Molecular alterations of human lumbar yellow ligament related to the process of intervertebral disk degeneration and stenosis.

PURPOSE: The objective of this study was to analyze the layers of yellow ligament in lumbar canal stenosis and disk herniation. METHODS: Eighteen ligaments were harvested from patients with lumbar spinal canal stenosis. Twenty-nine normal samples from lumbar spine disk herniation patients served as control. All surgical procedures were the same. Ligaments were stained in hematoxylin and eosin; picrosirius-hematoxylin for collagen; Weigert's resorcin-fuchsin for elaunin, oxytalan and elastic fibers; and transmission electron microscopy. Immunohistochemistry was performed for Il-6; Il-10; and CD-31, PGP9.5. Results are described in means and standard error (mean ± SE), and all analyses adopted the significance level of P < 0.05. RESULTS: Spinal stenosis ligaments were 2.5 × thicker. Control superficial ligaments presented a large number of thick, compact collagen fibers and a significant amount of oxytalan and mature elastic fibers. The deep layer presented a large number of mature elastic fibers. In the stenosis group, collagen was thinner and compacted in both layers. There was no difference in the interleukin profile among groups. The deep portion of the stenosis group presented a higher number of vessels and nerves. CONCLUSION: Two layers compose the elastic system of the normal ligamentum flavum, where the deep portion is mainly responsible for its elasticity (elaunin fibers), while its resistance depends on the concentration of oxytalan fibers, which are more present in the superficial layer. Ligamentum flavum in the stenosis samples presents more mononuclear infiltrate and more degraded elastic fibers with a higher number of vessels in its deep portion. These slides can be retrieved under Electronic Supplementary Material.

Histopathological changes in supraspinous ligaments, ligamentum flava and paraspinal muscle tissues of patients with ankylosing spondylitis.

OBJECTIVE: To examine the histopathological changes in spinal tissues of ankylosing spondylitis (AS) patients. METHODS: Tissue samples from 10 AS patients and 10 control subjects were obtained. Hematoxylin and eosin, picrosirius, Masson and van Gieson stainings were utilized to determine the pathological changes in tissues. Ultrastructural alterations were examined by electronic microscopy. Proteoglycan levels were assessed by enzyme-linked immunosorbent assays (ELISA). Matrix metalloproteinase-3 (MMP-3), transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) levels were evaluated by immunohistochemistry. RESULTS: Our results demonstrate that the density of collagen fibrils was reduced in the supraspinous ligaments of AS tissue and fibrils were loosely and irregularly organized as compared to a regular distribution of collagen fibrils in controls. In ligamentum flava from AS patients, activated fibroblasts with enlarged nuclei were detected, while the number of elastic fibers was greatly decreased. Paraspinal muscle tissues of AS patients exhibited increased collagen fibril accumulation and atrophy. Significantly decreased proteoglycan and elevated MMP-3 levels were found in supraspinous ligament samples from AS patients (P < 0.01). Additionally, the levels of TGF-ß1 in ligamentum flava and paraspinal muscle tissues of AS patients were increased (P < 0.01). The expression of TNF-α was also upregulated in the ligamentum flavum (P < 0.01), with no significant difference in the paraspinal muscle between control and AS patients (P > 0.05). CONCLUSIONS: Our findings reveal histopathological changes that occur in certain spinal tissues of AS patients and suggest that increased levels of MMP-3 and TGF-ß1 may contribute to the pathogenesis of AS.

Ultrastructural change of ligamentum flavum in galactosialidosis.

