Studies On The Relationship Between Cervical Dynamic Imbalanced And The Degeneration Of The Ligamentum Flavum

Objective:The manifestations of degenerated ligamentum flavum(DLF) included the proliferation,hypertrophy and ossification,the degeneration of the ligamentum flavum leading to the decline of its flexibility.The ligamentum flavum can be folded into the spinal canal when the spine extension,and the capacity of the spinal cave will be reduced significantly,the nerve roots and spinal cord will be compressed, especially when the DLF company with the protrusion of the intervertebral discs or proliferation and hypertrophy of the facet joints,it will decrease the effective space more obviously.This disorder will cause a series of spinal diseases,such as spinal stenosis,folding compression of cervical ligamentum flavum,cervical spondylosis, cervical ossification of the ligamentum flavum(OLF),cervical calcification of the ligamentum flavum,and emerge into the corresponding clinical symptoms.On the other hand,most of the stenosis sites were interdiscoligamentous space and this was the site where the nerves were compressed most commonly,therefore,the degeneration of ligamentum flavum was attended more and more by domestic and foreign scholars.Some people thought that fibrosis was the main reason of hypertrophy of the ligamentum flavum.The increase of the stress(accumulation of mechanical stress) to the ligamentus flavum is the main reason to cause the fibrosis,especially the dorsal aspect of the ligamentum flavum.Our preliminary studies found that transforming growth factor-β1 and BMP2 act an important regulatory role in the early stage of the hypertrophy of the ligamentum flavum.But the mechanism of the fibrosis and hypertrophy of the ligamentum flavum is not clear now.There have not been reported to the mechanism of the degeneration and the starting reasons.The resolution of these issues must be based on clear understanding of the pathogenesis of the DLF,therefore, it is important to study the mechanism of the cervical DLF from the different aspects.Some scholars have found from clinical observation that more than 80 percent of the patients with the DLF have engaged in manual work for a long time,the ligament hypertrophy have taken place in 60 percent of kyphosis patients.That means flexion of the spine can lead to ligamentum flavum suffered increased tension and it plays an important role in the degeneration of the ligamentum flavum.Therefore,based on the early research of us on cervical models,we have established the models of muscle imbalance of cervical vertebra on rats,to research the ligamentum flavum in the early stage(within two months) by electron microscopy scanning,immunohistochemistry, molecular biology,and other methods,compared with the normal control group and muscle stripping group,the relationship between cervical dynamic imbalance with the degeneration of the ligamentum flavum has been explored.Methods:Two months old healthy Wistar rats,the weight of 195～215 gms,were divided into three groups randomly,group A were posterior muscle resection,group B were posterior muscle stripping,group C for the control group.The incisions were from the center of the neck,about 2 cm in length,incised the skin and subcutaneous tissue.Posterior muscles resection group:incised the skin and subcutaneous,stopped bleeding,stripped the paraspinal muscles along the spinous process and bilateral laminae of vertebral arches about 2 cm,cut off the m.splenius,m.longissimus capitis, m.longissimus atlantis,m.longissimus cervicis,m.iliocostalis,m.semispinalis capitis,and with 1.5 cm in length,respectively,to prevent the muscles stump healing, sutured the incision layer by layer.Posterior muscle stripped group:incised the skin and subcutaneous,stopped bleeding,stripped the paraspinal muscles along the spinous process and bilateral laminectomy about 2 cm,and then sutured the incision.Control group:incised the skin and subcutaneous,stopped bleeding,sutured incision.The 8 rats of every group were taken for computer radiography films at the surgery day,30 days after the operation and 60 days after the operation.Evaluation of motor function:to carry out evaluation of motor function after 3d, 5d,15d,30d,60d among groups,the rat's longitudinal axis of body was arranged parallel with the oblique board,rats were test the biggest board angles which they counld stay 10 seconds with head-up and head-down separately,taking the average value as the experimental data.We have taken 3d,5d,15d,30d,60d a total of five points in time,three rats were prepared for the electron microscopy scanning,lavaged of blood,drawn the ligamentum flavum from cervical 5～6 and cervical 6～7 between laminae of vertebral arches under the microscope,and put small samples into the small bottles with pre-cooling glutaraldehyde,preservation at 4℃,ultra-thin slices for electron microscopy observation.We have taken 3d,5d,15d,30d,60d a total of five points in time,five rats were prepared and drawn the ligamentum flavum from cervical 5～6 and cervical 6～7 between laminae of vertebral arches under the microscope,biopsy specimens were sliced for HE staining,observed the pathological changes by light microscopy;dyed according to ABC dyeing methods,observed the endogenous TNF-αexpression in the ligamentum flavum;extracted the total RNA according to TRIzol Reagent statement method,tested its integrity of RNA,synthesized cDNA to randerm primer for primer, the PCR product purification,detection,calculated the