The Rat Model Establishment And Mechanism Research Of Ossification Of Ligamentum Flavum Induced By Cyclic Tension Stimulation

Ossification of ligamentum flavum(OLF)is one of the key factors leading to spinal stenosis and compression of the spinal cord and nerve roots.It is more common in the lower thoracic spine or thoracolumbar segment.There are many factors that induce OLF,but its specific mechanism is still unclear.The ligamentum flavum is mainly composed of fibroblasts.The increase of collagen in the cells is the initial pathological change of OLF,and the up-regulation of collagen type I(COL1)expression plays an important role here.Tissue inhibitor of metalloproteinase-1(TIMP-1)can upregulate the expression of COL1 by inhibiting matrix metalloproteinase-9(MMP-9),thereby promoting the occurrence and progression of OLF.Rab protein is mainly involved in protein transport between various organelles in the human body.It has been reported that Rab37 can increase the exocytosis of TIMP-1,and inhibit the metastasis of lung cancer cells through the TIMP-1-MMP-9 pathway.Through transcriptome sequencing,we confirmed that Rab L3 expression in fibroblasts of OLF patients was significantly enhanced.This suggests that Rab L3 may be an important factor in the occurrence of OLF,and it may play a role by regulating the expression of TIMP-1 and COL1.At present,the mechanism research of OLF mostly depends on in vitro cell experiments.However,as an important tool for in vivo research,animal models are rarely reported.It is reported that the OLF model is mainly established by implanting bone morphogenetic protein-2(BMP-2)and other biological factors on the surface of the ligamentum flavum.This method is scientific and effective and can focus on a certain stage in the OLF process.Tension stimulation is an important driving factor for the degeneration,hypertrophy and ossification of the ligamentum flavum,and the OLF model established by tension stimulation has not been reported.This subject intends to induce OLF through the production of cyclic tension stimulation of rat ligamentum flavum by self-made stress device,and conduct relevant experimental verification.Meanwhile,the animal model and in vivo experiments will be used to initially explore the expression changes and molecular mechanisms of Rab L3,TIMP-1 and COL1 during the occurrence and development of OLF.Part I:Tension Stimulation Method of Thoracolumbar Ligamentum Flavum in Rats and Safety TestObjective:To study and make induction method and stress device that can cause cyclic tension stimulation of the thoracolumbar ligamentum flavum in rats,to test the safety of nerve function,find the optimal tension stimulation frequency,and verify the safety of the thoracoabdominal organs.Methods:Researched and produced the stress device of thoracolumbar flexion in rat,which triggered the periodic flexion of the thoracolumbar spine,and naturally created the cyclic tension stimulation of the ligamentum flavum.Nine SD rats were randomly divided into 3 groups at random,and were subjected to tension stimulation at frequencies of 60/min,90/min,and 120/min respectively.Stimulate for 20 minutes daily for up to 12 weeks(5days/week).After the weekly tension stimulation was completed,the changes in limb movement function of the rats were evaluated by the BBB exercise assessment scale(normally 21 points),and the safety of tension stimulation was evaluated.The maximum frequency that did not cause a decrease in BBB score after 12 weeks was selected as the optimal tension stimulation frequency.The rats with 12 weeks of optimal tension stimulation were selected to dissect to verify the damage of their thoracoabdominal organs.Results:After 12 weeks of tension stimulation,60/min group and 90/min group did not develop limb dysfunction,and the BBB scores were all 21 points.Rats of 120/min group showed different degrees of motor dysfunction on the 5th,12th and 7th weeks(11 points,16points and 15 points respectively).After 12 weeks of tension stimulation at a frequency of 90times/min,the thoracoabdominal organs of the rats were not damaged.Conclusions:The rat thoracolumbar flexion method and its stress device can effectively induce cyclic tension stimulation of the ligamentum flavum.The optimal tension stimulation frequency is 90 times/min,which has good safety of nerve function and thoracoabdominal organs.Part II:Establishment