Establishment Of Rabbit Lumbar Intervertebral Disc Degeneration Model By Annulus Needle Puncture Percutaneously Under CT Guidance

BackgroundLow back pain (LBP) is a prevalent disease of musculoskeletal conditions, and is commonly encountered in clinical routine work. It was estimated that60-80%adults had the history of LBP in their lifetime, while about15%of the population underwent LBP at any one time. The factors that result to LBP are very complex, but the degeneration of intervertebral disc is thought to be the most important condition. Actually, diseases resulted from intervertebral disc degeneration(IDD) are among the main conditions that lead to functional incapacity of human beings, and a great number population lose their working abilities before their retirement for their suffering for LBP. For the pathophysiology of IDD is not yet completely understood, the pathway that IDD leads to LPB remains obscured. Recently, a serial of new treatments, including direct injections to intervertebral discs, carrying synthetic peptides or growth factors, genic segments, cells, and tissue engineering, aroused worldwide interest among researchers. These urge a thorough understanding concerning about the pathogenesis and pathophysiology of IDD. A further research focusing on IDD would be helpful for prevention and treatment of LBP, and furthermore, would do some good to our society.Intervertebral disc degenerates most easily among all tissues. Actually, it is the first tissue that degenerates as age increases. For the progress of IDD lasting for several decades, affected by a great number of factors, and concerning of principles of biomedical ethics, it is impracticable to perform direct researches in body of human beings in vivo. Inevitably, researchers tend to establish suitable animal models to investigate the pathogenesis and new approaches of treatment of IDD. The models would carry desirable degenerated intervertebral discs in vivo quickly, and the convenience of excision of the tissue for histopathologic analysis at any time. So it is feasible to reveal the pathophysiology in different stage of IDD, or to demonstrate the effect of a new treatment. However, it is very hard to develop a perfect animal model. First, the IDD progress of the animal model should be comparable with human’s. The second, the animal model must be controllable, reproducible and cost-effective. The third, ethical issues about animals have to be concerned on.Researchers prefer to use rabbits and rats to establish IDD model. They perform annular puncture as the most popular method to induce degeneration of animal disc For the easily reproducibility, directly visuality, exactly effectivity, they often underwent open surgery to injure annulus fibrosus. But the open surgical puncture brings severe injury to the animals, or even resulting to death. In addition, the excessive dissection would influence the result of the experiment. Currently, some scholars tried to develop animal models with minimal invasively methods to alleviate the disadvantages of open surgery. For example, some researchers succeeded to develop new models under fluoroscopy. They punctured the annular fibers or injected medicine to nucleus pulposus without open surgery. However, for the shortcoming of fluoroscopy, it just provides2-dementioned images about the position of the needle, leading to incorrect puncture. What’s more, fluoroscopic examination is helpless to show adjacent soft tissues of intervertebral disc. So it’s inconvienent to perform the operation, to some extent.The aim of our study was to develop a new minimal invasively rabbit lumbar IDD model. We performed percutaneous puncture of annulus fibrosus under CT guidance in vivo. To verify the progress and effectiveness of degeneration, radiographs and MR images were obtained preoperatively and at4,8,12weeks postoperatively. The rabbit were executed after radiologic examinations in24hours, the discs were isolated, decalcified, and stained. Histopathological and Immunohistochemical analysis were performed. The model we developed could be helpful for understanding the pathogenesis and pathophysiology of IDD, and could be applied to further study for new treatment.Based on the criteria described above, histological scores were summed and averaged between the two readers (Table2). The scores between injured discs and uninjured control discs were analyzed using the Mann-Whitney test.Objective:To establish a rabbit model of disc degeneration by puncturing annulus fibrosus under CT guidance, and to verify the progress and effectiveness of degeneration by radiology and pathology.Methods:1. Animals:Eighteen New Zealand white rabbits either sexes aged about3months weighing2.7-3.3kg were employed in our experiment.2. Grouping:L5/L6discs were stabbed group, which are the first discs in the cranial side of the line connecting bilateral iliac crests. L4/L5discs were taken as sham group, while L3/L4discs were control group. 3. Preoperative examination:All animals were taken lateral radiography and sagittal MR T2weighted images preoperatively, of which were excluded while there were lesions in the vertebrae or discs.4. Surgical procedure:Connected bilateral iliac crests with a line, and punctured subcutaneously by a22-gauge needle in parallel with the line, and the needle was used as metal loacator. Measured the distance between the targeted disc (L5/L6disc, which is the first disc in the cranial side of the line connecting bilateral iliac crests) and metal locator in CT located diagram, then marked the corresponding planar with a labeled line on the skin using a pen. In the lateral of the spine subcutaneously, a22-gauge needle was stabbed in parallel with labeled line, then measured the distance between the tail of the needle and the centre of the targeted disc, and confirm the projecting position on the skin. A18-gauge needle was stabbed though the projecting position, while the direction and position of the needle were adjusted under spiral CT guidance based on the axial images. When the pinhead touched the margin of disc, it was ensured that the extended line of the needle went though the centre of disc. The needle was gently stabbed into the annulus fibrosus. CT scan was performed to confirm to pinhead’s position, and the depth of about5mm of the needle was measured using the internal tools. The pinhead was pulled out carefully until it located in the margin of disc. The puncture procedure was repeated for3times. L4/L5disc was conducted with the same procedure, with the pinhead reaching