| Objective:To establish an animal model of chronic spinal cord compression in rabbits with angular kyphotic deformity(AKD).To explore the mechanism of histopathology and molecular biology on the disease of progressive chronic spinal cord compression through the study of the behavior score,imaging and pathological histology and molecular biology examination of the model animal.The apoptosis of the nerve cells in the chronic compression spinal cord was detected by TUNEL method and the expression of miRNA-21 and miRNA-370 in the compressed spinal cord tissue was detected by mi-RNA gene chip detection and real time quantitative polymerase chain reaction(Q-PCR),and to explore the mechanism of apoptosis and the significance of apoptosis in chronic spinal cord injury,which would provide theoretical basis for further research and clinical treatment.Methods:70 healthy New Zealand white rabbits of both genders(2.5-3.5 Kg,4 to 5 month old)were divided into five groups randomly:group A,group B,group C,group D and group E.Group A was the control group(n = 14)and the other groups were experimental groups.Group B,C,D and E were 2 weeks,4 weeks,6 weeks and 8 weeks after operation respectively,and 14 animals were included in each group.In the experimental groups,L2-3 intervertebral disc of animals was taken as the center,including the upper and lower vertebrae,’V’ shape resections were done to establish model.The range of excision included L2-3 intervertebral disc,2/5 of lower part of upper vertebral body and 2/5 of upper part of inferior vertebral body.The vertex of the ’V’ shape was the posterior edge of the L2-3 intervertebral disc,which pointing to the spinal canal and close to the dura mater.No fixation was done during and after the operation.Group A was a sham-operation group,the L2 and L3 vertebral bodies and the intervertebral discs in front of which were exposed only but without discectomy or excision of vertebrae.The BBB score of the motor function of the hind limbs was done.All of the animals were scored and evaluated after 2 weeks,4 weeks,6 weeks and 8 weeks after the operation respectively.Then the chronic spinal cord compression which caused by lumbar angular kyphosis was evaluated through examinations of gross observation,lumbar X-ray and MRI.The spinal cord specimens were removed after sacrificing the animals.The mi-RNA gene chip sequencing was done in the 2 specimens,each of which was from group A and group C.Two of the specimens were sliced 5 minutes after freezing at the-20℃,and then the spinal cord ischemia was observed after TTC staining.Six of the specimens were made of paraffin slices,and then the pathological changes of tissues were observed after HE staining by using TUNEL method.Six of the compressed specimens were used to detect the expression of miRNA-21 and miRNA-370 by Q-PCR.Results:1.Gross observation:In group A,there was no significant change in lumbar curvature at each time point before and after operation.In experimental group,there appeared visible kyphosis in lumbar operative segment 2 weeks after the operation.The angular kyphosis of the animals in the experimental group increased gradually along with the prolonged observation time and reached the peak at 6 weeks after the operation.The lumbar angular kyphosis of the animals progressed slowly and tended to stabilize at 8 weeks after the operation.The results of BBB score of animals showed:BBB score of group A was 20.93 ±0.18.In the experimental group,the degree of spinal cord compression increased along with the time,and the BBB score also decreased with the time.BBB score of group 2 weeks was 17.51±1.80,group 4 weeks was 15.61 ± 1.49,group 6 weeks was 13.75±1.47,group 8 weeks was 14±1.66.The difference between each experimental group and the group A was significant(p<0.001).In the experimental group,there was significant difference(P<0.01).between the other groups except for group D and E.2.X-ray and MRI imaging observation:The Cobb angle of lumbar kyphosis was measured and recorded by observing the X-ray lateral films of different groups.Statistical analysis showed that no obvious lumbar kyphosis was found in group A,the Cobb angle of which was(4.54±0.26)degrees.The Cobb angle of lumbar kyphosis in experimental group increased along with the prolongation of observation time.In the experimental groups,the Cobb angle was(30.08±3.66)degrees in 2 weeks’ group,the Cobb angle was(43.77±3.51)degrees in 4 weeks’ group,the Cobb angle reached the maximum of(47.13±2.09)degrees in 6 weeks’ group.The lumbar kyphosis process tended to be stable,the Cobb angle was(46.73±2.38).There was significant difference in Cobb angle between experimental groups(group B-E)and group A(P<0.001).There was significant difference(P<0.001)between the other groups except for group D and E(p=0.69>0.05).The anteroposterior diameter of L2-3 spinal canal was measured to calculate the spinal canal encroachment rate by observing the median sagittal MRI of lumbar vertebrae in different groups.The results of MRI examination showed that the spinal canal encroachment rate was(4.46±0.01)mm,no spinal cord compression could be found in group A.L2-3 spinal cord compression was found at 2 weeks after operation in the experimental group,the anteroposterior diameter of which was(3.95±0.02)mm and the spinal canal encroachment rate of which was(12.14±2.49)%.The compression gradually aggravated with the time goes on,the anteroposterior diameter was(3.06±0.07)mm and the spinal canal encroachment rate was(31.43±1.44)%at 4 weeks.The spinal cord compression reached a peak by 6 weeks,the anteroposterior diameter was(2.37±0.81)mm and the spinal canal encroachment rate was(49.12±8.90)%.The lumbar angular kyphosis of the animals progressed slowly and tended to stabilize at 8 weeks,the anteroposterior diameter was(2.50±0.21)mm and the spinal canal encroachment rate was(44.13±4.59)%.No definite progress could been found in spinal cord compression.The spinal cord compression at the apex of the angular kyphosis was most obvious.Variance analysis on the anteroposterior diameter of L2-3 spinal canal had been done and the results showed that there was a significant difference between the experimental group(B-E group)and the control group(A group)(p<0.001).There was difference between each group(p<0.05).Variance analysis on the spinal canal encroachment