Preliminary Study Of Zebrafish Intervertebral Disc Severely Damaged Led To Vertebrae Degeneration

Cervical spondylosis is a common chronic disease which troubled dentists. Research shows that the main reasons for cervical spondylosis is based on degenerative discs into cervical vertebra degeneration. Intervertebral disc degeneration constitutes the pathological basis of most musculoskeletal diseases of the vertebral column, including degenerative spinal stenosis, intervertebral instability, cervical spine syndrome, radiculopathy and myelopathy. Research shows that osteoporosis and intervertebral disc degeneration has certain correlation, osteoporosis causes even aggravate the intervertebral disc degeneration. However, the occurrence of intervertebral disc and vertebra development, degeneration and regeneration repair is a complicated process, we have not yet exactly understand the molecular biological mechanisms of intervertebral disc degeneration. For intervertebral disc and vertebra degeneration mechanism and self-repair mechanism research, which provide a novel theoretical guidance for degenerative disc disease prevention and treatment.Objective: We utilize zebrafish as a novel model animal to construct a new intervertebral disc damaged model. We intend to explore the intervertebral disc severe damage model in the research of intervertebral disc degenerative disease. To prepare for a further research which is the molecular regulation mechanism study of intervertebral disc degeneration and vertebral degeneration.Methods: Using molecular cloning techniques to construct recombinant plasmid pBluescript II-KS-twhh-Dendra2-NTR, through microinjection technique, inject 0.15 ng isolation of pure recombinant plasmid to the animal pole of zebrafish embryos during one cell stage. After three generations of consecutive batches, we cultivate the stable transgenic line Tg(twhh:Dendra2-NTR). We use the Mtz/NTR system to injury the zebrafish intervertebral disc severely in order to simulate the process on cervical disc degeneration. The spine histological change of zebrafish intervertebral disc degeneration and vertebrae degeneration through histological section observation. The spine Micro CT scanning to assess vertebral body bone microstructure difference. In situ hybridization detection of osteoblast and osteoclast vertebral body related gene expression changes.Results: We use laser scanning confocal microscope to scan the transgenic line Tg(twhh:Dendra2-NTR) in order to reveal the development process of zebrafish intervertebral disc. In the early larval, green fluorescent cells markers notochord cells and base plate, alizarin red staining vertebral mineralization is in the form of the ring of the vertebral body, from head to tail of notochord development gradually. In zebrafish youth period, green fluorescent cells markers the fibrous annulus and nucleus pulposus cells, alizarin red staining vertebral mineralization Began to backend forward and back ventral extension. Each spine contains three parts of vertebral body, neural arch, blood vessels. Neural arch and vascular arch are all in the edge of the side of the vertebral body. During zebrafish adulthood, green fluorescent cells markers Intervertebral disc fibrous annulus cells, alizarin red staining vertebral develop fully maturely, which put green fluorescent intervertebral disc embedded within it, and the spine is a distinct segmented mode.The innovation of the experiment is that Mtz/NTR system can specificity locate damage of intervertebral disc, and then construct a new animal model of zebrafish intervertebral disc degeneration by damaged the intervertebral disc severely. Through laser scanning confocal microscope scanning, severely damaged of intervertebral disc green fluorescent cells can’t repair by themselves. Red vertebrae were staining by alizarin red. The pathological phenotypes of vertebral degeneration is which vertebrae are closely linked, mineralization disorders, vertebral compression. Through the spine histological section observation, we found the dramatic change of cell morphological in intervertebral disc and vertebrae. The cellular structure of intervertebral disc degenerate obviously. TUNEL staining confirmed that in the treatment group only disc annulus fibrosus cells apoptosis, which is in accordance with the intervertebral disc degenerative disease significantly increased number of apoptotic cells. Combined with the Micro CT scanning, we could clearly reveal the pathological phenotypes of vertebrae degeneration, such as vertebrae fusion, smaller vertebrae, mineralization abnormity led to significantly shorter spine. By the technology in situ hybridization detection of spine, we could confirmed the osteoblast and osteoclast activity in down-regulation.Conclusion: The zebrafish intervertebral disc severely damage model could be used to study intervertebral disc degeneration. Severely damage intervertebral disc degeneration and vertebrae degeneration.