终板损伤致大鼠尾椎椎间盘退变模型的研究

A rat caudal vertebral intervertebral disc degeneration model based on cartilage endplate injury

中文关键词:  动物模型  软骨终板损伤  腰痛  椎间盘退变

英文关键词:Animal Models  Cartilaginous endplate injured  Low back pain  Intervertebral disc degeneration

中文摘要:

  【摘要】 目的：探索建立并验证一种操作简便、效果稳定的大鼠尾椎终板损伤致椎间盘退变的模型。方法：SD大鼠32只，年龄2个月，雄性，体重180~220g。随机挑选24只，通过腹腔注射麻醉后，使用手术刀沿尾6椎体下缘切入，损伤一侧软骨终板，分别在造模当天、造模2周与造模4周进行取材，分别为造模当天组、造模2周组、造模4周组。余8只大鼠作为假手术组，使用手术刀划开皮肤后缝合。通过Micro-CT对骨性终板进行观察，截取矢状面图像，测量并计算椎间盘高度指数。术段椎间盘切片后，分别行HE染色、番红固绿（safranin O-fast green，SO）染色、TUNEL染色（TdT-mediated dUTP nick-end labeling），通过HE、SO染色观察髓核、纤维环结构与终板损伤情况，通过SO染色对椎间盘进行组织学评分，评估椎间盘退变情况；通过TUNEL染色计数观察细胞凋亡情况。结果：Micro-CT显示造模2周后椎体骨性结构整体保持完整，终板厚度增加，骨性终板结构相对完整，但其中出现大片缺损，椎体出现扁平化倾向，周围出现大片骨赘。假手术组、造模当天组、造模2周组、造模4周组，椎间盘高度指数分别为（9.29±0.74）%、（9.20±1.64）%、（6.05±1.30）%、（5.10±1.15）%，造模组2周组、造模4周组椎间隙高度均显著下降，与假手术组相比，其差异具有统计学意义（P＜0.05）。SO、HE染色显示，造模当天组终板形态与假手术组相似；造模2周后，即可见软骨终板形态改变，孔隙率下降；造模4周时终板损伤更为明显，软骨终板向椎间盘侧突起，孔隙基本消失，纤维环、髓核呈退变表型，纤维环板层结构破坏紊乱，髓核细胞数量明显减少，基质纤维化。组织学评分显示：假手术组6.8±0.64分，术后当天6.8±1.36分，造模2周12.0±0.80分，造模4周13.0±1.20分。其中，造模后2周组和4周组椎间盘退变显著，假手术组、造模当天组与造模4周组间评分差异存在统计学意义（P＜0.05），余组间差异无统计学意义（P＞0.05）。TUNEL染色显示，假手术组TUNEL染色阳性细胞占比（4.43±0.46）%，造模当天组（5.82±0.90）%，造模2周（12.07±1.57）%，造模4周（98.26±4.34）%，两造模组相比于假手术组，其椎间盘细胞数量减少，凋亡细胞比例增加（P＜0.05）。结论：本研究构建得大鼠尾椎椎间盘软骨终板损伤模型构建方法可导致软骨终板损伤，进而诱发椎间盘退变，其造模效果快速稳定，是一种较为理想的用于终板损伤致椎间盘退变的研究模型。

英文摘要:

  【Abstract】 Objectives: To explore the establishment and validation of a simple and stable model of intervertebral disc degeneration(IDD) induced by endplate injury in the rat caudal vertebrae. Methods: 32 male Sprague-Dawley(SD) rats, aged 2 months, weighed 180-220g, were used. Among them, 24 rats were randomly selected. After anesthesia by intraperitoneal injection, the skin was incised and a scalpel was used to cut along the edge of the coccygeal 6(Co6) vertebral bone to injure the unilateral cartilaginous endplate. Specimens were harvested for study on the modeling day(modeling day group), at 2 weeks post-modeling(modeling 2-week group), and at 4 weeks post-modeling(modeling 4-week group). The remaining 8 rats served as a sham surgery group, and the skin was incised with a scalpel and then sutured. The bony endplate was observed using Micro-CT, and sagittal plane images were captured for measurement and calculation of the disc height index(DHI). Hematoxylin and eosin(HE) staining, safranin O-fast green(SO) staining, and TdT-mediated dUTP nick-end labeling(TUNEL) staining were carried on sample slices. The structure of the nucleus pulposus, annulus fibrosus, and endplate injury were observed via HE and SO staining. Histological scoring of the intervertebral disc was performed using SO staining to evaluate the degree of disc degeneration. Cell apoptosis was observed via TUNEL staining. Results: Micro-CT showed that 2 weeks after modeling, the overall bony structure of the vertebrae remained intact. The endplate thickness increased, and the bony endplate structure was relatively intact, but large areas of defect appeared within it. The vertebrae showed a tendency towards flattening, and large osteophytes appeared around them. The DHIs for the sham group, modeling day group, modeling 2-week group, and modeling 4-week group were (9.29±0.74)%, (9.20±1.64)%, (6.05±1.30)%, and (5.10±1.15)%, respectively. Disc space height significantly decreased in both the 2-week and 4-week post-modeling groups compared to the sham group(P<0.05). SO and HE staining showed that the endplate morphology in the modeling day group was similar to the sham group. At 2 weeks post-modeling, changes in cartilaginous endplate morphology and a decrease in porosity were visible. At 4 weeks post-modeling, endplate injury was more pronounced, the cartilaginous endplate protruded towards the disc side, and porosity was essentially absent. The annulus fibrosus and nucleus pulposus exhibited degenerative phenotypes: the lamellar structure of the annulus fibrosus was disrupted and disorganized, nucleus pulposus cell numbers were significantly reduced, and the matrix became fibrotic. Histological scoring revealed significantly increased degeneration scores in the 2-week (12.0±0.80) and 4-week post-modeling groups (13.0±1.20) compared to the sham (6.8±0.64) and modeling day groups (6.8±1.36). The intervertebral discs degenerated obviously in the 2-week post-modeling group and 4-week post-modeling group, and statistically significant differences(P<0.05) were observed between the sham, modeling day, and 4-week post-modeling groups, while no significant differences existed between the other group(P>0.05). TUNEL staining showed that the percentage of TUNEL-positive cells was (4.43±0.46)% in the sham group, (5.82±0.90)% in the modeling day group, (12.07±1.57)% at 2 weeks post-surgery, and (98.26±4.34)% at 4 weeks post-surgery. Compared to the sham group, both modeling groups showed a reduction in disc cell count and an increased proportion of apoptotic cells(P<0.05). Conclusions: The modeling method we constructed for rat caudal intervertebral disc cartilaginous endplate injury can cause cartilaginous endplate damage, thereby inducing intervertebral disc degeneration. Its modeling effect is rapid and stable, making it a relatively ideal research model for studying intervertebral disc degeneration induced by endplate injury.