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. 2018 Sep:70:102-122.
doi: 10.1016/j.matbio.2018.03.019. Epub 2018 Mar 29.

A novel mouse model of intervertebral disc degeneration shows altered cell fate and matrix homeostasis

Hyowon Choi  1 , Steven Tessier  1 , Elizabeth S Silagi  1 , Rutvin Kyada  2 , Farzad Yousefi  2 , Nancy Pleshko  2 , Irving M Shapiro  1 , Makarand V Risbud  3
Affiliations

A novel mouse model of intervertebral disc degeneration shows altered cell fate and matrix homeostasis

Hyowon Choi et al. Matrix Biol. 2018 Sep.

Abstract

Intervertebral disc degeneration and associated low back and neck pain is a ubiquitous health condition that affects millions of people world-wide, and causes high incidence of disability and enormous medical/societal costs. However, lack of appropriate small animal models with spontaneous disease onset has impeded our ability to understand the pathogenetic mechanisms that characterize and drive the degenerative process. We report, for the first time, early onset spontaneous disc degeneration in SM/J mice known for their poor regenerative capacities compared to "super-healer" LG/J mice. In SM/J mice, degenerative process was marked by decreased nucleus pulposus (NP) cellularity and changes in matrix composition at P7, 4, and 8 weeks with increased severity by 17 weeks. Distinctions between NP and annulus fibrosus (AF) or endplate cartilage were lost, and NP and AF of SM/J mice showed higher histological grades. There was increased NP cell death in SM/J mice with decreased phenotypic marker expression. Polarized microscopy and FTIR spectroscopy demonstrated replacement of glycosaminoglycan-rich NP matrix with collagenous fibrous tissue. The levels of ARGxx were increased in, indicating higher aggrecan turnover. Furthermore, an aberrant expression of collagen X and MMP13 was observed in the NP of SM/J mice, along with elevated expression of Col10a1, Ctgf, and Runx2, markers of chondrocyte hypertrophy. Likewise, expression of Enpp1 as well as Alpl was higher, suggesting NP cells of SM/J mice promote dystrophic mineralization. There was also a decrease in several pathways necessary for NP cell survival and function including Wnt and VEGF signaling. Importantly, SM/J discs were stiffer, had decreased height, and poor vertebral bone quality, suggesting compromised motion segment mechanical functionality. Taken together, our results clearly demonstrate that SM/J mouse strain recapitulates many salient features of human disc degeneration, and serves as a novel small animal model.

Keywords: Animal models; Hypertrophy; Intervertebral disc degeneration; LG/J; Matrix degradation; Nucleus pulposus; SM/J.

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Conflict of interest statement