ratio of PCR product of the TNF-αmRNA and 18 s RNA PCR product as relative content of TNF-αmRNA and a semi-quantitative analysis of value.Results:1 X-ray:Muscle resection group:Computer Radiography showed that the physiological curve of the cervical vertebra was normal at the day of operation,intervertebral spaces were not narrow,there were no osteophyte too;the physiological curve of the cervical vertebra was stiffness in a few rats at one month later,intervertebral spaces were no narrow and there were no osteophyte too;two months later,the physiological curve of the cervical vertebra with light stiffness in part of the rats,and the intervertebral space were slightly changed narrow in some of the rats,there were mild vertebral bone hyperplasia,facet joints were slightly hardened too.The control group and muscle tripped group:Computer Radiography showed no obvious abnormality with in 60 days.2 Evaluation of motor function:The oblique board angles were decreased in of the removal muscle group than muscle stripping group and the control group after 3 days and 5 days,p＜0.01,the difference was significant after 15 days,p＜0.05,but the difference were dissappeard among the 3 groups after 30 days and 60 days,p＞0.05.3 Ultramicrostructure:The control group:there were little changes at different time points,it showed a small number of fibroblasts,the nuclei were bigger,cytoplasm less,with a small number of organelle,mitochondria were naive state,showed that mitochondrial ridge unclear;elastic fibers with rules,dense,fiber cross striations of the fiber were regulation,bright and dark band were separated by each other,and dark band with the same pitch;we can saw the fiber bounds with the wave shaped.Muscle resection group:There were a large number of fibroblasts 3 days after the operation,the cells were circular,rich cytoplasm,endoplasmic reticulum increased thickening,regularly,mitochondria increased,ridge processes,size larger,both inside and outside film is not complete,a metabolic active state,some high-density electronic particles scattered in the cell.There were a small amount of collagen fibers around the fibroblasts,with disorder.Elastic fibers with uniform size,arrange regularly,there were cyclical dark with equal range,but elastic fibers were slightly thick than the normal control group,the substance among the fibers were increased. The fibroblasts were decreased at 5 days after the operation than 3 days,cells still thick appearance,rich cytoplasm,the form of the endoplasmic reticulum regularly,the high number of mitochondria,the ridge processes,both inside and outside membrane is not complete,a metabolic active state,There were a large amount of collagen fibers around the fibroblasts,with disorder;elastic fibers are still structured,with periodic stripes of bright and dark band,the fibers were still slightly rough than the normal control group.At 15 days,the number of fibroblasts was decreased,the cells become slim,but still rich cytoplasm,the number of organelle were declined too,the ridge within the mitochondria was not clear,a decline functional state,the new collagen fibers around the cells increased significantly,the structure dense and thick,fibers were disorder,with no regular stripes of bright and dark band,the gap of flexible fiber widened,with more matrix between fibers.At 30 days,the number of fibroblasts were decreased,the cells slender,deep-stained nuclei,cytoplasm less,fewer organelle, mitochondria ridge difficult to be distinguished,many field of vision had no fibroblasts.The new collagen fibers around the fibroblasts with varying thickness,but with periodic stripes of bright and dark band,arrange regularly,the fiber bounds with the wave shaped.At 60 days,the number and shape of the fibroblasts were similar with the normal ligamentum flavum,the number of cells,cytoplasm and organelle decreased significantly,the fiber bundles arranged regularly and larger than the normal control group,fiber gaps with more matrix.And the fibroblasts in stripping muscle group,were less than the muscle resection group,cell body slender,less cytoplasm,a small number of mitochondria,the ER was sparse bubbly.Elastic fibers are dense,rules,the dark band with the same pitch;can clearly show that fiber bounds with the wave-shaped.The elastic fibers have no obvious broken or faults,it was similar with the normal ligamentum flavum.4 HE staining results:The main component of the cervical ligametum flavum of rats was the fiber, collagen fibers were light pink,elastic fibers in a bright pink,it was very difficult to distinguish collagen fibers and elastic fibers,there are a small amount of the fibroblasts among the fibers.Muscle resection group:at three days,the number of fibroblasts in the ligamentum flavum were increased obviously,the nuclei were round or oval,fibrous tissue was scarce;at five days,the number of fibroblasts in the ligamentum flavum were still more,the nuclei were oval-shaped,surrounded by a few fibrous tissue;at 15 days, fibrous tissue gradually increased,at 30 days,the fibroblasts were dyed dark brown, the number were increased,like spindle,fibrous tissue was hypertrophy,with disordered,distribution irregularity;after 60 days,the fiber bundle was hypertrophy obviously,with disorder and thickening,distribution irregularity,lack of wave-running.Muscle stripping group and the control group:The characteristics of histology change a little.Fibers arranged regular,no fiber bundles broken.We can saw the fibrous tissue with regularity at 60 days after