Increased pain and sensory hyperinnervation of the ligamentum flavum in patients with lumbar spinal stenosis.

Nociceptive sensory nerve fibers have never been investigated in the ligamentum flavum (LF) of patients with LSS. The aim was to analyze nociceptive sensory nerve fibers in the ligamentum flavum (LF) of patients with LSS. A prospective study in patients with lumbar spinal stenosis (LSS) undergoing invasive surgical treatment for lumbar spinal stenosis (LSS) with flavectomy was performed. Patients with LSS were subjected to flavectomy and density of sensory and sympathetic nerve fibers, macrophages, vessels, activated fibroblasts, and cells were investigated by immunostaining techniques. A group of patients with acute disc herniation served as control group. We found a higher density of sensory nerve fibers in LSS patients versus controls. These findings support the role of LF in associated low back pain. Density of sensory nerve fibers in LSS, was positively correlated with typical markers of clinical pain and functional disability, but not with LF density of activated fibroblasts. Inflammation as estimated by macrophage infiltration and higher vascularity does not play a marked role in LF in our LSS patients. In the present study, compared to men with LSS, women with LSS demonstrate more pain and depression, and show a higher density of sensory nerve fibers in LF. This study shed new light on nociceptive nerve fibers, which are increased in LSS compared to controls. The findings speak against a strong inflammatory component in LSS. A higher pain levels in women compared to men can be explained by a higher density of nociceptive nerve fibers. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-7, 2019.

MiR-21 promotes fibrosis and hypertrophy of ligamentum flavum in lumbar spinal canal stenosis by activating IL-6 expression.

The molecular mechanism underlying the fibrosis of ligamentum flavum(LF) in patients with lumbar spinal canal stenosis(LSCS) remains unknown. MicroRNAs are reported to play important roles in regulating fibrosis in different organs. The present study aimed to identify fibrosis related miR-21 expression profile and investigate the pathological process of miR-21 in the fibrosis of LF hypertrophy and associated regulatory mechanisms. 15 patients with LSCS underwent surgical treatment were enrolled in this study. For the control group, 11 patients with lumbar disc herniation(LDH) was included. The LF thickness was measured on MRI. LF samples were obtained during the surgery. Fibrosis score was assessed by Masson's trichrome staining. The expression of miR-21 in LF tissues were determined by RT-PCR. Correlation among LF thickness, fibrosis score, and miR-21 expression was analyzed. In addition, Lentiviral vectors for miR-21 mimic were constructed and transfected into LF cells to examine the role of miR-21 in LF fibrosis. Types I and III collagen were used as indicators of fibrosis. IL-6 expression in LF cells after transfection was investigated by RT-PCR and ELISA. Patients in two groups showed similar outcomes regarding age, gender, level of LF tissue. The thickness and fibrosis score of LF in the LSCS group were significantly greater than those in LDH group (all P < 0.05). Similarly, the expression of miR-21 in LSCS group was substantially higher than that in LDH group(P < 0.05). Furthermore, the miR-21 expression exhibited positive correlations with the LF thickness (r = 0.595, P < 0.05) and fibrosis score (r = 0.608, P < 0.05). Of note, miR-21 over-expression increased the expression levels of collagen I and III (P < 0.05). Also, IL-6 expression and secretion in LF cells was elevated after transfection of miR-21 mimic. MiR-21 is a fibrosis-associated miRNA and promotes inflammation in LF tissue by activating IL-6 expression, leading to LF fibrosis and hypertrophy.

The involvement and possible mechanism of pro-inflammatory tumor necrosis factor alpha (TNF-α) in thoracic ossification of the ligamentum flavum.

Thoracic ossification of the ligamentum flavum (TOLF) is characterized by ectopic bone formation in the ligamentum flavum and is considered to be a leading cause of thoracic spinal canal stenosis and myelopathy. However, the underlying etiology is not well understood. An iTRAQ proteomics was used to reveal the involvement of inflammation factors in TOLF. TNF-α is a pro-inflammatory cytokine implicated in the pathogenesis of many human diseases. Protein profiling analysis showed that the protein level of TNF-α increased in the ossified ligamentum flavum of TOLF, which was confirmed by western blot. The effects of TNF-α on primary ligamentum flavum cells was examined. Cell proliferation assay demonstrated that primary cells from the ossified ligamentum flavum of TOLF grew faster than the control. Flow cytometry assay indicated that the proportions of cells in S phase of cell cycle of primary cells increased after TNF-α stimulation. To address the effect of TNF-α on gene expression, primary cells were derived from ligamentum flavum of TOLF patients. Culture cells were stimulated by TNF-α. RNA was isolated and analyzed by quantitative RT-PCR. G1/S-specific proteins cyclin D1 and c-Myc were upregulated after TNF-α stimulation. On the other hand, osteoblast differentiation related genes such as Bmp2 and Osterix (Osx) were upregulated in the presence of TNF-α. TNF-α activated Osx expression in a dose-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated TNF-α activation of Osx expression. This suggests that TNF-α activates Osx expression through the mitogen-activated protein kinase ERK pathway. Taken together, we provide the evidence to support that TNF-α involves in TOLF probably through regulating cell proliferation via cyclin D1 and c-Myc, and promoting osteoblast differentiation via Osx.

