Characterization of a Novel Model of Lumbar Ligamentum Flavum Hypertrophy in Bipedal Standing Mice

Objective To explore the main causes of hypertrophied ligamentum flavum (HLF) and the possibility of using bipedal standing mouse model to simulate the pathological changes in human HLF. Methods Thirty-two 8-week-old C57BL/6 male mice were randomly assigned to the experimental group (n = 16) and control group (n = 16). In the experimental group, mice were induced to adopt a bipedal standing posture by their hydrophobia. The experimental mice were maintained bipedal standing for 8 h a day with an interval of 2 h to consume food and water. The control mice were placed in a similar environment without bipedal standing. Eight 18-month-old C57BL/6 male mice were compared to evaluate the LF degeneration due to aging factor. Three-dimensional (3D) reconstruction and finite element models were carried out to analyze the stress and strain distribution of the mouse LF in sprawling and bipedal standing postures. Hematoxylin and Eosin (HE), Verhoeff-Van Gieson (VVG), and immunohistochemistry (IHC) staining were used to evaluate the LF degeneration of mice and humans. RT-qPCR and immunofluorescence analysis were used to evaluate the expressions of fibrosis-related factors and inflammatory cytokines of COL1A1, COL3A1, alpha-SMA, MMP2, IL-1 beta, and COX-2. Results The von Mises stress (8.85 x 10(-2) MPa) and maximum principal strain (6.64 x 10(-1)) in LF were increased 4944 and 7703 times, respectively, in bipedal standing mice. HE staining showed that the mouse LF area was greater in the bipedal standing 10-week-old group ([10.01 +/- 2.93] x 10(4) mu m(2)) than that in the control group ([3.76 +/- 1.87] x 10(4) mu m(2)) and 18-month-old aged group ([6.09 +/- 2.70] x 10(4) mu m(2)). VVG staining showed that the HLF of mice (3.23 +/- 0.58) and humans (2.23 +/- 0.31) had a similar loss of elastic fibers and an increase in collagen fibers. The cell density was higher during the process of HLF in mice (39.63 +/- 4.81) and humans (23.25 +/- 2.05). IHC staining showed that the number of alpha-SMA positive cells were significantly increased in HLF of mice (1.63 +/- 0.74) and humans (3.50 +/- 1.85). The expressions of inflammatory cytokines and fibrosis-related factors of COL1A1, COL3A1, alpha-SMA, MMP2, IL-1 beta, and COX-2 were consistently higher in bipedal standing group than the control group. Conclusion Our study suggests that 3D finite element models can help analyze the abnormal stress and strain distributions of LF in modeling mice. Mechanical stress is the main cause of hypertrophied ligamentum flavum compared to aging. The bipedal standing mice model can reflect the pathological characteristics of human HLF. The bipedal standing mice model can provide a standardized condition to elucidate the molecular mechanisms of mechanical stress-induced HLF in vivo.