injury gap.2.0ECs transplantation can promote the repair of spinal cord after SCI. BBB Score increases, the SEP latent period becomes shorter and potential amplitude increases, quantity and length of axons increase, axons can go through spinal cord injury gap.3.Better functional improvement appears if OECs and NGF are transplanted at the same time after SCI, higher BBB score, shorter SEP latent period and higher amplitude, more synapse, more axons appear in co-transplantation group.4. NGF can promote activity and survival of OECs , so that it can enhance the function of OECs. meanwhile, OECs can make axons go through the spinal cord injury gap so that it make NGF promote axons regenerate better. There is a synergistic effect between OECs and NGF.