Table 1.
Primers used for quantitative reverse transcription polymerase chain reaction.
Fig 1.
Histological analyses of the mouse lumbar spine and a mechanical stress loading device.
The application of mechanical stress to the mouse LF using a novel device. (A) HE and (B) EVG staining of the spine sections. (C) Photographs and (D) schematic illustrations of the device. (E) A coronal CT image showing that the iliac crest line corresponds to the L5-L6 disc level. (F) Sagittal CT images showing that mechanical stress was consistently applied at the L5-L6 level. Scale bars (A): 300 μm; insets: 50 μm; (B): 300 μm; insets: 100 μm. LF, ligamentum flavum; Ver, vertebral body; Du, dural tube; SAP, superior articular process; IAP, inferior articular process; La, lamina.
Fig 2.
Mechanical stress induced LF hypertrophy.
(A) Axial sections of the mouse LF with/without 12-week mechanical stress loading on EVG staining. (B and C) Bar graphs showing the cross-sectional area, width, thickness, and the area of collagen fibers and elastic fibers in the two groups. (D and E) High magnifications of (A). (F) Bar graph showing the ratio of collagen fibers to elastic fibers in the two groups. Scale bars (A): 500 μm; insets: 50 μm; (D and E): 10 μm. *p < 0.05, Wilcoxon’s rank sum test, n.s. = not significant (n = 5/group).
Fig 3.
Our mouse model histologically reflected mildly hypertrophied LF in humans.
(A) MRI and EVG staining of human samples: non-hypertrophy and mild and severe hypertrophy. The white broken lines indicate outlines of the LF. (B) Bar graph showing the ratio of elastic fibers to collagen fibers in the three groups. *p < 0.05, an ANOVA with the Tukey-Kramer post hoc test (n = 10/group). (C) EVG staining of the mouse LF with/without 12-week mechanical stress. (D) Bar graph showing the ratio of elastic fibers to collagen fibers in the two groups. *p < 0.05, Wilcoxon’s rank sum test (n = 5/group). Scale bars (A): 100 μm; (C): 50 μm.
Fig 4.
The changes in the cellular distribution in the hypertrophied LF of mice and humans.
(A and B) The number of cells significantly increased with LF hypertrophy in humans. *p < 0.05, an ANOVA with the Tukey-Kramer post hoc test (n = 10/group). (C-F) The comparison of the number of cells (Hoechst, blue), infiltrating BMDCs (EGFP, green), and proliferating cells (BrdU, red) in the mouse LF with/without 12-week mechanical stress. To detect BMDCs, we generated bone-marrow-chimeric mice by transplanting from CAG-EGFP mice. The white broken lines indicate the outlines of the LF. *p < 0.05, Wilcoxon’s rank sum test (n = 5/group). Scale bars (A): 100 μm; (C and E): 100 μm; insets: 10 μm.
Fig 5.
The mRNA expression of fibrosis-related factors in our mechanical stress mouse model.
(A-D) The gene expression of collagens, inflammatory cytokines, growth factors, and angiogenesis-related factors evaluated in the mouse LF with/without mechanical stress loading by qRT-PCR (n = 5/group). *p < 0.05, Wilcoxon’s rank sum test, n.s. = not significant. COL1A1, collagen type 1 alpha 1; TNF-α, tumor necrosis factor-α; IL, interleukin; CTGF, connective tissue growth factor; PDGF-A, platelet-derived growth factor-A; TGF-β1, transforming growth factor-β1; VEGF-A, vascular endothelial cell growth factor-A; MMP, matrix metalloproteinase.
Fig 6.
The influence of macrophage infiltration following micro-injury on the mouse LF.
(A) The presence of infiltrating macrophages (Iba1, green) and neovascular vessels (laminin, red) in the mouse LF with/without micro-injury. (B-D) Bar graphs showing the gene expression of collagens, fibrosis-related growth factors, and angiogenesis-related factors in the two groups. (E) The collagen synthesis in the injured area (COL1A1, green) at 2 weeks after micro-injury. (F) Sagittal sections of the mouse LF with/without micro-injury on HE staining at 6 weeks after micro-injury. The black broken lines indicate the outlines of the LF. The asterisk indicates the area to which micro-injury was applied. *p < 0.05, Wilcoxon’s rank sum test (n = 5/group). Scale bars (A): 50 μm; insets: 20 μm; (E): 20 μm; (F) 200 μm. COL1A1, collagen type 1 alpha 1; TGF-β1, transforming growth factor-β1; CTGF, connective tissue growth factor; PDGF-A, platelet-derived growth factor-A; VEGF-A, vascular endothelial cell growth factor-A; MMP, matrix metalloproteinase.