rate had been done and the results showed that there was a significant difference between the experimental group and the control group(p<0.001).There was difference between each group(p<0.01).3.TTC staining and HE staining.TTC staining of blood vessels showed that,compared with the control group,there were different degrees of ischemia in the spinal cord compression segment and its adjacent segments in the experimental group.In the experimental group,ischemia in the middle and posterior segment was the most serious.The spinal cord compression area showed small pallid area in group A and group B.The spinal cord compression area showed large pallid area in group C and group D.It showed that there was obvious ischemic necrosis.In the experimental group,histopathologic examination showed that the neurons were necrotic and decreased,white matter fibers were reduced,myelin sheath arranged in disorder,myelin sheath enlarged,nerve fibers demyelinated and foam cells formed in the anterior horn of the compressed spinal cord.It was aggravated with the degree of kyphosis and duration of spinal cord compression.4.Results of detection of TUNEL positive cells showed:The apoptosis rate of TUNEL positive cells in white matter and gray matter of group A were(10.03±0.60)%and(9.93±0.47)%respectively.In the experimental group,TUNEL positive cells were found in both gray matter and white matter of the spinal cord.The rate of apoptosis of TUNEL positive cells increased with the prolongation of compression time and the increase of the degree of spinal cord compression.In 2 weeks’ group,the rate of apoptosis of TUNEL positive cells was(27.64±2.12)%in white matter,(24.86±0.87)%in gray matter.In 4 weeks’group,the rate was(42.81±1.89)%in white matter,(30.05±1.19)%in gray matter.In 6 weeks’ group,the rate was(69.40±2.77)%in white matter,(49.31± 1.63)%in gray matter.In 8 weeks’ group,the rate was(59.62±1.83)%in white matter,(38.31 ±1.36)%in gray matter.Apoptotic cells of 2 weeks’ group increased compared with group A,reached peak at 6 weeks,and decreased at 8 weeks compared with 6 weeks.In WM(White Matter),the positive cell apoptosis rate of the experimental group(group B-E)was significantly different from that of the control group(group A)in TUNEL test(p<0.001).Results of pairwise comparison in multiple groups showed:there was significant difference between the other groups(P<0.001)except for group D and E(P=0.97>0.05).In GM(Gray Matter),the positive cell apoptosis rate of the experimental group was significantly different from that of group A(p<0.001).Results of pairwise comparison in multiple groups showed:there was significant difference between the other groups(P<0.001)except for group D and E(P=0.65>0.05).Paired sample T test method was used to compare the difference between WM and GM of that in each group.The results showed:there was difference between WM and GM in each group(P<0.05),the apoptosis rate of WM was higher than that of GM cells in each group.5.MicroRNAs gene sequencing and Q-PCR detection.1254 kinds of microRNAs were discovered in the microRNAs gene sequencing test of rabbit spinal cord,compared with the control group,there were 511 kinds of microRNAs changed in the spinal cord of experimental group animals,65 kinds of which changed significantly.The Ct values of miR-21 and miR-370 were detected by Q-PCR.The results of Q-PCR detection showed:In group A,the Ct values of miR-21 was 17.44±0.31 and the Ct values of miR-370 was 25.75±0.14.In experimental groups,the Ct values of miR-21 was 21.28±2.63 and the Ct values of miR-370 was 22.22±1.54 at 2 weeks.The Ct values of miR-21 was 24.34±0.71 and the Ct values of miR-370 was 21.60±2.86 at 4 weeks.The Ct values of miR-21 was 26.99±0.66 and the Ct values of miR-370 was 17.76±0.28 at 6 weeks.The Ct values of miR-21 was 26.73±0.21 and the Ct values of miR-370 was 18.50±0.35 at 8 weeks.In the spinal cord compression area of experimental group,statistical analysis on Ct value of miR-21 and Ct value of miR-370 showed that Ct value of MiR-21 and Ct value of miR-370 in the experimental group(B-E group)were significantly different from those in the control group(A group)(p<0.001).compared with the control group,miR-21 decreased,while miR-370 increased.In the experimental group,miR-21 gradually decreased and miR-370 gradually increased along with the extension of compression time and the aggravation of spinal cord compression.For the Ct value of MiR-21,there was no significant difference between group D and group E(P=0.48>0.05),there was difference between other groups(p<0.05).For the Ct value of MiR-370,there was no significant difference between group B and group C(P=0.48>0.05),there was no significant difference between group C and group D(P=0.40>0.05),there was significant difference between each of other groups(p<0.001).In the experimental group,the miR-21 gradually reduced in the first 6 weeks and increased at 8 week.The miR-370 increased gradually.However,the increase of miR-370 in the experimental group from 2 weeks to 4 weeks after the operation was not significant and the increase from 6 weeks to 8 weeks after the operation was not significant as well.Conclusion:1.This study has successfully established a stable animal model of chronic spinal cord compression caused by progressive thoracolumbar kyphosis deformity in rabbits.This model is easy and repeatable to establish,which could provide a practical and useful animal model for simulating clinical diseases to further study the pathogenesis and molecular biological changes of chronic spinal cord compression caused by spinal kyphosis.2.Chronic spinal cord compression leads to obvious ischemic changes in compression area and adjacent spinal cord area.Apoptosis plays an important role in the destruction of neurons caused by chronic spinal cord compression during this process,which also closely related to the degree and time of compression.3.For the first time,The study reported that miR-21 and miRNA-370 mediated neural signaling pathways play an important role in neuronal apoptosisIn of the chronic spinal cord injury.However,the increase of miR-370 in the experimental group from 2 weeks to 4 weeks after the operation was not significant and the increase from 6 weeks to 8 weeks after the operation was not significant. |
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