to the margin of disc but without stabbing into the annulus fibrosus. The L4/L5discs were used as control group without any operation.5. Evaluation of the effect of IDD:Each six rabbits were executed randomly at4,8and12weeks postoperatively respectively after radiographic and MRI examinations in24hours. The intervertebral space, the changes of vertebrea on radiography and signal intensity of discs on MR images in all groups were analyzed. The disc height relative value (DHRV) was calculated based on intervertebral space of pre and post operation (DHRV=postoperative intervertebral space/preoperative intervertebral space x100), and modified Thompson scoring based on MR images were performed in all groups. Histological, immunohistochemical features of disc and proteoglycan content of nucleus pulposus were assessed in stabbed, sham and control discs too.6. Statistical analysis:Data were presented as mean±standard deviation. The testing results were analyzed in SPSS software (Version17.0). One-Way ANOVA method were used to assess the values of DHRV and proteoglycan content. Kruskal Wallis tests were used to compare modified Thompson scoring of MRI. P<0.05was considered statistically significant.Results:1. Control and sham groups:At4,8,12weeks postoperatively, control and sham discs showed lack of decrease of the intervertebral space, without endplate sclerosis or formation of osteophyte of vertebrae, radiologically. On MR T2weighted images, the signal intensity remained bright in both control and sham discs. The nucleus pupposus presented as white gel-like material, surrounded by concentric fibrous lamellae, which could be clearly differentiated from each other. Histologically, nucleus pulposus contained a great number of cells with even distribution, and annulus fibrosus presented as concentric lamellae shape. Immunohistochemistry demonstrated strong positive type I and negative type II collagen staining of nucleus pulposus of control and sham discs at each time point. The proteoglycan content of control and sham groups at4,8,12weeks postoperatively were372.3±47.7,325.8±61.3,298.5±45.4μg/g, and346.7±74.6,273.6±50.9,269.0±29.5μg/g, respectively. They turned to continually decrease but without statistically difference(P>0.05).2. Stabbed group: ①At4weeks postoperatively, radiographs showed decrease of intervertebral space slightly (DHRV=70.78±4.55), and MR images demonstrated mild decrease of signal intensity, with5modified Thompson Ⅱ discs, and1modified Thompson III disc. Histologically, cell count of nucleus pulposus decreased slightly, while annulus fibrosus became disorganized. Immunohistochemical analysis exhibited that nucleus pulposus was stained lightly yellow in collagen type I immunostaining and moderate yellow in collagen type Ⅱ immunostaining. The proteoglycan content was233.9±16.6μg/g. Compared with control and sham groups, the DHRV of stabbed group decreased with modified Thompson scoring increasing, and the proteoglycan content decreased. The statistical result showed significantly difference of the above analysis.②At8weeks postoperatively, radiographs showed significant decrease of intervertebral space (DHRV=50.63±4.04), and endplate osteosclerosis was seen. MR images revealed moderate decrease signal intensity on T2weight images with4modified Thompson Ⅲ discs, and2modified Thompson IV discs. In morphologic pathology, the nucleus pulposus became atrophy without appearance of gel-like nature. It was found a great amount of fibrous tissue and small amount of cartilage tissue in the zone between the nucleus pulposus and annulus fibrosus. Histologically, nucleus pulposus was separated by proteoglycan matrix into multiple elliptical shaped cell islands, and fibrochondrocyte was found in nucleus pulposus. The concentric fibrous lamellae distorted, or even fractured. Immunohistochemically, collagen type Ⅰ immunostaining images showed that nucleus pulposus was stained yellow, and collagen type Ⅱ immunostaining images revealed moderate yellow stained nucleus pulposus. The content of proteoglycan of nucleus pulposus was184.9±11.6μg/g. The DHRV and proteoglycan content fell lower and, the modified Thompson scoring got higher, comparing with those of control and sham groups at the same time point and of stabbed group at4weeks postoperatively. The differences were of statistic significant.③At12weeks postoperatively, radiographs demonstrated successional decrease of intervertebral space (DHRV=44.78±4.55), with obvious endplate sclerosis and formation of vertebrae osteophyte. The signal intensity on T2weighted images of stabbed discs decreased continuously. There were1disc of modified Thompson III, and5discs of modified Thompson IV. Morphologically, the nucleus pulposus tissue almost disappeared, and was replaced by fibrous and osteoid tissues. Osteophyte was seen around the pinhole in annulus fibrosus. Histologically, nucleus pulposus was replaced by fibrocartilage tissue, while the fibrous lamellae led to fragmentations, and its architecture disintegrated. On immunohistochemical images, the nucleus pulposus was stained to dark yellow in collagen type I, and faint yellow in collagen type II immunostaining, respectively. The proteoglycan content of nucleus pulposus was143.5±8.5μg/g. The statistical result revealed that changes of DHRV, modified Thompson scoring, and proteoglycan content of stabbed group were differed from those of control and sham groups. In addition, the stabbed group showed progressive decrease of DHRV and proteoglycan content and continual increase of modified Thompson scoring at4,8,12weeks postoperatively. There exhibited significant difference between them.Conclusion:The percutaneous puncture of annulus fibrosus under CT guidance could induce lumbar disc degeneration in rabbit. The slowly progressive degeneration was confirmed by radiology and pathology. The advantages of this method included:1. Using minimal invasive technology, the rabbits were of high survival rate with little trauma to the adjacent tissues of disc. 2. The procedure was feasible, with accurately location of needle. This technology was not only suitable for investigating the physiopathology of IDD, but also could be applied as new therapeutic method of injection medicine.3. The progress of IDD of the model was progressive, so it provided a convenient method for acquiring different degree IDD for variant purposes.4. Compared with spontaneous models, normal discs could coexist with degenerated discs in a spine, which can be used as a control group in the further research of therapeutic methods.