Angiopoietin-like protein 2 promotes inflammatory conditions in the ligamentum flavum in the pathogenesis of lumbar spinal canal stenosis by activating interleukin-6 expression.

PURPOSE: Chronic inflammation is thought to cause ligamentum flavum (LF) degeneration and hypertrophy in lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is highly expressed in hypertrophied LF. Because Angptl2 regulates interleukin-6 (IL-6) expression in various tissues, we investigated whether IL-6 is expressed in hypertrophied LF and, if so, does Angptl2 induce IL-6 expression in LF fibroblasts. METHODS: LF tissue was obtained from LSCS patients and non-LSCS patients. Polymerase chain reaction (PCR) for Angptl2 and IL-6 genes and immunohistochemistry for IL-6 protein were performed in LF tissue. Fibroblasts from LF tissue were used for in vitro experiments. Expression of integrin α5ß1 (an Angptl2 receptor) and Angptl2 binding to receptors on LF fibroblasts were examined by fluorescence-activated cell sorter analysis and cell adhesion assays. After Angptl2 recombinant protein treatment, NF-κB activation and IL-6 expression in LF fibroblasts were investigated by immunocytochemistry, PCR, and enzyme-linked immunosorbent assay. RESULTS: IL-6 mRNA expression was increased in hypertrophied LF tissue from LSCS patients and positively correlated with LF thickness and Angptl2 mRNA expression. IL-6 protein was highly expressed in LF fibroblasts in hypertrophied LF tissue. In vitro experiments demonstrated integrin α5ß1 expression on LF fibroblasts and Angptl2 binding to cells via receptors. Angptl2 stimulation promoted NF-κB nuclear translocation and induced IL-6 expression and secretion in LF fibroblasts. CONCLUSIONS: Angptl2 promotes inflammation in LF tissue by activating IL-6 expression, leading to LF degeneration and hypertrophy.

Herniated intervertebral disk induces hypertrophy and ossification of ligamentum flavum.

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD). OBJECTIVES: To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines. METHODS: LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzyme-linked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed. RESULTS: Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1α, interleukin-6, tumor necrosis factor-α, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant. CONCLUSIONS: Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines.

Inflammatory cytokines induce fibrosis and ossification of human ligamentum flavum cells.

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and various inflammatory cytokines. OBJECTIVES: To examine the effect of inflammatory cytokines on LF cells and to identify their roles in the pathogenesis of LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by the degenerative processes (ie, increased collagen synthesis and chondroid metaplasia) of ligament fibroblasts. Degenerated intervertebral disk spontaneously produces inflammatory cytokines, which might affect the adjacent LF through local milieu of the spinal canal. METHODS: The interlaminar portion of the LF was collected during surgical spinal procedures in 15 patients (age range, 49-78 y) with lumbar spinal stenosis. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with various inflammatory cytokines: interleukin (IL)-1α, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO). Cytotoxicity was analyzed by MTT assays. DNA synthesis was measured with H-thymidine incorporation, and mRNA expression of types I, III, V, and XI collagen and osteocalcin were performed by reverse transcription-polymerase chain reaction. Histochemical stains such as Von Kossa were also performed to detect bone nodule formation. RESULTS: There was no cytotoxicity in the LF cells treated with each cytokine. There were significant increases in DNA synthesis and upregulated mRNA expression of types I, V, XI collagen and osteocalcin in LF cultures treated with various cytokines. LF cultures treated with IL-6, TNF-α, PGE2, and NO showed positive Von Kossa staining, indicating bone nodule formation from LF cells. CONCLUSIONS: Inflammatory cytokines (IL-6, TNF-α, PGE2, and NO) seem to play a crucial role in hypertrophy and ossification of LF. Degenerated, herniated intervertebral disks, and facet arthrosis may influence LF through inflammatory cytokines and cause hypertrophy and ossification of LF.

The "thickened" ligamentum flavum: is it buckling or enlargement?

BACKGROUND AND PURPOSE: Thickening of the LF is ascribed to buckling due to DSN. Uncertainty exists as to whether this can occur without DSN. Our primary hypothesis was that facet degenerative changes alone, independent of DSN, can thicken the LF. Our secondary hypothesis was that inflammatory changes surrounding degenerative facet joints may incite thickening. MATERIALS AND METHODS: Fifty-two patients were divided into 1 of 3 groups: group 1 (normal lumbar spine, n = 21), group 2 (LF thickening and FH with normal height of the L4-5 disk, n = 18), and group 3 (LF thickening and FH with decreased height of the L4-5 disk, n = 13). LF thickness measured on axial T1WI at the midpoint of the LF length was compared with that in group 1. Facet joints were evaluated for spurring, joint fluid, and cortical irregularity, indicating facet degeneration. Enhancement of the facet joints and LF thickening were also evaluated (n = 2). The Student t test was used to compare groups. RESULTS: Normal LF thickness (group 1) was 3.1 mm, whereas LF thickness averaged 4.9 mm in group 2 and 5.3 mm in group 3 (both P < .001). Patients with asymmetric LF thickness showed greater LF thickness on the side with greater FH. There was more LF enhancement on the side with greater facet degenerative disease. CONCLUSIONS: LF thickening can be secondary to facet degenerative changes, independent of DSN. Inflammatory changes may be an inciting factor for LF thickening.