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

English Keywords:Animal Models  Cartilaginous endplate injured  Low back pain  Intervertebral disc degeneration

English Abstract:

  【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.