The Establishment Of Animal Model Of Autoimmune Reaction In Vertebra And Its Related Risk Factors

Part ? The Distribution Characteristics and Risk Factors of Modic changes in Clinical PracticeObjective To investigate the risk factors and distribution characteristics of Modic changes in lumbar spine.Methods Retrospective analysis from July 2011 to September 2016 in Tianjin Hospital spine surgery department due to low back pain.All patients were evaluated by magnetic resonance imaging(MRI)and X-ray.The presence of Modic changes,the degree of degeneration of the disc,the degeneration or hernia of the intervertebral disc,the presence or absence of HIZ,the presence of the schmorl snode,age,height,weight,workload,driving volume,amount of smoking,exercise,low back pain and the degree of physical activity were collected.The analysis of the association between these risk factors and Modic's change was performed.Results All patients include 324 male and 297 females with an average age of 52.5 ± 14.9 years.The overall incidence of Modic change was 27.5%(n = 171),and the most common segment was L5/S1(46.2%).Univariate regression analysis showed that the risk factors such as age,sex,BMI index,smoking status,workload,intervertebral disc degeneration,intervertebral disc herniation and osteochondrosis of lumbar posterior vertebral edge were closely related to Modic.The intense of low back pain in Modic changes patients was more fiercely.In addition,almost all Modic changes patients have varying degrees of degenerative changes.Conclusions Age,sex,BMI index,smoking status,workload,intervertebral disc degeneration,intervertebral disc herniation,and posterior vertebral osteomalacia were associated with changes in the extent and duration of low back pain,Are likely to affect the incidence of Modic changes.Part ? The Establishment of Animal Model of Autoimmune Reaction in Vertebral BodyObjective To study the feasibility of establishing a rat model of autoimmune reactionin the vertebral body by using the needle puncture along the tail of the rat and destroying the intervertebral disc to make the nucleus pulposus accessible to the circulatory system.The reliability of the model was validated by imaging and histological analysis.Methods Twenty-four SD rats were randomly divided into control group,sham operation group and experimental group.Eight rats in each group were treated with needle puncture along the tail of the rat to destroy intervertebral disc to make the nucleus to contact the circulatory system.The sham operation group was treated with needle puncture along the transverse direction of the tail,only vertebral destruction,to retain the integrity of the disc and nucleus;control group is not puncture.The body weight of each group of experimental animals was measured and recorded every two weeks after the completion of the animal modeling.The effects of the operation on the general condition of the rats were compared.The rats in each group were sacrificed at 4 weeks,8 weeks and 12 weeks after operation.The histological changes of the vertebral body were observed and the changes between the signal changes and histological changes were observed.Results X-ray showed: 16 G needle can accurately penetrate the rat tail through the four segments of the vertebral body and adjacent three discs,the needle is located in the middle of the caudal vertebral body.Compared with the control group,the weight of the experimental group was decreased gradually,but the the sham group and the control group showed the opposite trend.At 4 weeks,8 weeks and 12 weeks after the experiment,MRI showed significant signal changes in the experimental group,while the control group and sham group did not observe significant signal changes.Pathological staining showed that the trabecular bone structure disorder in the experimental group,the number of bone and chondrocytes decreased with the increase of model time,while the control group and sham operation group of bone trabecular no significant change.Conclusions The rat model of autologous autoimmune response can be established by longitudinal puncture of rat vertebral body.The contact of the nucleus pulposus with the circulatory system has a certain influence on the body weight,bone condition and imaging changes.Part ? The Effect of Osteoimmunological Factors on Animal Model of Autoimmune Reaction in Vertebral BodyObjective The animal immunoreactive animals were analyzed by biomechanics,Micro-CT,Elisa,RT-PCR and Western-blot.The effect of the model revealed the pathogenesis of Modic changes.Methods 72 SD rats were randomly divided into control group,sham group and experimental group,24 rats in each group.In the experimental group,the needle was punctured along the tail of the rat to destroy the intervertebral disc and made the nucleus pulposus was exposed to the circulatory system.The sham operation group was punctured by the needle along the transverse of tail,only the vertebral body was damaged and the intact disc and nucleus were preserved.No puncture was performed in control group.All the experimental animals in the modeling after 4 weeks,8 weeks,12 weeks of nuclear magnetic resonance imaging.After imaging detection,the anesthetization was performed to rats in each group by using 10% chloral hydrate.After anesthesia satisfaction,according to the plan for each group of rats drawn,the specific steps are as follows:(1)Firstly,5-6ml blood of rat was havested,after standing at room temperature for 2 hours,3000 rpm centrifugal 15 minutes,take the supernatant after storage to-80 ? refrigerator spare.