| 1Research BackgroundAccording to statistics, low back pain is the most common orthopedic disease with up to4/5of all young and middle-aged groups suffering, and disc degeneration is generally believed by most scholars to be the most common cause. Disc degeneration, accompanied by progressive fibrosis, indicates loss of normal disc structure by means of gel-like nucleus pulposus lost, original clear boundaries between the nucleus pulposus and annulus fibrosus gradually disappeared and rough layer and fibrosis of the annulus fibrosus. Although there is still no exact conclusion for the etiology and mechanism of disc degeneration, most scholars believe that they are related to the biomechanics, genetics, disc nutrition, cell aging, autoimmune and other various factors. Currently, conservative operations, such as physical therapy and anti-inflammatory drugs, are applied for the treatment of disc-genetic low back pain, which are symptomatic rather than curring. Therefore, this study aims for the IVDD pathogenesis, starting from the causes to prepare a cause-oriented and fundamental treatment and even the prevention of IVDD.In recent years, emerging biological regeneration treatment, which takes disc degeneration as the target, causes as starting points and regeneration as tools, and includes molecular therapies, gene therapy, cell therapy and tissue therapy, has drawn a lot of focuses. The advantages of these biological therapies are to well keep the normal disc biomechanical stability, avoid damage to the surrounding anatomical structures and maintain the relative motions between the vertebraes. But to apply these methods from an experimental study to the clinical use, further studies are still required for the pathophysiology of IVDD pathogenesis. It would help the research for pathogenesis and prevention of IVDD to prepare a reliable and effective IVDD animal model.There are more than10kinds of animals used for the preparation of degeneration models, including mice, rats, rabbits, sheep, dogs, pigs, primates and other species. Rats and mice are smaller in size, which is convenient for the preparatin but difficult for intradiscal injection, therefore, larger animals should be chosen for therapeutic purposes. However, the prices of sheep, dogs and pigs are higher, which is difficult for the larger-amount investigation, which leaves rabbit as the ideal model animal. Also, intradiscal injection technique of rabbits has been reported. There has been reports on rabbits as lumbar disc degeneration model animals, but almost all of them need skin and muscle incision, downstream needle with naked eyes or incision of the annulus fibrosis, of which the process can cause a great deal of trauma to the peritoneum and surrounding soft tissues of the experimental animals, and increase the chance of infection and high postoperative animal mortality, resulting in a waste of experimental animals; both damages to the surrounding tissues of the lumbar spine during surgery and postoperative scar formation have impacts on test results, contrary to the imitation of the natural degeneration process. Minimally invasive method has not yet been adopted for the preparation of rabbit degenerative disc model. 2ObjectEstablish a new minimally invasive rabbit degenerative disc model to provide a better test object for the further studies of degenerative disc mechanisms and treatment.3Methods1.As for the preparation of animal models, a safe and minimally invasive method of puncturing is found and applied on the basis of thorough study of the rabbit lumbar spine and surrounding tissue anatomy, and repeated puncturing operation. The puncturing path is far away from the peritoneum and important blood vessels, and under the guidance of C-arm, so the operation is accurate, convenient with one step only, low in the possibility of accidental injuries, quick and simple to operate, minor in damages and chances of infection, high in animal survival rates, and good in repeatability. With this method,18rabbits were operated on the L3/4, L4/5, L5/6intervertebral disk, and randomly divided into groups A, B and C after surgery(n=6). MRI detection was adopted4,8and12weeks after 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,L4/5and L5/6intervertebral disc nucleus pulposus were taken out, and RT-PCR was applied to detect the expression of type II collagen, and fixed by4%formaldehyde solution at4℃, dehydrated, paraffin-embedded, tissue-sliced, then HE stained and type Ⅱ collagen immunohistochemical stained for observation, and compared before and after with L2/3and L6/7of the nucleus pulposus as normal controls. 4ResultModel Preparing:the rabbit lumbarMRI results before and8and12weeks after operation indicated decreasing tendency of the signal intensity before and after puncture of nucleus pulposus(L3/4, L4/5and L5/6). Pfirrmann grading method was applied by two professors in the magnetic resonance room to calssified the results, using text of two-way ordinal classification data, and P<0.05, which suggested the significant correlation between length of time and degeneration of the intervertebral disk, and the longer time is, the more obvious the model of disc degeneration would be.4,8and12weeks after the GM group disc injection, raw materials were detected using RT-PCR technique and found to be significant in the disc Collagen Type II protein expression, among which week4was the most and week8and12were gradually decreased. HE staining showed the control group of nucleus pulposus has a large number of nucleus pulposus cells, the number declined4and8weeks after modeling, and it was mainly fibrocartilage organization that was in the nucleus pulposus after12weeks, accompanied by very few amount of marrow nucleated cells. The control group and the groups after4,8and12weeks were analysed of their gray values after Ⅱ collagen immunohistochemical staining, and the single-factor ANOVA analysis results were:F=41.82(P<0.05), and comparison results between groups using LSD-t test all showed significant differences(P<0.05), indicating that the content of type Ⅱ collagen in the nucleus pulposus decreased with the lengthening of modeling time.5ConclusionThe new minimally invasive rabbit lumbar intervertebral disc degeneration animal model was successfully established. |
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