Mechanical stress contributes to ligamentum flavum hypertrophy by inducing local inflammation and myofibroblast transition in the innovative surgical rabbit model

Abstract

Background: Lumbar spinal canal stenosis (LSCS) ranks as a prevalent spinal disorder in senior populations. Ligamentum flavum hypertrophy (LFH) is a significant feature of LSCS, yet its cause is unclear. The purpose of this study was to create a novel animal model for LFH and explore the pathological mechanisms involved.

Methods: A novel rabbit model for intervertebral mechanical stress concentration was established through posterolateral fusion using steel wire. Radiological analysis and biological validation were used to determine the crucial role of mechanical stress in LFH and explore the effect of this animal model.

Results: After 12 weeks, the LF subjected to mechanical stress concentration exhibited a disruption and reduction in elastic fibers, collagen accumulation, increased thickness of LF, elevated LF cells, and increased levels of certain factors related to fibrosis and inflammation. These findings were histologically consistent to those found in human LFH. Furthermore, in vitro, mechanical stretch was discovered to enhance the conversion of fibroblasts into myofibroblasts by boosting TGF-β1 secretion in LF fibroblasts. In addition, compared to conventional internal fixation, this new surgical model provided advantages such as minor damage, decreased bleeding, and reduced technical difficulty and molding costs.

Conclusion: This novel rabbit model is able to replicate the moderate pathological features of human LFH. Mechanical stress is an independent factor leading to LFH, which can promote the TGF-β1 secretion in LF cells and some inflammatory cells, subsequently induce the myofibroblast transition, and finally result in collagen accumulation and LF fibrosis.

Keywords: TGF-β1; fibrosis; ligamentum flavum; mechanical stress; rabbit model.

Figure 1

Examination of rabbit spinal structure, implant and internal fixation method, and LF thickness evaluation. (A) Anatomical features of the rabbit spine. (B) Three-dimensional spinal image taken before surgery. (C) Fused spine at L3/4 and L5/6 with a locking plate, viewed from the lateral and posterior side. (D) Standard X-ray images of the spine fused with internal fixation using a locking plate. (E) Fused spine at L3/4 and L5/6 reinforced with steel wire, visible from lateral and posterior perspectives. (F) Typical plain X-rays showing the fused spine with steel wire internal fixation. (G) Graphic depiction of the bipedal standing rabbit. (H) The cutting level was established by slicing a cross section at the midpoint between the upper and lower edges of LF. (I) Method for assessing the LF thickness. LF, ligamentum flavum.

Figure 2

Dynamic evaluation of motion range with X-ray. (A) Typical plain X-ray images in posteroanterior, lateral, flexible, and extensional position. (B–D) Measurement of movement range of L3-4 (B), L4-5 (C) and L5-6 (D). (E) Measurement of the anterior L4-5 disc height in the flexion and extension position. ^**p<0.01,^***p<0.001, ns, no significance.

Figure 3

This rabbit model was similar in histology to the moderate LFH observed in humans. (A) MRI images and Masson staining display the human LF in states of normal, mild, moderate, and severe hypertrophy, with red lines highlighting the LF outlines. Scale bar, 100μm. (B) Images of rabbit LF samples in various groups stained with H&E, EVG, and Masson (n=12). Scale bar, 50μm. (C) Measurement and analysis of the LF area with Masson staining (n=12). (D) Analyzing the LF thickness among the four distinct groups (n=12). (E) Assessment of LF width in the four groups (n=12). (F, G) Evaluation of the area proportions of collagen and elastic fibers (n=12). (H) Comparison of the fibrosis scores in the four groups (n=12). (I) Rabbit LF ultrastructure was examined with transmission electron microscopy (n=4). Results are shown as mean ± standard deviation; ^**P< 0.01;^***P<0.001; ^#P<0.05; ^##P<0.01; ^###P<0.001. BS, bipedal standing; LF, ligamentum flavum; EF, elastic fiber; CF, collagen fiber; ns, no significance.

Figure 4

Variations in cell type and activity within the hypertrophied LF. (A) The representative image of H&E staining reveals alterations in cell density in rabbit and human LF samples across different groups. Scale bar, 100μm. (B) Immunohistochemical analysis of α-SMA-positive cells in LF samples from rabbits and humans. Scale bar, 25μm. (C) Quantitative evaluation of LF cell density across each field (n=5). (D) Quantitative assessments of α-SMA-positive cells in each high power field of human LF (n=5). (E, F) The proliferative activity of cells in rabbit LF identified by BrDu. Scale bar, 25μm. ^*P<0.05, ^**P<0.01; ^#P<0.05, ^##P<0.01, ^###P<0.001. BS, bipedal standing; NLF, normal ligamentum flavum; HLF, hypertrophied ligamentum flavum; ns, no significance.

Figure 5

Mechanical stress promotes LF fibrosis by triggering local inflammation. (A) Red immunofluorescence staining on LF sections displayed COL1A1, COL3A1, TGF-β1, α-SMA, TNF-α, and IL-6. Scale bar, 50μm. (B) Quantitative evaluation of the fluorescence density of COL1A1, COL3A1, TGF-β1, α-SMA, TNF-α, and IL-6. (C) The mRNA levels of inflammatory cytokines and fibrosis-related factors analyzed through RT-PCR. (D) Representative images showing M1 markers (iNOS) detected using immunohistochemical staining. Scale bar, 50μm. (E) Expression levels of M1 makers (iNOS) in rabbit LF tissues as detected by ELISA. (F) Analysis of the correlation between iNOS protein levels and LF thickness. (G, H) The levels of TNF-a and IL-6 in rabbit LF tissues identified through ELISA. ^*P < 0.01, ^**P<0.05, ^***P< 0.0001; ^#P<0.05, ^##P<0.01. BS, bipedal standing; ns, no significance.

Figure 6

Mechanical stress induce LF fibrosis through promoting the fibroblast-to-myofibroblast transition. (A) The distribution of α-SMA (red) and TGF-β1 (green) in LF from the Steel Wire and control groups shown by double-immunofluorescence staining. Scale bar: 10μm. (B) TGF-β1 levels in the supernatants of different modes detected by ELISA. (C) Expression of fibrosis-associated proteins (COL3A1, TGF-β1, and α-SMA) after mechanical-stretch stimulation in different time-points detected using RT-PCR. (D, E) Analysis of fibrosis-related protein expression using Western blot after mechanical stretch at several time points. (F, G) Double-immunofluorescence staining of fibroblast after stimulation by mechanical stretch reveals the TGF-β1 (green) and α-SMA (red). Scale bar: 25μm. Mean ± SD results are presented; ^*P <0.05, ^**P <0.01, ^***P <0.001, ^****P <0.0001; ^##P < 0.01, ^###P < 0.001. MS, mechanical stretch; ns, no significance.

Figure 7

Proposed model depicting the mechanical stress-induced mechanism of LFH. Continuous mechanical stress causes micro-injuries and local inflammation in the LF, resulting in increased TGF-β1 secretion by LF fibroblasts and certain inflammatory cells. Subsequently, the increased TGF-β1 induced the transition of fibroblasts into myofibroblasts, finally resulting in collagen accumulation and LFH.