PURPOSE: Galactosialidosis is an autosomal recessive lysosomal storage disease caused by deficiency of both α-neuraminidase and ß-galactosidase due to a defect of the protective protein/cathepsin A. Three clinical subtypes have been described, depending on the age of onset and severity of the symptoms: the early infantile, late infantile and juvenile/adult form. We report an adult-type patient who underwent surgery for galactosialidosis-related spinal deformity, and showed a favorable course thereafter. METHODS: The patient was a 50-year-old male, and he consulted our hospital with pain of the bilateral anterior thigh. Lumbar radiograph showed applanation and horn-like deformity of the L2 vertebral body, which is characteristic of this disease, narrowing of the L1/2 intervertebral space, and topical kyphosis. Fenestration between the L1/2, decompression of the L2 nerve root, and posterolateral fusion involving the T12 to L3 were performed. RESULTS: Immediately after surgery, pain of the lower limbs disappeared. During the 2-year postoperative follow-up, bone assimilation was achieved, showing a favorable course. Histological examination of the ligamentum flavum (LF) collected during surgery showed that the elastic fibers were thin, whereas the collagen fibers were abundant and dense. The ligament cells were swollen, and there were a large number of vacuoles in the cytoplasm. CONCLUSION: This is the first report on spinal surgery for adult-type galactosialidosis and histological examination of spinal LF.

Calcium pyrophosphate crystal deposition in the ligamentum flavum of degenerated lumbar spine: histopathological and immunohistological findings.

We investigated the histological and immunohistochemical features of degenerative changes in the ligamentum flavum of the lumbar spine with calcium crystal deposition. We investigated degenerative changes in 270 ligamentum flavum specimens harvested from 198 patients who underwent decompressive surgeries for lumbar spinal canal stenosis. En bloc sections of the ligamentum flavum were examined histologically. We also examined immunoreactivity for transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF), CD34, and CD68; immunoblot analysis for VEGF; and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end-labeling (TUNEL) method. The ligamentum flavum showed fragmented and disorganized elastic fiber bundles with increased collagen fibrils in the matrix. Calcium deposition, which was identified as calcium pyrophosphate dihydrate crystals, was evident in 72 of 198 patients and in 99 of 270 samples, and was associated with appearance of hypertrophic chondrocytes and new blood vessel formation. Areas of calcium deposits were surrounded by abundant hypertrophic chondrocytes (with marked immunoreactivity to TGF-beta and VEGF) and a significant number of TUNEL-positive chondrocytes. Calcium crystal deposition in the lumbar ligamentum flavum progresses with reduction in elastic fibers and accumulation of collagen fibrils in the matrix as well as expansion of chondrometaplastic areas.

An ultrastructural study on the ligamentum flavum of the cervical spine in patients with ossification of the posterior longitudinal ligament.

Some histological analyses of the ossification of the posterior longitudinal ligament (OPLL) have been reported, but no ultrastructural studies of the ligamentum flavum (LF) in patients with OPLL have been published to date. To understand the pathology of the ossification of the spinal ligament, we examined, by electron microscopy, ultrastructural changes in the LF in cases of OPLL and made a comparison with the LF in cervical spondylotic myelopathy (CSM). Subjects were three men and two women with cervical OPLL who underwent longitudinal spinous process-splitting laminoplasty. During surgery, a small piece of the LF was collected from C2-C3 to C7-T1 and was then analyzed by light and electron microscopy. We observed atrophic elastic bundles with a two-layer structure and disarrangement, a partially torn area, the disappearance of microfibrils, and an enlarged interstitium with an irregular alignment of collagen fibrils. We observed some properties of a cell preceding its death: the initial phase may be the disappearance of the plasma-membrane, followed by the scattering of many organellae around its degenerated nucleus. Finally, many extracellular plasma membrane-invested particles that resemble matrix vesicles remain there without phagocytosis. These results suggest that ultrastructural abnormalities exist in the spinal ligament in cases of ossification of the spinal ligament.

Histological investigation of rabbit ligamentum flavum with special reference to differences in spinal levels.