Disclosure: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. SM/J mice show severe intervertebral disc degeneration
(A, B) Safranin O/Fast Green staining of coronal section of 8- and 17-week-old LG/J and SM/J mouse intervertebral discs showed healthy discs of LG/J, and degenerated discs of SM/J (top row, scale bars=200 μm). High magnification view of NP tissue showed LG/J mice with vacuolated cells and a normal cellularity, while SM/J mice showed loss of vacuolated cells and presence of chondrocyte-like cells (black arrowheads) sparsely distributed within condensed and fibrotic matrix (white arrowheads, second row, scale bars=50 μm). In SM/J mice, tissue boundaries between NP and AF (third row, scale bars=50 μm), and between NP and endplate (bottom row, scale bars=50 μm) were blurred at 17 weeks (black arrow). (C) Histological grading using modified Thompson scale was performed. There was a significant difference between two mouse strains in the proportion of degenerated NP at both age groups, and in the proportion of degenerated AF at 17 weeks. Data were collected from 3 discs per mouse (n=6 mice per group). χ2 test. NS=not significant; ****, p≤0.0001.
Figure 2
Figure 2. SM/J mouse intervertebral discs have higher average Thompson grades
(A, B) Average modified Thompson score of NP and AF for each caudal disc level was shown as scatter plots. All three levels of NP and two levels of AF in SM/J discs at 17 weeks had significantly higher average Thompson grades, indicating increased severity of degeneration. There was no significant difference between these mice at 8 weeks. (C) Measurements of NP aspect ratio, determined by width divided by height of the tissue, showed significantly lower ratio at 8 weeks. Data were collected from 3 discs per mouse (n=6 mice per group). t-test. NS=not significant; *, p≤0.05; **, p≤0.01; ****, p≤0.0001.
Figure 3
Figure 3. NP cells in SM/J mice undergo apoptosis and lose their notochordal phenotype
(A) TUNEL assay (green) of LG/J and SM/J intervertebral disc sections showed increased cell death in NP of SM/J mice, especially at 8 weeks (top row; scale bars=300 μm). Corresponding high magnification images of the area within the box are shown (second and fourth column; Bars=150 μm). (B) Immunofluorescence staining of NP markers including CA3, GLUT-1, and KRT19 shows that all three markers were lost in the NP of 17-week-old SM/J mice. GLUT-1 and KRT19 levels were also decreased in SM/J mice at 8 weeks. SDC4 levels were low in SM/J mice at both age groups (scale bars=200 µm). Dotted lines were drawn to demarcate different tissue compartments within the disc. All staining was performed using at least 3 animals per group.
Figure 4
Figure 4. NP matrix of SM/J mice have increased collagen content
Lack of Picrosirius red staining in NP matrix of LG/J mice at both 8 and 17 weeks. SM/J mice showed little to no staining at 8 weeks, while discs of 17-week-old animals showed strong signal, indicating increased collagen content (top row). Collagen fibers visualized under polarized microscope (bottom row) supported drastic upregulation of collagen content in NP compartment in 17-week-old SM/J mice. Scale bars=200 pm. Staining was performed using 6 animals per group.
Figure 5
Figure 5. FTIR analysis show altered disc matrix composition in SM/J mice
(A) Safranin O/Fast Green staining of representative LG/J and SM/J discs at 17 weeks (left column), and corresponding spectral cluster analysis images (right column). Scale bars= 200 µm. Cluster analysis of infrared imaging spectra of the discs showed anatomic regions qualitatively similar to the histology images. (B) A representative of average inverted second derivative spectra generated from NP, AF, and CGP clusters of LG/J and SM/J mice. Mean absorbance values of peak at 1660 cm−1, 1338 cm−1, and 1156 cm−1 were used form total protein, collagen, and proteoglycan content respectively. (C, D) Average second derivative peak values for collagen content in different tissue compartments were represented with scatter plots. NP of SM/J mice had a significant increase in collagen content, while no difference was seen in AF, inner AF (IAF), and outer AF (OAF) between LG/J and SM/J mice. (E, F). Average second derivative peak values for proteoglycan content were significantly lower in NP of SM/J mice. No difference was seen in AF, IAF, or OAF between the two mouse strains. (G, H) Total protein content in NP of SM/J mice was significantly higher, while that in AF was statistically significantly but minimally lower. Data for all FTIR analysis were collected from n=6 mice per strain with 3 sections/disc/animal. Scatter plots shown as mean ± SEM. t-test. NS=not significant; *, p≤0.05; ***, p≤0.001; ****, p≤0.0001.
Figure 6
Figure 6. Aggrecan degradation is increased in the NP of SM/J mice
