Accumulated Spinal Axial Biomechanical Loading Induces Degeneration in Intervertebral Disc of Mice Lumbar Spine

Abstract

Objective: To investigate the effect of accumulated spinal axial biomechanical loading on mice lumbar disc and the feasibility of applying this method to establish a mice intervertebral disc degeneration model using a custom-made hot plate cage. In previous studies, we observed that the motion pattern of mice was greatly similar to that of humans when they were standing and jumping on their lower limbs. There is little data to demonstrate whether or not accumulated spinal axial biomechanical loading could induce intervertebral disc degeneration in vivo.

Methods: Twenty-four 0-week-old mice were randomly divided into model 1-month and 3-month groups, and control 1-month and 3-month groups (n = 6 per group). The model groups was transferred into the custom-made hot plate cage three times per day for modeling. The control group was kept in a regular cage. The intervertebral disc samples of the L₃ -L₅ were harvested for histologic, molecular, and immunohistochemical studies after modeling for 1 and 3 months.

Results: Accumulated spinal axial biomechanical loading affects the histologic, molecular, and immunohistochemical changes of mice L_3- L₅ intervertebral discs. Decreased height of disc and endplate, fissures of annulus fibrosus, and ossification of cartilage endplate were found in morphological studies. Immunohistochemical studies of the protein level showed a similar expression of type II collagen at 1 month, but a slightly decreased expression at 3 months, and an increased expression level of type X collagen and matrix metalloproteinase 13 (MMP13). Molecular studies showed that ColIIa1 and aggrecan mRNA expression levels were slightly increased at 1 month (P > 0.05), but then decreased slightly (P > 0.05). ColXa1, ADAMTS-5, and MMP-13 expression levels werer increased both at 1 and 3 months (P < 0.05). In addition, increased expression of Runx2 was observed.

Conclusion: Accumulated spinal axial loading provided by a custom-made hot plate accelerated mice lumbar disc and especially endplate degeneration. However, this method requires further development to establish a lumbar disc degeneration model.

Keywords: Biomechanical loading; Degeneration; Endplate; Intervertebral disc; Lumbar; Model.

Figure 1

The cage contained a shell with the bottom able to be heated up (A) and a separator (B) that prevented the mice from huddling. (C) The whole cage.

Figure 2

Illustrative description of the custom‐made hot plate cage, and the method of modeling. When mice were kept in a regular cage, the mice acted as usual; when in the cage heated to 50°C, the mice began to jump.

Figure 3

Alcian blue/orange G staining of lumbar samples. In both of the model samples, the IVD structure became disorganized. In the model groups, the height of disks (yellow arrows) was significantly decreased at 1 month when compared with the control group (×100, A, C, G), while the height of the endplate (red arrows) was decreased at all time points when compared with the control samples (×200; B, D, H); fissures (black arrows) were found in the outer layer of the anulus fibrosus of the lumbar sample after modeling at both time points, but were larger at 3 months (E, F) compared to 1 month (B, D). The cartilage endplate was gradually replaced by ossification. The staining of the nucleus pulposus of the 3 month model group was thinner compared to that of the control group. More endplate ossification was developed in the 3‐month group (E, F).

Figure 4

Immunohistochemical assessments of the protein level of type II collagen, type X collagen, and MMP13. Immunohistochemical study showed that the protein level of type II collagen was decreased slightly after modeling in both 1 month and 3 month groups compared with the control group. The collagen X positive staining reached to the outer annulus fibrosus and the nucleus pulposus, and that of the 3‐month group was stronger than for the 1‐month group. The positive staining of MMP‐13 of the 1‐month and 3‐month groups reached to the edge of the annulus fibrosus and nucleus pulposus, and was stronger for the 3‐month group than for the 1‐month group.

Figure 5

The mRNA expression of ColIIa1, aggrecan, ColXa1, MMP‐13, ADAMTS‐5, and Runx2. In the model group, the mRNA expression of ColIIa1 and aggrecan decreased but not significantly (A, C), while that of ColXa1, MMP‐13, and ADAMTS‐5 were significantly upregulated (B, D, E), and that of Runx2 also had the trend of upregulating compared with the control group at the same time after modeling (F). The columns represent the mean ± standard deviation of six independent experiments.