| ObjectivesTo establish an animal model of neuropathic pain after spinal cord injury using a novel percussion device(spinal cord precision percussion device(68099H)).To observe the expression of NG2 cells and their protein-chondroitin sulfate proteoglycan(CSPG)using immunofluorescence and Western-blot.To investigate the possible role of NG2 cells in the mechanisms involved in the generation of neuropathic pain after spinal cord injury.MethodsSeventy-five adult male Sprague-Dawlye rats,weighing 220-250 g,were randomly divided into three groups: control group without any treatment(Control group,n=15);sham-operated group with direct suture closure of the incision after surgical exposure of spinal cord T10(Sham group,n=30);neuropathic pain model after spinal cord injury group(SCI group,n=30),established after surgical exposure of spinal cord T10 using a new precision percussion device to strike T10.(1)The mechanical pain threshold of the hind limbs of the rats 1d before and 14 d,21d and 28 d after surgery was observed to assess whether the model was successfully constructed.The motor function of the rats after spinal cord injury by BBB score at 1d before and 1d,3d,7d,14 d,21d and 28 d after surgery was assessed.HE staining was performed at the relevant time points to observe the pathological changes.(2)On the basis of the first step of the experiment,immunofluorescence staining was performed on spinal cord tissues at four key time points to observe the proliferation and activation of NG2 cells.(3)The expression of CSPG in each group was observed at four key time point postoperatively using Western-blot.(4)Statistical analysis was performed using SPSS24.0 software,and the differences were considered statistically significant at p<0.05.Results I.Mechanical pain threshold,BBB score and HE staining after spinal cord injury in rats(1)The difference of preoperative BBB scores between three groups was not statistically significant(p>0.05).BBB scores of SCI group were all 0 at postoperative1 d and increased gradually at postoperative 3d(0.43±0.56),7d(4.03±1.00),14d(10.93±1.23),21d(15.57±1.65)and 28d(18.23±1.41),with statistically significant difference(F=1583.982,p<0.001).The differences in the trend of BBB scores between Control group and Sham group at each postoperative time point were not statistically significant(p>0.05).(2)The difference of preoperative mechanical pain thresholds of rats in the hind limbs between three groups was not statistically significant(p>0.05).In SCI group,mechanical pain threshold decreased from preoperative1d(16.33±2.40)g to postoperative14d(7.87±1.04)g,with a slight upward from postoperative 14 d to21d(9.87±1.57)g,and maintained at postoperative 28d(10.13±1.38)g(F=267.833,p<0.001).The difference between any two time points was statistically significant(p<0.05),except postoperative 21 d and 28d(p>0.05).There was no significant difference in mechanical pain threshold between Control group and Sham group at each time point(P>0.05).The change rate of mechanical pain threshold in SCI group was significantly higher than that in Control group and Sham group(F=37.441,p<0.001),but there was no significant difference between control group and sham group(p>0.05).(3)Proliferation and activation of glial cells corresponds to the behavior of spinal cord injury in rats.II.Activation and proliferation of NG2 cells in spinal cord of ratsIn SCI group,the number and proportion of NG2 cells increased from the preoperative 1d(40.60±8.68,4.53±0.50%)to postoperative 14d(184.10±41.38,16.63±1.59%),reaching the peak at postoperative 14 d.The number and proportion of NG2 cells decreased at postoperative 21d(123.90±18.78,12.19±2.37%)and maintained at postoperative 28d(114.00±30.90,11.96±2.13%).The difference was statistically significant(F=919.505,P<0.001).The difference between any two time points was statistically significant(p<0.05),except postoperative 21 d and 28d(p>0.05).Under fluorescence microscope,the soma of NG2 cells enlarged slightly at postoperative14 d,and enlarged significantly with the synapses increasing at postoperative 21 d.There was no significant change in soma and synapses at postoperative 28 d.The fluorescence intensity of NG2 cell at preoperative 1d,postoperative14 d,21d and 28 d was1.00,1.49,1.64and1.58 respectively.III.CSPG expression in spinal cord tissue of rats in each groupThe results of Western-blot showed that,the expression of CSPG increased from the preoperative 1d(1.00±0.06)to postoperative 14d(1.49±0.26),reaching the peak at postoperative 14 d,The expression of CSPG decreased at postoperative21d(1.38±0.18)and maintained at postoperative 28d(1.43±0.16).The difference was statistically significant(F=40.285,p<0.05).The difference between any two time points was statistically significant(p<0.05),except postoperative 21 d and 28d(p>0.05).The expression of CSPG was positively correlated with the proliferation and activation of NG2 cells after spinal cord injury(r=0.964,p<0.05).The expression of CSPG in SCI group(1.42±0.16),Control group(0.98±0.09)and Sham group(0.93±0.07)were statistically significant(F=2603.225,p<0.001).The expression of CSPG in SCI group was significantly higher than that of Control group and Sham group(p< 0.01).There was no significant difference of the expression of CSPG between Sham group and Control group(p>0.05).Conclusions(1)The animal model of neuropathic pain after spinal cord injury can be established by using a new type of percussion device(spinal cord precision percussion device(68099h)).This model has the advantage of avoiding the impact of secondary injury caused by traditional method of weight falling.(2)Proliferation and activation of glial cells corresponds to the behavior of spinal cord injury in rats.(3)Two weeks after spinal cord injury in rats is acute phase,and the pain comes to chronic phase from at 3 weeks after spinal cord injury.(4)After spinal cord injury,the proliferation and activation of NG2 cells increased gradually,with a proliferation peak at 2 weeks and activation peak at 3 weeks.The expression of CSPG was positively correlated with the proliferation and activation of NG2 cells after spinal cord injury.(5)NG2 cells may play an important role in the mechanism of neuropathic pain after spinal cord injury. |
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