and Verification of OLF Model Induced by Cyclic Tension in RatsObjective:To create the cyclic tension stimulation of the ligamentum flavum in rats and induce its ossification through thoracolumbar flexion method,and to establish a rat OLF model and perform experimental verification.Methods:Forty SD rats were evenly divided into 5 groups of 8 each:the blank group was not subjected to anesthesia and induction of tension stimulation;the anesthesia group was subjected to abdominal anesthesia(1 time/day,5 days/week)for 12 weeks,and no tension stimulation was induced;The other three groups were induced by tension stimulation for 4 weeks,8 weeks and 12 weeks,respectively.Tension stimulation induction frequency is90 times/minute,20 minutes per day,and 5 days per week(working day).The protein and m RNA expression of osteoblast-related molecules including CD44,BMP-2,Integrin?3,COL1,OPN,RUNX-2 and VEGF,in thoracolumbar ligament tissue,were detected by western blot and RT-PCR technology;Micro CT scan,reconstruction and ossification measurement,as well as pathological staining techniques(HE staining,toluidine blue staining)were used to observe the occurrence of OLF in thoracolumbar specimen.Results:The protein and m RNA expression of osteogenesis-related molecules in the ligamentum flavum in the three experimental groups were significantly higher than those in the two control groups;the protein expression of RUNX-2 showed a trend(the 12-week group>8-week group>4-week group),and m RNA expression up-regulation was most significant in the 12-week group.According to Micro CT scan and reconstruction,the4-week group and the 8-week group occurred single-segment OLF,and the 12-week group had a maximum of three consecutive-segment OLFs;the largest ossification area on horizontal CT section in the 4-week group,the 8-week group,and the 12-week group was1.42 mm~2,5.36 mm~2,and 7.28 mm~2,separately.Pathological staining results showed,that chondrocyte proliferation and woven bone formation occurred in the thoracic and lumbar ligamentum flavum in the 8-week group and the 12-week group,and that the woven bone formation was more significant in the 12-week group;the chondrocyte proliferation was found in the 4-week group,but the woven bone formation was not obvious;The blank group and anesthesia group had no chondrocyte proliferation and woven bone formation.Based on the above results,it is suggested that the OLF results:12 weeks group>8 weeks group>4weeks group.Conclusions:Applying cyclic tension to the ligamentum flavum in rats through thoracolumbar flexion can successfully induce OLF.The OLF induction result is time-dependent,that is,the longer the tension stimulation time,the more obvious the OLF performance.Part III:Study on the Expression Change and Mechanism of Rab L3 during OLFObjective:To study the expression changes of Rab L3,TIMP-1 and COL1 during the occurrence of OLF through in vivo experiments with animal models,and to analyze the regulatory role of Rab L3 in the occurrence of OLF and its possible molecular mechanism.Methods:Thirty-two SD rats were evenly divided into 4 groups of 8 each:the blank group was not subjected to anesthesia and induction of tension stimulation;the anesthesia group was subjected to abdominal anesthesia(1 time/day,5 days/week)for 12 weeks,and no tension stimulation was induced;The other two groups were induced by tension stimulation for 6 weeks and 12 weeks,respectively.Western blot,RT-PCR,immunohistochemical staining and quantitative techniques were used to detect the molecular expression of Rab L3,TIMP-1 and COL1 in the ligamentum flavum of four groups of rats.Results:Western blot results showed that Rab L3,TIMP-1,and COL1 protein expressions in the ligamentum flavum of rat were higher in the 6-and 12-week groups than in the blank and anesthetized groups.Relative m RNA expression:6-week group>12-week group>blank group/anaesthesia group;immunohistochemical staining and quantitative test results showed that immunohistochemical score of Rab L3,TIMP-1 and COL1 in ligamentum flavum:6-week group>12 Week group>Blank group/Anesthesia group.Conclusions:Rab L3 plays an important regulatory role in the process of OLF induced by tension stimulation.It inhibits the degradation of COL1 by caused MMP-9 through up-regulating the expression of TIMP-1,thereby promoting the degeneration,hypertrophy and ossification of the ligamentum flavum.