(2)After the rat tail was cut off,the steriled knife was used to obtain the caudal vertebrae of each section,and the muscles,ligaments and periosteum of the caudal muscle were stripped layer by layer.The Co7 vertebral body was put into RNA protection solution,4 ? refrigerator after standing overnight into the liquid nitrogen tank for real-time quantitative PCR;Co8 vertebral body was for immunoblot blot detection;(3)The Co9 vertebral body was wrapped in 0.9% Nacl gauze and placed in a-20 ° C refrigerator for biomechanical testing;(4)The Co10 vertebral body was placed in 4% paraformaldehyde and stored in vitro for Micro-CT analysis.Results Micro-CT results showed that compared with the control group,thetrabecular bone of the caudal vertebrae were significantly thinner in the experimental group and the sham operation group after 4 weeks of modeling,the trabecular bone structure disorder and the porosity increased significantly,Local or even visible vertebral trabecular part of the fracture or disappear,but the experimental group of animals is more obvious changes can be seen significant vertebral osteoporosis.After 8 weeks of modeling,the thickness of trabecular bone in the sham operation group was gradually restored,and the structure of the trabecular bone was basically normal.The trabecular bone of the experimental group was further thinner,the trabecular bone structure Porosity increased further,the trabecular part of the fracture and the degree of disappearance is more obvious than before.Twelve weeks after modeling,the trabecular bone of the sham operation group had basically recovered,and there was no obvious abnormality compared with the control group.In the experimental group,the bone trabeculae appeared more severe osteoporosis which performed.The results of mechanical tests showed that the biomechanical properties of the caudal vertebrae were significantly decreased after 4 weeks of modeling compared with the control group.Compared with the sham group and the difference was not statistically significant.After 8 weeks of modeling,the mechanical properties of the caudal vertebral body in the sham operation group were gradually restored,and the indexes were close to the values of the control group The Compared with the control group,the biomechanical properties of the caudal vertebral body in the experimental group showed a decreasing trend.After 12 weeks of modeling,the mechanical properties of the caudal vertebral body in the sham operation group were recovered.The three test indexes were close to the control group but still did not return to the control group.The rats in the experimental group Biomechanical properties continue to fall to low values.Western blot results showed that the factors such as Rank,Rankl,TNF-a,IL-17 and IL-1b were significantly higher in the experimental group,and the positive rate gradually increased with the increase of model time high.(P <0.05),while the expression of OPG and IFN-? were significantly inhibited,compared with the control group and sham operation group,the difference was similar significant.There was no significant difference in protein expression between the sham operation group and the control group.The serum of the experimental group were significantly higher thanthose of the control group and the sham group.The serum of the sham-operated group were higher than those in the control group(p> 0.05).The serum of the experimental group were significantly higher than those of the other two groups(p <0.05).The serum of the sham-operated group were decreased gradually at the 8th week,and gradually reached the level of the control group.The serum of the experimental group were significantly higher than those of the other two groups(p <0.05).The serum of the sham-operated group were significantly higher than those in the control group at the 12 th week.The results of RT-PCR showed that the levels of Rank,TNF-?,IL-17 and IL-1? in the vertebral body of the experimental group were significantly higher than those in the control group and sham operation group MRNA expression of OPG and IFN-? were significantly decreased.Compared with the control group,the m RNA expression of Rank,TNF-?,IL-17 and IL-1? m RNA in the caudal vertebrae of the sham group was higher than that of the control group,but the difference was not significant See significant statistical significance(p> 0.05).Compared with the control group and the sham operation group,the m RNA expression of Rank,TNF-?,IL-17 and IL-1? in the vertebral body of the experimental group continued to increase after 8 weeks of modeling The The expression of OPG and IFN-? decreased continuously.There was no significant difference between the sham operation group and the control group in the above indexes(p> 0.05).Compared with the control group and the sham operation group,the m RNA expression of Rank,Rankl,TNF-?,IL-17 and IL-1? in the vertebral body of the experimental group was still high after 12 weeks of modeling(P <0.05).The expression of OPG and IFN-? did not decrease,there was no significant difference in the m RNA expression of Rank,Rankl,TNF-?,IL-17,IL-1?in,OPG and IFN-? in the sham operation group compared with the control group(P > 0.05).Conclusions Osteoimmunological factors may play an important role in the pathological changes of autologous autoimmune animal models in rats,which may be one of the causes of local signal changes and osteoporosis.