The area was calculated 3 times and the average value was taken [ 5 ]. The algorithms for counting the cells were provided by the measurement software program Dynamic cell count BZ-H1c Keyence. To analyze the differences among three groups in the ratio of elastic fibers to collagen fibers and the cell count in human LF, ANOVA with the Tukey-Kramer post hoc test was performed. First, to evaluate whether the mouse was a feasible experimental animal model for studying LF hypertrophy, we performed histological analyses of the mice lumbar spine.
In the axial sections of the spine, HE staining showed that the mouse LF was located between the dural tube and facet joints Fig 1A. In the sagittal sections, EVG staining demonstrated that the LF ran between adjacent laminas and mostly consisted of black-staining elastic fibers Fig 1B.
These histological features were very similar to those of human LF. Therefore, we decided to use mice to examine the pathomechanism underlying LF hypertrophy.
The application of mechanical stress to the mouse LF using a novel device. C Photographs and D schematic illustrations of the device. To establish a LF hypertrophy mouse model, we initially developed a loading device by which the mouse LF was subjected to consecutive mechanical stress for the present experiment Fig 1C.
During the loading, the limbs were firmly strapped onto the bed under anesthesia. When the upper half of the bed was constantly moving, the spine was bent and extended repeatedly at the rate of 20 cycles per minute by the motor. To determine the appropriate loading, we performed a preliminary experiment with 12 weeks of mechanical stress loading for 1.
During this period, some mice in the 4. The controls were under anesthesia alone. To apply mechanical stress consistently at the L5-L6 level, we used the iliac crest line as an anatomical land mark of the level Fig 1E and confirmed that mechanical stress was adequately loaded to the mouse LF at the level by CT images Fig 1F. We found the sectional area in the stress group to be about 1.
Although the width was comparable between the two groups, the thickness of the stress group was significantly higher than that of the control group Fig 2B. These results indicated the successful establishment of a LF hypertrophy mouse model via mechanical stress. 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.
We then investigated the effect of mechanical stress on the extracellular matrix ECM. Previous observations in human samples demonstrated that the major ECM component was elastic fibers, while the minor component was collagen fibers in non-hypertrophied LF, whereas the ratio of collagen fibers was markedly increased in hypertrophied LF [ 4 , 5 ]. Similarly, in our mouse model, the area of collagen fibers was considerably higher in the stress group than in the control group Fig 2C.
Although the area of elastic fibers also increased, the density of elastic fibers decreased compared to that of collagen fibers Fig 2C—2F. We next evaluated the severity of LF hypertrophy in the mouse model in comparison to human samples. Non-hypertrophied LF of human showed a dense and regular bundle of elastic fibers, whereas severely hypertrophied LF showed thin, irregular, and fragmented elastic fibers in EVG staining Fig 3A.
In our mouse model, the elastic fibers in the control group were dense and aligned, whereas in the stress group, they were slightly degenerated, and a decreased elastin-to-collagen ratio was observed Fig 3C and 3D. These results indicated that our mouse model by mechanical stress was histologically identical to mildly hypertrophied LF of human. 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.
D Bar graph showing the ratio of elastic fibers to collagen fibers in the two groups. In addition to these ECM component changes, we examined the cellular distribution changes using human and our mouse samples. There were few cells in human non-hypertrophied LF, whereas a significantly increased number of cells was observed in human hypertrophied LF Fig 4A and 4B. Also in our mouse model, the number of Hoechst-positive LF cells was significantly higher in the stress group than in the control group Fig 4C and 4D.
Furthermore, the number of BrdU-positive proliferating cells was significantly higher in the stress group Fig 4E and 4F. We initially expected to observe BMDC infiltration by mechanical stress because macrophage infiltration was reported in human hypertrophied LF [ 21 ]. A and B The number of cells significantly increased with LF hypertrophy in humans. The white broken lines indicate the outlines of the LF. To examine the influence of mechanical stress on the activation of fibrosis-related factors in LF cells, we evaluated the gene expression of inflammatory cytokines, growth factors, and angiogenesis-related factors in the mouse model.
We therefore hypothesized that factors other than mechanical stress were involved in the progression of LF hypertrophy. To examine the pathological role of macrophages in LF hypertrophy, we induced macrophage infiltration by applying micro-injury to the normal mouse LF at the dorsal side. At 1 week after micro-injury, Iba1-positive macrophages had infiltrated around the injured lesion Fig 6A. Furthermore, laminin-positive micro-vessels and a significant increase in the levels of angiogenesis-related factors were also observed Fig 6A and 6D.