The structure of the ligamentum flavum has yet to be fully elucidated and no studies have investigated fine structural differences at different spinal levels in any animals. The aim of the present study was to clarify structural differences in the ligamentum flavum at different spinal levels (cervical: C3/4 and C5/6; upper thoracic: T2/3; lower thoracic: T9/10; lumbar: L3/4) using light and electron microscopy of rabbit specimens. Light microscopy using resorcin-fuchsin staining revealed that the distribution of elastic fibers was diffuse in the cervical and upper thoracic regions, but was generally dense in the lower thoracic and lumbar regions. Electron microscopy demonstrated that the cervical and upper thoracic regions were rich in collagen fibers. Conversely, the lower thoracic and lumbar regions were rich in elastic fibers. Quantitative image analyses displayed thick elastic fibers in the lower thoracic and lumbar regions, with high area ratios. Radiographic examinations revealed that ranges of motion were large at the cervical region, but small at the lower thoracic and lumbar regions. These findings suggest that structure of the ligamentum flavum varies at different spinal levels with respect to differences in motion.

Calcium crystal deposition in the ligamentum flavum of the lumbar spine.

OBJECTIVE: To investigate the histological and immunohistochemical properties of the lumbar ligamentum flavum with calcium crystal deposition. METHODS: We examined the histological characteristics of the ligamentum flavum of the lumbar spine containing calcium deposits, obtained from 16 surgical cases with symptomatic lumbar spinal stenosis. Sections of the ligaments were also immunostained for elastase, chymotrypsin and S-100 protein, and examined by energy dispensive X-ray microanalysis and scanning electron microscopy. The results were compared with those of ligaments without calcium deposits. RESULTS: The elastic fibres of ligaments with calcium deposits showed marked degeneration (irregular arrangement and fragmentation of the fibre bundles) and nodular granulomatous lesions. Calcium crystal deposits were present in this area and in the nodular granulomatous areas. The calcified areas were surrounded by neutrophils and new small blood vessels. Immunostaining for elastase and chymotrypsin was positive in these areas, and S-100 protein-containing chondrocytes were detected around and within the calcified areas. Calcium pyrophosphate dihydrate crystals were identified in the calcified areas in all patients. CONCLUSION: Nodular degeneration of the elastic fibres of the ligament is probably caused by the action of proteolytic enzymes. Degeneration results in small granulomatous areas that form the nidus for calcium crystal deposition. Accumulated neutrophils, and S-100 protein-containing chondrocytes appear to precipitate the deposition of calcium crystals in the granulomatous lesions of the degenerated lumbar ligamentum flavum.

Light and electron microscopy of hydroxyapatite depositions in the ligamentum flavum.

STUDY DESIGN: Ligamenta flava obtained from 24 patients who had undergone surgery for degenerative spinal diseases were investigated regarding the histopathology of hydroxyapatite crystal depositions. OBJECTIVES: Light and electron microscopy and energy dispersive x-ray microanalysis were done to clarify the cause and process of hydroxyapatite crystal depositions in the ligament. SUMMARY OF BACKGROUND DATA: No reports have been made regarding the cause of calcification in the ligamenta flava induced by hydroxyapatite crystal depositions. METHODS: Samples were fixed in 10% formalin and 2.5% glutaraldehyde for light and electron microscopy. Energy dispersive x-ray microanalysis was done to detect calcium and phosphate hydroxyapatite crystal deposition areas. RESULTS: Hydroxyapatite crystals, which occasionally formed clusters, existed in the connective tissue of the dural side around abundant profiles of growing capillaries. Hydroxyapatite crystals showed needle shaped figures, and the x-ray microanalysis revealed that the ratio of calcium to phosphate was about 1.66:1. A decrease or loss of collagenous and elastic fibers was prominent in these hydroxyapatite deposition areas. CONCLUSIONS: Hydroxyapatite crystal depositions occur around the growing capillaries in the dural side of the ligamenta flava.

[Histological investigation of the ligamentum flavum].

The micro- and ultrastructures of the human ligamentum flavum were investigated. It was found that the ligamentum flavum is made of chiefly of elastic fibers. This kind of structure provides a basis for the important physiological functions of the ligamentum flavum. So the load is transmitted between the vertebrae and spinal stability is maintained by means of this ligament. Then clinical relevance was discussed.