the muscle Stripping. Immunohistochemical staining:5 Immunohistochemical staining:Immunohistochemical staining showed that TNF-αhave no positive staining in the fibrous tissue,so most of the fibers can't show its organizational structure.Muscle resection group:the TNF-αhas strong expression in the ligamentum flavum at the third day of operation,the number of fibroblasts were increased,the membrane and cytoplasm of the cells was yellow or brown,but there were no significant expression of TNF-αin the stromal and fibrous tissue;the expression of TNF-αafter 5 days was strongest,the cytoplasm was brown,stromal of cells were positive expression at 15 days;the expression was weakening at the 15ths days,the cytoplasm was brown,stromal of cells stained with focal area;the expression of 30 days was weaker than 15 days,cytoplasm was light yellow,stromal was stained occasional;at 60 days,fibroblasts became slender,cytoplasm in some fibroblasts was still light yellow.Muscle stripping group and the control group:there have no significant positive expression of TNF-αin muscle stripping group and control group. 6 RT-PCR results:The RT-PCR product of the TNF-αgained by the agarose gel electrophoresis was 248 bps,the fragment was proved to be TNF-αby sequencing.The housekeeper geneβ-actin was amplified out at all samples showed that the samples have a good quality, condition and system of the RT-PCR were fit for requirements.The expression of TNF-αin the muscle group was positive,but the expression of muscle stripping group and control group was very weak.Relative expression volume:Relative expression volume of each specimen was the gray density of gene electrophoresis bands of each specimen divided by the gray density of correspondingβ-actin gene.The changes of TNF-αgene expression in each specimen of ligamentum flavum:Comparison within group:The length of the specific DNA fragment expression product of TNF-αgene gain by RT-PCR was 248 bps,the product was detected in all the samples with different level of expression.In muscle resection group,its expression was strongly positive at 3d,5d and 15d,the expression was gradually declining at 30d and 60d,but there was significant positive expression at 60 d after the operation.The expression at 3d,5d,15d was significant different from the expression at 30d and 60d(p＜0.05).Muscle stripping group(B) and the control group(C):there have no different within group B and group C(p＞0.05).Comparison between groups:Posterior muscle resection group had a significant difference(F=36.330,P＜0.01) with Group B and Group C,last two groups with a low expression,the difference between group B and C was not significant(P＞0.05).Conclusions:1.The structure and function of the ligamentum flavum in Wistar rats was similar with human beings,the rats presented a bow of the abnormal walking patterns when the posterior muscles of the neck were resected,the ligamentum flavum were over-stimulated by traction and we established a dynamic imbalance rats animal model successfully.It was resemble with the human who with a long-term bow work and at last caused the cervical spondylopathy.This model was simple,easy and ideal to research on the ligamentum flavum.2.The posterior muscles of the Wistar rats had a strong ability of regeneration,the rats were recome the normal gait and normal feeding after 2 months,the muscles regeneration complete,so this animal model was most suitable for observation of impact to the ligamentum flavum in the early stage of the cervical dynamic imbalance model,not suitable for the impact to the ligamentum flavum with a long-term cervical dynamic imbalance.3.The early changes of the cervical dynamic imbalance were stenosis of the intervertebral space,bone hyperplasia and sclerosis of the facet joints,but the change of the physiology curve of the cervical was later;the cervical imbanlance after removing the behind muscles can speed up the proliferation of fibrous tissue and with disorder significantly,it can cause hypertrophy and degeneration of the ligamentum flavum,it was an important reason to induce the degeneration of ligamentum flavum!4.The number of fibroblasts in the ligamentum flavum was increased at the early stage after the resection of the posterior muscles of the rats' cervical,there were a small amount of collagen fibers surrounding the fibroblasts,with disorder.As the time gone,the fibroblasts transforming into longer cells,the collagen fibers around the cells were increasing gradually,and at 12 months after the operation,the number of fibroblasts were decreased,plasma and organelle were decreased significantly,with regular and bulky fiber,there was more matrix among the space of the fibers.5.There were expression of TNF-αin the ligamentum flavum at each time points after resected the posterior muscles of the neck in rats,it was obviously in the early stage, and there was a downward trend of TNF-αafter 30 days.6.The level of the expression of the TNF-αwas very low in the muscle stripped group and control group in the ligamentum flavum of rats.It was failed to see the positive expression by the immunohistochemistry test,and there was a weak positive expression by the RT-PCR,the difference between the muscle resected group and the muscle stripped and control group was significently.7.The cervical dynamic imbalance of rats can cause the expression of TNF-αimproved markedly in the ligamentum flavum of rats,endogenous TNF-αmay have an important regulatory role to the degeneration of the ligamentum flavum of rats.