Indeed, excessive collagen synthesis without elastic fibers was observed in the injured area at 2 weeks after micro-injury Fig 6E , and LF hypertrophy was detected selectively in the micro-injury area at 6 weeks after injury Fig 6F. These results strongly suggested that macrophage infiltration was a significant factor involved in the progression of LF hypertrophy by activating collagen production. B-D Bar graphs showing the gene expression of collagens, fibrosis-related growth factors, and angiogenesis-related factors in the two groups.
The black broken lines indicate the outlines of the LF. The asterisk indicates the area to which micro-injury was applied. In this study, by applying consecutive mechanical bending stress to the mouse LF, we demonstrated that mechanical stress was one of the direct causes of LF hypertrophy. In the mouse hypertrophied LF, increased collagen fibers, proliferating cells, and the gene expression of several fibrosis-related factors were found.
In addition, macrophage infiltration with angiogenesis was induced by applying micro-injury to the mouse LF. These findings suggest that long-term mechanical stress and macrophage infiltration significantly influence the progression of LF hypertrophy. To date, previous studies have reported the common histological characteristics of human hypertrophied LF as follows: collagen deposition, elastic fiber fragmentation, and calcification [ 22 , 23 ]; inflammatory cell accumulation [ 21 ]; and an increased expression of fibrosis-related factors [ 6 — 8 , 24 , 25 ].
However, these pathological changes in human samples only indicated the advantaged stage of LF hypertrophy, and determining which factors contribute to the process of LF hypertrophy is difficult. Therefore, we established an experimental animal model to clarify its pathomechanisms. In our mechanical stress model, we confirmed the increases in collagen fibers, cell proliferation, and the fibrosis-related factors expression, in line with human LF pathology.
The main source of this cytokine was believed to be infiltrating macrophages in fibrosis [ 28 , 29 ]. Indeed, we found excessive collagen synthesis in the injured site at 2 weeks after micro-injury Fig 6E and confirmed LF hypertrophy at 6 weeks after micro-injury Fig 6F.
Another characteristic of human hypertrophied LF is angiogenesis. While the normal LF is non-vascularized, marked angiogenesis was observed in the area of collagen accumulation in the severely hypertrophied LF [ 21 ]. Several factors such as VEGF and MMPs are considered to be important for angiogenesis, and their actual expression has been mainly observed in fibrotic areas [ 30 ].
Therefore, the interplay between infiltrating macrophages and angiogenesis may also worsen the fibrotic pathology in LF hypertrophy progression. In our macrophage infiltration model, angiogenesis and the significantly increased expression of angiogenic factors were observed with infiltrating macrophages Fig 6A and 6D.
This macrophage infiltration model may help elucidate the effect of disrupting the cycle of macrophage infiltration and angiogenesis to prevent the progression to severe LF hypertrophy. Please Note: You can also scroll through stacks with your mouse wheel or the keyboard arrow keys. Updating… Please wait. Unable to process the form. Check for errors and try again. Thank you for updating your details.
Log In. Sign Up. Become a Gold Supporter and see no ads. Log in Sign up. Articles Cases Courses Quiz. About Recent Edits Go ad-free. Edit article. View revision history Report problem with Article. Citation, DOI and article data. Anonymized trial data would be published at www. Ligamentum flavum thickness was compared among groups using two-sample t-test or one-way analysis of variance. Paired comparison between groups was conducted using Student-Newman-Keuls test. Correlation of nationality, sex, height, age and weight with ligamentum flavum thickness was analyzed using Pearson's correlation coefficient.
Risk factors for ligamentum flavum hypertrophy were analyzed using multiple linear regression analysis. FDA Resources. Collecting lumbar CT imaging data of patients with lumbar spinal stenosis to observed the incidence of ligamentum flavum hypertrophy of patients with different nationalities, sexes, heights, ages, and weights and explored risk factors affecting ligamentum flavum hypertrophy.
Outcome Measures. Primary Outcome Measures : Ligamentum flavum thickness [ Time Frame: from May to May ] Changes in ligamentum flavum thickness were observed under different influential factors. Eligibility Criteria. Information from the National Library of Medicine Choosing to participate in a study is an important personal decision. Inclusion Criteria: Lumbar spinal stenosis revealed by Lumbar CT images Complained of low back pain Age between 45 and 80 years old Irrespective of sex Signed informed consent Exclusion Criteria: Spinal injury Spinal tumor Spinal infection Congenital or acquired spine deformity Pulmonary tuberculosis Severe systemic disease such as malignant tumor Cannot cope with the doctor due to mental illness or other causes.
Contacts and Locations. Information from the National Library of Medicine To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor. Please refer to this study by its ClinicalTrials. More Information. National Library of Medicine U. National Institutes of Health U. Department of Health and Human Services.
The safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Lumbar Spinal Stenosis.
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