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Development and Characterization of a Novel Bipedal Standing Mouse Model of Intervertebral Disc and Facet Joint Degeneration

Ao, Xiang, MD; Wang, Liang, MD; Shao, Yan, MD; Chen, Xulin, MD; Zhang, Jie, MD; Chu, Jun, MD; Jiang, Tao, MD; Zhang, Zhongmin, MD; Huang, Minjun, MD

Clinical Orthopaedics and Related Research®: June 2019 - Volume 477 - Issue 6 - p 1492–1504
doi: 10.1097/CORR.0000000000000712
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Background Intervertebral disc degeneration is a major cause of chronic low back pain, and excessive loading contributes to intervertebral disc degeneration. However, the lack of an effective bipedal in vivo animal model limits research about this condition.

Questions/purposes To evaluate the utility of a new type of bipedal standing mouse model for intervertebral disc degeneration, we asked: (1) Are there spinal degeneration changes in bipedal mice as determined by lumbar disc height, histologic features, and immunohistochemistry measures compared with control mice? (2) Are the bipedal mice comparable to aged mice for simulating the spinal degeneration caused by increased stress?

Methods Thirty-two 8-week-old male C57BL/6 mice were divided into experimental and control groups. Based on their hydrophobia, mice in the experimental group were placed in a limited water-containing space (5 mm deep) and were thereby induced to actively take a bipedal standing posture. This was conducted twice a day for a total of 6 hours a day, 7 days a week. Control mice were similarly placed in a limited but water-free space. Video surveillance was used to calculate the percentage of time spent in the bipedal stance for the two groups of mice. Compared with the control group, the percentage of time standing on both feet in the experimental group was higher (48% ± 5%, 95% confidence interval [CI], 42%–54% versus 95% ± 1%, 95% CI, 92%–97%; p < 0.001). Eight mice from both groups were then randomly euthanized at either 6 or 10 weeks and lumbar spine specimens (L3-L6) were collected. The lumbar disc height index (DHI%) of the two groups was compared using micro-CT measurements, and the extent of disc degeneration was assessed based on histologic staining (cartilage endplate height, disc degeneration score) and by immunohistochemistry (Col2a1,CollagenX, matrix metalloprotease-13 [MMP-13], osteocalcin [OCN]). In addition, the histopathologic features of spinal degeneration were compared with 12- and 18-month-old mice. A p value < 0.05 indicated a significant difference.

Results Lumbar disc degeneration was aggravated after 10 weeks with the DHI% decreasing (5.0% ± 0.4%; 95% CI, 4.6%–5.5% versus 4.6 ± 0.3%; 95% CI, 4.3%–4.9%; p = 0.011). Histologically, the cartilage endplate height of the experimental group was decreased compared with the control group (30 ± 6 μm; 95% CI, 24–37 μm versus 70 ± 7 μm; 95% CI, 63–79 μm; p < 0.001), and the disc degeneration score was increased (5 ± 1; 95% CI, 4–6 versus 1 ± 1; 95% CI, 0–2; p < 0.001). Expression of Col2a1, vimentin, and aggrecan in the experimental group was decreased compared with the control group, whereas the expressions of collagen X (60% ± 2%; 95% CI, 55%–66% versus 19% ± 3%; 95% CI, 17%–24%; p < 0.001), MMP-13 (54% ± 8%; 95% CI, 49%–61% versus 1% ± 1%; 95% CI, 1%–2%; p < 0.001), and OCN (41% ± 3%; 95% CI, 34%–49% versus 5% ± 1%; 95% CI, 2%–7%, p < 0.001) were increased. The spine degeneration caused by this model was primarily manifested in the degeneration of the annulus fibrosus and facet joints compared with aged mice, whereas the degree of degeneration in the nucleus pulposus tissue and cartilage endplates was mild.

Conclusions We believe we have established a noninvasive and effective in vivo bipedal mouse model for studying disc degeneration and biologic signal transduction comparable to that seen in intervertebral disc degeneration.

Clinical Relevance This in vivo mouse model of intervertebral disc degeneration can simulate the pathogenesis of spinal degeneration caused by increased stress and this can be used to study questions such as disc herniation in young adults.

X. Ao, L. Wang, Y. Shao, J. Zhang, J. Chu, T. Jiang, Z. Zhang, M. Huang, Department of Orthopaedics, the Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, PR China

X. Ao, L. Wang, Y. Shao, J. Zhang, J. Chu, T. Jiang, Z. Zhang, M. Huang, Academy of Orthopaedics, Guangdong Province, Guangzhou, Guangdong, PR China

X. Chen, Department of Orthopaedics, the First People’s Hospital, Jingdezhen, Jiangxi, PR China

M. Huang, Z. Zhang, Academy of Orthopaedics, Guangdong Province, 183 West Zhongshan Avenue, Guangzhou 510630 China, Email: hmjlceb@163.com, nfzzm@163.com

This work was funded by National Natural Sciences Foundation of China (81672228 [ZZ], 81874013[ZZ], 31801012[MH]) and Natural Science Foundation of Guangdong Province (2016A030313608 [MH]).

All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request.

Clinical Orthopaedics and Related Research® neither advocates nor endorses the use of any treatment, drug, or device. Readers are encouraged to always seek additional information, including FDA-approval status, of any drug or device prior to clinical use.

Each author certifies that his or her institution approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.

This work was performed at the Academy of Orthopaedics, Guangdong Province, Guangzhou, Guangdong, PR China; and the Department of Orthopaedic, the First People’s Hospital, Jingdezhen, Jiangxi, PR China.

Received August 08, 2018

Accepted February 15, 2019

© 2019 Lippincott Williams & Wilkins LWW
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