(A-D) Immunofluorescence staining of aggrecan, ARGxx, collagen I, and collagen II (red). (A) Aggrecan showed strong expression in NP and some expression in inner AF in both LG/J and SM/J mice at both age groups. (B) ARGxx, aggrecan neoepitope generated by ADAMTS-dependent degradation, was significantly increased in SM/J mice at 17 weeks. (C, D) At both ages, collagen I and II were mainly expressed in AF of both mouse strains. There was no discernable difference in the expression of these proteins between SM/J and LG/J mice. Dotted lines were drawn to demarcate different tissue compartments within the disc. Scale bar=200 µm. All staining was performed using at least 3 animals per group.
Figure 7
Figure 7. NP of SM/J mice have increased collagen X and MMP13 expression
(A) Immunofluorescence staining of collagen X showed minimal expression in NP and AF of both LG/J and SM/J at 8 weeks (left column; scale bar =300 µm). Hypertrophic zone of growth plate stained strongly for collagen X. At 17 weeks, there was a dramatic increase in collagen X within the NP tissue of SM/J mice (middle column; scale bar=300 µm). Images in the third column show the area within the box from the middle column in higher magnification (Scale bar =150 pm). (B) Immunofluorescence staining of LG/J and SM/J discs showed increased expression of MMP13, a marker of hypertrophic chondrocytes, in the NP and AF compartments of 17-week-old SM/J mice (Scale bar=200 µm). Dotted lines were drawn to demarcate different tissue compartments within the disc. All staining was performed using at least 3 animals per group.
Figure 8
Figure 8. NP cells of SM/J mice show evidence of hypertrophy
(A-Q) Real time RT-PCR analysis of NP tissue from 7-week-old LG/J and SM/J mice represented by scatter plots (mean ± SEM). (A-G) mRNA expression of markers of chondrocyte hypertrophy, including Col10a1, Ctgf, and Runx2, were significantly upregulated in SM/J mice. Although Mmp13 levels were not different, significantly higher percentage of SM/J mice expressed Mmp13. (E-G) Levels of Adamts4, Fmod, and Il6 were not different between LG/J and SM/J mice. (H) Expression of Col11a1 was significantly higher in SM/J mice. (I, J) Levels of Vegfa and Cdkn1a, pro-survival factors, were lower in SM/J mice. (K) Expression of a Wnt/B-catenin signaling target, Axin2, was lower in SM/J mice. (L, M) Expression of Bmp2, and its target, Msx2, was not different between LG/J and SM/J mice. (N, O) Levels of Ankh as well as Spp1 that are involved in modulating mineralization were not different between two mouse strains. (P, Q) Enpp1 level and the number of discs that expressed Alpl were significantly higher in SM/J mice. n=10 samples per group (10 mice for LG/J and 15 mice for SM/J). Significance of proportionality was tested using χ2 test (Mmp13, Adamts4, and Alpl). T test for normally distributed data, Mann-Whitney test for non-normally distributed data, Shapiro-Wilk normality test was done to check the distribution. NS=not significant; *, p≤0.05; **, p≤0.01.
Figure 9
Figure 9. Discs of SM/J mice have compromised biomechanical properties
(A) An example of a loading curve extrapolated at the final 20th cycle was redrawn so that the tension and compression curves were made to intersect before various parameters were measured. (B, C) Compressive stiffness was higher in SM/J mice while compressive ROM was not significantly different. (D, E) SM/J mice had significantly higher NZ stiffness and lower NZ ROM. (F) Creep displacement was significantly lower in SM/J mice. All of the parameters indicated that SM/J discs were stiffer than that of LG/J. Data are represented as scatter plots (mean ± SEM). N=1-3 motion segments for 5 animals per group. NZ: neutral zone; ROM: range of motion; t-test. NS=not significant; *, p≤0.05; **, p≤0.01.
Figure 10
Figure 10. SM/J mice demonstrate poor vertebral bone health
(A) Representative µCT scans of caudal motion segment (Ca6-7) of LG/J and SM/J at 17 weeks, showing a coronal cut-plane through 3D reconstructions. Scale bars=1 mm. (B-D) Disc height, vertebral length, and disc height index (DHI) measurements of LG/J and SM/J at both ages were shown as scatter plots (mean ± SEM). SM/J mice had significantly smaller disc height as well as vertebral length, resulting in higher DHI at 8 weeks. (E-J) Measurements of various vertebral trabecular bone parameters demonstrated that, compared to LG/J, SM/J mice had lower bone volume fraction (BV/TV), trabecular number (Tb. N.), and connectivity density (Conn. Dens.), but higher trabecular separation (Tb. Sp.), and structure model index (SMI), suggesting that SM/J mice had compromised vertebral bone quality. Trabecular thickness (Tb. Th.) was not significantly different between two mouse strains. All analyses were done with n=6 mice per group with 3 consecutive vertebrae/animal. Scatter plots shown as mean ± SEM. t-test. NS=not significant; *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001.
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