长期低头位与寒湿刺激建立颈型颈椎病动物模型的研究

Establishment of An Animal Model for Cervical Spondylosis by Prolonged Low Head Position and Cold-Damp Stimulation

目的 设计一种操作简便、适用性强的兔盒用于制备颈型颈椎病的动物模型并验证其可行性,为研究颈椎病的发病机制、防治方法及作用机制提供技术基础与实验载体。方法 24只雄性新西兰兔随机分为对照组与模型组,每组12只。使用自制兔盒以低头位结合寒湿刺激建立兔颈椎间盘退变模型。造模8周后,通过影像学检查观察兔颈椎生理曲度、椎间盘、椎体及颈肌的变化。影像学检查结束后,行空气栓塞法处死,立即取出肌肉与椎间盘组织。HE染色法观察椎间盘与肌肉组织形态学变化,Masson染色法观察肌肉组织纤维化程度,荧光定量PCR法检测肌肉组织中巨噬细胞M1与M2表达水平,免疫组织化学染色法检测椎间盘组织IL-1的表达水平。结果 与对照组相比,模型组颈椎生理曲度改变,椎体边缘骨质增生形成;MRI结果显示椎间盘高度降低、纤维环破裂、髓核信号降低呈灰黑色,提示髓核脱水严重;超声影像学结果显示肌肉厚度增加、结缔组织信号增强、血流信号增加;HE染色显示肌纤维溶解,肌膜欠完整,细胞核增多与炎性细胞浸润;椎间盘组织中髓核组织消失,纤维环排列不规则,出现裂隙,软骨终板不完整;Masson染色可见大量蓝色胶原纤维覆盖;荧光定量PCR可见肌肉组织中M1巨噬细胞标记物CD68与M2巨噬细胞标记物CD163、CD206mRNA表达显著增加(P<0.05);免疫组织化学染色法可见椎间盘组织中IL-1表达显著增多(P<0.05)。结论 使用自制兔盒造模,可致实验兔颈肌劳损与椎间盘变性,成功建立颈型颈椎病模型。

Objective To design a rabbit box with easy operation and good applicability for preparing the animal model of cervical spondylosis and verify its feasibility and provide the technical basis and experimental vector for studying the pathogenesis, prevention and treatment methods and the mechanism of action. Methods Twenty-four male New Zealand rabbits were randomly divided into control and model groups, with 12 rabbits in each group. A rabbit model of cervical disc degeneration was established using a home-made rabbit box in a low head position combined with cold-damp stimulation. After 8 weeks of modeling, the physiological curvature of the cervical spine, the intervertebral discs, the vertebral bodies and cervical muscles of the rabbits were observed by imaging. At the end of imaging, the patients were sacrificed by air embolism, and the muscle and disc tissues were immediately removed. The morphological changes of intervertebral disc and muscle were observed by HE staining, the fibrosis level of muscle tissue was observed by Masson staining, the expression levels of macrophage M1 and M2 in muscle tissue were detected by RT-PCR,and the expression level of IL-1 in intervertebral disc tissue was detected by immunohistochemical staining. Results Compared with those of the control group, the physiological curvature of the cervical spine was changed, and the edge of the vertebral body had osteophyte formation in the model group. MRI results showed the disc height was decreased and the annulus fibrosus was broken, and the decreased signal of the nucleus pulposus was grayish black, indicating the severe dehydration of the nucleus pulposus. Ultrasound imaging results showed the muscle thickness, connective tissue signal and blood flow signal were increased. HE staining showed muscle fiber dissolution, less intact sarcolemma and increased nuclei with inflammatory cell infiltration. The nucleus pulposus tissue disappeared in the intervertebral disc tissue, the fiber ring arrangement was irregular and the clefts appeared. The cartilage end plates were uncompleted. Massive blue collagen fiber coverage was visible with Masson staining. Fluorescent quantitative PCR showed a significant increase in the mRNA expression of M1 macrophage marker CD68 and M2 macrophage markers CD163 and CD206 mRNA in muscle tissue(P<0.05). The expression of interleukin-1(IL-1) in the intervertebral disc tissue was significantly increased by immunostaining(P<0.05). Conclusions The cervical muscle strain and disc degeneration in experimental rabbits can be triggered and a cervical spondylosis model can be successfully established using a self-made rabbit box model.