Study On The Correlation Between Osteoprotegerin Gene Polymorphism And Intervertebral Disc Degeneration And Rabbit Disc Degeneration Model

Objective: Intervertebral Disc Degeneration(IDD), also called degenerative disc disease(DDD), is the major cause of degenerative spinal disease, and one of the most common ailments that severely affects the quality of life. IDD is characterized by lower back pain, intervertebral disc herniation and spinal canal stenosis. The exact pathophysiology of IDD is not well understood, but both genetic and environmental factors play major roles in IDD. Osteoprotegerin(OPG) is a glycoprotein and a cytokine receptor containing 401 amino acid residues, which is also a osteoclastogenesis inhibitory factor or tumor necrosis factor receptor superfamily member. OPG is synthesized primarily by osteoblasts, but is also expressed in various organs and tissues, including heart,vessel wall, lung, kidney and bone. Previous reports suggest that factors affecting OPG gene regulation could be the major genetic factors influencing bone mass and increasing risk of fractures, osteoporosis and osteoarthritis.Earlier studies have revealed that increased bone mineral density may beimplicated in the etiology of IDD, and few studies have focused on a direct relationship between OPG gene polymorphisms and the risk of IDD. To address this issue, we performed the current study to investigate the correlations of OPG genetic polymorphisms, serum OPG levels, and IDD risk.Methods: A total of 200 IDD patients and 200 healthy controls were recruited. Genotype and haplotype frequency distributions of OPG polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) assay. Serum OPG levels were measured by enzyme-linked immunoassay(ELISA). SPSS 22.0 software was used to analyze all data.Results: Genotype and allele frequencies of OPG rs2073617 polymorphism were significantly higher in IDD patients compared to control group(P < 0.05).The C allele carriers showed a higher risk of IDD than T allele carriers(P <0.001). The genotypes and alleles frequencies of two other gene polymorphisms,rs2073618 and rs3102735, showed no statistical differences between patients and controls. Furthermore, OPG serum levels in IDD patients with TT, TC and CC genotypes in OPG rs2073617 polymorphism were markedly higher than control group(P < 0.05). The haplotype analysis showed that G-T-G haplotype associated with protection against IDD(P = 0.008), while G-C-G haplotype associated with elevated susceptibility to IDD(P = 0.007). Logistic-regression analysis showed that high serum levels of OPG positively correlated with IDD risk, while the T-C-A, T-G-A and T-G-G haplotypes negatively correlated with IDD risk(P < 0.05).Conclusion: OPG rs2073617 polymorphism is strongly connected with increased risk of IDD. We also find that up-regulated OPG serum level increasesIDD risk. By contrast, T-C-A, T-G-A and T-G-G haplotypes are protective factors for IDD.Objective: Intervertebral Disc Degeneration(IDD) is characterized by back pain or sciatica, which is one of the most common ailments that severely affects the quality of life and consumes a lot of medical resources. IDD is a complicated chronic process and participation by a variety of factors. The exact pathophysiology of IDD is not well understood. If we can better understand the pathophysiology of degeneration, we can work to directly prevent or slow the process. Further advances in the treatment of disc degeneration will require improved understanding of the pathophysiology of disc degeneration, together with testing the safety and efficacy by using suitable animal models that mimic disc degeneration in humans. Many methods to establish animal models of intervertebral disc degeneration requires an open surgical approach to the lumbar spine that is costly and bothersome to perform.The aim of the present study was to establish a simple, reproducible model of disc degeneration in rabbits through percutaneous puncturing the disc in a minimally invasive manner.Methods: Twenty four New Zealand white rabbits underwent percutaneous puncture of the L3/4、L4/5 and L5/6 discs by using 18G、20G and 22 G size needle under the guidance of C-arm, L6/7 disc as a control group. Use X-ray to observed the changes of intervertebral disc height, measured and compared the DHI% before and 4 weeks after percutaneous puncture surgery. MRI detection was adopted before and 4 weeks after percutaneous puncture surgery, and Pfirrmann grading method was applied as evaluation criteria for the before and after comparison. Rabbits were put to death after MRI detection, and L3/4 、L4/5 、 L5/6 and L6/7 intervertebral disc were taken out. Then HE stained,Masson stained, Safranine O stained and type II collagen immunohistochemical stained for observation. SPSS 22.0 software was used to analyze all data.Results: 24 New Zealand white rabbits were successfully operated, There were no death、paralysis and wound infection during or post operative.X-ray examination showed DHI% in the four group were no significant difference(P> 0.05). The rabbit lumbar MRI results before operation showed the signal intensity of disc were high. The rabbit lumbar MRI results 4 weeks after operation indicated decreasing tendency of the signal intensity in 18 G group.The MRI results were graded in each group by Pfirrmann grading method, using text of two-way ordinal classification data, and P <0.05. which suggested the significant correlation between the size of needle and degeneration of the intervertebral disc. HE staining showed that 18 G group of nucleus pulposus has less number of nucleus pulposus cell than other group. Masson staining showed the fissures in the annulus layers increased significantly in 18 G group than other group. Safranine O staining showed the proteoglycan was decreased significantly in 18 G group than other group. Immunohistochemical staining results showed that the type Ⅱ collagen synthesis and secretion decreasedsignificantly in 18 G group and 20 G group compared to 22 G group and control group(P<0.05).Conclusion: Minimally invasive percutaneous puncture disc inducing degeneration in the rabbit model was a safe and reproducible. The rabbit lumbar intervertebral disc degeneration animal model was successfully established by percutaneous puncture disc with the 18 G and 20 G needle. The rabbit lumbar intervertebral disc degeneration animal model was not established by percutaneous puncture disc with the 22 G needle. This study provide an ideal animal model of disc degeneration for the reseach on the pathophysiology of disc degeneration and disc regeneration.